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1.0.1Injury prevention initiative - FIFA 11+S. Della Villa1, M. Bizzini2, A. Junge2, J. Dvorak2

1Bologna/Italy, 2Zürich/Switzerland

Introduction: Football is the most popular sport in the world, played by approximately 200´000 professional and 240 million amateur players. Comparing the exposure-related incidence of injury in different team sports, ice hockey, handball, basketball, football and rugby are clearly types of sport with a relatively high risk of injury. The incidence of football injury has been investigated in several studies and varies substantially depending on the definition of injury, the characteristics of the investigated players and the research design. The majority of studies focus on adult male professional players. Epidemiological information on injuries in female and youth football players is limited. From the data presented, it is estimated that on average every elite male football player incurs approximately one performance-limiting injury a year.

Content: Several authors have described risk factors for football injuries, and discussed possibilities for prevention such as: warm-up (with more emphasis on stretching), regular cool-down, adequate rehabilitation with sufficient recovery time, proprioceptive training, protective equipment, good playing field conditions, and adherence to the existing rules. However, only few authors have reported results of preventive interventions in football players. Some studies have focused on the prevention of injuries in general, and others have evaluated the prevention of specific types of injury, namely ankle sprains, severe injuries of the knee and hamstring strains. Summarising the results of these studies, there is some evidence that multi-modal intervention programmes result in a reduction of injuries in general. Proprioceptive or neuromuscular training seems to prevent severe knee injuries and recurrent ankle sprains. The use of semi-rigid orthosis should be recommended for players with previous ankle sprains. The eccentric strength training of hamstrings has recently been shown to be effective in reducing hamstring strains. However, football injuries can be prevented only partly by improved physical condition of players. Knowing that a substantial amount of football injuries are caused by foul play, the observance of the laws of the game and especially the regard to Fair Play is an essential aspect in the prevention of injury. Recently, the effectiveness of specific exercise-based prevention programs in football has been examined. Research in amateur football has shown that specific programs were successfully implemented as “standard warm up” prior to the “routine” training. Recently, the effectiveness of specific exercise-based prevention program has been examined: the PEP program in the field of non-contact ACL injury, and moreover the 11 + program, were able to significantly reduce the injury incidence. “The 11+” injury prevention program was developed by F-MARC in cooperation with the Oslo Sports Trauma & Research Center and the Santa Monica Orthopaedics & Sports Medicine Center. The preventive exercises focus on core stabilisation, eccentric strength, neuromuscular control, agility and plyometrics. Good body control, and proper technique while performing the exercises are the key to enhance sensorimotor awareness and performance. The “11+” program represents an advanced version of the “11”, and is the result of a teamwork within the above mentioned research centers. In a recent published RCT study, Soligard et al showed that this program was effective in reducing the incidence of injuries by 1/3 in young female football players. The risk of severe injuries, overuse injuries, and injuries overall was significantly reduced. Another interesting finding in the study was that the results showed a trend toward a lower risk of injury among the most complaint players, which underlies the importance of compliance within the implementation of prevention programs. The teams in the intervention group performed regularly the “11+” as a routine warm-up, prior to the technical training. The program consists of three parts: a running part in the beginning and at the end to warm up, and six set of exercises focusing on core and legs strength, balance, plyometrics and agility (each exercises set with 3 levels of increasing difficulty). The “11+” should be performed at least two to three times a week, and takes about 20 minutes to be completed.

References:

Junge A, Dvorak J. Soccer Injuries. A Review on Incidence and Prevention. Sports Med 2004;34(13):929-938 Gilchrist G et al. A randomized controlled trial to prevent non contact anterior cruciate ligament injury in female collegiate soccer players. Am J Sports Med 2008 Soligard T et al. Comprehensive warm-up programme to prevent injuries in young female footballers: cluster randomized controlled trial. BMJ 2008

2.1.1A novel Polycarbonate-urethane meniscal implant: from bench to clinical useE. Linder-Ganz1, J.J. Elsner1, G. Zur1, A. Shterling1, R. Arbel2, V. Condello3, C. Zorzi3, F. Guilak4, E. Hershman5

1Netanya/Israel, 2Hod Hasharon/Israel, 3Negrar, Verona/Italy, 4Durham/United States of America, 5New York/United States of America

Introduction: The menisci play an important role in knee joint biomechanics. Clinical studies have shown that the loss of the meniscus leads to degenerative arthritis due to changes in cartilage load distribution [14]. In these cases, there is clearly a need to protect the articular cartilage by either repairing or replacing the meniscus. Meniscus replacement still represents an unsolved problem in orthopedics. Meniscal allografts have been shown to heal to the capsule and relieve pain [23]. However, besides problems related to availability, size matching, cost and risk of disease transmission, allograft menisci undergo remodeling after implantation, causing shrinkage and reduced mechanical strength [15,21]. These may lead to tearing of the allograft and contribute to uneven distribution of load, instability and recurrence of degenerative damage. Several meniscal substitutes based on synthetic and natural polymers have been described [3,5,10,20]. Most of these prostheses are based on biodegradable materials, which form temporary scaffolds that degrade in the body and are replaced gradually by newly formed tissue. Potential shortcomings of this approach include the lack of durability associated with most biodegradable materials under in vivo knee loading conditions [8,10], as well as the variability in the body response to the implant, limited age of the target population and the quality of the tissue formed. Traditional unicompartmental knee arthroplasty (UKA) is regaining popularity but requires significant bone resection and subsequent activity modification. Total knee replacement (TKR) is a reliable procedure but is not usually recommended for relatively young patients, less than fifty-five years of age, who will probably require subsequent revision surgery. The concept of a self-centering, non-fixed meniscal interpositional spacer that does not require osseous resection is an appealing alternative, designed to bridge the gap between the abovementioned approaches. Polycarbonate-Urethane (PCU) is a tough polymer with a low elastic modulus (10-100MPa, [16]). It is an attractive material for a meniscal implant application since it is durable and offers good mechanical and tribological properties that are comparable to those of natural cartilage [6,9,16,17]. Specifically, the natural meniscus is an anisotropic material and therefore it is suggested that circumferential reinforcement of PCU with high modulus UHMWPE fibers (Dyneema® Purity, DSM) be used to further improve its performance in this application. This is conceptually analogous to the structural characteristics of the natural meniscus where a highly orientated collagen fiber network supports the large hoop stresses to produce better distribution of contact pressures within the knee joint [1]. We hypothesized that a PCU-based meniscal implant, reinforced with high tensile modulus fibers, can provide better conformity in the knee joint as compared to previous approaches using hard materials as interpositional devices. Furthermore, we believe that such a compliant device can improve the load distribution by permitting local material deformation and thus delay degenerative changes and provide significant pain relief for the younger patient with osteoarthritis (OA). Therefore, the objectives of this study were (1) to develop a composite PCU-based meniscal implant that is able to restore the pressure distribution over the articular surfaces following meniscectomy, and (2) to apply a series of laboratory, computational and animal studies in order to evaluate the safety and effectiveness of the proposed implant towards clinical evaluation.

Content: The following sections will briefly review the development processes of a novel medial meniscal implant (Fig. 1a), composed of PCU reinforced with high modulus UHMWPE fibers (Dyneema® Purity, DSM). Implant Design As mentioned before, smart material design was considered a prime feature in the making of a compliant yet durable implant which is able to function as a load distributor on the medial cartilaginous surface. The implant was designed as a composite construct, reinforced circumferentially with UHMWPE fibers, embedded during the process of molding (Fig. 1a) to reproduce the structural characteristics of the natural meniscus which consists of a solid matrix embedded with a highly orientated collagen fiber network [1]. This combination of materials was chosen to enable the implant to withstand high impact forces while maintaining its form. The pliable matrix material should provide a damping effect and distribute pressure by permitting local material deformation whereas the reinforcement is designed to restrain matrix flow and bear a high portion of the stresses, i.e., compressive loads exerted on the

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implant transformed into tensile loads acting on the fibers. A representative geometric design for the meniscus shape was formed based on the analysis of more than 130 human knee MRI-scans. The lateral side of the implant body was restructured to form a full discoid shape by creating an artificial “bridge” along the gap between the original medial insertion points of the meniscus (the region of the inter-condylar notch). The preservation of the cruciate ligaments and prevention of undesired impingement were taken into account in the design, as well as knee alignment and stability. In-vitro biomechanical evaluation The biomechanical evaluation of the meniscal implant included more than 1500 in-vitro compression tests in 29 cadaveric knees, to assess the implants’ ability to distribute load on the tibial plateau. The compression test protocol described in detail elsewhere [12],. In brief, the implants were inserted into the medial compartment of cadaveric knees and were loaded under medial compression similar to the physiological load occurring during human gait cycle (1200N). Pressure distribution under the meniscal implant was measured utilizing flexible pressure sensors (Tekscan Inc., Boston, MA) and then compared to those attained for the natural meniscus prior to meniscectomy. Specifically, maximal pressure values and locations as well as pressure coverage area with respect to the natural meniscus were analyzed. A Peak-to-Average pressure relation (PAR) dimensionless ratio was defined, as an additional measure, which together with total contact area was used to compare the results to other related studies. Contact pressure distributions measured on the tibial plateau were in very good agreement to those measured under the intact natural meniscus of the specific knee (Fig. 1b). Peak and average pressures developed under the implant were compared to those measured under the natural meniscus and were found to be statistically indistinguishable (p≥0.05). Calculation of PAR (3.1±0.3) and contact area (658±135mm2) for the implant were also statistically indistinguishable compared to PAR (2.7±0.5) and contact area (642±96mm2) measured for the natural meniscus. Fifteen-million cycles fatigue test Cyclic mechanical compression/compression loading was applied to the implant according to fatigue tests requirements (ISO14243). The fatigue station was equipped with a flexible tube that maintained a continuous circulation of the heated test fluid (saline, 37°C). Six specimens of the smallest implant size were tested and considered the worst case scenario, since (i) it possesses the thinnest thickness to be subjected to the aforementioned compressive load, and (ii) the distance between the reinforcement fibers and the outer surface is the smallest. Thus, for the smallest implant, load would be distributed on a smaller area and may lead to the development of greater internal stresses. MRI-based UHMWPE replicas of the medial tibial plateau and femoral condyle were used as the compression surfaces. Fifteen-million loading cycles were applied on each specimen and the implant’s structure and functionality were examined before and after the test. The tests demonstrated that both of the implant’s components, PCU and UHMWPE fibers were not affected in the long term in respect to form, fiber-matrix bonding and structure-function relationship. Specifically, no significant dimensional changes were observed during the course of the test and pressure distributions post 15-milion loading cycles remained similar to those measured prior to the test. Finite element analysis A Finite element (FE) model of the medial knee with the PCU implant inside was developed and internal strains/stresses developed in the PCU bulk and UHMWPE fibers were calculated [11]. The model geometry was based on MR-scans of a cadaveric specimen and analyzed under 1200N compression: comparable to the biomechanical evaluation and other FE models. Peak stresses were compared to the allowed values supplied by the manufacturer for each material. The model was validated by comparing computational results to analogous tibial plateau contact pressures, measured in cadaveric knees in vitro [12]. Peak von-Mises, compressive and tensile stresses in the PCU were all lower than the maximal allowed stress (15MPa). Similarly, the peak tensile stress calculated in the fibers was significantly lower than the material’s yield stress (3.1GPa). Animal study All procedures were approved by the Institutional Animal Care and Use Committee of the Technion University Israel (#10-6-11-06) and are described in detail elsewhere [13]. Six ewes (1-2 years, 60-80 Kg) were allocated for the research. The sheep underwent a full meniscectomy of the medial meniscus of their left knee, and were implanted with a PCU meniscus substitute. Smaller joint tolerance in sheep required the release and reattachments of the MCL from the epicondyle to ease the insertion of the implant. Subsequent to the rehabilitation period, the sheep were relocated to a large pen and were allowed to ambulate freely. Functionality of the joint was assessed by measuring mobility and range-of-motion (ROM). Animals were euthanized at 3 (n=3) and 6 (n=3) months. Cartilage and the surrounding soft tissues of both knees were assessed macroscopically and microscopically, using a semi-quantitative histological analysis, based on a modified Mankin scale [4]. The contra-lateral knee served as control. From gross inspection, the PCU implant remained well-secured throughout the

experimental period and showed no visible signs of wear. Gross and microscopic examinations of the explanted PCU implant’s surfaces did not reveal any changes in their structural or material properties. Histological analysis showed relatively mild degenerative changes in the articular cartilage that were dominated by loss of proteoglycan content and cartilage structure. However, the total OA score did not significantly differ between the control and operated knees and there were no differences in the severity of degenerative changes between 3 and 6 months post surgery. Surgical procedure development The device was implanted under operating room conditions in >30 cadaveric knees, firstly in order to develop tools and a surgical procedure for the insertion of the device and secondly, as a part of a training program for surgeons. In general, the surgical procedure included standard arthroscopic meniscectomy, ~4 cm incision opening, a trial-based sizing confirmation, implantation, and closing. After insertion, the implant was found to independently self-center into its designated location due to its discoid shape. The insertion was initially performed by pushing the implant manually into the joint space, though at a later stage, when insertion tools were produced, the surgeon could decide whether to use the manual approach or a custom insertion tool. First clinical results Following all of the necessary laboratory tests and regulatory approvals (CE Marking 0473), the meniscal implant was implanted in 18 patients, of May 2010. The arthroscopic implantation procedure was short and uncomplicated. Arthroscopic observation assured that the device was located in its intended position between the medial femur and tibia. In addition, correct sizing was verified in terms of physical examination, e.g., the implant should rotate smoothly with the knee as the knee flexes and extends without undesired contact with the PCL or the ACL. The outcomes used in the study were the KOOS, VAS-Pain, Lysholm, and IKDC. MRI images were taken after 1.5, 12 and 24 months (Fig. 2a). The averaged KOOS results showed an improvement of ~65% in pain, ~12% in symptoms, ~35% in daily activity, ~30% in sports, and ~65% in quality of life, 1.5-months post implantation (Fig. 2b). When looking at the 12-months results, the averaged KOOS results showed an improvement of ~75%, ~35%, ~45%, ~250%, and ~120% in pain, symptoms, daily activity, sport and quality of life sections, respectively. DISCUSSION In the current study, we presented the development of a novel PCU meniscal implant for the medial compartment of the knee, along with an overview of essential tests. The main benefits claimed for this meniscal implant are pain relief and preservation of meniscal functionality. In a comprehensive review of synthetic meniscal implant solutions by van Tienen et al., (2009) it was concluded that the material requirements for artificial total meniscus replacement are not fully addressed to date, implying that cartilage damage in such case has not yet been prevented [22]. In the current work a new approach in the field of total meniscal replacement is presented. We suggest the use of a stable (non-degradable) PCU implant with exceptional mechanical and tribological properties to restore the missing functions of the meniscus following meniscectomy, e.g., shock absorption, pressure distribution and consequent chondroprotection. It is well agreed that the meniscus’s main biomechanical role is to distribute joint forces over a wider area to lower the pressure developed in the articular cartilage [18]. Good resemblance of the natural meniscus pressure patterns, as resulted from the current study, to other related works was observed. Specifically, PAR calculations (2.7±0.5) and contact area measurements for the natural meniscus (642±96mm2) were in accordance with ‘typical’ normative PAR values and contact area measurements [7]. Likewise, the implant’s PAR and contact area were in agreement with those measured for the natural meniscus. Interestingly, the meniscal implant did not display a focal stress concentration that was typically observed for natural menisci where direct femur to tibia contact occurred. This finding is due to the implant’s closed discoid shape, which is able to provide a larger, continuous bearing surface compared to the semi-lunar natural meniscus. The implication of these being that (a) the implant is able to reduce the overall cartilage load associated with meniscectomy by effectively distributing joint loads, and (b) the implant completely prevents contact between opposing cartilage surfaces. The in-vivo study of an analogous implant configuration in sheep showed that minimal changes associated with joint remodeling had occurred within the first 3 months. Such changes can be linked to the surgical procedure itself (e.g. joint opening and MCL release and reattachment from the epicondyle), and this is supported by the longer term (6 months) findings, which were consistent with the 3 months findings showing that on the whole, cartilage was preserved well [13]. On the other hand, it has recently been shown that total medial meniscectomy in a sheep model leads to extensive destruction of articular cartilage on the medial tibial and femoral condyles in as little as 3.5 months [8]. In light of these findings, the results of the current study can be considered exceptionally favorable, and support the hypothesis that a PCU meniscal implant may counter the occurrence of major degenerative cartilage changes following

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meniscectomy. Implantation of a non-fixed meniscal implant provides additional advantages compared to fixed implants reported to date. Without additional joint preparation or manipulation other than meniscectomy (e.g., drilling of bone tunnels or suturing to existing elements). The implantation surgical procedure is relatively short (20-25 min.), simple and straightforward, and does not require special expertise. It has been shown, for instance, that the performance of a fixed meniscus is very much influenced by the location and method of fixation [2,18,19], and calls for specialized training. Furthermore, preservation of existing bone stock is expected to reduce pain and rehabilitation time considerably and to increase the success rate of future revisions/interventions. In conclusion, this paper summarizes a series of studies carried out to design, create, evaluate, and implant a novel free-floating meniscal replacement. Preliminary clinical studies show a significant beneficial effect of the KOOS scores in patients receiving this implant. Our findings show that the device described here can significantly relieve pain post-injury and may delay the need for more aggressive procedures that require bone resection such as UKA or TKR among the goal-target population.

References:

1. Adams M, Hukins D. The extracellular matrix of the meniscus. New York, NY, Raven Press 1992.

2. Alhalki MM, Howell SM, Hull ML. How three methods for fixing a medial meniscal autograft affect tibial contact mechanics. Am J Sports Med 27: 320-328, 1999.

3. Buma P, van Tienen T, Veth R. The collagen meniscus implant. Expert Rev Med Devices 4: 507-516, 2007.

4. Carlson CS, Guilak F, Vail TP, Gardin JF, Kraus VB. Synovial fluid biomarker levels predict articular cartilage damage following complete medial meniscectomy in the canine knee. J Orthop Res 20:92-100, 2002.

5. Chiari C, Koller U, Dorotka R, Eder C, Plasenzotti R, Lang S, et al. A tissue engineering approach to meniscus regeneration in a sheep model. Osteoarthritis Cartilage 14: 1056-1065, 2006.

6. Elleuch R, Elleuch K, Salah B, Zahouani H. Tribological behavior of thermoplastic polyurethane elastomers. Materials & Design 28: 824-830, 2007.

7. Fukubayashi T, Kurosawa H. The contact area and pressure distribution pattern of the knee. A study of normal and osteoarthrotic knee joints. Acta Orthop Scand 51: 871-879, 1980.

8. Kelly BT, Robertson W, Potter HG, Deng XH, Turner AS, Lyman S, et al. Hydrogel meniscal replacement in the sheep knee: preliminary evaluation of chondroprotective effects. Am J Sports Med 35: 43-52, 2007.

9. Khan I, Smith N, Jones E, Finch DS, Cameron RE. Analysis and evaluation of a biomedical polycarbonate urethane tested in an in vitro study and an ovine arthroplasty model. Part II: in vivo investigation. Biomaterials 26: 633-643, 2005.

10. Kobayashi M, Toguchida J, Oka M. Development of an artificial meniscus using polyvinyl alcohol-hydrogel for early return to, and continuance of, athletic life in sportspersons with severe meniscus injury. II: animal experiments. Knee 10: 53, 2003.

11. Linder-Ganz E, Elsner JJ, Danino A, Zur G, Guilak F, and Shterling A. Design of a Polycarbonate-Urethane meniscal implant: Finite element approach. The 2009 ASME-SBC, June 17-21, Lake Tahoe, California, USA, 2009.

12. Linder-Ganz E, Elsner JJ, Danino A, Guilak F, and Shterling A. A novel quantitative approach for evaluating contact mechanics of meniscal replacements. J Biomech Eng 132: 024501, 2010.

13. Linder-Ganz E, Elsner JJ, Zur G, Shani J, Brenner O, Hershman E, Guilak F, Shterling A. Chondroprotective effects of a Polycarbonate-Urethane meniscal implant in a sheep model. The 2010 ORS meeting, March 6-9, New Orleans, Louisiana, USA, 2010.

14. McDermott ID, Amis AA. The consequences of meniscectomy. J Bone Joint Surg Br 88: 1549-1556, 2006.

15. Noyes FR, Barber-Westin SD, Rankin M. Meniscal transplantation

in symptomatic patients less than fifty years old. J Bone Joint Surg Am 86-A: 1392-1404, 2004.

16. Scholes SC, Burgess IC, Marsden HR, Unsworth A, Jones E, Smith N. Compliant layer acetabular cups: friction testing of a range of materials and designs for a new generation of prosthesis that mimics the natural joint. Proc Inst Mech Eng [H] 220:583-596, 2006.

17. Scholes SC, Unsworth A, Jones E. Polyurethane unicondylar knee prostheses: simulator wear tests and lubrication studies. Phys Med Biol 52:197-212, 2007.

18. Sekaran SV, Hull ML, Howell SM. Nonanatomic location of the posterior horn of a medial meniscal autograft implanted in a cadaveric knee adversely affects the pressure distribution on the tibial plateau. Am J Sports Med 30:74-82, 2002.

19. Tienen TG, Verdonschot N, Heijkants RG, Buma P, Scholten JG, van Kampen A, et al. Prosthetic replacement of the medial meniscus in cadaveric knees: does the prosthesis mimic the functional behavior of the native meniscus? Am J Sports Med 32:1182-1188, 2004.

20. Tienen TG, Heijkants RG, de Groot JH, Pennings AJ, Schouten AJ, Veth RP, et al. Replacement of the knee meniscus by a porous polymer implant: a study in dogs. Am J Sports Med 34:64-71, 2006.

21. van Arkel ER, de Boer HH. Survival analysis of human meniscal transplantations. J Bone Joint Surg Br 84227-231, 2002.

22. van Tienen TG, Hannink G, Buma P. Meniscus replacement using synthetic materials. Clin Sports Med 28:143-156, 2009.

23. Verdonk PC, Demurie A, Almqvist KF, Veys EM, Verbruggen G, Verdonk R. Transplantation of viable meniscal allograft. Survivorship analysis and clinical outcome of one hundred cases. J Bone Joint Surg Am 87:715-724, 2005.

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2.2.2Adult Stem Cells and Nanomaterials in Skeletal Tissue EngineeringR. TuanPittsburgh/United States of America

Introduction: Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the “regenerate tissue” needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This lecture will present the promises as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomaterial scaffolds. The biology of human adult mesenchymal stem cells, particularly the mechanisms regulating their proliferation versus differentiation into specific lineages, is intricately regulated by cell-cell interactions, signaling by extracellular bioactive factors, and transcriptional and epigenetic activities. More importantly, the extracellular matrix milieu provides critical cues, both architectural and structure-dependent, to guide cell-based tissue morphogenesis. We have developed biomimetic and biodegradable nanofibrous biomaterials to serve as scaffolds for cell-based tissue engineering. Information on the fabrication and biological basis of the scale-dependent bioactivities of the nanofibrous scaffold will be presented. Cell-nanofibrous constructs are currently being developed for the engineering of cartilaginous tissues, including articular cartilage and intervertebral disc. In conclusion, cell-based tissue engineering and regenerative medicine is an exciting, inter-disciplinary, and potentially high-impact research field that presents a natural platform for collaboration among life scientists, engineers, and clinicians.

Content: Nanoscale materials are the fundamental building blocks and functional subunits of cells, including subcellular organelles and extracellular matrix components. Currently, there is growing recognition of the importance of understanding and incorporating nanobiology into biomedical applications. This issue is of particular importance in the emerging field of regenerative medicine, the goal of which is to develop methods to repair, replace, and regenerate diseased, injured, or non-functional tissues. Towards this goal, stem or progenitor cells have been considered a highly desirable candidate cell type, because of their expandability and potential to be induced toward specific cell differentiation lineages. A key requirement in tissue engineering and regenerative medicine is that ultimately the “regenerate tissue” needs to be a three-dimensional structure. In weight-bearing musculoskeletal tissues, this requirement is particularly critical. Musculoskeletal disorders affect one out of seven Americans. This severe disease burden underscores the need to develop novel and effective treatment protocols. This lecture will present the promises as well as the challenges in the field of skeletal tissue engineering and regeneration, specifically the application of adult stem cells and nanomaterial scaffolds. The biology of human adult mesenchymal stem cells, particularly the mechanisms regulating their proliferation versus differentiation into specific lineages, is intricately regulated by cell-cell interactions, signaling by extracellular bioactive factors, and transcriptional and epigenetic activities. More importantly, the extracellular matrix milieu provides critical cues, both architectural and structure-dependent, to guide cell-based tissue morphogenesis. We have developed biomimetic and biodegradable nanofibrous biomaterials to serve as scaffolds for cell-based tissue engineering. Information on the fabrication and biological basis of the scale-dependent bioactivities of the nanofibrous scaffold will be presented. Cell-nanofibrous constructs are currently being developed for the engineering of cartilaginous tissues, including articular cartilage and intervertebral disc. In conclusion, cell-based tissue engineering and regenerative medicine is an exciting, inter-disciplinary, and potentially high-impact research field that presents a natural platform for collaboration among life scientists, engineers, and clinicians.

2.2.3Growth factors as regulators of chondrogenesis in vitro and in vivoG.J.V.M. Van OschRotterdam/Netherlands

Introduction: Cartilage defects do not heal well, despite the fact that chondrocytes in cartilage wound areas increase the expression of various growth factors (Bos et al 2001) and during surgical repair procedures, such as in micro-fracture, endogenous growth factors are being released. In order to improve the repair of cartilage defects using surgical techniques, application of growth factors, cells or bioactive materials more knowledge about the role of growth factors in cartilage repair is required. We have addressed the following questions:

· what is the effect of growth factors on chondrogenic differentiation of progenitor cells?

· what is the effect of growth factors on matrix synthesis and matrix assembly by chondrocytes? We focused our research on two well known growth factors in cartilage repair: Transforming Growth Factor-beta (TGF-beta) and Fibroblast Growth Factor (FGF).

Content: 0Growth factors and chondrogenic differentiation Mesenchymal progenitor cells derived from bone marrow stroma are well known to be able to differentiate to the chondrogenic lineage when cultured in the presence of TGF-beta. Human Bone Marrow Stroma-derived Cells (hBMSC) cultured in pellets go through phases similar to embryonic limb development: cellular condensation, early chondrogenic differentiation, finally inevitably leading to terminal differentiation (Hellingman et al 2010). Implantation of these cells in-vivo results in formation of bone tissue (Farrell et al 2009 and unpublished data). During chondrogenic differentiation of hBMSC in vitro FGF Receptors (FGFR) 1, 2, 3 are expressed in a stage specific manner that is comparable to embryonic limb development. During the condensation phase (N-cadherin expression)FGFR1 drops and FGFR2 showed in peak in expression. Differentiating chondrocytes (collagen II positive, collagen X negative) did not express any FGFRs. During hypertrophy (collagen X positive) all FGFRs were expressed. Different FGFs are known to have different binding affinities for the different FGFRs. To examine potential application of our findings, hBMSC were treated with FGF2 (high affinity for FGFR1) or FGF9 (High affinity for FGFR2 and FGFR3) in a stage specific manner (Hellingman et al 2010). When added during the entire culture period, both FGF2 and FGF9 treated pellets contained significantly less GAG at day 35 than control pellets. Pellets treated with FGF2 from day 3-14 had a lower GAG content at day 35 than control pellets whereas a trend towards a higher GAG content was seen when FGF 9 was added. Addition of FGF during hypertrophic differentiation is detrimental for cartilage-matrix production. Although all receptors are expressed during hypertrophy, FGF2 and FGF9 have differential effect when added from day 21-35. While FGF2 inhibits further matrix deposition, FGF9 increases matrix resorption. This suggests that the FGFRs have specific effects, even during hypertrophy when they are all expressed. We have also investigated the TGF-beta pathway in more detail (Hellingman et al unpublished data). TGF-beta signals through the canonical SMAD pathway (SMAD2/3) but recently it has been shown that also SMAD1/5/8 pathway can be targeted. We investigated the role of these different TGF-beta signaling pathways in chondrogenically differentiating BMSC. Terminally differentiated BMSC produced in vitro by addition of TGF-beta stained positive for both Smad2/3P and Smad1/5/8P, similar to cartilage in murine embryonic limbs. On the other hand, permanent hyaline cartilage that lacks expression for MMP13 and collagen X only expressed Smad2/3P. To investigate the role of Smad signaling pathways, Smad2/3 phosphorylation was blocked by addition of SB-505124 or Smad1/5/8 phosphorylation was blocked by addition of dorsomorphin in chondrogenically differentiation hBMSC. When either of them was added through-out culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P from day 14-35 resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time resulted in decreased expression of MMP13, collagen X and alkaline phosphatase without inhibiting further collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. Growth factors and cartilage matrix assembly TGF-beta and FGF2 have differential effect on matrix production by differentiated chondrocytes in alginate (Jenniskens et al 2006; Bastiaansen-Jenniskens et al 2010). FGF2 inhibits collagen and COMP deposition whereas TGF-beta inhibits proteoglycan deposition and

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collagen cross-link formation but stimulates COMP deposition. The distribution of extra-cellular matrix components over territorial and inter-territorial matrix is also differentially affected by these growth factors. In addition, modulation of glycosaminoglycan synthesis influences the assembly of collagen and vice versa. As a resultant of all these actions, growth factors can influence the mechanical properties of the produced matrix. We have demonstrated that the effect of a growth factor depends on the stage in which it is added in the medium. Since the effects of growth factors and modulation of intracellular signalling are mostly studied throughout culture, more attention to differentiation stage-specific effects and application may be warranted to improve cartilage repair. It is unpredictable what happens if different growth factors are present simultaneously (e.g. during wound healing in vivo). The growth factors may very well be counteractive. Knowledge about interactions between growth factors in the various stages and their mechanisms of action is largely absent. More specific, temporal control of growth factor signaling can help to optimize cartilage repair.

References:

Bastiaansen-Jenniskens YM, de Bart ACW, Koevoet W, Jansen KMB, Verhaar JAN, van Osch GJVM, DeGroot J. Stimulation of COMP Production in Cartilage Matrix Generation Decreases Collagen Fibril Diameter. Cartilage 2010 in press Bos PK, van Osch GJ, Frenz DA, Verhaar JA, Verwoerd-Verhoef HL. Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model. Osteoarthritis Cartilage. 2001 May;9(4):382-9. Farrell E, van der Jagt OP, Koevoet W, Kops N, van Manen CJ, Hellingman CA, Jahr H, O‘Brien FJ, Verhaar JA, Weinans H, van Osch GJ. Chondrogenic Priming of Human Bone Marrow Stromal Cells: A Better Route to Bone Repair? Tissue Eng part C Methods 2009 Jun;15(2):285-95 Hellingman CA, Koevoet W, Kops N, Farrell E, Jahr H, Liu W, Baatenburg de Jong RJ, Frenz D, van Osch G. Fibroblast Growth Factor Receptors in in-vitro and in-vivo chondrogenesis: Relating Tissue Engineering using adult mesenchymal stem cells to embryonic development. Tissue Eng Part A. 2009 Sep 3. Hellingman. CA , Blaney Davidson EN, Koevoet W, Vitters EL, van den Berg EB, van Osch GJVM, van der Kraan PM. Smad signaling determines chondrogenic differentiation of bone-marrow derived mesenchymal stem cells. Submitted for publication. Jenniskens YM, Koevoet W, de Bart AC, Weinans H, Jahr H, Verhaar JA, DeGroot J, van Osch GJ. Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFbeta2 and FGF2. Osteoarthritis Cartilage. 2006 Nov;14(11):1136-46.

Acknowledgments:

This research was financially supported by the Dutch Program for Tissue Engineering and the Dutch Arthritis Association.

2.3.1Enhancing tensile and compressive properties of self-assembled articular cartilageK. Athanasiou1, J. Hu1, D. Responte2

1Davis/United States of America, 2Houston/United States of America

Introduction: Due to articular cartilage’s low repair capacity, alternate strategies to effect a robust reparative process need to be developed, providing a strong impetus for tissue engineering. Tissue engineering strategies can be categorized as scaffold-based or scaffoldless. Scaffold-based tissue engineering uses a synthetic and/or natural polymer to create a temporary matrix for cells to populate. Various synthetic polymers1,2 and natural materials such as collagen,3,4 hyaluronic acid,5-7 and fibrin8,9 have been investigated for cartilage regeneration. Additionally, there are various scaffoldless approaches including pellet culture,10 aggregate culture,11 and self-assembly.12 The key advantage of these methods is increased cell-cell interaction, which has been shown to promote chondrocyte differentiation.13-15 Despite these various approaches, studies have n1ot yet produced neotissue with the same properties as native cartilage. To improve the functionality of constructs, various biochemical and biomechanical stimuli have been studied. In particular, members of the TGF-β superfamily have been shown to increase the deposition of GAGs16-18 and collagen.19,20 Insulin growth factor-1 (IGF-1) has been shown to increase GAG production in both explants21 and tissue engineered constructs.22-24 Mechanical stimuli can also be employed to advance tissue engineering efforts. Direct compression has been used to modulate matrix composition and concomitantly influence construct properties.25,26 In addition, hydrostatic pressure application has increased gene transcription,27 collagen production,28,29 and construct tensile properties.30 However, even with the administration of external stimuli, engineered cartilage has not yet attained the mechanical properties of native tissue. Our laboratory seeks to develop new scaffoldless strategies for articular cartilage engineering. In particular, a self-assembly process has been developed to grow functional neotissue that could potentially replace degenerated cartilage. Self-assembly acts as a simple, yet effective, platform technology that can be enhanced by applying various biochemical and biomechanical stimuli. Additionally, this versatile methodology can be employed to engineer a wide spectrum of cartilage types and geometries.

Content: Self-assembly: a new tissue engineering strategy

The problems associated with scaffolds including biocompatibility issues and exogenous degradation products spurred the investigation of a novel scaffoldless approach for functional tissue engineering. It was found that when chondrocytes were cultured at high density in non-adherent molds, the cells aggregated to form constructs that not only appeared to be hyaline cartilage-like but also had functional properties of the same order as native values. This self-aggregation or self-assembly of chondrocytes provided numerous advantages over scaffolds including increased retention of phenotype, increased cell-cell contact, and lack of degradation products. As a result, a comprehensive tissue engineering strategy was developed, based on self-assembly and a panoply of assays, to achieve the formation of cartilage constructs effectively and functionally. The objective continues to be the development of a clinically feasible method for engineering cartilage with the biochemical properties and mechanical integrity of native tissue. Self-assembly was developed based on the differential adhesion hypothesis to produce robust cartilage constructs (Fig. 1A). For example, on a dry weight basis these tissue-engineered constructs contained two thirds more GAG than native tissue. Collagen reached one third the level of native tissue, and the compressive stiffness reached more than one third of native tissue values.7 This progress toward achieving native biomechanical properties and matrix composition was exciting as it provided early validation for this scaffoldless approach.

These promising results spurred an investigation of the mechanism underlying self-assembly (Fig. 1B).19 Increased N-cadherin expression during neotissue formation suggested that differential adhesion mediated self-assembly. Also, several biochemical properties recapitulated cartilage development including an increased proportion of collagen II, decreased proportion of collagen type VI, decreased chondroitin 6- to 4- sulfate ratio, and localization of collagen VI to the pericellular matrix. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 weeks. These studies showed that the self-assembly method mimicked tissue development and maturation, suggesting that a set of exogenous stimuli could then be applied to augment tissue functional properties. Various growth factors, applied individually

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and in combination, were investigated to improve the functionality of self-assembled constructs. For example, a combination treatment of BMP-2 and IGF-I resulted in over 1-fold increases in aggregate modulus, accompanied by increases in GAG production. However, TGF-β1 was found to be the most potent growth factor, inducing 1-fold increases in both aggregate modulus and tensile modulus, and increasing GAG and collagen content.13 These findings are exciting as coupling application of select growth factors with the self-assembly process resulted in tissue engineered constructs with substantially improved functional properties.

Hydrostatic pressure stimulation was also advantageous for self-assembly. After applying hydrostatic pressure of different magnitudes, durations, and frequencies, a particularly effective regimen was identified. At the counterintuitive frequency of 0 Hz (i.e., static), 10 MPa, applied for 1 hour on days 10-14 of a 4 week culture, significantly increased the aggregate modulus by 1.4-fold. It was exciting to note that this regimen also affected functional properties that seem to be difficult to improve upon, namely tensile modulus and strength along with corresponding collagen content, which increased over 2-fold.20 For the first time, this study examined the immediate and long-term effects of hydrostatic pressure on biomechanical properties, and demonstrated that hydrostatic pressure has an optimal application time in construct development. The next logical step was to combine the optimal regimens of the growth factor and hydrostatic pressure stimuli. The combination further improved the properties of the tissue engineered constructs. Thus, the combination of 10 MPa static hydrostatic pressure, applied for 1 hour a day for 5 days, and 30 ng/ml TGF-β1 had an additive effect on the mechanical properties, increasing the aggregate modulus by 164% and the Young’s modulus by 231%, approaching 300 kPa and 2 MPa, respectively (Fig. 2A). Additionally, the combined treatment had a synergistic effect on collagen content, increasing it by 173%.18 Thus, the combination of hydrostatic pressure stimulation with growth factor application resulted in the formation of tissue engineered constructs with biomechanical and biochemical properties spanning native articular cartilage values.

Applying direct compression increases the aggregate modulus by 70%

Since hydrostatic pressure was proven to be such a potent stimulator, it became apparent that other biomechanical stimuli ought to be examined. Thus, a direct compression instrument was developed to compress samples at specific frequencies and magnitudes. By applying dynamic compression to medial meniscal explants, we showed that aggrecan was up-regulated by 108%.21 The beneficial effects of dynamic compression have also been observed in self-assembled constructs, where applying 17%, 0.1 Hz compression, for example, was found to increase the aggregate modulus by 70%. These results have shown the potential of direct compression to further improve tissue engineered constructs. The enzyme C-ABC was applied to deplete GAG content and subsequently improve biomechanical properties. C-ABC increased tensile properties of self-assembled articular cartilage (Fig. 2B) without compromising compressive properties, as GAG levels return post-treatment.22 Multiple C-ABC treatments further increased tensile properties, reaching values of 3.4 and 1.4 MPa for the tensile modulus and ultimate tensile strength, respectively.23 C-ABC represents an exciting method for engineering functional articular cartilage by departing from conventional anabolic approaches.

Intracellular Na+ and Ca2+ modulation increases the tensile properties

The effects of hydrostatic pressure are known to be mediated by various ion channels.24 Motivated by the mechanism of action of hydrostatic pressure, inhibitors of Na+ ion transporters and stimulators of intracellular Ca2+ were investigated as possible actors in the development of self-assembled constructs.25 We applied ouabain (Na+/K+-ATPase inhibitor), bumetanide (Na+/K+/2Cl- tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca2+ ionophore) to self-assembled constructs for 1 hour daily on days 10-14 of culture and examined the constructs at 2 weeks or 4 weeks. The results of these experiments showed that 20 µM ouabain, 0.3 µM ionomycin, or their combination increased the tensile modulus by 40-95%. Furthermore, the 20 µM ouabain treatment increased the ultimate tensile strength by 56-86% at 4 weeks. This study was the first to show that altering intracellular ion concentrations can increase the mechanical properties of engineered articular cartilage. In addition, these results have important relationships to hydrostatic pressure mechanotransduction.

Conclusions: Self-assembly shows great promise for cartilage tissue engineering and various stimuli can be used to improve the properties of constructs. Growth factor application induces 1-fold increases in both compressive and tensile properties. Mechanical stimuli also increase the mechanical properties of constructs; hydrostatic pressure increases tensile properties over two-fold. Furthermore, combining hydrostatic pressure and TGF-β1 synergistically increases functional properties. Additionally, the application of chondroitinase-ABC increases the tensile modulus by 80% without compromising compressive properties. By altering the concentrations of intracellular ions, the tensile properties can be increased. These studies illustrate how both mechanical and biochemical stimuli can be employed to improve the properties of self-assembled constructs. The straightforward approach of self-assembly and its versatility render it a highly translatable platform technology. Although results thus far show great promise, this process will need to be improved prior to clinical application. Current studies focus on continuing to optimize the process and improve the functional properties of constructs.

References:

1. Vacanti, CA, Kim, W, Schloo, B, Upton, J, Vacanti, JP. 1994. Joint resurfacing with cartilage grown in situ from cell-polymer structures. Am J Sports Med 22: 485-488.

2. Grande, DA, Halberstadt, C, Naughton, G, Schwartz, R, Manji, R. 1997. Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J Biomed Mater Res 34: 211-220.

3. Nehrer, S, Domayer, S, Dorotka, R, Schatz, K, Bindreiter, U, Kotz, R. 2006. Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair. Eur J Radiol 57: 3-8.

4. Kaplonyi, G, Zimmerman, I, Frenyo, AD, Farkas, T, Nemes, G. 1988. The use of fibrin adhesive in the repair of chondral and osteochondral injuries. Injury 19: 267-272.

5. Furukawa, KS, Suenaga, H, Toita, K, Numata, A, Tanaka, J, Ushida, T, Sakai, Y, Tateishi, T. 2003. Rapid and large-scale formation of chondrocyte aggregates by rotational culture. Cell Transplant 12: 475-479.

6. Stewart, MC, Saunders, KM, Burton-Wurster, N, Macleod, JN. 2000. Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J Bone Miner Res 15: 166-174.

7. Hu, JC, Athanasiou, KA. 2006. A self-assembling process in articular cartilage tissue engineering. Tissue Eng 12: 969-979.

8. Deng, Y, Zhao, K, Zhang, XF, Hu, P, Chen, GQ. 2002. Study on the three-dimensional proliferation of rabbit articular cartilage-derived chondrocytes on polyhydroxyalkanoate scaffolds. Biomaterials 23: 4049-4056.

9. Fortier, LA, Nixon, AJ, Mohammed, HO, Lust, G. 1997. Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1. Am J Vet Res 58: 66-70.

10. Smith, P, Shuler, FD, Georgescu, HI, Ghivizzani, SC, Johnstone, B, Niyibizi, C, Robbins, PD, Evans, CH. 2000. Genetic enhancement of matrix synthesis by articular chondrocytes: comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum 43: 1156-1164.

11. Sah, RL, Chen, AC, Grodzinsky, AJ, Trippel, SB. 1994. Differential effects of bFGF and IGF-I on matrix metabolism in calf and adult bovine cartilage explants. Arch Biochem Biophys 308: 137-147.

12. Blunk, T, Sieminski, AL, Gooch, KJ, Courter, DL, Hollander, AP, Nahir, AM, Langer, R, Vunjak-Novakovic, G, Freed, LE. 2002. Differential effects of growth factors on tissue-engineered cartilage. Tissue Eng 8: 73-84.

13. Elder, BD, Athanasiou, KA. 2008. Systematic assessment of growth factor treatment on biochemical and biomechanical properties of engineered articular cartilage constructs. Osteoarthritis Cartilage 18: 18.

14. Mauck, RL, Soltz, MA, Wang, CC, Wong, DD, Chao, PH, Valhmu, WB, Hung, CT, Ateshian, GA. 2000. Functional tissue engineering of

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articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng 122: 252-260.

15. Pei, M, Solchaga, LA, Seidel, J, Zeng, L, Vunjak-Novakovic, G, Caplan, AI, Freed, LE. 2002. Bioreactors mediate the effectiveness of tissue engineering scaffolds. Faseb J

16: 1691-1694. 16. Smith, RL, Lin, J, Trindade, MC, Shida, J, Kajiyama, G, Vu, T, Hoffman, AR, van der Meulen, MC, Goodman, SB, Schurman, DJ, Carter, DR. 2000. Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression. J Rehabil Res Dev 37: 153-161.

17. Hu, JC, Athanasiou, KA. 2006. The effects of intermittent hydrostatic pressure on self-assembled articular cartilage constructs. Tissue Eng 12: 1337-1344.

18. Elder, BD, Athanasiou, KA. 2008. Synergistic and additive effects of hydrostatic pressure and growth factors on tissue formation. PLoS ONE 3: e2341.

19. Ofek, G, Revell, CM, Hu, JC, Allison, DD, Grande-Allen, KJ, Athanasiou, KA. 2008. Matrix development in self-assembly of articular cartilage. PLoS ONE 3: e2795.

20. Elder, BD, Athanasiou, KA. 2008. Effects of Temporal Hydrostatic Pressure on Tissue-Engineered Bovine Articular Cartilage Constructs. Tissue Eng Part A 15: 1151-1158.

21. Aufderheide, AC, Athanasiou, KA. 2006. A direct compression stimulator for articular cartilage and meniscal explants. Ann Biomed Eng 34: 1463-1474.

22. Natoli, R, Revell, CM, Athanasiou, K. 2009. Chondroitinase ABC Treatment Results in Increased Tensile Properties of Self-Assembled Tissue Engineered Articular Cartilage. Tissue Eng Part A: doi:10.1089/ten.TEA.2008.0478.

23. Natoli, RM, Responte, DJ, Lu, BY, Athanasiou, KA. 2009. Effects of multiple chondroitinase ABC applications on tissue engineered articular cartilage. J Orthop Res.

24. Elder, BD, Athanasiou, KA. 2009. Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration. Tissue Eng Part B Rev 15: 43-53.

25. Natoli, RM, Skaalure, S, Bijlani, S, Chen, KX, Hu, J, Athanasiou, KA. Intracellular Na(+) and Ca(2+) modulation increases the tensile properties of developing engineered articular cartilage. Arthritis Rheum 62: 1097-1107.

Acknowledgments:

The authors acknowledge funding from NIH NIAMS R01 AR053286.

3.2.2Nutraceuticals in the treatment of OAJ. SteinmeyerGiessen/Germany

Introduction: Osteoarthritis (OA) is the most prevalent disease of the locomotory apparatus, affects people of all ethnic groups in all geographic locations, occurs more commonly in women, and is the most common cause of long-term disability in most populations of people over 65. The growing population of older age groups and an increase in risk factors for OA, primarily obesity and an inactive life style, causes further increase in the estimated number of total prevalent cases in the USA, Europe and Japan. The fact that OA is no longer seen as a “battle wound” of becoming old or as an unavoidable fate has created major demands on its therapy: drugs used for its treatment should if possible have a causal mode of action, and in addition, inhibit pain and inflammation, but should still have only few adverse effects in long-term use, in spite of age-related comorbidity and eventual polypharmacy.

Content: Alleviation of pain and inhibition of inflammation are the primary goal of pharmacotherapy whereby the objective is to return an active or transiently painful, decompensated OA to a latent (silent, pain-free) condition. This therapeutic goal can almost always be accomplished by using analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) or intra-articularly applied glucocorticoids. NSAIDs, despite serious adverse effects associated with their long-term use, remain among the most widely prescribed drugs for OA. In this context, there is a need for safe and effective alternative treatments while the absence of any cure reinforces the importance of prevention.

Such preventions and alternative treatment could come from nutritional factors and has created an enormous public interest in the relationship between nutrition and disease. The term “nutraceutical” was coined from “nutrition” and “pharmaceutical” in 1989 by DeFelice and was originally defined as “a food (or part of the food) that provides medical or health benefits, including the prevention and/or treatment of a disease” (1). In 1999, natural bioactive chemical compounds derived from whole food and available in a non-food matrix was used in a policy paper of Zeisel to describe nutraceuticals (2). Under this newer definition, nutraceuticals are thus functional ingredients sold as powders, tablets, dragées and other medicinal forms not generally associated with food. The FDA, however, uses instead the term dietary supplements (3). Numerous speculative lay publications advertise the use of a whole range of nutraceuticals, and health food stores offer a plethora of nutraceutical supplements represented as therapies for OA. Surveys suggest that up to 5-8% of adults in the US have used one or another of these products at some time with glucosamine and chondroitin together ranking third among all top-selling nutritional products in the US (4).

Nutrition is naturally better positioned to provide long-term rather than short-term health benefits. This is because, in most cases, a nutritional compound has only limited effects on its biological target and clinical relevant effects are only reached over time. For this reason, chronic diseases such as OA should, in theory, benefit more from nutrition than acute diseases. However, besides the general advice about healthy eating, dietary programs have played a secondary role in the management of this degenerative joint disease. Articular cartilage is critically dependent upon the regular provision of nutrients (glucose and amino acids), vitamins (particularly vitamin C), and essential trace elements (zinc, magnesium, copper). Depending on the nutraceutical as sold over-the-counter they can include a sole substance or a mixture of different agents from a wide variety of different chemical groups including sugars (glucosamine, chondroitinsulfate), vitamins (vitamin C, D, E, K), fatty acids (omega-3-polyunsaturated fatty acids, fish oil), proteins, peptides or amino acids (gelatin, collagen hydrolysate, lactalbon, glycine, histidin, lysin) and/or plant extracts.

In most cases, the marketing of nutraceutical products has been extensive and often based on minimal experimental data. It is clear from the literature that the subject of nutraceuticals is highly controversial and fraught with confusing and conflicting reports of efficacy, safety, and mechanism of action (for review see e.g. 4-7). Thus, it is difficult to form a balanced opinion about nutraceuticals. However, some nutritional factors seem to improve the symptoms of declared OA (glucosamine, chondroitinsulfate, collagen hydrolysate), whereas the role of nutraceuticals in slowing down progression of the disease remains to be seen. For instance, only very few randomized controlled trials, which used structure-modifying variables as primary endpoints, were unable to demonstrate a

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benefit for glucosamine, chondroitinsulfate and vitamin E, but the area deserves further investigation. Evidence for real efficacy is often lacking (e.g. gelatine, lactalbon, glycin, histidin, lysin, lycopene) or are based on epidemiological studies (e.g. vitamin C, D, K, calcium, zinc, boron, selenium); therefore, large, controlled, double-blinded, randomized clinical studies are required to determine the effect of nutraceuticals on symptom and structure modification in OA. Overall, it appears that there is some evidence to support claims for efficacy for some nutraceuticals, but given the lack of extensive well-designed clinical studies, we must be cautious in advocating their widespread use. Perhaps, at present, their best place in the clinical armory is as adjuncts to conventional therapy.

References:

(1) Kalra EK. Nutraceutical—definition and introduction. AAPS Pharm Sci 5, E25, 2003

(2) Zeisel SH. Regulation of “nutraceuticals”. Science 285, 1853-1855, 1999.

(3) Halstead CH. Dietary supplements and functional foods: 2 sides of a coin? Am. J. Clin. Nutr. 77, 1001S-1007S, 2003.

(4) McAlindon TE. Nutraceuticals: do they work and when could we use them? Best Practice Res. Clin. Rheum. 20, 99-115, 2006.

(5) Steinmeyer J, Konttinen YT. Oral treatment options for degenerative joint disease—presence and future. Adv. Drug Del. Rev. 58, 168-211, 2006.

(6) Rayman MP, Pattison DJ.. Dietary manipulation in musculoskeletal conditions. Best Practice Res. Clin Rheum. 22, 535-561, 2008.

(7) Ameye LG, Chee WSS. Osteoarthritis and nutrition. From nutraceuticals to functional foods: a systematic review of the scientific evidence. Arthritis Res. Ther. 8, R127, 2006.

3.2.3Can drugs affect cartilage regeneration? -Immunosuppressant FK506 promote chondrogenesis of synovial mesenchymal stem cells-N. Nakamura1, K. Tateishi1, W. Ando1, K. Kita2, K. Nakata1, H. Yoshikawa1

1Osaka/Japan, 2Suita, Osaka/Japan

Introduction: It is well known that the healing potential of articular cartilage is limited, in part, due to its avascularity. To compensate for such poor healing capacity, several cell-based approaches have been investigated to repair chondral lesions.

Mesenchymal stem cells (MSCs) were first identified in bone marrow and shown to have the ability to differentiate into a variety of connective tissue phenotypes including bone, cartilage, and adipose-tissue. Specifically, MSCs derived from synovium have been shown to have the ability to proliferate over many passages without losing their multipotency, and such expansion appears to be independent of donor age or cryopreservation. In addition, a recent study has shown that MSCs derived from synovium are superior to those from bone marrow or adipose tissue in terms of chondrogenic differentiation and it is a relatively easy and safe procedure to obtain cells from synovium with minimal donor-site morbidity. Taken together, synovium-derived MSCs could be a promising source for future cell-based cartilage repair.

FK506 (tacrolimus) is a widely used immunosuppressive agent with FDA approval, usually used to prevent graft rejection. The immunosuppressive effect of FK506 is believed to be related to its ability to inhibit calcineurin, an enzyme involved in activation of the nuclear factor of activated T cells (NFAT). Recent studies also suggest the involvement of NFAT in chondrogenesis. Furthermore, it has been shown that FK506 can induce chondrogenic differentiation of clonal mouse embryonic carcinoma cells (ATDC5), although the molecular target for its effect on chondrogenesis remains unclear. Such observations raise the possibility that FK506 might likewise promote chondrogenic differentiation of MSCs. To test this hypothesis, the effect of FK506 on chondrogenic differentiation of human synovial mesenchymal stem cells (hSMSCs) was investigated. Furthermore, the molecular mechanism (s) underlying the promotion of chondrogenic differentiation by FK506 were explored.

Content: FK506 promotes chondrogenic differentiation of SMSCs.

It has been reported that the degree of chondrogenic differentiation is related to pellet size and weight, based on increases in proteoglycan synthesis. SMSC pellets cultured in chondrogenic culture medium with FK506 were significantly larger than those cultured without FK506. Increases in pellet size were observed in a dose-dependent manner, but were most prominent at 1000ng/ml FK506 (mean of 27.5%). Exposure to FK506 also enhanced alcian blue staining of the cell pellets. Increases in staining were prominent in the center area of the FK506-treated cell pellets, and the staining intensity exhibited a dose dependency. Likewise, exposure to FK506 significantly increased GAG levels in the pellets; and again these exhibited a dose-dependency with increases of approximately 8-fold when FK506 was added at 1000ng/ml.

FK506 and BMP2/TGFbeta1 additively promotes chondrogenesis in human SMSCs.

To investigate whether the effects of FK506 on chondrogenesis were additive or synergistic with those observed for chondrogenic growth factors, the pellet culture system in the presence of growth factors (BMP2 or TGFbeta1) and several doses of FK506 was assessed. In combination with BMP2 or TGFbeta1, FK506 additively increased both the size of the cell pellet and GAG levels in a dose dependent manner. RT-PCR analysis showed that FK506 treated cell pellets exhibited increased mRNA levels for collagen II and aggrecan irrespective of the presence of growth factors, and the positive effects of FK506 on chondrogenesis were significantly promoted in combination with BMP2 or TGFbeta1. Type X collagen gene expression was not detected after 3 weeks of culture in any of the control or experimental conditions. However, after 6 weeks of culture, expression of the type X collagen gene in the BMP2/TGF beta1 supplemented groups was detected.

Involvement of Smad signaling pathway in FK506 induced SMSCs differentiation.

Since FK506 appeared to exhibit an additive effect with not only BMP2, but also TGF beta1, both of which are members of the TGF beta

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super-family, the potential involvement of Smad signaling pathway in FK506-mediated chondrogenesis was further investigated. The phosphorylation of Smad1/5/8 and Smad3 was readily detected following chondrogenic medium treatment of SMSCs. In FK506-treated cells, phospho-Smad1/5/8 and phospho-Smad3 levels were significantly higher than those in non-treated cells by 20-40% (p<0.01) after 30 minutes, 60 minutes, and 120 minutes of exposure to the drug. In addition, levels of phospho-Smad1/5/8 and phospho-Smad3 were significantly elevated in both the nuclear and cytoplasmic fractions after FK506 treatment. These data suggest that FK506 treatment has significant effects on the phosphorylation state of Smad proteins.

Discussion: The present studies demonstrate that a widely used immunosuppressant, FK506, promotes chondrogenic differentiation of human SMSCs and moreover, that FK506 additively enhances chondrogenic differentiation of hSMSCs in the presence of chondrogenic growth factors, such as BMP2 and TGF beta1. These results indicated that FK506 treatment especially promoted early chondrogenic differentiation of hSMSCs. These results indicate that the Smad signaling pathways are involved in the FK506 effects on SMSC differentiation towards a chondrogenic phenotype.

Although FK506 is already used patients clinically for therapeutic purposes, particularly to prevent graft rejection; With further optimization, FK506 could potentially become a unique therapeutic reagent to promote cartilage repair, alone or in combination with other modalities, either in vivo or in vitro.

References:

1. Nishigaki F, Sakuma S, Ogawa T, Miyata S, Ohkubo T, Goto T. FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5.: Eur J Pharmacol, 2002;437, 123-8.

2. Kugimiya F, Yano F, Ohba S, Igawa K, Nakamura K, Kawaguchi H, Chung U. I. Mechanism of osteogenic induction by FK506 via BMP/Smad pathways.: Biochem Biophys Res Commun, 2005;338, 872-9.

3. Tateishi K, Higuchi C, Ando W, Nakata K, Hashimoto J, Hart DA, Yoshikawa H, Nakamura N. The immunosuppressant FK506 promotes development of the chondrogenic phenotype in human synovial stromal cells via modulation of the Smad signaling pathway. Osteoarthritis Cartilage. 2007 15:709-18.

Acknowledgments:

This work is supported by the Grant-in-Aid for Scientific Research, Japan Society for the Promotion of Science, and the Nakatomi Foundation Research Grant

3.3.1Issues regarding clinical cartilage repair studies: Design, bias and interpretationG. KnutsenTromsoe/Norway

Introduction: Our group from Norway has lead a randomized trial aimed at comparing ACI and microfracture[1,2]. This journey has given me experience and reflections that I would like to share and discuss. Our study, widely acknowledged as a well organized and performed trial has also some weak points and certain bias, and for the designing of a new study today improvements would have been appropriate.

Content: Over the last two decades we have seen increasing amounts of efforts to develop approaches to promote biological repair of articular cartilage defects. The NEJM paper by Brittberg, Peterson et al[3] introduced the orthopedic community to ACI and since then cell based therapies have been a “hot” topic and tissue engineering therapies using cells, growth factors and scaffolds come in different and newer generations. Even though, Brittbergs study was not a RCT, it motivated basic and clinical scientists all over the world, and was also an important motivation factor when we planned our RCT. However, we still do not have enough evidence to say that newer generations and therapies are better than standard treatments like marrow stimulation procedures[4]. New is not synonymous with improved. It has been estimated that up to 90% of new treatments introduced in medicine in general are useless (Dr. Karim F Hirji, personal communication). Randomization Most of the clinical cartilage repair studies do not answer important therapeutic questions and do not bring us forward. We are still in the need of higher quality RCTs and registries. It is extremely important to design new studies so that we can get valid answers and a solid base for further improvements. On the other hand every type of study has a place, even case reports. One of the major problems we are facing in this field is that we have hundreds of studies not supporting necessary evidence for superiority. Additionally variations and newer generations of techniques are introduced before the older ones have been properly validated. The safety and complication rate have been well documented in clinical studies. Randomization may be accomplished using envelopes ore more sophisticated computer models. It is also important to have an appropriate comparator when new studies are designed. Bias, blinding and control It is also a fact that bias is a big problem in cartilage repair studies. Even if stated that no conflict of interest exists, problematic bias may be present. Orthopedic surgeons and researchers need to collaborate with the industry, but industry funded trials have a tendency to be biased. In general patient based questionnaires are preferred. However, a general observation is that overloading the participating patients with too many surveys and tests, the drop out rate may increase. The drop out rate of a study should be low. My experience is that by using phone, mail and email you can get information from most patients. Failures should have their last clinical score before revision-surgery as their final score (principle of intention to treat). Blinding, if possible should also as far as possible be used. There is a possibility to blind the assessor using stockings covering an operated knee. It is also possible to blind radiologists and pathologists even in case series. In general bias is reduced by having a control group, and blinding both assessors and patients. Independent assessors are of great value. Blinding of the surgeons is seldom used, but may be possible having e.g. products with and without cells. A major weakness of RCTs in this field (including our original study) is the lack of a non operative control group. The non operative control group could e.g. be offered a detailed rehabilitation and training program. Including a placebo surgery group is difficult, but very interesting[5]. It has been argued that placebo effect could reach up to 30-40% in surgery. It is also accepted that you need fewer patients (smaller sample size) in a placebo designed study. Power analyses are necessary to plan for optimal numbers of patients for each study. Multi center studies are often needed to be able to get enough power. However, multi center and multi surgeon trials create new issues to be considered. It is extremely important to standardize the protocols and provide adequate training for all surgeons. Some centers may not have the same interests in the trial compared to others and this may have high impact on the final outcomes. Motivation of the participating surgeons is essential for success. Having a non biased sponsor improves the quality enormously. Participating centers should have written regulations regarding all details including publication and use of the data. When designing a new study you should define one primary outcome. Secondary outcomes are also defined and described in detail before start of the study. Surrogate endpoints as MRI and histology are additionally clearly planned in advance. During the study period it is not a good

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practice to change the endpoints (it may even be unethical and could violate the study design). Analyzing subgroups could be problematic and may confuse the main results of the study. In our study we probably had too many endpoints and significant findings using analyses at a given time point could have been interpreted as a false positive result (Type I error). We concluded at the last follow up that there was no difference between the two treatments, and if in fact there was a difference this would have been a false negative result (Type II error). It is possible to note that non superior trials are less frequently published (publication bias). Journals also tend to favor studies showing superior results of new treatments compared to standard treatments. This fact indirectly represents a big hamper for progression. We sent our Norwegian randomized trial first to NEJM; however, this journal rejected the paper almost immediately. The following years have told us that our trial has been evaluated as one of the best so far and also rated as the trial with the least bias[6]. Clinical trials could give superior, non-inferior or equivalent profiles. It is not enough to have one excellent and well performed clinical RCT showing superiority to have the rights for changing the treatment strategies. However, surgery is complex and several different therapies may have equal outcomes for the patients. Before robot surgery has taken over we have to realize that the surgeon himself plays a very important role in the outcomes of an operation. Even though, we need to answer critical questions regarding introduction of new treatments as well as critical appraisal of standard treatments. Animal studies are necessary as well as pilot clinical studies followed by RCTs and registries. I believe it is critical to have easy access to databases and clinical questionnaires. We need to collaborate and share our knowledge to achieve clear improvements. I give value to the Socrates database and others, but would support that these instruments should be open and of free use for every clinician. I believe a common platform-database including large numbers of patients anonymously (both patients and surgeons) could have been useful. A properly validated patient-based score could have been the primary outcome, and secondary hard outcomes could have been failure (new resurfacing cartilage operation) and joint prosthesis. It is further for all clinical studies in this field essential to have a detailed rehabilitation protocol, and evaluate the patient compliance in this context. Interpretation We have so far not been able to regenerate native-like hyaline cartilage in adults, and our efforts results in repair cartilage of various qualities. Recently subchondral bone and the interface between bone and cartilage have come more into focus[7]. This might be an interesting arena for research regarding cartilage regeneration approaches. The safety and complication rate of biological clinically tested procedures seems to be acceptable. Most procedures seems to have acceptable short term results in younger active patients, however, osteoarthric defects in older patients not wanting joint prosthesis are problematic and long term results (more than 10 years) in quality RCTs are lacking for all defects. In evaluating clinical studies it is important to distinguish between acute, subacute and chronic lesions and also note size, depth and localization of the treated defects. Acute smaller chondral and subchondral injuries may become asymptomatic regardless of surgery or not, and particularly in studies including mainly acute lesions this has to be considered. In conclusion, I am optimistic, in light of the huge efforts worldwide from scientists and clinicians. Even though, we still have some steps to go.

References:

1. Knutsen G, Engebretsen L, Ludvigsen TC, Drogset JO, Grontvedt T, Solheim E et al.: Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. J Bone Joint Surg Am 2004, 86-A: 455-464.

2. Knutsen G, Drogset JO, Engebretsen L, Grontvedt T, Isaksen V, Ludvigsen TC et al.: A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am 2007, 89: 2105-2112.

3. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994, 331: 889-895.

4. Jakobsen RB, Engebretsen L, Slauterbeck JR: An analysis of the quality of cartilage repair studies. J Bone Joint Surg Am 2005, 87: 2232-2239.

5. Moseley JB, O’Malley K, Petersen NJ, Menke TJ, Brody BA, Kuykendall DH et al.: A controlled trial of arthroscopic surgery for osteoarthritis of the knee. N Engl J Med 2002, 347: 81-88.

6. Magnussen RA, Dunn WR, Carey JL, Spindler KP: Treatment of

focal articular cartilage defects in the knee: a systematic review. Clin Orthop Relat Res 2008, 466: 952-962.

7. Gomoll AH, Madry H, Knutsen G, van Dijk N, Seil R, Brittberg M et al.: The subchondral bone in articular cartilage repair: current problems in the surgical management. Knee Surg Sports Traumatol Arthrosc 2010, 18: 434-447.

8.1.2The posterolateral corner of the kneeT. Spalding1, J. Bird2

1Leamington Spa/United Kingdom, 2Coventry/United Kingdom

Introduction: THE POSTERO-LATERAL CORNER OF THE KNEE Tim Spalding FRCS Orth. and Jonathan Bird FRCS Orth. ICRS 2010 University Hospital Coventry, UK Introduction Isolated postero lateral corner (PLC) injuries are uncommon and probably represent less than 2% of knee injuries. There is however a higher incidence of PLC injuries in combination with ACL or PCL injuries. This is reported to be as high as 30-40% depending on the series. The postero lateral corner should always be examined when diagnosing an ACL or PCL injury as unrecognised PLC injury can be a cause for failure in outcome after ACL reconstruction.

Content: Evaluation of Postero-lateral Corner Accurate history is important for the mechanism of injury which can include: impact force to the antero medial aspect of the knee when it is nearly at full extension, non contact or contact hyperextension injury, varus load on a flexed knee, and knee dislocation. The patient will usually describe pain in the postero lateral area of the knee and it is important to ask for associated altered sensation in the lower leg which may have been transient at the time of injury. There may be giving way in a rotational activity or an instability feeling when going downstairs. Symptoms may not necessarily start immediately following injury but can progress over time as the PLC gradually ‘stretches out’. Examination Key examination features include those listed below. In evaluation of acute injury it may be necessary to rest the knee over a pillow in order to reduce guarding and make it more comfortable to examine the PLC.

Alignment – check for varus alignment and lateral thrust of the knee on walking.

Pain: there may be pain on hyperextension of the knee and a reluctance to straighten the leg as this pulls on the injured PLC.

Swelling and bruising: there may be isolated swelling around the postero lateral corner.

Footdrop: indicates nerve stretching.

Neurovascular examination looking for abnormal nerve function including numbness and the dorsum of the foot and first web space.

Detecting abnormal movement is the key to diagnosing PLC injury and the tests include:

Varus stress test at 0° and 30° with leg in neutral rotation.

Prone external rotation dial test at 30° and 90°

Isolated PLC injury will give increased rotation at 30° only and not at 90°

Increased rotation at 30° and 90° indicates combined PCL and PLC injury

External rotation recurvatum test

Reverse pivot shift test

These clinical tests are hard to do as positive findings are infrequent, making it difficult for the surgeon to build up expertise. The most sensitive test is the prone dial test and in this test the patient needs to lie fully prone and relaxed. Often an assistant is needed to hold the two knees together while the examiner supports the feet and ankle at 30° and 90°. A positive test is indicated when it is increased from the other side – usually by 15° or more. There is a fair amount of subjective interpretation required in this test. Management of Acute Injuries Once acute injury to the postero lateral corner is recognised then surgical management is best undertaken early before the

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tissue becomes too scared and difficult to reattach. The literature is clear that acute reconstruction is better than chronic reconstruction for isolated injury. (Noyes 1996) In multi ligament reconstruction for the acutely multi-ligament injured knee Stannard et al (2005) has identified a high failure rate for primary repair compared to anatomic reconstruction. Chronic Injuries Injuries beyond six weeks fall under the category of chronic injuries where significant scarring means that it is difficult to undertake any primary repair and the tissue is usually not suitable for repair. As previously described evaluation of gait and alignment along with the state of the other ligaments in the knee is critical to formulate a complete plan for management. Various surgical techniques have been described over the years and these have been broadly categorised into non anatomic and anatomic reconstruction. (Cooper et al 2006) Non anatomic reconstructions are generally based on the fibula head, reconstructing ligaments to the lateral epicondyle. The most popular repair is the Larson repair and there have been many modifications to this over the years (Larson 2001). These techniques are technically easier than the anatomic techniques and continue to be reported with good results (Khanduja 2006). Anatomic techniques attempt to reconstruct the primary anatomy of the postero-lateral corner including the elements of the three functional components – the lateral collateral ligament, the popliteus and the popliteo-fibula ligament utilising their anatomic insertion points. In addition plication of the postero lateral capsule is used to add to the repair. Two key techniques stand out: the technique published by La Prade et al (La Prade 2004) and by Arciero et al (Arciero 2005). Published results are scarce and generally tend to involve surgery for the postero-lateral corner in combination with posterior cruciate ligament injuries. The optimal technique is therefore unclear. Long term knee stability still remains an issue after reconstructive surgery. Yoon et al (2006) published improved results comparing anatomic against non anatomic reconstruction techniques. The decision however is difficult as anatomic techniques are more surgically demanding and may lead to over constraint of the knee.

La Prade technique: in this technique allograft tendons are used, either Achilles tendon with bone block at one end or hamstring tendons. An oblique tunnel is fashioned through the head of fibula from antero-lateral to postero-medial at the styloid process. An additional tunnel is made in the tibia from Gerdy’s tubercle to a point in the popliteal fossa just medial to the superior T-F joint emerging under the muscle belly of popliteus. The graft is passed from the lateral epicondyle, under IT band to the antero-lateral fibula head, through the fibula tunnel and through the tibial tunnel from posterior to anterior. A second graft is passed from the insertion of the popliteus on femur in a second tunnel, under the LCL reconstruction and through the tibia from posterior to anterior. Both grafts are then fixed with a staple and interference screw to the tibia.

Arciero technique: in this technique no tibial tunnel is drilled but the same fibula tunnel is drilled. A single graft is passed from lateral epicondyle, under IT band to antero lateral aspect of fibula, through fibula tunnel and obliquely anterior to the popliteus tunnel which is a separate tunnel located 12-15mm distal to the epicondyle (in the line of the femur).

Grafts are tensioned with the knee at 60° but care needs to be taken to ensure the knee is not over constrained. Post operative rehabilitation is slow and the knee is protected in a hinged knee brace. This is locked in extension when mobilising for the first six weeks. During that time period the brace is unlocked during the day for exercises. After six weeks the brace is removed and a sport protective brace may be used up to six months.

Complications The main structure at risk is the peroneal nerve with injury reported in 12-17% of acute cases. Fluid extravasation is a further complication in addition to residual laxity, persistent knee pain and arthrofibrosis (Noyes 2006). Importance Injury to the PLC is uncommon and difficult to detect, yet it is vitally important when reconstructing the ACL or the PCL and persisting joint laxity from a missed PLC injury will compromise the result of the more central ligament reconstruction. With practice, the surgical procedures can become straightforward enough to lead to increasing the indications for reconstruction and increased patient function.

References:

References Arciero RA. Technical Note Anatomic Posterolateral Corner Knee Reconstruction. Arthroscopy 2005;21: 1147-1148 Cooper JM, McAndrews PT, LaPrade RF: Posterolateral corner injuries of the knee: Anatomy, diagnosis, and treatment. Sports Med Arthrosc 2006;14:213-220. Khanduja V, Somayaji HS, Harnett P, Utukuri M,

Dowd GS: Combined reconstruction of chronic posterior cruciate ligament and posterolateral corner deficiency: A two- to nine-year follow-up study. J Bone Joint Surg Br 2006;88:1169-1172. Larson RV: Isometry of the lateral collateral and popliteofibular ligaments and techniques for reconstruction using a free semitendinosus tendon graft. Oper Tech Sports Med 2001;9:84-90. LaPrade RF, Johansen S, Wentorf FA, Engebretsen L, Esterberg JL,TsoA:An analysis of an anatomical posterolateral knee reconstruction: An in vitro biomechanical study and development of a surgical technique. Am J Sports Med 2004;32:1405-1414. Noyes FR, Barber-Westin SD: Treatment of complex injuries involving the posterior cruciate and posterolateral ligaments of the knee. Am J Knee Surg 1996;9:200-214.

Noyes FR, Barber-Westin SD, Albright JC: An analysis of the causes of failure in 57 consecutive posterolateral operative procedures. Am J Sports Med 2006;34:1419-1430. Stannard JP, Brown SL, Farris RC, McGwin G Jr, Volgas DA: The posterolateral corner of the knee: Repair versus reconstruction. Am J Sports Med 2005;33:881-888. Yoon KH, Bae DK, Ha JH, Park SW: Anatomic reconstructive surgery for posterolateral instability of the knee. Arthroscopy 2006;22:159-165.

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8.2.2Using Biomarkers to Monitor Joint Tissue Pathology and RepairV. Byers - KrausDurham/United States of America

Introduction: Osteoarthritis represents a heterogeneous group of conditions with a common endstage pattern of joint degeneration and maladaptive repair. The earlier the treatment likely, the greater the chance of substantively altering the course of the disease for the better. Biomarkers have the potential to provide an early warning of the initiation of matrix breakdown that could prompt earlier treatment to prevent the cartilage and bone destruction that leads to disability. Although there are currently no qualified biomarkers that can be considered as surrogate clinical endpoints, there are many biomarkers that are both commercially available and shown to be associated with one or more aspects of osteoarthritis and that fit one or more categories of the BIPEDS classification scheme. BIPEDS corresponds to Burden of disease, Investigational, Prognostic, Efficacy of Intervention, Diagnostic and Safety biomarkers. These six categories encompass the array of types of biomarkers that could be used for enhancing clinical trials and repair strategies.

Content: When using biomarkers, the principle tissue source(s) of a given biomarker should be identified as accurately as possible, so that the origin(s) of the epitope(s) is clearly understood. This in turn will facilitate the interpretation of clinical data when repair strategies are utilized in a trial setting. In this perspective the most proximal source of information is provided by synovial fluid analyses. A characterization of the synovial fluid biomarker patterns ensuing in early joint damage provides a signature of pathology that can be monitored to determine the efficacy of a repair strategy. An example will be provided from a recent pilot clinical trial. The OARSI FDA Biomarkers Working Group recommendations and current developments to enhance qualification of biomarkers for osteoarthritis will be described. It is hoped that these endeavors will provide valuable tools to assist the pursuit of cartilage repair strategies.

References:

Bauer DC, Hunter DJ, Abramson SB, et al. Classification of osteoarthritis biomarkers: a proposed approach. Osteoarthritis Cartilage. Aug 2006;14(8):723-727. [BIPEDS reference] Kraus, VB, B Burnett, J Coindreau, S Cottrell, D Eyre, M Gendreau, J Gardiner, P Garnero, J Hardin, Y Henrotin, D Heinegard, A Ko, S Lohmander, G Matthews, J Menetski, R Moskowitz, S Persiani, AR Poole, JC Rousseau, M Todman. 2010. Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis: OARSI FDA Osteoarthritis Biomarkers Working Group. Osteoarthritis Cartilage (in press).

8.2.3Can biomarkers be used as outcome measures in cartilage repair?S.L. LohmanderLund/Sweden

Introduction: The challenge of cartilage repair, be it the repair of a cartilage defect in an otherwise normal joint, or the repair/regeneration of cartilage in a joint with early-stage osteoarthritic changes, carries with it the need for outcome measures. Without appropriate outcome measures, results cannot be documented. Here, as in other conditions of the joint, we should consider patient-reported outcomes on symptoms, function and quality of life as the gold standard, the clinical endpoint. Other outcomes of interest may include functional tests to determine biomechanical tissue quality, imaging techniques to monitor structure and quality of joint tissues, and biomarkers (aka molecular markers) to reflect the turnover, structure and status of joint tissues. In common with osteoarthritis, studies on cartilage repair require long observation times and trials, and there is therefore a great need for measures that predict the long-term clinical outcome: surrogate markers of outcome. Considerable efforts are being invested in the development of biomarkers for osteoarthritis, and much of this work is relevant also when considering biomarkers for cartilage repair.

Content: For osteoarthritis biomarkers, a terminology named BIPEDS was proposed (Bauer et al. 26), which classifies these biomarkers into five categories corresponding to their proposed utility: Burden of disease, Investigational, Prognostic, Efficacy of Intervention, Diagnostic and Safety. Biomarkers that are likely to have the earliest beneficial impact on clinical trials fall into two categories. The first are those markers that would allow us to select for trials subjects that are most likely to respond and/or progress (prognostic markers) within a reasonable time for a clinical study, which for a cartilage repair or osteoarthritis study could be one to two years. The second category of biomarkers includes those that provide early feedback for preclinical decision-making and for trial organizers that an intervention has the desired effect on the primary target (efficacy markers). Both types of biomarkers are highly desirable in chronic conditions where conventional clinical outcomes may take years to present. These two categories could reduce the burden and risk of clinical trials by delivering essential early information, speeding up the product development cycle and making both osteoarthritis and cartilage repair more manageable targets. Many biomarkers are currently available that are being tested in relation to their utility for studies on osteoarthritis, existing in various states of validation. No biomarkers have been validated for cartilage repair. The term validation as used here refers to the evidence for a marker in support of its use as a surrogate endpoint, and includes verification of analytical performance characteristics and correlation of the biomarker with a specific biological process. Naturally, validation of a biomarker against a gold standard endpoint depends critically also on the performance and specificity of that gold standard endpoint. So what is the current evidence to support the use of biomarkers as surrogates of clinical outcomes in osteoarthritis, and might they be helpful as biomarkers of cartilage repair? A second useful classification system divides biomarkers into four categories according to their current level of qualification (Wagner et al. 27). Exploration level biomarkers are research and development tools with in vitro and/or preclinical evidence but without consistent information linking the biomarker to clinical outcomes in humans. Demonstration level biomarkers are associated with clinical outcomes in humans but have not been reproducibly demonstrated in clinical studies. Characterization level biomarkers are reproducibly linked to clinical outcomes in more than one prospective clinical study in humans. Surrogacy level biomarkers can substitute for a clinical endpoint, corresponding to ‘‘surrogate end point’’ as mentioned above, and requires agreement with regulatory authorities as an FDA registrable endpoint. The table below lists a selection of commercially available osteoarthritis-related biomarkers and an approximation with regard to level of qualification (van Spil et al. 2). However, that qualification is as always dependent on a specific context, limiting generalizability in the early development phase. There are currently no qualified biomarkers that can be considered as surrogate clinical endpoints in OA. Advances in the field will lead to rapid expansion of this list and more specificity with regard to qualification. Each of these biomarkers can be measured from easily available serum or urine samples. Challenges for the development and validation of biomarkers with utility for cartilage repair are similar to those for osteoarthritis, and will require the combination of reproducible and specific biomarker assays with large prospective, controlled clinical trials. To demonstrate utility of e.g. efficacy biomarkers, these trials will need to show structural joint protection, cartilage repair and/or clinically relevant efficacy. An additional challenge with regard

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to cartilage repair studies and biomarkers will be that the cartilage repair site often represents only a small localized process, as compared to a condition such as osteoarthritis which affects the whole or most of the joint.

Biomarker Proposed process BIPEDS Classification

urinary CTX-II type II collagen degradation BPED (characterization)

serum COMP cartilage degeneration BPD (exploration)

serum HA osteophyte burden, synovitis BPED (demonstration)

serum and urine C,2C types I and II collagen degradation D (exploration)

serum and urine C2C type II collagen degradation ED (demonstration)

serum and urine Coll2- &

Coll2-NO2 type II collagen degradation D,B,P (exploration)

serum CPII type II collagen synthesis D (exploration)

PIIANP collagen synthesis BPD (exploration)

urine/serum NTX- bone resorption P,E (demonstration)

urine/serum CTX- bone resorption B,D,P (exploration)

serum CS846 aggrecan synthesis /turnover P (exploration)

serum MMP-3 protease in joint tissue degradation E (characterization)

References:

Bauer, D. C. et al. Classification of osteoarthritis biomarkers: a proposed approach. Osteoarthritis Cartilage 2006;14:723-7.

van Spil, W., DeGroot, J., Lems, W., Oostveen, J. & Lafeber, F. Serum and urinary biochemical markers for knee and hip osteoarthritis: a systematic review applying the consensus BIPED criteria. Osteoarthritis Cartilage in press 2010.

Wagner, J. A., Williams, S. A. & Webster, C. J. Biomarkers and surrogate end points for fit-for-purpose development and regulatory evaluation of new drugs. Clin Pharmacol Ther 2007;81:104-7.

11.1.1Physical stimulation of cartilage repair by pulsed electromagnetic fieldsM. CadossiBologna/Italy

Introduction: Articular cartilage is a complex tissue characterised by a cellular component, chondrocytes, that are embedded within an organized dense extracellular matrix of collagen and proteoglycan, with a high water content: 75% of the weight of articular cartilage (Ulrich-Vinther M, 2003). Because of its high hydrostratic pressure, the articular cartilage can withstand a large number of repetitive strains during the lifetime; in fact, articular cartilage plays the fundamental role of facilitating movements between the articular heads, reducing friction, it facilitates the sliding of opposite articular surface, it absorbs and redistributes the mechanical loads on the joint (Buckwalter JA, 2002). Under physiologic condition, articular metabolism is slow, but under pathological condition turnover can increase and the matrix undergoes faster mechanical failure and deterioration, resulting in cartilage degeneration and development of arthritis, with a consecutively loss of the integrity of the collagen matrix. Among the causes that may damage the cartilaginous tissue, we should distinguish between a large number of stimuli, including metabolic, genetic, vascular and mechanical disorders. Mechanical injuries can occur from a single but severe load or repetitive but prolonged joint loading, that can cause microdamage, chondral or osteochondral fracture, ultimately exposing the subchondral bone tissue (Buckwalter JA, 2002; Radin EL, 1986). Moreover, modest damage of the articular cartilage, resulting from trauma or less invasive surgical procedure, produces an inflammatory reaction of the joint cartilage, that can cause irreversible degeneration through the increase of catabolic cytokines synthesis and the decrease of anabolic activity of chondrocytes. Pro-inflammatory cytokines increase the synthesis of matrix degrading enzymes and limit the production of proteoglycans (Schuerwegh AJ, 2003; Pellettier JP, 1999; Goldring SR, 2004). Being a hypocellular avascular tissue, the articular cartilage has minimal reparative potential: “cartilage when destroyed it is never recovered” (Hunter W, 1743). In the course of human life, the quality and mechanical properties of the articular cartilage can only decrease. In consideration of such premises, the availability of a therapeutic technique to prevent degeneration and to control local inflammation, both in the subchondral bone and in the articular structures, is of fundamental importance. Therapy has to directly affect the chondrocytes in the cartilage thickness, to prevent the catabolic effects of inflammatory cytokines and to favour anabolic activities and proteoglycans synthesis. The simultaneous treatment of cartilage, the subchondral bone tissue and the articular structures, is feasible only through physical means, such as pulsed electromagnetic fields, whose spatial distribution is able to expose uniformly the whole articular cartilage in its total thickness and extension. Massari et al.(Massari L, 2007), summarised the results of the translational research of the CRES (Cartilage Repair and Electromagnetic Stimulation) study group on the use of pulsed electromagnetic fields with specific parameters (I-ONE therapy, IGEA, Carpi, Italy) to control local joint inflammation and ultimately to have a chondroprotection effect on articular cartilage.

Content: Biophysical stimulation with I-ONE therapy In vitro and ex vivo effects: inflammation control and chondroprotection Preclinical studies for the cartilage repair have shown how biophysical stimulation with I-ONE therapy has to be considered as a therapeutic system, comparable to a pharmacological treatment. A recent ex vivo study, on articular cartilage explants, showed that the proteoglycan synthesis and the anabolic activity increased significantly when specific parameters of the pulsed electromagnetic fields were set: peak intensity of magnetic field 1.5 mT, minimum stimulation time 4 hours, I-ONE therapy (De Mattei M, 2007). These effective parameters were subsequently used in experiments on animal model. With these exposure parameters, the presence of growth factors in the microenvironment and environmental condition are able to increase the proliferation response of chondrocytes (Pezzetti F, 1999; De Mattei M, 2001). On cartilaginous bovine explants, I-ONE therapy acts in concert with the major anabolic growth factor (IGF-1) for cartilage to stimulate the synthesis of essential cartilage matrix components and to increase significantly proteoglycan synthesis (De Mattei M, 2003; De Mattei M, 2004). Articular cartilage explants cultivated in the presence of inflammatory cytokines (IL-1b) show an increase in catabolic activities, accompanied by the degradation of the cartilaginous matrix. However, if explants are exposed to biophysical stimulation with I-ONE therapy, the catabolic effect of the inflammatory cytokine on the matrix is totally inhibited and the integrity of the cartilaginous matrix is preserved (De Mattei M, 2003). The anti-inflammatory activity was also tested

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on human neutrophils cell membrane, on bovine chondrocytes and fibroblast-like synoviocytes exposed to I-ONE therapy, where an upregulation of the A2A adenosine receptors determined: inhibition of pro-inflammatory cytokine production, an increase of adenalyl cyclase activity and a reduction of superoxide anion production, as a result of upregulation of the A2A receptors located on the surface (Varani K, 2002; Varani K, 2008). Kinetics studies have shown that the anti-inflammatory effect is dependent on the width, frequency and shape of the wave of the biophysical stimulus created at the surface of the cell membrane (Varani K, 2002). Moreover, it has been demonstrated that drugs with A2A adenosine receptor agonist activity have shown to protect articular cartilage in animal models of induced osteoarthritis (Cohen SB, 2004). Together, these findings show that I-ONE therapy has an A2A adenosine receptor agonist activity, identifying the A2A adenosine receptor as the pharmacological molecular target of I-ONE treatment in inflammatory joint diseases. In a recent study, De Mattei et al. demonstrated how I-ONE therapy can strongly inhibit the release of PGE2 in bovine synovial fibroblasts exerting an anti-apoptotic effect on cells (De Mattei M, 2009). Moreover the effects of I-ONE therapy exposure on PGE2 production and COX-2 expression closely involved A1 and A2A adenosine receptors. These results are in line with the known anti-inflammatory activities of adenosine. The involvement of adenosine receptors and I-ONE therapy exposure in the inflammatory responses of synovial fibroblasts open perspectives to develop novel anti-inflammatory approaches in joint degenerative diseases. In vivo effects: chondroprotection and bone repair The in vitro results are confirmed by in vivo experiments. Subchondral bone, bone marrow, synovial cells, chondrocytes and synovial fluid contribute to the development of osteoarthritis and to the healing of defects of articular cartilage; for this reason the use of animal models is essential to understand the repair process. An animal model which develops spontaneous and asymptomatic age-related osteoarthritis of the knee, like Dunkin Hartley guinea pigs, was studied to show the strong chondroprotective effect of I-ONE therapy. The capability of biophysical stimulation to modify osteoarthritis progression was firstly demonstrated by Ciombor (Ciombor DM, 2005) and subsequently by Fini (Fini M, 2005; Fini M, 2008) in osteoarthritis lesions of different severity. The values of the cartilage and subchondral bone thickness, the fibrillation index and the quantification of the articular cartilage damage, according to Mankin’s score, highlight that I-ONE therapy exposure preserves the morphology of articular cartilage, inhibit subchondral bone sclerosis and retard the development of osteoarthritis lesions in the entire knee of aged osteoarthritic guinea pig. The degenerative processes observed in the articular cartilage with aging are inhibited by I-ONE stimulation. The health of the cartilage and the success of surgery is largely dependent on the characteristics of the subchondral bone tissue. Large osteochondral defect can be healed with many arthroscopic repair strategies, such as osteochondral grafts. In a sheep model, in which osteochondral grafts were performed, it has been showed that I-ONE therapy is able to enhance the new subchondral bone formation, in the interface between the graft and the host bone, favouring early graft stabilization. Moreover, a smaller incidence of cyst-like resorption areas, that can compromise the stability of graft and the success of the technique, was recorded in the stimulated animal. To support the in vitro results histological analysis of the synovial fluid were also performed in this animal model. The amount of inflammatory catabolic cytokines (IL-1b and TNF-a) in the synovial fluid of I-ONE treated animals was significantly lower than in control animals. On the contrary, TGF-b1 was significantly higher in stimulated animals than it was in controls. These results demonstrate not only the capability of I-ONE therapy to control the inflammatory reaction, but also its capability to favours cartilage anabolic activity (Benazzo F. J Orthop Res. 2008). Clinical trials The above results provide the rational to design clinical studies to demonstrate the possibility to transfer the treatment to humans. Two randomized, prospective, double-blind clinical trials (Level I) were conducted, one in patients treated by arthroscopy with condroabrasion and/or microfractures in the knee (Zorzi C. 2007) and the other in patients after anterior cruciate ligament reconstruction (Benazzo L. Knee Surg Sports Traumatol Arthrosc. 2008). Condroabrasion and/or microfractures. The first randomized, prospective, double-blind study was performed on patients suffering from cartilaginous degeneration in the knee that underwent a microfractures and chondroabrasion arthroscopic treatment. The patients were randomized in two homogenous groups: half of the patients were stimulated with active device (I-ONE group) and half with placebo one. All patients were instructed to use I-ONE device for 90 days, 4h per day. The patients were evaluated by the Knee injury and Osteoarthritis Outcome Score (KOOS) test before arthroscopy, and after 45 and 90 days. The use of NSAIDs to control pain was also recorded. Patients were interviewed for the long-term outcome 3 years after arthroscopic surgery. Thirty-one patients were

evaluated. KOOS value was the same for both groups at time zero (p=n.s.); at 45 and 90 days higher KOOS values were observed in the I-ONE group and the difference became significant at day 90 (p<0.05) and demonstrated that I-ONE therapy leads to complete patient’s recovery in a significantly shorter time. The percentage of patient who used of NSAIDs was 26% in the active group and 75% in the control group (p<0.05). At 3 years follow-up, the number of patients who completely recovered was higher in the I-ONE group compared to the control group (p<0.05). Anterior cruciate ligament reconstruction. Patients with rupture of anterior cruciate ligament at knee underwent arthroscopically assisted reconstruction with use of double-looped semitendinosus and gracilis tendon grafts using biodegradable interference fit fixation. They were randomised in placebo or active group according to age, sex, smoking status and pathology. All patients were instructed to use the device for 60 days, 4 hours per day: half of the patients were stimulated with active device (I-ONE group) and half with placebo one. The assessment was performed using the International Knee Documentation Committee form (IKDC) before, 30, 60 and 180 days after arthroscopy. The control of pain was also recorded by VAS (Visual Analog Scale). Patients were interviewed for long term follow-up at 1 and 2 years after arthroscopy. Sixty patients completed the study. Pre-operatively, no difference was observed between the 2 groups. At day 30 the percentage of patients requiring NSAID was significantly lower in the I-ONE group than in the placebo (p<0.05). The results showed a significant difference (P<0.0001) also between the I-ONE and placebo groups for VAS. At day 60, joint swelling was observed only in the placebo group (p<0.05) and limitation of knee range of motion was less frequent in the I-ONE group than in the placebo (p<0.05). After surgery, the SF-36 score increased faster in the I-ONE group than in the placebo (p<0.05). At the 2-year follow-up 86% of the patients in the I-ONE group and 75% in the placebo group reported complete functional recovery: no knee pain and return to sport activity. Conclusions. Joint injury involves synovial tissue, cartilage and subchondral bone leading to joint inflammation, swelling and pain. Inflammation can cause irreversible degeneration of the cartilage through increased catabolic functions and decreased anabolic activity of chondrocytes. Damage of this remarkable tissue decreases mobility and frequently causes pain with movement and, in the most severe instance, chronic pain. Our experimental and clinical results show how the specific biophysical stimulus, I-ONE, is able to inhibit inflammatory effects of catabolic cytokines and to favour anabolic activity of cytokines, in the whole extension and thickness of the articular cartilage. Moreover, I-ONE therapy acts like a drug, with a specific posology; in fact its efficacy is dependent on the field amplitude, frequency and duration. The preclinical findings are the scientific basis for the clinical use of I-ONE therapy immediately following surgery. I-ONE therapy is an effective chondroprotective treatment for patients, without any negative side effects, that limits inflammation, reduces recovery time and, ultimately, preserves a healthy articular cartilage of the knee.

References:

Benazzo F, Cadossi M, Cavani F, Fini M, Giavaresi G, Setti S, Cadossi R, Giardino R. Cartilage repair with osteochondral autografts in sheep: effect of biophysical stimulation with pulsed electromagnetic fields.J Orthop Res. 2008 May;26(5):631-42. · Benazzo F, Zanon G, Pederzini L, Modonesi F, Cardile C, Falez F, Ciolli L, La Cava F, Giannini S, Buda R, Setti S, Caruso G, Massari L. Effects of biophysical stimulation in patients undergoing arthroscopic reconstruction of anterior cruciate ligament: prospective, randomized and double blind study. Knee Surg Sports Traumatol Arthrosc. 2008 Jun;16(6):595-601. · Buckwalter JA. Articular cartilage injuries. Clin Orthop Relat Res. 2002 Sep;(402):21-37. Review. · Modification of osteoarthritis by pulsed electromagnetic field--a morphological study. Osteoarthritis Cartilage 2003;11(6):455-62. · Cohen SB, Gill SS, Baer GS, Leo BM, Scheld WM, Diduch DR. Reducing joint destruction due to septic arthrosis usingan adenosine2A receptor agonist. J Orthop Res 2004;22:427–35. · De Mattei M, Caruso A, Pezzetti F, Pellati A, Stabellini G, Sollazzo V, Traina GC. Effects of pulsed electromagnetic fields on human articular chondrocyte proliferation. Connect Tissue Res. 2001;42(4):269-79. · De Mattei M, Pasello M, Pellati A, Stabellini G, Massari L, Gemmati D, Caruso A. Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants. Connect Tissue Res. 2003;44(3-4):154-9. · De Mattei M, Pellati A, Pasello M, Ongaro A, Setti S, Massari L, Gemmati D, Caruso A. Effects of physical stimulation with electromagnetic field and insulin growth factor-I treatment on proteoglycan synthesis of bovine articular cartilage. Osteoarthritis Cartilage. 2004;12(10):793-800. · De Mattei M, Fini M, Setti S, Ongaro A, Gemmati D, Stabellini G, Pellati A, Caruso A. Proteoglycan synthesis in bovine articular cartilage

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explants exposed to different low-frequency low-energy pulsed electromagnetic fields. Osteoarthritis Cartilage. 2007;15(2):163-8. · De Mattei M, Varani K, Masieri FF, Pellati A, Ongaro A, Fini M, Cadossi R, Vincenzi F, Borea PA, Caruso A. Adenosine analogs and electromagnetic fields inhibit prostaglandin E(2) release in bovine synovial fibroblasts. Osteoarthritis Cartilage. 2009 Feb;17(2):252-62. · Fini M, Giavaresi G, Torricelli P, Cavani F, Setti S, Cane V, Giardino R. Pulsed electromagnetic fields reduce knee osteoarthritic lesion progression in the aged Dunkin Hartley guinea pig. J Orthop Res. 2005;23(4):899-908. · Fini M, Torricelli P, Giavaresi G, Aldini NN, Cavani F, Setti S, Nicolini A, Carpi A, Giardino R. Effect of pulsed electromagnetic field stimulation on knee cartilage, subchondral and epyphiseal trabecular bone of aged Dunkin Hartley guinea pigs. Biomed Pharmacother. 2008 Dec;62(10):709-15. · Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Rel Res 2004;427S:S27-S36. · Hunter W. Of the structure and diseases of the articular cartilages. Philosophical Transaction London. 1743;42:514-21. · Massari L, Benazzo F, De Mattei M, Setti S, Fini M; CRES Study Group. Effects of electrical physical stimuli on articular cartilage. J Bone Joint Surg Am. 2007;89 Suppl 3:152-61. Review. · Pellettier JP. The influence of tissue cross-talking on OA progression: role of nonsteroidal anti-inflammatory drugs. Osteoarthritis Cartilage1999;7:374-6. · Pezzetti F, De Mattei M, Caruso A, Cadossi R, Zucchini P, Carinci F, Traina GC, Sollazzo V. Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study. Calcif Tissue Int. 1999;65(5):396-401. · Radin EL, Rose RM: Role of subchondral bone in the initiation and progression of cartilage damage. Clin Orthop. 1986, Dec;(213):34-40. · Schuerwegh AJ, Dombrecht EJ, Stevens WJ, Van Offel JF, Bridts CH, De Clerck LS. Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage. 2003 Sep;11(9):681-7. · Ulrich-Vinther M, Maloney MD, Schwarz EM, Rosier R, O’Keefe RJ. Articular cartilage biology. J Am Acad Orthop Surg. 2003;11(6):421-30. Review. · Varani K, Gessi S, Merighi S, Iannotta V, Cattabriga E, Spisani S, Cadossi R, Borea PA. Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils. Br J Pharmacol. 2002;136(1):57-66. · Varani K, De Mattei M, Vincenzi F, Gessi S, Merighi S, Pellati A, Ongaro A, Caruso A, Cadossi R, Borea PA. Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields. Osteoarthritis Cartilage. 2008;16(3):292-304. · Zorzi C, Dall’oca C, Cadossi R, Setti S. Effects of pulsed electromagnetic fields on patients’ recovery after arthroscopic surgery: prospective, randomized and double-blind study. Knee Surg Sports Traumatol Arthrosc. 2007;15(7):830-4.

11.2.1New insights into cartilage biology and pathology using mouse cartilage proteomicsJ.F. Bateman1, R. Wilson2, S. Zivkovic2 1Victoria/Australia, 2Melbourne/Australia

Introduction: In recent years there has been a rapid expansion in the use of proteomics to discover biomarkers of joint disease, unravel new molecular mechanisms underlying cartilage degeneration and identify cartilage components and interactions that have evaded detection by conventional biochemical approaches. Our focus is to develop and apply techniques for proteomic analysis of mouse cartilage. Despite the limited amounts of material available, the benefits of mouse tissues outweigh the disadvantages. Specifically, the use of mouse models will facilitate comparison between wild-type tissue and cartilage lacking key elements of the degenerative machinery (e.g. ADAMTS5), cartilage lacking novel or important cartilage components or harbouring disease -causing mutations (eg., chondrodysplasia). 2-D electrophoresis (2-DE) remains a popular method for differential proteomics; however the composition of cartilage presents a unique challenge (1-5). Using centrifugal ultrafiltration to deplete aggrecan and hyaluronan from the media of femoral head (P21) explant cultures, we identified novel components of the cartilage “secretome”, including proteolytic fragments of perlecan/endorepellin, thrombospondin-1, connective tissue growth factor and chondromodulin. In addition to well-known markers such as MMP-3 and cartilage gp-39, new markers for cytokine-induced cartilage degradation, such as lipocalin-2, were identified and quantified using fluorescent labelling and difference in-gel electrophoresis (DIGE). More recently we have focused on proteomic analysis of cartilage tissue extracts using a novel fractionation approach based on differential protein solubility (6). Sequential extraction of mouse femoral head cartilage with 1 M NaCl followed by 4 M GdnHCl generates very distinct but, importantly, consistent 2-DE profiles. Identification of differentially abundant protein spots using HPLC-MS/MS (Agilent XCTplus ion trap) revealed effective partitioning of readily soluble cytosolic components (e.g. triosephsophate isomerase) from more tightly integrated, predominantly, matrix components (e.g. decorin, matrilin-1, lactadherin). Resolution by 2-DE effectively targets differentially abundant proteins and readily provides a visual map of a tissue proteome, including modified protein isoforms and proteolytic fragments. However, recent advances in high-resolution tandem mass spectrometry have enabled large-scale protein identification from complex peptide mixtures generated by in-solution tryptic digestion. Using nanoLC MS/MS (LTQ-Orbitrap) and spectral counting we identified 600 proteins at high confidence in extracts of 21 day old mouse cartilage, of which more than 250 proteins were significantly enriched in either the 1M NaCl or 4M GuHCl extract. Surprisingly, the solubility-based partitioning of proteins applied equally to cellular components (e.g. ribonuclear and proteasomal subunits) as ECM proteins and proteoglycans. Our fractionation approach therefore facilitates deeper mining of the cartilage proteome whilst retaining important biochemical information related to the proteins identified. We are currently using this approach to identify novel components of cartilage development, using high-density scaffold-free mouse chondrocyte cultures. While the resulting multi-layered “neo-cartilage” lacks the stratified organization of other systems (e.g. bovine), ultrastructural analysis by transmission EM revealed clearly defined pericellular and territorial zones, dense proteoglycan/ collagen networks and intricate chondrocyte-matrix contacts. Comparison of 3-week neo-cartilage with 3-day epiphyseal cartilage by SDS-PAGE revealed major differences in protein composition and extractability. In juvenile cartilage the greater proportion of proteins were readily soluble, whereas in the neo-cartilage a higher proportion of the extract was poorly soluble. Therefore, label-free quantitative MS/MS, combined with rigorous statistical and bioinformatic methods, was used to generate “extraction profiles” (NaCl-extracted versus GdnHCl-extracted) of the juvenile cartilage and neo-cartilage (7). We identified a panel of proteins involved in maturation of the neo-cartilage ECM, i.e. components with the greatest differential in extractability between the two sample types, including many pericellular and extracellular matrix components (e.g. collagen VI, nidogen-2, perlecan, matrilin-3 and COMP). Unexpectedly, one of the guanidine extract specific proteins in the mouse neo-cartilage was a serine protease inhibitor, protease nexin-1. We confirmed that PN-1 was a novel component of the developing articular cartilage in vivo by immunohistochemistry. The potential further application of cartilage “extraction profiling” in the context of cartilage repair will be discussed.

Extended Abstracts 135

References:

1. Belluoccio, D., Wilson, R, Thornton, D.J., Wallis, T.P., Gorman, J.J. and Bateman, J.F. (2006) Proteomic analysis of mouse growth plate cartilage. Proteomics 6, 6549-6553.

2. Wilson, R. and Bateman, J.F. (2008) Cartilage Proteomics: Challenges, solutions and recent advances. Proteomics: Clinical Applications 2 (2), 251-263

3. Wilson, R., Belluoccio, D and Bateman, J.F. (2008) Proteomic analysis of cartilage proteins Methods 45 (1), 22-31

4. Wilson, R., Belluoccio, D., Little, C.B., Fosang, A.J. and Bateman, J.F. (2008) Proteomic characterization of cartilage degradation in vitro.Arthritis and Rheumatism 58(10): 3120-3131.

5. Wilson, R., Whitelock, J.M. and Bateman, J.F. (2009) Proteomics makes progress in cartilage and arthritis research Matrix Biology 28(3):121-8.

6. Wilson, R. and Bateman, J.F. (2008) A robust method for proteomic characterization of mouse cartilage using solubility-based fractionation and two-dimensional electrophoresis. Matrix Biology 27(8):709-12

7. Wilson, R., Diseberg, A., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E., Gorman, J.J. and Bateman, J.F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Molecular and Cellular Proteomics. Feb 26. [Epub ahead of print]

Acknowledgments:

This work was funded by the National Health and Medical Research Council of Australia.

11.2.3Specific regions of genome contribute to articular cartilage regeneration and osteoarthritis resistance in miceM.F. Rai, S. Hashimoto, J. Cheverud, L.J. SandellSt. Louis/United States of America

Introduction: Articular cartilage injuries heal poorly which is thought to lead to cartilage degeneration and predispose to the development of osteoarthritis (OA) [1]. Many attempts have been made to restore or repair cartilage injuries through surgical interventions including tissue engineering but have shown unpredictable and variable outcomes. Recently, a mouse model of cartilage repair has been characterized which determined a direct correlation between articular cartilage healing and protection from OA comparing the healing DBA/1 mouse with the non-healing C57Bl/6 strain [2]. Also recently, it has been shown that MRL/MpJ strain of mice shows an ability to regenerate knee articular cartilage over time [3]. The MRL/MpJ strain has been produced by two final backcrosses of LG/J (Large, a congenic healer strain for ear wound healing) and shares 75% of its genome with this strain [4]. We were interested in exploring cartilage repair ability in healer and non-healer recombinant inbred (RI) lines generated from LG/J and SM/J (small) mice and thus to discover the genetic basis for cartilage repair. The RI lines were developed through brother-sister mating of LG/J x SM/J mice at F7 for over 20 generations [5]. Each strain of the RI line contains unique portions of the healing strain genome. We predict that the animals that are able to heal a wound in their ear are more able to repair damaged articular cartilage and may be more resistant to OA. As OA is considered to be a complex genetic disease, our present project creates a unique resource for study of genes that contribute to cartilage healing and thus to protection from OA in 4 strains of RI line and the parental strains. This will narrow the genetic site to 10-20 genes responsible for cartilage healing.

Content: An acute full-thickness cartilage injury was introduced in the trochlear groove of both knees of 8-weeks old mice from both sexes according to the method of Fitzgerald et al. [3]. Mice were sacrificed at 4, 12 and 16 weeks (strain 6 and 33) and at 12 weeks (strain 35 and N48). SM/J, DBA/1 and MRL/MpJ were used as controls and sacrificed at 12 weeks. At indicated time points, knee joints were harvested and promptly fixed in 10% neutral buffered formalin for 12-14 hours. Joints were decalcified using 10% formic acid in 5% formaldehyde for 48 hours followed by incubation in 0.01% EDTA for 4-6 hours. Samples were paraffin embedded, sagittally sectioned at 5 µm intervals and subsequently stained with 0.03% toluidine blue. Slides were graded for five parameters including cell morphology, staining intensity, surface regularity, thickness of hyaline cartilage and its integration with native cartilage tissue on a scale from 1 to 14. Selected sections from each strain were subjected to immunostaining using collagen type II and collagen type I antibodies detected by immunofluorescence. OA was induced by transection of the medial meniscal ligament.

Results indicated that strain 6 showed a partial filling of defects up to 12 weeks post-surgery, nonetheless, it demonstrated a significantly superior healing potential at 16 weeks when the defects were completely filled with new cartilage tissue. The most striking changes in the cartilage regeneration in this strain were observed between 12 and 16 weeks after surgery. Further, our preliminary results revealed that males in this strain show better healing than females at 16 weeks. In contrast, strain 33 showed poor or no healing at each time point and the defects remained unfilled even at 16 weeks (Fig. 1). Strain 35, N48 and SM/J did not show healing potential as well. As expected, DBA/1 and MRL/MpJ control mice showed a complete healing ability at 12 weeks with DBA/1 healing slightly faster than the MRL/MpJ. The outcome measures from histological grading based on five parameters mentioned above are summarized in Fig. 2. Immunostaining of collagen type II revealed that articular cartilage defects in strains 6, DBA/1 and MRL/MpJ were altogether filled with a hyaline-like cartilage. It was, however, interesting to note that the cartilage defect site in the non-healer mice showed no collagen type I staining. The composition of the repair cartilage is currently under investigation.

OA was induced by transection of the medial meniscal liagament in Strains 33 and 6. Strain 6 was protected from OA while strain 33 had severe OA.

Discussion. Our murine model of cartilage regeneration indicates that the outcome of full thickness cartilage lesions is strain, sex and age dependent in RI line. Amongst all RI strains, strain 6 males exhibited superior healing potential at 16 weeks. Based on our preliminary finding we have classified the mouse strains in to two categories,

Extended Abstracts136

super healers (high scorers) and poor healers (low scorers). Among the first category include strain 6, MRL/MpJ and DBA/1 while the second category includes strains 33, 35, N48 and SM/J.

We have also demonstrated that strain 6 is protected from OA when compared to strain 33, which is highly susceptible. Thus we have established in these recombinant inbred lines, that cartilage healing is associated with protection from OA. These new lines, 1 healing and 4 non-healing, establish a new set of models that will now be used to narrow the regions of the genome that contribute to cartilage repair and thus to OA.

References:

1. Buckwalter JA, Mankin HJ. Articular cartilage. 2: Degeneration and osteoarthrosis, repair, regeneration, and transplantation. J Bone Joint Surg 1997;79A:612–32.

2. Eltawil NM, De Bari C, Achan P, Pitzalis C, Dell’accio F. A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury. Osteoarthritis Cartilage. 2009;17:695-704.

3. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage. 2008;16:1319-26.

4. Murphy ED, Roths JB. Autoimmunity and Lymphoproliferation: Induction by Mutant Gene lpr and Acceleration by a Male-Associated Factor in Strain BXSB. Genetic Control of Autoimmune Disease, eds Rose NR, Bigazzi PE, and Warner NL (Elsevier, New York), 1979;207–20.

5. Hrbek T, de Brito RA, Wang B, Pletscher LS, Cheverud JM. Genetic characterization of a new set of recombinant inbred lines (LGXSM) formed from the inter-cross of SM/J and LG/J inbred mouse strains. Mamm Genome 2006;17:417–29.

11.3.1Cartilage repair by joint distraction in experimental models of joint degenerationT. IshiiTsukuba/Japan

Introduction: Cartilage regeneration in degenerative joint needs cells to make cartilage, a space for cell differentiation and making matrix, physiological conditionas growth factors and control of mechanical condition. We made an experimental model for large full thickness cartilaginous defect and control of mechanical condition using hinged joint external fixator.

Content: Experimental Model: Japanese white male rabbits those were more than 16 weeks old, were used for the experimental model. Under anesthesia, each rabbit was fixed external fixator, which was an originally constructed Ilizarov-type half-ring with a hinge using fine wires. After fixing, an operative approach to the joint was performed and cruciate and collateral ligaments were divided and the menisci resected. A 3 mm deep of full- thickness osteochondral defect was made by excising the entire surface of the tibial plateau using an oscillating saw. The space formed at the femorotibial joint by the resected osteochondral defect was maintained by the fixator. After the operation, voluntary movement was unrestricted.

Experimental groups:

(1) An effect of Joint Distraction and Motion. Control group (Group C) was killed at 1, 3, 6 and 12 weeks after the external fixation. Weight bearing group (Group W) was not distracted. Immobilization group (Group I) was locked the hinge joint. Those two groups were killed at 12 weeks.

(2) An effect of autologous culture expanded bone-marrow-derived mesenchymal cell transplantation (ACBMT). In transplantation group (Group T), ACBMT with atelocollagen gel was injected into the knee using an 18-gauge needle three weeks after operation. In vehicle group (Group V), only atelocollagen gel was injected at the same term. Those two groups were killed at 12 weeks. (3) An effect of concentrated autologous bone marrow aspirate transplantation (CABMAT). CABMAT group (Group CAB), CABMAT with autologous fibrin gel, which was prepared by 10 ml aspirated bone marrow blood of the femur, was injectd 7 days after the first operation. In another group, concentrated autologous peripheral blood cell with fibrin gel (Group PBC), which was prepared by 10ml of peripheral blood, was also injected in the same manner. Group GEL was underwent only fibrin gel transplantation from peripheral blood as same. Group CON was no transplantation. The animals were killed 8 or 12 weeks after the operation. (4) An effect of long term distraction. In Group A, the distraction and movement with the fixator were maintained for six months, and the animals were then killed.

In Group B, the distraction and movement were maintained for six

months. After which the devices were removed and without restriction for a further six months, rabbits were killed one year after operation.

(5) An effect of controlled mechanical stress. To clear the effect on mechanical stress to the regenerated cartilage in the experimental model, we develop the control system for hinged fixator with continuous passive motion (CPM) and gradual applied weight-bearing function. In GWB groups (6W-GWB and 9W-GWB), GWB was initiated 6 weeks in the 6W-GWB and 9 weeks in the 9W-GWB after operation. GWB (0.5 kg in the first week; 1 kg, second week; and 2 kg, third week) was applied with CPM for 2 hours every day. In CPM groups (6W-CPM and 9W-CPM), CPM with fixator was applied in the same manner but without GWB. The CONTROL groups (6W-CON and 9W-CON) received only joint distraction without GWB or CPM.

Evaluation: The proximal tibia was decalcified, and sagittal sections at the midportion of the medial and lateral tibial plateaus were obtained for histological analysis. These sections were stained with hematoxylin and eosin, safranin-O/fast green (SO), and a monoclonal antibody for type-II collagen (Col-II). The central one-third of the regenerated tissue was assessed and scored blindly using a grading

scale of the International Cartilage Repair Society (ICRS) visual histological assessment scale [3]. The area of regenerated soft tissue and that stained by SO and Col-II were measured, and the ratio of the area stained by SO and Col-II to the area of the regenerated soft tissue was calculated. Results:

(1) In regenerated process of Group C, immediately after forming the defect, the trabecular bone was exposed to the joint space. Seven days later, the articular surface was smooth and remodelled. Spindle-shaped mesenchymal cells were observed on the surface of the defect. At three weeks, there was synthesis of the cartilaginous

Extended Abstracts 137

extracellular matrix, as indicated by the staining of the matrix by SO and Col-I and II. The matrix was synthesised between the superficial fibrous stroma and the deeper area of a newly-formed woven bone. At six weeks, the

regenerated cartilage tissue had reached the surface of some areas and the cartilage matrix stained strongly with SO. The flat bone surface of the tibial plateau was remodelled into a concavity adapted to the shape of each femoral condyle. At 12 weeks, the cells had a columnar distribution. At six and 12 weeks, Col-II was observed in the matrix and Col-1 was less intense compared with Col-II. SO stained area was largest in Group C among three groups, which was significant

against Group I.

(2) Group T was significantly superior to Group V in ICRS scale II, scale III and SO-stained positive area. Comparing Group T with Group C of experimental group (1), there is no significant difference.

(3) Results were more eminent in 12 weeks than 8 weeks in general tendency. SO-stained positive area with respect to the regenerated soft tissue area was significantly highest in Group CAB to other groups.

(4) Group A was significantly inferior in SO and Col-II stained positive areas to Group C in 12 weeks. Comparing Group A with B, Group A was also significantly inferior in SO and Col-II stained positive areas.

(5) The area of regenerated soft tissue in the Group 9w-GWB was statistically larger than that in the 9w-CON group. The ratio of the SO-stained positive area in the Group 9W-GWB was the largest among all the groups and significantly larger than those in the Group 9w-CPM and 6w-GWB. The ratio of the Col-II-stained area in the 9w-GWB was similar to those of the SO-stained area. The ratios of the SO and Col-II-stained area in the 6w-CON were significantly larger than those in the 6w-CPM.

Discussion: Four essential factors for cartilage regeneration, cells to make cartilage, a space for cell differentiation and making matrix, physiological condition as growth factors and control of mechanical condition, are contained in our experimental model. Cells to make cartilage are introduced from bone marrow by spongialization. Cell transplantation technique as ACBMT or CABMAT slightly promoted quality of regenerated cartilage in the experimental model. In human, cartilage regeneration is difficult than rabbit model and some cell transplantation technique may be required. Three millimeters thickness of osteochondral resection of total tibial plateau makes a space for cell differentiation and making matrix. In large cartilage defects on weight-bearing joint, the space is lost by weight bearing without an external fixator. Without joint distraction, Group I were poor regeneration

especially at the weight bearing area. The space, in other words, is a culture box supplied growth factors from joint fluid in physiological articular condition. Control of mechanical condition is the most difficult understanding factor. Our experimental model is very interesting in an ability of control of mechanical stress.

Straight cut tibial plateau adapts to remodels in circular orbital shape centering at the hinged point. Too much time for distraction reduced the quality of cartilage as in Group A. On the other hand, excessive mechanical stress induces inflammation in the joint and progress the joint degeneration. In our gradual weight-bearing model, 9w-GWB was significant better quality than 6w-GWB. The result only means 9 weeks is more adequate regenerative condition for starting to begin weight

bearing than 6 weeks in the experimental model. On the stages of cartilage regeneration, adequate mechanical stress will be different in each stage, whose principle never knew.

Conclusion: Control of mechanical stress is essential for the cartilage regeneration of large cartilage defects. To better understanding of controlling the mechanical stress lead to clinical application of cartilage regeneration.

11.3.3Challenge of Repairing Large Cartilage Defects with Minimally Invasive TechniqueM. OchiHiroshima/Japan

Introduction: Several surgical approaches to repair cartilage defects have been reported such as reattachment of a detached osteochondral fragment to the lesion, microfracture, mosaicplasty and ACI. We treated eight cartilage defects with meniscal transplantation from 1990 to 1995. Then we started to perform transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (110 cases) as a second generation of chondrocyte transplantation from 1996(1). Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. Although the clinical results were satisfactory(2), we need the surgical approaches to treat large cartilage defects with minimally invasive technique.

Content: One of the less invasive surgical procedures to treat large cartilage defects is microfarcture or drilling. However, these techniques under arthroscopy are not sufficient to repair cartilage defects with hyaline cartilage. I think that there are two weak points such as insufficient number of mesenchymal stem cells and early overloading on the treated area. 1) One recent strategy is by the implantation of mesenchymal stem cells (MSCs)(3) . MSCs are the cell population of undifferentiated cells isolated from adult tissue that have the capacity to differentiate into mesodermal lineages, such as bone, cartilage, fat, muscle or other tissues. MSCs from bone marrow can be cultured and differentiated into the desired lineage in vitro with the application of specific growth factors or bioactive molecules.Intra-articular injection of too many MSCs, however, can generate free bodies of scar tissue(4) . We therefore developed a novel stem cell delivery system for cartilage repair using magnetically labeled MSCs and an external magnetic device to accumulate a relatively small number of MSCs to a desired area. Ferumoxides are dextran-coated super paramagnetic iron oxide nanoparticles approved by the US Food and Drug Administration as a magnetic resonance contrast agent for hepatic imaging of humans. By use of this ferumoxides, it is easy to make magnetically labeled MSCs(5) . Recently we demonstrated the ability to deliver magnetically labeled MSCs to a cartilage defect that is a desired place under arthroscopy in rabbit and swine knee joints using external magnetic device (0.6T) (5) . This result indicates that this minimally invasive system under arthroscopy can be applicable for a focal osteochondral defect in the knee joint. The next step is to examine if this external magnetic system is effective for osteoarthritis. We investigated if we could successfully regenerate a cartilage layer on degenerated human cartilage in vitro using this external magnetic system(6) . MSCs from human bone marrow were cultured and magnetically labeled. Degenerated human cartilage was obtained during total knee arthroplasty. The osteochondral fragments were attached to the sidewall of tissue culture flasks, and magnetically labeled MSCs were injected into the flasks. Using an external magnetic device, a magnetic force was applied for 6 hours to the direction of the cartilage, and then the degenerated osteochondral fragment was cultured in chondrogenic differentiation medium for 3 weeks. In the control group, a magnetic force was not applied. The specimens were evaluated histologically. A cell layer was formed on the degenerated cartilage as revealed by hematoxylin and eosin staining. The cell layer was also stained in Toluidine blue and Safranin O, and with anti-collagen type II immunostaining, indicating that the cell layer contained an abundant extracellular matrix. In the control group, a cell layer was not observed on the cartilage. In conclusion, we could demonstrate that our system could deliver MSCs onto degenerated human cartilage, and then form an abundant extracellular matrix on the degenerated cartilage in vitro. 2) Another technique is to reduce the load to the repaired area in the knee joint after bone marrow stimulation to protect immature tissue regenerated at the repaired area against destruction caused by overloading. We made a new distraction arthroplasty device (meira, Japan) which allows the range of motion with knee joint distraction(7)(8) . After drilling or microfracture under arthroscopy, the new external device was fixed with four 6-mm pins drilled into the distal femur and the proximal tibia. After the appropriate distractive tension was applied, the ROM and the postdistraction and predistraction tibiofemoral joint spaces at 30° of flexion were measured. Although this device is usually applied for 3 months, full weight bearing is allowed one month after surgery. Until now, this device has been demonstrated to function well to repair cartilage defects. I would like to show my technique using an external magnetic field to deliver precisely injected cells with magnetic beads to an articular defect and articulated distraction device for reducing the load for a large osteochondral defects or osteoarthritis.

Extended Abstracts138

References:

(1) Ochi M, Uchio Y, Kawasaki K, Wakitani S, Iwasa J: Transplantation of cartilage-like tissue made by tissue-engineering for the treatment of cartilage defects of the knee. Journal of Bone Joint Surgery[Br]. 84(4):571-578, 2002

(2) H. Tohyama, K. Yasuda, A. Minami, T. Majima, N. Iwasaki, T. Muneta , I. Sekiya, K. Yagishita, S. Takahashi, K. Kurokouchi, Y. Uchio, J. Wasa, M. Deie, N. Adachi, K. Sugawara, and M. Ochi: Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan. Journal of Orthopaedic Science. 14(5):579-588. Epub Oct 3, 2009

(3) Nishimori M, Deie M, Kanaya A, Exham H, Adachi N, Ochi M: Repair of chronic osteochondral defects in the rat: The bone marrow stimulating Procedure with cultured allogenic bone marrow mesenchymal stromal cells. The Journal of Bone and Joint Surgery [Br]. 88-B(9):1236-1244, 2006

(4) Muhammad Agung, Mitsuo Ochi, Shinobu Yanada, Nobuo Adachi, Yasunori Izuta, Takuma Yamasaki, Katsuhiro Toda: Mobilization of bone marrow-derived mesenchymal stem cells into the injured tissues after intraaricular injection and their contribution to tissue regeneration. Knee Surgery Sports Traumatology Arthroscopy. 14(12):1307-1314, 2006

(5) Kobayashi T, Ochi M, Yanada S, Ishikawa M, Adachi N, Deie M, Arihiro K: A novel cell delivery system using magnetically labeled mesenchymal stem cells and an external magnetic device for clinical cartilage repair. Arthroscopy. 24(1):69-76, 2008

(6) Kobayashi T, Ochi M, Yanada S, Ishikawa M, Adachi N, Deie M, Arihiro K: Augmentation of degenerated human cartilage in vitro using magnetically labeled mesenchymal stem cells and an external magnetic device. Arthroscopy. 2009 Dec;25(12):1435-1441. Epub Nov 6, 2009

(7) Ryoji Kajiwara, Osamu Ishida, Kenzo Kawasaki, Nobuo Adachi, Yuji Yasunaga, Mitsuo Ochi: Effective repair of a fresh osteochondral defect in the rabbit knee joint by articulated joint distraction following subchondral drilling. Journal of Orthopaedic Research. 23:909-915, 2005

(8) Masataka Deie, M.D., Mitsuo Ochi, M.D., Nobuo Adachi, M.D., Ryoji Kajiwara, M.D. and Atsushi Kanaya, M.D.: A new articulated distraction arthroplasty device for treatment of the osteoarthritic knee joint: a preliminary report. Arthroscopy. 23(8):833-838, 2007

15.0.3Clinical results with the use of biomaterials for cartilage repairS. Nehrer, F. HalbwirthKrems/Austria

Introduction: Cell based therapies in cartilage repair Autologous Chondrocyte Transplantation with the periosteal flap has changed the paradigm of the treatment of cartilage defects from repair to regeneration. This has been demonstrated in randomized trials proving the concept of regenerating tissue in a cell based therapy approach. However, the limitations of the periosteal flap concerning size and thickness and surgical demands with suturing and the variability of the biological reaction including hypertrophy, as well as the uncontrolled celltransplatation in a cell suspension has supported the introduction of biomaterials. The first attempt was to replace the periosteal flap by membranes which were sutured or glued to the defect combined with the cellsuspension injected or seeded onto the membrane during the surgery before implantation. Concerns about the phenotype of the cultured cells led to the use biomaterial assisted technologies in sponges or gels to maintain the chondrocytic function of the cells and allow a more controlled dispersion of the chondrocytes throughout the defect. The matrix characteristics concering biochemical composition, biophysical appearance in gel, foams or sponges, degradation dynamics and products, toxicity, immunological reactions and general biocompatibility are important parameters of biomaterial development. The cell-biomaterial interaction in a biological environment is lastly the decisive process of a successful cartilage regeneration.

Content:Biomaterials in clinical use Various materials have been tested in in-vitro laboratory studies and in-vivo animal experiment to evaluate the best material for human use. Most of the materials are able to support the chondrocytic phentotype, allow proliferation and cellmigration and facilitate production of cartilage specific substances. Animal models show in most cases that cell loaded constructs show superior results than unloaded matrices. Based on this results clinical studies were employed to prove feasibility and safety of these technologies. Most of the reported results are based on case studies only few randomized controlled trials were performed. There is still discussion about the approbiate control group in the study design, since microfracture is performed arthroscopically and basically a different approach, only very few studies compare matrix techniques with other cell transplantation methods, however the benchmark is still autologous chondrocyte transplantation with the periosteum patch. Collagen membrane The collagen membrane was the first biomaterial used in clinical studies. Based on positive results in animal studies, confirming the biological concept of matrix assisted cell technology a bilayer Type 1/3 collagen membrane was sutured instead of the periosteal flap to the defect site and the cell suspension is injected underneath. This method still requires suturing of the membrane and low control of the seeding process of the cellsuspension. The clinical results of five years follow-up are compareable to the periosteum technique. In biopsy studies hyaline-like tissue have been found. The preseeding of the membrane was the next development, allowing a more controlled distribution of the cultured cells and also allows glueing of the graft to the defect, in this technique the chondrocytes are seeded in a fibrin glue suspension, however the impact of fibringlue on chondrocytes is still controversial and experimental and studies on the stability of such fixation show minor mechanical properties. Hyaluronan Besides the collagen membrane the use of a hyaluronan based scaffold has been used in the indication of a chronic cartilage defect. The material consists of a non woven mesh of hyaluronan fibers modified by esterification by benzylesters to improve the biocompatibility of the material. Since the hyaluronan is a natural constituent of cartilage and the material desolves into hyaluronan, it is a promising candidate for chondrocyte transplantation. The invitro studies revealed the Hyaff 11 mesh as an appropriate scaffold for chondrocytes regarding the chondrocytic phenotype, however the animal studies on the material are sparse and no conclusive controlled data available. However, the clinical performance of the material in three- and five-years follow-ups document successful performance in over 80 % across the study population. Stageing the data according to age and severity of the cartilage defect, patients under the age of 35 and single circumscribed defects do a lot better with success rates over 90 %. The precultured hyaluronan matrix shows good attachment of the chondrocytes and also allows an arthroscopic implantation. The preformed shaping of the defect and the rough surface of the graft allows fixation without glueing and pressfit technique and reduces joint morbitiy through the arthroscopic approach. PLA/PGA membranes Simmilar arthroscopic techniques are performed with precultrred matrices consisting of PLA/PGA meshes, wich are fixed with technically demanding intraosseous suture-knot technique or lately with more easy pinfixation. The cellseeded construct shows compareable results to ACT in midterm followup in cohort studies and the stable, mechanically resistant graft allows also to treat circumscribed osteoarthritic lesions with satisfying outcomes. Recent techniques using minced cartilage pieces glued to the membrane have been developed, assuming that

Extended Abstracts 139

outgrowth of chondrocytes from the minced tissue provide adequate chondrogenic stimulation to facilitate cartilage regeneration. The minced tissue is harvested with a newly developed shaver system which allows automized seeding on the artificial membrane. The 2 years clinical follow up shows better results compared to microfracture. Collagen Gel The 3D-culture system in gels allows the chondrocytes to maintain the chondrocytic phenotype, especially when P1 cells are used. The distribution of the cells resembles more the sparsely dispersed cells in natural cartilage and a high bioactivity of the cell is achieved by vigourous cartilage specific protein production. The soft collagen gel needs a well debrided defect with stable walls to allow a pressfit stabilisation support by fibrin glue. The graft shows a complete fill of the defect and a perfect bonding to the adjacent cartilage. Clinical results of case controlled studies and multicenter data show comparable results to ACT and better performance with regards to effusion and surgery time. Fibrin Fibrin as a biomaterial was used in liquid form in a glue-cellsuspension or lately also in a spongelike matrix. The fibrin technique allows a homologous approach to the cell transplantation and the support of FGF growthfactor stimulates cellproliferation and differentiation to shorten the necessary culture period. First clinical case series show promising results of the material, the spongelike appearance serves as a more stable construct than liquid glues. Other combinations of gels and membranes are available to combine 3D environment with a biomechanical stable fixation technique. Clinical performance The clinical results of matrix techniques are comparable to the midtermresults of ACT, hence longterm results are not yet available. Esspecially circumscribed lesions on the condyle in the younger patients are doing good in all the studies. However, most studies are case series and only few are controlled trials including controls. The advantage of the use of biomaterials is the shorter surgery time, the easier handling and fixation, the secure and biological appropriate environment for the chondrocytes during the transplantation process, the smaller incision including arthroscopic techniques and the more predictable biological regeneration process avoiding overgrowth like hypertrophy of the periosteum graft. The safety reports reveal only minor adverse events most of the time and from the ethic standpoint no bridges are burned for further treatment options including revisions, mosaicplasty or redos of celltransplantation. The use of biomaterials without cultured cells is of course the logistical and legistical easier process and serves economical interest of companies and healthcare stake holders. All of the matrices were developed for the cellbased therapies and are now serving as scaffolds for the bloodclot techniques like microfracture. Some early midtermresults show increased healing response but –like microfracture alone- inadequate tissue formation and thinning of the surface layer by intracartilagenous bone formation. However clinical and experimental studies are on the way and hopefully will help to develop the optimal biomaterial based cartilage repair procedure. Up to now the evidence in experimental studies is low to suggest successful use of cell-free constructs and most animal experiments show advantages of the cell augmented techniques, however the addition of growthhormones, chondrocyte nutrients or application nanotechologic applications in biomimetic materials may allow such techniques in the future. However, cellbased therapies without biomaterials like chondrospheres or stemcells may be a relevant alternative to biomaterials and make the difference at last.

References:

Literature

1) Bartlett W, Skinner JA, Gooding CR, Canington RW, Flanagan AM, Briggs TW, Bentley G (2005) Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study. J Bone Joint Surg Br 87:640-645

2) Behrens P, Bitter T, Kurz B, Russlies M (2006) Matrix-associated autologous chondrocyte transplantation/implantation (MACT/MACI)-5-year follow-up. Knee 13: 194-202

3) Bentley G, Biant LC, Carrington RWT, Akmal M, Goldberg A, Williams AM, Skinner JA, Pringle J (2003) A prospective, randomized comparison of autologous chondrocyte implantation versus mosaicplasty for ostechondral defects of the knee. J Bone Joint Surg Br 85:223-230

4) Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Eng J Med 331:889-895

5) Dorotka R, Windberger U, Macfelda K, Bindreiter U, Toma C, Nehrer S (2005) Repair of articular cartilage defects treated by microfracture and a three-dimensional collagen matrix. Biomaterials 26:3617-3629

6) Erggelet C, Sittinger M, Lahm A (2003) The arthroscopic implantation of autologous chondrocytes for the treatment of full-

thickness cartilage defects of the knee joint. Arthroscopy 19:108-110

7) Gigante A, Bevilacqua C, Cappella M, Manzotti S, Greco F (2003) Engineered articular cartilage: influence of the scaffold on cell phenotype and proliferation. J Mater Sci: Mater Med 14:713-716

8) Gooding CR, Bartlett W, Bentley G, Skinner JA, Carrington R, Flanagan A (2006) A prospective, randomised study comparing two techniques of autologous chondrocyte implantation for osteochondral defects in the knee: Periosteum covered versus type I/III collagen covered. Knee 13:203-210

9) Hendrickson DA, Nixon AJ, Grande DA, Todhunter RJ, Minor RM, Erb H, Lust G (1994) Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects. J Orthop Res 12:485-496

10) Iwasa J, Ochi M, Uchio Y, Katsube K, Adachi N, Kawasaki K (2003) Effects of cell density on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Artif Organs 27:249-255

11) Jakobsen RB, Engebretsen L, Slauterbeck JR (2005) An analysis of the quality of cartilage repair studies. J Bone Joint Surg Am 87:2232-2239

12) Kimura T, Yasui N, Ohsawa S, Ono K (1984) Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin Orthop Relat Res 186:231-239

13) Knutsen G, Drogset JO, Engebretsen L, Grontvedt T, Isaksen V, Ludvigsen TC, Roberts S, Solheim E, Strand T, Johansen O (2007) A randomized trial of autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am 89:2105-2112

14) Marcacci M, Zaffagnini S, Kon E, Visani A, Iacono F, Loreti I (2001) Arthroscopic autologous chondrocyte transplantation: technical note. Knee Surg Spons Traumatol Arthrosc 10:154-159

15) Marlovits S, Singer P, Zeller P, Mandl I, Haller J, Trattnig S (2006) Magnetic resonance observation of cartilage repair tissue (MOCART) for the evaluation of autologous chondrocyte transplantation: determination of interobserver variability and correlation to clinical outcome after 2 years. Eur J Radiol 51:16-23

16) Nehrer S, Dorotka R, Domayer S, Stelzeneder D, Kotz R. Treatment of full-thickness chondral defects with hyalograft C in the knee: a prospective clinical case series with 2 to 7 years’ follow-up. Am J Sports Med. 2009 Nov;37 Suppl 1:81S-87S. Epub 2009 Oct .

17) Nehrer S, Spector M, Minas T. Histologic analysis of tissue after failed cartilage repair procedures. Clin Orthop Relat Res. 1999;365:149–162

18) Nehrer S, Minas T. Treatment of articular cartilage defects. Invest Radiol. 2000;35:639–646.

19) Nehrer S, Domayer S, Dorotka R, Schatz K, Bindreiter U, Kotz . R (2006) Three-year clinical outcome after chondrocyte transplantation using a hyaluronan matrix for cartilage repair. Eur J Radiol 57:3-8

20) Nehrer S, Chiari C, Domayer S, Barkay H, Yayon A. Clin Orthop Relat Res. 2008 Aug;466(8):1849-55. Epub 2008 Jun 5. 22) Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A (2002) Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med 30:2-12 23) Steinwachs M, Kreuz PC (2007) Autologous chondrocyte implantation in chondral defects of the knee with a type I/III collagen membrane: a prospective study with a 3-year follow-up. Arthroscopy 23:381-387 24) Zheng MH, Willers C, Kirilak L, Yates P, Xu J, Wood D, Shimmin A (2007) Matrix-induced autologous chondrocyte implantation (MACI): biological and histological assessment. Tissue Eng 13:737-746

21) Ossendorf C, Kaps C, Kreuz PC, Burmester GR, Sittinger M, Erggelet C (2007) Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results. Arthritis Res Ther 9:R41

Extended Abstracts140

16.1.1Biologic Unicompartmental Knee ReplacementJ. FarrIndianapolis/United States of America

Introduction: There have been incremental advancements in biologic unicompartmental knee replacement since the 2007 extended ICRS abstract on the topic.1 The term applies to resurfacing either one tibiofemoral compartment or the patellofemoral compartment. The techniques are extensions of standard cartilage restoration options applied to both sides of the compartment. That is, bipolar lesion treatments—often with large area of exposed bone that would classically be treated with metal and plastic unicompartmental arthroplasty. At present, biologic unicompartmental arthroplasty continues to offer limited success, but with growing hope for the future.

Content: For purposes of expanding discussion for algorithm development, the knee is typically divided into three uni-compartments: medial and lateral tibiofemoral and patellofemoral. The topics will group both tibiofemoral compartments together and the patellofemoral compartment separately. Biologic unicompartmental knee replacement requires optimization of the compartment’s biomechanical environment in addition to resurfacing the bone with articular cartilage. The patellofemoral (PF) pain patient is obviously very complex. As articular cartilage is aneural the pain originates in the bone and/or the soft tissues surrounding the compartment. An excellent point of reference for PF is a 2006 review by Gomoll et. al. titled “Treatment of Chondral Defects in the Patellofemoral Joint.”2 Two useful tools in evaluating PF compartment chondrosis are Fulkerson’s3 classification and Dejour’s4 dysplasia classification. Fulkerson’s classification may be summarized as: 1) the patella is located excessively laterally with reference to the trochlea, 2) the patella is excessively tilted 3) tilt and lateral patellar position 4) and those with a congruent PF joint. Dejour’s classification A through D detailed progressive extremes of trochlear dysplasia. In addition, each of these subtypes may have patellar alta or infera. There are a different set of consideration at the tibiofemoral (TF) compartment. The three main considerations at the TF compartment are the status of the meniscus, ligaments and coronal plane alignment (varus/valgus). For goal both the TF and PF compartments is to optimize the biomechanical environment for the cartilage implant. Results: Minas5 published his PF autologous cultured chondrocyte implantation (ACI) outcomes, which demonstrated better results than expected by anteromedialization alone as per the Pidoriano review of Fulkerson AMZ .6 Of these patients, 29 (64%) had a concomitant AMZ. His series of 45 patients included 24 bipolar cases with 71% good or excellent outcomes overall, yet without bipolar subset data analysis.5 Henderson reported a series with two subgroups: those with and without AMZ. Overall the groups have 54.5% and 86% good and excellent results for the ACI alone or with AMZ, respectively, but without subset analysis of bipolar lesions.7 In Peterson’s series, there were overall 84% good and excellent results while in the PF group had 65% good and excellent results without bipolar subset reporting.8 Farr9 in 2007 reported on 4 bipolar PF ACI patients out of 39 total PF ACI patients. 81% had good or excellent results. Again, in light of the small number of patients the bipolar treatments were not compared to monopolar patients. Cole’s10 subgroup of 5 bipolar PF ACI patients out of 62 total PF ACI patient were not subanalyzed, but the group as a whole had significant improvements over multiple knee scales. In all of these studies the decision to perform AMZ was empiric based on surgeon preference rather than using the tibial tuberosity trochlear groove distance. The role of tuberosity surgery in PF cartilage treatments remains under investigation noting that finite element analysis by Ateshian and Cohen demonstrated that AMZ candidates could have quite variable response to elevation and medialization.11 Their measured decrease in PF forces with AMZ was on the order of 20% rather than the historically quoted 50% reduction. Those patients with PF dysplasia and/recurrent patellofemoral lateral recurrent instability often have medial patellar facet and/or lateral femoral condyle chondral damage. The essential lesion for patellar lateral instability is the medial patellofemoral ligament (MPFL). The MPFL is repaired or reconstructed to prevent further instability episodes.12 The soft tissue envelope may be further balanced with a titrated lateral release or lateral retinacular lengthening. There is consensus that PF stability should be established using an anatomy based technique without over-constraint. There are few reports of PF bipolar osteochondral allografts. Bugbee reports a bipolar PF osteochondral allograft (OCA) shell success of approximately 60% in 12 bipolar patients compared with an approximately 80% success rate for PF monopolar patients.13 Bugbee did not alter the position of the tibial tuberosity. Teitge reported in 2006 on 14 patients, 12 of which were bipolar.14 His success rate for the bipolar OCA resurfacing was 58% while none of the monopolar transplants succeeded. In the TF compartments both OCA and ACI have lower success rates than unipolar treatments, but use the same basic techniques as monopolar treatments. The environment for the implant is optimized in regards to alignment, stability and meniscal function. Farr reported a similar modest positive result with combined meniscal allograft

transplantation and ACI, but did not separately report the bipolar outcomes.9 Minas and Peterson both have large ACI series that have subpopulations with bipolar TF salvage ACI restorations with good and excellent outcomes of on the order of 80-90% for monopolar lesions and 60-70% for bipolar lesions.17 Richardson reported on 8 bipolar ACI/MAT patients with 85% good and excellent results and 3 failures.18 In a non peer-reviewed article Gersoff reported acceptable results after ACI and MAT.16 Cole reported 16 patients treated with MAT combined with ACI and 15 patients treated with MAT combined with OCA but did not provide numbers of bipolar treatments.15 The ACI/MAT patient had 80% satisfaction and the OCA patients had 71% osteochondral allograft satisfaction. The bipolar treatment outcomes with OCA alone vary from the limited success reported by Gross who thus abandoned the technique,19 to Bugbee who has continued to use allograft in selected bipolar salvage patients with success, but with low patient numbers precluding statistically analysis20 Conclusion: Although the numbers are small for both PF and TF bipolar ACI and PF OCA, it appears that biologic arthroplasty does have a role in the treatment of patients who would otherwise be suboptimal candidates for metal and plastic endoprosthetics due to their young age. This group is comprised primarily of salvage situations in the young. In the future if the success rate of bipolar biologic treatments improves, the upper age range may be gradually extended.

References:

1. Farr J. Biologic Unicomparmental Knee Replacement. Interanational Cartilage Restoration Society; Warsaw, Poland. 2007.

2. Gomoll AH, Minas T, Farr J, Cole BJ. Treatment of chondral defects in the patellofemoral joint. J Knee Surg. 2006;19:285-295.

3. Fulkerson JP, Shea KP. Disorders of patellofemoral alignment. The Journal of bone and joint surgery. American volume. 1990;72:1424-1429.

4. Dejour H, Walch G, Neyret P, Adeleine P. [Dysplasia of the femoral trochlea]. Rev Chir Orthop Reparatrice Appar Mot. 1990;76:45-54.

5. Minas T, Bryant T. The role of autologous chondrocyte implantation in the patellofemoral joint. Clin Orthop Relat Res. 2005;30-39.

6. Pidoriano AJ, Weinstein RN, Buuck DA, Fulkerson JP. Correlation of patellar articular lesions with results from anteromedial tibial tubercle transfer. Am J Sports Med. 1997;25:533-537.

7. Henderson IJP, Lavigne P. Periosteal autologous chondrocyte implantation for patellar chondral defect in patients with normal and abnormal patellar tracking. Knee. 2006;13:274-279.

8. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A. Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med. 2002;30:2-12.

9. Farr J, Rawal A, Marberry KM. Concomitant meniscal allograft transplantation and autologous chondrocyte implantation: minimum 2-year follow-up. Am J Sports Med. 2007;35:1459-1466.

10. Pascual-Garrido C, Slabaugh MA, L’heureux DR, Friel NA, Cole BJ. Recommendations and treatment outcomes for patellofemoral articular cartilage defects with autologous chondrocyte implantation: prospective evaluation at average 4-year follow-up. Am J Sports Med. 2009;

11. Cohen ZA, Henry JH, McCarthy DM, Mow VC, Ateshian GA. Computer simulations of patellofemoral joint surgery. Patient-specific models for tuberosity transfer. Am J Sports Med. 2003;31:87-98.

12. Davis DK, Fithian DC. Techniques of medial retinacular repair and reconstruction. Clin Orthop Relat Res. 2002;:38-52.

13. Jamali AA, Emmerson BC, Chung C, Convery FR, Bugbee WD. Fresh osteochondral allografts: results in the patellofemoral joint. Clin Orthop Relat Res. 2005;:176-185.

14. Torga Spak R, Teitge RA. Fresh osteochondral allografts for patellofemoral arthritis: long-term followup. Clin Orthop Relat Res. 2006;444:193-200.

15. Rue JPH, Yanke AB, Busam ML, McNickle AG, Cole BJ. Prospective evaluation of concurrent meniscus transplantation and articular cartilage repair: minimum 2-year follow-up. Am J Sports Med. 2008;36:1770-1778.

Extended Abstracts 141

16. Gersoff W. Combined meniscal allograft transplantation and autologous chondrocyte implantation. Operative Techniques in Sports Medicine. 2002;10:165-167.

17. Peterson L, Minas T, Brittberg M, Nilsson A, Sjögren-Jansson E, Lindahl A. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop Relat Res. 2000;212-234.

18. Bhosale AM, Kuiper JH, Johnson WEB, Harrison PE, Richardson JB. Midterm to long-term longitudinal outcome of autologous chondrocyte implantation in the knee joint: a multilevel analysis. Am J Sports Med. 2009;37 Suppl 1:131S-138S.

19. Mahomed MN, Beaver RJ, Gross AE. The long-term success of fresh, small fragment osteochondral allografts used for intraarticular post-traumatic defects in the knee joint. Orthopedics. 1992;15:1191-1199.

20. Emmerson BC, Görtz S, Jamali AA, Chung C, Amiel D, Bugbee WD. Fresh osteochondral allografting in the treatment of osteochondritis dissecans of the femoral condyle. Am J Sports Med. 2007;35:907-914.

16.1.2Treatment of early osteoarthritis with cartilage regeneration techniquesA.W. GobbiMilano/Italy

Introduction: Articular cartilage is a thin layer of specialized connective tissue lining the articulations of diarthrodial joints, characterized by unique properties, which enable an almost frictionless joint movement and protect the underlying bone from excessive load and trauma, by dissipating the forces produced during movement. However, articular cartilage has a limited intrinsic healing potential due the presence of few specialized cells with a low mitotic activity; furthermore, cartilage is avascular and there is a lack of source of undifferentiated cells that can promote tissue repair. Once injury occurs, cartilage gradually degenerates leading to early Osteoarthritis (OA), which is characterized by full-thickness, focal loss of the cartilage compaction, sclerosis of the adjacent subchondral bone, formation of bone cysts, osteophytes and joint space narrowing thus surgical intervention is necessary in order to repair chondral defects and protect the joint from OA. The prevalence of chondral defects is frequent with sporting injuries, especially in over 40 years of age patients, leaving often-persistent pain. OA increases steadily with age, affecting 80% of the population over 75 years of age and 12.1% of the population between 25 and 74 years of age, being the leading cause of physical disability in people over the age of 65; furthermore community-based studies have shown that 10% of the population over the age of 55 have troublesome knee pain and, of those, 25% are severely disabled. The social impact of bone and cartilage pathologies entails high costs in terms of therapeutic treatments and loss of income: in the US OA therapies cost $5.31 billion dollars in 2007, (Intercontinental Marketing Service data) and muscoloskeletal conditions including OA results in nearly $86.2 billion per year in direct medical expenses i.e. total joint replacement procedures and loss of income and production. Accordingly, 2000 to 2010 have been called the “decade of bone and joint” to launch global awareness and promote further research in prevention, diagnosis and treatment of joint injuries. For these reasons, the trend is now going towards preventive interventions and therapeutic solutions that can lead to an enhancement of tissue regeneration and the reduction of degenerative mechanisms.

Content: Imaging: The standard radiographic evaluation should include a standing AP long-leg radiograph, including also hips and ankles, standing AP/lateral views of both knees, skyline patellofemoral and standing 45º bend knee views. Among the diagnostic imaging modalities used, Magnetic Resonance Imaging (MRI) has been developed for the earlier and more quantitative detection of articular cartilage changes, having a sensitivity which is > 95%. Aside from delineating the extent of the articular cartilage lesions, subchondral bone and associated ligament or meniscal injuries can also be assessed. The use of fast-spin-echo (with or without fat suppression) and/or fat-suppressed (or water-selective excitation) spoiled gradient-echo image for better resolution has been recommended. Signal properties of articular cartilage are dependent on: MR pulse sequence utilized, cellular composition of collagen, proteoglycans and water, orientation of collagen in different laminae of cartilage, and effective cartilage pulse sequencing. MRI techniques such as the delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC) and T2 relaxation time mapping have been available recently, in the evaluation of articular cartilage, providing the ability of the glycosaminoglycan content visualization, the measurement of collagen content and the mapping of anatomical zones of cartilage.

Special Considerations in Decision Making: The arthritic process may be a result of an inherent biologic failure or secondary to trauma, remaining knee instability and bone bruising predisposing to osteoarthritis. Moreover, existing tibio-femoral or patello-femoral malalignment accelerates the progressive joint degeneration, as there is asymmetrical distribution of loads. Bone defects following a subchondral fracture, osteonecrosis or osteochondritis dissecans, should be considered preoperatively and the depth of the defect should be estimated by radiography or tomography. Patients with ongoing inflammatory arthritis or multiple joint involvement might consider to be addressed with a biologic versus a prosthetic option. Conversely, patients suffering from tricompartmental arthritis or osteonecrosis should undergo a total knee replacement (TKR) rather than a biologic repair of cartilage lesions. Although prosthetic arthroplasty currently obtains better long-term results and the age at which it is performed decreases, it is desirable for the young patients who would like to remain highly active, to avoid it due to associated activity restrictions after TKR. Several options exist in the treatment of

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cartilage lesions in young patients with early osteoarthritis: any form of planned treatment should be based on patient characteristics and expectations, clinical symptoms and parameters such as lesion size, depth and associated issues like leg axis alignment, ligament and meniscal integrity and presence of bone deficiencies. Furthermore, other factors related to the patient (e.g. age, genetic predisposition, level of activity and associated pathologies) should not be ignored.

Operative treatment: The coexisting pathologies that need to be addressed for successful reconstruction are tibio-femoral axial alignment, patello-femoral alignment, ligamentous insufficiency, meniscal absence and bone deformities. Knee osteotomies are used to correct varus or valgus malalignment associated with unicompartmental osteoarthritis and can be combined with cartilage repair procedures. Opening wedge high tibial osteotomies are used extensively for the treatment of varus osteoarthritis as distal femur osteotomies are indicated mainly to correct valgus deformities. Correction of the axial deformity should be performed prior to cartilage defects repair, in a concomitant or staged procedure. When patello-femoral malalignment is present with a trochlear and/or patellar chondral lesion, a thorough evaluation must be carried out primarily in order to restore normal patellar tracking. Ligamentous insufficiency is a negative factor in chondral graft healing thus, ACL reconstruction, in a staged or concomitant procedure, must be performed to ensure graft protection and safe return in daily life activities or sports. Meniscal absence is also an important factor to consider and meniscal transplantation might be necessary furthermore, cartilage degeneration is associated with higher articular contact stress identified to occur following even partial meniscectomy. In the literature there is little evidence to support if concomitant meniscus transplantation following osteotomy will delay the recurrence of arthritic symptoms however, restoration of the meniscus it is supposed to be valuable.

Cartilage Repair Techniques: Traditional palliative techniques or newer reparative treatment options have been utilized to improve cartilage lesions healing and they have demonstrated variable results. Lavage and chondroplasty can provide symptomatic pain relief with no actual hyaline tissue formation. However, these techniques remove superficial cartilage layers, which include collagen fibers that are responsible for the tensile strength, creating a less functional cartilage tissue. Bone marrow stimulation techniques, such as subchondral plate drilling or microfracture have been reported to stimulate production of hyaline-like tissue with variable properties and durability compared to normal cartilage, decreasing in some cases pain and disability however recent studies demonstrated that these techniques produce fibrocartilaginous tissue, which degenerates with time. Osteochondral autologous transplantation and mosaicplasty can restore normal cartilage tissue, but they can be applied only to small defects and there are some concerns regarding donor site morbidity. Autologous chondrocyte implantation (ACI), which was first introduced by Peterson, has been proven to be capable of restoring normal hyaline-like cartilage tissue, which is mechanically and functionally stable even in athletes at long-term follow up. Defect size ranging from 2-12 cm2 has been shown to be favourable to regeneration. Osteochondritis Dissecans is not a contraindication for cartilage transplantation as long as the bone loss does not exceed 8 mm. However, this two-step procedure and showed local morbidity for periosteal harvest and uncertain distribution of chondrocytes solution. Additionally,the possible complication of periosteal patch hypertrophy prompted the scientific community to develop new techniques including second generation ACI. The use of a three-dimensional scaffold for autologous chondrocyte culture was developed with the aim to improve both the biological performance of chondrogenic autologous cells as well as renders the surgical technique easier and surgeons have been enabled to perform this procedure arthroscopically however this is still a two-step procedure with arthroscopic evaluation and biopsy followed by implantation, either using an arthroscopic technique or mini-arthrotomy. Since 2001, we have participated in an ongoing observational multicenter investigation to evaluate the long-term clinical outcomes of the treatment with Hyaluronic Acid (HA) scaffold (HYAFF 11® Fidia Advanced Biopolymers, Abano Terme, Italy). 141 patients with follow up assessments ranging from 2 to 5 years (average follow up time: 38 months) were evaluated. At follow up 91.5% of patients improved according to the International Knee Documentation Committee (IKDC) subjective evaluation 76% and 88% of the patients had no pain and mobility problems respectively assessed by the Euro Qol-EQ5D measure. Furthermore, 95.7% of the patients had their treated knee normal or nearly normal as assessed by the surgeon cartilage repair was graded arthroscopically as normal or nearly normal in 96.4% of the scored knees the majority of the second-look biopsies of the grafted site, histologically, were assessed as hyaline-like. A

very limited complication rate was recorded in this study. We also evaluated 32 patellofemoral full-thickness chondral treated with Hyalograft C the IKDC and EuroQoL EQ-5D scores demonstrated a statistically significant improvement (P <. 0001). Objective preoperative data improved from 6/32 (18.8%) with (IKDC) A or B to 29/32 (90.7%) at 24 months after transplantation. Mean subjective scores improved from 43.2 points preoperatively to 73.6 points 24 months after implantation. MRI studies at 24 months revealed 71% to have an almost normal cartilage with positive correlation with clinical outcomes. Second-look arthroscopies in 6 cases revealed the repaired surface to be nearly normal with biopsy samples characterized as hyaline-like in appearance. Characterized Chondrocyte Implantation (CCI) is a second-generation ACI procedure that uses ChondroCelect® (Ti-Genix NV, Haasrode, Belgium), which was developed to limit the chondrocytes&rsquo &ldquode-differentiation&rdquo and is an expanded population of chondrocytes that expresses a marker profile predictive of the capacity to form stable hyaline like cartilage in vivo in a consistent and reproducible manner. In a prospective, randomized controlled trial, that compared CCI versus microfracture as treatment for single symptomatic cartilage defects of the femoral condyle, CCI produced a superior type of tissue regenerate. It also showed clinical outcome similar in both treatments. These results suggest CCI may lead to an improved long-term clinical outcome. Although ACI has the potential to repair the damaged cartilage and remains one of the most promising technologies, the expansion of the cell population to obtain a sufficient cell number appears critical as there are some limitations: (a) if the damaged surface is particularly wide, the availability of healthy cartilage to harvest appears limited (b) in vitro expanded human articular chondrocytes reduce their chondrogenic potential after 5-6 cell doubling. Therefore, researches are currently underway to develop alternatives to this technique. Recent directions in cartilage repair are moving towards the possibility to perform one-step surgery several groups are analysing the possibility to use mesenchymal stem cells (MSC) with chondrogenic potential and growth factors (GF) avoiding the first surgery for cartilage biopsy and subsequent chondrocyte cell cultivation with a significant reduction of the cost of the total procedure. Some surgical techniques have been tested in animals for MSCs implantation including the simple injection of bone marrow aspirate concentrate cells (BMAC) into the lesion, which improved full-thickness cartilage repair compared to microfracture in an equine model of extensive cartilage loss. MSC have a self-renewal capacity and multi-lineage differentiation potential and they can be characterized by their cultivation behaviour and their differentiation potential into adipogenic, osteogenic and chondrogenic cells, as well as form bone, cartilage and fat therefore, once MSC are cultured in the appropriate microenvironment, they can differentiate to chondrocytes and form cartilage. Wakitani et al have used autologous culture expanded BMSC transplantation for repair of cartilage defects in osteoarthritic knees and they concluded that MSC was capable of regenerating a repair tissue for large chondral defects. Giannini et al. presented their one-step surgery procedure using MSC and scaffold. We use BMAC and GF combined with a biologic scaffold (ChondroGide®-Geistlich Wolhusen, CH) for full thickness cartilage defects repair as the porous structure of the scaffold facilitates adhesion, proliferation and differentiation of MSC. We prospectively followed up for 2 yrs 14 patients (15 knees) operated at our Institution for grade IV cartilage lesions of the knee all these patients have been transplanted using BMAC covered with a collagen membrane. Bone marrow was harvested from ipsilateral iliac crest and subjected to concentration and activation with Batroxobin solution (Plateltex®act-Plateltex SRO Bratislava, SK). The patients followed the same specific rehabilitation program for a minimum of 6 months. All the patients showed improvements in evaluation scores. Mean pre-op values were: VAS 5, IKDC subjective 41.73, KOOS Scores P=66.6/ S=68.3/ ADL=70/ SP=41.8/ QOL=37.2, Lysholm 65 and Tegner 2.07. At final follow up mean scores were: VAS 0.8, IKDC subjective 75.5, KOOS P=89.8/ S=83.6/ ADL=89.6/ SP=58.9/ QOL=68, Lysholm 87.9 and Tegner 4.1. No adverse reactions or post-op complication were noted. MRI showed good coverage of the lesions. Good histological findings were reported for all the specimens analyzed who presented many hyaline-like features. These data and other Italian authors (Giannini et al.) using MSC implantation with a one-step procedure seem to be promising, showing good clinical outcomes at early follow up. The plasma fraction just above the red and the white cells is called plasma rich in growth factors (PRGF): it is a plasma fraction with a high number of platelets - about three times the number of platelets in blood. Studies have been carried out using PRGF in articular cartilage lesion treatment can increase total collagen II synthesis and decrease degradation induce chondrogenesis of MSCs and promote chondrocyte proliferation, differentiation and adhesion. Kon et al. have studied a group of 30 patients with symptomatic degenerative disease of the knee joints treated with three PRP intra-articular

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injections weekly the follow up at 6 months showed positive effects on the function and symptoms. We also prospectively followed up a group of 50 patients with mean age 46.7, treated with 2 intra-articular injections (1 monthly) with autologous PRGF (RegenLab-PRP®). Follow up at 3 &ndash 6-12 months post-treatment showed improvement in all the scores. Mean pre-treatment values were: KOOS Scores: P=70.93/ S=69.13/ ADL=76.2/ SP=40.11/ QOL= 38.87, VAS 4, Tegner 3.39, IKDC 50.86 and MARX 3.24.At final follow up mean scores were: KOOS Scores: P=84.19/ S=82.89 / ADL=91.86/ SP=56.89 /QOL=61.08, VAS 2, Tegner 5.10, IKDC 70.37 and MARX 8. Recently authors have proven synergistic effects of PRP combined with MSC. Nishimoto et al suggested that simultaneous concentration of PRP and BMC could play important role in future regenerative medicine. Preliminary data are encouraging but further studies on clinical efficacy will clarify if simultaneous use of PRP and MSC could represents a real solution for regenerative medicine in cartilage repair.

Rehabilitation: The conservative postoperative rehabilitation program follows the principles of cartilage transplantation and healing of a concomitant osteotomy procedure should be established by radiographic criteria before making progress to full-weight bearing. Associated procedures as well as ligamentous reconstruction or meniscal transplantation should also be considered. Our rehabilitation protocol is based on functional rather than temporal criteria the progression of the recovery was related to the achievement of specific criteria that allow the patient to proceed to the next rehabilitative phase. The protocol after cartilage transplantation includes 4 stages:

Phase 1: Protection of the implant (0-6 weeks).

Phase 2: Transition and recovery of gait/ADL (6-12 weeks).

Phase 3: Maturation and functional recovery/running (12-24 weeks).

Phase 4: Turnover and sports recovery (24-52 weeks).

From a functional point of view in our protocol we identified different phases characterized by micro-objectives, which ended with the achievement of specific functional criteria as set out at the end of each stage. The first weeks after surgery are important for the healing process of the repaired chondral defect and continuous passive motion (CPM) is prescribed in order to avoid arthrofibrosis. We suggest a gradual increase of the knee flexion up to 90º and progressive recovery of range of motion. Partial weight bearing with crutches is allowed after the first few weeks and our protocol emphasizes the recovery of strength and proprioceptive abilities. An important part of the rehabilitation is the recovery of athletic general conditions and recovery of walking, running and sport activity are allowed according to specific clinical parameters and functional objectives.

Conclusions: Articular cartilage has limited intrinsic healing potential: therefore once injury occurs, may lead to progressive damage, joint degeneration and early OA. Critical in prevention of early OA is to restore any existing tibiofemoral axial deformity, normal patellofemoral tracking and knee stability as well as to address any bone or meniscal deficiency primarily, in a staged or concomitant procedure. A number of viable options have been available over the years to address problems concerning cartilage lesions each technique has its advantages and disadvantages. Furthermore, not all lesions can be addressed with a single technique retropatellar, tibial plateau and posterior condylar lesions remain a challenge. Development of better instruments and introduction of new techniques in the near future should be able to solve this dilemma. Biotechnology on the other hand is progressing at a rapid pace, allowing the introduction of numerous products for clinical application. An important issue to be addressed for the future perspective for cartilage repair is the quality of the bonding and the integration of the newly formed tissue to the native tissue. Among the most studied candidates, polypeptide growth factors have shown promise to enhance the structural quality of the repair tissue, showing less morbidities and complications inherent to cartilage surgical techniques however, medium-term prospective randomized studies are suggested to confirm these premature results. Numerous studies are currently under way to clarify some of the questions that still remain unanswered regarding the long-term durability of these procedures and the possible modifications that can still be made to achieve better results. However, carefully conducted randomised prospective studies for each of these innovations should be carried out to validate their efficacy for cartilage regeneration and prevention of early OA.

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Acknowledgments:

The Author acknowledges Georgios Karnatzikos, MD from 2nd Orthopaedic Dept. of GH “G.Papageorgiou” at Thessaloniki, Greece, fellow at OASI Bioresearch Foundation, for his contribution.

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16.2.1Growth factor-delivering scaffolds for cartilage tissue engineeringC. Brochhausen1, R. Zehbe2, B. Watzer3, C.J. Kirkpatrick1

1Mainz/Germany, 2Berlin/Germany, 3Marburg/Germany

Introduction: Tissue Engineering represents a highly complex interdisciplinary branch of science which brings the life sciences and engineering together to design innovative solutions for the treatment of critical tissue defects after traumatic or degenerative damage. To achieve this goal an interesting evolution has become apparent. Although originally understood as a principally biomaterial-based strategy using natural and synthetic materials as implants to be integrated into the neighbouring tissue, during the last two decades a change of paradigm has taken place which involves a movement away from the structural replacement of damaged tissue towards strategies to regenerate functional tissue (Kirkpatrick et al., 2006). In this context, modern tissue engineering approaches integrate the combined use of cells, scaffolds, and growth factors or signalling molecules respectively. Growth factors are polypeptides which are involved in the regulation of important cellular functions such as proliferation, differentiation, migration and cell adhesion as well as gene expression. Thus, the rationale for the use of signalling molecules and growth factors is the fact that such factors are able to stimulate the controlled proliferation and proper differentiation of the seeded cells in the scaffolds. Furthermore, growth factors should also enhance the migration, proliferation, and differentiation of cells from the edges of the treated defect. In general, such factors can serve to optimize tissue engineered cell-scaffold constructs in vitro by providing a suitable biomimetic microenvironment. Furthermore, growth factors could improve the performance of scaffold-cell constructs and their integration in vivo.

Content: To introduce growth factor-releasing scaffolds into the clinic some important requirements should be taken into account (Biondi et al, 2007). Since the half-life of growth factors is short, the physiological integration and release profile of such molecules is of crucial value for effective application. In this context it is of special interest to know at what time point the activity should reach its maximum – during the proliferation phase at the beginning of culture, during the differentiation stage after expansion of cells or during the integration of the scaffold-cell construct into the patient’s tissue. The expected time point for maximal efficacy is dependent on the growth factor itself and its mode of action on cells. To guarantee maximum release at this time point the release profile must be carefully controlled. The release profile is highly dependent on the mode of integration or binding of the growth factor to the scaffold material. Another important feature is the fact that the activity of the growth factor is dose-dependent, which means that for an optimal effect the physiological dose must be liberated at the correct time point. Furthermore, an accumulation of the growth factor within the scaffold or the surrounding tissue has to be avoided since there may be a risk of systemic effects. Taking these points together, it becomes obvious that for a clinical application of scaffold-cell-growth factor constructs the mode of action and the role of the candidate molecule should be well understood. To save time and reduce costs it would of great interest to use molecules which are well known and which are already available in pharmaceutical quality. Finally, during scaffold synthesis and integration a good incorporation efficacy is desirable to prevent loss of growth factor molecules. For the targeting of such potential signalling molecules and growth factors it is important to realize that regeneration in part recapitulates developmental processes. Therefore, it can be postulated that for the improvement of regeneration in the context of tissue engineering it is essential to understand the regulation of developmental processes (Reddi, 2003). With this purpose in mind our group demonstrated that the growth plate of the long bones represents a suitable developmental model to target interesting growth factors and signalling molecules for tissue engineering application of cartilage and bone (Brochhausen et al., 2009a). In previous work we and others could demonstrate the role of prostagladin E2 (PGE2) in the metabolism of chondrocytes (O’Keefe et al., 1998, Brochhausen et al. 2006). Until now, prostaglandin E2 has not played any significant role as a potential signalling molecule in tissue engineering applications. Although it has been reported as being beneficial in numerous tissue environments ranging from endothelial tissue to cartilage with a pronounced dose-dependent cell-regulatory mechanism, PGE2 has not yet achieved a major impact in tissue engineering. Therefore, one goal of our research was to develop different PGE2-based tissue engineering solutions. The first approach utilizes an emulsion-based route to synthesize polymeric (PLGA) microspheres with incorporated PGE2 (Brochausen et al., 2009b, Watzer et al., 2009). In this first approach we were able to demonstrate release kinetics

of biologically active PGE2 with a burst release of PGE2 over the first two days and further release over 8 days. These microspheres were used in a second approach to establish a three-dimensional scaffold system by distributing PGE2-PLGA-microspheres in a gelatinous suspension followed by freeze-drying and moderate chemical crosslinking in a formaldehyde-saturated atmosphere as briefly described elsewhere (Brochhausen et al. 2009c). The distribution of growth factor-loaded microspheres allows a gradient of growth factors due to the spatial distribution of the microspheres. Gradients of growth factors are of special importance for biphasic scaffolds, such as scaffolds for osteochondral defects (Wang et al., 2009). In a third tissue engineering system we developed a PLGA scaffold with direct integration of the biosignal. In this approach PLGA and PGE2 were prepared in an organic solvent, followed by supercritical fluid foaming. The integration rate of this scaffold was quite high, which predicts high release rates. Moreover, the incorporation efficacy of the fluid-foamed system was higher than different preparations of microspheres. This third approach demonstrated that that the direct incorporation of PGE2 into a polymer foam is possible without extensive loss of the growth factor. Currently, we are testing these tissue engineering systems extensively in vitro with first results already being promising with respect to the usefulness of PGE2 in cell stimulation assays. The development of growth factor-releasing scaffolds is highly interactive, as it combines aspects from the biomaterial and life sciences in interaction with important fields of clinical pharmacology. For clinical use of such systems extensive in vitro studies and the knowledge of the in situ situation in the human body are crucial. Therefore, human in situ models such as the growth plate are of special interest for the targeting of potential new candidates. In our projects we have used a well understood molecule –PGE2- which is available in pharmaceutical quality and offers significant advantage for a possible application in a clinically relevant scaffold-cell-release system. Future developments should focus on questions such as to what extent scaffolds are necessary for the regeneration of functionalized tissue in tissue engineering and regenerative medicine. In this context, it seems obvious that different tissues and defect sizes require different tissue engineering solutions (Chung & Park, 2007). Of particular importance are investigations which clarify how and under what conditions the amount of foreign material in scaffolds could be diminished by the use of controlled release systems which can trigger the endogenous regenerative potential of the patient’s tissues.

References:

Kirkpatrick, CJ, Fuchs, S, Peters, K, Brochhausen, C, Hermanns, MI, Unger, RE Visions for regenerative medicine: interface between scientific fact and science fiction. Artificial Organs. 2006; 30: 822-827. Biondi M, Ungaro F, Quagila F, Netti PA. Controlled Drug delivery in Tissue Engineering. Advanced Drug Delivery Reviews. 2007; 60: 229-242. Reddi, AH. Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Engineering. 2003; 6, 351-359 Brochhausen, C, Lehmann, M, Halstenberg, S, Meurer, A, Klaus, G, Kirkpatrick, CJ. Signalling molecules and growth factors for tissue engineering of cartilage – What can we learn from the growth plate? Journal of Tissue Engineering and Regenerative Medicine. 2009a; 3: 416-429. O‘Keefe RJ, Crabb ID, Puzas JE, Rosier RN: Influence of prostaglandins on DNA and matrix synthesis in growth plate chondrocytes. Journal of Bone and Mineral Research. 1992; 7: 397-404. Lowe GN, Fu YH, McDougall S, Polendo R, Williams A, Benya PD, Hahn TJ: Effects of prostaglandins on deoxyribonucleic acid and aggrecan synthesis in the RCJ 3.1C5.18 chondrocyte cell line: role of second messengers. Endocrinology. 1996; 137: 2208-2216. Schwartz Z, Gilley RM, Sylvia VL, Dean DD, Boyan BD: The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3‘,5‘-monophosphate and protein kinase C. Endocrinology. 1998; 139: 1825-1834. Brochhausen, C, Neuland, P, Kirkpatrick, CJ, Nüsing, RM, Klaus, G. Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – PGE2 dependent proliferation of growth plate chondrocytes. Arthritis Research and Therapy. 2006; 8: R78. Brochhausen C, Zehbe R, Watzer B, Halstenberg S, Gabler F, Schubert H, Kirkpatrick CJ, Immobilization and controlled release of prostaglandin E2 from poly-L-lactide-co-glycolide microspheres. Journal of Biomedical Materials Research Part A. 2009b; 91: 454 – 462. Watzer B, Zehbe R, Halstenberg S, Kirkpatrick CJ, Brochhausen C. Stability of prostaglandin E2 (PGE2) embedded in poly-D,L-lactide-co-glycolide microspheres - a pre-conditioning approach for tissue engineering applications. Journal of Material Science: Materials in Medicine. 2009; 20: 1357-1365 Brochhausen C, Lehmann M, Zehbe R, Watzer B, Grad S, Meurer A, Kirkpatrick CJ. Targeting und innovative Release Systeme für

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Wachstumsfaktoren und Signalmoleküle zur Anwendung im Tissue Engineering, Der Orthopäde. 2009c; 38: 1053-1061. Wang, X, Wenk, E, Zhang X, Meinel L, Vunjak-Novakovic G, Kaplan DL. Growth factor gradients via Microsphere Delivery in biopolymer scaffolds for osteochondral tissue engineering. Journal of Controlled Release. 2009; 134: 81-90. Chung HJ, Park TG. Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering. Advanced Drug Delivery Reviews. 2007; 60: 249-262

Acknowledgments:

This research was supported by the EU Network of Excellence, EXPERTISSUES, NovoNordisk and the German Research Foundation (DFG; Se 263/17-1; SCHu 679/27-1, SCHU 679/27-2).

16.2.3Towards cartilage grafts with zonal organizationJ. Malda, W. Schuurman, P.R. van Weeren, W.J.A. DhertUtrecht/Netherlands

Introduction: It is well established that articular cartilage has limited regenerative capacity and successful treatment of cartilage defects is still a challenge worldwide. The solution to this might be provided by regenerative medicine, a newly emerging area that aims to restore tissue function by combining principles of engineering and life sciences to develop biological substitutes. Functional reconstruction of articular cartilage defects has been demonstrated to be feasible using the patients’ own cells[1, 2]. Although such cell based techniques show superior histology[2] with more hyaline cartilage formation compared to other standard techniques, such as micro-fracturing, none of the current treatment modalities has demonstrated formation of a histologically optimal tissue repair that mimics the characteristic complex zonal architecture of hyaline articular cartilage and hence can be supposed to produce the desired optimal clinical long-term results[3].

Content: Articular cartilage is a hydrogel-like, matrix-rich tissue that contains only 5 - 10 % of chondrocytes, which maintain the structural and functional integrity of the matrix. The tissue is organised into characteristic depth zones, each with distinct physicochemical and biological properties and functions, which work together to impart low-friction, wear-resistant behaviour to diarthrodial joints. This “zonal” structure is typically divided into three zones; superficial (surface to 10-20% of thickness), middle (20%-70%), and deep (70%-100%). Cells in each of the different zones are organized distinctly and express zone-specific markers. In the superficial zone, the collagen network is aligned parallel to the surface, providing high tensile strength, whereas the glycosaminoglycan (GAG) content is low, resulting in compliant compressive properties. In this zone, the chondrocytes secrete proteoglycan 4 (PRG4 or lubricin), a molecule important for boundary lubrication and low-friction properties[4, 5]. In the middle zone, the collagen network is randomly oriented and the mechanical properties are intermediate to the adjacent zones. In the deep zone (deepest 30-50% of thickness), the collagen network is oriented perpendicularly to the calcified cartilage and bone, providing strong integration between dissimilar tissues, and the GAG content is high, resulting in stiff compressive properties (order of magnitude higher than the superficial zone [6, 7]). This layered design of articular cartilage is essential to provide the tissue with the (biomechanical) characteristics that are required for proper and life-long sustainable joint function. Providing constructs with zonal organization may improve long-term outcomes of attempts at cartilage repair via tissue engineering techniques, since chondrocyte-matrix interactions are essential for the maintenance of zonal chondrocyte phenotype[8, 9]. Moreover, it has been demonstrated that isolated and expanded chondrocytes from the different depth zones of the cartilage regain their zonal differences when they are redifferentiated in alginate beads, as evidenced by zone-specific reappearance of COMP and clusterin, as well as significantly higher GAG production by cells from the deep compared to the superficial zone[10]. To further mimic this zonal organization, an increasing number of investigations is currently directed towards the development of zonal tissue-engineered cartilage implants[11]. To achieve this, we are employing organ printing technology or bioprinting, which combines the deposition of specific cell populations with the simultaneous deposition of biomaterials [12]. This allows the development of zonal cartilaginous grafts and by using hydrogels a more physiological environment can be created. Biomechanical strength can be imparted and modified by mixing cell suspensions into in situ cross-linkable hydrogels (e.g., gelatin, agarose, alginate or PEG) in a cartridge, after which

the suspension is printed following a programmed 3D pattern[13-15]. Adding biologically active components, such as proteins, peptides, DNA, hormones and natural or synthetic polymers[16] to these water-based “bio-inks” will further enhance and direct the behaviour of the cells. We characterized the use of bioprinting technologies to design and build heterogeneous cell-laden 3D structures and evaluated these in vitro and in vivo.

References:

1.Vasiliadis, H., et al., Assessment of clinical outcomes 10-20 years after autologous chondrocyte implantation. Osteoarthritis Cartilage, 2010. 2.Saris, D.B., et al., Characterized chondrocyte implantation results in better structural repair when treating symptomatic cartilage defects of the knee in a randomized controlled trial versus microfracture. Am J Sports Med, 2008. 36(2): p. 235-46. 3.Haleem, A.M. and C.R. Chu, Advances in Tissue Engineering Techniques for Articular Cartilage Repair. Operative Techniques in Otrhopeadics, 2010. 20: p. 76-89. 4.Schumacher, B.L., et al., A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage. Arch Biochem Biophys, 1994. 311: p. 144-52. 5.Marcelino, J., et al., CACP, encoding a secreted proteoglycan, is mutated in camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Nat Genet, 1999. 23(3): p. 319-22. 6.Schinagl, R.M., et al., Depth-dependent confined compression modulus of full-thickness bovine articular cartilage. J Orthop Res, 1997. 15(4): p. 499-506. 7.Chen, A.C., et al., Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression. J Biomech, 2001. 34(1): p. 1-12. 8.Ng, K.W., G.A. Ateshian, and C.T. Hung, Zonal chondrocytes seeded in a layered agarose hydrogel create engineered cartilage with depth-dependent cellular and mechanical inhomogeneity. Tissue Eng Part A, 2009. 15(9): p. 2315-24. 9.Khanarian, N.T., et al., Zonal Chondrocyte Interactions Regulate Chondrocyte Calcification via PTHrP, in 56th Annual Meting of the Orthopaedic Research Society. 2010: New Orleans, LO. p. P3. 10.Schuurman, W., et al., Zonal chondrocyte subpopulations acquire zone-specific characteristics during in vitro redifferentiation. American Journal of Sports Medicine, 2009. 37: p. S97-S104. 11.Klein, T.J., et al., Tissue Engineering of Articular Cartilage with Biomimetic Zones. Tissue Engineering, 2009 in press. 12.Mironov, V., N. Reis, and B. Derby, Review: bioprinting: a beginning. Tissue Eng, 2006. 12(4): p. 631-4. 13.Cohen, D.L., et al., Direct freeform fabrication of seeded hydrogels in arbitrary geometries. Tissue Eng, 2006. 12(5): p. 1325-35. 14.Fedorovich, N.E., et al., Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing. Tissue Eng, 2007. 13(8): p. 1905-25. 15.Fedorovich, N.E., et al., Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing. Tissue Eng Part A, 2008. 14(1): p. 127-33. 16.Campbell, P.G. and L.E. Weiss, Tissue engineering with the aid of inkjet printers. Expert Opin Biol Ther, 2007. 7(8): p. 1123-7.

Acknowledgments:

Funding from the AO foundation, the Dutch Technology Foundation STW, Applied Science Division of NWO and the Technology Program of the Ministry of Economic Affairs is gratefully acknowledged.

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19.0.2Does cartilage have to be perfect for a good clinical outcome?E. Kon, G. Filardo, A. Di Martino, S. Patella, L. D’Orazio, B. Di Matteo, M. MarcacciBologna/Italy

Introduction: The ultrastructure of articular cartilage is unique: chondrocytes are sparsely distributed within the surrounding matrix, maintaining minimal cell-to-cell contact. The interaction between the cells, the collagen framework, aggrecan and fluid constitute a complex biomechanical feature of hyaline cartilage, making it difficult to repair. Moreover, its isolation from systemic regulation, and the lack of vessels and nerve supply contribute to the absence of a spontaneous healing process. The restoration of damaged cartilage is a difficult challenge for orthopaedic surgeons because of its limited spontaneous repair capacity and the difficulty in restoring the damaged areas with hyaline cartilage. Several techniques have been used in the operative treatment of traumatic and degenerative cartilage lesions: marrow stimulation techniques, osteochondral, periosteal or perichondral graft transplantation and the new procedures based on autologous chondrocyte implantation or the use of scaffolds to induce an in situ tissue regeneration.

Content: Marrow stimulation procedures, like drilling, abrasion and microfracturing have been proposed as an easy, rapid and inexpensive way to restore the articular damaged surface. The rational of these techniques is to stimulate the formation of fibrocartilage by facilitating access to the vascular system of the underlying bone, thus creating a clot populated with growth factors, platelets and bone marrow-derived progenitor cells capable of chondrogenesis. Steadman [1] reported highly satisfactory results at 11-year follow-up with the microfracture technique, but patients had to adjust their activity level to that of their knee function. However, the repair tissue response can be variable and unpredictable [2]. Moreover, most animal [3], MRI and histological studies [3,4] have shown the initial fibrocartilage formation with deterioration of the new –formed tissue over time. Nehrer [4] frequently observed soft, spongiform, fibrous tissue combined with central degeneration in the defect. The clinical failure was observed at an average follow-up of 21 months. Some authors have reported a significant decrease in clinical outcome at longer follow-up relative to short-term high satisfaction [5]. Osteochondral autograft transplantation (with an underlying bone) aims to replace the damaged area with hyaline cartilage and to capitalize on bone-to-bone healing, since the mature cartlagineous tissue has limited healing potential and heals completely with difficulty to surrounding cartilage [6]. However, this treatment has limited indications [7], due to the fact that a high number of grafts can promote the formation of a large amount of fibrous tissue filling the space between the plugs and also increase the incidence of technical problems, compromising the mechanical properties of the reconstructed zone. It is also difficult to obtain the physiological curvature of an articular surface, this could produce an abnormal stress distribution and subsequent suffering of the implant. Furthermore, excessive reaming can jeopardize the stability of the plugs in the subchondral bone, weakened by the fibrous tissue among them, and the fibrocartilage formed among plugs may reduce the biomechanical properties of the surface, thus contributing to the worst results observed in bigger lesions [7]. The limited indications and the worst results obtained with these reparative procedures have been mainly attributed to the formation of fibrocartilage, which presents low biomechanical properties and might not guaranty high and long-lasting clinical results. The more recent regenerative technologies aim to offer better results through the formation of hyaline-like cartilage. Autologous Chondrocyte Implantation (ACI) proposed by Brittberg in 1994 [8] involves the re-implantation of autologous cells isolated from cartilage harvested from the patient and expanded in vitro. Peterson [9] has shown that the early results obtained with the ACI technique are long-lasting. At 5 to 11-year follow-up, 51 of 61 patients had satisfactory results. The biomechanical evaluation of the grafted area by means of an indentation probe demonstrated greater than 90% normal cartilage with respect to stiffness measurements. ACI presents some advantages over other cartilage repair and reconstructive techniques, including the use of autologous engineered material, reduced donor site morbidity and no treatment limitations related to defect size. The most important theoretical advantage of this method is that it can restore the integrity of the damaged area with cartilage that has hyaline-like properties [9], whereas all the marrow-stimulation techniques provide fibrocartilagineous repair tissue that is likely to be less mechanically stable [10]. The more recently developed second-generation ACI technique uses a new tissue-engineering technology to create a cartilage-like tissue in a three-dimensional culture system, thus presenting all the advantages of the first

generation method without the problems that can be observed with standard ACI procedures, such as the difficulty in handling a delicate liquid suspension of chondrocytes at implantation surgery, the need to make a hermetic periosteum seal using sutures, the requirement of a second open surgery operation, the very long rehabilitation period, and possible complications associated with the use of a periosteal flap and large joint exposure. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for the in vitro growth of living cells and their subsequent transplantation into the defect site. However, despite first- and second-generation autologous chondrocyte implantations have emerged as promising therapeutic options, and several studies [11,12] have confirmed the good clinical results and durability of these treatments, the cell-based regenerative approach has not been clearly proven to be better than other procedures. At the time being, there is no agreement about the effective superiority of one technique to the others, and their indication and results are still controversial. In fact, most of the studies report clinical outcome at short- to medium-term follow-up, and many different techniques may appear satisfactory at the beginning, whereas the quality of the repair tissue might influence the long-term results. Knutsen et al. [13,14] didn’t report any differences in clinical outcome between ACI and microfracture at 2 and 5 years follow-up. Moreover, no statistically significant differences were detected in the macroscopic or histological results at 2 years (ACI biopsies tended to have a more hyaline-like appearance; p=0.08) and radiographic findings at 5 years follow-up, too. However, despite the lack of correlation observed between histological and clinical results, he also reported that none of the patients failed after microfracture presented high-quality repair cartilage, thus suggesting that a worst-quality repair tissue could increase the risk of failure or present a lower outcome over time. In another randomized study, Saris et al. [15,16] compared microfracture with characterized chondrocytes implantation (CCI). Similarly, comparable clinical outcome was found between the two procedures at short term follow-up, despite the superior histomorphometric and histologic score observed in the CCI group. However, the higher quality of the repair tissue seen with the structural analysis at short follow-up significantly influenced the later follow-up. In fact, at three years CCI offered a further improvement with better clinical results compared with microfracture, whose results reached a plateau after 18 months. Marrow stimulation procedures lead to the formation of a fibrous tissue that can not guaranty good results over time. Oppositely, ACI procedures regenerate a cartilaginous tissue that undergoes a remodelling process, thus leading to superior clinical results detectable only after at least 2 – 3 years follow-up. Therefore, it appears of primary importance to complete the clinical evaluation with the analysis of the regenerated tissue quality, but unfortunately only a few studies, due to practical and ethical difficulties, present a full histological analysis, thus contributing to the controversies and the lack of evidence about the potential of the different techniques and the correlation between histological and clinical results. Despite the importance of the objective evaluation offered by the histology analysis, the invasiveness of this procedure does not allow it to be performed routinely. An objective, non-invasive measure of the properties of the treated area would be very desirable and helpful for increasing the knowledge of the regenerative processes. At the time being, the gold standard method for the imaging evaluation of cartilage lesions is magnetic resonance. It represents a non-invasive, multi-planar technique capable of producing high-resolution, high-contrast images. MRI enables morphological assessment of the cartilage surface, as well as its internal structure, thickness, volume and the subchondral bone, other than the biochemical status of articular cartilage. Furthermore, in contrast to the small biopsy specimens used for histochemical assessment, which are from a discrete location and can offer only limited information, MRI can provide information on the whole area. Moreover, MRI images have been shown to correlate with cartilage histology, with the biochemical composition of cartilage in vivo and even in engineered cartilage generated in a bioreactor [17,]. The only qualitative MRI evaluation is not sufficient for quantifying the tissue repair characteristics, and a grading system is required to analyse the maturation processes and compare the results obtained with different techniques. For this reason, classification systems for describing articular cartilage repair tissue have been developed [17,18], one of the most important being that of Marlovits et al. [18] who in 2004 proposed a definition of pertinent parameters for the evaluation of chondral repair. This evaluation system (MOCART) has been shown to be reliable, reproducible, and with an excellent inter-observer variability [19]. Despite some preliminary results at a 12-month follow-up showing a poor correlation between MRI findings and clinical outcome, Marlovits and co-workers found a statistically significant correlation between the “filling of the defect,” the “structure of repaired tissue” and most of the subjective and objective outcome scores at two

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years after ACI. We previously used the MOCART score to evaluate the results obtained with second-generation, autologous chondrocyte implantation for the treatment of knee cartilage lesion after over 5 yrs of follow-up [20]. The MRI findings were interesting and somehow controversial. The overall MOCART score had a significant correlation with subjective IKDC scores, but among the variables, only the structure of the repaired tissue was significantly correlated to the clinical scores. Most likely, the signal of the repair tissue represents a most specific MRI parameter. In our previous experience analysing the long-term outcome of mosaicplasty technique, strong correlation between the signal of the repair tissue and the clinical outcome was detected even if a different surgical procedure was employed [21]. Signal of the repair tissue was confirmed to be a useful indicator of graft maturation, especially in comparison with the signal of adjacent normal articular cartilage. All other parameters did not show a significant correlation with the clinical outcome. This fact can suggest from one side that we need more precise tools for clinical and MRI evaluation, and on the other hand that the MRI findings can be better correlated with the clinical outcome at longer follow-up, as shown for the histological evaluation. Moreover, in a subsequent analysis of 78 knee MRIs [22], performed at least 24 months after second generation autologous chondrocyte implantation, the larger number of MRIs analyzed allowed to increase the power to detect any potential correlation between subchondral changes and clinical outcome, and indeed, the worst results were found in patients affected by bone marrow oedema, thus suggesting that the absence of correlations founded between clinical and MRI findings in some studies can be related to the low number of patients evaluated. Further longer follow-ups and larger high-level MRI and histological studies will solve these discrepancies. However, despite some controversial findings, a better quality tissue seems to be determinant for the long-term results, as shown by histology and imaging. In fact, in recent years there is an increasing awareness of the importance of the quality of the tissue obtained for achieving better and long-lasting results and several authors have even highlighted the need to treat the all osteochondral unit. The subchondral bone plays a role in the etiopathogenetic processes, and has to be carefully considered in the treatment of articular surface damage. Articular cartilage and its supporting bone functional conditions are tightly coupled since injuries of either adversely affects the all joint mechanical environment [23]. Certain defects, such as those resulting from osteochondritis dissecans, osteonecrosis and important trauma may in fact be osteochondral in nature with involvement of subchondral bone [24,25]. Moreover, in large cartilage lesions the subchondral bone is involved in the degenerative process as well, and even focal chondral defects, if left untreated, may increase in size over time and present concomitant changes of the underlying subchondral bone plate, either overgrowth or bone loss [26]. However, most of the bioengineered tissues used in clinical practice present the problem of promoting only the cartilage layer, but not the bone regeneration. The development of biphasic scaffolds aims to regenerate the all articular surface in the most anatomical way possible, reproducing the different biological and functional requirements for guiding the growth of the two tissues [27]. At the time being, despite all the pre-clinical studies reported [27,28], only two scaffold used for osteochondral regeneration are currently available for clinical application. Although preliminary results appear promising, there are no controlled studies, and only case reports have shown favorable results after implantation of these osteochondral substitutes [29, 30]. Long term randomized clinical, histological and MRI studies are need to confirm the potential of this approach, verifying the regenerative capacity and if the high-quality tissue obtained will also correspond to good long term clinical results. Concluding, despite some controversial results at short term follow-up, at the time being clinical, histological and MRI evaluation suggest that the higher-quality repair cartilage obtainable with the more ambitious regenerative approaches can offer better results at longer follow-up with respect to the traditional reparative procedures. Surgical goals should always try to re-establish the joint surface in the most anatomical way possible, restoring the physiological properties of the chondral tissue or, where necessary, of the entire osteochondral unit, in order to achieve a more predictable repair tissue that closely resembles native articular surface and remains durable over time.

References:

1. Steadman JR Outcomes of microfracture for traumatic chondral defects of the knee: average 11-year follow-up. Arthroscopy. 2003 May-Jun;19(5):477-84. 2. Shapiro F. Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. JBJS 1993; 75A:532-53. 3. Mithoefer K. The microfracture technique for the treatment of articular cartilage lesions in the knee. A prospective cohort study. JBJS Am. 2005 Sep;87(9):1911-20. 4. Nehrer S. Hystologic analysis of tissue after failed cartilage repair procedures. Clin. Orthop. Rel. R-es 365: 149-62, 1999. 5. Kreuz PC Is microfracture of chondral defects in the knee associated with different results in patients aged 40 years or younger? Arthroscopy. 2006 Nov;22(11):1180-6. 6. Hangody L: Autologous osteochondral mosaicplasty for the treatment of full-thickness defects of weight-bearing joints. J Bone Joint Surg Am 2003;85 A Suppl 2: 25-32. 7. Marcacci M, Kon E, Delcogliano M, et al: Arthroscopic autologous osteochondral grafting for cartilage defects of the knee: prospective study results at a minimum 7-year follow-up. Am J Sports Med. 2007 Dec;35(12):2014-21. 8. Brittberg M: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. New Engl J Med 331:889-895, 1994. 9. Peterson L Autologous chondrocyte transplantation. Biomechanics and long-term durability. Am J Sports Med. 2002 Jan-Feb;30(1):2-12. 10. Buckwalter J.A Articular Cartilage: composition, structure, response to injury and methods of repair. In: Ewin J. W., ed. Articular cartilage and knee joint function: basic science and arthroscopy. New York, Raven Press 19-56, 1990. 11. Kon E, et al: Matrix-assisted autologous chondrocyte transplantation for the repair of cartilage defects of the knee: systematic clinical data review and study quality analysis. Am J Sports Med. 2009 Nov;37 Suppl 1:156S-66S. 12. Kon E, et al: Second generation issues in cartilage repair. Sports Med Arthrosc. 2008 Dec;16(4):221-9. 13. Knutsen G, et al: Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. JBJS Am. 2004 Mar;86-A(3):455-64. 14. Knutsen G. A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. JBJS Am. 2007 Oct;89(10):2105-12. 15. Saris DB. Treatment of symptomatic cartilage defects of the knee: characterized chondrocyte implantation results in better clinical outcome at 36 months in a randomized trial compared to microfracture. Am J Sports Med. 2009 Nov;37 Suppl 1:10S-19S. 16. Saris DB Characterized chondrocyte implantation results in better structural repair when treating symptomatic cartilage defects of the knee in a randomized controlled trial versus microfracture. Am J Sports Med. 2008 Feb;36(2):235-46. 17. Roberts S, et al: Autologous chondrocyte implantation for cartilage repair: monitoring its success by magnetic resonance imaging and histology. Arthritis Res Ther. 2003;5(1):R60-73. 18. Marlovits S. Definition of pertinent parameters for the evaluation of articular cartilage repair tissue with high-resolution magnetic resonance imaging. Eur J Radiol 2004; 52, 310-319. 19. Marlovits S. et al. Magnetic resonance observation of cartilage repair tissue (MOCART) for the evaluation of autologous chondrocyte transplantation: determination of interobserver variability and correlation to clinical outcome after 2 years. Eur J Radiol 2006; 57, 16-23. 20. Kon E.: Eur j rad accepted for publication 21. Tetta C. et al. Knee Osteochondral Autologous Transplantation: Long-term MR findings and clinical correlations. Eur J Radiol. 2009 Jun 11. 22. Gomoll AH The subchondral bone in articular cartilage repair: current problems in the surgical management. Knee Surg Sports Traumatol Arthrosc. 2010 Apr;18(4):434-47. 23. Shirazi R, Computational biomechanics of articular cartilage of human knee joint: effect of osteochondral defects. J Biomech. 2009 Nov 13;42(15):2458-65. 24. Kocher MS, et al. Management of osteochondritis dissecans of the knee: current concepts review. Am J Sports Med, 2006 Jul 34(7):1181-91. 25. Pape D, et al: Disease-specific clinical problems associated with the subchondral bone. Knee Surg Sports Traumatol Arthrosc. 2010 Apr;18(4):448-62. 26. Gratz KR, et al. The effects of focal articular defects on cartilage contact mechanics. J Orthop Res. 2009 May;27(5):584-92. 27. Jiang CC Repair of porcine articular cartilage defect with a biphasic osteochondral composite. J Orthop Res 2007 Oct; 25(10):1277-90. 28. Kon E. et al: Novel nanostructured scaffold for osteochondral regeneration: pilot study in horses. J Tissue Eng Regen Med. 2010 Jan 4. 29. Kon E. et al. Novel nano-composite multi-layered biomaterial for the treatment of multifocal degenerative cartilage lesions. Knee Surg Sports Traumatol Arthrosc. 2009 Nov;17(11):1312-5. 30. Kon E. et al: A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesions: Technique note and an early stability pilot clinical trial. Injury. 2009 Dec 23.

Extended Abstracts 149

19.1.1The patello-femoral joint: What have we been doing so far?M. DrobničLjubljana/Slovenia

Introduction: The patello-femoral (P-F) joint has become problematic ever since the humanoids changed their way of walking to the fully extended knees. During the early extension patella rides proximal to the trochlear sulcus which makes it, in combination with the lateral pulling quadriceps forces, unstable in case any of the stabilizers (osseous-articular geometry, ligaments, retinacula, capsule, and muscles) fails [1]. High peak loads during activities are another contributing factor for early cartilage failure besides the intrinsic instability and incongruence [2]. This makes P-F joint the leader in the incidence of cartilage lesions encountered in more than 60% of arthroscopies [3]. In spite of stated problems, the P-F joint has long been considered as a forgotten compartment of the knee. Most of its symptoms were uncritically declared as “chondromalacia patellae”. We have fortunately witnessed a tremendous improvement in the biomechanical understanding, diagnostics, and therapies of P-F compartment over the last 2 decades with variable results. We still lack firm evidence that currently available operative procedures prevent further cartilage deterioration [1, 4].

Content: The unspecific anterior knee pain is predominantly treated conservatively. In general, the surgical means are mostly reserved for objective P-F instability (subluxation or luxation) and symptomatic cartilage injuries. The cartilage may be injured acutely during a direct anterior knee trauma (kissing lesions depending on the knee position) or dislocation (typical lateral trochlea and medial patella lesions), but also as chronic overload (central trochlea lesions) or maltracking (distal lateral patellar facet). Several realignment techniques are available for the treatment of objective P-F instability. In general, they can be divided into proximal procedures, distal procedures and trochleoplasties, or a combination of the above [1, 4, 5]. Although these procedures provide an instant increased stability of the P-F joint, they often result in an early osteoarthritis [1]. This early cartilage deterioration may be due to the previous injuries but also due to altered P-F biomechanics after surgery. We have reviewed the long-term outcomes of the P-F realignment procedures performed at our institution between the years 1963 to 1994 by various orthopedic surgeons using different techniques. Regardless of the realignment procedure a moderate/severe P-F osteoarthritis was confirmed in the majority of patients. A relative newcomer in the operative assortment of P-F instability is the reconstruction of the medial P-F ligament. The reconstructed ligament needs to be positioned isometrically and it is expected not to change P-F biomechanics significantly, but we are still awaiting the long-term outcomes [5].

Prior to the era of biological resurfacing the symptomatic cartilage degeneration was treated by cartilage debridement and P-F unloading. A widely used procedure at the time was anteriorization of the tibial tubercle as proposed by Maquet [6]. This procedure was due to a high rate of complications and due to the unpredictable outcome upgraded to antero-medialization (AMZ) popularized by Fulkerson [7]. AMZ offers simultaneous lateral-central unloading and medial realignment. The early results were reported to be clinically 86% to 97% successful; better results were demonstrated for younger patient with subluxations and without significant osteoarthritis [1]. The temptations for the cartilage biological resurfacing have been increasing since 90s; however, the initial results were inferior to the tibio-femoral compartments [8]. Of the cartilage repair techniques the only systematic studies on P-F joint are available for the autologous chondrocyte implantation (ACI). There have been no systematic published reports for microfractures alone for the P-F lesions, although they are frequently used. The results of ACI on P-F joint have heterogeneous results with good to excellent ranging from 62% to 90% [9-14]. The patients were not systematically included with regard to size, location, previous treatments, and additional procedures. The two published studies that compared patients with ACI alone to ACI with P-F realignment demonstrated superior outcome with the addition of AMZ [11, 13].

In spite of increasing medical evidence on P-F instability and cartilage repair, many issues remain unsolved: timing of combined procedures; treatment modifications with regard to lesion size, location, duration of symptoms, and patient’s age; degree of AMZ necessary; how to proceed with medial P-F lesions [15]. Until we get more answers the two questions need to be cleared and therapy accordingly modified for a successful cartilage repair: Why did the cartilage lesion occur? Is the negative impact on cartilage still active?

References:

1. Aglietti P, Giron F, Cuomo P. Disorders of the patellofemoral joint. In: WN Scott, ed. Disorders of the patellofemoral joint. Philadelphia, Pa: Churchill Livingstone Elsevier, 2006; 807-936.

2. Grelsamer RP, Dejour D, Gould J. The pathophysiology of patellofemoral arthritis. Orthop Clin North Am 2008;39:269-274.

3. Curl WW, Krome J, Gordon ES, Rushing J, Smith BP, Poehling GG. Cartilage injuries: a review of 31,516 knee arthroscopies. Arthroscopy 1997;13:456-460.

4. Teitge RA. Patellofemoral syndrome a paradigm for current surgical strategies. Orthop Clin North Am 2008;39:287-311, v.

5. Fithian D, Neyret P, Servien E. Patellar Instability: The Lyon Experience. Techniques in Knee Surg 2007;6:112-123.

6. Maquet P. Advancement of the tibial tuberosity. Clin Orthop Relat Res 1976:225-230.

7. Fulkerson JP. Anteromedialization of the tibial tuberosity for patellofemoral malalignment. Clin Orthop Relat Res 1983:176-181.

8. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889-895.

9. Gobbi A, Kon E, Berruto M, et al. Patellofemoral full-thickness chondral defects treated with second-generation autologous chondrocyte implantation: results at 5 years‘ follow-up. Am J Sports Med 2009;37:1083-1092.

10. Gomoll AH, Minas T, Farr J, Cole BJ. Treatment of chondral defects in the patellofemoral joint. J Knee Surg 2006;19:285-295.

11. Henderson IJ, Lavigne P. Periosteal autologous chondrocyte implantation for patellar chondral defect in patients with normal and abnormal patellar tracking. Knee 2006;13:274-279.

12. Minas T, Bryant T. The role of autologous chondrocyte implantation in the patellofemoral joint. Clin Orthop Relat Res 2005:30-39.

13. Pascual-Garrido C, Slabaugh MA, L‘Heureux DR, Friel NA, Cole BJ. Recommendations and treatment outcomes for patellofemoral articular cartilage defects with autologous chondrocyte implantation: prospective evaluation at average 4-year follow-up. Am J Sports Med 2009;37 Suppl 1:33S-41S.

14. Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A. Two- to 9-year outcome after autologous chondrocyte transplantation of the knee. Clin Orthop 2000:212-234.

15. Farr J. Autologous chondrocyte implantation and anteromedialization in the treatment of patellofemoral chondrosis. Orthop Clin North Am 2008;39:329-335.

Extended Abstracts150

19.1.2Osteochondritis Dissecans in the Knee: Therapeutic OptionsS. GörtzLa Jolla/United States of America

Introduction: Osteochondritis dissecans (OCD) is an acquired condition primarily affecting the subchondral bone of the femoral epiphysis which often manifests as progressive disease of the overlying articular cartilage, ultimately culminating in sequestration of osteochondral fragments as classified in the ICRS grading scale for OCD: OCD I - Stable, continuous: Area of softened subchondral bone covered by intact cartilage. OCD II - Partial discontinuity, stable on probing. OCD III - Complete discontinuity, ”dead in situ”, not dislocated. OCD IV - Dislocated fragment, loose within the bed or empty defect, with lesions of over 10mm in depth constituting the IVB sub-group.

Content: While the exact incidence and etiology of OCD remains somewhat controversial, a vast majority of lesions occur in the “classic” position on the lateral margin of the medial femoral condyle, with a considerable incidence of bilateral disease. Cumulative microtrauma and altered biomechanics are often considered causative for OCD, as evidenced by the association with repetitive impact loading and conditions such as discoid lateral meniscus. More recently, long axis varus and valgus malalignment has been implicated as being contributory to OCD of the medial and lateral femoral condyle, respectively. Generally, two distinct clinical entities of OCD are recognized, depending on the maturity of the distal femoral physis: Juvenile OCD characterizes lesions in skeletally immature patients while adult OCD describes lesions occurring at or after physeal closure, although many of these are thought to be progressive sequelae of unresolved juvenile cases. Traditionally, surgeons have relied on radiographic imaging to localize and characterize OCD lesions as well as to evaluate skeletal maturity as a prognostic factor. Standard imaging for OCD should include AP, lateral, Rosenberg (flexed knee PA) and Merchant (patellofemoral) views. MRI has become a routine diagnostic tool in the evaluation of OCD lesions, which has gained further prognostic value with the advent of advanced cartilage imaging protocols. Besides skeletal maturity and increased lesions size, a subchondral high T2 signal zone in lesions that present with loss of surface continuity on T1-weighted images have all been shown to be possible predictors of failure in non-operative management. Gadolinium enhancement and bone scintigraphy can be helpful in distinguishing if subchondral signal enhancement is owing to synovial fluid infiltration or formation of vascular granulation tissue. The former indicates disease progression due to loss of surface continuity and increasing instability (a negative prognostic sign) whereas the latter implies osseous reconstitution (a positive prognostic sign), especially in patients with open physes. For stable juvenile OCD lesions with intact cartilage surface (ICRS grade 1 or 2) and presenting without mechanical symptoms or effusion, conservative treatment has proven to be a reasonable initial step in management due to the favorable natural history. Non-operative treatment usually includes an initial four to six week period of no or protected weightbearing and range of motion exercises, followed by progressive low-impact rehabilitation if supported by favorable clinical presentation and serial imaging. Gradual return to supervised sport specific activities can be considered after three months if the patient remains asymptomatic. A repeat MRI can be helpful at that time to confirm healing and reossification before initiating impact loading. In juvenile OCD, operative management is indicated for stable lesions that have failed conservative management as well as more unstable lesions in patients who are nearing physeal closure. Stable grade 1 or grade 2 lesions with continuous articular cartilage surface are generally amenable to arthroscopic management via retrograde transarticular drilling. Antegrade drilling through the epiphysis is technically more challenging, but has the advantage of avoiding injury to the articular cartilage. Both techniques aim to stimulate revascularization of the sequestered bony fragment and have shown good results at short to medium follow up, with patient age and skeletal maturity being the most important predictors of outcome. The progressive nature of adult OCD generally justifies a more aggressive surgical approach, and the presence of unstable or detached fragments in either adult or juvenile disease necessitates operative intervention. Partially delaminated grade 1 or grade 2 lesions can usually be addressed arthroscopically by using bioabsorbable transchondral fixation devices. While interpositional fibrous tissue should be removed, extensive subchondral debridement should be avoided. Possible areas of subchondral bone loss can be packed with autologous bone graft before definite fixation. Salvageable sequestered or loose (grade 3 or grade 4) osteochondral fragments with sufficient (3 mm or more) adherent subchondral bone can be reattached either through an arthroscopic or mini-open approach if anatomically congruous. This usually involves removal of fibrous tissue, curettage and bone grafting of the femoral lesion, with drilling to invite neovascularization of the OCD bed. Different fixation methods have been described,

including cannulated screws, bioabsorbable devices, or autologous osteochondral plugs. While reported results have generally been favorable, these methods all have inherent disadvantages. Metal screws require eventual removal, bioabsorbable devices have been reported to cause host bone reaction, and both can damage juxtaposed articular surfaces or become loose bodies in case fixation is lost. While rare, donor site morbidity and hemarthrosis are possible complications of autologous osteochondral graft harvest. Grade 3 or grade 4 lesions with non-salvagable osteochondral fragments (due to fragmentation or lack of adherent bone) pose more of a surgical challenge. Simple loose body removal and debridement has been shown to lead to poor long-term clinical outcomes and arthritic disease progression and is not recommended. Due to the subchondral pathology including bone loss commonly encountered in OCD, marrow stimulating techniques are at a distinct disadvantage in treating this condition. While least invasive, microfracture generally leads to only fibrocartilagenous replacement tissue and has increasingly shown to negatively influence revision options due to induction of subchondral sclerosis and formation of intralesional osteophytes. Autologous chondrocyte implantation (ACI) has led to good clinical results at medium to long-term follow up in multiple studies. While the use of the ACI sandwich technique (with chondrocytes implanted between layers of periosteum over autologous bone graft) to address ICRS grade IVb lesions has been described in the literature, isolated outcomes of this variant have not been individually reported. Newer generation matrix-assisted ACI techniques employing different scaffolds promise to simplify the concurrent application of bone grafts for use in OCD. Likewise, the advent of multilayered acellular scaffolds holds promise as biomimetic conduits for true osteochondral replacement, with encouraging early results. Osteochondral grafting techniques are attractive for the treatment of OCD because of their ability to address both the osseous as well as the chondral component of the disease with a compound graft. Where available, fresh osteochondral allografts are generally considered the gold standard treatment for high grade OCD. Allografts reliably restore the appropriate joint anatomy by providing mature, orthotopic hyaline cartilage with an intact calcified cartilage tidemark on an osseous scaffold that can restore even advanced bone loss associated with ICRS grade IVb OCD lesion. Multiple authors have reported good clinical results with durable outcomes at long-term follow up for both autologous and allogeneic osteochondral grafts. Recently, renewed interest has been placed on optimizing the biological milieu of the joint in cartilage repair, including correction of instability and mechanical alignment. Since axial varus/valgus malalignment in particular has been implicated as contributory in the development of OCD, it stands to reason that failure to correct these factors will negatively affect outcomes, regardless of technique. Thus, adjuvant osteotomy is recommended to optimize the biomechanical environment of the knee with OCD, although this has not been conclusively borne out in the orthopaedic literature. In summary, osteochondritis dissecans is seen with increased frequency in adolescent and young adult patients, owing to increased athletic participation and advances in diagnostic modalities. Early diagnosis and treatment are important to optimize outcomes. An initial attempt at conservative treatment, focusing on activity restriction and modification, is warranted for stable OCD lesions in skeletally immature patients. Adult OCD lesions have limited healing potential and usually benefit from early operative treatment. Stable juvenile OCD lesions that fail conservative treatment, as well as unstable juvenile or adult OCD lesions, also require surgical management. Operative treatment options depend on lesion size, site, stage as well as patient age and include drilling, internal fixation, bone grafting, and different cartilage repair modalities with possible adjuvant corrective osteotomy.

References: Management of Osteochondritis Dissecans of the Knee : Current Concepts Review.

Mininder S. Kocher, Rachael Tucker, Theodore J. Ganley and John M. Flynn

Am J Sports Med 2006 34: 1181-1192

Treatment of Osteochondritis Dissecans of the Knee with Autologous Chondrocyte Transplantation: Results at Two to Ten Years.

Lars Peterson, Tom Minas, Mats Brittberg and Anders Lindahl

J Bone Joint Surg Am. 2003;85:17-24.

Fresh Osteochondral Allografting in the Treatment of Osteochondritis Dissecans of the Femoral Condyle.

Bryan C. Emmerson, Simon Görtz, Amir A. Jamali, Christine Chung, David Amiel and William D. Bugbee

Am J Sports Med 2007 35: 907-915

Extended Abstracts 151

19.3.3A 3D-culture model for the streamlined engineering of large-scale human cartilage graftsI. Martin, B. Tonnarelli, R. Santoro, A. Barbero, D. WendtBasel/Switzerland

Introduction: While Carticel® and Hyalograft-C® have been well established in the clinic for the treatment of traumatic focal cartilage defects, no tissue engineered product is currently available to treat large defects or those associated with advanced diseases such as osteoarthritis. Beyond the biological challenges that must be addressed to treat chronic joint disorders, it remains a significant engineering challenge to generate cartilage grafts with dimensions that would be sufficient for the repair of large, advanced, and deep defects, and moreover, to develop a manufacturing process which is safe, standardized, and ultimately cost-effective. We previously described a perfusion bioreactor system for seeding and culturing constructs of clinically relevant thickness and demonstrated that highly viable and homogeneous constructs could be generated [1]. In this work, we scaled-up our perfusion bioreactor to engineer human cartilage grafts in dimensions sufficient for uni-compartmental resurfacing of a human knee joint (i.e., 50mm diameter x 4mm thick). We next aimed to streamline the conventional cartilage tissue engineering production processes, by seeding, expanding, & differentiating isolated primary human articular chondrocytes (HAC) entirely within a porous 3D scaffold using a perfusion bioreactor system. The novel strategy would allow to eliminate the conventional and labor-intensive steps of 2D cell expansion in flasks prior to loading into the 3D scaffold, facilitating the development of a simple, automated, and closed bioreactor-based manufacturing process [2].

Content: A perfusion bioreactor system was first developed to generate a uniform velocity profile over the surface of 50 mm diameter scaffolds. Consistent with theoretical flow simulations, performed in collaboration with Prof. D. Lacroix (University of Catalonia), experimental assessments confirmed that cells could be uniformly seeded throughout the large scaffold volume. Following two weeks of perfusion culture in the bioreactor, viable, homogeneous and cartilaginous tissue constructs were generated, which were of similar quality to smaller “research-scale” control constructs, in spite of the 70-fold larger size (5.9cm3 vs. 0.085cm3). We next established a novel streamlined and bioreactor-based process, based on the definition of two different culture phases, namely related to chondrocyte proliferation and subsequent re-differentiation/extracellular matrix deposition. In five independent experiments, HAC were isolated from clinically representative sized biopsies (as obtained in the clinic for autologous chondrocyte implantations) and perfusion seeded onto Hyaff-11â non-woven meshes. Seeded meshes were subsequently perfused with “proliferating” culture medium (containing 1 ng/ml Transforming Growth Factor-beta1, TGFb, and 5 ng/ml Fibroblast Growth Factor-2) for approximately two weeks in order to colonize the scaffold volume and reach a defined cell density. Flow-through micro-oxygen sensors were integrated into the bioreactor system to monitor the oxygen levels in the media perfused through the constructs during bioreactor culture. After initially establishing a relation between the oxygen measurements and the number of cells within the 3D constructs, we applied this non-destructive method to quantitatively and non-invasively monitor the cell growth throughout the proliferation phase. After the initial proliferation phase, when the chondrocytes had been sufficiently expanded within the mesh, chondrocyte re-differentiation was then induced by perfusing “chondrogenic medium” (containing 10 ng/ml TGFb and 10 mg/ml insulin) under hypoxic conditions (5%O2) for an additional two weeks. Primary HAC could be seeded and extensively expanded (about 5 and 9 doublings in respectively 2 and 4 weeks) in the 3D scaffolds, densely populating the volume of the meshes. Immuno-staining for the Ki67 marker confirmed the presence of proliferating cells at day 14. Following subsequent chondrogenic conditions, constructs engineered by the streamlined bioreactor-based approach showed evidence of redifferentiation as significant increase in GAG content (up to about 4.5 ug/mg wet weight), immunohistochemical staining for collagen type II, and increased expression of collagen type II mRNA. The work describes a new paradigm for the generation of large-scale cartilage grafts, where the phases of cell expansion, cell seeding, and tissue culture are performed directly through 3D scaffolds and within a single closed perfusion bioreactor system. The limited number of chondrocytes obtained from a clinically representative sized biopsy could be extensively expanded directly in 3D scaffold under perfusion, reaching cell numbers similar to seeding densities used in conventional cartilage tissue engineering approaches based on cells previously expanded by 2D culture steps [3]. When constructs underwent following chondrogenic phase, we

then found that 3D expanded HAC could redifferentiate and generate cartilaginous grafts. Online monitoring of oxygen during perfusion bioreactor culture was used to estimate the number of cells within a 3D construct. Given that few non-destructive assays currently exist which can quantitatively characterize an engineered construct, this simple and non-invasive assay could represent a significant element in regulatory compliant manufacturing of tissue grafts meeting specific quality criteria. This novel streamlined cartilage tissue engineering strategy can serve as the basis of a manufacturing process requiring the minimal number of bioprocesses and unit operations, thereby facilitating simplified and compact bioreactor designs with limited automation requirements, and with the likely result of lower operating costs and increased compliance to safety guidelines [2].

References:

[1] Wendt D, Stroebel S, Jakob M, John GT, Martin I. Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions. Biorheology 43:481-488 (2006) [2] Martin I, Smith T, Wendt D. A roadmap for the bioreactor-based translation of tissue engineering strategies into clinical products. Trends Biotech 27(9): 495-502 (2009) [3] Moretti M, Wendt D, Dickinson SC, Sims TJ, Hollander AP, Kelly DJ, Prendergast PJ, Heberer M, Martin I. Effects of in vitro preculture on in vivo development of human engineered cartilage in an ectopic model. Tissue Eng 11:1421-1428 (2005)

Acknowledgments:

We acknowledge the EU for financial support (“STEPS”; FP6- #NMP3-CT-2005-500465)

Extended Abstracts152

20.2.3Preoperative radiographic evaluation of axis deformities around the kneeS. SpruijtWoerden/Netherlands

Introduction: The knee joint is the largest joint in the body and it has the longest lever arms. These arms produce substantial loading moments. Deformity in the frontal plane will increase the distance from the centre of the knee to the mechanical axis and thus create a moment arm at the knee joint. Axis deformities closer to the knee produce greater mechanical axis deviations than deformities near the ankle or the hip1. In a dynamic experimental loading study it was demonstrated that the position of the loading axis in the frontal plane has a strong effect on the tibiofemoral cartilage pressure distribution of the knee. The medial compartment is predominantly loaded in a varus knee; a neutral mechanical axis slightly loads the lateral more than the medial compartment. In valgus alignment, the main load runs through the lateral compartment2. It has been shown that frontal plane malalignment around the knee is associated not only with progression of knee osteoarthritis but also with the development of knee osteoarthritis3. Therefore axis deformities around the knee are regarded as pre-arthritic deformities. The goal of correctional osteotomies around the knee is the transfer of mechanical load from the diseased areas of the joint to areas with healthy, intact cartilage. Recent biomechanical work has shown that unloading of the medial compartment can be achieved in high tibial osteotomy by using a slight valgus overcorrection3, 4. Accurate preoperative evaluation of axial deformity is mandatory for the success of osteotomies around the knee. This requires detailed knowledge of normal limb alignment. Since the most frequent and relevant deformities around the knee occur in the frontal plane, this paper will primarily, but not exclusively, focus on the preoperative evaluation of axis deformities in the frontal plane.

Content: Radiographic work-up A thorough history and physical examination are absolutely mandatory in the preoperative workup for osteotomies around the knee. However, to understand the true nature of the deformity subsequent radiographic examination is indispensable. Preoperative evaluation for osteotomies around the knee includes anteroposterior weight-bearing views in full extension, weight-bearing PA tunnel views in 45° of flexion (Rosenberg view)5, lateral views and skyline views of the patella. These allow an assessment of the extent and localisation of knee osteoarthritis. The anteroposterior whole leg standing radiograph is considered the gold standard for measuring axial alignment and joint orientation and serves as the basis for the planning of osteotomies in the frontal plane. Standardized radiographs with full extension and correct anterior orientation of the patella are necessary for such measurements6, 7. For assessment of the sagittal plane of the knee joint strict lateral knee views are usually sufficient. The lateral whole leg standing radiograph in full extension is only indicated in the case of extra-articular deformity. Varus and valgus stress views are not absolutely necessary, but may be helpful to assess instability of the collateral knee ligaments or to define the degree of involvement of the less affected tibiofemoral compartment8. Magnetic resonance imaging (MRI) may be useful for assessment of meniscal and ligamentous lesions and cartilage damage. It is however not mandatory. Especially not if the osteotomy around the knee is preceded by an arthroscopy. Computer tomography (CT) is only indicated if torsional malalignment is suspected or in cases with posttraumatic defects. Scintigraphy is usually not indicated in the preoperative evaluation for osteotomies around the knee. Causes of axial deformities The joint orientation angles and axes can be pathologically altered in any plane (frontal, sagittal, transverse or oblique) and cause malalignment of the entire leg. The causes may be congenital or constitutional (skeletal dysplasia, achondroplasia). They may be develop during childhood as a result of malnutrition (rickets), metabolic bone disease (hypophosphatemic rickets), Blount’s disease, osteopathies (renal osteopathy), growth disorders with premature partial closure of the growth plate, obesity, iatrogenic, etc. The may also be acquired after trauma (e.g., malunited fracture). They may be related do systemic myopathic and neurogenic disorders. They may be the result of physical trauma. Furthermore secondary deviation of the axes can be caused by destruction of the joint surfaces as a result of bone necrosis, infection, bone tumour, inflammatory arthritis. The most frequent cause of secondary varus and valgus malalignment is secondary cartilage damage after meniscectomy9. Mechanical and anatomic bone axes In long bones anatomical and mechanical axes are distinguished. In clinical practice radiographs are standard made in the anteroposterior and lateral direction. Axis lines drawn on these radiographs are referred to as frontal and sagittal plane axes,

respectively. However, around the knee the deformities in the frontal plane are the most frequent and most relevant. The anatomical axis of a bone is the mid-diaphyseal line. The mechanical axis of a bone is the line that connects the joint centres of the proximal and distal bone-ends. The mechanical femoral axis in the frontal plane runs from the centre of the femoral head to the centre of the knee joint and forms a distally converging angle of 6 ± 1° with the anatomical axis of the femur9 In the tibia the frontal plane anatomical and mechanical axes are almost parallel. Physiologically, the angle between the mechanical axis of the femur and tibia is 1.2 ± 2.2° varus10. The anatomic tibiofemoral angle is physiologically 5-7° valgus11.The mechanical axis of the entire leg in the frontal plane (Mikulicz line or weight bearing line) is the line connecting the centre of the femoral head and the centre of the ankle joint. This line normally passes medial to the centre of the knee joint. Radiographically, this is approximated by the position of the medial tibial spine. The distance between the mechanical axis line and the centre of the knee in the frontal plane is the mechanical axis deviation (MAD). This deviation can be expressed in millimetres or as a percentage of the tibial plateau width (medial 0% and lateral 100%). The normal MAD is 8 ± 7mm medial to the centre of the knee joint12. Joint orientation angles The joint orientation line represent the position of a joint in a particular plane. These joint lines in the frontal and sagittal planes have a characteristic orientation to the mechanical and anatomical axes. Paley has standardised the nomenclature of these joint orientation angles. He has also reported on the normal values of these angles relative to the mechanical and anatomic axes in both the frontal and sagittal planes11, 12. The mechanical lateral proximal femoral angle (mLPFA) represents the hip joint orientation in the frontal plane and is defined as the angle between a line from the centre of the femoral head to the tip of the greater trochanter and the mechanical axis of the femur. Its standard value is 90° ± 5°. The distal femoral knee joint orientation is defined as the lateral distal angle between the mechanical femoral axis and the tangent to the distal femur (mLDFA) and has a standard value of 87°± 3°. The proximal tibial knee joint orientation is the medial proximal tibial angle (MPTA) and is the angle between the tangent to the tibial plateau and the anatomical or mechanical axes of the tibia and is 87°± 3°. The mechanical lateral distal tibial angle (mLDTA) represents the ankle joint orientation and is defined as the angle between the tangent to the dome of the talus and the anatomical or mechanical axes. Its standard value is 89°± 3°. The angle between joint orientation lines on opposite sides of the same joint is called the joint line convergence angle (JLCA). In the knee the JLCA is normally between 0-2° medial convergence. Increased JLCA-values due to ligament instability must be taken into account in correctional osteotomies to avoid overcorrection. The midjoint line (MJL) is a measure of joint line obliquity 13, 14. The MJL is centred between the knee base line of the femur and the baseline of the tibia plateau and forms femorolateral and mediotibial angles of 87°± 3° with the mechanical axis (Mikulicz line). The MJL thus has a slight varus inclination. This inclination is valuable because during gait the leg is held in slight adduction during the stand phase and consequently the knee joint line becomes parallel to the ground during weight bearing12. Correctional osteotomy may achieve correction of the mechanical axis, but may result in a pathological alteration of MJL if the correction was not performed at the site of the bone deformity. Pathological MJL leads to increased shear stresses al the joint line, which is not very well tolerated in the knee. The above described principles for joint orientation angles in the frontal plane are applicable to the sagittal plane as well. For the knee the posterior distal femoral angle (aPDFA = 83°±4°) and the posterior proximal tibial angle, also known as slope of the tibial plateau. (aPPTA = 81°± 4°) are important12. The sagittal joint orientation angles should not be increased or decreased during osteotomy in the frontal plane (valgus or varus correction) in patients with stable ligaments and normal range of motion. Conversely, increasing tibial plateau slope (flexion osteotomy) can be used to eliminate hyperextension, to unload the posterior tibial plateau and to reduce posterior instability. Alternatively, decreasing the tibial slope (extension osteotomy) can be used to eliminate extension deficit, to unload the anterior plateau and to reduce anterior instability15. Systematic evaluation of axis deformities The most frequent leg deformities occur in the frontal plane. Malalignment is present if the MAD exceeds its normal range and it indicates loss of collinearity of the hip, knee and ankle in the frontal plane. The normal range of MAD is 8 ± 7mm medial12. The source of MAD is identified with the “malalignment test” (MAT) which forms the basis in the evaluation of deformities around the knee11, 16. Hereto, the mLDFA, MPTA and JLCA are measured. If the mLDFA or MPTA exceeds its normal range respectively the femur or the tibia is contributing to the MAD. Abnormal values of the JLCA indicate ligamentous laxity or cartilage loss as causes of malalignment. Other sources of malalignment include knee joint subluxation and femoral or tibial condyle deficiency11, 16 The MAT

Extended Abstracts 153

just identifies which bone or joint contributes to the malalignment. It does not identify the level of the deformity. Neither does MAT identify any sagittal plane component of deformity. Malorientation of the knee very clearly leads to MAD and therefore The MAT is a “malorientation test” (MOT) of the knee. However, the MAT can be (almost) normal in the presence of malorientation of the hip and ankle. To recognize malalignment of hip and ankle it is therefore necessary to perform separate MOTs for these joint as well11, 16. The next step is to find the location of deformity within a particular bone. For this, the anatomical axes, using mid-diaphysial lines, for both femur and tibia are used. In a deformed bone the proximal and distal anatomic axes intersect. This point is known as the centre of rotation of angulation (CORA)17. The mechanical axis can also be used to determine the CORA. Hereto, the joint centre and a reference angle are used to draw the mechanical axis of the proximal or distal femur or tibia. A reference angle is drawn to a reference line. The two possible reference lines that can be used are the joint orientation line and the mid-diaphyseal line. If the CORA does not correspond to the obvious apex of angulation, there is either a translational deformity or there may be several (multiapical) deformities17. Furthermore rotational deformity may coexist. Deformity in the limb may occur in any plane, not just the “anatomical” sagittal or frontal planes. Uniapical angular deformities for which angulations are visible on both anteroposterior a lateral radiographs are often incorrectly referred to as biplanar deformities. These biplanar angular deformities are actually uniplanar deformities in an oblique plane (between the frontal and sagittal planes)18. The site of the deformity may be at the diaphysis, metaphysis or at the level of the joint. Any angular deformity should be described in terms of its magnitude, direction, plane and apex location. It is very important to realise that if angular deformities are corrected by an osteotomy that passes through CORA realignment occurs without translation. When the osteotomy is performed at another level than CORA the axis will not only realign by angulation, but translation will occur as well19. Genu varum and genu valgum A MAD that lies more medial than the normal range indicates a varus deformity and a MAD lateral to the normal range a valgus deformity. If the MPTA is decreased the varus malalignment is due to tibial deviation, whereas an increased mLDFA indicates a femoral cause. Conversely, if the mLDFA is smaller than the standard value, the cause of the valgus deformity is a femoral one. An increased MPTA indicates a tibial cause of valgus malalignment9. In a tertiary referral practice it has been demonstrated that only 31% of patients with varus joint degeneration deformity of the leg have osseous deformity at the tibia only. In 59% the varus deformity is located at the femur and in 10% both femur and tibia are affected. The situation for valgus deformity of the leg is similar. In these cases the deformity is located in the femur in only 22%. In 45% the valgus deformity is located at the tibia and in 33% it affects both femur and tibia. The frequently quoted tenet for correction of varus malalignment at the tibia and valgus malalignment at the femur is, consequently, incorrect for 50% of varus and valgus joint degeneration deformities13. This may lead to joint line obliquity. Varus and valgus deformities can also be caused by cartilage and bone loss and ligament instability. The JLCA will identify these pathologies. The tibial bone varus angle (TBVA) is a prognostic concept that has been introduced by Levigne in 1991 to differentiate between a constitutional extra-articular varus deformity of the proximal tibia and an acquired intra-articular tibial varus secondary to erosion of the medial plateau24. The TBVA is the angle between the epiphyseal axis and the mechanical axis of the tibia. The epiphyseal axis is a line connecting the centre of the tibial spines with the midpoint of a line connecting the two endpoints of the physis. In constitutional tibial varus, high tibial osteotomy (HTO) cures varus malalignment. In acquired tibial varus, HTO is palliative and creates a tibial valgus position with a poorer clinical outcome over time24. Patellofemoral joint and rotational deformity of the leg In the preoperative evaluation of axis deformities around the knee, the patellofemoral joint should be regarded as well. The height of the patella is changed in HTO. Using the open wedge technique, opening of the osteotomy causes distalisation and lateralisation of the tibial tubercle and the patella that can result in patella infera and excessive stresses associated with altered patellofemoral joint mechanics20, 21. In cases of a preexisting patella alta or in large corrections, an anterior osteotomy of the tibial tubercle extending distally should be combined with medial open-wedge HTO as this will leave patellar height unchanged20. Patellar malalignment can be present in combination with axis deformities around the knee and often indicates the presence of torsional abnormalities. Evaluation of patellofemoral and torsional malalignment is beyond the scope of this paper and the reader is referred to other sources22, 23. Conclusion Axial deformities around the knee can occur in all 3 anatomical planes of reference. There are frontal plane deformities (i.e. genu varum and genu valgum), sagittal plane deformities (i.e. genu procurvatum and genu recurvatum), and transverse plane

deformities (translation and torsion). Deformities can even occur in the so called oblique plane (i.e. in between the frontal and sagittal planes). Each axis deformity around the knee should be described in terms of its magnitude, direction, plane and apex location. Deformity correction should be performed at its source (femur, tibia or both) and if possible at the CORA. The success of realigning osteotomies around the knee therefore highly depends on an accurate preoperative evaluation of axial deformities.

References:

· McKellop HA, Sigholm G, Redfern FC, Doyle B, Sarmiento A, Luck JV (1991). The effect of simulated fracture-angulations of the tibia on cartilage pressures in the knee joint. J Bone Joint Surg Am; 73:1382-1391.

· Agneskirchner JD, Hurschler C, Wrann CD, Lobenhoffer P (2007). The effects of valgus medial opening wedge high tibial osteotomy on articular cartilage pressure of the knee: a biomechanical study. Arthroscopy; 23:852-61.

· Brouwer GM, van Tol AW, Bergink AP, Belo JN, Bernsen RM, Reijman M, Pols HA, Bierma-Zeinstra SM (2007). Association between valgus and varus alignment and the development and progression of radiographic osteoarthritis of the knee. Arthritis Rheum; 56:1204–11.

· Mina C, Garrett WE, Jr., Pietrobon R, Glisson R, Higgins L (2008). High tibial osteotomy for unloading osteochondral defects in the medial compartment of the knee. Am J Sports Med; 36(5):949-955.

· Rosenberg, TD, Paulos, LE, Parker, RD, Coward, DB, Scott, SM (1988). The forty-five degree posteroanterior flexion weight-bearing radiograph of the knee. J Bone Joint Surg Am; 70(10): 479-1482.

· Brouwer RW, Jakma TS, Brouwer KH, Verhaar JA (2007). Pitfalls in determining knee alignment: a radiographic cadaver study. J Knee Surg; 20(3): 210-5.

· Paley D. Radiographic assessment of lower limb deformities. In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 31-60.

· Gibson PH, Goodfellow JW (1986). Stress radiography in degenerative arthritis of the knee. J Bone Joint Surg; 68: 608-609.

· Galla M, Lobenhoffer P. Physiological axes of the lower limb. In: Lobenhoffer P, Van Heerwaarden RJ, Staubli AE, Jakob RP, eds, Osteotomies around the knee: indications-planning-surgical techniques using plate fixators. Davos, AO Publishing, 2008: 5-14.

· Hsu RW, Hemeno S, Conventry MB, Chao EY (1990). Normal axial alignment of de lower extremities and load bearing distribution at the knee. Clin Orthop Relat Res; 255: 215-27.

· Paley D, Tetsworth K (1992). Mechanical axis deviation of the lower limbs. Preoperative planning of uniapical angular deformities of the tibia or femur. Clin Orthop Relat Res; 280: 48–64.

· Paley D. Normal lower limb alignment and joint orientation. In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 1-18.

· Van Heerwaarden RJ, Wagenaar F, Hofmann S. Double osteotomies of the femur and tibia. In: Lobenhoffer P, Van Heerwaarden RJ, Staubli AE, Jakob RP, eds, Osteotomies around the knee: indications-planning-surgical techniques using plate fixators. Davos, AO Publishing, 2008: 167-184.

· Babis GC, An KN, Chao EY, Rand JA, Sim FH (2002). Double level osteotomy of the knee: a method to retain joint-line obliquity. J Bone Joint Surg Am; 84:1380-1388.

· Agneskirchner JD, Hurschler C, Stukenborg-Colsman C, Imhoff AB, Lobenhoffer P (2004). Effect of high tibial flexion osteotomy on cartilage pressure and joint kinematics: a biomechanical study in human cadaveric knees. Arch Orthop Trauma Surg;124:575-84.

· Paley D. Malalignment and malorientation in the frontal plane. In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 19-30.

· Paley D. Frontal plane mechanical and anatomic axis planning.

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In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 61-97.

· Paley D. Oblique plane deformities. In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 175-194.

· Paley D. Osteotomy concepts and frontal plane realignment. In: Paley D, ed. Principles of Deformity Correction. Berlin: Springer, 2005: 99-154.

· Gaasbeek RD, Sonneveld H, van Heerwaarden RJ, Jacobs WC, Wymenga AB (2004). Distal tuberosity osteotomy in open wedge high tibial osteotomy can prevent patella infera: a new technique. Knee;11:457-61.

· Stoffel K, Willers C, Korshid O, Kuster JM (2007). Patellofemoral contact pressure following high tibial osteotomy: a cadaveric study. Knee Surg Sports Traumatol Arthrosc;15:1094-100.

· Van Heerwaarden RJ, Koning P, van der Haven IB. Rotational osteotomies of the femur and the tibia. In: Lobenhoffer P, Van Heerwaarden RJ, Staubli AE, Jakob RP, eds, Osteotomies around the knee: indications-planning-surgical techniques using plate fixators. Davos, AO Publishing, 2008: 185-208.

· Fithian DC, Neyret P, Servien E (2007). Patellar instability: the Lyon experience. Tech Knee Surg; 6(2):112–123.

· Bonnin M, Chambat P (2004). Current status of valgus angle, tibial head closing wedge osteotomy in medial gonarthrosis. Orthopäde; 33:135-42 (in German).

24.2.2(EPIC) MicroCTI. PatschVienna/Austria

Introduction: Microcomputed-tomography (micro-CT) is a high-resolution imaging technique which is predominantly applied in osteopororsis research. Depending on geometry and properties of a dedicated scanner, the resolution can reach up to several micron (ex-vivo). Similar in-vivo devices with slightly decreased resolution are available for longitudinal small animal studies. In humans, HR-pQCT (high-resolution peripheral quantitative computed tomography) provides comparable image data of the distal radius and the distal tibia at a resolution of 82 micron. The scanners allow for quantitative assessment of bone microstructure and their use has significantly advanced osteoporosis research (e.g. by assessing treatment effects in animal studies, human iliac crest biopsies or non-invasive imaging studies of the distal radius or tibia). Microstructural osteologic parameters include trabecular and cortical thickness, trabecular number, trabecular separation and connectivity density. Using the structure model index, it is possible to classify trabecular bone as either plate-or rod-like. Owing to the outstanding quality of the images, finite element modelling as well as advanced texture analyses can be applied. Moreover, cortical porosity has become quantifiable. Linking 3D-bone-morphometry and biomechanics, micro-CT has had a major impact on osteoporosis research of the last decades.

Content: As cartilage can hardly be visualized in normal micro-CT, a specific technique termed EPIC (equilibrium partitioning of an ionic contrast) micro-CT has been developed for osteoarthritis research. Similar to the dGEMRIC (delayed gadolinium enhanced MRI of cartilage) technique, EPIC-micro-CT is based on the permeation of cartilage by a ioxaglate-based, negatively charged contrast agent. In EPIC micro-CT, the contrast material is an iodinated radioopaque agent, which distributes inversely to GAG content and density. Regions of low GAG content (i.e. damaged cartilage) will therefore be targeted by the charged contrast agent. Local increases in contrast agent deposition cause an increase in x-ray attenuation which is depicted as difference of a grey-scale image. Data processing includes sagittal reformation and subsequent semiautomated segmentation from subchondral bone and bone marrow. Parameters derived from EPIC micro-CT include cartilage thickness, surface area, volume and attentuation (i.e. local GAG density). Based on the isotropic, high resolution of micro-CT, 3D refomations can be easily obtained. These 3D reformations depict cartilage surface morphology as well as the spatial distribution of cartilage damage. Moreover, cartilage thickness maps can be generated .

EPIC micro-CT is applicable to cartilage explants, biopsy specimens, as well as whole cartilage in situ specimens . It has been used for longitudinal monitoring in a rat model osteoarthritis study . A recent study demonstrated the potential of EPIC-micro CT for monitoring cartilage repair and regeneration of tissue-engineered cartilage in sternum defects .

Whereas most EPIC research has been performed on the basis of a negatively charged (anionic) contrast agent, some researchers suggest the use of positively charged (cationic) agents. Cationic agents seem to display a higher sensitivity to GAG density than anionic agents. In a study performed on bovine cartilage plugs, the concentration of a cationic agent needed for appropriate contrast was much lower than the concentration of a comparable anionic agent .

Besides EPIC imaging, it is possible to visualize cartilage thickness and surface properties by simple micro-CT based arthrography. After injection into the joint space, the radioopaque contrast agent provides indirect display of the articular surface. However, micro-CT arthrography does not enable the assessment of GAG content and is therefore less sensitive to early osteoarthritic changes than EPIC-micro CT. However, the use of silicon-based contrast agents seems to aid in the differentiation of cartilage from synovial fluid and soft tissue components such as ligaments and menisci. The contrast between cartilage and adjacent joint fluid obtained from the injection of silicon-based contrast agents has been reported to be better than in ioxaglate-based studies .

Certain drawbacks of micro-CT of cartilage, irrespective of the type of contrast agent, are cartilage layer-dependant contrast up-take or diffusion into adjacent tissues. For successful application of this technique, specimens have to be incubated with adequate concentrations of diluted contrast agent. Optimal incubation times

Extended Abstracts 155

with the contrast agent seem to be species- and site-specific. Some researchers even suggest that the incubation times used in the majority of studies are by far insufficient. Most studies are focusing on the knee joint, but other locations might display different contrast media uptake kinetics and imaging characteristics. So far, no ex-vivo or in vivo studies on drug effects have been published. In addition the user needs to consider that the resolution of micro-CT depends on specimen size.

Cartilage and subchondral bone form a functional, metabolic and biomechanical unit. A certain benefit of EPIC micro-CT and other micro-CT based cartilage imaging techniques is the option of parallel imaging of cartilage and subchondral bone .

Cartilagemicro-CT has been carried out in various ex-vivo and also in some in vivo animal models, including mice, rats, rabbits and bovine tissue. Studies on human specimens are rare, in vivo data yet entirely missing. A Finnish research group has undertaken several approaches to perform EPIC-imaging on pQCT devices. One of these projects used human cadaveric knee specimens . However, HR-pQCT or similar high resolution devices which allow for scanning of larger samples than just biopsies or small animal joints have not yet been used in EPIC imaging.

To summarize, micro-CT offers a promising new approach to experimental cartilage and subchondral bone imaging. Longitudinal animal studies - especially those evaluating treatment options - are still to be published, and human data are rare. Various contrast agents are in use, but EPIC micro-CT, using negatively charged ioxaglate, is most frequently found in the literature.

The possible benefits of (EPIC)-micro-CT cartilage imaging are

1) the very high resolution,

2) the potential of parallel assessment of cartilage and subchodral bone, as well as

3) its biochemical aspect (i.e. GAG density measurement and mapping)

References:

Palmer AW. Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography. PNAS 2006;103:19255-260.

Xie L. Quantitative assessment of articular cartilage morphology via EPIC-uCT. Osteoarthritis Cartilage 2009; 17(3):313-20. Epub 2008 Sep 11.

Xie L. Nondestructive assessment of sGAG content and distribution in normal and degraded rat articular cartilage via EPIC-microCT. Osteoarthritis Cartilage. 2010; 18: 65-72.

Piscaer TM. Micro-CT Arthrography - A novel in vivo technique for longitudinal assessment of cartilage alterations in small animal models. 2010; 56th Annual Meeting of the Orthopaedic Research Society. Poster No. 1363.

Moyer HR. A new animal model for assessing cartilage repair and regeneration at a nonarticular site. Tissue Eng Part 2010; 16: 2321-30.

Stewart RC. The effect of cationic contrast agent concentration on GAG quantification using computed tomography. 2010; 56th Annual Meeting of the Orthopaedic Research Society. Poster No. 855.

Gu XI. Contrast Agent Enhanced High Resolution 3D Micro-CT Imaging of Hard and Soft Tissue in Rat Knee Joint. 2010; 56th Annual Meeting of the Orthopaedic Research Society. Poster No. 857.

Silvast TS. Diffusion and near-equilibrium distribution of MRI and CT contrast agents in articular cartilage. Phys Med Biol. 2009; 54:6823-36.

Aula AS. Simultaneous computed tomography of articular cartilage and subchonrdal bone. Osteoarthritis Cartilage. 2009; 17: 1583-8.

Silvast TS. pQCT study on diffusion and equilibrium distribution off iodinated anionic contrast agent in human articular cartilage - associations to matrix composition and integrity. Osteoarthritis Cartilage 2009; 17

24.2.3MRI (dGEMRIC and other techniques)S. TrattnigVienna/Austria

Imaging Technologies for MS Tissue Regeneration

MRI (dGEMRIC and other techniques) S.Trattnig MR imaging of the morphology of cartilage and cartilage repair tissue has significantly improved in recent years due to the development of clinical high-field MR systems operating at 3 Tesla. The improved performance has also been achieved as a result of the higher gradient strengths and the application of dedicated coils with modern configuration such as phased array coils. Additionally high resolution isotropic imaging provides identical resolution with reformatting in all planes. The combination of these technological advances now allows high-resolution imaging of cartilage within reasonable scan times. In addition to the evaluation of gross cartilage morphology by MRI, there is growing interest in the visualization of ultra structural components of cartilage by MR in two fields: - Osteoarthritis is manifested by significant changes in biochemical composition of articular cartilage. Loss of glycosaminoglycans (GAG) and increased water content represent the earliest stage of cartilage degeneration, while the collagenous component of the extracellular matrix remains mainly intact during this early phase of cartilage degeneration (1). - During the last years a lot of new surgical techniques for cartilage repair have been developed based on tissue engineering techniques such a autologous chondrocyte implantation (ACI) and matrix-associated autologous chondrocyte transplantation (MACT) (2). In addition to morphological MR imaging of cartilage repair tissue, an advanced method to non-destructively and quantitatively monitor parameters reflecting the biochemical status of cartilage repair tissue is a necessity for studies which seek to elucidate the natural maturation of MACT grafts and the efficacy of the technique. For example GAG are known to be responsible for stiffness properties of cartilage, which gains even more importance with cartilage implants and the content and organization of the collagen network reflects further mechanical properties of cartilage. Therefore, several MR techniques were developed, which allow detection of biochemical changes that precede the morphological degeneration in cartilage. To date, the most promising technique for visualizing the loss of GAG seems to be the delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) (3). It is based on the fact that GAG molecules contain negatively charged side chains which lead to an inverse proportionality in the distribution of the negatively charged contrast agent molecules with respect to the concentration of GAG. Consequently, T1 which is determined by the Gd-DTPA2- concentration becomes a specific measure of tissue GAG concentration. The dGEMRIC technique has provided valuable results in studies on hip dysplasia, in comparative studies with arthroscopically determined cartilage softening in early osteoarthritis, and demonstrated the positive effects of moderate exercise on glycosaminoglycan content in knee cartilage (4). However the main problem was that the standard dGEMRIC technique is either limited to single slices in 2D acquisition or is time consuming in 3D sequences such as 3D inversion recovery prepared fast spoiled gradient recalled acquisition in the steady state. This therefore limits the attractiveness of dGEMRIC for clinical use. To overcome these problems a fast T1 determination by using dual flip angle values in gradient echo based sequence was developed by one vendor. This allowed coverage of the whole knee joint with two different flip angles within the same sequence in about four minutes. The 3D dual flip angle dGEMRIC technique is comparable to standard T1 inversion recovery technique for T1 mapping and its application in patients after MACT surgery is feasible and can be performed in clinical acceptable scan times. We found that the difference between repair tissue and normal hyaline cartilage was statistically significant (p< 0.007) in patients between 3 and 13 months and patients between 19-42 months after surgery, the difference between these groups was not statistically significant (p=0.205). These findings show that the GAG content is significantly lower in repair tissue than in normal hyaline cartilage and does not change even after 3 to 4 years. While GAG content reflects stiffness properties of hyaline cartilage, the organisation of the collagen matrix in cartilage is important too, as failure within the collageneous fibre network is considered to entail further cartilage breakdown.. In a recent study of our group it was possible to evaluate T2 relaxation times in vivo in normal hyaline cartilage sites and in cartilage repair tissue after two different cartilage surgical procedures. Obtaining high resolution MR imaging it was also possible to assess zonal variations within the ROIs and use the measurement of the organization of articular cartilage as an additional tool to differentiate between cartilage repair tissues. We found no differences between deep, middle and superficial aspects within cartilage repair tissue

Extended Abstracts156

after microfracture therapy indicating disorganized and more fibrous tissue. After MACT, zonal variation could be measured, however compared to healthy cartilage sites the increase from deep to superficial zones was less pronounced. These findings may indicate that after MACT, cartilage repair tissue is, in terms of organization, hyaline like. Quantitative T2 mapping may help to better differentiate between normal maturation and development of abnormality in ACI and MACT. This technique may further help in the non-invasive, non destructive follow-up of patients operated on new generations of matrix-associated ACT using new scaffold or carriers. Better knowledge on the macromolecular organization in the implants may help in the planning of rehabilitative procedures after cartilage repair surgery. One encouraging alternative to these above mentioned sequence modalities for the evaluation of cartilage microstructure is the use of diffusion weighted sequences (9). Diffusion Weighted Imaging (DWI) is based on molecular motion that is influenced by intra- and extra-cellular barriers. Consequently, it is possible, by measuring of the molecular movement, to reflect biochemical structure and architecture of the tissue. Conventional DWI based on spin-echo (SE) sequences is relatively insensitive to susceptibility effects, but diffusion weighted SE sequences require a long acquisition time, which, for practical reasons, in a clinical examination is inapplicable. Echo planar imaging (EPI)-based diffusion sequences, the gold standard of DWI in neuro applications, suffer from image distortions due to susceptibility changes as well as from limitations in contrast due to rather long echo times needed. Both renders them impracticable for low T2 tissues like cartilage and muscles. Alternatively diffusion imaging can be based on steady state free precession sequences (SSFP) which realize a diffusion weighting in relatively short echo times. This is achieved by the application of a mono-polar diffusion sensitizing gradient, which, under steady-state conditions, leads to a diffusion weighting of consecutive echoes (spin echoes and stimulated echoes). For the assessment of diffusion weighted images, a three-dimensional steady state diffusion technique, called PSIF (which is a time reversed FISP (Fast Imaging by Steady State Precession) sequence), has been used (10). In order to allow a semi-quantitative assessment of the diffusional behaviour in the cartilage, the diffusion sequence protocol consisted of 2 separate but immediately consecutive measurements using none (0), and 75 mT*ms*m-1 monopolar diffusion gradient moments for Diffusion Weighted Imaging (DWI) and otherwise identical imaging parameters. For evaluation, the quotient image (non-diffusion weighted / diffusion-weighted image) was calculated on a pixel-by-pixel basis In a series of 15 patients after MACT we could demonstrate the feasibility of diffusion-weighted PSIF imaging with high resolution in vivo. The results show that in the follow up at different time points after MACT the diffusion behaviour of the transplants is changing. In the earlier period after MACT the diffusion was higher restricted which declined in the later follow up, but even after a period of up to 42 months there was still a difference in diffusion values between repair tissue and normal hyaline cartilage. Diffusion weighted imaging using the semi-quantitative approach can practicably complement the information obtained from approaches which rely on relaxation properties such as dGEMRIC or T2-Mapping. In comparison to dGEMRIC imaging of cartilage and cartilage repair procedures, no contrast medium is needed ,coverage and resolution is improved and scan times reduced. It may be another tool of biochemical evaluation of cartilage transplants in the near future and could be added in a clinical setting to dGEMRIC and T2 Mapping for evaluation of cartilage repair outcomes.

24.3.2Small animal models in cartilage research: stimulating repair versus inhibiting degenerationC.B. LittleSt. Leonards, NSW/Australia

Introduction: Small animals (mice, rats, guinea pigs, rabbits) have been used extensively for studies of cartilage degeneration in arthritis, and more recently for repair of osteochondral defects. The pros and cons of the different animal species (small and large) for study of repair of isolated cartilage defects have recently been well reviewed (1, 2). In small animals, factors including the physical size of the joint, articular cartilage cellularity, thickness and ratio of non-calcified to calcified zones, thickness of subchondral bone, lack of growth plate closure (mice and rats), and spontaneous repair of cartilage defects in certain strains (mice, rats, rabbits), are all factors that must be considered when evaluating use of small animal models and the translation of findings to humans. Nevertheless, the availability of immune-compromised mice and rats for xenotransplantation experiments, and the array of genetically modified mice provide unique tools to explore specific research questions. Rather than re-covering the information that has already been presented in these excellent available reviews, the current presentation will focus on the relationship between cartilage degradation versus repair and how the use of genetically modified mice and models of osteoarthritis (OA) might inform research on cartilage regeneration strategies.

Content: Degradation and erosion of articular cartilage is a central pathological event of all arthritic conditions. The maintenance of any tissue is a balance between anabolism and catabolism, both of which are inevitably occurring simultaneously as part of normal homeostasis. In musculoskeletal tissues such as cartilage and bone where the extracellular matrix makes up such a large proportion of the tissue volume and is key to the critical mechanical properties, this balance is particularly important. In diseases such as OA, despite evidence for increased biosynthesis of cartilage matrix components at least early in the disease, the catabolic processes predominate and matrix breakdown and cartilage loss occurs. Targeting cartilage degradative mechanisms has not surprisingly therefore, been a major focus of OA research, and significant advances have been made in recent years in identifying important events in OA cartilage breakdown. Many of these advances have come from using small animal models and particularly genetically-modified (GM) mice. A review of the burgeoning literature on cartilage repair highlights that while the majority of the studies in animal models (both small and large) have involved evaluation of repair strategies in isolated chondral or osteochondral defects in otherwise normal joints, a major focus of this research area is actually targeted towards repair and/or regeneration of cartilage in OA. It is widely accepted that cartilage has a limited ability to repair particularly with advancing age, and this may be even more compromised in diseases such as OA. A significant research effort has therefore been directed towards improving the reparative potential of chondrocytes with the view that this can redress the anabolic/catabolic imbalance in OA cartilage and facilitate repair. To this end the effect of numerous growth factors (e.g. IGF-1, TGF-beta, PDGF, CTGF, FGF-2, OP-1) on cartilage metabolism have been extensively studied. The potential of these agents to (re)induce a chondrogenic phenotype in chondrocytes or mesenchymal stem cells for potential repopulation of isolated cartilage defects or OA is the subject of extensive ongoing study. Similarly, the chondrogenic potential of different natural and synthetic scaffolds, ± seeding with different cells with or without mechanical loading has been investigated. Surprisingly however, very little attention has been paid in the “cartilage repair literature” to directly controlling the catabolic pathways that are activated in diseased joints in order to potentially facilitate cartilage repair. Given the importance of balance between anabolism and catabolism in maintenance of cartilage, a valid approach to identifying potential agents to enhance cartilage repair, may be to identify those molecules that have been shown to significantly inhibit or enhance cartilage degeneration in spontaneous or induced models of OA in genetically modified mice. Particularly in the case of cytokines, growth factors and enzymes, such proteins would be obvious therapeutic moieties for cartilage repair, either by augmenting or inhibiting their function. In a previous study examining the relationship between cartilage damage and pathology in other joint tissues in OA, a search in Medline was done using the terms “mouse/murine/mice” and “osteoarthritis” (3). This data has now been updated and reexamined with a view towards cartilage repair, and additional search terms “cartilage repair” and “cartilage regeneration” were included. A total of 798 entries retrieved dating from 1952 to 2010. Papers describing GM mice, defined as strains in which specific genes were either knocked out (KO) or mutated, as

Extended Abstracts 157

well as those where specific proteins were transgenically (TG) or virally over-expressed, were further examined to determine if spontaneous or induced joint disease and/or cartilage degradation and cartilage repair/regeneration were reported. Studies where only inflammatory arthritis (e.g. collagen-induced or antigen-induced) was reported, were excluded except where they provided a useful comparison for GM mice in which non-inflammatory OA-like cartilage degradation/disease and repair had also been evaluated in the same or other reports. No studies were identified where cartilage repair/regeneration alone was studied (i.e. in the absence of analysis of cartilage degeneration and OA). A total of 88 different GM mice were found where an effect on articular cartilage degradation and OA was specifically described. In 37 of the 88 GM mice, OA was induced (usually by some form of surgical destabilization of the joint), while spontaneously occurring joint disease was evaluated in the remainder. The 88 GM mice do not represent mutations in separate proteins, as in some cases different mutations in the one gene/protein, multiple gene KOs, or both increased and decreased expression of a gene have been reported, and are therefore considered separately. Over 90% of the GM lines identified were reported since the year 2001, consistent with the recent explosion in the use of GM mice as a research tool. The GMs were classified as those involving cartilage extra-cellular matrix (ECM) proteins (n = 24), matrix degrading enzymes, enzyme inhibitors or mutations in enzyme cleavage sites in substrates (n = 17), growth factors, cytokines, effector molecules or their receptors and converting enzymes (n = 23), cellular proteins and transcription factors (n = 19), and spontaneous mutations in unknown genes (n = 4). The GM mice were further separated into those where the manipulation “increased” (n = 58), “decreased” (n = 10) or had “no effect” (n = 20) on OA cartilage damage. In only 3 of these GM strains of mice was any analysis of actual “cartilage repair” as opposed to cartilage degradation examined: tenascin C KO, MRL/MpJ and Jaffa mice (see below). Recent advances have been made in defining degradative mechanisms in cartilage, in particular the enzymes responsible for breakdown of the two principle structural matrix proteins, aggrecan and type II collagen. Catabolism of aggrecan is an important early event in pathological cartilage breakdown that likely precedes and may be prerequisite for collagenolysis. Cleavage within the interglobular domain (IGD) by the aggrecanases at ..EGE-ARG... appears to be primarily responsible for aggrecan loss from articular cartilage in vitro and in vivo particularly in diseased states. Mice with inactivation of ADAMTS-5 were found to be resistant to IL-1 stimulated aggrecanolysis in vitro and cartilage degradation following induction of both inflammatory arthritis and OA in vivo (4, 5). Similarly, mice in which the IGD sequence was mutated to render it resistant to ADAMTS-cleavage (but not when made resistant to MMP-cleavage) also have significant inhibition of cartilage breakdown both in vitro and in induced models of arthritis (6). Late stage cartilage degeneration is characterized by extensive proteolysis of the type II collagen network. Much of the collagenolysis in OA cartilage is due to the action of MMPs, particularly MMP-13, and mice deficient for MMP-13 shown chondroprotection in surgically-induced OA (7). Significant upregulation of MMPs and ADAMTS expression occurs in focal diseased cartilage in OA. Importantly in the context of cartilage repair strategies however, we have found that expression of many of these enzymes is significantly increased in cartilage throughout the joint, adjacent to and well removed from the focal erosive lesions. Subsequent activation/release of the latent MMPs and ADAMTS sequestered in cartilage may significantly impact on cartilage surrounding a focal repair as well as the repair tissue itself. In a diseased joint with increased inflammatory cytokines and/or abnormal biomechanical loading, cells implanted to effect repair of a defect will have induction of catabolic pathways, and imunostaining of tissue generated following autologous chondrocyte implantation (ACI) in humans shows increased MMP-cleavage of collagen and proteolysis of aggrecan by ADAMTS. There is little/no published information on the effect of direct “anti-catabolic strategies” (e.g. proteinase inhibitors, anti-cytokine therapies) on actual cartilage repair in vivo. As noted above, in only 3 GM mice where OA degeneration was examined has cartilage regeneration/repair been described. Following induction of an acute inflammatory arthropathy in which almost complete and equivalent loss of aggrecan occurs by day 7 in both wild type mice and those with aggrecan IGD resistant to ADAMTS or MMP cleavage, subsequent cartilage degeneration or repair was monitored. We found significant restoration of cartilage proteoglycan at day 28 only in the Jaffa mouse strain with aggrecan resistant to ADAMTS cleavage (6). Furthermore, this correlated with significantly less cartilage fibrillation and structural damage in these mice compared with the two other strains. MRL/MpJ (“healer”) mice have a significant inhibition of cartilage proteoglycan loss and erosion following intra-articular fracture (8). This protection against post-traumatic OA in MRL/MpJ mice was associated with significantly lower circulating levels compared with C57BL6 mice of IL-1alpha (but

not IL-1beta, TNFalpha or IL-6) both pre- and at 4 and 8 week post-injury. In a separate study, we found that this same strain of MRL/MpJ mice had significantly enhanced cartilage regeneration of an isolated osteochondral defect in the femoral condyle compared with C57BL6 animals (9). In contrast, there was no difference between strains in repair, or lack of repair of cartilage injuries that did not penetrate into the subchondral bone. These two studies support the hypothesis that factors that inhibit cartilage degeneration in models of OA may also augment bona fide cartilage repair or regeneration. In the case of the tenascin C KO mice the corollary was also found to be true i.e. that a factor (in this case ablation of tenascin C) that increases cartilage damage in spontaneous (age-related) or surgically induced OA, also significantly inhibited repair of surgically-induced isolated osteochondral defects (10). There has often been a poor overlap between research and publications focused on cartilage degradation mechanisms in OA and those on cartilage repair. The above examples highlight that better cross-fertilization between these two research arms is needed, and further evaluation is warranted of molecules identified as significantly affecting cartilage degeneration in models of OA (and even inflammatory arthritis) in terms of their ability to influence chondral and osteochobdral repair. In some cases e.g. FGF2 and TGFbeta signaling, there is independent evidence of a coordinated link between increased cartilage degeneration in GM mice where these molecules/signaling pathways have been ablated and beneficial effects on repair of osteochondral defects in another animal species through administration of the growth factor. Despite the caveats about use of small animals described in the opening paragraph, methods to introduce and evaluate healing of well-controlled osteochondral defects in mice have recently been reported (11). The current literature survey has identified at least another 35 GM strains of mice in which a factor that has a significant effect on OA cartilage degeneration and could readily be targeted as a therapeutic (i.e. enzyme, cytokine, growth factor or transcription factor), where cartilage repair/regeneration should be directly examined. Such studies may provide a significant and rapid advancement in identifying targets to improve cartilage repair and tissue engineering.

References:

1. Ahern BJ, Parvizi J, Boston R, Schaer TP. Preclinical animal models in single site cartilage defect testing: a systematic review. Osteoarthritis Cartilage 2009;17(6):705-13. 2. Chu CR, Szczodry M, Bruno S. Animal models for cartilage regeneration and repair. Tissue Eng Part B Rev 2010;16(1):105-15. 3. Little CB, Fosang AJ. Is cartilage matrix breakdown an appropriate therapeutic target in osteoarthritis--insights from studies of aggrecan and collagen proteolysis? Curr Drug Targets 2010;11(5):561-75. 4. Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature 2005;434(7033):644-8. 5. Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker CT, et al. ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 2005;434(7033):648-52. 6. Little CB, Meeker CT, Golub SB, Lawlor KE, Farmer PJ, Smith SM, et al. Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair. J Clin Invest 2007;117(6):1627-36. 7. Little CB, Barai A, Burkhardt D, Smith SM, Fosang AJ, Werb Z, et al. Matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development. Arthritis Rheum 2009;60(12):3723-33. 8. Ward BD, Furman BD, Huebner JL, Kraus VB, Guilak F, Olson SA. Absence of posttraumatic arthritis following intraarticular fracture in the MRL/MpJ mouse. Arthritis Rheum 2008;58(3):744-53. 9. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage 2008. 10. Okamura N, Hasegawa M, Nakoshi Y, Iino T, Sudo A, Imanaka-Yoshida K, et al. Deficiency of tenascin-C delays articular cartilage repair in mice. Osteoarthritis Cartilage 2009. 11. Eltawil NM, De Bari C, Achan P, Pitzalis C, Dell’accio F. A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury. Osteoarthritis Cartilage 2009;17(6):695-704.

Extended Abstracts158

24.3.3Skeletal maturity does not affect the repair quality of damaged articular cartilage using a scaffold-free tissue-engineered construct (TEC) derived from synovial mesenchymal stem cellsN. Nakamura1, K. Shimomura1, W. Ando1, K. Kita2, H. Fujie3, K. Nakata4, K. Shino4, H. Yoshikawa4

1Osaka/Japan, 2Suita, Osaka/Japan, 3Tokyo/Japan, 4Suita/Japan

Introduction: It is widely accepted that chondral injury does not usually heal spontaneously due to its avascular surroundings and unique matrix organization. Therefore, a variety of approaches have been assessed to improve cartilage healing. Among them, stem cell therapies have been tested to facilitate regenerative cartilage repair. Mesenchymal stem cells (MSCs) have the capability to differentiate into a variety of connective tissue cells including bone, cartilage, tendon, muscle, and adipose tissue. These cells may be isolated from various tissues such as bone marrow, skeletal muscle, synovial membrane, adipose tissue, and umbilical cord blood. MSCs isolated from synovial membrane may be well suited for cell-based therapies for cartilage based on the relative ease of their harvest and their strong capability of chondrogenic differentiation. Recent implantation studies have reported successful cartilage repair using synovial membrane-derived MSCs.　One of the crucial factors that may affect the results of cell-based therapies is the age of the donors and recipients. Specifically, the results of implantation surgery could be potentially affected by both the characteristics of the cells and the local environment in the lesions following implantation. Therefore, it is important to elucidate the influence of skeletal maturity on these two factors. Regarding the cell proliferation and differentiation capacities of MSCs, it is controversial as to whether they are age-dependent or not. In terms of the host tissue reaction, natural healing responses of osteochondral defects has been compared between immature and mature animals using rabbit models, and in this species the studies demonstrated better healing responses in immature animals. On the other hand, there have been no studies which compared the results of cell-based repair of chondral defects between immature and mature animal models.

As a potential MSC-based therapeutic method, we have developed a scaffold-free three dimensional tissue-engineered construct (TEC) composed of allogenic mesenchymal stem cells (MSCs) derived from the synovium and extracellular matrices (ECMs) synthesized by the cells, and demonstrated the feasibility of the resultant TEC to facilitate cartilage repair in a large animal study. In the present study, we compared the results of TEC implantation to the chondral lesions morphologically and biomechanically in order to elucidate the influence of the skeletally maturity on subsequent cartilage repair with the TEC.

Content: Cell proliferation and chondrogenic differentiation capacity of immature and mature porcine synovial MSCs

Cell number assessments, as well as WST-1 assays demonstrated that there were no significant differences in the proliferation capacity of porcine synovial MSCs derived from immature or mature animals. In terms of chondrogenic differentiation potential, there were no significant differences in expression of collagen II detected by RT-PCR, in Alcian blue staining of the cell pellets, or in GAG synthesis levels. These results suggested that maturity may not significantly affect the chondrogenic differentiation capacity of porcine synovial MSCs.

Histological assessment of the repaired tissue: Regardless of skeletal maturity, the chondral defect of in the control group showed osteoarthritic change with loss of cartilage and destruction of subchondral bone. Conversely, TEC-treated defect was repaired by chondrogenic tissue with positive Safranin O staining in both immature and mature models. Higher magnification view showed that there was good tissue integration to the adjacent cartilage obtained and the defect treated with the TEC was repaired with hyaline-like cartilage in both models. In histological scoring, the TEC group had significantly higher scores than the control group in all the criteria categories in the immature model. In the mature model, the TEC group had significantly higher scores than the control group in all the categories but the category “Matrix” and “Cell distribution”.

Mechanical properties of the repaired tissue: Articular cartilage is a biphasic viscoelastic material that exhibits strain-rate-dependent mechanical behaviors. This means that the viscoelasticity of cartilage which retains interstitial water might be mainly reflected by faster compression test (@100µm/s) outcomes, while the matrix viscoelasticity without interstitial water should be mainly reflected by slower compression test (@4µm/s) outcomes. Regardless of

skeletal maturity, there was no significant difference between the modulus of normal cartilage and that of repaired tissue treated with TEC at either 4µm/s or 100µm/s.

Discussion:

Previous study showed that in vitro expandability and differentiation capacity of human synovial mesenchymal stem cells do not depend on donor ages. While, natural healing response of rabbit osteochondral defect was better in immature model than in mature model. Therefore, although there has been no study demonstrating the age dependent healing response of cartilage in large animal model, skeletal maturity might likewise affect the repair and differentiation process following the implantation of the TEC. The results of the present study showed the TEC implantation effectively contributed to chondrogenic repair of chondral defect with similar viscoelastic properties to normal cartilage. Notably, such repair response was equivalently observed in both immature and mature pigs. This study could be the first demonstration of the comparison of healing response of cartilage between skeletally mature and immature large animals. It is notable that the allogenic stem cell-based approach was proved to be feasible to cartilage repair regardless of the skeletal maturity. The use of allogenic stem cells could be advantageous in time- and costsaving without tissue sacrifice of host tissue in comparison with autologous cell-based approach and therefore the results of the present study would support the clinical application of this strategy to promote cartilage repair and regeneration.

References:

1. Ando W, Tateishi K, Katakai D, Hart DA, Higuchi C, Nakata K, et al. In vitro generation of a scaffold-free tissue-engineered construct (TEC) derived from human synovial mesenchymal stem cells: biological and mechanical properties and further chondrogenic potential. Tissue Eng Part A 2008 Dec;14(12):2041-2049.

2. Ando W, Tateishi K, Hart DA, Katakai D, Tanaka Y, Nakata K, et al. Cartilage repair using an in vitro generated scaffold-free tissue-engineered construct derived from porcine synovial mesenchymal stem cells. Biomaterials 2007 Dec;28(36):5462-5470.

Acknowledgments:

This study was supported by a grant from the New Energy and Industrial Technology Development Organization, Japan, and Grant-in-Aid for Scientific Research (B), Japan Society for the promotion of Science, Japan.

Free Papers 159

9.1.2Predictors of Osteochondral Allograft FailureW. Bugbee, A. DeYoung, S. Görtz La Jolla/United States of America

Purpose: To determine risk factors for clinical failure of osteochon-dral allografting.Methods and Materials: Between 1983 and 2009, 527 osteochon-dral allografting procedures of the knee were performed in 467 pa-tients. Cases were prospectively entered into a clinical database, including patient demographics, diagnosis, graft characteristics, clinical outcome, and reoperations. A logistic regression model was used to determine variables predicting failure of the osteochondral allograft. Failure was defined as removal (i.e., conversion to knee arthroplasty) or revision of the allograft. The study included 279 males (59.7%) and 188 females (40.3%) with a mean age of 33.6 years (range, 14–68 years). Primary diagnoses included traumatic cartilage injury (35.1%), osteochondritis dissecans (29.8%), degen-erative cartilage lesions (12.0%), osteonecrosis (7.8%), failed al-lograft (5.7%), and osteochondral fracture (3.2%). Anatomic graft locations included medial femoral condyle (26.8%), lateral femoral condyle (20.1%), patella (5.7%), trochlea (4.2%), lateral tibial pla-teau (4.7%) and medial tibial plateau (0.9%). 28.5% of cases in-volved multiple grafts to two distinct anatomic locations and 9.1% of cases involved grafting of three separate surfaces. Average total graft area was 9.7 cm2 (range, 1.2–57.5 cm2).

Results: Sixty-five knees (15.3%) were reoperated for clinical fail-ure at a mean of 38.1 months (range, 3–107 months). Forty-four were converted to arthroplasty and 21 underwent repeat allograft-ing. Gender, age, total graft area, and number of grafts were signifi-cant (p<0.05) risk factors for failure. Females were 2.2 times more likely to fail than males. Compared to patients <30 years, those who were 30–40 were 5.1 times more likely to fail; those >40 were 6.4 times more likely to fail. Grafts >10 cm2 were more likely to fail than grafts <5 cm2 (OR=5.1) and knees with two grafts were more likely to fail than knees with only one graft (OR=58.7).Conclusions: Understanding the impact of clinical variables on outcome of osteochondral allografting is useful in clinical decision making.

9.1.3The Optimal Temperature for the Long Term ex-Vivo Cartilage Storage: 11°C is Superior to 4°C, 23°C, and 35°CA. Alibegovic, R. Blagus, M. Drobni Ljubljana/Slovenia

Purpose: Several studies confirmed prolonged ex-vivo viability of human chondrocytes under various environmental conditions. The longest recorded follow-up of 2 months showed 38% viable cells at 4°C. The objective of presented study was to determine the longest surviving period of chondrocytes in ex-vivo explants at four ambi-ent temperatures. Methods and Materials: Osteochondral cylinders were procured bilaterally from femoral condyles from 3 male donors (19, 21, and 33 years) within 24 hours after death. They were stored in the cell culture media (DMEM/F12) parallel at 4°C, 11°C, 23°C, and 35°C. The middle chondral zone was first stained with Live/Dead Viabil-ity/Cytotoxicity Kit and analyzed for cell viability by the confocal laser scanning microscope. Initial analysis was performed 24-36 hours after death, and then repeatedly every 3 days for each tem-perature until all cells died. Analysis at the individual temperature level was discontinued after three consecutive viability analyses failed to detect surviving cells. Results: The detection of viable chondrocytes was lost after do-nor’s death as following: 35°C on day 28, 23°C on day 40, 4°C on day 76, and 11°C on day 100. The statistical analysis with a two way repeated measures ANOVA performed until day 28 revealed significant time and ambient temperature dependent chondrocyte viability. At 28 days were the viabilities following: 20.20% at 35°C, 87.03% at 23°C, 91.38% at 11°C, and 68.90% at 4°C. Conclusions: The presented study re-confirmed that chondrocyte viability is dependent of ambient temperature and time of cartilage ex-vivo storage. Moreover, its results demonstrated the cell sur-vival up to 100 days, which is much longer as previously known. A superior survival times at 11°C in comparison to the other three tested ambient temperatures suggest, that this temperature level might better preserve osteochondral allografts in tissue banking.

9.1.4Long-Term Survival of Concurrent Meniscus Allograft Transplantation and Articular Cartilage Repair: A Prospective 2- to 12-Year Follow-Up EvaluationK.R. Stone, W. Adelson, J.R. Pelsis, A. Walgenbach, T. TurekSan Francisco/United States of America

Purpose: The purpose of the study was to evaluate the effective-ness of combined meniscus allograft transplantation and articular cartilage repair, in patients with meniscus-deficient knees and se-vere articular cartilage damage, over the course of long-term follow up (2 - 12 yrs).Methods and Materials: Clinical examination and subjective pa-tient outcome assessments were conducted pre-operatively and at 2, 3, 5, 7, and 10-year post-operative intervals (N = 119). Procedure survival was determined using Kaplan-Meier survival analysis. Cox proportional hazards model was used to assess the effect of covari-ates on survival. Secondary analysis of validated subjective patient outcomes was accomplished using the Wilcoxon rank-sum test.Results: Average follow-up was 5.80 years (2.1 months - 12.3 years). 25 procedures failed (20.1%) with a mean failure time of 4.65 ± 2.99 years (2.1 months -10.37 years). Age at time of surgery and number of previous surgeries were significant covariates. Procedure sur-vival was not affected by sex, severity of cartilage damage, axial alignment, degree of joint space narrowing, or medial vs lateral allograft. Cox proportional hazards model showed significant fac-tors on relative odds of allograft failure were patient age at time of meniscus allograft (h=1.061;p=0.006) and number of previous surgeries to the affected knee (h=1.528;p=0.026). Patients expe-rienced significant improvements in subjective outcome measures over the course of follow-up (p < 0.05), with exception the 7-year Tegner Index score.Conclusions: This study demonstrates that meniscus allograft transplantation in combination with articular cartilage repair is ef-fective in patients with severe articular cartilage damage. Outcome was not affected by the classic contraindications of age, severity of arthritis, joint space narrowing, and axial alignment. Biologic joint reconstruction may be an appropriate first step for many people with severe articular cartilage damage.

9.1.5Immunosuppressed versus non immunosuppressed bipolar fresh osteochondral allograft in the treatment of end stage ankle arthritis: a clinical and histological comparisonF. Vannini, R. Buda, M. Cavallo, A. Ruffilli, R. Ghermandi, B. Grigolo, S. Neri, S. GianniniBologna/Italy

Purpose: Post-traumatic ankle arthritis in young patients rep-resents a challenge. Fresh bipolar shell osteochondral allograft (FBOA) is a treatment option, nevertheless radiographic arthritis occurrence at follow-up is cause of concern and may be correlated to an immunoreactive response to transplanted cartilage. Aim of the study is to compare two groups of patients who received FBOA in association or not to immunosuppression. Methods and Materials: 2 groups of 16 patients each underwent FBOA. Only one group (group-B) received an immunosuppressive therapy. Patients evaluation was carried out clinically by AOFAS, radiographically by X-Rays, CT scans and MRI up to 2 years and a II look with biopsy was performed at 1 year follow-up. Samples were examined histochemically by Safranin-O staining and immunohis-tochemically by collagen type I, II and caspase-3 evaluations. The evaluation of the transplanted tissues was performed following the ICRS II scoring system.Results: In group-A pre-operative AOFAS score was 26.2 ± 8.2, while at 24 months was 70.6 ± 17.9 (p<0.005). In group-B pre-operative AOFAS score was 27.3 ± 6.9 and 72.7 ± 8.5 (p<0.005) at follow-up. No statistically significant difference in the clinical outcome be-tween the 2 groups was found. A better morphology of the grafts was observed in group-B which presented an higher score (mean ~68%) compared to the other (mean 40%)(p<0.05) . In group-B, cartilage samples showed a regular surface, oval chondrocytes sur-rounded by metachromatic matrix in the middle and deeper layers, integration to subchondral bone, moderate positivity for collagen type II and negativity for caspase-3. Group-A presented instead car-tilage with low proteoglycan content and hypocellularity.Conclusions: Although clinical results were comparable in the two groups, histological results showed that samples from immuno-suppressed group possess morphological and biochemical charac-teristics more similar to hyaline articular cartilage compared to the other group. Further studies are needed to confirm these data.

Free Papers160

9.1.6Isolated Osteochondral Allografting of the Femoral TrochleaW. Bugbee, A. DeYoung, S. GörtzLa Jolla/United States of America

Purpose: Cartilage disease of the patellofemoral joint represents a difficult clinical challenge. Although osteochondral allografting has been shown to be effective in the treatment of chondral and osteo-chondral lesions of the femoral condyle and tibial plateau, there are few clinical studies on osteochondral allografting of the patel-lofemoral joint. In this study, we report on the outcome of isolated osteochondral allografting of the trochlea.Methods and Materials: Twenty patients (20 knees) underwent isolated osteochondral allografting (OCA) of the trochlea. Two were lost to follow-up and the remaining 18 patients (18 knees) comprise the current study population. The mean age was 31 years (range, 14–46 years) and 67% were male. 94% had previous sur-gery (range, 1–13 procedures), including four patients who had sur-gery for realignment of the extensor mechanism. Major preopera-tive diagnoses were traumatic chondral injury and osteochondritis dissecans. Mean allograft area was 6.6 cm2 (range, 2.3–20.0 cm2).Results: One patient (6%) failed and was converted to a total knee arthroplasty at 92 months. 11% had further surgery (arthroscopy) with retention of the allograft. Mean follow-up of the non-failures was 59 months (range, 26–195 months). Mean IKDC pain scores improved from 5.7 to 3.4 postoperatively (p= 0.06). Mean IKDC function scores improved from 3.6 to 7.0 (p< 0.01). 88% reported less pain and 94% reported better function. 88% reported improve-ment, were satisfied with their results, and would choose allograft surgery again under similar circumstances.Conclusions: Isolated osteochondral allografting of the femoral tro-chlea resulted in significant functional improvement and less pain. The majority of patients were satisfied with their treatment. Failure and reoperation rates were low. We conclude that osteochondral allografting is a useful procedure for treatment of chondral and os-teochondral lesions of the femoral trochlea.

9.1.7Particulated juvenile cartilage allograft transplantation for repair of high grade chondral lesionsJ. Farr1, J. Kercher2, M. Salata2, J. Yao3

1Indianapolis/United States of America, 2Chicago/United States of America, 3Austin/United States of America

Purpose: As articular cartilage defects do not spontaneously heal, multiple techniques are used to restore lesions. Techniques are typically based on classical treatments such as marrow stimula-tion, osteochondral grafting, and ACI. This study presents the pre-liminary results of a new repair technique that utilizes particulated juvenile cartilage allograft transplantation.Methods and Materials: 48 patients with ICRS grade 3a,b,c,d or 4a focal chondral defects of the knee were treated with particulated juvenile cartilage allograft transplantation by a single surgeon. These lesions were debrided to a stable rim and the calcified cartilage layer was removed without violating the subchondral bone (in those with grade 3 lesions). The particulated juvenile cartilage (DeNovoNT from IsTo [St. Louis, MO USA distributed by Zimmer, Inc-Warsaw, IN USA]) was placed in a monolayer and secured with fibrin glue. For larger le-sions, the cartilage fragments were protected with a BioGide scaffold (Geistlich Surgery, Switzerland). A standardized ACI rehabilitation protocol was utilized. Visual analog scale (VAS) and British Satisfac-tion (BS) scores were used to evaluate outcomes.Results: 41/48 patients (85%) were available for follow-up at an average of 20 months (range 12-35mos). There were 19 right knees and 22 left knees in the cohort. There were 28 females and 13 males with a mean age of 36 years (range 17-50). A 1-3 bilayer collagen patch augmentation was performed in 39/41 knees. 18/41 patients underwent a concomitant bony realignment procedure. 28 knees contained unipolar lesions and 13 knees contained multiple lesions. Average VAS resting scores improved from 2.1 to 0.8 and max pain scores improved from 6.3 to 4.3. The average BS score improved from 2.1 to 3.3 post-operatively. Conclusions: The preliminary results of particulated juvenile carti-lage allograft transplantation for the treatment of high grade chon-dral lesions appear to be promising in the short-term and compares favorably to the currently established methods of chondral restoration.

9.1.8Chondral defect repair with particulated juvenile cartilage allograftJ. Farr1, J. Yao2 1Indianapolis/United States of America, 2Austin/United States of America

Purpose: This clinical study was conducted to evaluate a cartilage repair procedure of transplanting particulated juvenile cartilage tissue allograft.Methods and Materials: A multi-center, prospective, single arm, twenty-five-subject clinical study was designed to evaluate clinical outcomes such as IKDC and KOOS scores as well as extent and qual-ity of repair with MRI and optional biopsies at various time points post-implantation (up to five years). Currently, 19 patients with 1 or 2 chondral lesions on the femoral condyles and trochlea have been enrolled and treated in the prospective study.Results: The first group of nine patients has been evaluated with up to 18 months post-op follow-up. No revision has been performed to date. Significant improvements in clinical outcomes over the pre-op baseline data have been observed. MRI data indicated good de-fect filling. No optional biopsy has been obtained to date.Conclusions: The present report describes, for the first time, clini-cal data of a cartilage repair procedure of transplanting particu-lated juvenile cartilage tissue allograft to treat cartilage lesions of the femoral condyles and/or trochlea. Clinical outcome data as measured at up to 18 months post-implantation and MRI data in-dicate early positive outcomes and suggest that this technique is capable of filling chondral defects and improving symptoms. Con-tinued clinical evaluation of this technique is needed with longer follow-up durations.

9.1.9Skeletal maturity does not affect the healing response of articular cartilage using scaffold-free tissue-engineered construct (TEC) derived from allogenic synovial mesenchymal stem cells. -A large animal study-K. Shimomura1, W. Ando1, K. Tateishi1, R. Nansai2, H. Fujie2, D.A. Hart3, T. Kanamoto1, T. Mae1, K. Nakata1, K. Shino1, H. Yoshikawa1, N. Nakamura4

1Suita/Japan, 2Tokyo/Japan, 3Calgary/Canada, 4Osaka/Japan

Purpose: One of the crucial factors that may affect the results of mesenchymal stem cell (MSC)-based therapies is the age of donors and recipients. However, no basic or clinical research has addressed this point in detail. Specifically, there have been no controlled stud-ies which compared the results of MSC-based repair of cartilage be-tween skeletally immature and mature animal models, particularly in a large animal model. As a MSC-based therapeutic method, we previously developed a scaffold-free three dimensional tissue-en-gineered construct (TEC) composed of synovial membrane-derived MSCs from immature animals and demonstrated the feasibility of such TEC to facilitate cartilage repair using similarly aged skeletally immature recipients in a chondral defect model.Methods and Materials: Synovial membrane-derived MSCs were isolated from immature (3-4 months of age) and mature (>12 months of age) domestic pigs. The proliferation and chondrogenic differentiation capacity of these MSCs were compared. The TEC from these MSCs were implanted into equivalent chondral defects in the medial femoral condyle of both immature and mature pigs, respectively. The results of TEC implantation were morphologically and biomechanically evaluated at 6 months postoperatively.Results: Cell proliferation and chondrogenic differentiation capac-ity showed no significant differences between the MSCs from imma-ture and mature pigs. Histologically, the TEC implantation effective-ly contributed to chondrogenic repair of both immature and mature pigs with good tissue integration to the adjacent cartilage (Fig.1). The resultant repair tissue exhibited similar viscoelastic properties to normal cartilage in both immature and mature recipients (Fig.2), with no evidence of immune reaction to the allogenic TEC implants.Conclusions: The present study is the first demonstration of the direct comparison of cartilage repair responses mediated by a cell-based therapy between skeletally mature and immature large ani-mals. These findings suggest the use of immature pigs to test the feasibility of cell-based therapies in chondral lesions has validity.

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9.2.2Chondrocyte gene expression is affected by VSOP-labeling in long-term in vitro MRI trackingC.B. Foldager, M. Pedersen, S. Ringgard, C. Bünger, M. LindAarhus/Denmark

Purpose: Autologous chondrocyte transplantation is an essential part of many treatment procedures for cartilage repair. The aim was to investigate the effect and dose-response of very-small iron ox-ide particle (VSOP) labeling of human chondrocytes for long-term in vitro MRI tracking.Methods and Materials: Chondrocytes were isolated from cartilage biopsies from four patients. The cells for the dose-response study were labeled with 25, 50 or 100 µg/mL VSOP. Quantitative gene expression and cellular proliferation were compared to unlabeled controls at day 1, 3, and 7. The cells suited for MRI tracking were labeled with 50 µg/mL VSOP and embedded in alginate beads, followed by MRI (using T2-weighted sequences) at day 0, 1, 3, 7, 14, 21, 28, and histology was performed at each time-point.Results: Histology revealed that VSOP particles were intracellularly confined at all time-points, whereas no extracellular VSOP™s were observed. A mean reduction in T2-value of 25.1 ms (±SD 3.5 ms) was found on T2-maps. The chondrocyte-specific genes aggrecan, colla-gen type 2, and sox9 were all affected by labeling, the two latter in a dose-dependent manner. VSOP™s had no effect on proliferation.Conclusions: VSOP-labeling of chondrocytes affected gene expres-sion but not proliferation. The labeled chondrocytes could be rec-ognized by MRI for 4 weeks without significant changes in the T2 relaxation time.

9.2.3Modelling of tissue formation in chondrocyte filter culturesC.J. Catt1, W. Schuurman2, B. Sengers1, C.P. Please1, W.J.A. Dhert2, J. Malda2 1Southampton/United Kingdom, 2Utrecht/Netherlands

Purpose: The filter culture is a frequently used three-dimensional (re-)differentiation model to investigate chondrocyte behavior in cartilage research. In this model, expanded chondrocytes are redif-ferentiated on collagen coated filters to form a cartilaginous tissue over time. The understanding of the governing processes involved in the formation of such a cartilaginous multi-layered structure is limited. Therefore, this study aims to provide further insight into these processes by the means of an applied mathematical approach. Methods and Materials: Filter cultures were generated using ex-panded adult equine chondrocytes and maintained for up to seven weeks. Samples were processed for histology and image analysis was performed. In addition, glycosaminoglycan (GAG) and DNA con-tent were determined biochemically. A mathematical model was then constructed based on the analysed data. This consisted of partial differential equations explaining the distribution of cells and GAG, as well as the transport of nutrients and overall height of the tissue. Results: The generated tissue in the filter cultures had a glassy hya-line appearance and tissue height increased at a constant rate over a seven week culture period. GAG content of the cultures increased uniformly, although considerable release of GAG was observed. Af-ter an initial settling period for cells, growth was restricted to a re-gion in the upper half of the tissue whilst the density of cells on the filter membrane and top surface remained constant (Figure 1). The mathematical model showed that cell behaviour and consequently growth, was restricted to the concentration of nutrients within a cell’s local environment. Conclusions: Redifferentiation of chondrocytes in the filter cul-ture system resulted in the formation of cartilaginous tissue. The developed mathematical model could predict the experimental observations and revealed that the cell’s behaviour is dependent on its position within the tissue structure, as well as on the local nutrient concentration.

9.2.4A novel 3D tissue assembly approach for cartilage tissue engineering applicationsS. Stroebel1, J. Malda2, T.B.F. Woodfield1

1Christchurch/New Zealand, 2Utrecht/Netherlands

Purpose: Cell-scaffold based cartilage tissue engineering strategies provide the potential to restore long-term function of damaged articu-lar cartilage. A major hurdle in such strategies is the adequate (uniform and sufficient) population of porous 3D scaffolds with cells. We de-scribe a novel approach to engineer cartilage grafts using pre-defined micro-mass cartilage pellets, integrated into specifically designed 3D plotted scaffolds. Methods and Materials: Expanded (P2) human nasal chondrocytes (HNCs) from 3 donors (age 47-62 years) were micro-mass cell pellet cul-tivated at 5x105 cells/pellet for 4 days. Subsequently, micro-mass pel-lets were integrated into 3D plotted poly(ethylene glycol)-terephtha-late/poly(butylene)-terephthalate (PEGT/PBT) scaffolds with 1mm fibre spacing. Constructs were cultured dynamically in spinner flasks for a to-tal of 21 days. As a pellet-free control, expanded HNCs were spinner flask seeded into PEGT/PBT fibre plotted scaffolds. Constructs were assessed via histology (Safranin-O staining), biochemistry (glycosaminoglycan (GAG) and DNA content) and collagen type I and II mRNA expression. Results: After 4 days, micro-mass cultured pellets could be success-fully integrated into the fibre plotted scaffolds. DNA content of pellet integrated constructs was 4.0-fold±1.3 higher compared to single seed-ed constructs. At day 21, cartilage tissue was uniformly distributed throughout pellet integrated scaffolds, with minimal cell necrosis ob-served within the core. GAG/DNA and collagen type II mRNA expression were significantly higher (2.5-fold±0.5 and 3.1-fold±0.4 respectively) in pellet versus single cell seeded constructs. Furthermore, compared to single cell, the pellet seeded constructs contained significantly more total GAG and DNA (1.6-fold±0.1 and 3.1-fold±1.0 respectively). Conclusions: We developed a novel 3D tissue assembly approach for cartilage tissue engineering which significantly improved the seeding efficiency, as well as tissue uniformity and integrity compared to more traditional seeding approaches using single cell suspensions. Further-more, the integration of micro-mass cell pellets into 3D plotted PEGT/PBT scaffolds significantly improved the amount and quality of tissue engineered cartilage.

9.2.5Stem cell surface marker SSEA-4 levels correlate with enhanced chondrogenic potential of cultured human osteoarthritic chondrocytesK. Schrobback1, T.B.F. Woodfield2, R. Crawford1, D.W. Hutmacher1, T. Klein1

1Brisbane/Australia, 2Christchurch/New Zealand

Purpose: One important challenge for cartilage tissue engineering is to produce a clinically relevant number of cells with consistent chondrogenic potential. In vitro expansion of autologous chondrocytes results in a het-erogeneous population of dedifferentiated cells and variable amounts of chondroprogenitors. Identification and isolation of chondroprogenitors could lead to more consistent cartilage formation. Unfortunately, reliable chondroprogenitor markers have not been identified. Therefore, we eval-uated SSEA-4, a marker of embryonic and mesenchymal stem cells, as a potential chondroprogenitor marker in cultured human chondrocytes. Methods and Materials: Osteoarthritic cartilage was obtained from four consenting patients (age >60yrs.) undergoing knee replace-ment. Chondrocytes were isolated from different sites of the joint (macroscopically normal [ICRS grade 0-1] and degenerated [grade 2-4]) and zones (“normal” superficial and middle/deep) and ex-panded in monolayers. Immunophenotype was determined by flow cytometry at passage 3. Propagated cells from two donors were red-ifferentiated in pellet cultures over four weeks. Glycosaminoglycan (GAG) and DNA contents were quantified biochemically and expres-sions of aggrecan, collagen II, I and X were determined by qRT-PCR. Results: Flow cytometry revealed heterogeneous expression of SSEA-4 in CD44+/CD45-/CD105+ chondrocyte populations (A). We observed no differences in surface marker expressions between cells from dif-ferent sites (B). However, the percentage of SSEA-4-positive cells after expansion correlated positively with expression levels of chondrogenic markers aggrecan and collagen type II at day 7, and GAG/DNA levels at day 28 of pellet culture. Further, SSEA-4 levels were inversely cor-related with expression of hypertrophic marker collagen type X (C). Conclusions: We report for the first time that subpopulations of hu-man osteoarthritic chondrocytes express the stem cell surface antigen SSEA-4 during in vitro propagation. Our observations suggest that ex-pression levels of SSEA-4 can be predictive of the cell’s chondrogenic differentiation capacity. Ongoing research is focussed on the cellular characterisation of SSEA-4-positive cells to confirm their superior chondrogenic potential in vitro and in vivo.

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9.2.6Polyglycan promotes the intrinsic damage-repair response of chondrocytes in equine articular cartilage explants.A. Getgood1, L. Barr1, D. Caborn2, N. Rushton1, F.M. Henson1 1Cambridge/United Kingdom, 2Louisville/United States of America

Purpose: The aim of this study was to investigate the effect of polyg-lycan (PG) (hyaluronic acid, N-acetyl glucosamine, chondroitin sul-phate solution) on the intrinsic damage-repair (IDR) response in ar-ticular cartilage explants following single impact load (SIL) in-vitro. Methods and Materials: Equine articular cartilage explants, with-out subchondral bone, had a SIL of 500g applied from a height of 2.5cm. Explants were then cultured in DMEM in the presence of 10%, 5%, 2%, 1% and 0% PG compared to 50ng/ml FGF-2 as a posi-tive control for up to 28 days. Unimpacted discs served as negative controls. Histological and immunohistochemical techniques were used to quantify and characterise the chondrocyte response to damage and to the effect of each of the treatments. Results: Evidence of an IDR was seen in all impacted discs (Fig.1). This was significantly upregulated by 1% PG compared to FGF-2 and untreated controls, particularly by day 28 (p<0.05); large amounts of chondrocyte repair cells noted on the cartilage surface (Fig.2). Within the cartilage both type II collagen and pericellular type VI collagen was seen to by upregulated following SIL, and maintained with the addition of 1% PG. The most striking finding was a significant reduction in dead cells (as detected using TUNEL staining) with 1% PG (15%) compared to the SIL control group (50%) (p<0.05). This was not seen in the higher concentrations of PG. Conclusions: This study again confirms the potential ability for chondrocytes to migrate to an area of injury and mount an IDR fol-lowing SIL. The addition of 1% polyglycan has been shown to upreg-ulate the IDR, to an even greater degree than FGF-2 and to maintain type II and VI collagen within the cartilage. The addition of polygly-can to synovial joints following impact injury may have protective effects by stabilising the extracellular matrix and preventing chon-drocyte apoptosis thereby preventing early degenerative change.

9.2.7Redifferentiation in Hyaff scaffolds affects the chondrogenic potential in vivoC. Brantsing1, H. Stenhamre1, S. Concaro1, M. Brittberg2, A. Lindahl1 1Göteborg/Sweden, 2Kungsbacka/Sweden

Purpose: It has been shown that the second generation of autolo-gous chondrocyte implantation (ACI) has great potential in order to regenerate cartilage injuries. The purpose of this study was to evaluate if the degree of redifferentiation of in vitro cultured scaf-folds could have an impact on neocartilage in vivo in a mice model. Further, the signaling pattern occurring during the in vitro chondro-genesis was studied as well as potential chondrogenic markers. Methods and Materials: Adult human chondrocytes were seeded into Hyaff scaffolds and cultured for 1, 7, 14 and 21 days in chondro-genic media and then implanted subcutaneously in Balb C nu/nu mice model. The constructs were further assessed by histological, immunohistochemical and molecular methods Results: The in vitro culture revealed that longer culture time in redifferentiation culture gave a more mature tissue. The gene ex-pression during the in vitro showed an uppreglation of Collagen IIA, Collagen IIB, Aggrecan, Sox 9, SerpinaA3 and COMP. Whereas Col-lagen I, Versican, Cathepsin B and EGR1 were down regulated. The markers Tenacin C and Fibronectin increased the first days of cul-ture and were then down regulated during the further maturation of the chondrocytes. The in vivo experiments reveal that in vitro redifferentitaion is needed for further maturation.Conclusions: The results indicate that a redifferentiation of in vitro expanded human articular chondrocytes is required to obtain good quality redifferentiation in vivo. Furthermore the results show that the chondrocytes cultured in Hyaff scaffolds express a signaling pattern similar to the one seen during the chondrogenesis during fetal development. Furthermore, the results might indicate that Cathepsin B, EGR1 and SerpinA3 are potential markers to monitor the differentiation status of 3D tissue engineered constructs.

9.2.8The tumor gene survivin is expressed in primary human chondrocytes – a novel mechanism of cartilage protection and regeneration?P. Lechler1, S. Doukas1, T. Renkawitz1, S. Anders1, J. Grifka1, S. Balakrishnan2, R.H. Straub3, J. Schaumburger1

1Bad Abbach/Germany, 2London/United Kingdom, 3Regensburg/Germany

Purpose: The smallest member of the inhibitor of apoptosis (IAP) gene family survivin is considered to be of critical importance for regulation of mitosis and maintenance of cell viability. As such it is essential for embryonic development and highly relevant in cancer. Little knowledge exists whether survivin has any physiological or pathophysiological role in adult differentiated tissues. Methods and Materials: Survivin expression in primary human chondrocyte cultures was studied by immunoblotting and real-time PCR. Subcellular localization was visualized by immunofluorescence. Knock-down and over-expression experiments were performed to study survivin function, i.e. cell cycle and apoptosis assays. Results: Survivin mRNA and protein is expressed in primary human chondrocytes. Subcellular localization of survivin protein during interphase showed a predominantly cytoplasmic signal, while in-volved in the formation of the spindle apparatus during mitosis. RNA-interference targeting survivin resulted in a G2/M arrest of the cell cycle and increased the rate of apoptosis in cartilage cultures. Over-expression of wild-type survivin in primary human chondrocytes in-creased rates of proliferation and cell viability. During subculturing of primary chondrocytes, growth stimulatory effects of TGF-beta in early passages were associated with an up-regulation of survivin. Conclusions: In summary, we demonstrate for the first time that survivin is expressed in primary human chondrocytes on mRNA and protein level. Functional analyzes indicate that survivin is an important factor for cell division and cell viability in primary chon-drocytes. In contrast to the reported biologic effect of TGF-beta sig-naling on malignant cells, in primary human chondrocytes TGF-beta treatment resulted in a survivin dependent stimulation of prolifera-tion. Modulation of survivin gene expression should be carefully evaluated for interventional strategies in cartilage protection and tissue regeneration.

9.2.9Beta-1 integrins are crucial for structural organization of the articular cartilage but dispensable for chondrocyte metabolism.A. Raducanu1, M. Stolz2, I. Drosse3, E. Hunziker4, A. Aszodi1 1Martinsried/Germany, 2Southampton/United Kingdom, 3München/Germany, 4Bern/Switzerland

Purpose: To explore the role of beta-1 integrins in articular cartilage (AC) and joint function, we generated mice in which the floxed beta-1 integrin gene was ablated in early limb bud mesenchymal cells using the Prx1cre transgene.Methods and Materials: AC of mutant and control mice was exam-ined by brightfield, confocal and electron microscopy. Chondrocyte apoptosis and proliferation were evaluated by TUNEL assay and PCNA immunohistochemistry (IHC), respectively. The expression of matrix proteins, proteases, degradation products and signaling molecules were investigated by IHC and/or ELISA assays. Surface topograpy and AC mechanical properties were assessed by indentation-type atomic force microscopy (IT-AFM).Results: The mutant AC was structurally disorganized accompanied by defects in chondrocyte proliferation, survival and terminal differ-entiation. Electron micrographs revealed disorganized layers of col-lagen fibrils in the superficial zone. IT-AFM showed thickened fibrils, surface roughening and decreased nanostiffness of mutant samples. No significant differences in cartilage degradation, in the expression of proteases or in the exposure of matrix degradation neoepitopes were observed between genotypes. We found no evidence for disturbed activation of MAP kinases in beta-1-null chondrocytes. Mutant mice exhibited abnormal gait, reduced flexibility of the knee joints and de-creased mobility. We frequently observed chondrification of the syn-ovium and the large vessels. A mouse line with incomplete deletion of the beta-1 integrin gene exhibited normal motility and joint usage. The AC of these mice showed mild structural disorganization, soft-ening and increased destruction compared to the AC of control mice. Conclusions: Our study indicates that 1) the lack of beta-1 integrins on AC chondrocytes has no obvious impact on cartilage homeostasis; 2) beta-1 integrins are pivotal for proper development and structural organization of joint tissues; 3) structural changes lead to softer AC; 4) softening of the AC increases OA severity only upon mechanical loading; 5) beta-1 integrin signaling is not a good candidate for inter-vention in osteoarthritis.

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9.3.2Expression patterns of proinflammatory cytokines and matrix metalloproteinases before and after autologous chondrocyte transplantation (MACT) in a leporine animal model: The influence of varying in-vitro settingsM. Sauerschnig1, G.M. Salzmann2, M. Berninger1, T. Sütfels1, M. Schönfelder1, S. Vogt1, P.B. Schöttle1, A.B. Imhoff1 1München/Germany, 2Freiburg/Germany

Purpose: We hypothesize that mRNA-expression of TNF-α, IL-1β, MMP-1 and MMP-3 before and after matrix assisted chondrocyte transplantation (MACT) could be influenced by variations of the in-vitro parameter cell-count, cell-passage and time-on-matrix. Methods and Materials: Chondrocytes of post puberty New Zea-land White Rabbits were harvested from articular cartilage, cul-tured under varying combinations of cell-passage (P1,P2,P3) and cell-count (C: 2x105/matrix=C1, 1x106/matrix=C2, 3x106/matrix=C3)

and seeded on 3D-matrices (Chondrogide, Geistlich) with varying time-on-matrix (T: 24 hours=T1, 2 weeks=T2) forming 18 different groups. Each time two identical cell-seede-matrices (CSM) where assembled of which one (CSM-i) was analyzed for TNF-α, IL-1β, MMP-1 and MMP-3 via qPCR directly prior to autologous press-fit-implantation of the duplicate (CSM-e) into cartilage defects of the trochlear (n=2/animal; contralateral to harvest-site knee). After 12 weeks CMS-e were explanted and expression of the parameters were assayed again using qPCR. Data were statistically compared using a mixed linear model, multiple regression analysis. Signifi-cance was set P<.05 for all tests. Results: CSM-i showed higher expression of MMP-1, MMP-3 and IL-1β compared to CSM-e while expression of TNF-α was found higher in CSM-e. General linearity between CSM-i and CSM-e values was found. Expression levels of all parameters showed significant dif-ferences between all groups in CSM-i while in CSM-e groups this was found true for MMP-3 only. Comparing CSM-i to CSM-e the difference for all parameters was significant. MMP-1 and MMP-3 showed stronger expression at T2 combined with P1 while different C took mild effect. IL-1β expression increased with higher P and T while for TNF-α the reverse was true.Conclusions: Results demonstrate expression-levels of cytokines and proteinases within regenerate-tissue to be influenced by vary-ing in-vitro-settings and offer routing of inflammatory processes by optimizing cell-culture and seeding.

9.3.3The combination of IL-4 and IL-10 protects against blood-induced cartilage damageM.E.R. van Meegeren, N.W.D. Jansen, G. Roosendaal, F.P.J.G. Lafeber Utrecht/Netherlands

Purpose: Blood-induced cartilage damage can occur after a joint trauma, during or after major joint surgery, or in haemophilia pa-tients. We have previously shown that interleukin (IL)-10 limits in vitro blood-induced cartilage damage. In addition, IL-4 has been suggested to have cartilage protective properties. Hence our aim was to study whether IL-4 can support IL-10 in the prevention of blood-induced damage.Methods and Materials: Human and canine articular cartilage ex-plants were cultured for 4 days in the presence or absence of 50% v/v homologous blood. IL-4, IL-10, or a combination of both was added in a concentration of 10 or 100 ng/ml during blood exposure. Cartilage matrix turnover was determined 12 days later.Results: Human cartilage cultured in the presence of blood showed a decrease of proteoglycan synthesis rate of 70% and an increase of proteoglycan release of 60%, resulting in a decrease of proteoglycan content of 20% (p<0.05). This blood-induced damage of the carti-lage matrix was limited by IL-4 in a similar way as IL-10 does: proteo-glycan synthesis rate was approximately 50% enhanced compared to exposure to blood and the release was 50% reduced (p<0.05, dose-dependently). The combination of the two cytokines was even more protective against damage caused by blood. This was espe-cially evident for the proteoglycan synthesis rate which showed a twofold increase (p<0.05). Limiting blood-induced cartilage dam-age by IL-4 and IL-10 was also confirmed in canine cartilage.Conclusions: Besides IL-10, as shown previously, also IL-4 protects against blood-induced cartilage damage. The combination of these two cytokines is clearly the most protective. This justifies further evaluation of the combination of IL-4 and IL-10 in prevention and treatment of blood-induced cartilage damage.

9.3.4Platelet-Rich Plasma intra-articular injections versus viscosupplementation as treatment for early osteoarthritis: a comparative studyE. Kon1, B. Mandelbaum2, R. Buda1, G. Filardo1, M. Delcogliano3, A. Timoncini1, A. Di Martino1, S. Patella1, S. Giannini1, M. Marcacci1

1Bologna/Italy, 2Santa Monica/United States of America, 3Roma/Italy

Purpose: Aim of this study is to evaluate and compare the efficacy of Platelet Rich Plasma (PRP) and Viscosupplementation (HA) i.a. injections for the treatment of severe chondropathy of the knee.Methods and Materials: The study involved 150 patients affected by chondropathy and either early stage or severe osteoarthritis. Fifty symptomatic patients were treated with 3 autologous Platelet-Rich Plasma (PRP) intra-articular injections and evaluated prospec-tively. All patients were clinically evaluated at the enrolment, after the treatment and at 6 months follow up. The results were also com-pared with two homogeneous groups of patients treated by HA in-jections in two different centers (High Molecular Weight Hyaluronan in one group, Low Molecular Weight Hyaluronan in the other). IKDC and EQ-VAS scores were used to clinically evaluate the patients, while their satisfaction and functional status were recorded.Results: Neither complications nor other major adverse events oc-curred; only some minor adverse events were detected, as mild pain reaction and effusion after the injections, but they just lasted for a couple of days maximum. At the follow-up evaluations, all groups showed a significant improvement in terms of function and quality of life. The comparison between the outcomes of the three groups showed a statistically significant difference (p<0.05), reporting a superiority of the PRP group results.Conclusions: PRP is a simple, low cost and minimally invasive ap-proach to osteoarthritis; it leads to a natural concentrate of autolo-gous growth factors directly from the blood. Our clinical results are encouraging and suggest PRP may be used to treat the degen-erative articular pathology of the knee. Autologous PRP injections demonstrated a longlasting and better efficacy than HA injections in recovering articular function and reducing symptoms in patients affected by knee degeneration. Long-term and randomized con-trolled studies will be needed to confirm the reliability and evaluate the durability of this promising procedure.

9.3.5Comparing the chondroprotective capability of curcumin (Curcuma longa) and curcumin derivatives against IL-1β-mediated chondrolysisM.J. Goldstein1, N.V. Shah1, R. Greenwald1, P. Razzano1, L. Golub2, D. Grande1 1Manhasset/United States of America, 2Stony Brook/United States of America

Purpose: The pro-inflammatory cytokine interleukin-1β (IL-1β) stimulates articular chondrocytes to produce matrix-degrading enzymes, contributing to the pathogenesis of osteoarthritis (OA) by promoting lysis of extracellular matrix (ECM) compounds. Tur-meric-derived curcumin (Curcuma longa) has demonstrated inhi-bition of inflammatory signaling pathways activated during IL-1β stimulation. However, poor solubility and rapid metabolism yields low plasma and tissue levels of curcumin. In this study, we attempt to identify an improved chondroprotective agent against IL-1β-mediated chondrolysis by comparing several novel curcumin de-rivatives. We hypothesize that a curcumin derivative will enhance chondroprotection compared to curcumin.Methods and Materials: Full-thickness articular cartilage cores were isolated from femoral condyles of bovine knee joints. Car-tilage explants were pulse-labeled with S-35 sulfate for 24 hours and separated into seven experimental groups: negative control (growth media alone/D-MEM), positive control (D-MEM+IL-1β, 10 ng/ml), curcumin (10 µM) + IL-1β, and four novel curcumin deriva-tives (10 µM) + IL-1β, labeled H1.1, H1.2, H2.5, and H2.7. Degree of S-35 release was evaluated at 24, 48, and 72 hours post-treatment. Following each time-point, supernatant media was scintillation-counted. After 72 hours, explants were lyophilized to obtain dry weights (mg). Counts per minute (CPM)/mg dry weight was calcu-lated; mean and standard deviation for each group were derived for statistical analysis.Results: Media-alone controls (negative controls) displayed a baseline level of S-35 release throughout the 72-hour period. IL-1β-challenged explants (positive controls) demonstrated an early release of S-35 significantly higher than that of negative controls at all time-points. Curcumin derivatives, specifically H2.5 and H2.7, exhibited significant chondroprotective effects compared to other derivatives at all time-

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points. Interestingly, native curcumin displayed a similar low release; however, this is likely attributed to toxicity issues, not chondroprotec-tive mechanisms based on most recent data.Conclusions: Results demonstrate improved chondroprotection by novel derivatives against IL-1β-induced chondrolysis, compared to no treatment. Curcumin derivatives that improve bioavailability and solubility may further enhance chondroprotective capability.

9.3.6Lack of disease modifying activity of celecoxib in osteoarthritis: a randomized clinical trialS.C. Mastbergen1, T.N. De Boer1, A.M. Huisman2, A.A. Polak2, J.W.J. Bijlsma1, F.P.J.G. Lafeber1 1Utrecht/Netherlands, 2Rotterdam/Netherlands

Purpose: Treating human osteoarthritic (OA) cartilage in vitro and ex vivo demonstrated a direct chondroprotective (DMOAD) effect of celecoxib (celebrex), one of the selective COX-2 inhibitors. The present study aims to validate these findings in a blinded random-ized in vivo (clinical) study, treating patients with severe knee os-teoarthritis prior to joint replacement surgery with subsequent ex vivo detailed evaluation of joint degeneration.Methods and Materials: Patients (n=172) with knee osteoarthritis were randomized and treated for at least 4 weeks prior to knee replace-ment surgery with celecoxib 2dd200mg, celecoxib 2dd200mg stopped 3 days prior to surgery, naproxen 2dd250mg, or received no treatment. WOMAC questionnaires were used to evaluate pain, stiffness and func-tion before and after treatment. Cartilage and synovial tissue were col-lected during replacement surgery and analyzed in detail ex vivo.Results: Age, gender, weight (BMI) and cartilage damage (X-ray, macro-scopic or histological) was not different between the treatment groups. In contrast to all previous findings celecoxib treatment showed no effect on the proteoglycan release, the primary outcome, of the cartilage com-pared to the untreated group. Also naproxen showed no effect. Similar lack of effect was found for proteoglycan content compared to the no-treatment group. Prostaglandin-E2 and nitric oxide (NO) produced by the cartilage showed no changes compared to the no-treatment group. However celecoxib treatment showed an improvement of WOMAC-pain (p<0.01) compared to the no-treatment group. The ex vivo release of IL-1β, TNFα, prostaglandin-E2 and NO by the synovial tissue only shows a statistical significant decrease in NO levels (p<0.04) in the celecoxib treated group compared to the no-treatment group. Conclusions: Despite an improvement in WOMAC score no DMOAD effect of celecoxib could be demonstrated in this blinded randomized well-powered study.

9.3.7Treatment of painful bone marrow edema syndrome with intravenous bisphosphonatesC. Bartl1, G.M. Salzmann2, A.B. Imhoff3, R. Bartl3

1Ulm/Germany, 2Freiburg/Germany, 3München/Germany

Purpose: The aim of the study was to assess the efficacy of intrave-nous bisphosphonates (Ibandronate and Zoledronate) in the treatment of bone marrow edema (BME) syndrome around the knee and ankle in a prospective, randomised MRI-controlled off-label use study. Methods and Materials: 30 patients with an ankle-BME and 40 patients with a knee-BME were randomised to receive either Ibandronate-infu-sion (3x6 mg – group G1) or zoledronate-infusion (1x 5mg – group G2) in an outpatient setting following BME diagnosis by MRI. All patients gave written informed consent and agreed to participate in the study. Patients were followed-up clinically at baseline, 1, 3 and 6 months after the first infusion using the Mazur-foot score, the Larson-knee score and a VAS pain scale. MRI (T1-, T2- and STIR-images) was performed before and 3 month after the first infusion. Results: The mean VAS pain-score decreased from 8.1 at baseline to 2.3 at 6 months in G1 (7.9 to 2.1 in G2 respectively – each p<.05). Both the Mazur (Ankle) and the Larson-score (Knee) increased significantly from baseline to 6 months (Mazurscore: av. from 57p. to 89p- no sig. diff be-tween G1 and G2 – each p<.05)( Larsonscore: av. from 52p. to 90p- no sig. diff between G1 and G2 – each p<.05). MRI revealed a significant reduction of BME-size or complete normalization in 70% of the cases in both groups. Acute phase reactions with flue-like symptoms were higher in group G2 (G1:14% - G2:23%). We did not observe cases of kid-ney dysfunction or osteonecosis of the jaw during the study period. Conclusions: Both intravenous bisphosphonates (Ibandronate and Zoledronate) were very effective for the treatment of BME in the knee and ankle. After the infusion therapy the majority of patients showed a fast regain in function and the infusion therapy seems to shorten the natural course of the disease.

9.3.8Overexpression of SIRT1 inhibits osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytesH. Sasaki, T. Matsushita, R. Kuroda, K. Takayama, K. Ishida, N. Fujita, S. Kubo, T. Matsumoto, M. KurosakaKobe/Japan

Purpose: Interleukin-1β (IL-1β) has been suggested to be involved in the pathogenesis of osteoarthritis (OA) by increasing catabolic enzymes or enhancing apoptosis. Recently it has been reported that SIRT1, a mammalian homologue of longevity factor sir2, inhib-its apoptosis and promotes survival of human chondrocytes, sug-gesting that SIRT1 plays a protective role in human chondrocytes. Nevertheless, protective roles of SIRT1 in human chondrocytes are not yet fully understood. In this study, we examined the effect of overexpression of SIRT1 on IL-1β-treated human chondrocytes to explore protective roles of SIRT1 in human chondrocytes. Methods and Materials: Normal Human Articular Cartilage-knee (NHAC-Kn) was used as normal human chondrocytes. NHAC-kn was transfected with either control plasmid or SIRT1 expression plas-mid by a lipofection technique. After the transfection, NHAC-Kn was treated with 10ng/ml IL-1β for 24 hours. The effects of IL-1β treatment and the overexpression of SIRT1 were examined by real-time PCR and western blotting.Results: The effect of SIRT1 overexpression was confirmed by the increased SIRT1 protein, decreased acetylated P53 and decreased acetylated p65 (a member of NF-kB ) in the SIRT1-plasmid-trans-fected chondrocytes. The stimulation of IL-1β upregulated MMP-13 and ADAMTS-5 while downregulated aggrecan and COL2A1. The overexpression of SIRT1 inhibited the upregulation of MMP-13 and ADAMTS-5 caused by the stimulation of IL-1β. On the other hand, the overexpression of SIRT1 inhibited the downregulation of aggre-can and COL2A1 caused by the stimulation of IL-1β. In addition, the overexpression of SIRT1 alone significantly increased the expres-sion of aggrecan and COL2A1. Conclusions: SIRT1 overexpression counteracted against the up-regulations of expressions of catabolic factors induced by IL-1β while promoted cartilage extracellular matrix gene expressions. Our observations suggested that SIRT1 can play a protective role for chondrocytes and SIRT1 overexpression might be a new thera-peutic approach for OA.

9.3.9Initial Phase 1 safety of retrovirally transduced human chondrocytes expressing TGF-β1 (TG-C)C. HaSeoul/Korea, Democratic People‘s Republic of

Purpose: TissueGene-C (TG-C) represents a cell-mediated gene therapy for localized delivery of allogeneic chondrocytes express-ing TGF-β1 directly to the damaged knee joint. Untransduced hu-man chondrocytes (hChonJ cells) have also been incorporated into the TG-C product in a 3:1 ratio with TGF-β1 expressing chondrocytes (hChonJb#7) in order to help fill in the defect and as target cells for the actions of the expressed TGF-β1. A Phase 1 dose escalating clinical trial was performed to evaluate the safety and biological activity of TG-C in patients with advanced osteoarthritis of the knee joint (full thickness cartilage defect) that was refractory to existing nonoperative therapies.Methods and Materials: Following a single intraarticular injection into the joint space of the damaged knee, patients were monitored for safety, and an evaluation was performed to assess the pharma-cokinetics and biological activity of TG-C.Results: There were no treatment related serious adverse events. Swelling, effusion and minor localized reactions such as warming sensation or itching were observed in a dose-dependent manner at the injection site. MRI results and knee evaluation scores seem to indicate a dose-dependent trend toward efficacy; however patient numbers were not sufficient to determine statistical significance. Conclusions: Overall, there were no significant safety issues re-lated to the administration of TG-C, with only some minor injection site reactions observed. Additionally, the preliminary data from the MRI and knee scroring analyses indicate a possibility that TG-C may contribute to regeneration of articular cartilage and/or improve-ment of arthritic symptoms. More study is warranted to further evaluate the safety and determine the potential efficacy of TG-C.

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9.4.2Mechanically Induced in vitro Chondrogenesis under Simulation of Different Chondrocyte Implantation TechniquesN. Wang1, S. Grad1, M. Stoddart1, M. Alini1, N. Sudkamp2, G.M. Salzmann2

1Davos/Switzerland, 2Freiburg/Germany

Purpose: Advanced cell-based cartilage regeneration techniques are represented by three generations of autologous chondrocyte implantation (ACI) procedures, which aim to deliver new cells ca-pable of chondrogenesis into the lesions. This study investigated the effect of dynamic compression and sliding surface motion on chondrogenesis in a joint-specific bioreactor to mimic and compare the performance of first and third generation ACI (matrix-assisted chondrocyte transplantation, MACT) in vitro. Methods and Materials: Primary (P0) or third passage (P3) bovine chondrocytes were seeded in fibrin-polyurethane scaffolds. Con-structs were cultured either free swelling, under mechanical loading for 2 or 4 weeks, or free swelling for 2 weeks followed by 2 weeks of loading (F2L2 group). The latter group simulates the MACT method, while the directly loaded group mimics conventional ACI. Samples were collected for DNA and glycosaminoglycan (GAG) quantifica-tion, expression of chondrogenic marker genes, and histology. Results: Mechanical loading stimulated GAG synthesis, although a significant proportion of GAG was released from loaded scaffolds into the medium. The F2L2 group retained more GAG in scaffolds than the immediately loaded group. Generally, P0 chondrocytes ap-peared more responsive to load than P3 cells. Chondrogenic gene markers, including collagen II, aggrecan, COMP, and lubricin, were up-regulated after loading in both P0 and P3 chondrocytes; the F2L2 group demonstrated most favorable chondrogenic gene expression results. Load also increased matrix staining, while most intense staining was observed in the F2L2 group of P0 chondrocytes. Conclusions: Different duration and mode of mechanical load can increase matrix synthesis and chondrogenesis. Our data indicate that free swelling culture followed by loading (simulating in vivo MACT) may be most favorable for chondrogenesis. It is likely that a pre-existing extracellular matrix improves the responsiveness of the chondrocytes to mechanical stimuli. This in vitro study suggests that MACT procedures may be preferred, while primary cells show better responsiveness regarding matrix synthesis than passaged cells.

9.4.3Chitosan-Glycerol Phosphate/Blood Implants Alter Maturation and Location of Chondrogenic Foci in Marrow Stimulated Cartilage RepairA. Chevrier1, C.D. Hoemann1, J. Sun2, M.D. Buschmann1 1Montreal/Canada, 2Laval/Canada

Purpose: Bone-marrow stimulation initiates cartilage repair through the formation of chondrogenic foci. Previous studies have shown that chitosan-glycerol phosphate/blood implants improve the quantity and quality of bone marrow derived cartilage repair. The purpose of this study was to characterize these chondrogenic foci and to investi-gate the effects of chitosan-GP/blood implants on their formation.Methods and Materials: Thirty-three New Zealand White rabbits re-ceived bilateral cartilage defects with four microdrill holes. One knee per rabbit was treated by applying a chitosan-glycerol phosphate/blood implant over the defect. Cell morphology and distribution of glycosamin-oglycan and collagen type II, I and X were characterized. Chondrogenic markers and proliferation were identified with Patched and Ki-67.Results: Chondrogenic foci were categorized as nascent (small, ho-mogenous, GAG and col II positive), mature (larger, col II positive, stratified cell morphology) or resorbing (extensive vascular invasion and endochondral bone formation). In mature foci, glycosaminogly-can and collagen type II were present throughout while the upper zone expressed collagen type I and the lower zone collagen type X (Fig 1). Patched and proliferating cells were immunodetected within foci (Fig 1). Treatment with chitosan-glycerol phosphate/blood im-plants led to three important and statistically significant modifica-tions of chondrogenic foci: 1) the initial appearance of foci occurred later (Day 21 for treated versus Day 14 for control) 2) more nascent and mature foci were produced than resorbing and 3) foci were closer to the articular surface (Fig 2).Conclusions: Chondrogenic foci bear some similarities to growth car-tilages and can give rise to a repair tissue that has similar zonal strati-fication as articular cartilage. Treatment with chitosan-GP/blood implants produce a greater fraction of nascent and mature foci that are closer to the articular surface and thereby contribute to a more effective cartilage repair.

9.4.4Presolidified chitosan-based implants for osteochondral repair in sheepA. Bell1, M. Lowerison1, J. Sun2, V. Lascau-Coman3, M.B. Hurtig1, C.D. Hoemann3 1Guelph/Canada, 2Laval/Canada, 3Montreal/Canada

Purpose: To explore the use of novel pre-solidified chitosan formu-lations as therapeutic implants for osteochondral repair, using Jam-shidi bone biopsy as an alternative marrow stimulation approach.Methods and Materials: Three chitosan formulations (10kDa, 40kDa and 150kDa MW) were mixed with autologous ovine blood to create pre-solidified cylindrical implants. Under general anaesthesia using aseptic technique, six, 2-8 mm deep holes were made with a 2 mm di-ameter Jamshidi biopsy needle (Cardinal Health) in each medial femo-ral condyle of five sheep. Each chitosan implant was placed in dupli-cate holes while controls were left to bleed. Sheep were sacrificed at 1 (n=1), 21 (n=2) or 93 days (n=2). A correlative study was undertaken using histology images of Safranin O-stained sections and MicroCT imaging (GE Explore Locus scanner, 27µm resolution) of bone remod-elling in biopsy holes. Software was created in Python (ver 3.1.1) and R (www.r-project.org) to determine radial gradients in tissue mineral density (TMD) and bone volume fraction (BVF) from the central axis of the hole to beyond the repair tissue-bone interface.Results: A gradient of increasing BVF and TMD towards the repair tissue-bone interface was seen after 93 days with a significant dif-ference compared to day 1 (p<0.05, Fig. 1). There was a significant difference (p<0.05) in TMD and BVF between the 10kDa and 150kDa formulations inside the repair tissue-bone interface. The 10kDa chito-san treatment had the highest increase in BVF and TMD, and control defects had the lowest increase (Fig. 1). BVF beyond the repair tissue-bone interface generally decreased after 93 days. Both treated and control defects developed a mixed angiogenic granulation-cartilage repair tissue after 93 days (Fig. 2). Conclusions: These results highlight the potential use of chitosan-based implants to induce subchondral bone repair of Jamshidi-gen-erated marrow-stimulation channels. Repair periods longer than 93 days will be required in this model to observe cartilage repair in treated defects.

9.4.5An In Vivo Study to Clarify Mechanisms of Spontaneous Cartilage Regeneration Induced by Implantation of a Novel Double-network Gel: Determination of the Effect of the Two Component Gels on the RegenerationM. Ogawa1, N. Kitamura2, K. Arakaki3, S. Onodera2, T. Kurokawa2, J.P. Gong2, Y. Tanaka1, Y. Takakura1, K. Yasuda2

1Kashihara/Japan, 2Sapporo/Japan, 3Naha/Japan

Purpose: We have developed a novel therapy for spontaneous carti-lage regeneration using the originally developed PAMPS/PDMAAm double-network (DN) hydrogel. This DN gel was composed of two single-network (SN) gels; poly(2-acrylamide-2-methylpropanesul-fonic acid) (PAMPS) and poly(N,N’-dimetyl acrylamide) (PDMAAm). To clarify the mechanisms for regeneration, we have evaluated the in vivo effect of these SN gels on the cartilage tissue induction in comparison with the effect of the DN gel. Methods and Materials: An osteochondral defect of 4.3-mm diam-eter was created in the bilateral femoral trochlea of the rabbit knee. The treatment groups consisted of: 1) PAMPS/PDMAAm DN gel (Group-DN); 2) PAMPS gel (Group-PAMPS); 3) PDMAAm gel (Group-PDMAAm); 4) untreated defect (Control). A cylindrical gel plug was randomly implanted into the defect so that having 2-mm depth re-mained. At 4 weeks, the regenerated tissue was stained with HE, Sa-franin-O and immunohistochemical stain (type-2 collagen), and was quantitatively evaluated with the Wayne’s grading scale. The real time PCR for type-2 collagen, aggrecan, and Sox9 was performed.Results: Group-DN was filled with the proteoglycan-rich tissue stained with Safranin-O and type-2 collagen. Group-PAMPS was filled with hyaline/fibrocartilage tissue inhomogenously, while Group-PDMAAm and Control were filled with the fibrous and bone tissues. Wayne’s scores were significantly higher in Group-DN and Group-PAMPS than in Group-PDMAAm and control. The histology score was significantly higher in Group-DN than in Group-PAMPS. Each mRNAs were highly expressed in Group-DN and Group-PAMPS. Conclusions: The present study demonstrated that the negatively charged PAMPS gel may play an important role in induction of the cartilage regeneration, although the degree of the cartilage regen-eration was significantly greater in Group-DN than in Group-PAMPS. We speculate that the unique mechanical properties of the DN gel improved by the PDMAAm gel enhanced the effect of the PAMPS gel on spontaneous cartilage regeneration.

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9.4.6Testing tissue time dependant mechanical properties by AFM: experimental procedures and finite element modeling of soft agarose model gels and articular cartilageR. Gottardi1, E. Landini1, D. Carnelli2, M. Taffetani2, P. Vena2, R. Raiteri1

1Genova/Italy, 2Milano/Italy

Purpose: We recently showed that the atomic force microscope (AFM) can readily image articular cartilage (AC) morphology and measure its elastic properties at the nanometer scale, i.e. the scale at which bio-chemical processes occur and pathological lesions start. This opens the possibility for detecting pathological features of AC at their early stages and/or for using AFM as a quality control tool of engineered tissue (functionality and structure) prior to implantation. Aim of this study was to develop AFM-based methods for time dependant me-chanical characterization at different length scales of model materials (agarose hydrogels) and animal AC through a combined experimen-tal/numerical approach.Methods and Materials: An AFM with spherical indenter tip (r=7.5µm, k=8N/m) was used to test agarose gels (0.8%, 1.5%, 3% w/v) and na-tive and enzyme (hyaluronidase) treated bovine AC in physiological conditions. The viscoelastic response was assessed by: (a) frequency sweep (FS) - a pre-load is applied by the AFM probe followed by an os-cillation (<10% of the pre-load) over a range of hundreds of hertz; (b) stress relaxation (SR) - a step-like load is applied by the AFM probe and the progressive stress relaxation monitored; (c) creep (CR) - a step-like load is applied and the piezo displacement necessary to maintain the load constant is monitored. Results were interpreted with the aid of an axi-symmetric finite element model to simulating both preload phase and the cyclic load phase during indentation. Equivalent mass, stiffness and damping were simulated by a lumped parameter model. Poroelastic properties have been preliminarily disregarded.Results: Rate-dependant effects related to different gel concentra-tions were observed for all methods. FS comparison with numerical modelling suggests a significant influence of mechanical resonance due to wave propagation and reflexion through the gel. CR testing fol-lows the same models as millimetre scale creep testing, SR requires additional modelling to account for AFM force-based controls.Conclusions: Our preliminary findings open the possibility of using AFM-based mechanical testing, complemented by appropriate con-stitutive modelling, in the field of tissue characterization, in particular testing the functional properties and possible pathologies of both na-tive and engineered AC.

9.4.7The application of a chondroitin sulfate-bone marrow adhesive towards meniscal repairJ.A. Simson, I. Strehin, J. Elisseeff Baltimore/United States of America

Purpose: Meniscal injuries may lead to joint degeneration and the development of post-traumatic osteoarthritis. There are few tech-nologies beyond sutures available for surgeons to repair the menis-cus, and sutures inherently cause additional damage to the tissue. Adhesives that work to repair meniscus while fixing the tissue in place offer an appealing alternative solution. Here, we demonstrate the use of a NHS-functionalized chondroitin sulfate (CS-NHS) bio-adhesive for use in meniscal repair.Methods and Materials: 10% CS-NHS was mixed with 10% Polyeth-ylene Glycol (PEG) in a 1:1 ratio, and with BM in ratios of 3:7 (70% BM), 1:1 (50% BM), and 7:3 (30% BM). Meniscus cells were encapsulated in gels to quantify in vitro tissue generation, and bovine meniscus explants were glued to observe meniscus cell migration into the ad-hesive. Constructs were analyzed at one and three weeks using live/dead, H&E staining, and Hoescht dye DNA assays. Glued tissue ex-plants were sectioned and stained with H&E at two and four weeks. Results: Live/dead and Hoescht DNA assays showed statistically significantly higher viability at both time points in CS-BM gels in comparison to CS-PEG. At one week normalized DNA levels in-creased with BM concentration, but this effect diminished by week three. However, extensive clustering of cells was observed at three weeks in CS-BM gels, indicating cell proliferation. In the explant study cells were observed proliferating on the surface of 70% BM at week two, and at week four cells were seen within the gel prolif-erating and depositing matrix.Conclusions: These findings indicate that meniscal cell viability in the CS-BM gel remains high after several weeks in culture, they proliferate within the gel, and that meniscal cells are capable of migrating from meniscal tissue first onto the hydrogel surface, and later into BM gels. These are promising preliminary results for the use of CS-BM adhesive in regenerating meniscus tissue.

9.4.8Effects of combined 3D- and hypoxic culturing on cartilage-specific gene expression in human chondrocytesC.B. Foldager, A.B. Nielsen, S. Munir, M. Ulrich-Vinther, K. Søballe, C. Bünger, M. LindAarhus/Denmark

Purpose: In vitro expansion of autologous chondrocytes is an es-sential part of many clinically used cartilage repair treatments. Na-tive chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. The aim of this study was to inves-tigate conventional monolayer culturing compared to combined 3D and hypoxic culturing using quantitative gene expression analysis. Methods and Materials: Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were divided to either monolayer or scaffold culture. The scaffold was a clinically available MPEG-PLGA scaffold (ASEED). After harvesting cells for baseline investigation, the re-mainders were divided into three groups for incubation in normoxia (21% oxygen), hypoxia (5% oxygen) or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2 and 6 days after the baseline time-point respectively. Quantitative RT-PCR was performed using assays for collagen type 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyd-3-phosphate dehydrogenase rela-tive to two hypoxia stable house keeping genes. Statistics were performed using three-way ANOVA.Results: Sox9, aggrecan and collagen type 2 expression increased sig-nificantly with lowered oxygen. The expression of collagen type 2 was higher after 6 days in 3D compared to monolayer at all levels of oxygen.Conclusions: These new results suggest a combined positive effect of 3D and hypoxic culturing on cartilage-specific gene expression. The positive effects of 3D culture alone were not present until day 6, suggesting a benefit of long-term scaffold culturing for matrix-assisted chondrocyte implantation.

9.4.9Biomechanical Optimization of a Novel Polycarbonate-Urethane Meniscal ImplantJ.J. Elsner1, E. Linder-Ganz2, G. Zur2, R. Arbel3, F. Guilak4, A. Shterling2

1Tel Aviv/Israel, 2Netanya/Israel, 3Hod Hasharon/Israel, 4Durham/United States of America

Purpose: Meniscus replacement represents an unsolved problem in orthopedics. Graft availability, sizing, and complexity of repairs limit the use of allografts and may contribute to uneven distribution of load and degenerative damage. A synthetic substitute, available in a number of sizes/shapes, offers significant advantages for meniscal replacement. In this study we present a novel optimization method for meniscal implant design, and employ it in the development of a polycarbonate-urethane (PCU) meniscus implant in an ovine model.Methods and Materials: A gross structure was constructed ac-cording to MRI scans. Insertion attachments were added later to the horns (Fig.1). Samples based on this design were produced for testing. Additional modifications (geometry (surfaces), fibrous re-inforcement and fixation) were also considered. An experimental evaluation of the biomechanical performance of different designs was performed by tibial-plateau (TP) pressure distribution score (Linder-Ganz et al. 2010), reflecting on the magnitude of peak pres-sure and contact area coverage with respect to the natural menis-cus under load (45° flexion, 330N compression). Results: The all-PCU implant showed limited ability to distribute pressure. Its pressure score (37%) reflects on a small contact area (151mm2) subject to high pressures (>1.85MPa, Fig.2b). The pres-sure distribution improved when reinforcement fibers were added. A fixation tension of 20N increased the contact area (273mm2) and al-though a region of focal pressure concentration existed (Fig.2c), the pressure score increased (77%). Inspection of different pre-tension values showed that optimal pressure distribution (87%) is attained when a pre-tension of 30-50N is applied (Fig.2d). In this configura-tion, peak pressures and coverage area (1.65MPa and 310mm2) are similar to those of the natural meniscus (1.61MPa and 373 mm2). Conclusions: The current device can be used as a practical solution for patients suffering from severe meniscal injury. We conclude that peripheral reinforcement of the implant, in addition to pretension of 30-50N, can significantly improve TP pressure distributions.

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12.1.2The small heat-shock protein HSP27, shows a decreased abundance in OA and mediates IL-1b induced IL-6 secretion in human articular chondrocytesS. Lambrecht1, K.F. Almqvist1, P.C. Verdonk2, G. Verbruggen1, D. Deforce1, D. Elewaut1

1Gent/Belgium, 2Gent-Zwijnaarde/Belgium

Purpose: Based on two proteome analyses, the differential expres-sion of two small-heat shock proteins, HSP27 and alphaBcrystallin, was discovered in osteoarthritis-affected chondrocytes. Recently, we reported the differential expression and biological function of alphaBcrystallin. Based on a proteome analysis and the functional and structural relationship between alphaBcrystallin and HSP27, we further performed differential expression analysis on HSP27. We aimed to achieve further insights in the involvement of small heat-shock proteins, more specific HSP27, in the biology of chondrocytes. Methods and Materials: Western blot and real-time RT-PCR analy-sis were performed to determine the expression levels of HSP27 in healthy and OA chondrocytes cultured in alginate beads. RNA-interference mediated gene knock-down was used to explore the role of this small heat shock protein in IL-1b activated pathways by transfecting low concentrations of siRNA in cultured chondrocytes. Upon knock-down of HSP27, cells were stimulated by IL-1b and IL-6 concentrations were determined by ELISA. Results: Western blot of healthy and OA chondrocyte lysates showed a decreased abundance of HSP27 in OA. Moreover, real-time RT-PCR confirmed the differential expression at the mRNA-lev-el between chondrocytes isolated from visually intact and visually damaged zones of OA cartilage. The pro-inflammatory cytokines IL-1beta and TNF-alpha, both down regulated HP27 expression. Transfection of low concentrations siRNA in cultured chondrocytes resulted in an efficient knock down of HSP27 gene expression. This decreased HSP27 expression results in a reduced secretion of IL-6, in response to IL-1b (figure 1).Conclusions: This study adds to the evidence that small heat-shock proteins may be important mediators in chondrocyte biology during the development of OA. Our study showed the involvement of HSP27 in IL-1b induced IL-6 secretion in human articular chondrocytes.

12.1.3Effect of IL-1β on the proteome of chondrocytes derived from human osteoarthritic cartilage- a pharmacoproteomics approach for drug screeningH. Zwickl1, E. Niculescu-Morzsa1, V. Haudek2, C. Gerner2, F. Halbwirth1, M. Berger1, S. Nehrer1 1Krems/Austria, 2Vienna/Austria

Purpose: Inflammation plays a pivotal role in cartilage tissue de-struction in osteoarthritis. IL-1β, a key mediator, affects the bal-ance of biosynthesis and degradation of extracellular matrix (ECM) constituents by chondrocytes. Indeed, exuberant synthesis of ma-trix metalloproteinases and reduced expression of collagen II and aggrecan are well-known cytokine-mediated hallmarks of osteoar-thritis. In addition, IL-1β induces the production of the pain media-tor prostaglandin E2 via cyclooxygenase which decisively contrib-utes to joint dysfunction. Today’s treatment options are restricted to symptome-modifying drugs. However, analgesics or anti-inflam-matory drugs partially show just limited efficacy with respect to pain relief or cause undesirable side effects. Hence, the identifica-tion of disease-modifying and efficient symptome-modifying drugs is a main challenge in osteoarthritis research.Methods and Materials: Pharmacoproteomics is a promising ap-proach for drug screening. In order to establish a reference system for monitoring substance effects, we performed proteome profil-ing of human osteoarthritic chondrocytes. Alterations of the secre-tion performance and the metabolism of chondrocytes due to IL-1 β treatment were assessed by 2D-PAGE and shotgun proteomics combined with the mass spectrometrical identification of proteins.Results: A special focus was laid on the determination of interin-dividual differences existing per se as well as those arising due to stimulation by IL-1β. The proteomes were functionally character-ized and biomarkers/functions reflecting the cytokine effect were gathered. The resulting database was used to assess the effect of different derivatives of hyaluronic acid in order to evaluate the ap-plicability of this approach.Conclusions: Our approach enabled insights into molecular altera-tions due to cytokine and substance effects at a comprehensive systemic level. Furthermore, it is well-suited to identify “real” substance effects against the background of patient- and disease-derived heterogeneity of samples.

12.1.4Phenotypic differences between in vitro expanded articular chondrocytes and mesenchymal stem cells: a proteomic study.M. Polacek, I. MartinezTromsø/Norway

Purpose: One of the remaining conflicts in the field of cartilage re-construction is linked to the question of which cell type is more suit-able for attempting cell-based therapies or for the ex-vivo engineer-ing of cartilage implants. This study was undertaken to compare the spectrum of proteins released to the spent medium by either human articular chondrocytes (AC) or bone marrow-derived mes-enchymal stem cells (MSCs) after cell expansion for several weeks. Methods and Materials: Cartilage signature gene expression was measured by real-time PCR on AC and MSC from 6 different patients after 4 weeks of growth in monolayer cultures. Stable isotope label-ling of aminoacids in cell culture (SILAC) was applied to investigate differences between proteins secreted by chondrocytes and MSCs. Proteins in cell supernatants were resolved by 1D gel electropho-resis and analysed by LC/MS-MS. The spectrums of identified pro-teins were contrasted and the results corresponding to relevant proteins were validated by specific immunoblotting. Results: Our results show comprehensive list of proteins secreted by ACs and MSCs after 4 weeks in culture. Qualitative comparison shows that a larger number matrix components (48% vs. 42%) and growth factors (15 % vs. 11%) were released into the media by chondrocytes. However MSCs expressed lower number of matrix catabolic agents (7% vs. 10%) and higher number of matrix anabolic agents (12% vs. 5%). In addition some proteins like MMP3, Clusterin, Mimecan, Proteoglycan 4, Tenascin and Sushi (SVEP1) were expressed only by chondrocytes, while proteins like serpins, bone morphogenic protein 2 and galectins were expressed exclusively by MSCs. Conclusions: Our results show that both cell types have developed a de-differentiated phenotype, and express a similar phenotype after long term culture periods. However some differentially ex-pressed proteins could be identified in the culture supernatants. The biological relevance of the differences revealed in this study remains an area of investigation.

12.1.5The effect of cellular passage, quantity and in vitro 3-D matrix culturing time on gene expression profiles before and after matrix-assisted chondrocyte transplantation (MACT) in a leporine animal modelG.M. Salzmann1, M. Sauerschnig2, M. Berninger2, T. Sütfels2, M. Schönfelder2, P.B. Schöttle2, A.B. Imhoff2 1Freiburg/Germany, 2München/Germany

Purpose: Matrix-assisted chondrocyte transplantation (MACT) still lacks any standardization in its execution.Methods and Materials: Post puberty NZW-rabbit knee articular chondrocytes were seeded within 3-D matrices (Chondrogide, Gei-stlich, Switzerland) at different passages (P 1, 3, 5); cellular yields (C: 2x105/matrix=C1, 1x106/matrix=C2, 3x106/matrix=C3) and in vit-ro membrane-holding times (T: 24 hours=T1, 2 weeks=T2) to define 18 different groups. Each time, two cell-matrix-constructs (CMC, n=6/group) were in identical duplicate whereof one was for in vitro (CMCi) analysis directly prior to re-implantation of the other duplicate which was press-fit implanted (autologous) (CMCe) into trochlear full-thickness chondral defects (n=2/animal) of the biopsy-contral-ateral knee, reproducing a MACT situation. 12 weeks postimplan-tation the regenerates were analysed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 mRNA expression. Data were statistically compared using a mixed linear model, significance was at P<0.05 for all tests. Results: Generally, CMCi values were higher than CMCe values for all differentiation targets, while the opposite was true for dedifferenti-ation targets. There appeared a general linearity between CMCi and CMCe values to potentially predict the CMCe outcome based on CMCi quality. Typically, animals improved, target-specific, a disadvanta-geous CMCi profile into an advantegeous CMCe expression, while generally advantageous CMCi values persisted within CMCes. CMCi values were significantly different between groups for all targets analysed, while the difference was not significant for Collagen-1,-10, Aggrecan among CMCes. Usually, interacting P and T resulted in significant different results, while this was true with much lesser frequency for C. A combination of low P, of medium C and short T ap-peared to result in an optimal CMCi and CMCe outcome, while gener-ally P3, more pronounced P5 and foremost T2 (as well their combina-tion) strongly impaired the outcome, with much lesser impact of C. Conclusions: The results demonstrate that both in vitro and in vivo performance are strongly affected by cellular passage, density, membrane-holding time.

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12.1.6Grem1, FRZb and DKK1 are highly specific markers for articular cartilage which prevent hypertrophic differentiationJ. Leijten1, J. Emons2, C. Sticht3, E. Dekker4, G. Rappold4, J.M. Wit2, C. Van Blitterswijk1, M. Karperien1 1Enschede/Netherlands, 2Leiden/Netherlands, 3Heidelberg/Germany, 4Mannheim/Germany

Purpose: Chondrogenically differentiated human mesenchymal stromal cells (hMSCs) are prone to undergo endochondral ossifica-tion. Under normal circumstances, this process is limited to growth plate cartilage (GP) but not articular cartilage (AC). The molecular mechanisms that prevent AC from terminal differentiation and ossi-fication are largely unknown. Our key objectives were i) to identify genetic markers that distinguish AC from GP, and ii) to identify molec-ular mechanisms that prevent AC from endochondral ossification. We hypothesized that this information can be utilized to optimize tissue engineering strategies to obtain AC from differentiating hMSCs.Methods and Materials: Gene expression profiles of healthy AC and GP from the same patients were compared in a genome wide micro-array study.Results: We identified differential expression of 2915 genes between GP and AC, from which we generated a marker-set able to distinguish the two hyaline cartilage subtypes. Furthermore, we showed that differentiating MSCs acquire a genetic fingerprint resembling GP, but not AC. Interestingly, the three most differentially expressed genes, being enriched in the AC, are Wnt- and BMP antagonists: FRZb, DKK1 and Gremlin1. Immunohistochemistry demonstrated the presence of these antagonists throughout the entire AC. In the GP their expres-sion was mainly restricted to the resting zone. Chondrogenically dif-ferentiated hMSCs did not express the 3 antagonists and underwent hypertrophic differentiation. Remarkably, the addition of recombi-nant Grem1, FRZb or DKK1 did not affect chondrocyte differentiation but prevented their hypertrophic differentiation.Conclusions: Micro-array analysis has identified genetic finger-prints highly specific for the two types of hyaline cartilage, GP and AC, which can be used to optimize tissue engineering strategies. We have identified the Wnt- and BMP-antagonists, FRZb, DKK1 and Grem1 as specific markers for AC. We show that these antagonists prevent hypertrophic chondrocyte differentiation. Our data suggest that Wnt- and BMP-antagonists play a prominent role in establish-ing a joint micro-environment, which prevents AC from undergoing endochondral ossification.

12.1.7Intraarticular platelet rich plasma ( PRP) therapy evaluated in 42 sport horses with Osteoarthrosis (OA)M. Prades1, I. Abellanet2

1Cerdanyola/Spain, 2Barcelona/Spain

Purpose: To assess the clinical progression of OA treated by in-traarticular injection of PRP. Methods and Materials: 42 sport horses were included in the study and divided into 2 groups: Group 1:12 control horses chronically lame unresponsive to conventional therapy . Group 2: 30 horses with both acute (10 cases) and chronic conditions (20 cases). Com-plete lameness examination and radiography were performed. Horses were classified according to chronicity of clinical disease and OA radiographic findings. All patients were reexamined before each treatment and at 45 days, 2 months and 4 months following treatment. Follow-up was from one year up to 3 and a half years. Preparation of PRP was done by manual double centrifugation . A paired Student’s T test was used to compare the progression of lameness before and after treatment and an ANOVA was used to see the influence of “radiographic changes”, “chronicity” and “number of PRP doses”. Results: In group 1, 75% (9/12) returned to previous athletic per-formance and of these, 33% (3/9) relapsed while in competition. In group 2 (10 acute lesions and 20 chronic lesions), 70% (21/30) regained previous athletic level and 9,5% (2/21) relapsed. There was a negative effect of “chronicity” (P<0.019) and “radiographic changes” (P<0,021) on athletic prognosis. There was a significant difference (p<0.005) between 1 or more doses but not between 2 or 3 doses (p<0,532) of PRP on final outcome. Conclusions: PRP is a safe, effective, simple treatment that allevi-ates joint pain as evidenced by lameness examination and clini-cal parameters.

12.1.8Serum cartilage oligomeric matrix protein (sCOMP) is elevated in patients with knee osteoarthritis: a systematic reviewJ.M. Hoch, C.G. Mattacola, J.S. Howard, C. Lattermann Lexington/United States of America

Purpose: Serum cartilage oligomeric matrix protein (sCOMP) has been implicated to be a potential biomarker for osteoarthritis (OA) diagnosis and progression. The purpose of this systematic review is to assess whether sCOMP is elevated in patients with radiographically diagnosed knee osteoarthritis (OA) compared to healthy controls.Methods and Materials: Data Sources: A systematic search of the literature was completed from 1966 to March 2010 utilizing 4 dif-ferent databases: CINAHL, PEDro, Medline, and Sports Discus. Keywords: knee, osteoarthritis, serum COMP, radiography. Study Inclusion Criteria: Written in English, comparison of healthy con-trols and radiographically diagnosed knee OA, Kellgren/Lawrence (K/L) classification, measured sCOMP in an adult population, and reported adequate statistics for calculation of effect sizes (means and associated variability). Data Extraction: Effect sizes (Cohen d) were calculated. Values were interpreted as weak if they were less than 0.40, moderate if between 0.41-0.70, and strong if greater than 0.71. 95% Confidence Intervals (CI) were calculated for each point estimate.Results: Five studies met the inclusion criteria resulting in the calculation of 15 effect sizes and 95% CIs (Figure). All effect sizes indicated sCOMP was elevated in those with radiographically diag-nosed OA (range 0.07-5.8). However, the CI at six point estimates crossed zero indicating a level of caution when interpreting these results. Level B evidence exists that sCOMP is elevated in patients with knee OA. This recommendation was reached based on consis-tent level 3 evidence or higher (as defined by Centre for Evidence Based Medicine).Conclusions: Serum COMP is consistently elevated in patients with knee OA and is sensitive to OA progression as indicated by greater effect sizes. In conjunction with other properties, such as its responsiveness to acute knee injury in younger adults, sCOMP may be useful in identifying early cartilage damage prior to the development of fully established OA.

12.1.9Synovial fluid changes related to pathologyR. Esparza1, J. Moya-Angeler2, P. Martinez de Albornoz1, R. Sanchez Hidalgo1, I. Zapero1, F. Forriol Campos1

1Majadahonda/Spain, 2Madrid/Spain

Purpose: Synovial fluid (SF) is a joint lubricant that also nourishes joint tissues and collects catabolic factors that may contribute to the degradation of cartilage and meniscus.Methods and Materials: SF was extracted from 123 patients, males, between 19 and 58 years of age who were operated on dif-ferent knee pathologies grouped into cartilage, meniscal and liga-mentous injuries and as well sub classified as acute injuries, if less than 6 months of evolution, subacute, between 6 and 12 months evolution and chronic beyond a year of evolution. Aspiration re-sulted negative in 53 knees. All patients signed informed con-sent documents. Extracted liquid concentration was performed with Centricon ® tubes. We used ELISA technique to detect the presence of IL-2, IL-10 and TNF-a, using an antibody detection kit (Biolegend). Fluid was incubated with capture antibodies for 16 h according to instructions. To detect the signal at the plate TECAN team used a 540 nm wavelength. A multivariant analysis was per-formed to establish the relation between time, type of injury and amount of cytokines.Results: We found no differences in the detection of cytokines (IL-2, IL-10) with the evolution time, but an increased of TNF-a in the SF was observed in the group of more than a year of evolution. Neither difference between meniscus and ligament injuries was observed, although we note a trend for increased TNF-a in liga-mentous injuries.Conclusions: TNF-a is a pro-apoptotic factor related to knee inflam-matory processed which seems to be elevated in lesions of more than one-year evolution and also in ligamentous injuries.

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12.2.2Two-year clinical and radiological evaluation following a prospective, randomized comparison of traditional and accelerated approaches to post-operative rehabilitation following matrix-induced autologous chondrocyte implantationJ. Ebert, M. Fallon, W.B. Robertson, D.G. Lloyd, T.R. Ackland, M.H. Zheng, D.J. WoodPerth/Australia

Purpose: Policies on post-operative load bearing rehabilitation following autologous chondrocyte implantation (ACI) are varied and the subject re-mains controversial. This study aims to determine the safety and efficacy of an ‘accelerated’ post-operative weight bearing (WB) regime following matrix-induced autologous chondrocyte implantation (MACI). Methods and Materials: A randomized controlled study design was used to assess clinical and radiological outcome in 70 patients (45 males, 25 females) who underwent MACI to the medial or lateral femoral condyle, in combination with either ‘traditional’ or ‘accelerated’ approaches to post-operative WB rehabilitation. Apart from the gradient and time to full WB, components of both rehabilitation interventions were the same. Under the ‘accelerated’ protocol, patients reached full WB at eight weeks post-sur-gery, compared to 11 weeks for the ‘traditional’ group. Clinical outcomes were undertaken pre-surgery and at 3, 6, 12 and 24 months post-surgery. These included the Knee Injury and Osteoarthritis Outcome Score (KOOS), the visual analogue pain scale (VAS), the six-minute walk test, knee range of motion and activity level. High resolution magnetic resonance imaging (MRI) was undertaken at 3, 12 and 24 months post-surgery.Results: A significant effect (p<0.05) for time existed for all clinical measures, demonstrating improvement up until 24 months in both groups. A significant interaction effect (p<0.05) existed for pain sever-ity and the six-minute walk test, with accelerated group patients report-ing significantly less severe pain and demonstrating superior six-minute walk distance over the period. While there was a significant group effect (p<0.05) for maximal active knee extension range in favor of the accel-erated regime, no further significant differences existed. Both groups sig-nificantly improved (p<0.05) in an MRI composite score and pertinent descriptors of graft repair throughout the post-operative timeline, up until 24 months post-surgery, while there were no differences (p>0.05) observed between the two groups.Conclusions: The ‘accelerated’ WB approach was not detrimental to graft development at any stage throughout the post-operative assessment timeline up until 24 months post-surgery, and may accelerate patient return to normal function, whilst reducing post-operative muscle loss, intra-articular adhesions and associated gait abnormalities.

12.2.3Comparison of knee strength pre-operatively, and at 6 and 12 months post-operatively following autologous chondrocyte implantation(ACI)C.G. Mattacola, J.S. Howard, J.M. Hoch, C. LattermannLexington/United States of America

Purpose: The goal of rehabilitation programs involve returning patients to pre-injury or pre-operative status. Achieving pre-operative strength levels following surgical intervention is necessary to return patients to their previous levels of function. Our purpose was to compare knee ex-tension and flexion strength at pre, 6, and 12 months post ACI surgery. Methods and Materials: 18 (36.6yrs,174cm,89.09kgs) patients under-going ACI surgery participated. Concentric and eccentric knee exten-sion (KE) and flexion (KF) were tested on a BTE (Baltimore MD) isokinet-ic dynamometer at 60 deg/sec. Testing of the involved and uninvolved limb was performed pre-operatively

(n=18), 6

(n=12), and 12

(n=7) months post-

operatively. Dependent variables were peak force(N) and percentage of strength of the involved/uninvolved limb. Paired T-tests and Bonferroni corrections were performed to compare differences between limbs at pre, 6 and 12 monthsResults: Concentric KE strength was significantly different when the involved and uninvolved limb were compared at pre (331.28 ±148N vs. 430.53 ±148N) and 6 months, (266.51 ±75.4N vs. 433.4 ±91N,) (P<.01) but not 12 months, (284.4 ±140N vs. 383.8 ±118N) (P=.09) . The involved KE strength was less than 60% of the uninvolved leg 12 months post-surgery(Figure). Concentric KF strength was signifi-cantly different when the involved and uninvolved limb were com-pared at pre (284.6 ±119N vs. 337.4.53 ±129N) and 6 months(258.8 ±80vN vs. 344.54 ±67N,) (P<.01) but not 12 months (313.15 ±95N vs. 352.5 ±98N) (P=.12). The involved KF strength was less than 80% of the uninvolved leg 12 months post-operatively (Figure). Conclusions: Although patients may report in the clinic that they subjective-ly feel stronger 3 and 6 months after ACI, their quadriceps strengths remains compromised (<80% of uninvolved side) past 1 year postoperatively. The clinician and patient need to be aware of this and quadriceps strength train-ing may need to be emphasized and continued past one year.

12.2.4High patient compliance to an identical rehabilitation guideline after characterized chondrocyte implantation or microfracture is related to improved patient functional outcome at 2 yearsD. Van Assche1, D. Van Caspel2, F. Staes1, D.B. Saris2, J. Bellemans1, J. Vanlauwe1, F. Luyten1

1Leuven/Belgium, 2Utrecht/Netherlands

Purpose: Rehabilitation following cartilage repair procedures and compliance to rehabilitation are often stated as important, but rarely studied. The major aim of the study was to explore the effect of patient compliance on the objective functional outcome up to 2 years after surgery. Therefore an identical rehabilitation program was implemented and followed-up in the RCT comparing character-ized chondrocyte implantation (CCI) versus microfracture (MF). Methods and Materials: In a cohort design patient compliance to an identical rehabilitation protocol was studied. An electronic re-port form including 18 physiotherapy variables was used to com-pare physiotherapy management. Patients’ objective functional outcome was assessed using the pooled symmetry index (SI) based on one strength and 3 hop tests. The objective outcomes were evaluated pre-surgery, at 1 and 2 years post-surgery. Results: During the first 3 months 85% of the physiotherapists reported weekly. Overall physiotherapists adhered very consistently and showed comparable management to the protocol in both treatment groups. In contrast in both treatment groups patients showed great variability in the amount of time (minutes per day) they were active in low load activi-ties. Two cohorts were created. Patients with good compliance to the rehabilitation protocol (Good Comp, n=21) and patients with poor com-pliance (Poor Comp, n=17) performed activities in low load conditions for a mean of 45 minutes a day and 7 minutes a day respectively. These cohorts were not significantly different for any other parameter. At 24 months after surgery the Good Comp cohort performed the objective functional tests significantly better compared to the Poor Comp cohort. The mean pooled SI of the Good Comp cohort was 92.4% compared to 78.2% for the Poor Comp cohort (95%CI 1.8 to 26.2).Conclusions: High patient compliance to the rehabilitation protocol, which reflects a high amount of low load activities post-surgery, appears beneficial for the objective functional outcome at 24 months after surgery.

12.2.5Changes in functional performance during walking, squatting, rising, and stepping following autologous chondrocyte implantation (ACI)J.S. Howard, C.G. Mattacola, J.M. Hoch, C. LattermannLexington/United States of America

Purpose: To document the recovery of functional performance over 6 months following ACI. Methods and Materials: Participants were 18 (37.6yrs, 172cm, 90.03kgs) patients undergoing ACI to the knee. All patients completed functional tests simulating walking, squatting, rising from sitting, stepping-up and stepping-down using the NeuroCom Long Forceplate(Clackamas, OR), preoperatively and 3, 6, and 12 months postoperatively. Repeated Mea-sures ANOVAs were used to compare performance(p<.05).Results: For walking, improvements from preoperative levels were seen in stride length, width, and speed 3 months post ACI with sig-nificant increases in stride length(19%) and decreases in stride width(8%) at 6 months. During squatting(30°, 60°, and 90°of flexion) asymmetries in weight distribution were minimal preoperatively(3%, 4%, -2%, respectively) but increased significantly 3 months postop-eratively(8%, 10%, 6%) and persisted 6 months postoperatively(6%, 7%, 5%(Figure1.). The force generated bilaterally to rise from sitting increased significantly at 6 months(22%>preoperative). Side-to-side comparison of rise force demonstrated greater force production by the uninvolved-limb relative to the involved-limb by 5% preopera-tively, 14% at 3 months, and 11% at 6 months. There were significant increases in force generated when stepping-up at 3 months(24%) and at 6 months(33%) relative to preoperative force(Figure2.). Step-down impact forces increased above preoperative measures at 3(13%) and 6(21%) months, representing a loss of eccentric control as patients lowered themselves. Preliminary data from 8 subjects at 12 months suggests continued improvements in speed and stride length and some reduction in side-to-side differences; however, between limb discrepancies for weight distribution and force production remain greater than preoperative observations. Conclusions: Despite early improvements in some functional tasks, 6 months post-ACI side-to-side differences in weight distribution and force production during squatting, rising, and stepping-down persist-ed. Even 12 months postoperatively some elements of function, par-ticularly those associated with eccentric control and limb symmetry, remain below preoperative levels, and further emphasis of these ac-tivities during rehabilitation is necessary.

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12.2.6ACI vs Mosaicplasty for Symptomatic Articular Cartilage Lesions in the Young Adult Knee. Ten-year Results of Randomized Comparison StudyG. Bentley1, L.C. Biant2, S. Vijayan3, S. MacMull3

1London/United Kingdom, 2Edinburgh/United Kingdom, 3Stanmore/United Kingdom

Purpose: Autologous chondrocyte implantation (ACI) and mosa-icplasty (MP) are two methods of repair of symptomatic articular cartilage defects in the adult knee. This study represents the only long-term comparative clinical trial of the two methods.Methods and Materials: A prospective, randomized comparison of the two methods involving 100 patients with symptomatic articular cartilage lesions was undertaken. Patients were followed for ten years. Pain and function were assessed using the modified Cincin-nati score, Bentley Stanmore Functional rating system and visual analogue scores. ‘Failure’ was determined by pain, a poor outcome score and arthroscopic evidence of graft disintegration. Patients had a mean age at index operation of 31. There was a long mean pre-op duration of symptoms of seven years, and the defects had an average of 1.5 operations (excluding arthroscopy) to the ar-ticular cartilage lesion prior to the cartilage repair surgery. The aeti-ology of the articular cartilage defects was mainly trauma, some pa-tients had osteochondritis dissecans or chondromalacia patellae.Results: Five patients were lost to follow-up. A total of 23 out of 42 mosaicplasty patients failed, 10 out of 58 ACI patients failed.Conclusions: At ten years, patients who underwent cartilage re-pair using ACI fared significantly better than those who under-went mosaicplasty.

12.2.7Prospective Evaluation of Meniscal Transplantation Procedure: Minimum 7-year Follow-UpE. Daley, S. Bajaj, J. Kercher, E. Strauss, P.B. Lewis, B.J. Cole Chicago/United States of America

Purpose: Meniscal transplantation is an accepted method of treat-ment for meniscal deficient patients. Although mid-term results have been promising there is limited long-term clinical follow-up data. The purpose of this study is to report post operative out-comes of meniscal allograft transplantation by a single surgeon at a minimum of 7 year follow-up. Methods and Materials: Clinical results from 20 patients who under-went meniscal allograft transplantation were evaluated at a mean follow-up of 8.23 years. Average patient age at the time of surgery was 32.2 years. 10 meniscal transplants were performed in medial compartment and 10 in the lateral. Post-operatively, patients were assessed using the International Knee Documentation Committee (IKDC), Tegner-Lysholm, Knee Injury and Osteoarthritis Outcome Score (KOOS), and San Francisco – 12 (SF-12) outcome measures. Results: The mean IKDC score improved from 50.49 to 64.13 (p<0.05). Similarly mean Lysholm improved from 55.83 to 71.84 (p<0.05). SF-12 physical scores improved from 42.24 to 49.74 and SF-12 mental improving from 45.84 to 48.72. In the KOOS scor-ing system, pain (63.72 to 79.81) (p<0.05), symptoms (63.69 to 67.03), activities of daily living (80.15 to 89.7), and quality of life (71.15 to 75.0) improved. Subjectively, on a scale of 1-10, with 10 being completely satisfied with surgical outcome, the mean post-operative satisfaction rate was 8.0. Additionally, 100% of the pa-tients would elect to have this surgery again if they had the same problem on the opposite knee. Conclusions: The average IKDC, Lysholm, SF-12, and KOOS scores demonstrated improved patient outcomes at a minimum 7-year fol-low up. Based upon this data, meniscus transplantation is a viable treatment option for meniscus deficient patients and can reducing pain, increase range of motion and improve patient function.

12.2.8Indication of articular cartilage formation inside Trufit® plugs one year after implantation as analyzed by delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC)J.E.J. Bekkers, L.B. Creemers, W.J.A. Dhert, D.B. Saris Utrecht/Netherlands

Purpose: Implantation of Trufit® plugs is an option for treatment of focal osteochondral lesions. This study analyzed the, quality of, cartilage formation in Trufit® plugs using a dGEMRIC MRI protocol. Methods and Materials: A total of 5 patients were analyzed by dGEM-RIC at 12 months (range 9-15 months) after treatment of an osteo-chondral lesion in their femoral condyle using Trufit® plugs (Smith & Nephew, UK). Patients were injected with 0.2mmol/kg Magnevist (Bayer, Germany) 90min prior to scanning and asked to walk for 15min to facilitate contrast uptake into the knee cartilage. A 3D T1 protocol with 5 inversion times (50, 150, 350, 650 and 1650 ms) and 3 mm slice thickness on a sagittal image acquisition at 1.5T was used. The T1gd was calculated by averaging the T1gd across the manually segmented region-of-interest (ROI) using in-house developed soft-ware. Five different ROIs were defined; the Trufit® plug, cartilage either surrounding or directly opposing the Trufit® plug, non-artic-ulating tibial cartilage and femur cartilage (Figure 1). Differences in T1gd were analyzed by a one-way ANOVA and post-hoc-LSD test. Results: One year after implantation the T1gd in the Trufit® plug (391±81 ms) is comparable (p=0.812) to the T1gd in the femur car-tilage (415± ms) and the cartilage surrounding (392±77 ms) the Trufit® plug (Figure 2). In addition, the T1gd for the directly ar-ticulating (opposing) cartilage (543±53 ms) at the tibia and non-articulating tibia cartilage (508±45 ms) is comparable (p=0.535). Conclusions: This study showed a comparable T1gd for femur car-tilage and the Trufit® plug, which suggests the presence of carti-laginous tissue inside the Trufit® plug one year after surgery. In addition, articulation between the Trufit® plug and tibia cartilage surface does not introduce damage in the articulating tibial carti-lage as shown by the similar T1gd signal between the tibial carti-lage that was articulating against the implant and non articulating tibial cartilage.

12.2.9Chondroprotective effects of a polycarbonate-urethane meniscal implant in a sheep modelG. Zur1, J.J. Elsner2, E. Linder-Ganz1, J. Shani1, O. Brenner1, E. Hershman3, F. Guilak4, A. Shterling1 1Netanya/Israel, 2Tel Aviv/Israel, 3New York/United States of America, 4Durham/United States of America

Purpose: Meniscus injury leads to alterations in the knee load trans-fer, associated with degenerative osteoarthritis. While allograft replacement can improve joint stability and function, it often pro-vides little benefit in preventing osteoarthritic changes. Thus, the development of artificial meniscus could provide therapeutic po-tential for treatment meniscal injury. We hypothesized that a novel Polycarbonate-Urethane (PCU) meniscus implant could protect the cartilage under loading.Methods and Materials: 6 ewes underwent a full medial meniscecto-my and were implanted with PCU implants (Fig.1). Joint functionality was assessed by measuring mobility and joint ROM. Animals were sacrificed at 3 and 6 months. Cartilage was assessed macroscopical-ly and by H&E and Saf-O staining using a semi-quantitative modified Mankin score. The contralateral knee served as control. The score incorporated cartilage structural changes, proteoglycan loss, chon-drocyte cloning, osteophyte formation and subchondral bone thick-ening in the tibial plateau (TP), femoral condyles (FC) and patella. Results: The sheep tolerated the operations well. Full ROM and no signs of distress were observed. Examinations of the explanted implants revealed no structural or property changes. Macroscopi-cally, cartilage in contact with the implant was well preserved. Remodeling was observed by the presence of minimal to mild ir-regularity of the medial rim of the FC and the medial TP. Mild his-tological changes were observed at 3 and 6 months post-op. At 3 months, Mankin scores for the whole joint were increased from 10 in controls to 17 in operated limbs (p=0.05). However, at 6 months, no progression was observed and modified Mankin scores were similar to 3 months, and did not significantly differ from control (p=0.08), (Fig.2). Conclusions: Histologic changes were generally mild, as previous animal studies found significant loss of cartilage structure and properties post meniscectomy. Our findings suggest that a PCU im-plant may protect, but not completely prevent, cartilage degenera-tive changes.

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12.3-9Repair tissue assessment after matrix associated autologous chondrocyte transplantation with CaReS in the knee – Quantitative MRI evaluation at 3.0 TeslaD. Stelzeneder1, S. Domayer1, S. Nehrer2, T. Luksch2, R. Dorotka1, S. Goed1, T.C. Mamisch3, G.H. Welsch1, S. Trattnig1 1Vienna/Austria, 2Krems/Austria, 3Berne/Switzerland

Purpose: T2-mapping and delayed Gadolinium-Enhanced-MRI of Carti-lage (dGEMRIC) are helpful for the assessment of cartilage repair tissue. The purpose of this investigation was to provide biochemical MRI data for repair tissue after matrix associated autologous chondrocyte trans-plantation (MACT) with CaReS in the knee. Methods and Materials: Fourteen knees of 14 patients (age 38.1±10.0 years [mean±standard deviation]) with a mean follow-up of 2.0±0.9 years (range 0.9-3.6) were investigated. Twelve defects were located on the medial femoral condyle, 1 at the lateral femoral condyle, and 1 at the tro-chlea. All MR-examinations were performed on a 3.0-Tesla MR-unit. In addition to morphological imaging, T2-mapping and a dGEMRIC (T1Gd) with 60 minutes delay were performed. The region of interest (ROI)-evaluation was performed manually on 3 adjacent slices covering the whole transplant-area. Reference regions were evaluated in the healthy cartilage of the posterior condyle. The deep and superficial layers were assessed separately, each measuring 50% of full thickness. Repair tissue area was measured on morphological sequences. The clinical evaluation was performed on the day of the MRI using the IKDC knee form, IKDC sub-jective form, Lysholm score, and a modified Cincinnati rating. Descriptive statistical data is shown as mean±standard deviation or as median and interquartile range (IQR). Paired t-tests were used for the analysis of zon-al differences (deep vs. superficial). Results: The mean repair tissue size was 3.9±1.4cm2. The median of IKDC rating was B (IQR A;B), IKDC subjektive 68.3 (IQR 43.4;82.2), Cincinnati 7.5 (IQR 4.5;8) and Lysholm 85.5 (IQR 49.5;95.3). Mean T2-values in millisec-onds for the repair tissue and reference cartilage were as follows (deep/superficial, p-values for zonal differences are given): 34.8±6.7/39.8±5.7 (p=0.003) and 38.5±5.1/40.7±5.4 (p<0.001). The respective T1Gd-values were: 630±170/499±114 (p<0.001) und 885±236/697±162 (p<0.001). Conclusions: Our results suggest a zonal organisation of repair tissue after MACT with CaReS, with zonal differences in collagen structure, water and proteoglycan content.

12.3.2Development of a Flexible Tissue Engineered EarM.A. Randolph1, C.A. Sundback1, I. Pomerantseva1, D.A. Bichara1, X. Zhao1, K.M. Kulig1, L. Zhou1, E. Bassett1, N. Hwang2, R. Langer2, D.G. Anderson2, M. Cheney1, T. Hadlock1, J. Vacanti1 1Boston/United States of America, 2Cambridge/United States of America

Purpose: Current standard approaches for complete auricular recon-struction include carved autologous rib cartilage and alloplastic im-plants provide rigid reconstruction of the ear with suboptimal aesthetic outcomes. Considerable progress in producing tissues from isolated cells and degradable scaffolds makes engineered cartilage a feasible option for auricular repair. One of the largest challenges is maintain-ing the complex 3D shape of the auricle and avoiding distortion and shrinkage when supporting scaffold is resorbed and the neocartilage matures. Degradation products of synthetic materials elicit a host in-flammatory response negatively affecting neocartilage formation in immunocompetent animals. Fibrillar collagen, although biocompatible, is promising but does not possess required strength to withstand scar contraction forces exerted by skin. The objectives of this study were: 1) to explore neocartilage formation in an immune competent sheep with FDA-approved materials, and 2) evaluate a flexible internal metallic structure to maintain 3D shape.Methods and Materials: Commercially available porous fibrillar colla-gen were provided by Kensey Nash Corp. and porous poly(L-lactide)/poly(DL-lactide-co-caprolactone) (PLA/PCL) scaffolds were synthe-sized by our MIT team. Sheep auricular chondrocytes were seeded onto the scaffolds. Formulations were screened in a nude mice. Prototypes of human-shaped ears were manufactured from fibrillar collagen with an embedded permanent framework to help retain the shape during neocartilage formation. Seeded constructs were implanted subcutane-ously on the back of nude mice and in the neck in sheep.Results: Neocartilage formation was demonstrated in nude mice and, most importantly, sheep at 6 wks (Fig.1). A surgical model for implanta-tion of human size and shaped auricles was developed in sheep (Fig.2). No extrusion of engineered ears was observed. Retention of ear shape was demonstrated in nude mice and sheep.Conclusions: Autologous cartilage was successfully engineered in sheep. Early results show that our enhanced scaffold prevents distor-tion and allows retention of the original ear shape.

12.3.3MR evaluation of repair tissue in osteochondral defects following treatment with acellular scaffolds: high resolution MR-histological correlation in a goat modelC.S. Winalski, M.F. Freire, F.A. Sakamoto, E. Schneider, R. Midura, A. Vasanji Cleveland/United States of America

Purpose: Describe morphologic appearances and quantitative MR measures of repair tissue following treatment of osteochondral defects with an acellular scaffold, and correlate MR findings with histology to determine if MR can predict repair tissue type.Methods and Materials: 12 goats with surgically created 7mm di-ameter 6mm deep osteochondral defect were treated with TruFitCB acellular scaffolds (Smith&Nephew). 6 goats per group were sacri-ficed at 3 or 9 months postoperatively. Anesthetized animals were imaged in vivo at 1.5x0.2x0.3mm resolution. Following sacrifice the excised femoral condyles were imaged at 7T with PDW, T2, and T1-mapping at 1.0x0.1x0.1mm resolution. Condyles were fixed in for-malin and dGEMRIC was performed with T1 maps repeated before and after 48 hrs equilibration in 1mM GdDTPA. Specimens were processed for histology. T1 and T2 maps were created on a pixel by pixel basis using Matlab and MRImapper software (MIT/BIDMC).Results: At 3 months, 3 specimens had deep defects and 3 nearly filled. All had bone openings with a new bone rim covered by hyaline cartilage repair tissue. Central repair tissue had low glycosamino-glycan (GAG) on histology. At 9 months, 4 of 6 defects showed near complete bony closure with overlying repair tissue slightly below native cartilage. Repair tissue stained for GAG similar to native cartilage on histology. dGEMRIC values were similar to adjacent cartilage. Repair tissue T2 had a bilaminar pattern with lower deep layer T2, similar to, but less uniform than native cartilage. Repairs with deep defects at 9 months appeared similar to 3 month group. All specimens had bone cavities larger than initial surgical defects. Hyaline-like (high-GAG) repair tissue showed significantly longer post-GdDTPA T1 on dGEMRIC than fibrous-like repair at all time-points, 525ms vs 383ms, respectively (p<0.01). Conclusions: MR accurately tracked healing of defects treated with acellular scaffolds accurately showing bone defect closure, tissue fill, and cysts. Quantitative techniques may predict tissue type.

12.3.4Liposomes-enriched fibrin scaffold in cartilage regeneration.E. Filova1, M. Rampichová1, A. Lytvynets1, A. Míčková1, J. Uhlík1, L. Vajner1, L. Martinova2, E. Prosecká1, D. Usvald3, J. Motlík3, E. Amler1 1Prague/Czech Republic, 2Liberec/Czech Republic, 3Liběchov/Czech Republic

Purpose: Nanofibers are suitable for cell proliferation. Liposomes have already been used as drug delivery systems.(1) The aim of the study was to study cartilage regeneration in minipigs using fibrin scaf-fold with liposomes-enriched nanofibres containing growth factors. Methods and Materials: Polyvinyl alcohol nanofibers containg lipo-somes were incubated with basic fibroblast growth factor and in-sulin, and mixed with Tissucol®. The liposome-enriched composite scaffold was implanted into eight load-bearing osteochondral de-fects in miniature pigs. As a control, the defects in the left knees were left untreated. Animals were sacrified 12 weeks after the sur-gery, and examined histologically. Results: In a scaffold group, a regular formation of isogenic lines of chondrocytes near the defect bases and differentiation towards hyaline cartilage were observed. Fibrocartilage was found on the defect surface. The middle and basal zones of the defects were pre-dominantly alcian blue positive. Type II collagen was positive in the non-cellular transient zone in the newly formed cartilage and on the border of young isogenic groups. Weak positivity was in the centre of the defect. In a control group, fibrocartilage or unorganized fi-brous tissue with isogenic groups of chondrocytes situated at the borders, and fibrous tissue on the surface of the defect were ob-served. The differentiation was predominantly accompanied by vas-cularization. Alcian blue was positive in the upper part of defects. Immunohistochemical examination for collagen II was positive in newly formed cartilage. Conclusions: The therapy of osteochondral defects with liposomes-enriched scaffolds supported the hyaline cartilage formation. The scaffold is suitable for cartilage regeneration. References (1) Panwar P, et al. Int J Nanomedicine. 9;5:101-8, 2010. Acknowledgements Sup-ported by Grant Agency of AS CR grant No. IAA500390702, MSMT CR grants No. 1M0510 (1M6798582302) and NPV II 2B06130, AV0Z – ASCR, No. AV0Z50390512 and AV0Z50390703, EU project Bioscent ID number 214539, Grant Agency of Charles university No. 119209.

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12.3.5One stage osteochondral repair with cartilage fragments in a hyaluronic acid/fibrin glue/platelet rich plasma scaffold: in vitro human and in vivo rabbit and goat animal modelA. Marmotti1, F. Castoldi1, R. Rossi1, C. Realmuto1, M. Bruzzone1, D.E. Bonasia1, U. Cottino1, B. Peyrone2, M. Mauthe von Degerfeld2, P. Rossi1 1Torino/Italy, 2Grugliasco/Italy

Purpose: Our study evaluate a “one-stage” procedure combining minced autologous cartilage fragments, as a viable cell source, with a resorbable scaffold composed of hyaluronic acid (Hyaff-11), fibrin glue and platelet rich plasma. Methods and Materials: In vitro: minced cartilage fragments from human knees (<35y and >50y) and rabbit and goat knees were cultured onto the scaffold for 1, 2 and 3 months and histo-logically evaluated. Human cultures were also stimulated with TGF-beta(10ng/ml) and characterized by immunofluorescence. In vivo unilateral trochlear model: osteochondral defects were created in 32 adult rabbit knees and 15 goat knees and were either treated with cartilage fragments embedded in the scaffold (Group “one stage”) or loaded with scaffold alone (Group “scaffold”) or left un-treated (Group “empty”). Rabbit were sacrificed at 1, 3, 6 months, goats at 3, 6, 12 months. Repair process was evaluated with histol-ogy (modified ICRS and O’Driscoll scores) and immunohistochemis-try. In goat, nanoindentation of defects have also been performed.Results: In vitro, chondrocytes from cartilage fragments migrate and proliferated into the scaffold better in animal than in human explant cultures. In human, outgrowth was time-dependent and age-dependent; TGF-beta increased outgrowth at 1 and 3 months; migrating cells were positive for sox9, CD151 and CD49c and nega-tive for CD105. In vivo, Group “one stage” showed better quality of repair tissue than control groups and some typical features of articular cartilage, as presence of collagen type II in extracellular matrix. Nanoindentation of goat “one stage” samples showed me-chanical properties similar with normal trochlear goat cartilage. Conclusions: Minced cartilage fragments in a HA/fibrin/PRP scaf-fold provide a viable cell source for one-stage cartilage repair in rabbit and goat model. In-vitro human cultures showed some limi-tations, as the age-dependence, and some interesting features, as the positive stimuli of TGF-beta. Further studies are required to op-timize human model.

12.3.6Giant cell foreign body reactions to osteobiologic implants: case reportsS.K. Tabet Albuquerque/United States of America

Purpose: This paper reports two confirmed cases of giant cell for-eign body reaction to osteobiologic implants and offers discussion of an additional three similar cases which occurred in a larger co-hort of seventy-two cases. Articular surface defects are a major cause of joint degeneration. They tend to be progressive resulting in serious debilitation. A number of techniques are aimed at restor-ing cartilage but none produce the structure of native cartilage. Some are done at the time the defect is discovered others require multiple surgeries and a long time to recovery. Failure of any of these requires extraordinary measures to correct. Methods and Materials: Having encountered failure of osteo-biologic implants (Tru-fit, Smith-Nephew), we retrospectively reviewed charts, x-rays, MRI and path reports of our series of seventy-two cases done over a period of four years. We found five cases in which the mechanism of failure was similar. Two of these cases had pathology examinations showing giant cell foreign body reaction. We offer case reports of the two cases that had path confirmation and a summary of the other three. Results: In two cases pathology confirmed giant cell reactions. We have found only one other report of this in the literature. Conclusions: We suspect that all five cases discussed, because of their similarity, may have involved foreign body reaction. Failure of the implant is a serious complication resulting in further joint degenera-tion and requires at least two further surgeries to achieve resolution. Awareness of this problem may lead to early recognition or prevention.

12.3.7Use of a biphasic copolymer scaffold for the treatment of isolated osteochondral defects of the femoral condyleF.A. Petrigliano, H. Su, I. Solsky, L.F. Foo, S. Rodeo, T.L. Wickiewicz, R.F. Warren, H.G. Potter, R.J. Williams, IIINew York/United States of America

Purpose: The purpose of this study was to report the outcomes of patients treated with the TruFit biphasic plug for isolated os-teochondral defects of the knee, and to assess the morphological characteristics of these scaffolds using cartilage-sensitive MRI.Methods and Materials: Thirty patients (31 knees; mean age 44.3 years, range: 18 to 66) underwent TruFit scaffold implantation for isolated osteochondral lesions. Lesions were located on the MFC (17), trochlea (11), and LFC (3). An average of 3 plugs (range: 1 to 6; size 7-11 mm) were used per lesion. The mean follow-up inter-val was 32.8 months (range: 24-48). Subjective outcomes were assessed using the Activity of Daily Living Score (ADL), Marx Ac-tivity Scale, and IKDC Score. The mean followup for MRI evalua-tion was 20.8 months (range: 12-40). Implants were evaluated for cartilage signal, interface morphology, displacement, hypertrophy, subchondral edema, bony overgrowth, percentage fill, and degree of incorporation. ANOVA and linear regression analysis were per-formed with significance set at p<0.05. Results: The mean ADL score increased from 62.7 to 82.4 (p<0.001). The mean IKDC score increased from 45.1 to 71.3 (p<0.001). There was no significant change in Marx score(p=0.13). MRI evaluation demonstrated that 90% of plugs were isointense or hyperintense. 69% of plugs demonstrated near complete to complete cartilage fill. Moderate to good bony incorporation was noted in 64% of plugs, with 49% demonstrating a flush morphology. Subchondral edema was minimal to absent in 72% of plugs. Plug hypertrophy, displacement, and bony overgrowth were rare. MRI appearance of plugs did not correlate with clinical outcomes. There was a trend towards poorer outcomes with larger plug configurations. Conclusions: Treatment of isolated femoral cartilage lesions with the TruFit biphasic implant results in an improvement in clinical outcome measures at a minimum two-year follow-up. The majority of the implanted plugs demonstrated a favorable appearance using cartilage-sensitive MRI.

12.3.8Nano-composite biomaterial used as salvage procedure for the treatment of massive osteochondral lesions.M. Delcogliano, D. Marotta, F. Pezzillo, C.F. De Biase, R. Giacomi, M. Manili, A. DelcoglianoRoma/Italy

Purpose: Large osteochondral articular defects are difficult to treat and present a high rate of complications, such as post-traumatic joint congruity, axial misalignment and possibly arthritis. In the last 20 years many different approaches have been proposed to restore the cartilage lesions, and satisfactory results have been obtained. However, the treatment of osteo-cartilaginous lesions is even more problematic, because tissue damage is extended also to the sub-chondral bone, involving two different tissues characterized by dif-ferent intrinsic healing capacity. The objective of this clinical study was to test safety and performance of a newly developed type-I collagen-hydroxyapatite (HA) nanostructural bio-mimetic osteo-chondral (O.C.) scaffold which reproduces cartilage-subchondral bone morphology.Methods and Materials: A gradient composite O.C. scaffold, based on type-I collagen-HA, was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles at physiological conditions. 15 cases with large knee osteochondral lesions and osteochondritis dissecans were treated as salvage procedure with scaffold im-plantation. The lesions size went from 3 cm2 to 8 cm2. All patients achieved minimum 1 year follow up and were clinically evaluated using the International Repair Cartilage Society score.Results: IKDC objective score improved after 1 year showing a nor-mal or nearly normal knee in 80% of patients. Similar results were obtained with the IKDC subjective score MRI evaluation demon-strated good bone and cartilage formation.Conclusions: This open one-step surgery was used for the treatment of large osteochondral defects. The results of this technique at short follow-up are very encouraging and show satisfactory results even in massive ostechondral defects where other techniques had filed.

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12.4.2Characterized Chondrocyte Implantation versus microfracture for the treatment of symptomatic cartilage defects in the knee in patients with short onset of symptoms, 60 month follow-upJ. Vanlauwe1, D.B. Saris2, J. Victor3, K.F. Almqvist4, J. Bellemans1, R. Verdonk4, F.P. Luyten1 1Leuven/Belgium, 2Utrecht/Netherlands, 3Assebroek/Belgium, 4Gent/Belgium

Purpose: A five-year evaluation of clinical efficacy of Characterized Chondrocyte Implantation (CCI) compared to microfracture (MF) for the repair of symptomatic cartilage defects of the femoral condyles. Methods and Materials: In a prospective, randomized, controlled, multi-center trial, CCI was compared to MF in patients aged 18-50 years with a single symptomatic ICRS grade III-IV lesion of the knee. Clinical outcome was measured 60 months post-surgery by means of the Knee injury and Osteoarthritis Outcome Score (KOOS) in subgroups with early onset of symptoms. Results: In patients with time since onset of symptoms <3 years, the mean improvement from baseline to 60 months in Overall KOOS was significantly greater with CCI than MF (26.02+/-3.42 vs. 14.26 +/- 3.01 respectively and p 0.012). There were no apparent differ-ences between treatments in the subgroup of patients with 3 years or more since onset of symptoms. Conclusions: Clinical benefit of CCI over MF in patients with short duration of symptoms was noted at 3 years post-surgery. Time to treatment has shown to affect the outcome at 5 years post-surgery, i.e. early treatment with CCI improves long-term clinical outcome.

12.4.3The full arthroscopic technique of autologous chondrocyte transplantation using chondrospheres - safety study with 2 years follow upS. Roessing1, T. Schreyer2, P. Baum3, H. Thermann4, H. Paessler4 1Mannheim/Germany, 2Darmstadt/Germany, 3Gundelfingen/Germany, 4Heidelberg/Germany

Purpose: The most matrix based procedures in ACT require an open surgical procedure for the cell transplantation. The aim of the study was to prove the feasibility and safety of the full arthroscopic ap-proach using spheroids (ACT3D/Codon). Methods and Materials: Prospective cohort study; Level of evi-dence 3. multi center study.Mean follow-up was 2 +/- 0.1 years. 42 patients (mean 38,3 years, 18 to 50 yrs) underwent full arthroscopical autologous chondrocyte implantation with spheroids (ACT3D Codon) of full size defects in the knee with a mean defect size of 4,7 cm² (range 2,5 to 9.5cm²). Knee function was measured by Lysholm- Womac-D score and VAS. All patients underwent an one year follow up MRI. For MRI imaging the MOCART score was used.Results: The study showed no increased risk or severe adverse events in the arthroscopic application. The two year follow up showed significant improved results in the Lysholm, Womac D and tegner activity scoring. No revision surgery was needed during fol-low up time. We implanted in the mean 38,5 spheroids/cm². This is corresponding to 7,7 million cells/ cm². The threshold dose of clas-sical ACT of 1 million cells /cm² (5/spheroids/cm²) was not under runned. The intraoperative loss of spheroids was estimated by the surgeons underneath 10% (5 Spheroids). MRI revealed that in the majority of cases, filling of the defect was accomplished with a non homogeneous surface and structure of the graft.Conclusions: The full arthroscopic application of autologous chon-drocyte implantation using chondrospheres is a safe procedure that yields to significant functional improvement in the 2 years follow up time. An arthrotomy causing prolonged rehabilitation and increased postoperative pain can be avoided. During opera-tion no conversion to open procedure was necessary. The risk of intraoperative spheroid loss can be controlled by an experienced arthroscopic surgeon. Even retropatellar defects can be reached without everting the patella.

12.4.4Effect of gender in the follow-up after matrix-associated autologous chondrocyte transplantation (MACT) as assessed by morphological and biochemical magnetic resonance imagingG.H. Welsch1, S. Werner1, D. Stelzeneder1, T.C. Mamisch2, F. Hennig3, S. Domayer1, L. Zak1, S. Marlovits1, A. Kautzky-Willer1, S. Trattnig1

1Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany

Purpose: By means of MRI, the morphological constitution of the re-pair tissue can be assessed by the magnetic resonance observation of cartilage repair tissue (MOCART) score, whereas the biochemical collagen ultra-structure and the water content can be quantified by T2 mapping. The objective of this study was to determine gender related differences in the repair tissue after MACT as assessed by advanced morphological and biochemical MRI.Methods and Materials: 40 patients (20 female, 20 male) after MACT of one femoral condyle of the knee were included. Both groups were matched by age (female:35.1±13.7; male:35.2±8.7 years) and post-operative follow-up (female:34.0±17.4; male:34.2±16.8 months). At 3.0 Tesla-MRI, morphological MOCART scoring including nine variables was based on high-resolution PD-TSE, Dual-FSE and TIRM sequences; biochemical T2 mapping was based on a multi-echo spin-echo sequence. A zonal (deep and superficial) region-of-inter-est analysis was performed to assess the quantitative T2 values in native control cartilage and cartilage repair tissue.Results: The MOCART score was comparable in female (69.4±20.0) and male (69.3±13.0) patients (p=0.982). Seven of the nine vari-ables did not differ significantly. Whereas the structure of the re-pair tissue was more homogeneous in female patients (p=0.026), changes in the subchondral bone and bone marrow edema were less often visible in male patients (p=0.007). Quantitative T2 values (ms) showed comparable results for the native control cartilage (fe-male: deep=47.7±9.6, superficial=53.1±9.2; male: deep=49.8±12.6, superficial=53.7±10.5; p=0.445(deep), p=0.826(superficial)); whereas the repair tissue showed significant shorter T2 values of the deep cartilage layer in female compared to male patients ((fe-male: deep=43.5±9.8, superficial=48.5±9.8; male: deep=48.2±7.7, superficial=52.6±11.0; p=0.009(deep), p=0.052(superficial)).Conclusions: Our initial results suggest that there are differences in the ultra-structure of the repair tissue between female and male pa-tients after MACT. Based on the morphological and the biochemical results, these differences might be due to changes in the subchon-dral bone and the adjacent deep cartilage layer.

12.4.5Time-line for self-reported changes in function following autologous chondrocyte implantation (ACI) over one yearC. Lattermann, J.S. Howard, J.M. Hoch, C.G. MattacolaLexington/United States of America

Purpose: Functional improvement following ACI is of great interest to clinicians and patients. The purpose of this study is to describe the pat-tern of self-reported recovery for patients undergoing ACI to the femo-ral condyle or patellofemoral joint and provide comparative data for individuals who failed treatment.Methods and Materials: Prospectively, 7 patients(38yrs, 176.5cm, 80.2.4kg) undergoing ACI for chondral defects to the femoral condyles and 10 patients (33yrs, 1746cm, 91.2kg) undergoing ACI for patell-ofemoral defects were enrolled. All patients completed the IKDC and Lysholm Scale preoperatively, 3, 6, and 12 months postoperatively. Re-peated measures ANOVAs were used to evaluate differences between defect locations and between patients considered clinical successes (n=13) or clinical failures(n=4, 2 each location) at 12 months. Results: There were no main effects for location. There were significant differences(p<.05) between scores for successes and failures (Figures 1&2). At 3 months, IKDC scores for successes improved 8% but decreased 2% among failures. At 6 months, IKDC scores in the success group im-proved 27% and decreased 4% in the failure group from preoperative levels. At 12 months success patients improved 35%, while failure pa-tients decreased 2% from preoperative IKDC scores. For Lysholm scores successes improved 20% while failures decreased 3% at 3 months. At 6 months successes improved 38% while failures decreased 2% below preoperative scores. At 12 months, success patients improved 43% and failure patients improved 1% from preoperative Lysholm scores. Conclusions: Using the IKDC and Lysholm scale, unique, although not statistically different, recovery patterns can be observed between fem-oral and patellofemoral ACIs (Figures1&2). While the overall number of failures is small, they demonstrate significantly lower IKDC and Lysh-olm scores as early as 3 months postoperatively. Though there may be some debate regarding when clinical failure occurs, evidence of failure was seen as early as 3 months postoperatively, prompting consider-ation to follow early self-reported outcomes closely.

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12.4.6What is the impact of untreated severe cartilage damage on clinical outcomes and osteoarthritis progression in the knee? A natural history study.W. Widuchowski1, G. Kwiatkowski2, R. Faltus2, A. Mroczka2, J. Widuchowski2 1Katowice/Poland, 2Piekary Śląskie/Poland

Purpose: Deep cartilage lesions, left with no treatment are considered to cause permanent knee deterioration and to advance the develop-ment of osteoarthritis. But, is it always like that? We aimed to evaluate whether and to what extent isolated deep cartilage lesion localized with-in tibiofemoral joint and patellofemoral joint has an impact on the clin-ical outcomes and osteoarthritis progression when it is left untreated. Methods and Materials: 37 patients with single isolated chondral lesion of Outerbridge grade 4 located within weight-bearing ar-eas of femoral and tibial condyles (FT group) and patella (P group) were included in the study. The lesion size ranged from 2 to 4 cm2. In 26 cases (70.3%) the lesion was left without any treatment. In other cases following procedures were performed: debridement of the lesion, loose body removal, shaving. The mean follow-up was 15.3 years. Outcomes were collected reported using Lysholm score, Tegner activity scale and Womac score. Radiographic eval-uation was performed according to Kellgren-Lawrence scale. Results: Osteoarthritic changes were found in 39% patients. There was no difference in OA frequency and severity between injured and unin-jured knee. In patients of FT group there was a relationship between the incidence of tibiofemoral OA and patellofemoral OA (p = 0.00075). Both groups demonstrated good overall clinical results. Worse out-comes according to all scores were observed in P group (p<0.05). Conclusions: Severe isolated single chondral damage left with no treat-ment has a limited influence on clinical outcomes and development of osteoarthritis. Lesions located within patella may predispose to ad-vanced knee deterioration. The overall osteoarthritis progression must be the result of the influence of different local and systemic factors.

12.4.7Correlation of tissue histomorphometry with ICRS histology scores in biopsies obtained from a randomized controlled clinical trial comparing BST-CarGel™ versus microfractureC.D. Hoemann1, N. Tran-Khanh1, S. Methot2, G. Chen1, C. Marchand1, V. Lascau-Coman1, E. Rossomacha2, C. Jarry2, A. Restrepo2, W.D. Stanish3, P. MacDonald4, N. Mohtadi5, P. Marks6, M. Malo1, R. McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10, F. Macule11, M.S. Shive1, M.D. Buschmann1 1Montreal/Canada, 2Laval/Canada, 3Halifax/Canada, 4Winnipeg/Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada, 8Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain, 11Barcelona/Spain

Purpose: BST-CarGel™ (BioSyntech Inc, Laval, Canada) is a novel thera-py where an in situ-solidifying implant of chitosan and autologous blood is applied to fresh microfracture defects, to stabilize the blood clot and stimulate marrow-derived repair. In an 80-patient randomized clinical trial comparing BST-CarGel™ to microfracture to treat condylar lesions, an interim analysis was carried out that included 21 osteochondral bi-opsies obtained from patients with informed consent after 13 months post-treatment. The aim of this study was to correlate repair tissue histological quality with histomorphometric staining characteristics. Methods and Materials: Biopsies were obtained with REB-approved pro-tocols from the middle of the original condylar lesion at 13 months post-treatment (N=21), fixed in formalin, decalcified, processed in paraffin and 5 µm sections stained with H&E and Saf-O/Fast Green (SafO), or immu-nostained for collagen type I (Col1) and collagen type II (Col2). 3 readers carried out analyses on blinded sections including histological scoring (ICRS II Overall on a scale of 0=worst to 100=normal hyaline cartilage) and histomorphometry for each stain (percent stained tissue area). Overall scores were correlated by calculating Pearson’s correlation coefficients. Results: A range of repair quality was obtained that could be classified based on ICRS scores into good/acceptable/poor categories (Fig. 1). A subgroup within the “poor” category for ICRS scores was found to have strong SafO and Col2 staining yet very poor structural integrity. When these 3 disintegrated samples were removed from the analysis, strong correlations were obtained between histomorphometry and ICRS evaluations (Fig. 2). These data showed that overall histological score correlated positively with SafO and Col2 staining and negatively with Col1 (p<0.05).Conclusions: Strong correlation between histological scores and quan-titative histomorphometry in cartilage repair can be obtained when overall tissue integrity is accounted for.

12.4.8BST-CarGel™ treatment shows trend of improved collagen architecture and stratified hyaline structure compared to microfracture in 13 month biopsies from an interim analysis of a randomized controlled clinical trialA. Changoor1, M. Nelea1, N. Tran-Khanh1, S. Methot2, A. Restrepo2, W.D. Stanish3, P. MacDonald4, N. Mohtadi5, P. Marks6, M. Malo1, R. McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10, F. Macule11, M. Shive2, M.D. Buschmann1 1Montreal/Canada, 2Laval/Canada, 3Halifax/Canada, 4Winnipeg/Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada, 8Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain, 11Barcelona/Spain

Purpose: The biodegradable and natural polymer of glucosamine, chi-tosan, can be used to physically stabilize an autologous blood implant above a debrided and microfractured cartilage lesion. This concept is the basis of the BST-CarGel™ product (BioSyntech Inc., Laval, Canada) that has been compared to microfracture in a randomized controlled clinical trial. Here we report biopsy analyses of collagen architecture and zonal stratification using a novel polarization light microscopy (PLM) method.Methods and Materials: An interim analysis was carried out on 21 elective condylar biopsies (12 BST-CarGel™, 9 Microfracture) obtained from the RCT at 13 months post-treatment. They were fixed, decalcified, embedded and sectioned to 5µm. Blinded consecutive sections were de-paraffinized and examined unstained by 3 readers using PLM and Scan-ning Electron Microscopy (SEM) after metallic coating. An ordinal scale to grade collagen architecture and zonal stratification was applied using PLM birefringence characteristics: 0=no organization, 1= vertical deep zone apparent, 2= deep zone well developed, 3= three zones present, 4= normal zonal proportions. Parametric or non-parametric statistical tests were used to compare groups depending on distribution normality. Results: The novel PLM and SEM methods and scores were sensitive to re-pair tissue collagen structure (Fig. 1) A majority of the BST-CarGel™ treated biopsies (7 of 12) revealed a well-organized and stratified collagen architec-ture with a PLM score > 2 (median 2.67) while this desired structure was only occasionally observed with microfracture (2 of 9) (median 1.33) (Fig. 2). Due to small sample sizes (12 BST-CarGel™, 9 Microfracture), the effect of treatment on the PLM score was not statistically significant (p=0.099). Conclusions: Augmentation of bone-marrow derived cartilage re-pair using a chitosan scaffold stabilized autologous blood implant could improve zonal stratification and collagen architecture at 13 months post-treatment compared to microfracture alone. Since collagen structure is the main determinant of tissue durability, this treatment may sustain benefits longer than microfracture.

12.4.9Comparison of the size of the cartilage femoral condyle lesions in CARTIPATCH® phase III clinical trial versus mosaicplastyF. Dubrana1, J. Potel2, C. Bussiere3, E. Servien3, H. Robert4, E. Stindel1

1Brest/France, 2Toulouse/France, 3Lyon/France, 4Mayenne/France

Purpose: The tolerance and efficacy of CARTIPATCH®, autologous chondrocyte implant containing an agarose/alginate gel is fur-ther demonstrated through a prospective, randomized, controlled trial versus mosaicplasty, for treatment of femoral condyle lesions. Methods and Materials: 64 patients, between 18 and 50 years old, with osteocartilaginous single lesion (2.5 to 7.5cm2) are randomized for first-line treatment, either into the CARTIPATCH® or the mosaicplasty arm (Arthrex instruments). CARTIPATCH® superiority is expected on the basis of IKDC subjective scoring at 24 months. For size evaluation (MRI and arthroscopy), the lesions were assimilated to rectangles. Results: 47 patients underwent surgery, 25 in CARTIPATCH® arm and 22 in mosaicplasty arm. The population mean age (15 women, 33 men) was 28 years old. At inclusion, the mean IKDC subjective scores were 41 (26-55) for CARTIPATCH® arm and 36 (18-53) for mosaicplasty arm. In CARTIPATCH® arm, the lesion size, evaluated either by the surgeon or by an independent expert ranged respectively between 2.3-7cm2 (mean: 3.7cm2) and 1.6-4.9cm2 (mean: 2.9cm2, 14 patients). In mosaicplasty arm, the sizes were respectively between 2.5-7.0cm2 (mean: 4.0cm2) and 1.0-5.9cm2 (mean: 3.1cm2, 18 patients). During arthroscopy, the mean size of the lesion was 3.8cm2 for both arms (CARTIPATCH®: 0.5-8.0cm2, 13 patients; mosaicplasty: 2.0-7.0cm2, 11 patients).Conclusions: The preliminary characterization of the trial population shows that a wide range of lesion sizes and associated clinical signs is represented in this young population of patients. MRI evaluation of the lesion size seems to be globally lower when evaluated blindly as compared with the surgeon estimation. The lattest is closer to the size measured during arthroscopy. This hazard was prevented in the ongoing phase III clinical trial CARTIPATCH® versus microfracture as the lesion size is measured during arthroscopy.

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17.1.2ICRS histology scores of biopsies from an interim analysis of a randomized controlled clinical trial show significant improvement in tissue quality at 13 months for BST-CarGel™ versus microfractureS. Méthot1, C.D. Hoemann2, E. Rossomacha1, A. Restrepo1, W.D. Stanish3, P. MacDonald4, N. Mohtadi5, P. Marks6, M. Malo2, R. McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10, F. Macule11, M.S. Shive2, M.D. Buschmann2 1Laval/Canada, 2Montreal/Canada, 3Halifax/Canada, 4Winnipeg/Canada, 5Calgary/Canada, 6Toronto/Canada, 7Vancouver/Canada, 8Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain, 11Barcelona/Spain

Purpose: BST-CarGel™ (BioSyntech Inc., Laval, Canada) is a mix-ture of a physiological solution of the natural polymer chitosan (polyglucosamine) and autologous blood that is applied on top of a debrided and microfractured cartilage lesion to improve cartilage repair from subchondral bone. The purpose of this study was to compare the histological quality of cartilage repair by BST-CarGel™ versus microfracture at 13 months in a randomized clinical trial. Methods and Materials: Voluntary osteochondral biopsies (12 BST-CarGel™, 9 microfracture) from the central region of the original lesion on the femoral condyle were obtained after 13 months post treatment from a total of 41 patients in an interim analysis. They were fixed in For-malin, decalcified, and 5 µm paraffin sections stained with H&E and Saf-O/Fast Green. Both the ICRS I and ICRS II histological scores for car-tilage quality were obtained by 3 readers that were blinded to sample identity. Scores were unblinded and statistically analysed using para-metric or non-parametric tests depending on distribution normality. Results: The ICRS II score contains 14 parameters on a continuous scale and is an expanded version of the ICRS I system which has 6 parameters evaluated on an ordinal scale. Improvements in qual-ity that exceeded 10% (range of improvement 10% - 80%) for the repair tissue of BST-CarGel™ versus microfracture were detected in 10 of the total 20 parameters (Table 1). Only one parameter was better in microfracture (Table 1), while 9 were similar (< 10% difference). Several tissue characteristics were significantly im-proved (p<0.05) for BST-CarGel™ versus microfracture includ-ing the Overall Score and Superficial Zone score from ICRS II and the Surface Score and Cell Viability Score from ICRS I (Figure 1). Conclusions: A statistically significant improvement in the quality of cartilage repair was found in patients treated with BST-CarGel™ com-pared to microfracture, suggesting a greater durability of repair and sustained clinical benefit.

17.1.3Mid-term results of Autologous Matrix Induced Chondrogenesis (AMIC) for treatment of focal cartilage defects in the kneeJ. Gille1, J. Wimmer2, E. Schuseil2, J. Gellissen1, S. Wallstabe1, P. Behrens1

1Hamburg/Germany, 2Lübeck/Germany

Purpose: Autologous Matrix Induced Chondrogenesis (AMIC) is an innovative treatment for localized full-thickness cartilage defects combining the well-known microfracturing with collagen scaffold and fibrin glue. The purpose of this prospective study was to evaluate the medium-term results of this enhanced microfracture technique for the treatment of chondral lesions of the knee.Methods and Materials: Thirty-two chondral lesions in 27 patients were treated with AMIC. Within the context of clinical follow-up, these patients were evaluated for up to 5 years after the intervention. Five dif-ferent scores (Meyer scoreMeyer score, Tegner score, Lysholm score, ICRS score, Cincinatti score) were used for outcome analysis. Articu-lar resurfacing was assessed by magnetic resonance imaging (MRI). Results: The average age of patients (11 females, 16 males) was 37 years (range 16 to 50 years). The mean defect size of the chondral le-sions was 4.2 cm2 (range 1.3 to 8.8 cm2). All defects were classified as grade IV according to the Outerbridge classification. The follow-up period was between 24 months and 62 months with a mean of 37 months. Twenty out of 23 individuals (87%) questioned were subjec-tively highly satisfied with the results after surgery. Significant im-provement (p<0.05) of all scores was observed as early as 12 months after AMIC and further increased values were notable up to 24 months postoperatively. MRI analysis showed moderate to complete filling with a normal to incidentally hyperintense signal in most cases. Conclusions: AMIC is an effective and safe method of treating symp-tomatic full-thickness chondral defects of the knee in appropriately selected cases. However, further studies with long-term follow-up are needed to determine if the grafted area will maintain structural and functional integrity over time.

17.1.4TruFit plugs for cartilage repair in the knee. 4 year results and refining the indicationsT. Spalding1, P. Thompson2, M. Dhillon2, J. Bird2

1Leamington Spa/United Kingdom, 2Coventry/United Kingdom

Purpose: We report the four year results of TruFit plugs for osteo-chondral and chondral repair of articular cartilage defining the indi-cations and outlining the gradual regeneration of articular cartilage surface by ingrowth from the surrounding healthy cartilage and the from the marrow elements Methods and Materials: From a consecutive prospective series of 40 patients, 27 have minimum 2 year followup and form the study group, with clinical scores 6 monthly and MRI scans annually. Mean defect size was 1.8cm square and the mean patient age was 35. Three patients had osteochondral defects. Results: Mean IKDC subjective score improved from 42.5 preop to 69 at 12 months, 76 at 18 months and 81 at 24 months. Tegner ac-tivity score improved from a mean of 2.8 preop to 5.7 at 2 years without deterioration. Lysholm score improved from 53 preop to 83 at 24 months. All results are significant (p<0.05). All modalities of the KOOS score were improved particularly sport and quality of life domains. MRI showed persistence of bone marrow oedema in 20% up to 12 months and gradual remodelling of subchondral bone over 24 months. Second Look arthroscopy showed two patterns of regeneration – ingrowth from surrounding cartilage and from the subchondral bone marrow elements. No patients were revised in this series but two patients developed persistent effusions requir-ing treatment. In both, results improved between 18 and 36 months post implantation. Conclusions: TruFit CB plugs remain soft for up to 9 months and are not indicated where there is no surrounding bone for support. MRI scans show reformation of the subchondral lamina with no bony overgrowth. Results are maintained to 4 years without reduction challenging the results of microfracture which is indicated in similar size lesions. The plugs have defined indications and are not indi-cated for larger areas.

17.1.5New nanostructured biomimetic scaffold for the treatment of ostechondral defects: pilot clinical study at 3 years follow-upE. Kon, G. Filardo, A. Di Martino, S. Patella, S. Zaffagnini, L. D’Orazio, G. Altadonna, M. Marcacci Bologna/Italy

Purpose: The ideal osteochondral graft should be an off-the-shelf product. For the clinical pilot study we performed, a newly devel-oped nanostructured biomimetic scaffold was used to treat chon-dral and osteochondral lesions of the knee; its safety and manage-ability, as much as the surgical procedure reproducibility and the clinical outcome, were evaluated in order to test its intrinsic poten-tial without any cell culture aid.Methods and Materials: 30 patients (9F, 21M, mean age 29,3yy) affected by either chondral or osteochondral lesions of the knee underwent the scaffold implantation. The sizes of the lesions were in between 2 and 6 squared cm. All patients and their clinical out-come were analyzed prospectively at 6, 12, 24 and 36 months using the Cartilage standard Evaluation Form as proposed by ICRS and an high resolution MRI.Results: We observed a statistically significant scores improve-ment and function recovery comparing the pre-operative to the follow-up parameters evaluated. Moreover, we noticed a bet-ter improvement from 12 to 24 months follow-up while the good results gained at 2yy were confirmed at 3yy follow up evalua-tion. The MOCART scoring scale was used to analyze the MRIs. In most of cases we obtained a complete filling of the cartilage de-fect and in some patients we even appreciated articular surface congruency. In this series we report 1 failure followed by a re-oper-ation with different technique.Conclusions: This new minimally invasive one-step surgical approach to osteochondral defects seems to be an easy and effective procedure.

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17.1.6Influence of the cell differentiation at the timepoint of transplantation on the clinical outcome of matrix-associated chondrocyte transplantation (MACT)C. Albrecht, L. Zak, B. Tichy, S. Hosiner, V. Vecsei, S. Marlovits Vienna/Austria

Purpose: Chondrocytes lose their specific properties and become fibroblast-like, when they are propagated in monolayer culture for MACT. This process is called dedifferentiation. A lot of efforts have been made to develop different stratagies in order to redifferenti-ate the cells prior transplantation. The aim of this study has there-fore been to analyse the effect of chondrocyte differentiation at the timepoint of transplantation on the clinical outcome of MACT after 6, 12 and 24 months. Methods and Materials: Gene expression of chondrocyte markers (Col1, Col2, aggrecan, versican and Gdf-5) were analysed in trans-plant samples (HYAFF, Bio-Gide, Cares) of 90 patients by quanti-tative real-time PCR. For the evaluation of cell differentiation two differentiation indices were calculated as the ratio of Col2/Col1 and aggrecan/versican expression. From the same patients clinical scores (Brittberg, IKDC, KOOS, Noyes, Tegner, and VAS pain) were obtained 6, 12 and 24 months after surgery.Results: The three scaffolds significantly differed in gene expres-sion. However, no correlation between the analysed factors and the clinical outcome was found. Also the differentiation indices showed no significant correlation with clinical outcome. Only a slight tendency was found that low matrix production resulted in better IKDC and KOOS scores.Conclusions: The data of the analysed factors demonstrate that the differentiation of cells at the time point of transplantation does not affect the clinical outcome after MACT. In fact other parameters - such as biological conditions in the joint, weight bearing and after-treatment - seem to play a much more important role for the suc-cess of the MACT procedure.

17.1.7Prospective Evaluation of Autologous Chondrocyte Implantation Procedure: Minimum 7-year Follow-UpE. Daley, S. Bajaj, J. Kercher, M. Salata, B.J. ColeChicago/United States of America

Purpose: Autologous chondrocyte implantation (ACI) is a common method for the treatment of full thickness cartilage lesions; how-ever long-term clinical follow-up data is limited. The purpose of this study is to report clinical results following ACI from a single sur-geon at a minimum of 7 years follow-up. Methods and Materials: Data was collected prospectively on 21 pa-tients who underwent ACI by a single surgeon. The mean follow-up time was 8.15 years. Post-operatively, patients were assessed us-ing the International Knee Documentation Committee (IKDC), Tegn-er-Lysholm, Knee Injury and Osteoarthritis Outcome Score (KOOS), and San Francisco – 12 (SF-12) outcome measures.Results: The mean IKDC score improved from 38.82 to 61.65 (p<0.05). The mean Lysholm score showed an increase from 45.78 to 71.31 (p<0.05). SF-12 physical scores improved from 45.06 to 47.48, with SF-12 mental improving from 40.77 to 44.71 (p<0.05). In the KOOS scoring system, pain (53.10 to 73.92), symptoms (49.29 to 71.43), activities of daily living (66.18 to 86.39), and qual-ity of life (61.16 to 81.25) improved. Subjectively, on a scale of 1-10, with 10 being completely satisfied with surgical outcome, the mean post-operative satisfaction rate was 8.18. Additionally, 93% of the patients would elect to have this surgery again if they had the same problem on the opposite knee.Conclusions: The average IKDC, Lysholm, SF-12, and KOOS scores demonstrated improved patient outcomes at a minimum 7-year fol-low up. Based upon this data, ACI is a viable treatment option for patients with symptomatic full thickness cartilage lesions and can reduce pain, increase range of motion and improve patient function.

17.1.8Articular cartilage repair using collagen type I hydrogels (CaReS-technology)– Results of a multicenter studyU. NöthWürzburg/Germany

Purpose: Different technologies are available for matrix-based au-tologous chondrocyte implantation (ACI). Since 2003, over 2000 patients have been treated with a collagen type I hydrogel (CaReS® technology) in Europe. This study investigated the outcome of ar-ticular cartilage repair in the knee using collagen type I hydrogels.Methods and Materials: We performed a prospective multicenter study in 9 centres including 116 patients to test the outcome of the CaReS- technology. The International knee documentation score (IKDC), as well as the patient and doctor satisfaction have been in-vestigate before, 3, 6, 12, 24, 36, 48, and 60 months after surgery. Results: The mean follow-up was 30.7 months with an IKDC of 70.5. Patients with osteochondral defects showed an IKDC of 80.4, pa-tients with chondral lesions only an IKDC of 68.2. Patients with defect sizes more than 4 cm2 showed an IKDC of 72.8, while patients with defects smaller than 4 cm2 showed an IKDC of 69.6. For the defect site, best results were found at the condyles (IKDC 66.7) compared to defects of the patella (IKDC 61.7). The patient and doctor satis-faction showed in 79.4% and 85.3% very good and good results. Conclusions: Patients with osteochondral lesion showed bet-ter clinical results compared to chondral lesions. Concerning the defect size (< 4 cm2 or > 4 cm 2) the results were not statisti-cally significant different. Defects located at the condyles showed significant better results when compared to defects located at the patella. In summary, the results are comparable to those reported with other scaffold materials for ACI.

17.1.9Comparison in clinical outcome of first generation ACT with ACT of third generation with spheroidsT. SchreyerDarmstadt/Germany

Purpose: The purpose of this retrospective study was to ex-amine if the better clinical outcome in patients after ACT with spheroids in arthroscopical technique is due to the new kind of cells (spheroids) or due to the different operations procedure. Therefore we compare the clinical outcome of ACTs with perioste-um flaps to those with spheroids in open procedure and those with spheroids in arthroscopic procedure.Methods and Materials: Between 1998 and 2004 we treat-ed 40 patients with cartilage defects grade 3 -4 acc. to Outerbridge classification with ACT with perosteum flap technique (chondrotransplant, codon)= first generation. Between 2005 and 2009 we treated 15 patients with spheroid tech-nique (chondrosphere, codon, Teltow, Germany) in open procedure and 16 patients with arthroscopic procedure = third generation. We examined after 3 months, 6 months, 12 months and afterwards every 12 months from 3-7 years with 5 different scores (DGKKT, HSS, Lysholm, Tegner and Cincinnati - Score).Results: We show that the improvement in clinical outcome in the arthroscopic group is much faster than in the open spher-oid procedure and much faster than in the periosteum group. The patients of the periosteum group reached almost full score points after 6 monhts, whereas with open spheroid techique and with perois-teum technique it lasted up to two years until the get the same level. Furthermore we can show that the longevity results of spheroid technique are as good as with the periosteum technique as far as we can oversee up to now.Conclusions: We conclude that the big difference in clinical out-come between the three techniques compared results from the different operations technique (arthroscopic - open) and not so much from the different status of the chondrocytes used. The greater number of cells per scm seems to be subordinate. We expect the longtime results at least as good as the ones with the periosteum technique.

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17.2.2Autologous Chondrocyte Implantation for Cartilage Lesions with Malalignment of the Patellofemoral Joint -Long Term ResultsH.S. Vasiliadis1, M. Brittberg2, A. Lindahl3, L. Peterson3 1Ioaninnina/Greece, 2Kungsbacka/Sweden, 3Gothenburg/Sweden

Purpose: The aim of our current study is to present the long term follow up of patients with cartilage lesions of the patellofemoral joint, treated with Autologous Chondrocyte Implantation (ACI) with the use of periosteum.Methods and Materials: 92 patients having patella or trochlea le-sion participated in our study. Lysholm and Tegner-Wallgren ques-tionnaires were filled 12.6 years in average after the surgery. They patients were asked whether they feel better, worse or unchanged compared to previous years and whether they would do the operation again. Complications or subsequent surgeries were also assessed. Results: Tegner-Wallgren score was 7.1, improved by 0.95 com-pared to preoperative values (p=0.01). Lysholm score was 68.1, improved by 9 points in average (p=0.3). Seventy two % of the pa-tients were better or unchanged while 93% would do the operation again. Patients with no kissing lesions appeared to have a better prognosis. Patients with malalignment or instability that had a re-alignment procedure had comparable outcomes to the cases which did not need any additional surgery. Realignment procedures in-creased the incidence of serious complications but they were as-sociated with decreased incidence of periosteal hypertrophy. No association was found between the age at the time of the ACI or the size per lesion and any of the clinical outcomes.Conclusions: It seems that correcting the coexisting background factors with realignment, stabilizing or unloading procedures, along with the treatment of cartilage lesions, is improving the re-sults over time. ACI provides a satisfactory result even for the dif-ficult cases with concomitant patellar instability. Our long term fol-low up study reveals preservation of good results and of high level of patients’ activities, even 10 to 20 years after the implantation, in both isolated trochlea and patella lesions and also in multiple and in kissing lesions where an intervention could be considered as a salvage procedure.

17.2.3Cartilage load before and after opening-wedge high tibial osteotomyM. Lind1, J. Mcclelland2, K. Webster2, J. Wittwer2, T. Whitehead3, J. Feller2

1Aarhus C/Denmark, 2Bundoora/Australia, 3Richmond/Australia

Purpose: Medial opening wedge high tibial osteotomy (MOW-HTO) is widely used in the treatment of medial compartment osteoarthri-tis of the knee. The purpose of the procedure is to alter the coronal alignment of the knee to a slightly valgus alignment, thereby re-ducing medial compartment cartilage loading and symptoms. The aim of this study was to investigate the functional biomechanical consequences of this alteration in alignment.Methods and Materials: Eleven male patients with medial com-partment osteoarthritis underwent 3D gait analysis during level walking before and 12 months after MOW-HTO. Nine male control subjects of a similar age were also tested with the same protocol. Sagittal and coronal angles and moments in both knees were com-pared. Pre- and postoperative radiographic coronal plane align-ment was also measured. Results: Preoperatively the mean maximum varus angle during stance was 13.5. This reduced to 5.4 postoperatively (p = 0.0001) and was not different from control subjects (6.8). The mean maxi-mum adduction moment also reduced from 3.5 to 2.7 (% Bw/ht, p=0.02), compared to 3.6 in control subjects. Interestingly, the adductor moments in the non-operated knee increased postopera-tively. The mean radiological mechanical alignment was changed from 8.7 varus preoperatively to 0.1 valgus postoperatively. Conclusions: Gait analysis is a useful tool for functional assessment following MOW-HTO. The procedure resulted in normalisation of various aspects of dynamic knee function and specifically reduced the adduction moment at the knee, which will reduce cartilage me-dial load and subsequent osteoarthritis development. The finding of an increased adduction moment in the non-operated knee could indicate increased cartilage wear in the non-operated knee which could increase the risk for development of OA in this joint.

17.2.4Patella cartilage – relationship between T2 mapping and measurements of patello-femoral joint alignmentG. Scheurecker1, M. Vlychou2, S. Apprich1, D. Stelzeneder1, A. Messner1, G.H. Welsch1, S. Trattnig1

1Vienna/Austria, 2Larissa/Greece

Purpose: To evaluate the relationship between T2 relaxation times of patella cartilage and the sulcus angle and the patella tilt angle Methods and Materials: IRB approval and informed consent was obtained. Twenty subjects (mean age 27.7y, range 16 - 48y; 10 fe-males, 10 males) with an ICRS grade 0 cartilage on the whole patella based on MRI examination. All subjects were examined on a 3T MRI unit. We employed an axial multi-echo spin-echo sequence for T2 mapping. The T2 maps were analyzed with ROIs for both the deep and superficial cartilage layer of the medial and lateral patella facet. We further used an isotropic sagittal PDw sequence for morpholog-ic analysis of the cartilage according to the ICRS criteria and for vol-umetric measurements of the sulcus angle of the trochlea and the patella tilt angle, the latter defined as angle between the posterior condyle line of the femur and the bony medial-lateral axis of the pa-tella. Linear regression was used to look for trends in the relationship between T2 relaxation times and the sulcus or the patella tilt angle. Results: The regression lines show trends for increased T2 relax-ation times of patella cartilage with both higher angles of the sulcus and the patella tilt. These trends were stronger for the lateral versus the medial facet, the deep versus the superficial layer of the lateral facet, and the superficial versus the deep layer of the medial facet. Conclusions: Increased angles of patello-femoral joint alignment tend to put more strain on the cartilage of the patella as quantified with T2 mapping.

17.2.5Psychometric evaluation of the Knee Injury and Osteoarthritis Outcome Score (KOOS) subscales for patients with articular cartilage lesions of the knee using pilot study data of the Cartilage Autograft Implantation System (CAIS)L. Nelson1, L.D. McLeod1, M.M. Mordin1, J. Farr2, B.J. Cole3, S. Uddin4, L. Engelhart5 1Research Triangle Park/United States of America, 2Indianapolis/United States of America, 3Chicago/United States of America, 4Raynham/United States of America, 5Milford/United States of America

Purpose: Changes in symptoms, pain, and functioning as mea-sured by the KOOS subscales are important endpoints in clinical trials evaluating treatment effectiveness for knee-related injuries and disease. Psychometric analyses were conducted to provide evidence of the reliability, validity, responsiveness, and minimal important difference estimates for the five KOOS subscales (i.e., Symptoms, Pain, Activities of Daily Living, Sports/Recreation, and Quality of Life) for use in clinical studies evaluating patients with articular cartilage lesions. Methods and Materials: Two multicenter, randomized pilot studies evaluated the safety and performance of the Cartilage Autograft Implantation System (CAIS) for primary surgical treatment of ar-ticular cartilage lesions of the knee (ranging from ≥ 1 cm2 to ≤ 10 cm2). A total of 54 patients aged 18 to 55 years with articular dam-age on the femoral condyle were randomized to receive the CAIS or a microfracture procedure. Data were collected prior to surgery and at seven post-surgical follow-up visits for up to 12 months. The internal consistency reliability, construct validity, responsiveness, discriminating ability, and interpretation statistics were evaluated for all KOOS subscales. Results: Participants were predominantly male (59.3%), white (92.6%) with an average age of 34.1 years. The KOOS subscales showed excellent internal consistency reliability, ranging from 0.74 to 0.97 at baseline. Construct validity results were supportive of hypothesized relationships, with statistically significant correla-tions (r ≥ 0.50) in the expected direction. Responsiveness analy-ses demonstrated highly satisfactory sensitivity to change, with standardized response means ranging from 0.8 to 1.2. Discriminant tests confirmed the utility of the KOOS subscales. Minimal impor-tant differences were approximately 10 points for each subscale. Conclusions: Despite the small sample size, the psychometric anal-yses strongly support the reliability, validity, responsiveness, and utility of the KOOS subscales in assessing knee-specific improve-ments in patients after receiving CAIS or microfracture surgery for treatment of articular cartilage lesions.

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17.2.6Improved life quality in patients with hip Osteoarthritis treated with an implant of cartilage artifical matrixE. Hermida, L.F. Hermida Mexico City/Mexico

Purpose: The purpose of this study is to evaluate the clini-cal outcome of patients with hip Osteoarthritis (OA) after treat-ment with an intraarticular implant based on cartilage matrix. Another purpose of this study is to evaluate if a lower cost, less invasive, ambulatory procedure is a strong option for patients with I-II hip OA. Methods and Materials: This is a prospective study of 30 patients with II hip OA that have been treated with NSAID, weight loss and physical therapy without a good outcome. The treatment consisted in 5 hip intraarticular injections with a mixture of chondroitin sulfate and sodium hyaluronate sulfate as follows: day 0,15,30,60 and 90. This was done in the office. No NSAID was allowed during the treat-ment, just acetaminophen like analgesic. The follow up was 2 years. We evaluated the outcome with a patient satisfaction questionare and the Harris hip score, at the beginning and at the end of follow up. Results: Of the 30 patients, 2 failed to final follow up. Of the 28 pa-tients followed, 22 were women and 6 men. Average age was 58.4 years old. The final follow up was 2 years after the first injection and a physical exam was performed at the office in order to asses the outcome. Initial Harris Hip Score was 53.25 and at final follow up was 87.05. Regarding the satisfaction questionare 92% would definitely go through the treatment all over again. Conclusions: Although improvement in the range of mo-tion was not constant in our study (only 40% improved range of motion), the pain improvement of the hip was present in all but 1 patient of the study. The fundamental difference lies in the medium and long term response given by the chondro-genic induction and Stromelysin enzyme synthesis inhibition with the implantation of an artificial matrix like device.

17.2.7Preliminary result of repairing articular cartilage defect by autogenous chondrocyte implantation: the feasibility study of a novel biphasic osteochondral compositeH. Chiang1, C. Jiang1, C. Liao2, C. Shen1, C. Shieh1, Y. Huang1 1Taipei/Taiwan, 2Hsinchu/Taiwan

Purpose: Autologous chondrocyte implantation (ACI) has been re-cently used to treat cartilage defects. We had previously developed a biphasic osteochondral composite as the matrix for ACI. We fur-ther conducted this feasibility study of such device to treat patients with osteochondral lesion of the knee joints. Methods and Materials: Nine patients with symptomatic isolated osteochondritis at the femoral condyle were treated by replacing the pathological tissue with autologous chondrocyte-laden bipha-sic cylindrical plug of DL-poly-lactide-co-glycolide, with its lower body impregnated with b-tricalcium phosphate as the osseous phase. The osteochondral lesion was drilled to fashion a pit of iden-tical size and shape as the plug. The chondrocyte-laden plug was inserted by press-fitting to fill the pit. Outcome of repair was exam-ined by functional evaluation with KOOS score at 3 and 6 months postoperatively, and tissue sample was collected with second-look arthroscopic needle-biopsy at 12 months. The primary outcome parameter was the postoperative change of KOOS score; and the secondary outcome parameter was the regeneration of cancellous bone and hyaline cartilage, in their respective phases, at the repair site. Mean KOOS scores were compared with paired t-test. Results: The mean KOOS score at 3 months postoperatively sig-nificantly improved than the pre-operative baseline. The score kept improving thereafter, but the change from 3 to 6 months postop-eratively was insignificant. At 12 months, gross appearance of the repair site under arthroscopy showed full-filling of the grafted site, with the surface of regenerate cartilage flush with the surrounding native joint surface. Microscopic examination of the regenerate tis-sue showed formation of hyaline cartilage. Conclusions: The preliminary result of ACI with the novel biphasic matrix showed successful regeneration of hyaline cartilage and cancellous bone to repair the osteochondral lesion. The matrix was feasible for ACI to treat such lesion in the femoral condyle.

17.2.8Treatment of osteochondritis dissecans of the knee by arthroscopic one-step repair technique with bone marrow-derived cellsM. Cavallo, F. Vannini, R. Buda, A. Ruffilli, R. Ghermandi, C. Cavallo, B. Grigolo, S. Giannini Bologna/Italy

Purpose: Etiology of osteochondritis dissecans (OCD) of the knee is still unclear, recognizing a predisposition due to areas of hypo-vascularization. Although non-surgical management is still a good option, surgery is often required. Good results were obtained by fixation of the detached fragment, or by Mosaicplasty and Autolo-gous Chondrocytes Implantation when a reconstructive procedure was required. Arthroscopic One step repair technique with bone marrow derived cells was previously used with satisfactory re-sults. This option may be particularly suitable for OCD treatment, since the bone marrow contains different types of cells, including hemopoietic precursors, useful for neo-angiogenesis. Aim of this study is to present the application of the “one-step” technique for the treatment of OCD of the knee and evaluate the results.Methods and Materials: From April 2006 to May 2007 6 patients with OCD underwent the One-step procedure. The mean age was 19 yrs (15-24); 4 cases affected the medial condyle and 2 cases the lat-eral condyle. Bone-marrow was harvested from the posterior iliac crest, and the cells were concentrated in the operating room and implanted at the lesion site on a hyaluronan-based scaffold after detached osteochondral fragment removal. Platelet Rich Fibrin was added providing growth factors.Results: The mean preoperative IKDC score was 48±7. The IKDC at 6 months follow-up was 57±6, at 12 months was 83±7 and at 24 months was 90±5. The control MRI at 12 and 24 months follow-up showed a good regeneration of the subchondral bone and the carti-laginous tissue. A biopsy of the regenerated tissue performed at 12 months showed a cartilaginous tissue in organization and a newly formed subchondral bone.Conclusions: These results demonstrated that the one-step tech-nique represents a good option for the treatment of OCD in the knee joint. Further studies are necessary to confirm the angioge-netic effect of the multipotent cells present in the bone marrow.

17.2.9Effect of hyperbaric oxygen and compression on chondrocyte proliferationB. Sievers1, N. Hoechsmann1, F. Dueren2, C. Melcher1, S. Mayer1, P.E. Müller1 1München/Germany, 2Bernau-Felden/Germany

Purpose: Hyperbaric Oxygen (HBO) has been recognized for de-cades as an appropriate treatment modality for more than a dozen clinical conditions ranging from decompression-sickness and arte-rial gas embolism to the treatment of wound problems. Further-more in in vivo examinations the administration of HBO has also been shown to have a protective effect on chondrocytes in carti-lage regeneration. However, the protective in vivo effect of HBO treatment on chondrocytes has not been examined in vitro. The purpose of this study was to examine the effect of HBO treatment on chondrocytes in an in vitro cell culture model concerning growth and gene expression pattern.Methods and Materials: Chondrocytes from the 2 passage were transferred to a HBO chamber and exposed daily to 100% oxygen for 7 consecutive days. Compressions of 1 and 2atm were used. A WST-1 assay was performed at 1,3,5, and 7 days after HBO treat-ment. Gene expression pattern of apoptosis markers as well as car-tilage specific proteins were detected by real-time-PCR.Results: In vitro administration of HBO inhibited growth of chon-drocytes. When the applied compression was increased up to 2atm the amount of chondrocytes was reduced by half at days 3 and 7. In association an up regulation of the apoptosis markers PARP and Casp3 was observed and an increase of cartilage specific proteins Collagen II and COMP was detected. No significant differences were monitored between the different times of daily treatment.Conclusions: In this study the growth of chondrocytes was in-hibited in vitro by HBO treatment. This inhibitory effect was in-creased by elevating the applied compression. The molecular re-sults showed that the administration of HBO may lead to reduced chondrocytes`growth due to apoptosis. However the increased compression induced the expression of cartilage specific proteins, which might cause a redifferentiation of chondrocytes.

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17.3.2Human cultured fibroblasts isolated from anterior cruciate ligament have different growth rates depending on the age of the patient and the type of lesionP. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos, M. Guillen-Vicente, E. Santos, F. Garcia-Gomez, T. Fernandez-Jaen, J.M. Lopez-Alcorocho Madrid/Spain

Purpose: In the last years, a common approach for replacement of injured tissue involves cell therapy. An example of a clinical appli-cation that may benefit from these techniques is the reconstruction of anterior cruciate ligament (ACL). With this aim, some preliminary studies have been carried-out in animal models with two main ap-proaches: a) to determine the efficacy of cells from different sourc-es and b) the use of new biomaterials combined with cultured cells to promote the ACL repair by generating a new tissue with the ad-equate biomechanical properties. The present study compares the features and behaviour of monolayer cultured fibroblasts extracted from ACL of patients with different ages and types of lesion. Methods and Materials: ACL biopsies from 100 patients with ACL rupture. Forty nine patients had acute ACL rupture (elapsed time from rupture to surgery shorter than 1 month), twenty nine had chronic ACL (elapsed time from rupture to surgery longer than 1 month). Normal ACL samples from 22 patients were included as controls. The median age of these 62 patients was 35 years (range: 18 – 52 years). Fibroblasts were isolated; the number of cells was estimated and cultured in monolayer. Results: The fibroblast culture could be established in all cases. First passage was done at the day 15th and the mean growth rate was dif-ferent among the three groups: 108.3 ± 47.7 in acute lesions, 79.5 ± 65.5 in healthy ACL and 76.6 ± 41.7 in chronic lesions. Statistical anal-ysis showed that differences were significant. Negative correlation was observed between the age of the patient and the growth rate. Conclusions: The growth rate of ACL fibroblast in culture is higher in young patients and in patients with acute ACL rupture.

17.3.3Long-term clinical and radiological follow-up of viable meniscal allografts.P.C. Verdonk1, J. van der Maas2, T. Tampere2, K.F. Almqvist2, R. Verdonk2

1Gent-Zwijnaarde/Belgium, 2Gent/Belgium

Purpose: There is growing evidence in literature that meniscal allograft transplantation performed with the right indications results in signifi-cant pain relief and functional improvement of the involved joint. Long-term data on clinical and radiological outcome are however scarce. Methods and Materials: We evaluated 89 transplants (53 lateral and 36 medial) in 87 patients. Mean time of follow-up was 15,5 ± 2,85 years (range 9,9 - 20,4), mean age at surgery was 35,2 years (range 22 - 50). Clinically, the patients were evaluated using a KOOS, SF-36, HSS, VAS, Tegner and Lysholm score. HSS scores were compared to pre-operative and mid-term follow-up data. Each patient received radiographs (AP, profile and Rosenberg view). Ra-diological outcome parameters were joint space width narrowing and Fairbank changes and were scored according to IKDC. Failures were defined as patients who were converted to an arthroplasty. Results: HSS-scores improved significantly from 119 ± 27 pre-oper-atively to 160 ± 40 at long-term follow-up. Lysholm-score was 69 ± 22, which was defined as a fair result. Mean VAS-score was 3,4 ± 3, mean Tegner was 4 ± 2. There were no significant differences be-tween following subgroups: left or right knee, medial or lateral al-lograft, combined procedure with a high tibial osteotomy and male or female. Nine (25%) of the thirty-six medial and ten (19%) of the fifty-three lateral grafts failed after a mean of 9,9 years.Conclusions: Transplantation of a viable meniscal allograft can significantly relieve pain and improve function of the knee joint. Survival analysis showed that this beneficial effect remained in ap-proximately 70% of the patients at fifteen years. This study proves that meniscal allograft transplantation is a beneficial procedure to postpone total knee arthroplasty for more than 10 years in young active patients.

17.3.4The suture of menisceal tears in the avascular zone enforced by a collagen I/III membrane: Clinical experience over 6 years and animal experimentsR. Jakob1, M. Jacobi1, D. Nesic2, P. Mainil-Varlet2 1Fribourg/Switzerland, 2Bern/Switzerland

Purpose: Due to the limited healing potential of meniscal tissue differ-ent efforts have been done to enhance healing of sutured meniscus. Methods and Materials: The meniscus wrap technique is based on the experience with a technique developed by the senior au-thor (RPJ) and first applied in 2003. Wrapping the meniscus with a collagen I/III matrix might create some kind of bioreactor, guiding cell ingrowth and improving suture stability. Thirty patients with tears in the red-white or white-white zone, complex tears, delayed traumatic tears with degenerative aspects, or repeat sutures, were treated with this technique.Results: After a mean follow-up of 2.5 years (range 1-5) three pa-tients had a symptomatic failure (10%). In two of them partial me-niscectomy was performed and in the other patient (a 20-year-old female with a second suture of a bucket-handle tear) a third suture combined with an unloading osteotomy was performed, which ul-timately led to clinical meniscal healing. All other 27 cases (90%) were asymptomatic. Additionally, the following complications were noted: arthrofibrosis requiring mobilization under anaesthesia (one patient), saphenous nerve entrapment necessitating revision (one patient), and ACL rerupture after reconstruction and a new trauma, with the meniscus remaining intact (one patient).Results of an animal experimental study (goats) shall be reported Conclusions: This repair enhancement technique seems to improve the chances of healing, even in unfavourable conditions. Although the evaluation did not include a second-look arthroscopy, 90% of patients remained asymptomatic after a mean follow-up of 2.5 years. Similar to fascia sheath coverage, this technique has the dis-advantage of being technically demanding and time-consuming.

17.3.5TKAMen: A new source for meniscus reconstructionT. Brune, C. Martin, L. Barnouin Mions/France

Purpose: Allogeneic transplants and substitutes produced in vari-ous biomaterials are currently used for partial or complete menis-cus reconstruction. To combine the advantages of these two so-lutions (complex matrix structure of allografts and good handling and safety of substitutes), we explored the opportunity of using decellularized allogeneic menisci. So as to get a continuous sup-ply we studied the possibility of considering lateral menisci derived from total knee arthroplasty (TKAMen) as a potential source.Methods and Materials: Relying on over 15 years experience in tis-sue banking, we started in 2006 collecting TKAMen. Around 110 tissues were already collected, fully characterized and classified in 3 different groups according to criteria describing their integrity and surfaces aspect. To assess the relevance of our approach, we compared these macroscopic observations with histological and mechanical properties for a group of 16 menisci. We determined their Young modulus by using a compression-testing machine, while histological sections underwent a zone specific analysis after standard haematoxylin-eosin staining.Results: According to our classification, 29% of the tissues are con-sidered as slightly osteoarthritic, 53% moderately osteoarthritic and 18% belong to the severely osteoarthritic group. Regarding the mechanical resistance, we showed a significant decrease of the mean Young modulus for the severely osteoarthritic tissues (10,62 +/-11,09 N/mm2) compared to both other groups (49,93 +/-8,25 N/mm2 and 41,54 +/-11,39 N/mm2). Menisci from slightly and mod-erately osteoarthritic groups displayed a histological pattern simi-lar to normal tissues or those derived from multi-organ retrievals. Conversely, severely osteoarthritic TKAMen presented many de-generative signs, correlating with the macroscopic observations.Conclusions: Comparing macroscopic observations with mechani-cal and histological data, we demonstrated the relevance of select-ing tissues according to well-defined morphological criteria. Our results showed that 80% of TKAMen display high mechanical prop-erties and a histological pattern similar to healthy tissues. Further studies will now investigate the possibility to use them for menis-cus reconstruction.

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17.3.6Lateral Collagen Meniscus Implant (Menaflex): Prospective European Multicenter Study with 2 Year Minimum Follow-UpJ.C. Monllau1, R. Crespo2, S. Zaffagnini3, M. Marcacci3, R. Berbig4, D.G. Holsten5, P. Bulgheroni6, K. Lagae7 1Barcelona/Spain, 2Alcazar de San Juan/Spain, 3Bologna/Italy, 4Zürich/Switzerland, 5Koblenz/Germany, 6Varese/Italy, 7Antwerp/Belgium

Purpose: Our purpose was to determine if Menaflex Collagen Menis-cus Implants are safe and effective to treat loss of lateral meniscus tissue and if the implant functions whether or not tissue is missing at the popliteal hiatus. Methods and Materials: 60 patients with irreparable lateral meniscus tears or meniscus loss requiring surgical treatment were enrolled at 7 European centers. Patients were deemed “acute” (no prior surgery) or “chronic” (1 to 3 prior surgeries on the involved meniscus). Inclusion criteria included stable knee ligaments, neutral knee alignment and no untreated Grade IV chondral defects. Menaflex implantation was performed arthroscopically with all-inside suture fixation +/- inside-out sutures anteriorly. Patients underwent standardized rehabilitation with clinical follow-ups (FU) at 6, 12, and 24 months after implantation.Results: 49 patients (mean age 30.5 years) underwent lateral Mena-flex implantation. All patients had minimum 24 months FU (mean, 34 months). 11 (22%) patients were acute and 38 (78%) were chronic. 11 (22%) patients had no tissue spanning the popliteal hiatus AT TIME OF Menaflex implantation. All patients returned to activities of daily living 3 months after surgery. Postoperative scores improved significantly at 1 and 2 years from preoperative. Mean Tegner scores improved from 3.0 to 5.6 at 2 years, mean Lysholm improved from 63 to 93, mean pain decreased from 36 to 7, and 94% were satisfied. MRIs revealed no changes to articular surfaces or joint space height postoperatively; however, the new tissue was not yet mature. Popliteal hiatus tissue deficiency did not influence device placement or clinical outcomes. 5 patients (10%) underwent reoperation due to pain and swelling. Conclusions: At 2-year FU, 90% of patients had improved clinical out-comes compared to preoperative. Menaflex reoperation rates were similar to reported lateral meniscus repair. Longer FU continues in or-der to determine extent and duration of the benefits.

17.3.7Feasibility study of adapting the delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC) technique for the meniscusJ.E.J. Bekkers1, B. Claassen1, L.B. Creemers1, W.J.A. Dhert1, A.P. Hollander2, D.B. Saris1

1Utrecht/Netherlands, 2Bristol/United Kingdom

Purpose: The dGEMRIC technique has successfully been applied to quantify the quality of articular cartilage. This study analyzed the feasibility of delayed gadolinium enhanced MRI of meniscal carti-lage (dGEMRIM).Methods and Materials: From Feb-Dec 2009, a total of 30 patients underwent a dGEMRIC for articular cartilage pathology. Magnevist (Bayer, Germany) was injected 90 min prior to scanning (0.2 mmol/kg). A 3D T1 MRI protocol, at 1.5T, with 5 inversion times (50, 150, 350, 650 and 1650 ms) and 3 mm slice thickness and sagittal image acquisi-tion was used. The T1gd (dGEMRIM index) was calculated by averag-ing the T1gd across the manually segmented region-of-interest (ROI) using in-house developed software. A total of five different ROIs were defined for both lateral and medial menisci (Figure1). The menisci were defined as healthy, degenerated or meniscectomized by con-ventional MRI and arthroscopy. Statistical analysis was performed by Pearson correlations and one-way ANOVAs with post-hoc LSD tests. Results: The meniscus T1gd showed good correlations to the medial (R=0.699, p=0.001) and lateral (R=0.729, p=0.000) femur cartilage T1gd. The meniscus as a whole showed no difference (p>0.279) in the T1gd between those defined healthy or unhealthy. In most types of menisci there were statistically significant differences in T1gd between the inner and outer (p=0.064) and upper and lower (p=0.002) regions. Healthy and degenerate menisci also showed a statistically significant difference in dGEMRIM index between me-dial and lateral compartments (p=0.017), however these compart-ments did not differ after medial meniscectomy (p=0.847). Conclusions: The meniscus quality (dGEMRIM) shows good cor-relation with dGEMRIC. The varying T1gd across healthy and de-generated menisci indicates a heterogeneous glycosaminoglycan distribution within the tissue. The decrease in T1gd after medial meniscectomy, compared to healthy lateral menisci, could be due to leakage of glycosaminoglycans. We conclude that the dGEMRIM technique offers a new approach to assessing degeneration of the meniscus after meniscectomy.

17.3.8New process for the production of an acellular meniscus scaffoldT. Brune, A. Schirlin, C. Martin, L. Barnouin Mions/France

Purpose: Allogeneic transplants and substitutes produced in vari-ous biomaterials are currently used for meniscus reconstruction. To combine the advantages of these two solutions we studied the possibility of developing an acellular meniscus scaffold. Therefore we compared the effect of several chemical treatments in combina-tion with freeze-drying and gamma radiation to find out a process maintaining the extracellular matrix complexity and leading to a safe product, sterile and ready to use.Methods and Materials: Initially, selected chemicals were tested to remove peripheral tissues as part of a preliminary cleaning. Then we analyzed the capacity of different chemicals to partly extract gly-cosaminoglycans (GAG) and eliminate cell residues while preserving the complex matrix structure of the tissue. This was mainly achieved by performing biochemical assays (hydroxiproline measurement and GAG release) and wide-ranging histological studies including Masson’s trichrome, Feulgen-Rossenbeck and Safranin O staining. We also addressed the ability of these treatments to inactivate well-defined viruses. Finally we considered modifications of the menisci size and weight as a consequence of the process.Results: Our results suggest that ethanol offers a satisfying solu-tion to clean out the tissues. Histological data showed that sodium hydroxide in combination with hydrogen peroxide promotes a com-plete meniscus decellularization without denaturing collagen struc-ture, similar to what was obtained with sodium dodecyl sulfate. Preliminary data also indicated that such a combination turned out to be effective to inactivate viruses. Moreover the whole process, especially freeze-drying and sterilization, severely affected menisci weight and dimensions. However the tissues recovered their initial physical characteristics following extended rehydration.Conclusions: Based on a combination of biochemical data, macro-scopic and histological observations we demonstrated that there are several alternatives to process successfully menisci with the intention of getting them acellular. In the ongoing studies we are now investigating the consequences of such processes in terms of biocompatibility and alteration of the mechanical properties.

17.3.9An Ex Vivo Organ Culturing and Loading System for Large Species Intervertebral Discs; a Feasibily Study With Caprine Lumbar Discs.C.P.L. Paul, H.A. Zuiderbaan, B. Zandieh Doulabi, A.J. Veen van der, T.H. Smit, M. Helder, B.J. Royen van, M.G. Mullender Amsterdam/Netherlands

Purpose: Mechanical loading is a natural stimulus to chondrocytes and regarded essential for maintenance and repair of cartilage ex-tracellular matrix (ECM), yet loading is considered a major extrinsic component in degenerative disc disease (DDD). To further investi-gate the interaction between mechanical loading and cell and ma-trix response we have developed the Loaded Disc Culture System (LDCS); this ex vivo bioreactor conserves IVD cells in their native environment and allows close control of tissue loading conditions. The purpose of the current study is to investigate the feasibility of culturing goat IVDs with maintenance of their native cellular and ex-tracellular properties in the LDCS over a 7- and 14-day culture period. Methods and Materials: IVD’s (L1-6; N=40) were harvested from goats (N=8) under sterile conditions. IVD’s were cultured in the LDCS either without loading or with a simulated diurnal physiologi-cal load. Cell viability was assessed in the nucleus (NP) and annu-lus (AF) regions. Also, water, glycosaminoglycan (GAG) and total collagen content of the extracellular matrix (ECM) were measured. Results: Fresh discs (day 0) displayed a mean cell viability of 79.4% (NP) and 77.7% (AF). At 7 and 14 days, cell viability of the unloaded group dropped by half in both NP and AF, while physiological load-ing maintained cell viability (fig. 1). In the ECM, water-, GAG- and collagen content of the physiologically loaded IVD’s did not change significantly after 7 and 14 days, when compared to day 0 (fig. 2). Conclusions: We were able to maintain the native properties of goat discs in the LDCS over a 14-day culture period by applying a simulated diurnal physiological load. The LDCS may serve as a valu-able platform to study the processes involved in degenerative disc disease (DDD) along with new treatment strategies for cartilage repair. Acknowledgements: This study was supported by ZonMw grant number 11400090. No conflict of interest.

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17.4.2Local anesthetics exposure leads to mitochondrial oxidative stress and oxidative damage in human chondrocytes.V. Grishko1, A.W. Pearsall, IV2, G.L. Wilson2

136688/United States of America, 2Mobile/United States of America

Purpose: Purpose: Several different mechanisms have been pro-posed to explain toxicity of local anesthetics including the block-ade of potassium channels and mitochondrial injury. The purpose of this study was to investigate whether oxidative stress is involved in toxic effects of lidocaine, bupivacaine, and ropivacaine on nor-mal and OA chondrocytes. Methods and Materials: Materials and Methods: Primary chondro-cytes cultures, generated from cartilage from patients undergoing total knee replacement and normal donors, were exposed for 1 hr to saline, 2% and 1% of lidocaine, 0.5% and 0.25% of bupivacaine, 0.5% and 0.2% of ropivacaine. Enhanced mitochondrial superoxide production, oxidative damage to mitochondrial DNA and mitochon-drial proteins were evaluated immediately after exposure or follow-ing 72 h of recovery. Mito SOX Red fluorescent dye, highly selective for mitochondrial superoxide, was used to study the mitochondrial ROS levels. Protein carbonylation as the result of oxidative stress was studied by Protein Carbonyl assay. Oxidative damage to mtD-NA was evaluated using Quantitative Southern blot analysis. Results: Results: When normal and OA chondrocytes were exposed to local anesthetics, only small amount of mitochondrial ROS was accumulated in only OA chondrocytes immediately after exposure. Following 72 h after treatment, mitochondrial ROS production was enhanced. OA chondrocytes exhibited significantly higher levels of mitochondrial superoxide compare to normal donors. Enhanced mi-tochondrial ROS production in OA chondrocytes dose-dependently correlated with oxidative damage to mitochondrial DNA and proteins. Conclusions: Conclusion: The present results demonstrate for the first time that mitochondrial oxidative stress is involved in toxic ef-fects of local anesthetics on human chondrocytes. Moreover, OA chondrocytes exhibit higher susceptibility to this oxidative stress then chondrocytes obtained from normal donors. These differenc-es are likely due to already compromised mitochondrial function and mitochondrial damage in OA chondrocytes.

17.4.3Matrilin-1 and matrilin-4: new disease causing loci for multiple epiphyseal dysplasia?M. Ristiluoma1, A. Aszodi2

1Oulu/Finland, 2Martinsried/Germany

Purpose: Matrilins (matrilin1-4) are adaptor proteins of connective tissues and mutations in the matrilin-3 gene (MATN3) are associ-ated with multiple epiphyseal dysplasia (MED), a skeletal disorder characterized by mild dwarfism and early-onset osteoarthritis. Most MATN3 MED mutations are missense and cause the retention of the missfolded protein in the rER. MED is genetically heteroge-neous, and in addition to MATN3, mutations in the genes encoding COMP, COL9A1-A3 and DTDST have been identified, however, the causative gene in about 50%-80% of the cases is not known. Tak-ing into account the conserved structure of matrilins, MATN1 and MATN4 are good candidates for additional MED loci.Methods and Materials: Human MATN3-like MED mutations were generated into conserved sites of tagged mouse Matn1, Matn3 and Matn4 cDNAs and transfected into 293-EBNA cells or primary chondrocytes. Cellular and medium samples were collected and analyzed by Western blot, or the cells were immunostained using matrilin- or tag-specific antibodies.Results: Wild type and polymorph Matn1, Matn3 and Matn3 con-structs were expressed at high levels in 293-EBNA cells and were efficiently secreted. MED-type Matn3 mutations were expressed at lower levels and displayed secretion defects. MATN-3 MED muta-tions in Matn1 and Matn4 exhibited three secretion patterns: most mutant proteins were 1) completely missing in the medium or 2) only moderately secreted; whereas a few 3) were apparently nor-mally deposited into the supernatant. In chondrocytes, the MATN3 A219D-equivalent Matn1 and Matn4 mutations were present in the cell fraction but were absent in the medium. Immunofluorescence analysis revealed that while control constructs were clearly visible extracellularly, most mutant proteins retained in the cell and ac-cumulated in the rER. Conclusions: Most MATN3-MED mutations in Matn1 and Matn4 lead to secretion defect and accumulation of the mutant proteins in the ER. These results imply the involvement of MATN1 and MATN4 in MED via a similar pathomechanism as described for MATN3.

17.4.4Obesity affects the chondrocyte responsiveness to leptin in patients with osteoarthritisP. Francin1, S. Pallu2, C. Guillaume1, P. Pottie1, D. Mainard3, N. Presle1

1Vandoeuvre les Nancy/France, 2Orleans/France, 3Nancy/France

Purpose: As hyperleptinemia disrupt the physiological function of the adipokine in obese individuals, the current study has been un-dertaken to determine whether elevated leptin levels found in the joint from patients with osteoarthritis (OA) induces also a defect in leptin action in chondrocyte.Methods and Materials: Chondrocytes isolated from OA patients with various body mass index (BMI) were treated with 100 or 500 ng/ml of leptin. The expression of cartilage-specific components (aggrecan, type 2 collagen), as well as regulating factors (IGF-1, TGFβ, MMP-13, TIMP‑2) was investigated by real-time PCR to evaluate chondrocyte responsiveness to leptin.Results: The leptin activity was shown to be critically dependent on both the concentration and the BMI of the patients. A negative as-sociation between the activation of regulated genes and BMI was found for the lowest dose of adipokine. Conversely, the cell respon-siveness increased with the BMI when 500 ng/ml of leptin was used to stimulate chondrocytes. Additionally, chondrocytes collected from normal or overweight patients displayed most responsive-ness with the low concentration of leptin while an elevated dose of leptin was required to change gene expression in cells obtained from obese patients. Interestingly, the gene encoding MMP-13 was identified as a target of leptin for obese patients only. Beside, the expression of TIMP-2 was up-regulated in leptin-treated cells ob-tained from OA patients with the lowest BMI.Conclusions: Our results clearly showed that chondrocytes from obese OA patients behave differently from chondrocytes isolated from normal or overweight patients. The cells from obese patients required elevated levels of leptin to be responsive and the BMI-de-pendent effect of leptin on the expression of TIMP-2 and MMP-13 may explain why obesity is associated with an increased risk for OA.

17.4.5Autologous Matrix-Induced Chondrogenesis (AMIC) on the patella plus periosteal coverage on the trochlea combined with mechanical realignment- A New Treatment Option in Symptomatic Isolated Femoropatellar Osteoarthritis due to Subluxation of the PatellaT. Kusano, C. Marti, M. Jacobi, R. Jakob Fribourg/Switzerland

Purpose: Symptomatic isolated femoropatellar osteoarthritis is re-ported for 8% of women and 2% of men over the age of 55 years. Surgical options include osteotomy of the tibial tubercle with patel-lar debridement and realignment (1), patellar hemiarthroplasty and femoropatellar arthroplasty and patellectomy. We present our re-sults of a new biological treatment option, in which (1) is combined with the AMIC procedure (autologous matrix-induced chondro-genesis) and periosteal resurfacing of the lateral trochlea femoris. Methods and Materials: Only symptomatic isolated lateral femoro-patellar osteoarthritis with an unsuccessful conservative treatment was included in the study. The surgery consisted of an AMIC proce-dure described by Behrens on the retropatellar cartilage defect and a periosteal coverage of the trochlear cartilage defect, both naked surfaces prepared by 1, 1 mm drillings combined with resection of the lateral pole of the patella with lateral release, medial reefing and a tibial tubercle medialisation and advancement. Lack of im-provement and need for reoperation were defined as endpoints. Results: From 2003 to 2009 a total number of 21 patients underwent this described new procedure of which two were operated bilater-ally. 1/4 of patients had a lack of improvement, thus 3/4 were satis-fied. One patient who underwent bilateral surgery was satisfied at the last follow-up but died later of an unrelated cause. The other patient with bilateral surgery had persistent anterior knee pain on the one knee and a good result on the other. One patient had a reoperation with the AMIC procedure and is now satisfied. Another patient underwent partial meniscectomy. His arthroscopy showed fibrous cartilage coverage. One patient had total knee replacement. The oldest patient in this series showed radiographic signs of pa-tellar osteolysis. Patients over the age of 75 years all were failures. Conclusions: The proposed combination of mechanical and biolog-ic treatment modalities has proven to be a valid alternative to deb-ridement and alignment alone or to arthroplasty in patients under the age of 75 years. It shows that there is a chondroplastic potential in osteoarthritis and advanced age.

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17.4.6Treatment with chondroitin sulfate reduces cartilage volume loss in knee oa patients assessed by MRI: a randomized, double-blind, placebo controlled pilot studyJ.P. Pelletier1, L.M. Wildi1, J.P. Raynauld1, A.D. Beaulieu2, L. Bessette3, F. Morin4, M. Dorais5, J. Martel-Pelletier1

1Montreal/Canada, 2Quebec/Canada, 3Sainte-Foy/Canada, 4Trois-Rivières/Canada, 5Notre-Dame de l’Ïlle-Perrot/Canada

Purpose: To determine the effect of chondroitin sulfate (CS) on ex-tent of cartilage loss, subchondral bone marrow lesion (BML) size and severity of synovitis in patients with knee OA using magnetic resonance imaging (MRI).Methods and Materials: This pilot study was a multicentre, ran-domized, double-blind, controlled trial of CS (Condrosan®, Bioi-bérica, Spain) 800 mg daily vs. placebo for 6 months followed by an open label period of 6 months with all patients receiving CS 800 mg daily. MRI was performed at baseline, 6 and 12 months. The cartilage volume was quantified using 3D reconstruction of the MR images. The BML size, severity of synovitis and meniscal extrusion were assessed on a semi-quantitative scale.Results: 70 patients were enrolled. Patients receiving CS for 12 months compared to placebo (6 months placebo followed by 6 months CS) experienced a reduction in cartilage volume loss in the lateral compartment at 6 (-1.1±3.3%, -3.0±4.5%; p=0.05) and 12 months (-1.0±3.8%, -3.9±4.5%; p=0.013). These differences were more pronounced in OA patients without medial meniscal extrusion (6 months, -1.1±3.4%, -3.6±4.3%; p=0.02; 12 months, -1.1±3.8%, -4.3±4.3%; p=0.008). Patients on CS treatment presented a reduc-tion in BML at 6 months and a marked reduction at 12 months (glob-al knee, -0.7±2.6, 0.6±2.1; p=0.06; lateral compartment, -0.4±1.1, 0.7±1.4; p=0.002). Patients from CS group, who received concomi-tant NSAIDs demonstrated a reduction in synovial membrane thick-ness compared to placebo (1.3±0.3 mm, 1.6±0.3 mm; p=0.03), and a reduction in synovitis score (3.0±1.9, 4.5±1.6; p=0.09), as well as lower incidence of joint swelling (p=0.09).#Conclusions: CS was found to reduce cartilage volume loss in knee OA starting as early as 6 months treatment. This was asso-ciated with a reduction in BML size supporting the hypothesis of their role in cartilage volume loss. CS treatment combined with NSAIDs also reduced severity of synovitis. These findings support the DMOAD effect of CS.

17.4.7Osteoarthritis after intense sports activityM.I. Iosifidis, D. Neophytou, T. Liakos, I. Melas, K. Apostolidis, T. Kyriakidis, A. Kyriakidis Thessaloniki/Greece

Purpose: Intense sport activity, mostly by elite athletes, causes in-creased loads in the joints. It is hypothesized that the high impact and torsional loading and also the repeated low magnitude loading cause cartilage degeneration. We investigated the prevalence of lower extremities osteoarthritis (OA) in former elite athletes.Methods and Materials: We studied 218 former athletes (soccer players, skiers, volleyball players, martial arts athletes, track and fields athletes, basketball players/ range 40-84 years, mean 50.67 years, SD±10.04) who participated in national championships of premier league, and in international games. The control group was 181 males (range 40-77 years, mean 50.13 years SD±8.48) who did not have systematic sport activity. The participants in the study had not diagnosed or/and operated for significant limb injury. OA was recorded through questionnaire, clinical examination and ra-diological evaluation (full weight bearing limbs xrays).Results: After adjusting the age, height, weight and body mass index (BMI), the statistical analysis (SPSS, independent samples t-test, z-test, CI=95%) showed no difference in OA prevalence in former ath-letes compared with the general population. However, the intensive sport activity was a significant risk factor for the athletes, as in com-parison with the control group their body weight increased less, their occupation was lighter, and also they developed OA in younger age. Interestingly, the x-rays of the former athletes showed increased osteoarthritic signs compared with control group (p<0.001).Conclusions: Even if there was no statistically significant difference in OA prevalence between elite athletes and general population, the intensive sports was the major predisposing factor for degen-erative joint disease in former athletes.

17.4.812 year follow-up of ACL Reconstruction: Long Term Outcomes of Prospectively Studied Osseous and Articular InjuriesB.T. Hanypsiak1, K. Spindler2, R. Parker3, G. Calabrese3, B. Richmond3, C. Rothrock4, T. Herrenbruck5 1Naples/United States of America, 2Nashville/United States of America, 3Cleveland/United States of America, 4St. Louis/United States of America, 5Salina/United States of America

Purpose: Bone bruises and articular cartilage injuries sustained at the time of initial ACL injury would not resolve. Our secondary hypothesis was that the presence of a bone bruise or articular car-tilage injury originally identified on magnetic resonance imaging would not be associated with long-term outcomes after anterior cruciate ligament reconstruction evaluated by the International Knee Documentation Committee questionnaire.Methods and Materials: Cohort study (prognosis); Level of evi-dence, 1. METHODS: We attempted to contact all patients from an original cohort (N = 54) for follow-up evaluation, which included repeat radiographs, magnetic resonance images, physical exami-nation, and International Knee Documentation Committee ques-tionnaire more than a decade postoperatively.Results: Forty-four patients (82% of the original cohort) returned for on-site follow-up. No patient with a bone bruise identified on original magnetic resonance imaging had one identified at 12-year follow-up. The mean ( +/- SD) International Knee Documentation Committee score at follow-up with no bone bruise originally pres-ent was 70.6 ( +/- 12.7) versus 70.0 ( +/- 8.1) when a bone bruise was observed (P > .05). No consistent association was observed between the presence of an initial articular cartilage lesion with a lesion on follow-up magnetic resonance images. The mean ( +/- SD) International Knee Documentation Committee score at follow-up with no articular cartilage injury was 69.0 ( +/- 11.9) versus 72.8 ( +/- 12.0) with articular cartilage lesion (P > .05).Conclusions: All bone bruises identified in our study with magnetic resonance imaging at the time of initial injury had resolved at 12-year follow-up. The presence of a bone bruise at the time of initial injury did not significantly alter the patient-oriented outcome by In-ternational Knee Documentation Committee after anterior cruciate ligament reconstruction. Additionally, articular cartilage abnormal-ity on magnetic resonance imaging did not influence the Interna-tional Knee Documentation Committee score.

17.4.9Type II collagen fragment HELIX-II is a marker of early cartilage damage but is insensitive to predict progression of cartilage destruction in human knee joint synovial fluidX. Wei1, K. Li2, L. Wei3 1Taiyuan/China, 2Taiyuan City/China, 3Providence/United States of America

Purpose: Objectives: To determine whether the level of type II col-lagen fragment HELIX-II in the knee joint synovial fluid is correlated to the severity of the articular cartilage damages determined by arthroscopy or surgical observation directly. Methods and Materials: Methods: 83 patients who had undergone knee arthroscopy treatments or total knee replacements were en-rolled in the study (49% women, age 14-78 year old, mean 49.5 year old). The content of type II collagen fragment HELIX-II was measured by enzyme-linked immunosorbent assay (ELISA) in 42 patients with knee osteoarthritis (OA), 25 patients with meniscus injury, and 14 patients with anterior cruciate ligament (ACL) injury or posterior cruciate ligament (PCL) injury. Cartilage damage of knee joint was classified with Outbridge cartilage damage severity scoring system by arthroscopy or surgical observation directly. Maximum damage score was defined as the highest score among the six area of the knee joint cartilage and cumulative score was defined as the sum of the scores of the six area of the knee joint cartilage. We investi-gated the relationships between synovial HELIX-II and the cartilage destruction measured by Outerbridge scoring system. Results: Results: The level of HELIX-II in the synovial fluid was cor-related with the cumulative scores (r =0.807, p<0.05) and the maximum scores (r =0.794, p<0.05) respectively. The level of HELIX-II in the severe cartilage damage groups (Outbridge score: 2 to 4) was much higher than the slightly cartilage damaged groups (Outbirdge score: 0 or 1) but there was no significance difference among the severe groups. Conclusions: Conclusions: Elevated HELIX-II level is correlated with cartilage structure changes and is a biomarker for the early diagno-sis of cartilage damages but is insensitive to predict the severity of cartilage damage in the knee joint synovial fluid.

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25.1.2Function and structure of cartilage defect repair with frozen and fresh osteochondral allografts in the goatA.L. Pallante, S. Görtz, A.C. Chen, S.T. Ball, D. Amiel, R.L. Sah, W. Bugbee La Jolla/United States of America

Purpose: Fresh-vs-frozen allografts represent best-vs-worst cases with respect to cell viability, but difficult-vs-simple with respect to ac-quisition and distribution. The objective of this study was to compare load-bearing, geometrical, biochemical, and cellular properties of such allografts for defect restoration in the adult goat model.Methods and Materials: Adult Boer goats (n=7, 3yo) were operated in one knee, with one FROZEN and one FRESH site-matched osteochon-dral allograft (d=8mm, h=5mm) implanted into alternating medial femoral condyle (MFC) and lateral trochlea (LT) sites. Contralateral knees were NON-OPerated controls. At 6months, joint structure was assessed by micro-Computed Tomography (mCT), quantifying carti-lage thickness, cartilage contour-fill at the defect site, cartilage sur-face location, and cartilage-bone interface location. Cartilage stiff-ness was assessed by indentation testing at the allograft center and matched Non-Op site. Cellularity was assessed by Live/Dead® stain-ing overall and in superficial, middle, and deep zones. Matrix fixed charge was assessed (inversely) with Hexabrix™-enhanced-mCT (HE-mCT). Effects of treatment (NON-OP, FRESH, FROZEN), site (and zone) were assessed by ANOVA. Results: Cartilage cellularity varied with treatment (p<0.001), be-ing similarly high in NON-OP and FRESH, and ~99% lower in FROZEN allografts. Cellularity also varied with zone (p<0.001), decreasing with depth. Cartilage stiffness and HE-mCT absorption varied with treatment (p<0.001). Cartilage stiffness was ~80% lower in FROZEN than NON-OP or FRESH allografts. Concomitantly, deep zone HE-mCT absorption was ~60% higher in FROZEN than NON-OP and FRESH. Cartilage thickness varied with site (MFC=1.44mm, LT=0.86mm), but was not affected by treatment (p=0.4). Other allograft geometry parameters were affected by treatment (p<0.01). In FROZEN MFC allografts, cartilage fill was less (~50%) in association with surface depression (~0.7mm), and the cartilage-bone interface tended to be depressed (~0.9mm, p=0.06). Conclusions: Maintenance of cartilage load-bearing properties in al-lografts is associated with maintenance of cartilage cellularity and fixed charge. Frozen or non-viable allografts may fail due to both car-tilage softening and graft subsidence.

25.1.33D-Printing of cells in gels: a new concept for constructing osteochondral graftsW. Schuurman, N.E. Fedorovich, H.M. Wijnberg, H. Prins, P..R. van Weeren, J. Malda, J. Alblas, W.J.A. Dhert Utrecht/Netherlands

Purpose: Osteochondral defects are prone to induce osteoarthritic de-generative changes. Current approaches for osteochondral grafting suf-fer from poor tissue formation and compromised integration. 3D-print-ing is a novel technique that fabricates organized, cell-laden scaffolds by layer-by-layer deposition. The aim of this study was to test the appli-cability of the use of 3D-printing for the production of heterogeneous cell-laden 3D hydrogel structures as a model for osteochondral grafts. Methods and Materials: We assessed the effect of the extrusion of cell-laden hydrogel on cell viability. To construct scaffolds containing two different cell types, alginate hydrogel was mixed with either human chondrocytes or human multipotent stromal cells (MSCs) (both fluo-rescently labeled). Subsequently, porous scaffolds (20x10x1mm) with in one part MSCs and in the other part chondrocytes were produced. Extracellular matrix formation was analyzed using (immuno)histology. Results: The cells survived the deposition process, and were as via-ble as non-printed controls (mean 89% and 88.9%, respectively). The MSCs and chondrocytes remained in their printed part of the scaf-fold for up to three weeks when cultured in vitro. Moreover, heteroge-neous and cell specific matrix formation occurred in vitro, as shown macroscopically and by immunocytochemistry. In the chondrocyte-laden part, cell clusters were observed, as well as cartilage-specific collagen type II and type VI staining, none of which were noted within the MSC-laden part. In the MSC-laden part, we showed the presence of alkaline phosphatase and positive Alizarin red staining, indicative for respectively osteogenic differentiation and calcium deposition. Conclusions: Our results demonstrate that 3D-printing can be used to fabricate viable, heterogeneous cell-laden hydrogel grafts with for-mation of cartilage-like matrix in the chondrocyte-laden part of the construct and osteogenous matrix differentiation in the MSC-laden part, suggesting the potential to produce osteochondral grafts with this novel technology.

25.1.4Regeneration of osteochondral lesions in the knee with a combined MACI / Bone Graft - Sandwich TechniqueE. Basad, B. Ishaque, H. Stürz Giessen/Germany

Purpose: Osteochondral defects in the knee are primarily based on pathologic changes of the subchondral bone, which affect the chon-dral surface. Repair depends on reconstruction of cartilage and bone. The Matrix-associated Chondrocyte Implantation (MACI) is a third generation cartilage regeneration technique where chondrocytes are seeded onto a collagen I/III membrane. We combined the MACI tech-nique with an autologous bone graft (BG) in a two-step sandwich pro-cedure (MACI-BG). Goal of our prospective observational study was to evaluate the outcomes after two years.Methods and Materials: In 32 patients with isolated lateral or medial condylar osteochondral defects (> 3 cm2) first an arthroscopic car-tilage biopsy was taken and a deep debridement of the sclerotic sub-chondral bone was performed through a mini arthrotomy, followed by a press-fit filling of the defect with impacted cancellous iliac bone. In a second operation, a double-layer MACI was fixed onto the consoli-dated bone graft with fibrin glue. The clinical outcome was measured using the Tegner and Lysholm scores after 6, 12 and 24 monthsResults: The median Tegner score improved from Level 2 to Level 4 (p= 0.0001). The mean Lysholm score median improved from 57.8 ±21.1 to 94.25 ±11.1 (p= 0.0001). There was no significant difference between the lateral or medial condyle (p=0.349). MRI follow-ups showed a bi-polar regeneration without dislocation of impacted bone or hypertrophy of the regenerate tissue.Conclusions: Cartilage and subchondral bone perform as an interact-ing unit. Treatment of osteochondral lesions without regeneration of the subchondral bone is likely to fail. The combination of MACI with BG is one strategy that addresses this particular problem. Our study shows promising results pointing out that a rebuilt subchondral bed plays an important role for the success. The results may also imply that an early detection and proper treatment of subchondral alterations is important for the outcome of cartilage regeneration techniques.

25.1.5Comparison of the results of immature articular cartilage allograft and mature autograft in osteochondral defect treatment- an experimental study with ratsA. Koroglu1, K. Memisoglu2, B. Muezzinoglu2, S.U. Muezzinoglu2

1Sakarya/Turkey, 2Kocaeli/Turkey

Purpose: To address the effect, advantage and superiority of the im-mature allograft in the treatment of osteochondral defects in rats. Methods and Materials: A 4x2 mm deep osteochondral lesion was created in the knee joints - femoral condyles- of 30 mature rats and re-paired with immature osteochondral allografts and with autografts. Results were evaluated with Modified Mankin’s histological score at 8 weeks after surgical implantation of the grafts. Results: All wounds healed without complication. Grossly, almost all de-fects were filled with healing tissue, but specimens from autograft group showed rough appearences on the transplantation sites. (p<0.001)

Group 1(Autograft)

Group 2 (Ä°mmature)

Mean ± SD Mean ± SD p value

Articular cartilage structure 8.39±1.66 5.68±2.51 0.001

Superficial cells 2.21±1.55 1.43±0.57 0.001

radial cells 7.36±2.92 4.61±3.2 0.002

Toluidine blue staining 2.96±0.84 1.89±0.99 0.001

Pannus formation 2.29±0.9 1.57±1.07 0.01

Tide mark zone 2.32±1.06 1.75±1.21 0.07

Total 25.54±6.29 17.29±7.9 0.001

Conclusions: Immature cartilage contains high amount of undifferenti-ated multipotent elements with a marked mitotic activity, paracrine and autocrine signaling among the cells and high susceptibility to the grow-ing factors and cytokines. Moreover, the immature cartilage has a double source of nutrition, it can survive with metabolic support of the synovial fluid and vascular connection from the base of the graft. Immature carti-lage allografting group scores differ superiorly from the autograft group with a very low p value ( p< 0.001). Immature allogenic cartilage with histopathologino immune response, and due to the superior properties and well integration with the surrounding tissue the immature cartilage allograft may be an appropriate graft alternative for chondral defects.

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25.1.6Evaluation of a Biphasic Graft for Osteochondral Repair in an Equine ModelL.A. Fortier1, T.M. McCarrel1, G. Bradica2, E. Castiglione2, R. Saska2, R.C. Lehman3 1Ithaca/United States of America, 2Exton/United States of America, 3Kirkwood/United States of America

Purpose: Osteochondral (OC) and full thickness chondral lesions heal with biomechanically inferior fibrocartilage. The objective was to evaluate a biphasic graft (KNC CRD) for OC repair. Our hypoth-esis was that KNC CRD would be safe and improve repair compared to microfracture.Methods and Materials: Twelve horses (2-5 years) had 10mm diam-eter x 10mm deep OC defects created in the lateral femoral trochlear ridge arthroscopically. KNC CRD was hydrated in bone marrow and press-fit into the defect. In the control/contralateral limb, 10mm di-ameter full thickness chondral defects were created and microfrac-tured. Radiographs were obtained pre-operatively, post-operative-ly, and 2-, 4-, and 12-months post-operatively. Repair tissue was evaluated arthroscopically at 4- and 12-months post-operatively. Results: No horse had radiographic evidence of lysis, bone cysts, or osteophytes. KNC CRD had significantly increased sclerosis (p=0.003) compared to microfracture at all post-operatives time-points. At 12-months post-operative, the % of defect replaced with bone for both treatments was significantly improved compared to all other time points (p<0.001). Percent replaced by bone was sig-nificantly better for control defects than for KNC CRD at 12-months (p=0.021). There was no evidence of infection, device fragmen-tation, synovial inflammation, or blood within any joint at 4- and 12-month arthroscopies. There was no significant difference by treatment or time for patella, color, surface area repaired, integra-tion, surface, or total safety score. KNC CRD had significantly better fill (p<0.0001), total efficacy (p=0.0021), and total arthroscopic scores (p=0.0069) at 4 months compared to control. Neither treat-ment changed significantly from the 4- to 12-months.Conclusions: The study suggests that KNC CRD improves defect fill earlier, as compared to microfracture, and is able to maintain re-pair out to 12-months post-operative. Further evaluation to 2 years post-operative will provide valuable quantitative MRI, biomechani-cal, and biochemical information regarding the long-term repair tis-sue formed in KNC CRD or microfracture treated lesions.

25.1.7Degenerative change and donor-site deterioration of patella-femoral joint after the harvest of plug in osteochondral autologous transplantationS. Mukai, Y. Nakagawa, M. Kobayashi, R. Arai, T. Nakamura Kyoto/Japan

Purpose: There are very few reports on the radiographic findings of the donor site of the osteochondral autologous transplantation(OAT). Our aim is to determine if the donor harvest directly affect the progression of osteoarthritic(OA) change of pa-tello-femoral(PF) joint. Methods and Materials: The materials are patients treated by OAT against the various knee disorders and followed more than 5 years, which include 43 persons (17 men, 26 women) 53 knees. The mean age at the operation was 42.3 ( 14 -76 ) and the follow up was 74 months(60 - 140). We retrospectively evaluated the OA change by Kellgren-Lawrence classification of PF joint during the post-opera-tive period. Degree of progression was determined as severe, mild and no-significant-change (NSC). Then each donor-site was also evaluated. We assessed age, the size and the location of injured area, numbers of donor plugs, and primary disorder. Results: Twenty-one knees (40%) were severe and 16 knees(30%) were mild, and 16 (30%) were NSC. All cases of steroid-induced osteonecrosis(5/5) were severe. But excluding these cases, the patients with NSC were significantly younger. Donors were har-vested from 49 lateral and 32 medial condyles, and 25 sites were left as un-operated. The mild or severe OA changes were found on 29 of 49 lateral, 20 of 32 medial donor-sites, but also found on 15 of 25 un-operated sites. Twenty-eight knees were harvested from both sides, and only 8 of them were bilaterally NSC. It is difficult to distinguish degenerative change and donor-site deterioration. We suppose that steroid-induced osteonecrosis strongly concern to the OA progression. More than half of donor sites deteriorate, however, more than half of un-operated sites also become worse.Conclusions: Progression of OA findings in PF joint depends on the primary disorder. The OA change tend to deteriorate in older peo-ple. Non-harvested sites as well as donor sites show OA findings after the donor harvest.

25.1.8Knee mosaicplasty prospective evaluation: assessment of functional outcome and joint degeneration progression at 10 years minimum follow-upG. Filardo, E. Kon, S. Patella, A. Di Martino, L. D’Orazio, B. Di Matteo, M. Marcacci Bologna/Italy

Purpose: Articular cartilage lesions, because of their limited heal-ing potential, are still a challenge for the orthopaedic surgeon. Dif-ferent approaches have been proposed to treat these lesions, while both their indications and clinical efficacy are still controversial. Purpose of this study is the evaluation of the outcome of osteo-chondral autografts for the treatment of femoral condyle cartilage lesions at long-term follow-up. Methods and Materials: We prospectively evaluated 25 patients (mean age 29.4) affected by full-thickness chondral lesions of the knee (<2.5 cm2) treated with arthroscopic autologous osteochon-dral transplantation. 12 out of 25 patients underwent previous sur-gery. 16 patients underwent associated procedures. All patients were clinically evaluated at 2 and 7 years and at the final follow-up (10 to 12 years) using the International Knee Documentation Committee form and the Tegner score. X-rays were also taken at the final follow-up and used to assess the progression of the articular degeneration. Results: The International Cartilage Repair Society objective evalu-ation showed 76% of patients had normal or nearly normal knees at the latest follow-up. The International Knee Documentation Com-mittee subjective score significantly improved from preoperative (37.3) to the final follow-up (76.2). The Tegner evaluation showed a significant improvement after the surgery at 2- and at the last fol-low-up (from 2.9 to 6.1 and 5.2, respectively); however, we noticed reduced sports activity from 2 years to the last follow-up. Conclusions: The results of this technique at long-term follow-up are encouraging. This arthroscopic one-step surgery appears to be a valid solution for treatment of small, grade III to IV cartilage defects.

25.1.9Osteochondral allograft: is it an active tissue or just a scaffold?S. Kirk1, D. Ruta1, A. Meininger1, G. Filardo2, A.A. Hakimiyan1, L. Rappoport1, B.J. Cole1, A. Kurz3, S. Chubinskaya1 1Chicago/United States of America, 2Bologna/Italy, 3Centennial/United States of America

Purpose: Evaluate the effect of pro-inflammatory cytokines on the metabolism and survival of osteochondral allograft cartilage (OA) as compared to fresh cartilage (FC).Methods and Materials: Nine OA specimens (previously refrigerat-ed) and six fresh hemicondyles (collected within 24h of death) were obtained from normal donors. 4mm explants from OA and FC were divided into: cultured control, IL-1b (0.1ng/ml), IL-6 (3ng/ml), IL-1b (0.1ng/ml)+IL-6, IL-1b (10ng/ml), IL-1b (10ng/ml)+IL-6. IL-6 soluble receptor (5ng/ml) was added to all IL-6-containing cultures. Cell vi-ability, apoptosis, histology, proteoglycan (PG) synthesis and con-tent were tested on days 0, 2, 7, and 14.Results: At day zero, the viability of OA chondrocytes was 24% lower and contained 29% more apoptotic cells than fresh chondro-cytes (p<0.05). Cytokines did not significantly induce cell death or apoptosis in OA cartilage. In FC, high dose IL-1 alone or in com-bination with IL-6 decreased chondrocyte viability by day 14 (up to 20%; p=0.047 and p<0.005) and increased apoptosis by more than 2-fold (p=0.05 and p<0.05) when compared to day 0 or day 14 controls. At day 0, FC showed 2.5 times greater PG synthesis (p<0.05) and only half as much release of newly synthesized PGs (p<0.01) when compared to OA chondrocytes. However, FC was more sensitive to cytokine treatments: by day 14 high dose IL-1 alone or combined with IL-6 inhibited PG synthesis by more than 8-fold (p<0.02) vs 4-fold in OA (p<0.02) and induced higher PG release, which resulted in elevation of Mankin score for FC.Conclusions: The viability and metabolism of OA cartilage is sig-nificantly lower than FC; however, OA cartilage is more resistant to cytokine treatment. Hypothetically, the reduced metabolism of OA cartilage might enable its better performance in inflammatory environment suggesting that OA cartilage acts more like a scaffold rather than an active tissue.

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Free Papers 185

25.2.2Hybrid morphological and biochemical T2 evaluation of cartilage repair tissue based on a recently described double-echo at steady-state (DESS-T2d) approachG.H. Welsch1, T.C. Mamisch2, L. Zak1, A. Mauerer3, S. Apprich1, S. Marlovits1, S. Trattnig1 1Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany

Purpose: Recently, the widely used 3D Double-Echo Steady-State (DESS) sequence was reported to enable, in addition to the morphological infor-mation it provides, the generation of biochemical T2 maps in one hybrid sequence. The aim of this study was to use this new DESS-T2d approach to assess the morphological Magnetic resonance Observation of Carti-lage Repair Tissue (MOCART) score, as well as biochemical T2 values in patients after matrix-associated autologous chondrocyte transplantation (MACT) of the knee. Furthermore, to correlate this new hybrid approach to standard morphological sequences as well as to standard T2 mapping.Methods and Materials: Fifty consecutive MR scans during clinical rou-tine standard follow-up intervals at 3.0 Tesla in patients (36.1±9.3 years) after MACT of the knee joint were prospectively included. MOCART scoring was prepared by i) a set of standard MR sequences and ii) the morphologi-cal images of the new DESS-T2d sequence. T2 relaxation times were as-sessed by region-of-interest analysis on basis of i) a standard multi-echo spin-echo sequence and ii) the biochemical T2d images of the DESS-T2d sequence. Analysis-of-variance and Pearson correlation was performed.Results: The MOCART score correlated (0.945; p<0.001) significantly as assessed with standard morphological sequences (68.8±13.2) and the morphological images of the DESS T2d sequence (68.7±12.6). T2 and T2d relaxation times (ms) were comparable in between the control carti-lage (T2: 52.5±11.4; T2d: 46.6±10.3) and the repair tissue (T2: 54.4±11.4; T2d: 47.5±13.0) (T2: p=0.157; T2d: p=0.589). As expected, T2d values were lower than the MSME-T2 values, however both functional relax-ation times correlated significantly (Pearson:0.429; p<0.001).Conclusions: Comparable information on the repair tissue could be achieved at enormous time-savings, with ~20 minutes for the stan-dard sequences and ~5 minutes for the hybrid DESS-T2d sequence. Hence the presented approach provides the possibility to combine morphological and biochemical MRI in one fast 3D sequence, and thus, may attract for the clinical use of biochemical MRI.

25.2.3Advanced morphological 3D- magnetic resonance observation of cartilage repair tissue (3D-MOCART) scoring using different new isotropic 3D sequences and their multi-planar reconstructionG.H. Welsch1, L. Zak1, T.C. Mamisch2, A. Mauerer3, S. Marlovits1, S. Trattnig1 1Vienna/Austria, 2Berne/Switzerland, 3Erlangen/Germany

Purpose: To use the capabilities of the new isovoxel sequences and their three-dimensional (3D) multiplanar-reconstruction (MPR) features in the monitoring after cartilage repair procedures, the 3D magnetic resonance observation of cartilage repair tissue (MOCART) scoring system was recently introduced. The purpose of this study was to eval-uate a new isotropic 3D proton-density, turbo-spin-echo (PD-SPACE) sequence compared to an isotropic 3D true-fast-imaging with steady-state-precession (True-FISP) sequence and to a set of 2D standard MR sequences in their depiction of the 3D-MOCART score.Methods and Materials: Sixty consecutive MR scans on 37 patients (32.8±7.9 years) during clinical routine at standard follow-up intervals after matrix-associated autologous chondrocyte transplantation (MACT) of the knee at 3 Tesla were prospectively included. The new 3D-MOCART score with eleven variables was assessed using the standard 2D sequences (~15minutes) and the multiplanar-reconstruction (MPR) of both isotropic sequences (3D-PD-SPACE~7minutes; 3D-True-FISP~6minutes). Statisti-cal correlation as well as subjective quality analysis was performed.Results: The correlation between the 3D-MOCART scoring performed by the different sequences was highly significant for the variables 1)de-fect fill, 2)cartilage interface, 3)bone interface, 4)surface, 7)subchondral lamina, 8)chondral osteophyte, and 11)effusion (Pearson-coefficients 0.514 to 0.865 (p<0.001)). The variables 5)structure, 6)signal intensity, 9)bone marrow edema, and 10)subchondral bone showed lower correla-tions with best results in between the standard sequences and the 3D-PD-SPACE sequence and in between the 3D-True-FISP sequence and the 3D-PD-SPACE sequence (0.307 to 0.633 (p=0.016 to p<0.001). Grading of subjective quality revealed good results for all sequences (p≥0.05). Ar-tifacts were most often visible on the 3D-True-FISP sequence (p<0.05).Conclusions: Different isotropic sequences can be used for the 3D evaluation of cartilage repair tissue with the benefits of isotropic 3D-MRI, MPR and a significantly reduced scan time. The 3D-PD-SPACE sequence reveals best results due to a better performance in the sub-chondral bone and because of the suppression of susceptibility arti-facts produced by implantation and previous surgeries.

25.2.4Sodium MRI at 7.0 Tesla in patients after matrix associated autologous chondrocyte transplantation and microfracturing in the knee: correlation to morphological scoring and clinical data - preliminary resultsD. Stelzeneder1, S. Zbyn1, L. Negrin1, G.H. Welsch1, V. Juras1, P. Szomolanyi1, S. Domayer1, R. Dorotka1, T.C. Mamisch2, S. Trattnig1

1Vienna/Austria, 2Berne/Switzerland

Purpose: Sodium imaging at ultra-high field-strength is able to depict proteoglycan content in cartilage. The aim was to correlate the rela-tive sodium content of cartilage repair tissue to morphological MRI (MOCART score) and clinical evaluation (IKDC subjective form) in pa-tients after matrix-associated autologous chondrocyte transplanta-tion (MACT) and microfracturing (MFX). Methods and Materials: Seventeen patients (mean age 36.7) after MACT (N=12) and MFX (N=5) were investigated on a 7.0 Tesla whole-body MR-unit. Mean follow-up time after surgery was 4.7 years, lesion size 3.0 cm2, and BMI 24.4. In addition to morphological sequences, so-dium imaging was performed using a dedicated 23Na-knee-coil with an optimized 3D-gradient-echo sequence. Sodium evaluations were per-formed on equally sized repair tissue and native cartilage regions. The normalized sodium-ratio (NSR) was calculated: NSR=healthy cartilage/repair-tissue. The MOCART-point-scale was evaluated on morphologi-cal proton-MRI. Clinical assessment was done on the day of MRI using the IKDC subjective form. Pearson’s correlation was applied. Results: The mean NSR (±standard deviation) was 1.64±0.37 (range 1.02; 2.42), with no difference between MACT (1.62±0.43) and MFX (1.67±0.27). The mean MOCART score was 76.4±15.0 and IKDC sub-jective was 71.7±23.2. There was no correlation between IKDC sub-jective score and NSR (r=0.18; p=0.48), however there was a trend for a moderate inverse association of the MOCART-point-scale and the NSR (r=-0.46; p=0.055). Conclusions: Sodium imaging at 7.0 Tesla is able to differentiate be-tween repair tissue and native cartilage. The NSR-values higher than 1.0 reflect a lower sodium content within the repair tissue in compari-son to healthy cartilage. Our results suggest a trend for an association of high NSR (i.e. low sodium and proteoglycan content) with low MO-CART scores. We found no association between the IKDC subjective score and the NSR. This experimental study correlates sodium imaging at ultra-high field-strength to clinical parameters and morphological grading in patients after cartilage repair surgery for the first time.

25.2.5Correlation between MRI- and clinical assessment after matrix-associated autologous chondrocyte transplantation on the femoral condyle of the knee jointL. Zak1, G.H. Welsch2, S. Trattnig2, S. Marlovits2

1Baden/Austria, 2Vienna/Austria

Purpose: The aim of this study was to develop a pointing-scale for the previous published 3D-MOCART-score, to evaluate the correlation between clinical and MRI-results after matrix-associated autologous chondrocyte transplantation of the knee (MACT). Methods and Materials: 41 MRI Follow-up measurements of 41 patients, with a minimum follow-up of 12 months (Mean: 41.1 month) after MACT on the medial or lateral femoral condyle of the knee joint were studied by means of MRI and different clinical Scores. The MRI-measurements were performed on a 3.0 Tesla unit. A proton-density Turbo Spin-Echo (PD-TSE) sequence, a T2-weighted Dual Fast Spin Echo (Dual-FSE) se-quence and a T1-weighted Turbo Inversion Recovery Magnitude (TIRM) sequence were used for assessing the MOCART-score, and an istotropic 3D-True FISP sequence was used for the 3D-MOCART-score. The sub-jective clinical assessement was evaluated at the same point than the MRI measurement, whereas the state of health and state of activity was ascertained by the Brittberg-Scale, the IKDC-Score, the KOOS-Score, the Noyes Sports Scale, the Tegner & Lysholm Score and the VAS-Score for pain. The weighting of the variables in the 3D-MRI-assessement was calculated by the Regression-coefficients. Furthermore a pointing-scale for the 3D-MOCART-score was developed and correlated with the origi-nal MOCART-score as well as with the clinical data.Results: The comparison between the two scores resulted in a good correlation with a Spearman Correlation Coefficient of 0.59 (p<0,005). The MRI-evaluation and clinical scores showed medium, mostly signifi-cant correlations with coefficients between 0.24 and 0.48 in the 2D-evaluation and values between 0.26 and 0.5 for the 3D-MOCART. Conclusions: Within the present approach the newly introduced 3D-MOCART score was adapted to a point-scaling system based on ac-cepted clinical parameters. The given results imply that besides the ac-cepted 2D-MOCART, the 3D-MOCART can be exploited for statements about the clinical condition of MACT in the knee joint.

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25.2.6T2 mapping MRI characterization of articular cartilage in the ankle joint after bone marrow-derived cells transplantation repair procedureM. Battaglia, F. Vannini, R. Buda, M. Cavallo, A. Ruffilli, C. Monti, S. Giannini Bologna/Italy

Purpose: Qualitative evaluation of postoperative outcome in carti-lage repair techniques is an issue. Recently T2 mapping MRI is becom-ing an increasingly popular instrument to provide information about the histological and biochemical contents of healthy or reparative tis-sue. Aim of this study was to investigate by the use of T2 mapping the quality of the regenerated tissue obtained after bone marrow-derived cells transplantation (BMDCsT) repair procedure in the ankle.Methods and Materials: 20 patients affected by osteochondral le-sions of the talus and previously treated by arthroscopic BMDCsT, were evaluated by T2 mapping at 24 months post-op. DPFSE with or without fat suppression, T2FSE with or without fat suppression, 3D SPGR and T2-Mapping using a dedicated phased array coil and 1.5 T MR scan-ner were used. 20 healthy patients were used as a control. MRI results were correlated with clinical score (AOFAS) in all the cases. Results: The regenerated cartilage showed in all the cases a mean T2 value of 45 ms (range 34-50) with no significant difference com-pared to that of normal hyaline cartilage. A reduced mean T2 value <30 msc expression of fibrocartilage features, was shown in lim-ited foci at repair sites. A clinical score >90 was correlated with the presence of hyaline cartilage (p=0,05). Foci of fibrocartilage within the repaired area negatively affected the clinical results (p=0,045). Conclusions: T2 mapping MRI evaluation showed that BMDCsT is able to provide a repair cartilage tissue closely similar to healthy hyaline cartilage, confirming the validity of the technique. Nev-ertheless the regenerated cartilage was not qualitatively homo-geneous in the whole repaired area. Limited foci of fibrocartilage within the repaired area were shown to be detected by the T2 map-ping and to significatively correlate with the clinical result. T2 map-ping showed to be a valuable tool for qualitative cartilage evalua-tion in the ankle joint.

25.2.7Delayed Gadolinium Enhanced MRI technique for the long term evaluation of patients treated with Autologous Chondrocyte ImplantationH.S. Vasiliadis1, B. Danielson2, M. Ljungberg2, B. McKeon2, A. Lindahl2, L. Peterson2 1Ioannina/Greece, 2Gothenburg/Sweden

Purpose: Our purpose is to show whether MRI with dGEMRIC tech-nique (delayed Gadolinium Enhanced MRI of Cartilage) can give valuable information regarding the quality and quantity of the re-paired cartilage lesion after treatment with Autologous Chondro-cyte Implantation.Methods and Materials: Thirty six knees in 31 patients were as-sessed 9 to 18 years after treatment with Autologous Chondrocyte Implantation. All patients had isolated lesions, 27 on a femoral con-dyle, 1 on the trochlea and 8 on the patella. The knees were evalu-ated clinically with KOOS score and with the dGEMRIC (delayed Gadolinium Enhanced MRI of Cartilage) technique. The T1 value was measured for two representative regions of interest (ROI), one in the repair tissue area (ROI1) and one in the surrounding cartilage (RΟΙ2), giving information of the content of proteoglycans in the tissue.Results: The T1 value in ROI1 was measured to be on average 467.5 ms (range 440-495) while the mean in ROI2 was 495.3 (range 462.3-528.2). There was no significant difference between these mean values of ROI1 and ROI2, suggesting comparable levels of proteogly-cans in the repair tissue and the surrounding cartilage (p>0.05). Intralesional osteophytes were found in 64% of the lesions, mainly in younger patients with osteochondritis dissecans lesions or with the history of subchondral bone surgeries (abrasion, drilling, mi-crofractures) (p<0.05). Bone marrow oedema was found in 39% of the knees. Mean Mean KOOS score was 80 for pain, 85.6 for ADL, 70.7 for symptoms, 48.3 for sport/rec and 57.8 for QOL. There was no correlation with any of the MRI findings.Conclusions: MRI with dGEMRIC gives valuable information for the macroscopic appearance and micro-molecular quality of the repair tissue after ACI. Nine to 18 years after the ACI, the quality of the repair tissue is similar to the surrounding normal cartilage. The de-fect area is restored in most of the cases.

25.2.8Ultrasonic probe is useful for in-vivo quantitative assessment of medial femoral condyle articular cartilageT. Shimizu1, S. Wakitani2, Y. Tanaka2, Y. Yonetani3, Y. Shiozaki2, S. Horibe2 1Gifu/Japan, 2Osaka/Japan, 3Sakai/Japan

Purpose: Although objective evaluation of the articular cartilage is important to assess the outcomes of surgical treatment, a reliable method has not yet been developed. It has recently been reported that quantitative ultrasound could assess living human cartilage. The purpose of this study was to investigate whether quantitative ultrasound can detect subtle change of the articular cartilage, as well as age-related changes in the normal cartilage. Methods and Materials: Thirty patients with an injured knee un-derwent ultrasonic evaluation of the articular cartilage during ar-throscopy. During the ultrasonic evaluation, the ultrasonic probe was inserted into the knee joint using standard anterolateral and anteromedial portals. The reflex echogram from the cartilage was transformed into a wavelet map using wavelet transformation. As quantitative index on the wavelet map, the maximum magnitude was selected. Whether the cartilage was damaged or not was judged from the arthroscopic view of the articular surface. The nor-mal sites (27 sites) and the damaged lesion (outerbridge grade I-II, 10 sites) were measured.Results: An accurate measurement was possible in the medial fem-oral condyle, where the ultrasonic probe was easily to be put on perpendicularly under arthroscopic control. The average maximum magnitude values of normal and damaged cartilage were 4.06 ± 1.58 and 1.40 ± 0.62, respectively. The maximum magnitude was significantly higher in the intact cartilage compared to the injured one (P < 0.01). The maximum magnitude for intact cartilage of me-dial femoral condyle showed a significant correlation with patient age (R2 = 0.434, p < 0.01).Conclusions: Our ultrasound measurement system offers potential for the detection of subtle change of the cartilage. The maximum magnitude is useful for quantitative assessment of especially me-dial femoral condyle articular cartilage. Care should be taken in interpreting results because the maximum magnitude values de-crease with age.

25.2.9Cartilage repair evolution in post-traumatic osteochondral lesions of the talus: from autologous chondrocyte to bone-marrow-derived cells transplantationF. Vannini, R. Buda, M. Cavallo, A. Ruffilli, F. Di Caprio, B. Grigolo, S. Giannini Bologna/Italy

Purpose: Cartilage repair in osteochondral lesions is now more than ever an hot topic for research. Although autologous chondro-cyte implantation gave well documented satisfactory results, the need for two operations and high costs has prompted a search for alternatives. The purposes of this study are to describe evolution in cartilage repair from open field autologous chondrocyte implan-tation to regeneration by arthroscopic bone-marrow-derived cells (BMDCs) “one step” technique; to present the results of a series of patients consecutively treated and to compare in detail the differ-ent techniques used in order in order to establish the advantages obtained with the evolution in cartilage regenerative methods. Methods and Materials: 81 patients (mean age 30±8 years) were treated between November 1996 and January 2007. Patient evalu-ation included clinical AOFAS score, X-Rays and MRI preoperatively and at different established follow-ups. All the lesions were > 1.5 cm2 and received open autologous chondrocyte implantation (10 cases), arthroscopic autologous chondrocyte implantation (46 cas-es), and “one step” arthroscopic repair by BMDC transplantation (25 cases). For arthroscopic repair techniques a hyaluronic acid mem-brane was used to support cells and specifically designed instrumen-tation was developed. Patients of all the 3 groups underwent a sec-ond arthroscopy with a bioptic cartilage harvest at 1 year follow-up. Results: Mean AOFAS score before surgery was 57.1 ± 17.2 and 92.6 ±10.5 (p<0.0005) at mean 59.5 ± 26.5 months. A similar pattern of AOFAS improvement in results was found in the 3 different tech-niques. Histological evaluations highlighted collagen type II and proteoglycan expression. Conclusions: The described techniques were able to provide a repair tissue which closely approximates the characteristics of the naive hyaline cartilage. Evolution in surgical technique, new biomaterials and more recently the use of BMDCs permitted a marked reduction in procedure morbidity and costs up to a “one step” technique able to overcome all the drawbacks of previous repair techniques.

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25.3.2Quantitative Evaluation of Spontaneously and Surgically Repaired Rabbit Articular Cartilage using Ultrasound Measurements in situT. Viren1, S. Saarakkala1, H.J. Pulkkinen1, V. Tiitu1, P. Valonen1, I. Kiviranta2, M. Lammi1, J. Jurvelin1, J. Töyräs1 1Kuopio/Finland, 2Helsinki/Finland

Purpose: In this study we investigated the ability of intravascular ultrasound (IVUS) device to assess structural changes in spontane-ously and surgically repaired rabbit articular cartilage.Methods and Materials: Repair tissue of osteochondral lesions (di-ameter 4 mm) in the rabbit knee joints were investigated within two groups: 1) surgically repaired (recombinant human type II collagen gel with chondrocytes (10×106/ml), n=8), and 2) spontaneously healed (n=5) lesion sites. Intact adjacent tissue (n=13) was used as a con-trol. Histological integrity of the samples was determined with the O’Driscoll grading, light microscopy (Safranin O staining of proteo-glycans and immunohistochemical staining of type II collagen) and polarized light microscopy (Parallelism index). Ultrasound imaging of the samples was conducted with a high frequency (40 MHz) clini-cal IVUS (ClearView Ultra, Boston Scientific Corporation, San Jose, CA, USA) device. Reflection coefficient (R), integrated reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI) were determined from radiofrequency ultrasound signal. Results: URI and AIB was significantly higher in both repair groups than in intact cartilage (p < 0.05). The reflection parameters (R and IRC) were significantly lower in surgically repaired cartilage (p < 0.05) than in intact cartilage. O’Driscoll grading indicated that surgically and spontaneously repaired tissue were similar with and structurally inferior to healthy cartilage. Further, repaired tissue exhibited abnormally organized collagen network, rough surface and lower type II collagen and proteoglycan contents. The repro-ducibilities (sCV) of the ultrasound parameters were 1.4 – 9.1%.Conclusions: Surface roughness and integrity of the intact and re-paired rabbit cartilage could be quantitatively evaluated with the IVUS technique. Furthermore, qualitative information about integration of the repair tissue, integrity of the surface of the repair tissue and the internal structure could be extracted from the ultrasound images.

25.3.3The influence of microfracture hole spacing and depth on cartilage repair in chronic chondral defects: Preliminary resultsM.B. Hurtig1, R. Whiteside2, P. Marks2

1Guelph/Canada, 2Toronto/Canada

Purpose: Approximately 40% of patients with 2-4 cm2 chondral defects do not respond to microfracture. Chronicity and tissue degeneration must play a role. This experiment was designed to identify operative parameters that could optimize the repair response after microfrac-ture in chronic cartilage lesions accompanied by abnormal bone. Methods and Materials: Adult female sheep (n=24) underwent bi-lateral femoral condyle contusive impact injuries and partial me-niscectomy. Three months later the injury sites were debrided to 10 mm in diameter, and three patterns of microfracture holes were created as per Table 1. The control treatment represents the stan-dard of care recommended by Steadman et al (2001). Animals were sacrificed at 3 and 6 months post-microfracture ((n=4/group/time period). Cartilage repair was assessed by biochemistry (cartilage total sGAG), ICRS 2 scoring of histological sections and morphom-etry to determine base attachment, integrative attachment and de-fect fill. A paired T-test with the significance level set at p<.1 was used to illustrate differences between treatments.Results: All condyles developed degenerative cartilage lesions and sclerotic subchondral bone. Treatment A disrupted of subchondral bone, and caused cyst formation that delayed elaboration of repair tissue at both time points. Treatment B improved defect fill at 3 and 6 months (p<.08) as well as early basal attachment of repair tissue (p<.03).Conclusions: Dense sclerotic subchondral bone had a negative effect on repair tissue formation and attachment after microfrac-ture. Careful resection of the subchondral bone plate followed by shallow (2 mm deep) microfracture should be considered as an al-ternative to the standard method. Aggressive microfracture using closely spaced deep holes is contra-indicated. Drilling may be less disruptive but without resection of sclerotic bone, attachment may be a problem.

25.3.43-D metrics of structure and function of autologous osteochondral grafts and surrounding host cartilage and bone in the goat knee at 6 and 12 monthsE.F. Chan1, I. Liu2, E. Semler3, H. Aberman4, T. Simon4, A.C. Chen2, K. Truncale3, R.L. Sah2 1San Diego/United States of America, 2La Jolla/United States of America, 3Edison/United States of America, 4Los Alamitos/United States of America

Purpose: Cartilage defect repair is traditionally analyzed by 2-D (im-muno)histochemistry and biomechanical testing at single locations. However, bone and cartilage remodeling may vary substantially within and surrounding the repair site. The objective of this study was to determine 3-D metrics of repair with autologous osteochon-dral graft (AOCG) in the goat model.Methods and Materials: Full-thickness AOCGs (Ø=3.5mm, h=6mm) were harvested from the lateral trochlea and press-fit into defects of the medial femoral condyle (MFC) in one knee of adult (3-4yo., n=8) Spanish goats. Animals were euthanized at 6 and 12 months (n=4 each), and OPerated and NON-OPerated knees were harvested. Cartilage function was mapped in 63 locations per knee by rapid in-dentation testing (Ø=0.4mm indenter) and stiffness normalization based on cartilage thickness. Cartilage structure was determined by micro-computed tomography (mCT, (45µm)3 voxels) and calcula-tion of 3-D metrics (volume, thickness, curvature). Data (mean±SD) were analyzed by 2-way repeated measures ANOVA.Results: In regions distant from the graft site, OP and contralateral NON-OP MFC had similar geometry, with bone surface locations dif-fering by only 0.071±0.023 mm and cartilage thickness differences of 0.036±0.067 mm. Within and surrounding the OP graft, carti-lage and bone were different in 3-D geometry from NON-OP. The OP cartilage surfaces had subsided relative to NON-OP, with carti-lage volume diminished by 21.3±6.15 mm3 (p<0.05) and cartilage thickness decreased from 0.92±0.18 to 0.56±0.18 mm in the graft and from 0.94±0.18 to 0.77± 0.11 mm in the adjacent host cartilage (p<0.05). The OP bone surfaces were variably elevated relative to NON-OP and surrounding host bone. Cartilage indentation stiffness was lower in the OP than NON-OP knees by 68% at the graft site and 28% at the proximal surrounding host site (each, p<0.05). Conclusions: 3-D analysis may provide increased sensitivity to ef-ficacy of cartilage defect repair as well as mechanisms underlying success or failure.

25.3.5Zonal characterization of Equine Osteochondral Tissue from the Medial and Lateral Femoral CondyleK.E.M. Benders1, T. Klein2, J.C. de Grauw1, D. Hutmacher2, P..R. van Weeren1, M. Kik1, D.B. Saris1, W.J.A. Dhert1, J. Malda1 1Utrecht/Netherlands, 2Brisbane/Australia

Purpose: The equine species is becoming increasingly popular as a model for the in vivo evaluation of orthopaedic approaches. As there is also an increasing body of data suggesting that successful lasting tissue recon-struction requires an implant that mimics tissue organization, there is a clear need to investigate characteristics of equine osteochondral tissue. Therefore, we undertook a comparative study of the characteristics of equine, human and primate osteochondral tissue of the stifle (knee) joint. Methods and Materials: Osteochondral cores (4-8 mm) were taken from the summits of both medial and lateral femoral condyles of horses (n=16, mean age: 10.5), primates (two Gorillas, a Barbary Macaque and a Baboon) and humans (n=4, n=8 for cartilage thickness, mean age: 79.0±9.6 yrs). Osteochondral cores were either fixed in 10% formalin (for histology and image analysis) or frozen at -20°C and sectioned in 200mm slices for zonal biochemical quantification of DNA, glycosamin-oglycans (GAG) and collagen.Results: The average Mankin score of the equine samples was 0.9±0.9, whereas human and primate samples had considerably higher average scores (3.9±1.1 and 3.5±1.3, respectively). The average equine cartilage thickness was 2.37±0.58 mm (medial) and 1.30±0.21 mm (lateral). GAG content was significantly lower in the superficial zone and DNA content decreased significantly with depth through the articular cartilage. Im-age analysis of cell distribution yielded a similar distribution with depth. Consequently, GAG per DNA increased steadily with depth. Bone density decreased with depth from the bone-cartilage interface. Human and pri-mate samples demonstrated comparable trends, although more varia-tion was observed, which was associated with the higher Mankin scores. Conclusions: Equine and human osteochondral tissue possess similar geometrical (thickness) and organizational (GAG, DNA distribution, bone density distribution with depth) features. These similarities fur-ther highlight the potential value of the equine model in the evaluation of zonal approaches for cartilage repair.

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25.3.6Abnormal tibial torsion may cause an earliest detectable deformity in STR/OrtCrlj osteoarthritis mouse modelK. Naruse, K. Urabe, K. Uchida, M. Toyama, M. Itoman Sagamihara/Japan

Purpose: Morbidity caused by osteoarthritis of the knee joint (knee OA) enormously expands medical expense for the treatment. Physi-cians and surgeons as well as researchers are anxious to develop treat-ments for OA patients at early stages of knee OA. Reported model ani-mals develop detached anterior cruciate ligament or excised menisci, which mainly reflects post-traumatic late stages of OA. Lack of choice is apparent among the available models to be used for testing the ef-ficacy of potential drugs, which target the early stages. A few reported models, which may be suitable for studying early stages, are those of spontaneous knee OA in guinea pigs, C57BL/6J and STR/ort mice. Our histological observation as well as soft x-ray and micro-three dimen-sional computed tomography (3DCT) profiles supported that early changes typically observed among OA patients including the internal tibial torsion are reproduced in STR/ OrtCrlj mice. Abnormality was detectable even in the 10- to 20-week-old animals.We believe that the internal tibial torsion reflects mechanisms through which osteoarthritis develops in STR/OrtCrlj mice.Methods and Materials: STR/OrtCrlj mice were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan) and analyzed several weeks of age. The mice were euthanized by intraperitoneal injection of sodium pentobarbital, and bilateral lower limbs were excised from each hip joint and cleaned. The excised limbs were immediately reduced with ice-cold 4% paraformaldehyde in phosphate buffer solution (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) at 4oC for 48 hours. Samples were analyzed by X-ray system (SOFTEX-CMB4). The tibiae were ana-lyzed by a high-resolution micro-CT system (MCT-100CB). For the his-tochemical studies, dissected bilateral lower limbs were further decal-cified. Slides were prepared with 4-µm-thick sections, embedded in paraffin, and stained with classical hematoxylin/eosin or safranine O.Results: Except for one out of 24 knees in 12 animals of 35-week and 2 out of 4 knees in two 45-week-old mice, all the animals showed subchon-dral bone sclerotic changes in FT joints. Histological abnormality was a greater difference in cartilage degeneration between 26 and over 35 weeks. Alterations of cell density and gradient and the decreased stain-ing with Safranin O were ubiquitously observed in PF joint cartilage of all the knees of-10 week-old mice. At 20 weeks, the loss of Safranin O stain-ing was moderate to severe. In 20-week-old STR/OrtCrlj mice with age-matched C57BL/6J mice as a control, radiographic and micro-CT observa-tions pointed out internal rotation of tibiae. Morphological measurements of tibial torsion by micro-CT 3D analysis showed that typical STR/OrtCrlj tibiae at 35 weeks were twisted by 10 to 27 degrees when the two axes, the proximal posterior condyle and bimalleolar axes are superimposed. Conclusions: In conclusion, the earliest histological OA change was ob-served in the patellofemoral joint prior to similar observations in the femo-rotibial joint. Internal tibial torsion may be a cause of OA in the patellofem-oral joints, which leads to the development of medial femorotibial OA.

25.3.7Efficacy of bi-weekly intra-articular injections of BMP-7 in a rabbit ACLT/MMT model of osteoarthritisD. Lickorish, M. Haskell, S. Wood, A. Collett, D. Schrier, E. Goad, C. Flory Hopkinton/United States of America

Purpose: To evaluate the effect of bi-weekly, intra-articular injections of BMP-7 on articular cartilage morphology and bony architecture in an ACLT/MMT model of osteoarthritisMethods and Materials: Twenty nine New Zealand White rabbits under-went unilateral anterior cruciate ligament transection and medial meniscec-tomy. Intra-articular injections of either control vehicle (n=9) or 5mcg BMP-7 (n=20) were given bi-weekly (every other week) for 8 weeks. Femoral artic-ular cartilage morphology was assessed by india ink staining and decalci-fied histology. Osteophyte volume and trabecular bone volume/thickness in the medial and lateral femoral condyles were quantified using MicroCT.Results: Compared to vehicle only controls, BMP-7 treated animals showed consistently superior cartilage lesion scores (Figures 1a, 1b). Marked fissuring of the articular cartilage and erosion to subchondral bone was observed in controls but not BMP-7 treated joints. MicroCT analysis of joints demonstrated no radioopaque material in either the joint space or surrounding soft tissues in any of the BMP-7 treated ani-mals. Osteophyte formation, predominant along the medial border of the medial femoral condyle in control animals, was significantly reduced or absent in BMP-7 treated animals. Femoral trabecular bone volume (BV/TV) and trabeculae thickness in both medial and lateral condyles were both higher in the BMP-7 treated group compared to controls.

Conclusions: Bi-weekly dosing of 5mcg BMP-7 over 8 weeks prevented progression of cartilage damage in the rabbit ACLT/MMT model and did not elicit either intra-articular or extra-articular bone formation. Changes observed in femoral osteophyte formation and the femoral trabecular bony compartment suggest altered loading conditions of the BMP-7 treated joints.

25.3.8Subchondral bone repair following marrow stimulation in a mature rabbit modelH. Chen1, C.D. Hoemann1, J. Sun2, W. Ouyang1, A. Chevrier1, L. Dragomir1, V. Lascau-Coman1, N. Tran-Khanh1, M.D. McKee1, M.S. Shive1, M.B. Hurtig3, M.D. Buschmann1 1Montreal/Canada, 2Laval/Canada, 3Guelph/Canada

Purpose: Subchondral bone repair in osteochondral defects following bone marrow stimulation was evaluated at 3 months post-op using sur-gical techniques of microfracture and drilling to different hole depths.Methods and Materials: Trochlear cartilage defects 4 mm x 4 mm were prepared bilaterally in 16 skeletally mature rabbits. Microfracture holes were made to a depth of 2 mm, and drill holes made to either 2 mm or 6 mm depth under cooled irrigation. Animals were sacrificed 3 months post-operatively and joints scanned by micro-CT. Bone repair was as-sessed in a region of interest (ROI) of 3 mm x 3 mm x 2 mm within the defects and compared to un-operated controls. Results: Although almost all surgical holes were partially filled with mineralised tissue, there was incomplete regeneration of normal bone structure in defects. Abnormalities such as residual holes, cysts and bony overgrowth were observed (Fig. 1). Subchondral defect repair led to an average bone volume density (BV/TV) of 53.3%±8.89%, similar to that in the control (57.1%±7.88%), but the repair bone was more po-rous and branched as shown by significantly higher bone surface area density (BS/TV) and connectivity density than native bone (Fig. 2). No significant differences were found between microfracture and drilling techniques for BV/TV, BS/TV and connectivity density (Fig. 2, compare MFX2/I to DRL2/I). However, deeper drilling induced a repair response in a larger region than shallower drilling (5.4±2.04 mm2 vs 4.4±2.17 mm2), suggesting a greater level of bone remodeling, which also led to a higher BV/TV in defects (Fig.2A, compare DRL6/II to DRL2/II). Conclusions: Reconstitution of normal bone structure was incomplete at 3 months following bone marrow stimulation. Surgical techniques of microfracture versus drilling had similar effects on subchondral bone repair. In comparison with shallow drilling, deep drilling appeared to be advantageous for subchondral bone repair through increased marrow access and bone remodeling.

25.3.9Intra-Articular therapy of Hyrdogel scaffold with Chondroitin Sulphate in porcine cartilage defect modelK. Kannan, R. Xiafei, A. Mohd Hassan, J.H.P. Hui, E.H. Lee Singapore/Singapore

Purpose: Autologous chondrocyte implantation (ACI), currently, is the only useful surgical approach for full-thickness cartilage defects in patients, which utilizes injection of cultured cartilage cells into a debrided cartilage defect beneath a specially created lid of perioste-um. Chondroitin sulphate (CS) in hydrogel presents a novel and safe method of non-cell based treatment modality for cartilage defects. CS is a direct stimulator of human chondrocytes growth. We studied the optimal delivery of CS to the defect site using biodegraded injectable hydrogel medium. A long term release carrier like the hydrogel pro-vides a scaffold for a more local and direct source of CS to the joint.Methods and Materials: 18 adolescent male pigs were utilized in the study that involved the intra-articular injection of CS-hydrogel mix-tures (CSH) and CS-phosphate buffered solutions (CSPBS) as con-trols. Full thickness cartilage defects measuring 7mm were created in the medial distal femoral condyles and the defects were injected. Histological sectioning of the operated as well as the un-operated side was performed at 6 weeks and at 12 weeks. Results: Early results showed significant improvement in cartilage regen-eration with more native features of hyaline cartilage in the CSH group. There was no hyaline cartilage detectable at 12 weeks in the CS PBS con-trol group. Only a few chondrocyte clusters were observed in the control group while the CSH group exhibited increasing chondroctye morphology. There were marked improvements in the Wakitani histological scores de-noting the quality of the repair tissue in both time points of the CSH group. Conclusions: We conclude that CS delivered in an injectable, in situ gelable and biodegradeable hydrogel is a safe, convenient and effec-tive away to treat cartilage defects. The long term and slow release of CS in the joint could significantly enhance cell proliferation and cel-lular migration from cartilage and bone to the defect site.

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25.4.1A randomized, multicenter clinical trial comparing BST-CarGel™ to microfracture in repair of focal articular cartilage lesions on the femoral condyle: interim results from 41 patientsW.D. Stanish1, A. Restrepo2, P. MacDonald3, N. Mohtadi4, P. Marks5, M. Malo6, R. McCormack7, J. Desnoyers8, S. Pelet9, F. Lopez-Olivia10, J. Vaquero10, F. Macule11, M.D. Buschmann6, M.S. Shive6 1Halifax/Canada, 2Laval/Canada, 3Winnipeg/Canada, 4Calgary/Canada, 5Toronto/Canada, 6Montreal/Canada, 7Vancouver/Canada, 8Greenfield Park/Canada, 9Quebec City/Canada, 10Madrid/Spain, 11Barcelona/Spain

Purpose: BST-CarGel™ (BioSyntech Inc., Laval, Canada), a chitosan-based medical device, is applied in conjunction with bone marrow stimulation procedures. A randomized clinical trial was conducted to evaluate the efficacy and safety of BST-CarGel™ treatment compared to microfracture alone for repair of articular cartilage lesions, and an in-terim analysis was carried out on the first 41 patients (20 BST-CarGel™, 21 microfracture). Methods and Materials: The international (Canada, Spain, Korea), mul-ticenter trial enrolled 80 patients, aged 18 to 55, with BMI≤30 and single symptomatic grade III/IV focal lesions on femoral condyles. Patients were randomized (1:1) at the time of surgery to receive either BST-CarGel+microfracture or microfracture alone, and followed standard-ized 12 week rehabilitation. The primary endpoint was cartilage repair structure (quantity and quality), assessed using quantitative MRI at 12 months and analysis of elective biopsies after 13 months. Secondary endpoints were safety, evaluated through adverse events, and clinical benefit, determined with WOMAC (VAS) questionnaires. Results: At 12 months, MRI analyses found increased fill and quality of repair tissue in BST-CarGel-treated lesions compared to microfracture. ICRS macroscopic grading of cartilage repair at the time of biopsies was statistically better (p<0.05) for BST-CarGel™. (Fig1) Analyses of 21 biopsies (12 BST-CarGel™, 9 microfracture) demonstrated statistical improvement (p<0.05) for BST-CarGel™ compared to microfracture for Surface Architecture and Cell Viability parameters (ICRS I), the Over-all Score and Superficial Zone (ICRS II), and repair tissue thickness by histomorphometry. (Figures 1-2) Collagen organisation via Polarization Light Microscopy was also improved for BST-CarGel™. WOMAC data yielded equivalent improvements for both groups for pain, stiffness and function. Adverse events showed BST-CarGel™ to be comparably safe to microfracture alone. Conclusions: BST-CarGel™ treatment improved lesion filling and qual-ity of repair tissue at 12 months compared to microfracture in this inter-im analysis. This improvement in tissue quality should result in greater longer term durability of repair and sustained clinical benefit.

25.4.2Evaluation of Safety & Efficacy of Mesenchymal Stem Cell Composite for the Regeneration of Articular Cartilage Defect of Human Knee JointC. HaSeoul/Korea, Democratic People‘s Republic of

Purpose: This study is to evaluate the safety and efficacy of human umbilical cord blood derived mesenchymal stem cell composite for the regeneration of articular cartilage defects of human knee joint. Methods and Materials: Arthroscopically proven ICRS grade 4 lesions were included in this study. MSC concentration of 0.5 × 107cells/ml were implanted to the lesion as 0.5ml/㎠. Safety parameters included physical exam (swelling, tenderness, range of motion, pain), vital signs, lab tests and any advere events which were evaluated by the WHO common toxicity criteria. Efficacy was evaluated according to the ICRS Cartilage Repair Assessment , Pain VAS. and histological assessment. Second look arthroscopy and biopsy was performed in 2 out of 7 cases in this clinical trial after informed consent was obtained.Results: There were no significant adverse event of more than grade 3 according to the WHO toxicity criteria. The overall Repair assessment was improved in 67%. The biopsy result showed highly hyaline like re-generative tissue by H&E, Saf-O staining and Col II immunostaining. Conclusions: From the result of this study, the safety of the application of human umbilical cord derived mesenchymal stem cell composite in the regenerative treatment of articular cartilage defect of human knee joint was assured. The efficacy was assessed as reasonable. The prom-ising result of this study warrants further investigation in this strategy of articular cartilage repair.

25.4.3Agarose-alginate hydrogel as a scaffold for an allogeneic Cartipatch®T. Brune, N. Tan, L. Laganier, L. Barnouin Mions/France

Purpose: Current strategies for cartilage repair aim at introducing chondrocytes into scaffolds. An allogeneic source of cells would be interesting as there would be no donor site morbidity and it would allow a single-step surgical approach, without any timeout between cartilage harvest and tissue graft. Therefore we studied the oppor-tunity to grow arthroplasty-derived chondrocytes in comparison with chondrocytes from young pathological donors (indicated for autologous Cartipatch) in a characterized agarose-alginate hydro-gel for cartilage repair. We also addressed the possibility to main-tain the constructs throughout extended incubation.Methods and Materials: Chondrocytes were isolated from macro-scopically healthy cartilage harvested exclusively in non-weight-bearing zones, either from young patients (with traumatic lesions or osteochondritis dissecans) or from total knee arthroplasties. They were monolayer-expanded, suspended in the agarose-alginate hydrogel and incubated for 1 to 17 months under orbital agitation at 37 C. We achieved immunohistochemical staining and RT-PCR analysis in order to assess the potential of cell redifferentiation. Vi-ability was also estimated by propidium iodide staining.Results: Incubation in the hydrogel promoted redifferentiation of both arthroplasty-derived (n=6) and young donors chondrocytes (n=23). Indeed histological sections displayed cells with a spheri-cal morphology surrounded by an extracellular matrix positively stained for aggrecan and type II collagen. Preliminary RT-PCR data showed an important increase of type II/I collagen ratio and a slight increase of aggrecan expression throughout the process, both higher for arthroplasty-derived chondrocytes. Assessing the expression of matrix metalloproteinases evidenced the matrix turnover. Finally following long-term incubation inside the hydrogel viability remained stable (n=6) and the cells did not proliferate.Conclusions: This agarose-alginate scaffold allows extracellular matrix deposition and cell redifferentiation, in a roughly similar pattern for arthroplasty-derived and young donors chondrocytes, which had both partly lost their phenotype due to monolayer ex-pansion. It also maintains cell viability for months, thus offering a promising “on the shelf” solution for cartilage repair.

25.4.4Arthroscopic autologous chondrocyte transplantationA. Di Martino1, E. Kon1, G. Filardo1, S. Patella2, L. D’Orazio1, F. Balboni, C. Montaperto, M. Marcacci Bologna/Italy

Purpose: The incidence of articular cartilage pathology has grown due to the marked increase in sports participation and greater em-phasis on physical activity in all age groups. Unfortunately, articular cartilage lesions, with their inherent limited healing potential, are hard to treat and remain a challenging problem for orthopaedic sur-geons. In the last years matrix autologous chondrocyte transplan-tation has become a possible solution in the treatment of chondral lesions. We used a biodegradable, hyaluronian-based biocompat-ible scaffold for cell proliferation (Hyalograft C). The easy handling of Hyalograft C has permitted to develop an arthroscopic procedure for chondrocyte implant. Methods and Materials: Arthroscopic technique has been used from December 2000 in more than 150 cases. All the patients have been prospectively clinically evaluated using the International Car-tilage Repair Society score. Actually 83 patients achieved at least 7 years follow-up. Tegner score was applied to evaluate the sport activity level. MRI evaluation was also performed. Results: At the 7 years follow-up evaluation we had 8 failed pa-tients, who were re-operated for the chondral lesion. However, all the scores analysed still presented good results with a significant improvement compared to the basal level. The mean IKDC subjec-tive score obtained was 77.5 at 7 years. Self-assessment of quality of life, assessed by EQ-VAS, showed a statistically significant im-provement, too: 85/100. Conclusions: This matrix autologous chondrocyte transplantation procedure avoids the use of periosteal flap, simplifies the surgical procedure and permits to perform the arthroscopic implant reduc-ing the morbidity of the procedure. The medium-long term clinical and MRI results at 7 minimum years follow-up are positive, confirm-ing the positive results previously obtained and the perseverance of the beneficial outcome offered by this bioengineered approach.

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25.4.5Ambulatory treatment of recurrent tenopathies using infiltration of autholog blood growth factorM.V. Fernandes Dias, C.H. Bittencourt, P. Bittencourt, C.F. Bittencourt Niterói/Brazil

Purpose: .O {font-size:149%;} The use of growth factor on walk-in patients is considered by us a way of simplifying these procedures with total safety for patients and also for quality of expected tissue regeneration. Costs are lowered considerably. Patients become less anxious as the method is less complex. The procedure is con-sidered a simple treatment. Methods and Materials: .O {font-size:149%;} Simplified technique of infiltration in recurrent tenopathies (Achilles, patellar, elbow, ischial tuberosity and rotator cuff) using local anesthetic after evaluation of lesioned area. Seventy six patients were submitted to this treatment from June 2007 to March 2008. Method consists in drawing approximately 60ml of total blood from patient. Blood then undergoes centrifugation for 15 minutes at 3500rpm followed by removal of gel rich in platelets which are annulled of clotting functions, adding calcium chlorate. Solution resulting of this pro-cess is then infiltrated in lesioned area where platelets will release protein contents during first `hour.Results: .O {font-size:149%;} Patients informed complete improve-ment of symptoms in 82% of cases and the other 18% informed 50% to 70% reduction of pain. Significant improvement was seen when comparing MRIs before treatment and three months after. Conclusions: .O {font-size:149%;} Chronic tenopathies occur spe-cially due to repetitive effort lesions associated to deficient irriga-tion of tendons. Our method proved to be extremely efficient as it concentrates an extraordinary amount of proteins in the area and after local gene differentiation, cells multiply and regenerate these tendons, thus reducing pain and local inflammatory condition, al-lowing the patient an early return to physical activities.

25.4.6Image-guided surgical techniques for cartilage repair – an animal trialM. Kunz1, M.B. Hurtig2, S. Waldman1, S. Devlin1, J. Rudan1, D. Bardana1, J. Stewart1 1Kingston/Canada, 2Guelph/Canada

Purpose: We investigated whether image-guided surgical techniques can improve the clinical outcome of mosaic arthroplasty. While mo-saic arthroplasty is a valuable reconstruction option for cartilage repair, the accuracy to harvest and deliver osteochondral grafts re-mains problematic. Here we report on the use of two image-guided systems (opto-electronic and patient-specific instrument guides) to improve clinical outcome of mosaic arthroplasty in an animal model. Methods and Materials: Fifteen sheep were randomized into three groups with impact cartilage defects created in each knee. After three months, the defect of one knee was repaired using the: (i) conventional manual surgical approach, (ii) opto-electronic track-ing, or (iii) patient-specific instrument guides. For both image-guid-ed groups (ii, iii) harvest and delivery sites were pre-operatively planned using custom-made software. During opto-electronic guid-ance (group ii), instrument position and orientation were tracked and superimposed onto the surgical plan. For the patient-specific instrument guides (group iii), plastic templates were manufactured which incorporated mirror images of the articular surface to allow an exact fit between template and anatomy. Cylindrical holes within the template guided surgical tools according to the plan. Three months post-surgery, both knees were harvested and the curvature of the reconstructed cartilage was compared to the cartilage surface of a pre-defect arthrogram CT scan. For each repaired defect, macro-scopic (ICRS) and histological repair (ICRS II) scores were assessed. All results were statistically compared between the three surgical approaches using either non-parametric or parametric ANOVA tests. Results: There were no significant differences found in cartilage surface reconstruction and macroscopic scores between the im-age-guided and the conventional surgeries. However, both image-guided surgical approaches resulted in significantly better histol-ogy scores compared to the conventional approach (improvement by 55%, p=0.02).Conclusions: The results suggest that image-guided systems can improve the clinical outcome of mosaic arthroplasy for the repair of cartilage defects.

25.4.7The biological reconstruction of a chondral defect of the patella-femoral joint using dry arthroscopy.B. Sadlik1, A. Solecki1, T. Bielecki2, R. Brzoska1, A. Blasiak1

1Bielsko-Biala/Poland, 2Sosnowiec/Poland

Purpose: The tissue engineering technique using MSC are promising for patellar chondral lesions where the blood supply from the sub-chondral bone is insufficient. We present our method for the biologi-cal reconstruction of chondral defect of the patello-femoral(PF) joint with a dry arthroscopy technique (DA). This technique allows us to perform the AMIC technique in the patella-femoral joint enhanced by concentrated bone marrow without an open approach. An arthros-copy conducted with neither a liquid filled joint cavity nor gas hyper-presure we call dry arthroscopy and this allows us to perform a dry procedure with a collagen matrix stick into the chondral defect. Such an MSC implantation needs to be conducted in a dry environment.Methods and Materials: The lateral border of the patella is sus-pended by four threads to an Arthromast designed by us to allow sufficient space for arthroscopic surgery without hyperpressure in the PF joint. The membrane soaked with MSC is introducted into the joint using a specially designed sleeve and fixed by Tissucol glue to the defect crater. Results: In this preliminary trial five patients ( 2 men and 3 women, mean age 41Y ) with symptomatic chondral lesion (average 2,3cm2) in the patello-femoral joint underwent the above described proce-dure. Clinical results were assessed after 6 and 9 months by clinical examination IKDC2000 score , VAS scale and MRI. Good and very good results were obtained and confirmed by MRI in all 5 cases. Conclusions: We have showed the clinical ability to perform AMIC procedure with MSC transplantation as an arthroscopic procedure with good early clinical results. Development of the dry arthrosco-py technique will allow further application of more dry procedures which have, up to now, been performed as open techniques.

25.4.8‘Selective Osteoporosis of Medial Tibial Condyle’, one of the culprit for onset of Medial Compartment Osteoarthritis Knee.D. Goyal, A.D. Goyal, V. Thakkar Ahmedabad/India

Purpose: Majority of Degenerative Osteoarthritis of Knee begins with Medial Compartment. Medial overload can be due to; Varus de-formity, Weight bearing axis passing thru Medial Joint in Non-varus knee, deformities at other bone/joints etc. Literature speaks a lot about association of Medial Compartment Osteoarthritis (MCOA) and Varus deformity. But, association between Selective Osteopo-rosis of Medial Tibial Condyle (MTC) and Varus deformity has never been reported. Author believes that ‘Selective Osteoporosis of Me-dial Tibial Condyle’ occurs in some, leading to Proximal Tibia varus deformity. This in turn causes medial compartment overload, lead-ing to onset of MCOA. The paper aims to find out if such entity as “Selective Medial Tibial Condyle Osteoporosis” exists?Methods and Materials: Presence of Genu Vara overloads the Me-dial Compartment, thus starting/advancing medial arthritis. MCOA can also cause Genu vara deformity. So either of them can be pre-cursor/successor of other. Presence of “Selective Osteoporosis of MTC” can lead to gradual collapse of MTC architecture causing ‘Proximal Tibial Metaphyseal Varus Deformity’. This deformity, oc-curring few centimetres below medial joint line causes medial over-load. The Medial compartment continues to bear the load, till carti-lage reaches a threshold, and starts degenerating. All Consecutive patients coming to Senior Author’s Knee Clinic, with Medial Knee Pain, from Jan2009-Jan2010 were included in study. Patterns of Os-teoporosis, if present in Proximal Tibia, were studied. Cases with ra-diological evidence of multiple compartment involvement, Sclerotic joint margins, multiple osteophytes were excluded from the study.Results: 27% of cases studied had ‘Selective Osteoporosis of MTC’. 65% of such cases had associated “Proximal Tibial Metaphysis Varus Deformity”. There is a distinct group with overloaded Medial Joint, that has Osteoporotic Medial Tibial Condyle as culprit. Conclusions: “Selective Osteoporosis of Medial Tibial Condyle” does exist. However, How many progresses to “Proximal Medial Tib-ial Metaphyseal Vara” and MCOA; remains to be studied further?

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25.4.9Implantation of a novel synthetic, biodegradable Scaffold for Meniscus Tissue RegenerationR. Verdonk1, W.C.J. Huysse1, R. Forsyth1, P.C. Verdonk2

1Gent/Belgium, 2Gent-Zwijnaarde/Belgium

Purpose: To assess the safety and performance of a meniscus scaffold designed to restore the function of the meniscus after meniscectomy. Methods and Materials: This was a prospective, non-randomised, single-arm, multicentre clinical investigation in patients with an ir-reparable medial or lateral meniscal tear or partial meniscus loss, with an intact rim and the presence of both horns. The scaffold is designed to support the body’s own physiological pathways of tis-sue repair. Clinical efficacy was assessed at up to 24 months post-surgery using the following scores: Visual Analog Scale (VAS), the International Knee Documentation Committee (IKDC), the Knee and Osteoarthritis Outcome Score (KOOS) and the Lysholm score. Follow-ing implantation, DCE-MRI was performed to assess tissue ingrowth at 3 months, and anatomic MRI was performed to assess meniscal defect filling and cartilage scores at 1 week, and 3, 12 and 24 months. Results: Clinically and statistically significant improvements in all outcome scores were observed at 6, and 12 months post-implanta-tion. MRI scans show tissue gain was evident in 97.5% of subjects, and that there was no evidence of cartilage damage related to the device, up to 24 months. No safety issues related to the scaffold were reported. It is anticipated that the complete 24 month data will be presented here.Conclusions: Implantation of the meniscal scaffold resulted in statistically significant and clinically relevant improvements in all subjective clinical outcome scores at 12 months post-surgery. Tis-sue ingrowth and stable or improved cartilage scores were demon-strated by MRI assessments performed up to 24 months after im-plantation. Actifit Study Group: R Verdonk, P Beaufils, J Bellemans, P Colombet, R Cugat, P Djian, H Laprell, P Neyret, H Paessler

Posters192

P3Labral transplantation for hip instability after labrectomyJ.I. Erquicia, J. Miquel, M. Tey Pons, X. Pelfort, J.C. Monllau, E. Cáceres PalouBarcelona/Spain

Purpose: Acetabular labrum is important to maintain hip joint integrity. There is an increasing interest in hip joint related procedures to preserve the labrum and joint stability. A labral transplant case is presented.Methods and Materials: A 27 years old man was visited because of hip pain. and instability. He was able of daily life activities but he couldn’t perform heavy works or sports. On clinical exam impingement test and apprehension test were positive. He was previously treated because of femoroacetabular impingement through arthroscopic femoral osteoplasty for three consecutives times. X ray showed residual cam lesion in the Dunn axial view. MRI showed absence of anterosuperior labrum. Intraoperative findings confirmed radiological images. Degenerative labrum lesion around the defect was eliminated with a 3,5 cm final defect. Acetabular bone rim was debrided with a round burr. Labral defect was measured and transplant was fixed with 5 suture anchors. Acetabular labral transplantation and anterior femoral re-osteoplasty was performed by an anterior approach.Results: At two years follow-up patient is pain free and refers no hip instability. He has returned to sport activity. Postoperative non-arthritic hip score achieved is 95% and WOMAC score is 90.6%.Conclusions: At two years follow-up, labral transplantation offers an excellent result in this case. Effects of labral debridements need to be considered as a potential risk of hip instability.

P4Increased articular loading induced by uphill running cause cartilage lesion: An adjustable cartilage mechanical loading model in vivo and histological observationK. Li1, X. Wei2 1Taiyuan City/China, 2Taiyuan/China

Purpose: To develop a new adjustable in vivo cartilage loading model and study different effect of uphill and horizontal running on articular cartilageMethods and Materials: The training took place on a self-made adjustable treadmill. 15 rats were divided into three groups: control group (ambulate freely in cage, n = 5); horizontal running group (1 km/d, 45 days, n = 5); and uphill running group (1 km/d, horizontal running for 15 day for precondition and then uphill running for 30 days, n = 5).After 45 days, the rats were killed and articular cartilage of loading area of the knee was observed with Safranin-O staining and immunohistology.Results: The cartilage surfaces were intact after the horizontal running exercise. But the glycosaminoglycan concentration was decreased in the superficial and middle zones. Pathologic signs consistent with osteoarthrosis were developed after uphill running exercise. The OARSI score significantly worsened after uphill running.Conclusions: The increased mechanical loading during horizontal running decreased the glycosaminoglycan concentration, and the more increased mechanical loading induced by running on uphill slops induced cartilage degeneration. Running on different slops is an effective way to modulate the in vivo loading on cartilage.

P5Efficacy and security of a bioengineered meniscus implant. Experimental study in rabbit model of OA.A. Izaguirre1, R. Gomez-Garcia1, F. Izaguirre2, G. Gonzalez1, Z. Garcia3, G. Luna-Barcenas4, H. Lecona1, H. Garcia-Campillo1, H. Villegas1, L. Solis-Arrieta1, C. Pineda1, C. Velasquillo1, C. Ibarra1 1Mexico City/Mexico, 2Tampico/Mexico, 3Queretaro/Mexico, 4Mexico/Mexico

Purpose: To evaluate the efficacy and security of a bioengineered meniscus implant and meniscus transplant using meniscectomy as a control in an OA animal model. Methods and Materials: Our sample included fifteen New Zealand rabbits, five in each group. After simple randomization they were assigned to a control group of lateral meniscectomy (MMx), lateral Bioengineered Meniscus Implant with a composed chitosan/PVA scaffold (BEMI), or lateral Meniscus Allograft Transplantation

P1Histological and Biochemical Characterization of Fresh vs. Frozen Osteochondral Allografts: A Six Month In Vivo Study.D. Chase1, A.L. Pallante2, R. Healey2, S. Görtz2, A.C. Chen2, S.T. Ball2, W. Bugbee2, R.L. Sah2, D. Amiel2

18894/United States of America, 2La Jolla/United States of America

Purpose: Osteochondral allografting is a popular articular cartilage repair procedure. It is empirically believed that the use of fresh allografts conveys a longevity benefit, as compared to frozen allografts, owing to maintained tissue homeostasis secondary to preserved chondrocyte viability. This in vivo study aims to characterize the structural and biochemical differences between fresh and frozen allografts in a goat model. Methods and Materials: Frozen (-70°C) osteochondral allografts (8mm diameter and 5mm deep) and fresh (48hr post-mortem) allografts were transplanted (site-matched) into the distal femur of seven adult Boer goats, either in the weightbearing medial femoral condyle (MFC) or the patella femoral groove (PFG). At six months the allografts along with contralateral controls were harvested (28 grafts total). The retrieved allografts and controls underwent gross and histological analysis with H&E staining. GAG content was assessed qualitatively with Safranin-O staining. Radioactive Sulfate (35S) uptake was analyzed as a marker of metabolic activity and cell viability and was expressed as CPM (Counts Per Million)/mg. dry weight. Histomorphometry to assess surface roughness using computer generated idealized curves was also performed.Results: Grossly, two of the three frozen MFC allografts were markedly depressed. GAG content was nonexistent in all of the frozen allografts yet was qualitatively robust in all of the fresh allografts and controls (Figure 2). 35S uptake was significantly decreased in the frozen allograft group as compared to the fresh allografts (p<0.01) and controls (p<0.05, (Figure 1). Surface roughness was significantly increased in the frozen MFC allografts compared to the fresh MFC allografts (p<0.05). However, roughness was not significantly different between the fresh and frozen non-weightbearing PFC grafts (p=0.19).Conclusions: In our study, freezing osteochondral allografts prior to implantation resulted in gross depression, decreased GAG content, decreased 35S uptake and increased surface roughness. Therefore, freezing osteochondral allografts is indeed detrimental to the biochemical and structural integrity in vivo.

P2Normalization of Glenohumeral Kinematics in Curved vs. Flat Soft Tissue Interposition Graft: Implications of Glenoid ReamingN. Ghodadra1, K. McGill1, M.T. Provencher2, A.A. Romeo1, B.R. Bach, Jr1, N.N. Verma1

1Chicago/United States of America, 2San Diego/United States of America

Purpose: Resurfacing of the glenoid with biologic interposition of soft tissue and isolated reaming of the glenoid to recreate glenoid concavity have been used to treat young patients with glenohumeral arthritis. The goal of this study was to determine the change and location of glenohumeral contact pressures in curved vs. flat biologic interposition arthroplasty in a glenoid arthritis model. We hypothesized that biologic interposition with meniscal allograft will lead to the best normalization of glenoid contact pressure. Methods and Materials: Twelve cadaveric shoulders were tested in static positions of humeral abduction (30, 60, 60 with 90 degrees external rotation) with a 440N compressive load. Glenohumeral contact area and pressure were determined for 1) intact glenoid, 2) glenoid with cartilage removed, 3) placement of interpositional lateral meniscus allograft, 4) placement of interpositional achilles allograft, 5) arthritis model with concentrically reamed glenoid, 6) reamed glenoid with interpositional lateral meniscal allograft, 7) reamed glenoid with interpositional Achilles allograft. Results: Concentrically reamed glenoid with biologic interposition of lateral meniscal allograft restored mean contact pressure to 80% (p<0.03) of the intact state compared to 62% (p<0.05) for the Achilles allograft. Use of the curved allograft (meniscus) in reamed glenoid resulted in decreased contact pressure in the anteroinferior quadrant compared to flat allograft (Achilles). Glenoid with cartilage removed demonstrated statistically higher peak pressures than intact glenoid and glenoid with interpositional allograft. Concentric reaming of the glenoid led to decreased peak pressure and edge loading on the periphery of the glenoid compared to the arthritic glenoid model. Conclusions: Glenohumeral contact pressure is optimally restored with biologic interposition of lateral meniscal allograft compared to Achilles allograft. Our findings suggest that concentric reaming of the glenoid plays a pivotal role in evenly distributing glenohumeral contact pressure compared to the unreamed glenoid.

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volume of the proteoglycan-rich tissue stained with Safranin-O and type-2 collagen. On the other hand, the untreated defect was filled with the fibrous and bone tissues (Fig 1). Wayne’s scores were significantly higher in the DN gel-implanted knees than the untreated control concerning all gross appearance, histology and total scores at 12 weeks (Fig 2).Conclusions: This study demonstrated that implantation of the PAMPS/PDMAAm DN gel at the bottom of an osteochondral defect can induce spontaneous hyaline cartilage regeneration in vivo in a large animal (sheep) model.

P8Development of a new canine model of osteoarthritis: the groove modelS.C. Mastbergen, F.P.J.G. Lafeber Utrecht/Netherlands

Purpose: The frequently used anterior cruciate ligament transection (ACLT) model of osteoarthritis in the dog makes use of a permanent trigger (joint instability) for inducing degenerative changes. The present study evaluates a relatively new canine model of osteoarthritis, which is induced by a one-time trigger: the groove model. Methods and Materials: Articular cartilage of the weight-bearing areas of the femoral condyles in one knee of 30 Beagle-dogs was damaged by making grooves without damaging the subchondral bone. Surgery was followed by 3, 10, or 20 weeks intensified loading of the affected joint. The severity of osteoarthritis was evaluated at 3 (n=10), 10 (n=10), 20 (n=5) and 40 weeks (n=5) after surgery. Cartilage integrity, chondrocyte activity, and synovial inflammation were determined by macroscopy, histochemistry and biochemistry. Results: Ten weeks post-surgery osteoarthritic features were found. Proteoglycan synthesis, release of newly formed and resident proteoglycans, and amount of denatured collagen were enhanced, whereas proteoglycan content was diminished (all p<0.05). Based on these parameters there was a slow progression over time from 20 and 40 weeks after induction. Importantly, three weeks post-surgery these characteristics of osteoarthritis were not yet evident. In contrast, synovial inflammation was mild at 3 and 10 weeks and diminished slightly in time. Conclusions: The present results show that characteristics observed at 10 weeks or later post-induction of osteoarthritis in the groove model: · comparable to those found the canine ACLT model. · slowly progressive over time in the first year. · not (primarily) mediated by synovial inflammation. · induced by a one-time trigger. · not just the expression of the surgically applied damage. In this model the effect of treatment of cartilage damage is not counteracted by permanent joint instability or hampered by inflammation. Therefore, the groove model might be more sensitive to effects of therapy, aimed at cartilage protection and repair.

P9In vitro effect of Hyaluronic acid, Carnosine and Hyaluronic Acid+Carnosine on Bovine Articular Cartilage after oxydative stress (Preliminary Study).A. Migliore1, E. Bizzi1, U. Massafra1, V. Benetti2, M. Polzella2, D. Benetti2 1Rome/Italy, 2Perignano (PI)/Italy

Purpose: To evaluate the effects of Hyaluronic Acid (HA) alone or in combination with Carnosine on the release of Glucosaminoglycans (GAG) from a culture of bovine cartilage cells challenged with hydroxyl radicals. Methods and Materials: Fragments of articular cartilage were harvest from 12 months old bovine ankle and cultered in Ham/F12 medium supplemented with antibiotics, fetal calf serum (1%) and incubated for 24h in a mix of air/CO2 (19:1 v/v). Six different cultures were then separated. For five cultures the medium was changed with other mediums containing HA (10 or 50 mg/ml), or Carnosine (350 mg/ml) or HA (10 or 50 mg/ml) and Carnosine (350 mg/ml). Cartilage was challenged with hydroxyl radicals produced by Femton reaction with minor modifications and incubated for other 24 hours. At the end of incubation period, GAG were assayed by DBD method. Results: Hydroxyl radicals induced a significant release of GAG in the culture without modified medium, while cultures in medium containing HA at 10 and 50 and Carnosine 350 mg/ml showed a reduction of GAG release of 18% and 31% and 15.5% respectively. When HA and Carnosine were employed together, the efficacy was more evident and statistically significant at HA 50 mg/ml and Carnosine 350 mg/ml.

(MAT). We evaluated the following outcomes: 1) surgical technique reproducibility; 2) efficacy in terms of Macroscopic Jackson OA scale, Histologic Modified Mankin OA scale; 3) security in terms of Electron Microscopy and ultrasonographic assesment of the implants. We conducted non parametric hypothesis tests with 2-tailed significance set at (p<0.05). Results: We standardized the fibrochondrocyte cell culture technique and proved adhesion to the scaffolds. Surgical technique was feasible for MMx, BEMI, and MAT. We detected no differences in the OA evaluation between MMx or MAT, and the BEMI group presented significant inflammatory response. Conclusions: We standardized surgical techinques of MMx, MAT and BEMI in a rabbit model of OA. BEMI was neither efficacious nor secure in preventing OA in this model.

P6Effect of Hyaluronic Acid on impact-induced chondrocytes apoptosisR.B. Barreto, M.U. Rezende, G.B. dos Santos, A.C.F. Bassit, D. Sadigursky São Paulo/Brazil

Purpose: To evaluate the suppressive effect of intraarticular injection of hyaluronic acid immediately after blunt trauma on the progress of chondrocytes apoptosis in rabbits knees.Methods and Materials: An experimental model of traumatic-induced chondrocytes apoptosis was used in a total of twenty mature New Zealand white rabbits of the study. The rabbits was anesthetized and in the supine position the Mazieres contusion model ( Â 1 kg weight released from the top of a cylinder of one-meter length) was reproduced 3 times in each knee. Immediately after the trauma was injected in one knee 2ml of Hyaluronic Acid (HA knees) and in the contralateral knee was injected 2ml of Saline (saline knees). The medication intervention was repeated 5 times within intervals of 4 days. Postoperatively, the animals was permitted cage activity without any immobilization. One-week after the last injection, they were euthanized and articular cartilage fragment harvested. Apoptosis was detected using the ApopTag® Peroxidase Kit that uses the TUNEL method. TUNEL-positive cells was counted under light microscopy. Results: Two rabbits died before the end of the experiment. There was no significant difference in gross appearance of cartilage surface between the HA and saline knees. Blinded histologic analysis revealed that the HA knees had significant decrease of the rate of chondrocytes opotosis (apoptosis rate mean + SD, 33.08 + 1.68) compared with the saline knees (54.49 + 3.61) (P<0.001).Conclusions: This study suggest that the multiple injections of HA, starting immediately after the trauma, decrease the rates of impact-induced chondrocytes apoptosis.

P7The efficacy of a novel double-network hydrogel for spontaneous articular cartilage regeneration in a sheep modelM. Yokota1, N. Kitamura1, K. Arakaki2, E. Kondo1, S. Onodera1, T. Kurokawa1, J.P. Gong1, K. Yasuda1 1Sapporo/Japan, 2Naha/Japan

Purpose: We have developed an innovative method to induce spontaneous hyaline cartilage regeneration by implanting an originally developed PAMPS/PDMAAm double-network (DN) gel at the bottom of an osteochondral defect in a rabbit model. Prior to clinical application of this method, however, we need to clarify whether the spontaneous hyaline cartilage regeneration occurs in vivo in a large animal model.Methods and Materials: A total of 5 mature female sheep (Suffolk) were used in this study. An osteochondral defect having a 6.0-mm diameter was created in the femoral groove of the right patellofemoral joint. A cylindrical DN gel plug was implanted into the defect so that a defect having 3-mm depth remained after surgery. In the left knee, an osteochondral defect was created and remained without any treatment. At 12 weeks after surgery, the tissue regenerated in the defect was stained with HE, Safranin-O and immunohistochemical stain (type-2 collagen). The macroscopic and histological scores were quantitated using the Wayne’s grading scale.Results: The defect treated with a DN gel plug was mostly filled with a white opaque tissue at 12 weeks, while the untreated (control) defect was filled with white patchy tissues. Histologically, the defect in the DN gel-implanted knee was filled by a sufficient

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P12Articular cartilage repair by novel CCK2/gastrin receptor antagonist “AG-041R” using Drug Delivery SystemM. Kubo1, S. Imai1, T. Mimura1, K. Nishuzawa1, S. Araki1, H. Kitamura2, K. Noda2, Y. Hirakura2, Y. Matsusue1 1Otsu/Japan, 2Tokyo/Japan

Purpose: AG-041R was a novel indolin-2-one derivative and originally synthesized as a CCK2/gastrin receptor antagonist to treat gastric ulcers. Oral administration of a high dose of AG-041R on rats was unexpectedly found to stimulate systemic cartilage hyperplasia. Beneficial effects on chondrocytes in vitro and also for cartilage defects in vivo were proven. In this study, we applied AG-041R for rabbit cartilage defect using Drug Delivery System (DDS) and examined the effectiveness histologically. Methods and Materials: Full-thickness articular cartilage defects (5mm-diameter) at the patellar groove of the rabbit knee were used as cartilage defect model. PLA microspheres containing several amounts of AG-041R were prepared by the solvent-evaporation method. At first, in vivo release test was performed. In actual study, PLA microsphere containing 40% AG-041R and PLA microsphere uncontaining AG-041R (control ) embedded collagen gel were grafted into the left knee (AG group) or right knee (control group), respectively. The repair tissues were histologically examined by subsequent staining, Toluidine Blue (TB), the immunostaining of Bromodeoxyuridine (BrdU), type I and II collagen, and TUNEL staining, 1, 2, 3, 4, 8, 12 weeks after the surgery. Results: By in vivo release test, AG-041R was hardly detected in synovial fluid, however remained in repair tissue for four weeks. Repair tissue evaluation by TB staining showed significant improvement of AG group’s repair tissue at 12 weeks compared with control group’s one. Repair tissues were negative-stained with type I collagen and positive-stained with type II collagen immunostaining, i.e. hyaline cartilage. Proliferation cell number by BrdU staining was significantly large in AG group’s repair tissue at 3 weeks compared with control group’. Apoptotic cell number by TUNEL staining was not significantly different with each other. Conclusions: The application of AG-041R using DDS was proved to be effective for articular cartilage repair.

P13Experimental Osteoarthritis in a Stable Knee Joint Using a Critical-Size Defect in an Ovine ModelM. Schinhan1, M. Gruber1, P. Vavken1, R. Dorotka1, L. Samuoh1, C. Chiari1, S. Nehrer2 1Vienna/Austria, 2Krems/Austria

Purpose: Animal models simulating osteoarthritis are frequently associated with irreversible changes in biomechanics. Although these models successfully induce osteoarthritis, the results of experimental repair procedures are impaired by biomechanical problems. The aim of this study was to define the critical size of a chondral lesion to induce osteoarthritis in a stable joint, thus permitting correct assessment of cartilage repair procedures.Methods and Materials: 16 mature Austrian mountain sheep with a physiological joint were randomly divided into four treatment groups. In each group, a full-thickness chondral cartilage defect was created in the weight-bearing zone of the right medial femoral condyle. The diameter of the defect was 7 or 14 mm. The sheep were mobilized and permitted full weight-bearing for 6 and 12 weeks. Osteoarthritis was determined by gross assessment, India ink staining, biomechanical testing, histology (Mankin and OARSI scores), and COMP was chronologically monitored by ELISA.Results: In the six-week group, only minor osteoarthritis was registered for either defect size. After 12 weeks, the seven-millimeter defect created focal monocompartmental osteoarthritis at the medial femoral condyle and minor degenerative changes at the corresponding tibia. The 14-mm defect induced minor osteoarthritis at the femoral condyle and caused significant degenerative changes at the tibial articular cartilage and the meniscus.Conclusions: In an animal model, a 7-mm full-thickness chondral defect with a weight-bearing regimen of 12 weeks may be considered suitable to induce local osteoarthritis in an otherwise stable joint. We conclude that this approach is well suited for cartilage repair experiments.

Conclusions: This study seems to demonstrate the effects of HA and Carnosine on cultures of bovine cartilage cells on the release of GAG caused by oxidative stress induced by hydroxyl radicals. Such results seem to suggest a biologic role of HA and Carnosine on bovine cartilage cells in protecting cells from oxidative stress.

P10Investigation of hyperlipidemic property in STR/Ort miceK. Uchida, K. Naruse, M. Itoman, K. Urabe Sagamihara/Japan

Purpose: STR/Ort mice have unique characteristics including osteoarthritis and hyperlipidemia, and may be a useful model for investigating the effect of dyslipidemia on the underlying mechanism of primary osteoarthritis. We investigated the serum biochemical parameters, and serum adiponectin concentration in STR/Ort mice. Methods and Materials: Serum biochemical parameters and serum adiponectin concentration in STR/Ort mice were mesured, and compared the results with those for C57BL/6J, CBA/JN, and ICR mice, the genetically obese, diabetic mouse strain BKS.Cg-m+/+Lepr db/J (db/db) and lean control mice (m/m and db/m)Results: The serum concentrations of total cholesterol in STR/Ort mice were higher than those in other strains at 10 weeks. Serum triglyceride concentrations in STR/Ort mice were also significantly higher than those in ICR, CBA/JN, m/m, db/m and db/db mice at 10 weeks of age. Serum concentrations of insulin in STR/Ort mice were higher than those in C57BL/6J, CBA/JN, ICR, m/m, and db/m mice at 10 weeks of age. The serum concentration of adiponectin in STR/Ort mice was significantly lower than in other strains at 10 weeks.Conclusions: STR/Ort mice have human hyperlipidemic patient-like symptoms such as high serum total cholesterol, high serum triglyceride, hyperinsulinemia, insulin resistance, dysregulation of NEFA and low serum adiponectin. We hope this information will be useful for researchers investigating lipid metabolism and primary osteoarthritis using STR/Ort mice.

P11In vivo stabilization of chondrogenically differentiated mesenchymal stem cells by immune-isolationK. Kleinschmidt1, D. Lichtenberg1, W. Brehm2, E. Steck1, W. Richter1 1Heidelberg/Germany, 2Leipzig/Germany

Purpose: To determine the cartilaginous potential of human MSC, immune-deficient mice are used, but their acquirement is expensive. We investigated if the cartilage developing properties of hMSC can be analysed in immune-competent mice using encapsulation techniques. Furthermore, we aimed to analyse whether the reduction of nutrient supply and thus an approximation to the joint situation may reduce the mineralisation of MSC based cartilage constructs. Methods and Materials: MSC pellets were pre-differentiated chondrogenically for 4 weeks and encapsulated in alginate (AP), dialysis tubes (DT) or diffusion chambers (DC) in parallel to non-encapsulated controls. Constructs were transplanted into SCID and immune-competent BDF1 mice. Explants were analysed by alcian-blue, alizarin-red, and collagen-II (Col-II) staining as well as by human specific ALU hybridisation and evaluated using double-blinded histology scoring. Results: Control constructs in BDF1 mice surprisingly showed no signs of destruction and 4/5 spheroids were positive for alcian and Col-II. In parallel, the degree of mineralisation was reduced in BDF1 compared to SCID (p=0.03). Alginate hampered mineralisation in BDF1 (0/8) whereas in SCID 7/8 constructs calcified (p=0.001, Figure A+B). AP resulted in better histological scores for Col-II (p=0.04) and reduced mineralisation (p=0.03). In addition in SCID alginate conferred higher alcian levels compared to controls (p=0.003). DC was inapplicable since constructs lost their cartilage-like properties. Only in SCID the use of DT resulted in an enhancement of Col-II (p=0.03) and a reduced calcification (p=0.004). Conclusions: The BDF1 mouse is an attractive cost-reducing alternative to evaluate in vivo stability of MSC based cartilage constructs which obviously conferred a reduced mineralisation compared to SCID. Possibly, the in vitro generated cartilage matrix constitutes an environment that inhibited destruction of transplanted constructs. Whether the cartilage stability is promoted by an intact immune-system or impaired by a high NK-cell activity reported for SCID mice has to be evaluated by further studies in additional mouse models.

Posters 195

Here, we established an OA model of C57BL/6 mice that is more reproducible and amenable to therapeutic intervention by controlling their movement.Methods and Materials: OA was induced in 9 week-old C57BL/6 mice by destabilizing the medial meniscus (DMM). the mice were then placed in the big cage for free movement (Group I) or in the confined cage (4.5x7x20 cm) with no exercise (Group II), with an exercise of 400 m/day (Group III), and with an exercise of 800 m/day (Group IV), respectively. The mice were sacrificed 1, 2, and 4 weeks after the surgery. Macroscopic and histological evaluations with ICRS scoring of cartilage lesions were performed on the medial femoral condyles and tibial plateaus cartilages. Results: The Group II mice showed slightly severer but more reproducible OA lesions than those of Group I mice. The mice in Groups III and IV showed that the OA lesions were getting severer depending of the amount of exercise. In all Groups, the degree of OA lesions increased along with the analysis times of 1, 2 and 4 weeks. Overall, the individual variation of OA lesions was much less significant in the Groups of the confined cage than in the Group with the free movement. Conclusions: The OA lesion of mice could be more reproducible and regulated by the enforced and periodic exercise of mice in the confined cage. We speculate that our OA model of mice with controlled movement could be a useful tool to study the OA process and efficacy of OA therapy.

P17Healing process of the fibrocartilaginous insertion of a supraspinatus tendon tear in primatesH. Ueba, S. Imai, S. Araki, K. Nishizawa, Y. Matsusue Otsu/Japan

Purpose: It is well known that supraspinatus tendon has a prominent zone of fibrocatilage at its attachment site. And it is suggested that it may have some important biomechanical role to protect the tendon. Microfracture procedure represents a frequently used technique for repairing of articular cartilage defects. With this procedure, marrow-derived mesenchymal stem cells were led to produce a fibrocartilage repair at the defect site. We compared healing process of the microfracture-group which had microfracuture procedure after transection of the supraspinatus tendon to the control group. We chose cynomolgus monkeys as a model animal because they use their forelimbs for grasping or holding things rather than walking unlike other animals. Methods and Materials: Eight cynomolgus monkeys were used in this study. The shoulders were divided into 4 groups, which were determined by the size of supraspinatus tear and subsequent microfractures: 4mm+, 4mm-, 8mm+, and 8mm-. In the 4mm+ and 4mm- groups, the tendon was transected in 4mm length. In the 8mm+ and 8mm- groups, the tendon was transected in 8mm length to be detached completely from its insertion. Microfracture was then performed in the 4mm+ and 8mm+ groups. The monkeys were euthanized on the 6, 12 and 24 weeks after surgery. The specimens were evaluated with hematoxylin-eosin and Toluidine blue staining and types I and III collagen immunostaining. Results: In all groups, torn sites were surrounded by granulation tissue. Histological analysis revealed progressive maturation and reorganization of the bone-tendon interface. Compared to the 4mm- group, more collagen fiber continuity and more fibrocartilage at the insertion site was seen in the 4mm+ group. Same trend was seen in comparison the 8mm- group to the 8mm+ group. Conclusions: The result of the present study indicated Marrow-derived mesenchymal stem cells may play an important role in the process of rotator cuff tear healing to the bone interface.

P19Intervertebral disc repair using adipose tissue derived stem and regenerative cells: Experiments in a canine modelH. Meisel1, T. Ganey2, W. Hutton2, M. Hedrick3, B. Strem3 1Halle/Germany, 2Atlanta/United States of America, 3San Diego/United States of America

Purpose: Disc injury can lead to disc degeneration. However, if a damaged disc could be repaired in the early stages, before the onslaught of degeneration then the process may be slowed down. Our goal was to test the hypothesis that repair of a damaged disc is possible using autologous adipose tissue derived stem and regenerative cells (ADRCs).

P14Tissue Engineering in Osteoarthritis- Cartilage Repair in an Ovine Model using a Hyaluronan MatrixM. Schinhan1, M. Gruber2, R. Dorotka1, N. Roessler1, S. Nehrer2 1Vienna/Austria, 2Krems/Austria

Purpose: This animal model is aiming to prove the concept of tissue engineering in a stable osteoarthritic joint in by the use of a hyaluronan matrix (Hyalograft-C-plus) as a scaffold for chondrocyte transplantation.Methods and Materials: Osteoarthritis on the medial femoral condyle was induced using a critical size defect of 7mm and a weight bearing regime of 12 weeks. At this index procedure cartilage biopsies were taken for cell culture in the tissue engineering group. At the second procedure a defect of 20x10mm was created in the arthritic region of the medial condyle including an abrasion of the subchondral bone to provide a trough for the implant. Then the 18 sheep were divided into 3 groups with 6 sheep each: G1: implantation of a hyaluronacid matrix seeded with chondrocytes, G2: implantation of an unseeded hyaloronan matrix, G3: only the bone preparation by abrasion. Sheep were euthanized and analyzed 16 weeks after the second procedure. Gross examination and Indian Ink Staining were performed. Histological sections were stained with HE and Safranin O and assessed with the Mankin and the O’Driscoll Score.Results: Comparing the medial femoral condyle gross assessment showed significant better results in G1 than G2 (p<0,009), which was worse than G3 but not significant. Histological analysis using the Mankin score for the regeneration area examined significant better results of G1 compared to G2 und G3. Scoring the regeneration area with the O´Driscoll score showed a high significant difference (P<0,0001) between G1 (15,920±0,519) and G2 (5,042±1,491) and G3 (6,583±0,550). Conclusions: The four-month results in our ovine model suggest that matrix associated chondrocyte transplantation is a valid strategy for osteoarthritis in an early stage.

P15Modelling of chondral and osteochondral injuries in sheep: a tool for preclinical studies in regenerative medicineJ. Barrachina1, D. Codina1, D. Peris2, C.S.F.R. Da Fonseca2, M. Caminal3, X. Moll2, A. Morist2, R. Rabanal2, F. García2, J.J. Cairó2, F. Gòdia2, J. García3, A. Pla3, J. Vives2 1Sant Cugat del Vallés/Spain, 2Bellaterra/Spain, 3Barcelona/Spain

Purpose: Animal studies are critically important to developing effective treatments for cartilage injuries. We herein present our experience, in an ovine model, on the technical aspects of generation of chondral and osteochondral lesions and their application for testing the therapeutical potential of new approaches in regenerative medicine. Methods and Materials: 2-year old ewes were used in all the studies. To reduce the number of animals, we first used frozen legs in order to determine the most convenient tools for each type of lesion. In living animals, chondral and osteochondral lesions were generated arthroscopically in the femoral medial and lateral condyles of the two posterior legs. Animals were monitored by MRI, echography and X ray and a full necropsy and histological analysis was performed at the end of the studies. Results: We describe our arthroscopic technique for the generation of lesions of different depth for the study of chondral and osteochondral regeneration. The reproducibility and the clinical evolution of the lesions were assessed by MRI, ecography and X ray. Also, macroscopic and histological analysis were performed at the end-point of the studies to confirm the extent of the lesions and the effect on surrounding tissue. Conclusions: We demonstrated that we can generate reproducible lesions in sheep with the same extension and depth for the study of both partial thickness and full thickness chondral repair, as well as osteochondral repair.

P16Establishment of osteoarthritis model using C57BL/6 mice by controlling their movementB.J. Kim1, B.H. Choi2, S.R. Park2, D. Minh1, B. Min1 1Suwon/Korea, 2Incheon/Korea

Purpose: Many osteoarthritis (OA) models have been developed in mouse to understand the OA progress and evaluating new therapies. However, the individual variation of the joint lesions remains as a critical problem in most of the current OA models.

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monolayer culture before treatment. The most likely contaminantes in the cultures are synoviocytes due to synovial overgrowth of the biopsy area. The aim of the study was to (i) study chondrogenic differentiation of synoviocytes and to (ii) identify genes that could specifically determine purity regarding synoviocyte contamination. Methods and Materials: Synoviocytes were isolated from human tissues (n=5) and the cells were expanded in monolayer culture (ML) followed by RNA preparation or seeding to a hyaluronan scaffold (HYAFF 11, Fidia Advanced Polymers) subsequently cultured for 14 days in a modified differentiation media. The scaffolds were analyzed regarding handling properties, morphology and histology. Messenger RNA from ML cultures was subjected to gene expression analysis using oligonucleotide microarray (Affymetrix). Expression data was compared to previous microarray data from human chondrocytes in ML (n=5). Candidate genes selected from the microarray analysis were confirmed by real-time PCR. Results: When comparing monolayer chondrocytes with synoviocytes no differences were observed microscopically and synoviocyte seeded scaffolds showed similar handling characteristics as chondrocyte seeded scaffolds. However, the histology results showed slightly higher matrix production in the chondrocyte seeded scaffolds. The gene expression comparison identified a distinct set of 4 genes (designated Syn 1- 4) that was barely detected in chondrocytes but highly expressed in synoviocytes. Conclusions: Although the articular cartilage and the synovium tissue can be easily identified in biopsies it is impossible to exclude potential contaminating synoviocytes in the subsequent culture process. We here demonstrate that three of the identified candidate genes have the potential for identifying chondrocyte purity regarding synoviocyte contamination.

P22T2 mapping - Does the choice of imaging sequence matter when using T2 phantoms?J.K. Riek Rochester/United States of America

Purpose: The purpose of this study is to examine the use of different T2 mapping sequences on different manufacturers’ MR scanners and determine if similar results can be obtained by using T2 phantoms.Methods and Materials: Two different T2 mapping sequences were utilized on a Philips Panorama HFO 1T magnet, and on a GE Signa HDxt 1.5T magnet. On the Philips magnet, a 7-echo sagittal T2 mapping sequence was used (TR=2000ms, TE=16,32,48,64,80,96,112ms, ETL=7, 3.5mm slices, FatSat), and a dual-echo sagittal FSE (TR=2000ms, TE=8.9, 85ms, ETL=12, 3.5mm slices, FatSat). On the GE Magnet, a dual-echo sagittal FSE (TR=3200ms, TE=22.3ms, 78.1ms, ETL=8, 3.5mm slices, FatSat). T2 maps were calculated by performing a linear regression on the natural log of the intensity of the images. For the sequences with more than two echoes, where the signal intensity was not different than the background noise, the data point was ignored. Vials with varying concentrations of copper sulfate were placed on the knee. These vials had known T2 relaxation times of 30, 60, 121 and 231 ms. Polynomial interpolation was used to adjust the calculated T2 values in the image so that the mean T2 relaxation in the vials corresponded to the known values.Results: The average T2 value within the femoral cartilage on one sagittal slice was calculated from both sequences on the Philips machine. For the dual-echo sequence, the uncalibrated value was 68.1ms. The calibrated value was 77.8ms. For the seven-echo sequence, the uncalibrated value was 48.4ms, and the calibrated value was 71.01ms. The calibrated dual-echo value on the GE machine was 69.5ms. Conclusions: It has been demonstrated that reasonably similar T2 values can be calculated across multiple pulse sequences and multiple vendors utilizing T2 phantoms. Resolution and SNR become the most important factors, not the pulse sequence.

P23Expression of microRNAs in peripheral blood mononuclear cells as a novel biological marker for knee osteoarthritisA. Okuhara, T. Nakasa, H. Shibuya, T. Niimoto, N. Adachi, M. Deie, M. Ochi Hiroshima/Japan

Purpose: MicroRNA (miRNA) is a family of non-coding RNA that plays an important role in human diseases, including osteoarthritis (OA). Several studies demonstrated that OA was a systemic disease with an aggregation of immune cells in synovium. The objective of

Methods and Materials: Twelve dogs underwent a partial nucleotomy at three lumbar levels (L3-L4, L4-L5, L5-L6); adjacent levels served as non-operated controls. The animals (or discs) were allowed to recover from the surgery for six weeks. At that time subcutaneous adipose tissue was harvested and adipose tissue derived stem and regenerative cells (ADRCs) were isolated. The three experimental discs that had undergone a partial nucleotomy were randomized to receive: 1) ADRCs in hyaluronic acid carrier (Cells/HA); 2) HA only; or 3) No Intervention. Assessments of the three experimental discs plus the two adjacent untouched discs were made using MRI, radiography, histology and biochemistry. The animals were euthanized at 6 months, and at 12 months.Results: Repair in this study was specifically demonstrated through histology and biochemical analysis. Disc levels receiving ADRCs more closely resembled the healthy controls as evidenced in matrix translucency, compartmentalization of the anulus, and in cell density within the nucleus pulposus. Matrix analysis for Type-II collagen and aggrecan demonstrated evidence of a statistically better regenerative stimulation to the disc provided by ADRCs when compared to either the HA only or the No Intervention treatments. Conclusions: Autologous adipose tissue derived stem and regenerative cells, as used in this disc injury model, were effective in promoting disc regeneration, as evidenced by disc matrix production and overall disc morphology.

P20The early administration of PRGF in the ovine ACL-transected knee osteoarthritis modelM. Sanchez, E. Anitua, J. Guadilla, R. Prado, F. Muruzabal, G. Orive, N. Fiz, J. Azofra Vitoria/Spain

Purpose: We assessed the ability of PRGF (plasma rich in growth factors) to influence OA progression in sheep-model following trauma to the joint caused by ACL rejection. The treatment was initiated immediately after the surgery. PRGF system is a 100% autologous preparation characterized by a 2-3 fold platelet concentration from whole blood, and is a leukocyte-free plasma.Methods and Materials: Seven Latxa sheep (4-6 years) and weighing 61±6.1 kg were used. Body condition score was evaluated estimating the level of muscling and fat deposition. Neiker-Tecnalia Animal Care Committee approved all procedures used. Surgeries were performed bilaterally in the hind limbs. A medial parapatellar incision was made and the patella was reflected laterally in order to expose the ACL, which was subsequently transected. Ultrasound-guided infiltrations were initiated ten days after ACL transection. Sheep were treated with a series of ten biweekly intra-articular injections of 4 mL of PRGF in one stifle, and 4 mL of saline in the contralateral. The platelet concentration was measured. Sheep were sacrificed 32−37 weeks post-surgery. Knees were macroscopically studied and the cartilage and subcondral-bone of the femoral condyles were evaluated by two histological scales. ANOVA analysis was performed to assess the differences between the control and PRGF-treated animals. Age, weigh and platelet number were used as covariables.Results: Body condition scoring ranged from 2.0 to 3.5 (2.8673±0.5). PRGF platelet mean content was 319x106 (±93x106) platelets/mL and MPV 4.0±0.3 fL. The macroscopical findings were evident signs of osteoarthritis, but no significant differences were found in the scores. In the histological evaluations, the PRGF-treated group presented an improvement in internal condyle scores (p<0.05) compared with the controls. Both histological scales showed a high correlation (Pearson correlation=0.922, p<0.01).Conclusions: These preliminary data suggests that there may be a therapeutic benefit associated with intra-articular injection of PRGF in secondary osteoarthritis, following traumatic injury to the knee.

P21Potential gene specific markers to proof the purity of human articular chondrocytes in monolayer cultureC. Concaro1, H. Sterner1, C. Brantsing1, M. Leander1, J. van der Lee1, M. Brittberg2, T. Dehne3, J. Ringe3, A. Lindahl4 1Gothenburg/Sweden, 2Kungsbacka/Sweden, 3Berlin/Germany, 4Göteborg/Sweden

Purpose: Due to the new EC regulations, No 1394/2007 on Advanced Therapy Medical Products, there is a need to establish markers to proof the purity of human articular chondrocytes in

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novel biomaterials based on biodegradable polylactide polymers in combination with the gel. With these constructs we aim to provide suitable environment for the maintenance of chondrocyte phenotype within a matrix that also provides structural stability.Methods and Materials: Chondrocytes were harvested from patellofemoral groove cartilage of bovine knees. To fabricate collagen gels, rh type II collagen (FibroGen Europe, Helsinki, Finland; 3mg/ml in 10mM HCl) was combined with growth medium (DMEM/F12 with supplements) containing chondrocytes. After gelation the gel-cell constructs were moved onto two different type of non-woven PLDLA-scaffolds (PLDLA 96/4, Tampere University of Technology, Tampere, Finland), and the hybrids cultured for 9 to 21 days. Different protocols were used to maximize infiltration of the gel into the PLDLA-scaffold. Cell viability and depth of gel penetration were analyzed. Chondrocytes were also cultured on the PLDLA-scaffolds without the rh type II collagen gel, and analyzed for penetration, viability and collagen type II production.Results: When cultured in PLDLA-scaffolds without the rh type II collagen gel, majority of the chondrocytes were alive and maintained their rounded phenotype while growing parallel to the PLDLA-fibers. The cells secreted collagen type II, suggesting preservation of chondrocyte phenotype. Chondrocytes grown inside the gel retained rounded, and the gel infiltrated into the PLDLA-scaffold. Penetration depth of the chondrocytes within or without rh type II collagen gel was 100-500µm. This indicates the tightness of the PLDLA-scaffolds to allow free infiltration of the gel into the scaffold.Conclusions: These results suggest that rh type II collagen gel within biodegrable PLDLA-scaffold can provide mechanically rigid chondrogenic structures for repair of large articular lesions. Further studies are needed to increase the penetration of the gel into the PLDLA-scaffold and to investigate cartilage producing capacity of the hybrid implants.

P26Chondroinductive potential of subchondral nacre implant in the sheep kneeM. Rousseau1, O. Delattre2, P. Netter2, P. Gillet1, E. Lopez3 1Vandoeuvre les Nancy/France, 2Fort de France/Martinique, 3Paris/France

Purpose: One of the major challenges of orthopedic surgery is the restoration of bone and cartilage losses by using biomaterials. Until now, only few molecules and biomaterials have shown their potential ability to restore osteochondral structure and function. The present study was thus designed to analyze the intra-articular behaviour of nacre, a natural biomaterial, when implanted in the subchondral bone area in the sheep knee. Preliminar results suggest that this natural biomaterial may provide a potential alternative source of chondroinductive molecules. Methods and Materials: We implanted nacre blocks in sheep trochlea by replacing the halfth of the femoral trochlea (nacre group). For comparison we used complete cartilage resection (resection group) down to the subchondral bone. Sheep were sacrificed after 3, 6 and 9 months for histological and radiological evaluations of the cartilage/nacre interface. Results: In the “nacre group”, implants were well tolerated without any synovial inflammation. In addition, we observed centripetal regrowth of new hyaline cartilage in 6/9 cases after 3 months as radiography showed osteointegration. In the “resection group”, no chondral regrowth was observed, but, in contrast, a thin layer of fibrous tissue formed. After 6 and 9 months, a new layer of cartilage covered the nacre implant and cartilaginous islets of hyaline cartilage were observed in the deep layers. No spontaneous osteochondral regeneration was found in the “resection group” at these keypoints. Conclusions: Nacre exerts chondroinductive potential as a subchondral implant for cartilage regrowth. This new result opens new perspectives for the use of nacre as a biomaterial for cartilage repair and regeneration.

P27Ibuprofen delivery microspheres : efficacy on cyclooxygenase in activated cartilage and synovial membranesL. Bédouet1, L. Moine2, F. Pascale3, M. Wassef1, M. Bonneau3, V. Nguyen2, D. Labarre2, A. Laurent1 1Paris/France, 2Châtenay-Malabry/France, 3Jouy-en-Josas/France

Purpose: Biodegradable microspheres (40 – 100 µM) containing ester linked ibuprofen were prepared to provide a sustained release of the anti-inflammatory drug in the joint cavity for intra-articular

this study was to investigate the expression pattern of miRNAs in peripheral blood mononuclear cells (PBMCs) and explore the feasibility of using miRNAs as a biomarker to diagnose OA. Methods and Materials: Thirty six patients with OA, and 10 healthy controls were included in this study. We obtained PBMCs and analyzed the expression of miRNA(miR)-146a, 155, 181a, and 223, which express in immune cells and regulate immune function and inflammation, in PBMC using quantitative reverse transcription-polymerase chain reaction. The expression of miRNAs in OA was compared with that in healthy subjects. We also investigated the expression pattern of miRNAs expression in OA progression, and its relationships with parameters that include age, body mass index (BMI), femoro-tibial angle (FTA), and keratan sulfate. Results: The average relative expression levels of miR-146a, miR-155 and miR-223 were 5.8-, 7.6-, and 12.6-fold, respectively, higher for OA patients than for healthy subjects. As for the relative expression levels of miR-146a, miR-155 and miR-223 according to Kellgren-Lawrence classification, that of miR-146a, 181a and miR-223 were intensely expressed in OA PBMCs with a low grade of the Kellgren-Lawrence classification. There was a significant correlation between the expression of miR-223 and the serum concentration of the keratan sulfate. Conclusions: The present study demonstrated that the expression levels of miR-146a, miR-155, miR-181a, and miR-223 in the PBMC of OA patients could be a novel biomarker for diagnosis of early stage OA and its prognosis. This evidence might lead to the elucidation of OA pathogenesis and a novel therapeutic strategy for OA.

P24Tailor-made scaffolds for stem cell chondrogenesis? Different cross-linking of scaffolds from marine collagen affects their mechanical and physical properties.J. Renger1, H. Notbohm1, R. Wendlandt1, C. Koch2 1Lübeck/Germany, 2Kiel/Germany

Purpose: Stiffness of scaffolds for tissue engineering is known to impact stem cell fate by influencing focal-adhesion structure and cytoskeleton. For tailor-made scaffolds, one seeks to deterministically change this parameter to find the optimal growth conditions for chondrogenesis. From preliminary analyses we can show an initial model for the impact of UV-irradiation on stiffness and associated physical properties. Methods and Materials: Beside decreasing its solubility, cross-linking of collagen by UV-irradiation, heat, or chemicals results in a higher stiffness. Collagen concentrations and collagen superstructure (i.e. molecular or fibrillar collagen) have further impact on stiffness of a matrix. By variing these parameters, one can take this advantage for tailor-made collagen scaffolds. Pilot study: Scaffolds from molecular and fibrillar-like marine collagen were produced from several collagen concentrations. Cross-linking was induced by UV-light at 254 nm in different doses optionally followed by heat fixation. Levels of cross-linking were estimated by measuring solubility, enzyme resistance and heat stability. A mechanical testing machine (Zwick/Roell) was used to determine the influence of cross-linking degree and scaffold composition on its mechanical properties, i.e. its elastic modulus. Results: Increasing levels of cross-linking by UV-light showed an increase in elastic modulus of matrices, whereas solubility decreased. Heat fixation (1h) itself had a comparable effect to UV-irradiation, but scaffolds irradiated with 106 kJ were insoluble even without heat fixation. Differential scanning calorimetry showed an increase in collagen denaturation after UV-irradiation, matching the lower trypsin resistance. Conclusions: Our studies show that by optimized cross-linking by UV-irradiation, marine collagen scaffolds can be adjusted to a required stiffness guiding stem cells to differentiation to chondrocytic lineage. Cross-linking of the material can be observed by measuring its solubility, heat- and enzyme resistance. Additionally, fibrillar-like collagen scaffolds represent nano-designed matrices for tissue engineering. This approach might be a further step to tailor-made scaffolds from marine collagen.

P25Cartilage tissue engineering with recombinant human type II collagen gel infiltrated in PLDLA-scaffoldsV. Muhonen1, E. Järvinen1, E. Siren1, M. Forsten2, V. Ellä2, M. Kellomäki2, I. Kiviranta1 1Helsinki/Finland, 2Tampere/Finland

Purpose: Recombinant human (rh) type II collagen gel has been shown to be eligible 3D-matrix for chondrocytes. We have used

Posters198

P29Surface Modification of Extracellular Matrix Biomembrane for CytophilicityB.R. Song1, Y.J. Kim1, M.S. Kim1, J.H. Yoon1, S.R. Park2, B. Min1 1Suwon/Korea, 2Incheon/Korea

Purpose: Cell delivery has been getting more popular for tissue regeneration so that many biomaterials are being developed for cell delivery vehicle. In our previous study, we developed extracellular matrix biomembranes with higher biocompatibility, but it needed a higher cell-compatibility to be used as a vehicle for the cell delivery. In this study, we examined whether surface charge of extracellular matrix biomembrane can be changed to be a suitable cell delivery vehicle by enhancing cytophilicity. Methods and Materials: The surface charge of the Artifim® (RegenPrime Co., Ltd., Korea) measured to confirm any changes of chondrocyte attachment rate/forced with the alternated surface charge. The surface charge of the Artifim® was changed by the chemical bond of poly L Lysine and glutaric anhydride. For binding of electrically positive polymer, poly L Lysine, on Artifim® surface, the Artifim® was treated with 50mM EDC/NHS for 12hr and reacted with the poly L Lysine for 24hr. Negative charges were introduced on Artifim® surface by treating the glutaric anhydride in 0.8M sodium phosphate buffer (pH8) for 24hr. Results: Before modification, average surface charge of Artifim® was -55mV. After modification, poly L lysine 5uM group showed -30mV and glutaric anhydride 0.45M group showed -66mV. The glutaric anhydride group significantly enhanced chondrocyte attachment rate/force and those were getting more along with higher concentration of glutaric anhydride. However Artifim® treated with highly concentrated poly L Lysine decreased chondrocyte attachment rate/force. Conclusions: The attachment rate/force of chondrocytes can be affected by the changes of surface charge. Especially the more negatively charged surface provided suitable condition for the chondrocyte attachment. From this study, we could modify the surface of the extracellular matrix biomembrane with improving cytophilicity.

P30Substrate Elasticity Modulates TGF beta Stimulated Re-differentiation of Expanded Human Articular ChondrocytesD. Vonwil, A. Barbero, A. Trüssel, O. Haupt, I. Martin Basel/Switzerland

Purpose: Culture of mesenchymal progenitor cells on substrates with different elasticity has been shown to modulate cell fate/commitments. We aimed this study at investigating whether substrate elasticity modulates TGFβ stimulated chondrogenic re-differentiation of expanded/de-differentiated human articular chondrocytes (HAC).Methods and Materials: Expanded HAC from 4 donors (43-77 years) were seeded onto Type I collagen (CI) functionalized poly acrylamide (PA) films (100-150 μm thickness) with a Young’s modulus of 0.26±0.08 kPa (soft), 21.32±0.79 kPa (intermediately stiff) and 74.88±5.13 kPa (stiff) and induced to re-differentiate in a defined serum free medium containing or not TGFβ-3 for 7 days. CI coated tissue culture treated plastic was considered as an infinitely stiff substrate and HAC aggregate cultures served as a standard re-differentitation control. HAC were assessed for attachment, proliferation, morphology and mRNA expression (type I and II collagen).Results: In the presence of TGFβ-3, HAC attached similarly on the different substrates and accomplished less than one total doubling within 7 days. On intermediately stiff to infinitely stiff substrates HAC assumed a fully spread fibroblastic morphology (shape factor Φ = 0.23-0.27), whereas on the soft substrate, they remained more spherical (Φ = 0.35±0.02) and had a reduced spreading area (up to 3.2-fold). F-actin organization on the soft substrate was restricted cortically, while on the stiffer substrates, F-actin assembled into stress fibres. Type II collagen mRNA expression on the soft substrate was similar to that in aggregate culture and 18.1-fold higher than on infinitely stiff substrates.However, in the absence of TGFβ-3, type II collagen mRNA remained at levels expressed by expanded/de-differentiated HAC.Conclusions: Substrate elasticity modulated the re-differentiation response of expanded/de-differentiated HAC to the chondrogenic stimulus TGFβ-3, and thus underscores that mechanical compliance in combination with appropriate soluble signals is an important parameter in designing biomaterials for cartilage repair.

treatment of inflammation and pain during osteoarthritis. The objective of the study was to determine on cartilage and synovial membrane explants the efficacy ie the cyclooxygenase (COX) inhibition of the ibuprofen loaded microspheres. Methods and Materials: Ibuprofen release from microsphere Biodegradable microspheres conjugated to ibuprofen (40-100 µM) were incubated in PBS at 37°C. Ibuprofen was measured using HPLC. Anti-inflammatory assay of microspheres In order to prove the efficacy of ibuprofen released from microspheres on COX activity inhibition, cartilage and synovial membrane explants from sheep shoulder joint were co-cultured (n = 4) and inflammation was induced with lipopolysaccharides (LPS 10 μg/mL). To LPS-activated explants were added three doses (25 – 50 and 75 mg) of microspheres conjugated to ibuprofen. As positive control for COX inhibition, control explants were treated with free ibuprofen (1 - 10 - 50 and 100 µM). At day 2, PGE2 in the explant supernatants was measured by ELISA. Statistical analyses were performed on StatView SAS 2000 (SAS institute, Cary, NC).Results: HPLC indicated that ~1 % of ibuprofen was released after one month of incubation in PBS without initial burst release. Anti-inflammatory testing on joint explants indicated that after two days of culture, a significant reduction of PGE2 synthesis was measured at 10 µM (p = 0.0033), 50 µM and 100 µM (p = 0.0008) of free ibuprofen and at every dose of microspheres (p = 0.0008). Conclusions: HPLC analysis of the culture medium showed that 50 mg of microspheres released ibuprofen in medium at a concentration of 35 μM which was sufficient to inhibit PGE2 synthesis in explants, authorizing efficacy studies of the biodegradable microspheres loaded with ibuprofen in an animal model of osteoarthritis.

P28Cartilage tissue engineering for auricular reconstruction in based polyvinyl alcohol polymersM.I. Garnica1, V. Martinez-Lopez1, Z. Garcia1, C. Ibarra2, F.E. Villalobos Cordova2, G. Luna-Barcenas1, C. Velasquillo1 1Mexico/Mexico, 2Mexico City/Mexico

Purpose: The biomaterials used for tissue engineering have evolved over the last few decades. Natural and synthetic biopolymers have been investigated for the development of tissue-engineered cartilage. One obstacle that cartilage tissue engineering faces is the development of a suitable environment for growth and cell adhesion, able to promote retention of the chondrogenic phenotype, production of ECM, and integration of the scaffold to the native tissue. The objective of this study was to analyze the viability, tissue organization and cellular adhesion of auricular chondrocytes seeded onto polymers of PVA-HA-EDGE (polyvinyl alcohol [PVA], hyaluronic acid-ethylene glycol diglycidyl ether), Q-PVA-EDGE (chitosan-PVA-EGDE) and Q-PVA-ECH (Q-PVA-epichlorohydrin), as a step for the clinical application of tissue engineering in the construction of a pinna. Methods and Materials: Three polymers, were synthesized as films: PVA-HA-EDGE, Q-PVA-EDGE and Q-PVA-ECH. Auricular cartilage was harvested from young New Zealand rabbits. The perichondrium was carefully removed. Samples were digested to isolate and to culture chondrocytes at high confluence. The three aforementioned polymeric materials were seeded and cultured in standard in vitro conditions for 10 days. The cell viability was determined by calcein, and morphology characteristics was studied by hematoxylin staining.Scanning electron microscopy (SEM) was used in order to analyze cell adhesion to the polymer. Results: Q-PVA-ECH constructs exhibited growth and cell adhesion and ECM accumulation, whereas the remained polymers retained their construct size but induced death cell and did not provide support to cell adhesion. After 10 days of culture, analysis by SEM and hematoxylin indicates cell adhesion to Q-PVA-ECH and calcein test show survival of chondrocytes seeded onto this film.Conclusions: According to the results, Q-PVA-ECH is the most viable to form an auricular structure to be used for reconstruction of external ear . Nevertheless it is still necessary to perform the mechanical characterization to determine if it keeps the properties of the native auricular cartilage.

Posters 199

P35A Novel Double-network Hydrogel Induces Spontaneous Articular Cartilage Regeneration In A Large Osteochondral DefectN. Kitamura1, K. Arakaki2, T. Kurokawa1, J.P. Gong1, F. Kanaya2, K. Yasuda1 1Sapporo/Japan, 2Naha/Japan

Purpose: We have developed an innovative method to induce spontaneous hyaline cartilage regeneration in vivo by implanting a double-network (DN) hydrogel composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N,N´-Dimetyl acrylamide). The purpose of this study is to quantitatively evaluate the regenerated cartilage with this method in comparison with the untreated control.Methods and Materials: A total of 23 mature rabbits were used in this study. An osteochondral defect having a 4.3-mm diameter was created in the femoral groove of the right patellofemoral joint. A cylindrical DN gel plug was implanted into the defect so that a defect having 2-mm depth remained after surgery. In the left knee, a defect having 2-mm depth was created and remained without any treatment. Five rabbits were sacrificed at 1, 2, 3, and 4 weeks after implantation, respectively. Their knee joints were used for histological (HE, Safranin-O) and immunohistochemical (collagen-II) evaluations. The remaining 3 rabbits were sacrificed at 4 weeks and served for real time PCR analysis.Results: A proteoglycan-rich tissue appeared in a localized zone close to the bony wall at 2 weeks, increasing at 3 weeks. The regenerated tissue was rich in proteoglycan and type-2 collagen at 4 weeks. On the other hand, the untreated (control) defect was filled with the fibrous and bone tissues even at 4 weeks. Wayne’s scores were significantly higher in the gel-treated knees than in the untreated control (p<0.01). In the real time PCR analysis, type 2 collagen, aggrecan, and Sox9 mRNAs were highly expressed, while it was seldom seen in the tissues regenerated in the untreated defect.Conclusions: The present study demonstrated that the implantation of the DN gel plug could induce spontaneous hyaline cartilage regeneration in vivo. We speculate that the in vivo biochemical and biomechanical environment created by existence of the DN gel may differentiate some undifferentiated cells including MSCs into chondrocytes.

P37Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan based scaffoldsM. Alves da Silva1, A. Martins2, A.R. Costa-Pinto2, V.M. Correlo2, P. Sol2, M. Battacharya3, S. Faria4, R.L. Reis1, N.M. Neves1 1Caldas das Taipas/Portugal, 2Guimarães/Portugal, 3Minessota/United States of America, 4Braga/Portugal

Purpose: Herein we studied the effect of the stimulation provided by a home-made flow perfusion bioreactor in the chondrogenic differentiation of human bone marrow derived mesenchymal stem cells (hBM-MSCs). We intended to ascertain if the shear stress caused by the medium perfusion trough the cell seeded constructs was capable of augmenting the differentiation process. Methods and Materials: Human BMSCs were isolated from bone marrow aspirates from informed consent donors. Cells were characterized by flow cytometry. After expansion, hBM-MSCs were seeded statically onto fiber meshes scaffolds, consisting of a blend of 50/50 chitosan and Poly(Butylene Terephtalate Adipate)– CPBTA. Constructs were cultured for 4 weeks in a flow perfusion bioreactor, using complete medium for chondrogenesis supplemented with TGF-β3. Samples were taken at different time points for DNA content quantification, SEM analysis, histology, imunolocalisation of collagens type I and type II and RT-PCR (specific primers for the marker genes collagen type I, type II, type X, aggrecan, Sox9, Runx2 and reference gene GAPDH).Results: We observed enhanced ECM deposition and collagen type II production in the bioreactor samples, when compared to the static controls. Moreover, it was observed that hBM-MSCs, in static cultures, take longer to differentiate. ECM accumulation in these samples is lower than in the bioreactor sections, and there is a significant difference in the expression of collagen type I.Conclusions: We found that shear stress has a beneficial effect on the chondrogenic differentiation of hMSCs. Thus, shear stress generated in the bioreactor had a positive influence in the chondrogenic differentiation of hBM-MSCs cultured in chitosan-based scaffolds.

P31The Effect of Joint Immobilization On The Spontaneous Hyaline Cartilage Regeneration Induced By PAMPS/PDMAAm Double-network HydrogelN. Kitamura1, K. Arakaki2, T. Kurokawa1, J.P. Gong1, F. Kanaya2, K. Yasuda1 1Sapporo/Japan, 2Naha/Japan

Purpose: We have developed an innovative method to induce spontaneous hyaline cartilage regeneration in vivo by means of implanting an originally developed PAMPS/PDMAAm double-network (DN) gel. However, the mechanisms have not been clarified as of yet. We have studied the effect of joint immobilization as a kind of mechanically un-physiological environment on the spontaneous hyaline cartilage regeneration induced by the PAMPS/PDMAAm DN gel.Methods and Materials: A total of 20 rabbits were used in this study. An osteochondral defect having a 4.3-mm diameter was created in the femoral groove of the patellofemoral joint, and a cylindrical DN gel plug was implanted into the defect so that a defect having 2-mm depth remained after surgery. The right knee was immobilized by a previously validated method, whereas the left knee was left mobile. Ten rabbits were sacrificed at 4 and 12 weeks after implantation, respectively. The knee joints were used for histological (HE, Safranin-O) and immunohistochemical (collagen-2) evaluations, and real time PCR analysis. The tissue regenerated in the defect was quantitatively evaluated with the Wayne’s score.Results: The regenerated tissue was rich in proteoglycan and type-2 collagen at 4 and 12 weeks, whereas no staining with Safranin-O or type-2 collagen was found in the defect of the immobilized knees. Wayne’s scores were significantly higher in the non-immobilized knees than in the immobilized knees (p<0.05) at 4 weeks. At 12 weeks, the gross appearance and total scores were significantly higher in the non-immobilized knees than in the immobilized knees (p<0.05). The mean relative values of type-2 collagen, aggrecan, and Sox9 mRNAs in the regenerated tissue were higher in the non-immobilized knees than in the immobilized knees. Conclusions: The present study suggested that the mechanically physiological environment is one of the essential factors to induce the in vivo spontaneous hyaline cartilage regeneration by means of implanting the PAMPS/PDMAAm DN gel.

P34Hydrophobic/Hydrophilic Hydrogels for Use as Artificial Cartilage MaterialsB. Thomas, B. Mimnaugh, H. Brinkerhuff Warsaw/United States of America

Purpose: Cartilage is comprised of both a hydrophobic collagen segment and a hydrophilic GAG segment. We hypothesized that hydrogels combining both hydrophilic and hydrophobic structures may have better mechanical properties than those of produced by a similar process containing only a hydrophilic segment and that the hydrophobic segment would not adversely affect the wetability of these materials. Methods and Materials: The polymers and chemicals used in this study included: polyvinylalcohol; poly(ethylene-co-vinyl alcohol); poly(vinyl pyrrolidone); and dimethyl sulfoxide. A melt processable miscible blend was created of the polymers utilizing a Leistritz twinscrew extruder and molded into test samples. Compressive modulus was measured on an Instron 3345. Contact angle measurements were performed on a KRÜSS DSA100 Drop Shape Analysis System using deionized water and bovine serum and a drop size of 10µl. Bovine cartilage for the study was obtained from fresh-frozen femoral heads. Results: Figure 1 shows the compressive modulus of various hydrogels. Figure 2 shows the contact angle for varying hydrogels produced from the extruder/injection molding process as compared to fresh-frozen bovine cartilage. Conclusions: The data presented demonstrates the usefulness of producing hydrogels with hydrophilic/hydrophobic segments. These materials exhibit some of the desired mechanical properties to be useful as cartilage replacement materials. The introduction of hydrophobic groups in the materials has provided a positive effect on mechanical properties. The contact angle has shifted from 32° for the pure hydrophilic hydrogel to around 60° for the hydrophobic/hydrophilic hydrogels. Therefore, the materials produced in this study are more similar in hydrophobic/hydrophilic nature to that of natural bovine cartilage.

Posters200

P41Wear Rate Evaluation of Polycarbonate-Urethane Cushion Form Bearings in Artificial Hip JointsJ.J. Elsner1, Y. Mezape2, G. Zur2, E. Linder-Ganz2, A. Shterling2, N. Eliaz1 1Tel Aviv/Israel, 2Netanya/Israel

Purpose: Polycarbonate-urethane (PCU) is an elastic material with a high resistance to tear. It offers excellent mechanical and tribological properties, comparable to those of natural cartilage. Thus, it is an attractive alternative to Polyethylene in load bearing applications (Fig. 1). The current study aimed to evaluate the wear performance of a PCU acetabular buffer over 10 million gait cycles. The wear rate was evaluated using gravimetric, Bio-Ferrography and filtration methods, simultaneously. Methods and Materials: Five 46mm buffers were tested, coupled with 40mm CoCr spherical femoral heads, in anatomical positioning. Four independently-controlled motions were induced according to ISO 14242-2 gait pattern for 10 million cycles (Mc). The PCU implants were examined every 1Mc and weighed to evaluate gravimetric changes due to wear. The amount of wear particles released into the lubricant (3:1 diluted bovine serum) was evaluated using a novel Bio-Ferrography method and conventional filtration. Results: The wear rate demonstrated by the PCU material in the M-O-PCU configuration was found to reach a constant rate after ~2 Mc. The initial ‘run-in’ particle generation rate of 1x108 particles/Mc was reduced gradually to 1-3x106 particles/Mc after 2Mc and remained steady thereafter. Gravimetric and Bio-Ferrography methods produced a steady-state volumetric wear rate of 8-10mm3/mc, whereas filtration yielded a lower wear rate: 5mm3/mc. The wear particles isolated by filtration and Bio-Ferrography were similar and mean sizes were found to be 10-14µm Conclusions: PCU displays promising wear characteristics. The wear rate was found to be lower than that of UHMWPE and x-UHMWPE, lying closer to that of MOM bearings (Table 1). The mean PCU wear particle size was larger than that of UHMWPE, metal and ceramic, and mostly not in the biologically active range (0.1-10µm). The number of particles generated was found to be 5-8 orders of magnitude lower than that reported for the aforementioned materials.

P43Magnetic scaffold for advanced osteochondral tissue engineeringA. Russo1, S. Panseri1, V. Goranov2, D. Casino1, T. Shelyakova1, A. Tampieri3, M. Sandri3, C. Dionigi1, V. Dediu1, M. Marcacci1 1Bologna/Italy, 2Minsk/Belarus, 3Faenza/Italy

Purpose: Tissue engineering has recently emerged as a multidisciplinary approach for the treatment of bone/osteochondral defects. Scaffolds with the potential to circumvent the limitations of autologous and allogenic tissue repair are employed to restore tissue functions. This project propose magnetic scaffolds that via magnetic guiding will be able to attract and take up in vivo growth factors/stem cells bound to magnetic nanoparticles.Methods and Materials: Magnetic scaffolds are prepared following two different methods. In the first strategy, apatite/collagen porous scaffolds are infiltrated with ferrofluids leaving magnetic nanoparticles entrapped in the construct. The second approach is based on the direct nucleation of biomimetic apatite on self-assembling collagen fibrils in presence of magnetite nanoparticles, thus realizing the magnetization of the scaffold material in situ. The scaffolds become magnetic maintaining their specific porosity and shape. In vitro study is performed with human mesenchymal stem cells (hBMSCs) to test the biocompatibility of magnetic scaffolds. In vivo biocompatibility of magnetic scaffolds is tested in a rabbit model. The cylindrical scaffolds are implanted bilaterally in the tibial diaphyses and femural epiphyses of the animals.Results: In vitro analysis shows the ability of these new magnetic scaffolds to sustain cell adhesion and proliferation, since there are no significant differences in the level of living/dead cells between control scaffolds and magnetized scaffolds. hBMSCs adhere and attach firmly to the scaffold surfaces and are shown to penetrate inside the scaffold. In vivo preliminary results show good biocompatibility and bone integration with no inflammation reaction even on organs biopsies.Conclusions: The proposed scaffolds will work like magnetic local field amplificators: their relatively strong magnetization can be aligned in the same direction by moderate external field. For magnetic targeting, funtionalized nanoparticles with growth factor/cell introduced in the body will be concentrated in the target area by means of an externally applied magnetic field.

P38Double-Network Hydrogels: candidate Cartilage Repair Materials with high dynamic Stiffness, Suture Tear Resistance and Tissue adhesive Bond StrengthM. Arnold1, D. Daniels2, S. Ronken2, N.F. Friederich1, T. Kurokawa3, J.P. Gong3, D. Wirz2 1Bruderholz/Switzerland, 2Basel/Switzerland, 3Sapporo/Japan

Purpose: In focal repair of joint cartilage and meniscus, initial stiffness and strength of repairs is generally much less than surrounding tissue. This increases early failure potential. Secure tissue attachment is also a problem. Acrylamide polymer double network (DN) hydrogels are candidate improved repair materials. DN gels incorporate a soft, ductile molecular network within a rigid one. Compared to a single network gel, an optimized DN gel is markedly stronger and tougher. Previous studies demonstrated physico-chemical stability and tissue compatibility, plus ability to foster cartilage formation. Exploratory studies reported here on PAMPS/PDMAAm and PAMPS/PAAm DN gels determined mechanical properties related to surgical use.Methods and Materials: Two different double-network (DN) hydrogels, known as PAMPS/PDMAAm and PAMPS/PAAm, as provided from the Japanes Co-authors were tested. Dynamic stiffness was measured by fast (gait mode) and slow (nutrition-mode) mm-scale micro-indentation. Suture tear-out and tissue adhesive forces were measured with an MTS test machine.Results: The results further support the clinical potential of the DN-Gel concept. Remarkably these 90+% water, DN gels exhibited dynamic impact stiffness (E*) values (~1.1 and ~1.5 MPa) approaching swine meniscus (~2.9 MPa). Dynamic impact energy-absorbing capability was much lower (median loss angles ~2º) than swine meniscus (>10º), but it is intriguing that 90+% water materials can efficiently store energy. Also, fine 4/0 suture tear-out strength approached cartilage (~2.1 N and ~7.1 N vs. ~13.5 N). Initial strength of attachment of DN gels to cartilage with acrylic tissue adhesive was also high (~0.20 and ~0.15 N/mm2).Conclusions: DN gel strength and toughness properties stem from optimized entanglement of the two network components. DN gels have obvious structural parallels with cartilagenous tissues.

P39Tribological properties of poly(HEMA-co-MMA) materials as meniscus substitutes.J.L. Sague Doimeadios1, S. Honold1, Y. Loosli1, J. Vogt2, R. Luginbuehl1 1Bettlach/Switzerland, 2Flüh/Switzerland

Purpose: Based on their biocompatibility and chemical-physical properties, hydrogels have found a prominent place in modern orthopaedic applications in recent years. Most hydrogels under investigation are composed of a uniform polymeric body. We demonstrate the potential of tuning hydrogels to comply with mechanical and tribological requirements for meniscus substitute materials.Methods and Materials: Samples were prepared by reacting 2-hydroxyethyl methacrylate (HEMA), methyl-methacrylate (MMA), distilled water (10%w/w) and 0.01mol% AIBN, as reaction initiator. Equilibrium water content (EWC) and mechanical characterization were determined on samples of 10mm height and 12mm diameter. The unconfined-creep-modulus (Ec) was measured using a Zwicki Z5.0 (Zwick-Roell, Germany). Tribological measurements were done in PBS with an in-house-built pin-on-plate instrument using a CoCrMo plate as counterpart.Results: The EWC decreased linearly as a function of increasing MMA concentration in the poly(HEMA-co-MMA) (R2=0.99). The Ec values exhibited a bi-modal relationship: samples with less than 35mol% MMA had a very low Ec and the influence of MMA was minor whereas at higher MMA contents, the Ec increased significantly and the MMA influence was substantially stronger (Fig 1a). The apparent dynamic friction, µ, increased exponentially as a function of MMA content up to 45mol% MMA. 45mol% MMA content marked a maximum for µ as it decreased for higher MMA contents significantly.Conclusions: It was found that an increase in the MMA concentration yielded higher Ec and µ and lower EWC values as expected for hydrogels based on PHEMA. Samples with 35mol% MMA content exhibits a transition point in the Ec values, whereas at MMA content above 50mol% the µ values were similar to PMMA. These HEMA–co–MMA polymers cover a broad range of Ec and their tribological performance can be further optimized by mixing different co-polymers. These approaches allow for tuning the mechanical and tribological properties to mimic those of human menisci.

Posters 201

content of cartilage-specific proteins. Chondrocytes initiated col-II-secretion, whereas col-I-secretion gradually decreased. Apoptosis levels after initial preparatory compression remained constant, while proliferation was slightly elevated in the course of bioreactor cultivation. The average aggregate modulus did not deteriorate under cyclic loading, but declined in controls. The transmitted forces have been recorded and correlated with matrix remodeling.Conclusions: By compressive cyclic loading engineered cartilage replacement tissue steadily develops a more cartilage-like appearance. In the course of tissue remodeling biological outcomes are well-correlated with mechanical characteristics.

P46Effects of Serial Sectioning and Repair of Radial Tears in the Lateral MeniscusG.E. Ode, S. McArthur, G.S. Van Thiel, J. Kercher, J. Dishkin-Paset, E. Shewman, V.M. Wang, B.J. Cole Chicago/United States of America

Purpose: The structural foundation of a functioning meniscus is the longitudinally oriented peripheral meniscal fibers which transmit hoop stresses. Radial transection of these fibers could theoretically render a meniscus non-functional with clinical sequelae. We hypothesize that sequential radial sectioning of the lateral meniscus will increase contact pressures, and repair with either an all-inside or an inside-out technique will restore meniscal function. Methods and Materials: Ten paired cadaver knees were dissected down to the capsule. Each tibia was placed in a Taylor-Spatial-Frame and the femur was affixed in a custom jig. A vertical osteotomy of the lateral femoral condyle was performed to access the lateral compartment. Tekscan sensors were placed sub-meniscal in both compartments. Knees were loaded at 800N in both extension and 60 degrees of flexion in an MTS machine. The compartment was reopened and sequential radial sections approaching the periphery (50%, 75%, 100% of radial width) were made in the lateral meniscus, posterior to the popliteal hiatus. Complete sectioning was followed by a pair matched repair using an all-inside or an inside-out technique. Each condition was loaded twice to ensure reproducibility and repaired knees were cycled 50 times to test structural integrity. Results: ANOVA testing was completed. We observed overall no significant difference in contact area or pressure with radial sectioning up to 75% of the meniscal width but a significant change from 75% to 100% (p<.05). The two repaired constructs showed improved pressure profiles when compared to the sectioned state, but no differences between each other. Conclusions: Radial meniscal tears that violate the periphery benefit from repair with either all-inside or inside-out repair at time zero. Violation of the meniscal periphery results in a pressure profile identical to total meniscectomy and repair at time zero approximates the intact condition.

P48Effect of the sample pre-conditioning on the evaluation of cartilage mechanical propertiesV.A. Acosta, I. Ochoa, J.M. García-Aznar, M. Doblaré Zaragoza/Spain

Purpose: The mechanical characterization protocol of the articular cartilage needs to be homogeneous to compare the parameters in degradated and regenerated cartilage. For the determination of the cartilage mechanical properties, experimental uniaxial confined and unconfined compression tests were used to estimate the Aggregate Modulus, Young Modulus and Poisson’s ratio. To perform the compression test, a preconditioning protocol has to be carried out achieving an optimum contact between the test tool and the sample and eliminating the non-linearities of the cartilage geometry. This process allows developing all the experiments with a similar initial strain, obtaining repeatability in the estimated mechanical properties. Methods and Materials: The results of applying three different precondition protocols for load-relaxation tests were presented: A 4 % and 10% predeformation of the total thickness of the sample, and a preload equivalent to 12.5 kPa. After the precondition protocol, a mechanical experiment consisting on making a stress–relaxation ramp with six controlled displacement and relaxation of 15 minutes until 20% of strain defined from the unloaded thickness.

P44Comparison of human and animal chondral propertiesS.D. Taylor, E. Tsiridis, E. Ingham, Z. Jin, J. Fisher, S. Williams Leeds/United Kingdom

Purpose: Investigations into tissue preserving orthopaedic treatments have assessed cartilage tribology; and used samples from animal joints. However, little is known about the mechanical differences between human and animal articular cartilage. This study aimed to characterise the differences in geometry and mechanical properties of human and animal cartilage samples.Methods and Materials: Creep indentation was performed on plugs taken out of the weight bearing portions of porcine (3–6 months old), bovine (18–24 months old), ovine (≈4 years old) and human femoral heads. The human femoral heads were obtained from surgery due to femoral neck fracture. Cartilage thickness was measured by monitoring the resistive force change as a needle traversed the cartilage and bone at a constant feed rate using a mechanical testing machine. The percentage deformation over time was determined by dividing deformation by thickness. A biphasic finite element model was used to obtain the intrinsic material properties of each plug. Results: The femoral head diameters for all animal samples were considerably different compared to human and all animal cartilage thickness was remarkably less compared to human (Table 1). When comparing between animals, the femoral head size was positively correlated with cartilage thickness, agreeing with previous studies. Human cartilage showed the least percentage total deformation, however, ovine cartilage showed the highest modulus and lowest permeability (Table 1). Ovine showed the most similar mechanical behaviour to human. Table1: Geometric and mechanical results (mean±standard deviation). Up to six repeats were tested.

Head Diameter (mm) Thickness (mm) Deformation (%) Young‘s

Modulus (MPa)

Human 46.84±4.34 1.82±0.15 6.87±0.68 4.95±0.69

Porcine 35.63±1.15 1.22±0.04 28.82±2.57 1.18±0.17

Bovine 64.36±4.96 1.32±0.12 22.47±2.36 1.86±0.42

Ovine 23.25±2.31 0.57±0.07 18.59±1.37 6.73±0.08

Conclusions: Large differences in anatomy and mechanical properties were established between different species and human cartilage. Therefore, it is important to consider these differences to enable more clinically relevant investigations.

P45Cyclic mechanical stimulation in the generation of cartilage replacement materialS. Nebelung1, K. Gavenis1, A. Ladenburger1, M. Stoffel1, R. Müller-Rath2 1Aachen/Germany, 2Neuss/Germany

Purpose: Adequate cartilage replacement remains a challenge to orthopedics. Tissue engineering is a promising alternative treatment for cartilage defects. By specifically stimulating chondrocytes, e.g. through unconfined dynamic compressive loading, engineered tissue can be modulated. Numerous bioreactor systems applying compression have been developed; however, to our knowledge none is measuring the actual force the probe is exposed to. We developed a bioreactor system that allows for recording and analysis of the transmitted forces during the cultivation period and investigated the influence of cyclic compressive loading on a chondrocyte-seeded collagen-type-I gel (see Figure 1). Methods and Materials: Chondrocytes were harvested from human knees, enzymatically released and seeded in a rat tail collagen type-I gel at a density of 2x105 chondrocytes/ml gel. The cell-seeded scaffolds were condensed by factor 20, punched into disks and cultivated in the afore-mentioned bioreactor system under continuous cyclic compressive loading (strain: 10 %, force: 30 mN, frequency: 0.3 Hz, duration: 14 days) under standardized conditions (37° C, 5 % CO2, humidified atmosphere). A load cell beneath the cultivation chamber recorded the transmitted forces. Probes were analyzed by means of histology (HE, Safranin O), immunohistochemistry (col-I, col-II, Ki 67, TUNEL), molecular biology (RT-PCR of aggrecan, col-I, col-II, MMP-13) and biomechanical evaluation of tensile and compressive characteristics.Results: Cyclic compressive loading modulates chondrocytes’ viability, gene expression and biosynthetic activity and thereby the

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Results: The resistive force varied based on the location in the knee. For instance, the inner region of the medial femoral condyle had a lower resistive force than the outer region. The area covered by the meniscus on the tibial plateau had a higher resistive force than those uncovered by the meniscus. The ranges per zone are shown. Similar values were found by Lyrra et al. Conclusions: The result of this study provides valuable insights into how mechanical properties vary throughout the knee joint. In particular, the regions noted to have lower resistive forces are also regions typically noted to primarily contain osteoarthritic lesions. With a range of values now established for the normal knee, testing can be performed on osteoarthritic cartilage to further understand the mechanical changes caused by this disease.

P51The Mechanical State of the Human Hip Joint, Implanted with a Pliable Polycarbonate-Urethane Acetabular BufferS. Portnoy1, E. Linder-Ganz1, G. Zur1, Y. Mezape1, F. Guilak2, N. Shasha3, A. Shterling1 1Netanya/Israel, 2Durham/United States of America, 3Tel Aviv/Israel

Purpose: Replacing the hip joint with a prosthetic implant is the conventional treatment for degenerative hip disorders. Recently, an acetabular buffer device, made of pliable Polycarbonate-Urethane (PCU), was developed (TriboFit®, Active Implants Co., Memphis, TN; Fig. 1a). The implant’s design aims to mimic the natural behavior of the cartilage. However, the mechanical state of the pelvis, implanted with a PCU buffer directly on the acetabular bone, has never been quantified. Our goal was therefore to compare between three pelvic states: natural pelvis, pelvis implanted with PCU buffer, and the buffer on an implanted metal shell. Methods and Materials: We developed subject-specific 3D CT-based finite element (FE) models of the natural and implanted cadaveric human hip joint (Fig. 1b). The models simulated in vitro experimental trials on the cadaveric sample, where the natural and implanted hip joint was loaded. Surface pressures between the acetabulum and the femoral head were measured using pressure films. Surface strain distributions on the pelvic bone were visualized using photoelasticity. Principal strains in discrete locations were measured using strain gauges. The measured and calculated acetabular contact pressures and surface strains upon the pelvic bone were compared for model validation and further analyses of stress/strain distribution in the implanted hip joint (Fig. 2). Results: The results show similar strain distributions at the natural acetabulum, compared to the hip joint, implanted with the PCU buffer. The strains on the bone surface of the hip joint implanted with the metal shell were distributed across a larger area, compared to the natural pelvis and buffer-implanted pelvis. The pressure distribution at the acetabulum-buffer surface was similar to the pressure distribution in the natural hip joint.Conclusions: We conclude that the mechanical state of the pelvis, implanted with a PCU buffer, is similar to the mechanical state of the natural pelvis.

P52The effect of clearance to cartilage wear in hip hemiarthroplastyJ. Lizhang, J. Fisher, Z. Jin, S. Williams Leeds/United Kingdom

Purpose: Hip hemiarthroplasty is used in elderly patients following femoral neck fracture. However, longevity of this treatment is limited by the metal or composite prosthesis degrading the cartilage over time. Inappropriate sizes of implant in hip hemiarthroplasty may cause early cartilage wear (Beksaç et al, 2008). This study investigated the effect of clearance (between acetabular cartilage and metal head) on cartilage wear area and volume. We hypothesised that the outcome of hemiarthroplasty could be improved if the clearance was optimised. Methods and Materials: Acetabulums (from 6-month old porcine hips) were dissected and mounted at 45º in a pendulum friction simulator. Cobalt Chrome heads were set homocentric to the acetabulums and applied a dynamic (75~800N) load and a flexion-extension motion (±15º) in 25% bovine serum for 2 hours. Components were set up to allow the different radial clearances: Small (≤0.6mm), Medium (>0.6 & ≤1.2mm), Large (>1.2 & ≤1.8mm), Extra Large (>1.8mm), n=3 per group. A silicon rubber replicate was made of the acetabular surface after testing to

Results: The 4 and 10% of predeformation have a same tendency for confined and unconfined tests. For the 10% of predeformation, the difference is the high degree of deformation and stress. Although the implementation of preload achieves the contact, the load-relaxation test starts with different levels of strain what could alter the results trends. The preconditioning protocols showed similar tendency where Aggregate Modulus was higher than Young Modulus. However the variability of the experimental data obtained by preload shows a statistical difference for Aggregate Modulus compared with the 10% results. Conclusions: Performing a predeformation of 4% ensures contact with the lowest percentage of deformation on the sample, and makes the compression tests start with a similar initial strain level. Moreover, despite of the thickness variation, this protocol allows obtaining a normalized experiments and repeatability in the estimated mechanical properties.

P49MRI Analysis of Anteroposterior Stability in the Normal Human KneeS. Arno, M. Chaudhary, R. Forman, P. Glassner, R. Regatte, P. Walker New York/United States of America

Purpose: We hypothesize that the knee achieves anteroposterior (AP) stability on the medial side and simultaneously achieves a high range of flexion by rollback on the lateral side. We also wish to show that the medial meniscus plays an important role in maintaining this stability.Methods and Materials: A Siemens 7 Tesla MRI with T1 weighted non-fat suppressed settings and isotropic resolution of 0.6mm was used to scan eight healthy male volunteers with the knee at 15 degrees of flexion. Two scans were obtained: One with an axial force of 667 N along the tibial long axis and a second with the axial force and a shear force of 36 N. The forces applied were 25% of the forces at heel strike determined by D’Lima and Colwell (2005, 2006). Using 3D-Doctor (Able Software Corp, Lexington, MA), solid models were created of the femur, tibia, and menisci. The solids were imported into RapidForm (Inus Technology, Seoul, S. Korea) and a deviation analysis was then performed to determine the movement of the femur and menisci.Results: Overall, the lateral side displaced anteriorly (range: 0.5 - 2.5mm) and the medial side even displaced posteriorly (range: 0 - 1.25mm), indicating an axial rotation point between the lateral and medial condyle, but closer to the medial. In addition, the medial meniscus was found to exhibit at most 0.5mm of anterior displacement whereas the maximum anterior displacement of the lateral meniscus ranged from 0.5mm to 1.00mm, suggesting that the medial meniscus contributes to medial stability.Conclusions: These findings can have important implications in the design of treatments for the restoration of normal knee mechanics. In addition, the above data stresses the important role of the medial meniscus in maintaining stability in the knee. Future studies will look at how a damaged medial meniscus can lead to increased AP displacements causing long-term damage to cartilage.

P50Stiffness of cartilage in the normal human knee as determined by a novel indentation deviceL. Kuo, D. John, S. Arno, P. Walker New York/United States of America

Purpose: The purpose of this study is to determine the resistive force of normal cartilage on the distal femur and proximal tibia. To do so, an indentor was modeled and constructed after the Artscan 200 and Actaeon Probe, optimized for lab setting usage to conduct short duration indentation testing of articular cartilage surface layer. Methods and Materials: The indentor was first validated on samples of agarose and rubber with known properties to show its capability of providing reproducible readings independent of the operator. It was then used to test the distal femur (inner and outer region) and proximal tibia (area covered and uncovered by the meniscus) of seven fresh-frozen cadaveric knees with visually normal cartilage. A total of six measurements were made by two different users and the resistive force was recorded via SensoCom software. The Friedman test was then used to establish a range of resistive force per zone tested.

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(N=4) post-surgery, the femoral ends were retrieved and scanned by micro-computed tomography. Histological sections were Hoechst counter-stained to show nuclei and residual implant in a representative field with peak chitosan-RITC fluorescence intensity or stained with Safranin O to detect glycosaminoglycan. Results: Hoechst staining revealed considerable chitosan-mediated cellular attraction (Fig. 1). Repair tissue at 21 days in treated defects had significantly more blood vessels, improved integration and less areal GAG-positive repair tissue (~0.3% vs ~22.1% control; p=0.0012) which is, after three weeks, a good indicator of future repair enhancement. More residual 150kDa implant was detected compared to 10 kDa implant (p=0.019), however comparison of hole geometry with both day 1 and day 21 drill-only controls revealed that all implants elicited a similar level of subchondral bone remodelling at the edges of the drill holes (Fig. 2). Conclusions: These results confirm that after three weeks in aged rabbits, this novel pre-solidified chitosan/NaCl-blood implant can deliver resident chitosan and stimulate a biological activity in regenerating subchondral bone that is consistent with previously successful therapies.

P55Complete Hole Placement with Microfracture Improves Cartilage Repair as Compared to Limited Central Hole PlacementM. Suri1, J. Rodrigo2, S.B. Singleton3, D. Wyland4, J. DesJardins5, M. Hoyle4, P. Boatwright6, M. McDonough7 1Harahan/United States of America, 2Spartanburg/United States of America, 3Greer/United States of America, 4Greenville/United States of America, 5Clemson/United States of America, 6Cleveland/United States of America, 7Washington, DC/United States of America

Purpose: Microfracture is a mainstay in the treatment of articular cartilage injuries. However, in the cases that do not do well there may be inadequate surgical technique. We hypothesized that there would be improved cartilage healing after microfracture in a rabbit full-thickness trochlear defect model if more holes were distributed evenly throughout the defect vs. fewer holes placed centrally.Methods and Materials: Full-thickness cartilage defects measuring 3 x 10mm were made in the articular cartilage of the trochlear grooves of 30, 52-week old New Zealand white rabbits. Three groups were assigned and all were sacrificed at 4 months. One group (n=10) received a 6-hole microfracture, with holes evenly distributed extending to periphery. The second group (n=10) received a 4-hole microfracture that was central in the lesion, representing inadequate technique. The third group (n=10) was abrasion only controls. Photographs of lesions at time of surgery and at sacrifice were used to calculate percentage fill of the regenerate. H&E stained slides were graded by the ICRS scoring system and were used to measure quality of healing. Safranin-O stained slides were graded to determine the percentage of the regenerate cartilage in each group that had positive proteoglycan staining. Results: Percentage fill of the regenerate was significantly (p<0.05) improved in the 6-hole group compared to the 4-hole group and the control group. ICRS scores were improved between the 6-hole group (13.7) and the 4-hole group (9.7) (p<0.05), but not between the 4-hole group (9.7) and the control group (7.7) (p<0.05). All of the 6-hole group demonstrated positive proteoglycan staining as compared to 57% of the 4-hole group and 43% of the abrasion group (p<0.05).Conclusions: Placement of microfracture holes extending to the periphery of the cartilage defect significantly improved percentage fill and quality of regenerate after microfracture.

P56Migration of subchondral mesenchymal progenitor cells in microfracture: Impact of human serum and synovial fluidM. Endres, G. Kalwitz, K. Andreas, K. Neumann, M. Sittinger, T. Haeupl, C. Kaps Berlin/Germany

Purpose: In clinical routine, bone marrow stimulating techniques like drilling, abrasion, or microfracture (Mfx) are frequently used for small size focal cartilage defects. In Mfx, the introduction of multiple perforations into the subchondral bone of the cartilage defect leads to bleeding, allows mesenchymal stem and progenitor cells from the subchondral bone to enter the defect and induces formation of cartilaginous repair tissue. The cellular mechanisms underlying stem cell migration into the defect and subsequent

investigate wear. The wear area was traced onto flexible film and the area of different cartilage damage grades (followed ICRS wear grades) measured. Additionally, the replica surface was measured by a two-dimensional profilometry across the wear area with approximately 12 traces, and this was multiplied by the wear area length to provide a volume (Figure 1). Results: Different cartilage wear grades (1~3) were seen in different clearances. When clearance increased contact area decreased, hence the percentage of unworn cartilage area increased. Wear volume increased significantly with XL due to the increase in contact stress above a critical threshold and the increase of cartilage wear depth clearance (Figure 2). Conclusions: Cartilage wear in hip hemiarthroplasty is affected by clearance which influences both wear area and wear grade, and significantly higher wear is produced with extra large clearance.

P53Loading Effects Intervertebral Disc Cell viability in the Loaded Disc Culture System.C.P.L. Paul, H.A. Zuiderbaan, B. Zandieh Doulabi, A.J. Veen van der, T.H. Smit, M. Helder, B.J. Royen van, M.G. Mullender Amsterdam/Netherlands

Purpose: We have developed a novel Loaded Disc Culture System (LDCS) to study the effects of loading on large species IVDs in relationship with degenerative disc disease (DDD). We are able to culture and load goat IVDs in the LDCS with maintenance of their native cellular and extracellular properties over a 14 day culture (data not published). The purpose of the current study is to investigate the influence of different loading regimes on goat IVDs cultured in the LDCS over a 7- and 14-day period. Methods and Materials: IVD’s (L1-6; N=175), were harvested from goats (N=35) under sterile conditions. IVD’s were assigned to 1 of 4 diurnal loading groups (fig. 1a). Discs were cultured for 7 or 14 days in the LDCS. Cell viability was measured in the nucleus (NP) and annulus (AF) regions. IVD stiffness was calculate from loading force and IVD displacement. Water, glycosaminoglycan (GAG) and total collagen content were analysed to assess the condition of the extracellular matrix (ECM). Results: Static loading showed a drop in cell viability only in the AF region, when compared to day 0. Low dynamic load maintained cell viability, whereas high dynamic and prolonged high dynamic loading showed increasing cell death (fig. 1b). IVD stiffness increased initially within each cycle, but no significant changes were observed over the 7 and 14 day culture period (figure 2). In the ECM, water-, GAG- and collagen content did not change significantly after 7 and 14 days in any experimental group when compared to day 0 (data not shown). Conclusions: Caprine IVD cells show different responses in cell viability depending on the applied loading condition. Low dynamic loading maintained cell viability, whereas unloaded, static and higher dynamic loads induce pathological changes within 14 days on a cellular level. To determine loading effects on the ECM, longer culture duration may be needed.

P54Novel pre-solidified chitosan/blood implant provides local bone-marrow stimulation-associated biological activity in skeletally aged rabbits after a three week treatmentC. Lafantaisie Favreau1, J. Guzman-Morales1, J. Sun2, A. Harris1, T.D. Smith1, A. Carli1, J. Henderson1, W.D. Stanish3, C.D. Hoemann1 1Montreal/Canada, 2Laval/Canada, 3Halifax/Canada

Purpose: The purpose of this study was to analyze the biological impact of different formulations of a novel pre-solidified chitosan/NaCl-blood implant on subchondral bone and cartilage repair after three weeks in skeletally aged rabbits. We tested the hypothesis that pre-solidified implants degrade in situ with molecular weight-specific kinetics, and stimulate subchondral angiogenesis and bone remodeling, acute reactions previously tied to hyaline cartilage repair. Methods and Materials: Hybrid chitosan-autologous blood implants were made with 150kDa, 40kDa and 10kDa chitosan (80% DDA) with fluorescently labelled chitosan. Three 1.5mm osteochondral drill holes were generated bilaterally in femoral trochlea of N=5, 2.5 year old NZW rabbits. One implant of each molecular weight was press-fit into the 3 drill holes of one knee, while the contralateral trochlea was used as drill-only controls. After 1 day (N=1) or 21 days

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Methods and Materials: Normal (n=4) and OA (Outerbridge 1-2, n=4) human medial femoral condyles as well as normal animal condyles (n=4 each species) were obtained. All underwent microCT imaging at 45µ resolution and the resulting images were analyzed using regions of interest (ROIs) created in the SBPs to measure BMD (bone mineral density), TMD (tissue mineral density), and BVF (bone volume fraction). A depth-wise analysis of mineral density was done by expressing the mean voxel densities in each horizontal slice by depth (Python 3.1.1 and R (www.r-project.org). Results: Human SBP BMD, TMD was significantly lower (p<.05) than all animal species except the sheep and dog. Human BVF was the lowest of all species. Rate of change in BMD by depth showed that the dog and human have a similar mineralization profile. In early human osteoarthritis the thickness (>2mm) and density of the SBP was significantly (p<.01) expanded. Conclusions: In normal human femoral condyles, the SBP is comprised of the condensation of approximately two horizontal trabeculae. No animal species approximates the very thin and porous normal human SBP; however, the dog and human have a similar mineralization gradients while other species have properties approximating sclerotic human bone. In osteoarthritis the sclerotic SBP presents a substantial barrier to marrow stimulation procedures.

P59Advance trans-medullary stimulation of mesenchymal stem cells enhances spontaneous repair of full-thickness articular cartilage defects.K. Nishizawa, S. Imai, T. Mimura, M. Kubo, S. Araki, S. Shioji, Y. Takemura, Y. Matsusue Otsu/Japan

Purpose: Mesenchymal stem cells (MSCs), especially those close to cartilage defects, are an important cell source for cartilage regeneration. We assumed that a larger number of MSCs would become available, should the bone marrow at the immediate vicinity of the subchondral bone be stimulated for MSCs in advance of the microfracture, i.e., creation of the full-thickness articular cartilage defects in this study. Methods and Materials: We stimulated the immediate vicinity of the subchondral bone from outside of the joints 4 days prior to the creation of cartilage defects (Advance Trans-medullary Stimulation (ATS) group). In another setting, bFGF was administered through the trans-medullary passageway in order to augment the stimulation of the MSCs (ATS + bF group). Finally, we designed a negative control group (non-ATS group). The rabbits were sacrificed at 1, 2, 3, 4 and 8 weeks after the creation of cartilage defects. The triple staining of Bromodeoxyuridine (BrdU), CD44 and CD45, histological evaluation and RT-PCR assay for assessment of the regenerated cartilage were performed. Results: A considerable proportion of the proliferating cells were identified as bone marrow-derived MSCs. The BrdU-positive cell density of the ATS group increased to a larger extent than that of the non-ATS group. The recruitment of BrdU-positive cells to the cartilage defects was even more pronounced in the ATS + bF group than that in the ATS group. The histological grading score of the ATS + bF group was superior to the other groups. Type II collagen mRNA expression also tended to be higher in ATS + bF group. Conclusions: The advance stimulation of the bone marrow at the immediate vicinity of the subchondral bone increased the available MSCs and enhanced the regeneration of the chondral defects at the early follow-up time point.

P60Interaction of microfracture awl design with subchondral bone in early osteoarthritisS. Allendorf1, M. Lowerison1, A. Bell1, C.D. Hoemann2, P. Bursac3, M.B. Hurtig1 1Guelph/Canada, 2Montreal/Canada, 3Alachua/United States of America

Purpose: To explore the relationship between awl design and performance in osteoarthritic human femoral condyles. Our hypothesis was that compression and condensation of the subchondral bone plate may create a barrier to remodeling and repair in sclerotic bone. Femoral condyles from animals provided a convenient model of subchondral sclerosis. Methods and Materials: Human and horse femoral condyles were scored macroscopically (Outerbridge system 0-5) and microCT imaged at 27µm voxel resolution. A 2 cm2 full thickness chondral defect was made, then 3 mm deep microfracture holes were made

tissue formation are not fully understood. In this study, we verify the assumption that mesenchymal progenitor cells from the subchondral bone (CSP) can be recruited by chemotactic factors like blood serum, synovial fluid from normal donors and donors with rheumatoid arthritis (RA) or osteoarthritis (OA) and selected chemokines. Methods and Materials: The presence of chemokines in blood serum and synovial fluid could be demonstrated using human chemokine antibody membrane arrays. Human blood serum, synovial fluids and selected chemokines were tested in combination with CSP cells in a 96 well chemotaxis assay.Results: Human blood serum as well as synovial fluid from healthy donors and OA donors recruited CSP cells. Remarkably, synovial fluid from RA donors recruited significantly less cells than synovial fluid from healthy donors or OA donors. Additionally, distinct chemokine candidates were able to recruit CSP cells. Conclusions: In conclusion, human serum as well as synovial fluid recruits CSP during Mfx. For cartilage repair approaches, these results suggest to use chemotactic factors like serum or potentially chemokines to improve stem and progenitor cell migration to sites of defective cartilage.

P57Analysis of the mid-term effects of chitosan-NaCl/blood pre-solidified implants in an in vivo osteochondral repair modelJ. Guzman-Morales1, C. Lafantaisie Favreau1, J. Sun2, G. Rivard1, C.D. Hoemann1 1Montreal/Canada, 2Laval/Canada

Purpose: Strategies are needed to enhance osteochondral repair with increasing age. Recently, our group developed novel pre-solidified chitosan/NaCl-blood implants to stimulate revascularization of subchondral bone in skeletally aged animals. After 3 weeks, this implant elicits angiogenesis and subchondral bone remodeling. The purpose of this study was to determine whether bone repair of treated drill holes is achieved after a longer repair period.Methods and Materials: With Institute-approved protocols, 2 osteochondral drill holes (1.5mm diameter, 2mm deep) were created bilaterally in femoral trochlea of six skeletally mature NZW rabbits (12 or 32 months old). Drill holes were treated with pre-solidified implants consisting in a homogeneous mixture of 80% DDA chitosan-NaCl (40 kDa or 10 kDa), autologous blood and fluorescent chitosan tracer. Contralateral microdrill holes were left to bleed as surgical controls. During the 10 week repair period, post-operative knee inflammation was evaluated daily using an in-house scoring system (0=no inflammation to 4=significant effusion) with a cumulative score for each day of effusion. At necropsy repair tissues were assessed macroscopically; femoral ends were fixed and scanned by micro-computed tomography to quantify 3-D drill hole bone repair. Results: Post-operative knee effusion was attenuated in chitosan implant-treated knees compared to contralateral controls (12 vs 26 average cumulative score, Fig. 1). Treated drill holes were macroscopically filled with more tissue (p<0.05) compared to the drill-only controls, and no macroscopic traces of fluorescent implants remained in treated defects after 10 weeks. New bone growth filled on average 50% of the untreated drill holes starting from the base of the hole. Chitosan-treated defects were repaired with approximately 25% new mineralized bone in the drill holes especially in the superficial subchondral bone plate (Fig. 2).Conclusions: Biodegradable pre-solidified chitosan/NaCl-blood implants suppress post-operative effusion and permit new bone repair in microdrill holes with a delayed kinetics compared to drill-only controls.

P58Subchondral bone plate characteristics in human and animal bone: Implications for cartilage repairM. Lowerison1, S. Allendorf1, A. Bell1, L.M. White2, R. Whiteside2, P. Marks2, M.B. Hurtig1 1Guelph/Canada, 2Toronto/Canada

Purpose: To characterize the subchondral bone plate (SBP) in normal and osteoarthritic (OA) femoral condyles. Our hypothesis was that increased subchondral bone plate thickness in osteoarthritis may make it necessary to make deeper microfracture holes. Animals were also considered since they are used as models of cartilage repair.

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in NPCs. These results support the hypothesis that IL-1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disc degeneration.Conclusions: HBO treatment-induced increase of the anabolic factor (TIMP-1) /catabolic factor (MMP-3) ratio may provide a therapeutic approach to slow the course of intervertebral disc degeneration.

P63Digestion-isolated chondrocytes demonstrate superior yield and chondrogenic potential versus cells migrating from minced cartilageM. DiMicco1, S.J. Duguay2, J.P. Wicks2, G. Matthews1 1Framingham/United States of America, 2Cambridge/United States of America

Purpose: Autologous chondrocyte implantation (ACI) techniques currently involve chondrocyte isolation and expansion in culture. Methods utilizing cell migration from undigested, uncultured minced cartilage could confer handling and cost advantages to ACI, perhaps allowing a one-step procedure. Our objective was to evaluate the suitability of substituting minced cartilage for cultured, digestion-isolated chondrocytes in cartilage repair procedures.Methods and Materials: Minced human cartilage (2-3 mm3) was evaluated ± enzymatic digestion for cell yield at baseline and after 11 days of culture. Separately, minced bovine cartilage was sieved by size (< 500µm, 500-1000µm, and >1000µm) and cultured. Cell viability in fragments was observed, and cell migration out of fragments was assessed at 7 and 12 days. To better understand the contribution of cell density toward chondrogenic behavior, aggrecan and collagen II gene expression were determined using enzymatically-isolated cells seeded at 0.5-4 million cells/cm2 and cultured on a collagen scaffold.Results: No cells were detected in supernatants immediately after mincing. When minced tissue was enzyme-treated for up to 2.5 hours, cell yield was 555±414 cells/mg (mean±SD), approximately 10% of that typical of overnight digestion. Yield from culture of 100-200 mg of minced cartilage (at 10-11 days) was 45,500±40,046 cells, while a 2-hour enzyme treatment of these pieces before culture increased yield nearly 9-fold. However, these values are still 2-18% of those expected following overnight enzyme digestion and culture. Mincing resulted in a zone of cell death at cut tissue edges, giving smaller pieces more dead cells per unit volume. After seeding onto a collagen membrane, cells at higher densities had higher collagen II gene expression, while aggrecan expression did not vary with density.Conclusions: Using minced cartilage for cartilage repair is expected to give fewer implanted cells than enzymatic digestion of tissue, with potential consequences on the chondrogenic behavior of these cells.

P64Human cartilage fragments in a hyaluronic acid/fibrin glue/platelet rich plasma scaffold for single stage cartilage repair: how to optimize cell outgrowth. In vitro studyA. Marmotti, C. Realmuto, F. Castoldi, R. Rossi, M. Bruzzone, D.E. Bonasia, C. Tarella, G. Collo, A. Maiello, P. Rossi Torino/Italy

Purpose: Cartilage fragments provided a viable cell source for single stage cartilage repair in previous rabbit and goat models. Nevertheless, human in vitro explant cultures showed some limitations as age-dependence, time-dependence and reduced cell migration and outgrowth from cartilage fragments, compared to animal model. Purpose of our study is to optimize cell outgrowth into a hybrid HA/fibrin/PRP scaffold under TGF-beta and G-CSF exposure and evaluate their effect on chondrocyte behaviour. Methods and Materials: Articular cartilage from human knees(< 35y and > 50y) was minced into small fragments and loaded onto the scaffold; constructs were cultured for 1, 2 and 3 months both in standard culture medium and under exposure to TGF-beta(10ng/ml) and/or G-CSF(10ng/ml). Explant cultures were evaluated histologically, with immunohistochemistry and with immunofluorescence. Results: Cell migration and outgrowth was time-dependent and age-dependent; with older donors, increased migration was observed under exposure to TGF-beta, reaching values similar to unstimulated younger donors(p<0.05); with younger donors, outgrowth increased at 1 month in a ratio of 1,6:1 with exposure

with four awls: a 20° Arthrex, a 30° Linvatec, and a 30o Sontec and half-size 30° Sontec. MicroCT scans were repeated to measure the depth and shape of each hole. Bone compaction was measured by a radial analysis of BMD from the central axis of the microfracture holes at the bone surface and at 0.5 mm intervals mm from the cartilage surface.Results: Bone compaction in the microfracture hole wall was evidenced by increased BMD in the adjacent bone. In human condyles with low (grade 2 & 3) Outerbridge scores the Linvatec awl created increased BMD profile from the surface to a depth of 2 mm whereas other awls were associated with less bone compaction. The Arthrex awl created larger diameter holes at the surface (1.67 mm) compared to the other 3 designs (1.12-1.08mm). The dense subchondral bone plate of the horse resulted in incomplete holes (<2mm deep) holes that were partially filled with mineralized debris. Conclusions: All microfracture awls penetrated to the marrow space of bone affected by early OA but as bone sclerosis increased, bone compaction and debris resulted in holes with poor connectivity to marrow spaces. This may lead suboptimal repair tissue formation and attachment.

P61Cartilage defects pretreated with microfracture and covered with a Artelon scaffold give a hyaline like repair tissue.S. Concaro1, H. Stenhamre2, A. Lindahl3, L. Peterson3 1Göteburg/Sweden, 2Gäteborg/Sweden, 3Gothenburg/Sweden

Purpose: Microfractures represent the first line of treatment for cartilage lesions up to 2-3 cm2. This bone marrow stimulating technique results in a repair tissue that is fibrous. The objective of this study was to evaluate whether a scaffold implanted into the defect at the time of microfracture treatment gave a more hyaline like tissue than microfracture alone in a full-thickness articular cartilage defect of rabbit. Methods and Materials: Scaffolds made out of ArtelonÒ, a poly(urethane urea), was placed in the full-thickness articular cartilage defects (n=3). Defects treated with microfracture only served as controls (n=3).Results: After two weeks of implantation cellular infiltration was seen in the scaffolds as well as in the controls. Close contact between the surrounding cartilage and the implanted scaffold was observed. Six months after implantation, cartilage implants and controls were assessed by macroscopic evaluation and histological scoring. ICRS macroscopic evaluation and histological scoring documented an improvement of repair tissue formation when the defects treated with microfracture were covered with the Artelon® scaffold, compared to controls treated with only microfracture.Conclusions: Implanting a cell-free polymer-based scaffold into microfractured defects might be a treatment option for cartilage defects and thus an option to use to improve the regeneration of articular cartilage.

P62Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disc cells via suppression of IL-1 β and p38 MAPK signalC. Niu, S. Lin, L. Yuan, C. Yang, W. Chen 1Kweishan, Taoyuan/Taiwan

Purpose: Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)-1, nitric oxide (NO), prostaglandin E2 (PGE-2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disc. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs.Methods and Materials: NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor-kinase array kit. RNA was isolated for real-time quantitative polymerase chain reaction (Q-PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL-1β, NO, PGE-2, MMP-3, and TIMP-1 in the conditioned media were quantified by enzyme-linked immunosorbent assay (ELISA).Results: Our data showed that HBO treatment decreased expression of IL-1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE-2, and MMP-3, and increased TIMP-1 expression

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blue and Alizarin red staining after having stimulated the cells with specific activating factors. Histological, immunohistochemical and molecular biological analyses were performed on cells grown in monolayer and onto the constructs. Results: Despite significant differences in their cellular composition BMCs express the same markers as isolated ones but at higher percentage. They are able to differentiate in osteogenic and chondrogenic sense after opportune stimuli. The differentiation process is favoured by the growth of the cells onto the hyluronan-derivative scaffold. Conclusions: BMCs represent the ideal candidate to be used in autologous transplantation to regenerate osteochondral tissues since are able to differentiate in their main cell populations. This could be particularly useful for the “one step” procedure allowing to directly implant the cells in operatory room. The employment of a hyaluronan scaffold could improve the strategy.

P67The effect of low-intensity pulsed ultrasound on scaffold-free chondrocyte plate in rabbit model.K. Uenaka1, S. Imai2, Y. Matsusue2 1Otsu City/Japan, 2Otsu/Japan

Purpose: The aim of this study is to evaluate the effect of low-intensity pulsed ultrasound (LIPUS) for scaffold-free chondrocyte plate in rabbit full-thickness defect model. Methods and Materials: Chondrocytes were collected from articular cartilage of Japanese white rabbits. The cells were then condensed to the density at 107cells/cm2 (passage 1, P1) on synthetic membranes with 0.2µm pore. The LIPUS application group was stimulated for 20 min/day. To investigate effect LIPUS stimulation on the matrix-synthesis of the constructs, mRNA expression of type II collagen, aggrecan and type I collagen was studied using real-time polymerase chain reaction. Synthesis of type II collagen and proteoglycan was also assessed histochemically in vitro. Moreover we tried to repair full-thickness defect model using the scaffold-free plates stimulated LIPUS or not in vivo. Results: The chondrocytes(P1) prepared at 107cells/cm2 detached from the membranes to form a plate of chondrocytes around the 7th day (day 7) of starting P1 culture. The expression of type II collagen and aggrecan mRNA was significantly higher in the group by stimulation of LIPUS (LIPUS group) than the group by no stimulation (sham group). The constructs had resultant matrix of type II collagen and proteogrycan which confirmed histochemically. The tissues were stained strongly with safraninO, and there were partially columnar pattern. Immunohistochemical analysis revealed that type II collagen was abundantly deposited in the tissue. Interestingly, LIPUS group was also more strongly stained than sham group by histology in vitro. However In vivo study, histology of repair tissue with the chondrocyte plate at 2week was not significant different between LIPUS group and non-treated LIPUS (sham) group. Conclusions: As a result for application of LIPUS, we could stimulate the matrix synthesis of the scaffold-free chondrocytes plate in vitro study. In vivo study, we could not show the effect of LIPUS to stimulate the matrix synthesis of the plate.

P68Detailed evaluation of Autologous bone marrow-derrived mesenchmal cells transplantation in Cynomolgus Monkeys.S. Araki, S. Imai, M. Kubo, T. Mimura, K. Nishizawa, H. Ueba, Y. MatsusueOsu/Japan

Purpose: In the field of articular cartilage repair, 5 mm full-thickness chondral defect is demonstrated to be relatively well repaired in small animal models (i.e. rabbit models) with tissue engineering techniques. Although attempts to repair human chondral defects with tissue engineered methods has been made, details of histological process is still unknown because therapeutic and ethical issues make it unable to extract and evaluate human tissue. In addition, no report of spontaneous repair of primate full-thickness cartilage defects has ever been made. We have reported regeneration of full-thickness cartilage defect in cynomolgus monkey model in our previous studies. In these studies, we have attempted to determine the critical size that is repairable. Two mm diameter full-thickness cartilage defect has been demonstrated to be repaired with hyaline-like cartilage. Three mm diameter

to TGF-beta (compared to unstimulated cultures), in a ratio of 2,1:1 with exposure to G-CSF and in a ratio of 1,9:1 with exposure to both factors(p<0.05); immunofluorescence of migrating cells was positive for sox9, CD151, CD49c and negative for CD105, consistent with a predominant chondrogenic phenotype; G-CSF Receptor was detected on migrating cells with immnunofluorescence; exposure to G-CSF slightly decreased SOX-9 expression and increased PCNA, beta-catenin and pan-cadherin expression. Conclusions: Cell outgrowth into the HA/fibrin/PRP scaffold increased under exposure to TGF-beta or G-CSF. Higher values have been obtained with G-CSF, suggesting a possible role of G-CSF in increasing chondrocyte outgrowth. Further studies are required to better ascertain the role of G-CSF on chondrocytes migrating from articular cartilage fragments. These promising results could have relevance for improving in-vivo single stage cartilage repair with minced human cartilage fragments.

P65Reduced chondrocyte viability is associated with the use of marker pen ink.A. Getgood1, I. Mcnamara1, S. Kili2, T. Bhullar3, F.M. Henson1 1Cambridge/United Kingdom, 2Shrewsbury/United Kingdom, 3Peterborough/United Kingdom

Purpose: The aim of this study was to investigate whether the addition of two different marker pen inks to a collagen membrane would affect chondrocyte viability, and therefore be potentially detrimental to articular cartilage repair in autologous chondrocyte transplantation. Methods and Materials: Human chondrocytes were applied to Chondro-Gide® collagen membranes at a volume of 12 million cells/ml. Two sterile marker pens were used to mark the membranes; one contained methylene blue, the other contained crystal violet inks. Three groups of membrane were tested in duplicate for each pen. Group A consisted of no ink mark, group B had only the uppermost ‘smooth’ layer marked, and group C had the lower ‘porous’ layer marked. All membranes were then cultured in standard growth media for 24 hours. Cell viability was assessed at 24 hours on all membranes using a live/dead cell viability assay. Cell viability was quantified with florescent microscopy with mean number of cells compared to control membranes using the students t test (p<0.05). Results: Table 1 shows the mean number of viable cells in each group. Control membranes (group A) with no ink showed excellent cell viability. A statistically significant reduction in cell viability with both methylene blue (p<0.0001) and crystal violet (p<0.0001) was found adjacent to the ink mark on the smooth side (group B) and on the porous side remote from the ink (group C). Marked cell death was seen with both dyes (p<0.0001) adjacent to the ink on the porous side. A small number of cells cultured with crystal violet were live – no live cells were detected in the presence of methylene blue. Conclusions: Chondrocyte viability is significantly reduced when cells are cultured in-vitro on collagen membranes marked with methylene blue and crystal violet pen ink. Surgeons should be aware of the potential negative impact of marker pens in cell based therapies.

P66Bone marrow “niche”: evidences which support its use in vivoB. Grigolo, C. Cavallo, G. Desando, R. Buda, F. Vannini, M. Cavallo, S. Giannini, A. Facchini Bologna/Italy

Purpose: Mesenchymal Stem Cells (MSCs) have been indicated as a new option for regenerative medicine because of their ability to differentiate into various lineages. However, isolation procedures are crucial for the functional activity of the transplanted cells and not easily viable in operatory room. The use of concentrated bone marrow cells (BMCs) enables to implant a cell population surrounded by its microenvironment (or “niche”). The cells of the niche regulate stem cell behaviour through direct physical contact and paracrine signalling. The aim of the research was to characterize BMCs in vitro to validate their use to regenerate osteochondral tissues. Methods and Materials: BMCs were obtained from bone marrow harvested from iliac crest of 20 patients operated on for focal osteochondral lesions of the talar dome by “one-step” procedure. Bone marrow was concentrated in a cell separator (BMAC®; Harvest Technologies Corp) and BMCs were successively analyzed by FACS using specific antibodies. BMCs were also grown onto a hyaluronan based scaffold already used in clinical practice. Chondrogenic and osteogenic potential were evaluated by Alcian

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from knees of deceased patients at the department of pathology. Cartilage was digested overnight in 0.15% collagenase. Isolated chondrocytes were, either directly (P0) or after expansion (P2), pelleted by centrifugation. After 4 weeks of culture, pellets were digested and analyzed for glycosaminoglycan (GAG) and DNA content, using a DMMB and Picogreen assay respectively. In addition, pellets were also paraffin embedded for collagen I, II and X immunohistochemistry and Safranin-O histology. Differences in GAG/DNA were analyzed by a one-way ANOVA with post-hoc LSD test. A p<0.05 was considered statistically significant. Results: After 4 weeks of culture, P0 pellets from grade III and FWB chondrocytes contained more (p<0.000) GAG/DNA when compared to pellets from grade IV and NWB chondrocytes (Figure 1). After expansion, P2 pellets contained less GAG/DNA than their P0 counterparts and GAG/DNA was not different anymore between the donor sites. Immunohistochemistry for collagen II and Safranin-O staining appeared to be increased in the P0 pellets compared to the P2 pellets. Collagen I and X staining was slightly positive in the P2 pellets. Conclusions: The chondrogenic potential of freshly isolated FWB and chondrocytes from Grade III lesions is higher, and after expansion similar, compared to NWB and Grade IV chondrocytes. Thus for ACI based on expanded chondrocytes, cells both from grade III and grade IV lesions are a suitable alternative for cells from non-weight bearing areas.

P71Scaffold-Free, Three-Dimensional Chondrocyte Constructs for Regeneration of Articular cartilageJ. Lee1, J. Lee1, E. Lee2, J.H. Hwang1, Y. Son2 1Seoul/Korea, Democratic People’s Republic of, 2Yongin/Korea

Purpose: In this study, we evaluated the potentiality of scaffold-free pellet-type chondrocytes for regeneration of articular cartilage.Methods and Materials: Rabbit costal chondrocytes were expanded at passage 8, and then the expanded cells were seeded onto commercial collagen scaffold (Ch-COL) or centrifuged to make scaffold-free pellet constructs (Ch-PEL). After 2-week chondrogenic three-dimensional culture, the Ch-COL or the Ch-PEL was transplanted onto cartilage defect of rabbit knee. Fibrin glue was used as a control. The ability of these constructs to cartilage defect repair was evaluated by histological and immunohistological examination of regenerative tissue at 6 and 12 weeks after transplantation.Results: After chondrogenic culture, the dedifferentiated chondrocytes formed homogenous hyaline cartilage-like structure in the scaffold-free pellet culture system and the diameter of the pellet was approximately 1.0 mm. However, in the Ch-COL construct, a lacunae-occupied chondrocyte and ECM-rich structure was only detected at the surface of the construct. In vivo study, transplantation of Ch-COL as well as Ch-PEL restored hyaline cartilage in the articular cartilage defect. However, undegradable scaffold frames were detected in the regenerative tissue of Ch-COL group even at 12 weeks after transplantation.Conclusions: In scaffold-free pellet culture system, the cells generate their own ECM similar to the natural hyaline cartilage and transplantation of these pellet constructs to articular cartilage defect is useful to make a good-quality hyaline cartilageous tissue without immune response.

P72Repair of partial-thickness chondral defects by gene plugsA. Ivkovic1, A. Pascher2, D. Hudetz1, D. Maticic1, M. Jelic1, S.C. Dickinson3, M. Loparic4, M. Haspl1, R. Windhager5, M. Pecina1 1Zagreb/Croatia, 2Graz/Austria, 3Bristol/United Kingdom, 4Basel/Switzerland, 5Austria/Austria

Purpose: The aim of this translational study was to test whether an abbreviated ex vivo protocol utilizing vector-laden, coagulated bone marrow aspirates for gene delivery to partial-thickness cartilage defects may be feasible for clinical application.Methods and Materials: Autologous bone marrow harvested from sheep was transduced with adenoviral vectors containing cDNA for GFP or TGF-β1. The marrow was allowed to clot forming a gene plug and implanted into partial-thickness defects created on the medial condyle. After 6 months the quality of repair was evaluated using biochemical, histological, and biomechanical parameters.

full-thickness cartilage defect was repaired with fibrocartilage. In contrast, 5 mm diameter full-thickness cartilage defect was repaired with fibrous tissue. Although up to 3 mm diameter full-thickness cartilage defect in rabbit models have demonstrated to be repaired spontaneously, 2 mm diameter is the limit for repair in primates. Methods and Materials: With this basic data, in the present study we assessed the effectiveness of autologous bone marrow-derived mesenchymal cells transplantation to repair a full-thickness cartilage defect of 3 mm and 5mm in a cynomolgus monkey model. Nine skeletally mature Cynomolgus monkeys were used in this study. The monkeys were anesthetized and approached through medial parapatellar incision. Full-thickness osteochondral defects were created in patella groove(3-mm wide, 5-mm deep) and in medial chondyle(5-mm wide, 5-mm deep). The animals were devided into three groups: MSC, Gel, and Defect. In the MSC group, the defects were filled with MSCs that were harvested from bone-marrow and cultured 4 weeks embedded in collagen gel. In the Gel group, the defects were filled with Type I collagen. Animals were sacrificed at 6, 12, and 24 weeks after operation. Samples were examined histologically. Results: In the MSC groups 3-mm and 5-mm diameter full-thickness cartilage defect was repaired with hyaline-like cartilage. Another 2-groups in contrast repaired with fibrocartilage or fibrous tissue. Conclusions: We decided that Cynomolgus monkeys were useful in the fields of cartilage repair.We considered that the present study was useful for regeneration of cartilage defects using bone marrow-derrived MSCs for clinical cases.

P69Tendon regeneration using cell seeded scaffolds in a rat modelM.F. Pietschmann, B. Frankewycz, P. Schmitz, D. Docheva, M. Schieker, V. Jansson, P.E. Müller Munich/Germany

Purpose: Irreparable tendon ruptures constitute a grave clinical problem. Our study investigates the effects of scaffold-based tendon regeneration using different cell types (mesenchymal-stem-cells/MSC, bone-marrow-stromal-cells/BMSC and tenocytes/TC) in a rat model. A polyglycol acid (PGA) and a collagen scaffold were used.Methods and Materials: A 2-3 mm full-thickness-defect in the rats achilles tendon was created, and filled, with either cell-seeded or not cell-seeded scaffolds. Cells were harvested from male rats and then implanted into female rats. After 12 weeks the maximum tensile strength was determined and histological stainings performed. Implanted cells were traced by DNA PCR of male Y-chromosomes.Results: Macroscopically the regenerated tendons were bigger in diameter, firmer and less elastic than normal tendons. In the MSC and TC groups the implanted cells could be clearly identified by DNA PCR. The biomechanical examination revealed a comparable tensile strength for collagen and PGA without cell-seeding. No positive influence of using BMSC or MSC on the mechanical stability of the regenerated tissue could be found. Using tenocytes the highest pull out strength was found in both scaffolds. Histologically there was a variable amount of bone formation in all groups, but considerably less in the TC groups. An inflammatory reaction was seen in PGA groups but not with collagen.Conclusions: Our findings indicate that both collagen and mesenchymal stem cells have stimulating effects on bone formation. The use of TC improves mechanical stability and histological appearance of the tendon regenerates. BMSC and MSC are inferior to TC.

P70Articular cartilage debrided from focal lesions as a cell source for cell-based cartilage therapyJ.E.J. Bekkers, L.B. Creemers, M. Rijen, W.J.A. Dhert, D.B. Saris Utrecht/Netherlands

Purpose: Currently, ACI is based on the expansion of chondrocytes harvested from a non weight-bearing area in the knee. This study evaluated the chondrogenic potential of chondrocytes from a focal lesion as an alternative cell-source for ACI. Methods and Materials: Articular cartilage was obtained from focal lesions of patients (n=17) undergoing cartilage surgery and classified according to the ICRS grading for focal cartilage lesions. Macroscopically healthy cartilage was harvested from full-weight bearing (FWB, n=5) and non-weight bearing (NWB, n=5) regions

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magnitude was more than 60% and echo duration was almost equal compared to normal cartilage in magnetic force group (P=0.3734). Conclusions: This study showed that we could obtain better hyaline-like cartilage regeneration by accumulating relatively small number of m-MSCs to the cartilage defect using external magnetic force. We believe that our cell delivery system using external magnetic force is a promising option for cartilage regeneration.

P75Meniscal Repair with Chondrocyte-seeded Flexible ScaffoldsJ.J. YOO1, D.A. Bichara2, X. Zhao2, M.A. Randolph2, T.J. Gill2 1Seoul/Korea, Democratic People’s Republic of, 2Boston/United States of America

Purpose: A cell-based tissue-engineered construct can be used for healing of intractable meniscal lesions. Our aims were 1) to assess the culture conditions (static versus dynamic) and the healing capacity of the chondrocyte-seeded flexible scaffolds; and 2) to assess in vivo cell labeling for tracking cells during the reparative process.Methods and Materials: Swine articular chondrocytes were labeled with PKH 26 or DiI dye and seeded onto flexible PLGA scaffold (VicrylTM, Ethicon Inc.) in dynamic oscillating conditions for 24 hours. Half of cell-seeded scaffolds were cultured in static conditions and the remaining half were cultured in dynamic conditions for 7 days. During culture period, live/dead assay and DNA measurements were performed. Chondrocyte-seeded scaffolds were placed between devitalized swine meniscal discs and sutured in place (A, dynamically cultured, B, statically cultured, C, acellular, and D, no scaffolds). All constructs were implanted subcutaneously in nude mice for 6 weeks and evaluated histologically.Results: Labeled chondrocytes covered all scaffolds completely in both dynamic and static culture. DNA measurement showed no statistically significant difference in the two culture conditions. Histologic analysis demonstrated a continuous fibro-cartilagenous interfacial tissue between meniscal discs in all 12 dynamically cultured scaffold constructs and 9 of 11 statically cultured scaffold constructs. There was no evidence of meniscal healing between discs in both acellular and no scaffold constructs. Both PKH 26- and DiI-labeled cells were present along the entire interface of the meniscal discs.Conclusions: Culture conditions after seeding may not affect healing outcome. The articular chondrocyte-seeded flexible PLGA scaffolds induced healing of meniscal discs in nude mice. These cells are responsible for the healing process in the interface. Based on these encouraging results, swine experiments are underway to assess biomechanical bonding properties and immune responses after meniscal repair with aotologous and allogeneic chondrocyte-seeded scaffolds.

P76Optimized Alkylated cyclodextrin polysulphates restore osteoarthritic chondrocyte extracellular matrix metabolism in vitro and in vivoS. Groeneboer1, S. Lambrecht1, A. Dhollander1, P. Jacques1, B. Vandercruyssen1, K. Devreese1, D. Elewaut1, G. Verbruggen2 1Gent/Belgium, 2GHENT/Belgium

Purpose: To compare the ability of different cyclodextrin polysulphate derivatives to affect human articular cartilage cell metabolism in vitro and to act as Disease Modifying OsteoArthritis Drugs in vivo. Methods and Materials: OA chondrocytes were cultured in gelled alginate and exposed to 5 µg/ml (2-carboxyethyl)-β-cyclodextrin polysulphate (CE-CDPS), or β-cyclodextrin polysulphate (CDPS) during 5 days. Effects on IL-1-driven chondrocyte extracellular matrix (ECM) metabolism was assayed by analysis of the accumulation of aggrecan in the interterritorial matrix and by the release of IL-6 in the culture supernatant. The compounds were analyzed for their in vitro effect on coagulation and their ability to activate platelets in an in vitro assay to detect possible crossreactivity with HIT (heparin-induced thrombocytopenia) antibodies. CE-CDPS was selected for further qPCR analyses, in order to investigate whether this compound directly influences the gene expression of IL-6 and aggrecan. The effectiveness of CE-CDPS was further assayed in collagenase-induced OA of the knee, by injecting 1 mg/kg CE-CDPS or saline subcutaneously once weekly in a mouse model. Knee joints were scored semiquantitatively on inflammation, fibrosis, osteophyte growth and loss of articular cartilage and Safranin-O staining.

Results: Assessment of repair showed that the groups treated with gene plug transplantation contained more cartilage-like tissue than untreated controls. Improved cartilage repair was observed in groups treated with unmodified bone marrow plugs and Ad.TGF-β1 transduced plugs, but the repaired tissue from TGF-treated defects showed significantly higher amounts of collagen II (p<0.001).Conclusions: The results confirmed that the proposed method is fairly simple technique for application in clinical settings. It is a single-step procedure that can be easily implemented in standard clinical settings, avoids the usual drawbacks associated with gene therapy because it is administered locally, and excludes the expensive in vitro production of autologous and engineered tissues. Gene plugs are sufficient to facilitate articular cartilage repair of partial thickness defects in vivo. Further studies should focus on selection of transgene combinations that promote more natural healing.

P73Repair of articular cartilage defects with Sox9 gene modified bone marrow mesenchymal stem cellsZ. Yang, X. Wei Taiyuan/China

Purpose: to observe the influence of Sox9 gene overexpression on the chondrogenesis of BMSCs and to repair articular cartilage defects with Sox9 gene modified BMSCs embedded in alginate.Methods and Materials: Bone marrow mesenchymal stem cells of New Zealand Rabbit were transfected with recombinant eukaryotic expression Sox9 plasmid. The chondrogenesis of Sox9 overexpressing BMSCs were analysised by RT-PCR, western blot, immunohistology and GAG assay. Cartilage defects of the knee joints were filled with Sox9 gene modified MSCs, MSCs or alginate alone. The samples were evaluated histologically with a semi-quantity scoring system for the repairing of the cartilage . Results: The Sox9 overexpressing BMSCs expressed chondrogenic marker molecules and synthesis more GAG than the control groups . The defects filled with Sox9 gene modified BMSCs showed hyaline like cartilage tissue with smooth surface and good integrating with the adjacent cartilage; while most of the defects filled with BMSCs in alginate showed a fibrocartilage-like regenerate tissue and most of the defects filled with alginate only showed with fibrous tissues. The histological scoring system showed that the cartilage repairing of the experiment groups were better than the two control groups with statistical significances. Conclusions: The overexpression of exogenous Sox9 gene can trigger the chondrogenic differentiation of BMSCs in vitro. Sox9 gene modified BMSCs embedded in alginate may regenerate hyaline like cartilage in vivo. Gene enhanced tissue engineering may be a promising way to repair cartilage defects.

P74Articular cartilage repair with magnetically labeled mesenchymal stem cells and external magnetic device using porcine modelG. Kamei, N. Adachi, M. Deie, T. Kobayashi, H. Shibuya, S. Ohkawa, M. Ochi Hiroshima/Japan

Purpose: The purpose of this study is to evaluate the utility of new cell delivery system using an external magnetic force to accumulate a relative small number of magnetically labeled mesenchymal stem cells (m-MSCs) to the desired area using porcine model.Methods and Materials: MSCs were cultured from bone marrow of 6 months porcine and labeled with ferucarbotran(superparamagnetic iron oxide). We made a full-thickness cartilage defect (6mm diameter) in the center of the patella. 4 weeks after creation of cartilage defect, for magnetic force group, m-MSCs (5×106 cells) were accumulated to the cartilage defect using an external magnetic force (1.5 Tesla) for 10 minutes. For injection group, the patella was faced upward, filled with MSCs (5×106 cells) and held for 10 minutes. For the control group, PBS was injected. Porcine were sacrificed at 3 and 6 months, and macroscopic and histological evaluation was done. We evaluated the maximum magnitude (articular cartilage stiffness) and echo duration (macroscopic surface roughness) of the repaired area using ultrasonic pulser system. Results: Macroscopic and histological findings showed better cartilage regeneration in magnetic force group at 6 months after MSC injection. The mean maximum magnitude and echo duration were 75.1±9.4% and 0.46±0.05㎲at 6 months. The maximum

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P79Interleukin-1-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in human OA chondrocytes is associated with histone H3K4 methylationF. El Mansouri, N. Chabane, N. Zayed, J. Martel-Pelletier, J.P. Pelletier, H. Fahmi Montreal/Canada

Purpose: Increased expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 plays a key role in the pathogenesis of osteoarthritis. Methylation of lysine 4 on histone H3 (H3K4) was shown to be of fundamental importance in the regulation of gene expression. In the present study, we investigated the role of H3K4 methylation in interleukin-1b (IL-1)-induced COX-2 and iNOS expression in human OA chondrocytes Methods and Materials: Chondrocytes were stimulated with IL-1 for various time periods and the expression of iNOS and COX-2 mRNAs and proteins were evaluated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. H3K4 methylation at the iNOS and COX-2 promoters was evaluated using chromatin immunoprecipitation (ChIP) assays. The role of histone methylation was further evaluated using the methyltransferase inhibitor, 5’-deoxy-5’(methylthio) adenosine (MTA). Results: IL-1 induced iNOS and COX-2 mRNA and protein in a dose- and time-dependent manner. The induction of iNOS and COX-2 expression by IL-1 was associated with H3K4 di- and trimethylation at the iNOS and COX-2 promoters, whereas the levels of H3K4 monomethylation remained unchanged. Treatment with MTA inhibited IL-1-induced H3K4 methylation as well as IL-1-induced iNOS and COX-2 expression. Conclusions: These results indicate that H3K4 methylation contributes to IL-1-induced iNOS and COX-2 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of osteoarthritis.

P80Glial Fibrillary Acidic Protein Maintains Nuclear Morphology And Supports the Resistance to Severe Mechanical Stress in the Chondrocytes of Elastic CartilageS. Kanazawa1, S. Nishizawa2, T. Takato2, K. Hoshi2 1Bunkyo-ku/Japan, 2Tokyo/Japan

Purpose: Glial fibrillary acidic protein (GFAP) is a intermediate filament that is expressed in astrogrial cells, but is also localized in chondrocytes of elastic cartilage which is present in auricles or epiglottis. Among various kinds of cartilage, the elastic type can maintain tissue function without collapse, even after repeated bending. This suggests that GFAP may contribute to the resistance to the severe mechanical stress in the cells and should conserve the structures and functions of cytoplasmic organelles.Methods and Materials: To verify the functions of GFAP in the chondrocytes, we compared the morphology in cultured chondrocytes of mouse auricles between wild type (Gfap+/+) and GFAP-deficient mice (Gfap-/-). We also examined the cell responses to stretch stress for these chondrocytes by cell-stretch devise.Results: In the auricular chondrocytes (passage 3) of Gfap+/+, GFAP was densely accumulated in the perinuclear areas, while the vimentin was generally present in cytoplasmic areas. Nuclei were shaped columnar, with the size of approximately 12 micron in diameter and 4 micron in height. In contrast, the Gfap-/- chondrocytes (passage 3) significantly showed the flattening and the irregularity of nuclei (approximately 14 micron in diameter and 3 micron in height). When the passage was repeated by passage 8, this tendency was exaggerated in the Gfap-/- chondrocytes, and, moreover, the multinucleation became evident in that type. In the cell-stretch studies (30% strain and 24 h), although most of the Gfap+/+ chondrocytes maintained the cell attachment (approx. 96%) and cell viability (approx. 100%), the Gfap-/- counterparts were detached at the rate of 60% and decreased the cell viability to 83%.Conclusions: Based on these results, GFAP seems to play pivotal roles in the conservation of cell structures and functions thought the maintenance of nuclear morphology, and support the resistance to severe mechanical stress in the tissue which physiologically suffers from stretch overload.

Results: The polysulphated cyclodextrin CE-CDPS at 5 µg/ml concentrations, significantly induced aggrecan production and repressed IL-6 release by the chondrocytes in culture. Five µg/ml of CE-CDPS, in contrast to other polysulphated cyclodextrins, did not significantly activate platelets and thus showed no potential to induce HIT thromboembolic accidents in vivo. Therefore, CE-CDPS was selected for further qPCR analyses. These analyses confirmed the anabolic and anti-catabolic profile of CE-CDPS. CE-CDPS was therefore tested on collagenase–induced knee OA in mice and was shown to prevent cartilage proteoglycan depletion but not osteophyte formation in the OA knee joints of these mice. Conclusions: CE-CDPS is a new, structurally adjusted sulphated β-cyclodextrin derivative with preserved chondroprotective capacity and a promising safety profile.

P77Pellet culture induces cartilage specific gene expression but does not alter DNA methylation patterns in bone marrow stromal and synovial cellsD. Dono, S.J. Duguay, S. Rapko Cambridge/United States of America

Purpose: To evaluate the capacity of a pellet culture system to induce chondrocytic gene expression and genomic DNA methylation patterns in adult human cell cultures derived from bone marrow stroma, synovium, and hyaline cartilage. Methods and Materials: Monolayer cultured bone marrow stromal cells (BMSCs), synovial cells (SCs), and chondrocytes were seeded into pellet cultures with sampling at days 0 and 20. Gene expression and DNA methylation levels of cartilage specific genes were examined using PCR methods. Results: Gene expression levels of cartilage specific genes were low or undetectable in all of the day 0 cultures, but significantly upregulated by day 20 of pellet culture. DNA methylation patterns in the day 0 cultures were specific to the tissue of origin for each culture and remained steady throughout the pellet culture period. DNA methylation did not strongly correlate with expression, as methylated genes were expressed by MSCs and SCs during pellet culture.Conclusions: Pellet culture induced the expression of cartilage specific genes in BMSCs, SCs, and chondrocytes, but did not alter their original tissue specific DNA methylation patterns. These observations suggest that MSC and SC DNA methylation patterns are highly stable. As cartilage possesses specific DNA methylation patterns presumably established during chondrogenesis, the results of this work have implications for the selection of cell types and culture conditions used for cartilage tissue engineering applications.

P78Valproic acid suppresses interleukin-1b-induced microsomal prostaglandin E2 Synthase-1 expression in chondrocytesN. Zayed, N. Chabane, F. El Mansouri, J. Martel-Pelletier, J.P. Pelletier, H. Fahmi Montreal/Canada

Purpose: Microsomal prostaglandin E2 Synthase (mPGES)-1 catalyzes the terminal step in the biosynthesis of PGE2. Early growth response factor-1 (Egr-1) is a key transcription factor in the regulation of mPGES-1. In the present study we examined the effects of valproic acid (VA), a histone deacetylase (HDAC) inhibitor, on interleukin (IL)-1b-induced mPGES-1-expression in human chondrocytesMethods and Materials: Chondrocytes were stimulated with IL-1 in the absence or presence of VA, and the level of mPGES-1 protein and mRNA expression were evaluated using Western blotting and real-time reverse-transcription polymerase chain reaction, respectively. The mPGES-1 promoter activity was analyzed in transient transfection experiments. Egr-1 recruitment to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation (ChIP) assaysResults: VA dose-dependently suppressed IL-1b-induced mPGES-1 protein and mRNA expression as well as its promoter activation. Treatment with VA did not alter IL-1-induced Egr-1 expression, nor its recruitment to the mPGES-1 promoter, but prevented its transcriptional activity.Conclusions: Our study demonstrates that VA inhibits IL-1-induced mPGES-1 expression in chondrocytes. The suppressive effect of VA was not due to reduced expression or recruitment of Egr-1 to the mPGES-1 promoter.

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P83Evaluation of Proliferation and Synthesis of osteoarthritic Chondrocytes with and without pericellular MatrixM. Rothdiener, Q. Wang, A. Badke, W.K. Aicher, B. RolauffsTübingen/Germany

Purpose: The pericellular matrix (PCM) surrounding chondrocytes is responsible for structural and functional connections of cell and extracellular matrix (ECM) of articular cartilage. PCM is considered to affect proliferation, phenotype-maintenance and matrix-synthesis. The aim of this project was the evaluation of chondrocytes with intact PCM for biological therapy in osteoarthritic (OA) patients. The objectives of the current studies were a) to investigate the metabolism of OA chondrocytes under preservation of the intact PCM b) cultivation and proliferation in the presence or absence of FBS and c) gene expression profiling. Methods and Materials: Human chondrocytes from knee-replacement patients (mean: 62 years) were isolated in a collagenase/dispase digestion at different concentrations and time periods to maintain/remove PCM. Cells were cultivated as monolayers for 30d (F12:DMEM 1:1, 10% FBS, 0,1% ascorbic acid) and counted every second day. mRNA was isolated at d0, cDNA was synthesized and quantitative PCR was performed using the Roche Lightcycler system. OA grades were assessed with the Kellgren-Lawrence-Score. Results: Without FBS, chondrocytes +/-PCM showed no proliferation but remained their typical phenotype. In the presence of FBS, cells showed spread morphology. With intact PCM, rapid proliferation was observed (at day 10: 400% of initial) whereas chondrocytes without PCM remained under 100%. Within this group, fast-growing and slow-growing populations occurred but the proliferation rate did not correlate with the OA grades. Gene expression profiles showed a significantly higher expression of matrix proteins COMP and aggrecan in chondrocytes with intact PCM. However, no significant differences in collagen expression occurred. With intact PCM, the chondrogenic marker FGF-2 was also significantly higher expressed. In contrast, the cartilage degeneration marker IL-1β was expressed similarly. Conclusions: Proliferation of OA chondrocytes was vastly superior with intact PCM. Synthesis of matrix components and chondrogenic markers were also enhanced with maintained PCM suggesting that OA cells with PCM may be further evaluated for biological therapy.

P84Co-culturing of Human Bone Marrow Mononuclear Cells and Primary Chondrocytes to Improve Cartilage FormationJ.E.J. Bekkers, D.B. Saris, A.I. Tsuchida, W. Verra, W.J.A. Dhert, L.B. Creemers Utrecht/Netherlands

Purpose: The combination of freshly isolated chondrocytes with mesenchymal stem cells has previously been advocated as a possible alternative for the use of expanded chondrocytes in autologous chondrocyte implantation (ACI). This study aimed to investigate whether co-culture of the mononuclear fraction from freshly isolated human bone marrow mononuclear fraction (MNF) and human primary chondrocytes (hPC) stimulates chondrogenesis. Methods and Materials: Knee cartilage was obtained at autopsy, bone marrow aspirates from patients undergoing hip arthroplasty and human fibroblasts from the foreskin (hFB). Cartilage was digested overnight in 0.15% collagenase. After erythrocyte cell lysis, the MNF cells were combined with hPCs at different ratios (containing 0%, 2%, 5%, 10%, 15%, 20% 50% and 100% hPCs) and pelleted by centrifugation. Pellets were also created between PCs and hFB in similar ratios as the PC MNF pellets. In addition, PCs were also pelleted in quantities identical to those in the co-culture pellets, however, without adding other cells (PC Alone group). After 4 weeks of culture, the pellets were digested and analyzed for GAG and DNA content, using a DMMB and PicoGreen assay respectively. Differences in GAG content per DNA were analyzed by a one-way ANOVA and post-hoc-LSD test. Results: The amount of matrix produced per cell (GAG/DNA) after 4 weeks of culture was higher (p<0.05) in the PC/MNF co-cultured pellets when compared to the PC Alone pellets, in the range 15-100%hPC (Figure 1). This was not observed in co-cultured pellets containing PCs and hFB (Figure 2). Surprisingly, low amounts of chondrocytes (PC Alone 2-5%) show more efficient GAG/DNA production compared to co-cultured hPCs and MNFs. Conclusions: Co-culturing of the nonexpanded MNF and hPCs enhanced matrix producing activity. This stimulation was not achieved combining hPCs with hFB cells suggesting specific interactions between PCs and MNF cells. This finding may further pave the way towards a one-step cell-based regenerative cartilage therapy.

P81Trophic effects of mesenchymal stem cells on chondrocytes increase cartilage formation in co-culturesL. Wu1, J. Leijten1, N. Georgi2, C.A. van Blitterswijk1, M. Karperien1 1Enschede/Netherlands, 2Enchede/Netherlands

Purpose: Autologous Chondrocyte Implantation (ACI) requires a large number of chondrocytes. Replacement of part of the chondrocytes with Mesenchymal Stem cells can significantly reduce the number of chondrocytes required for ACI. Previously, it was shown that cartilage matrix formation is significantly increased in co-cultures of MSCs and chondrocytes compared to cultures of pure cell populations. To explain the beneficial effect of MSCs in co-cultures, it is hypothesized that increased matrix formation is either due to i) chondrogenic commitment of MSCs induced by chondrocytes; ii) increase of cartilage matrix formation by chondrocytes stimulated by the MSCs, or iii) a combination of both.Methods and Materials: To study cell fate by species specificity, we used bovine chondrocytes and human MSCs. Pellets were cultured in a 96-well plate in chondrocyte proliferation medium for 4 weeks. Glycosaminoglycans (GAG) assay, DNA assay and histological staining were performed to evaluate cartilage matrix formation. Real time PCR was carried out to determine the origin of cartilage matrix. Cell proliferation and apoptosis were also examined.Results: Co-culture of bovine chondrocytes and human MSCs in pellets significantly increased glycosaminoglycan production per initial % of seeded chondrocytes as expected. To avoid donor variation, we used immortalized MSCs (iMSCs) in the subsequent experiments. Species specific quantitative PCR indicated disappearance of human cells after 4 weeks of co-culture. This was confirmed by species specific qPCR of cartilage matrix components showing its bovine origin. In co-cultures, chondrocyte, but not MSC, proliferation was significantly enhanced compared to cultures of pure chondrocytes. Inversely, TUNEL assay indicated preferential death of MSCs, most likely by apoptosis, in co-cultures only at week 1 and week 2. Finally, we show that increased proliferation of chondrocytes in co-cultures is, at least in part, caused by soluble factors secreted by MSCs.Conclusions: Our data indicate that trophic effects of MSCs increase cartilage matrix formation by chondrocytes in co-cultures.

P82Prostaglandin D2 enhances interleukin -1beta- induced cyclooxygenase-2 expression in osteoarthritic chondrocytesN. Zayed, N. Chabane, F. El Mansouri, J. Martel-Pelletier, J.P. Pelletier, H. Fahmi Montreal/Canada

Purpose: To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1beta (IL-1beta)-induced cyclooxygenase (COX)-2 expression in human chondrocytes and the signalling pathways involved in these effects.Methods and Materials: Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of COX-2 protein was evaluated by western-blotting. Messenger RNA (mRNA) expression was analyzed by real-time reverse transcription-polymerase chain reaction. The role of the PGD2 receptors D prostanoid receptor 1 (DP1) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) was evaluated using specific agonists.Results: PGD2 increased in a dose-dependent manner IL-1-induced COX-2 protein and mRNA expression. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by DK-PGD2 and Indomethacin, selective agonists of CRTH2, but not by BW245C, a selective agonist of DP1. Furthermore, treatment with an anti-CRTH2 antibody reversed the effect of PGD2, indicating that the stimulatory effect of PGD2 is mediated by CRTH2. Activation of CRTH2 is consistent with the activation of a receptor coupled to a phosphoinositide-specific phospholipase, suggesting that the effect of PGD2 is mediated by the CRTH2/PIP2/PKC.Conclusions: PGD2 enhances IL-1-induced production of COX-2 by chondrocytes through the CRTH2/PIP2/PKC signalling pathway.

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P88Acid ceramidase, chondrocyte survival and transplantationS. Sachot, Y. Ge, X. He, E. Eliyahu, E.H. Schuchman, C.M. Simonaro New York/United States of America

Purpose: The mucopolysaccharidoses (MPS) are inherited joint and bone disorders due to mutations in specific genes degrading glycosaminoglycan (GAGs). We have previously shown that GAG storage in the MPS disorders results in a complex sequence of downstream events, including the release of numerous inflammatory cytokines and proteases, and alterations in the metabolism of several lipid signaling molecules. Acid ceramidase (AC) is a key enzyme in sphingolipid metabolism, helping to maintain the critical balance between three important signaling lipids, ceramide, sphingosine, and sphingosine-1-phosphate (S1P). The purpose of this research was to evaluate whether AC could be used to improve the production of bone marrow-derived chondrocytes for the eventual treatment of MPS patients. Methods and Materials: Bone marrow-derived mesenchymal stem cells (BM-MSC) were prepared from normal and MPS VI (Maroteaux-Lamy disease) rats using standard methods ongoing in our laboratory, and grown with and without recombinant AC (rAC). Cell viability, apoptosis rates and stem cell properties, including the ability of the expanded cells to form chondrocytes, were evaluated. Results: Addition of rAC to BM-MSC culture media permitted their growth and expansion for more than 15 passages. Importantly, these cells maintained their stem cell properties and could be differentiated into chondrocytes and other lineages.Conclusions: rAC can be used to improve the manufacturing of chondrocytes for transplantation in the MPS disorders and other diseases with joint injury. Further characterization of these cells is currently underway in the MPS VI rat model.

P89Autophagy modulates osteoarthritis-related gene expressions in human chondrocytesK. Takayama, T. Matsushita, R. Kuroda, K. Ishida, N. Fujita, S. Kubo, T. Matsumoto, H. Sasaki, M. Kurosaka Kobe/Japan

Purpose: Autophagy, an evolutionarily conserved process for the bulk degradation of cytoplasmic components, serves as a cell survival mechanism. The purpose of this study is to elucidate the role of autophagy in human chondrocytes and pathophysiology of osteoarthritis (OA). Methods and Materials: Autophagy in articular cartilage tissues and primary chondrocytes derived from patients with OA was assessed by immunohistochamistry and immnoblotting using antibodies for atuophagy markers, LC3II and beclin1. The state of autophagy in normal chondocytes under catabolic (IL-1β and NO) and nutritional stresses were examined by real-time PCR. We also examined the effects of inhibition or induction of autophagy under the stimulation with IL-1β. Autophagy was inhibited by small interfering RNA (siRNA) targeting Atg5, an autophagy essential gene and autophagy was induced by rapamycin under IL-1β stimulation. The effects of inhibition or induction of autophagy were examined by real-time PCR for MMP13, ADAMTS5, aggrecan and COL2A1 mRNA. Results: Osteoarthritic cartilage strongly expressed LC3II as compared with non-arthritic cartilage. Catabolic and nutritional stresses caused increased autophagy. The inhibition of autophagy by Atg5siRNA under the stimulation of IL-1β caused significant upregulation of MMP13 and ADAMTS5 expressions while the induction of atuophagy by rapamycin reduced these gene expressions. Additionally, the inhibition of autophagy downregulated aggrecan and COL2A1 while the induction of autophagy uperegulated the expressions of aggrecan and COL2A1. Conclusions: Autophagy was increased in osteoarthritic cartilage. In addition, the catabolic and nutritional stresses increased autophagy in normal chondrocytes. Furthermore the inhibition of autophagy caused OA-like gene expression changes while the induction of autophagy prevented. These observations suggested the increased autophagy was an adaptive response to protect cells from stresses and autophagy may play protective roles in chondrocytes. Further studies about autophagy in chondrocytes will provide novel insights into the pathophysiology of OA.

P86To establish a method for increasing cell seeding rate in PLGA scaffoldC. Hsieh, J. Tsai-Wu, H. Chiang, W. Lee, C. Jiang Taipei/Taiwan

Purpose: The application of three-dimensional scaffolds in tissue engineering has been shown to be important in cell differentiation induction and extracellular matrix production. Effective cell seeding and uniform cell distribution throughout the scaffold are the major issue for the success of three-dimensional culture. Our purpose is to establish a quick, convenient and efficient method to increase cell-seeding rate in the 3D sponge scaffold composed of poly DL-lactic-co-glycolic acid (PLGA).Methods and Materials: The mesenchymal stem cells (MSCs) from the green fluorescent protein (GFP) transgenic pigs were used for cell seeding. Four different methods, including the static surface seeding, orbital shaker seeding, vacuum seeding and modified vacuum seeding were examined. The seeding efficiency was compared by cell survival and distribution of GFP-MSCs after 3-day culture.Results: The distribution of GFP-MSCs was limited to the cell loading region by the static surface seeding method, and the seeding rate was very low. The seeding rate was greatly enhanced by using the orbital shaker seeding method; however, the cells were not uniformly distributed. By applying the vacuum seeding method, the cell distribution was more uniform and the seeding rate was as high as the orbital shaker seeding. The cell seeding rate and distribution were further improved by using the modified vacuum seeding method.Conclusions: Our results showed that the modified vaccum seeding method was the better method for 3D scaffold culture regardless the irregular structure of the scaffold. This method could be a useful tool for the cell-seeding to irregular scaffolds in the future study.

P87Tumor necrosis factor -α (TNF-α) antagonist inhibits matrix mettalloproteinase-3 (MMP-3) secretion induced by TNF-α in human articular chondrocytes in vitroS. Abe, H. Nochi, T. Ruike, T. Matsuno Asahikawa/Japan

Purpose: Mouse monoclonal anti-TNF-α antibody, infliximab, has been used to control inflammation and inhibit bone and joint destruction in rheumatoid arthritis (RA) We explore the effects of TNFα-antagonist, infliximab, on human articular chondrocytes (ACs) in vitro. Methods and Materials: 1. ACs were isolated from Osteoarthritis (OA) and RA knee joints under the informed consent. ACs were cultured in DMEM with 10% human serum and they were used at the passage 1 or 2. 2. To characterize cultured ACs, flow cytometric analysis was performed. 3. To measure cell proliferation, [3H] thymidine was added at the following culture condition. (1) ACs were cultured with several concentration infliximab (Remicade®, Centcor Inc.) for 4 days. (2) ACs were treated with 20 ng/ml of TNF-α and infliximab for 4 days. (3) ACs were treated with 20 ng/ml of TNF-α and infliximab were added 24 hours later, then they were cultured for 4 days. 4. The concentration of MMP-3 in conditioned media at the above assay was determined using ELISA kits. Results: 1. CD34, CD45, HLA-DR/DP/DQ, CD80 and CD86 were negative on ACs. HLA-ABC, CD73, CD90, CD105 and CD166 were positive on ACs. These patterns on surface makers in ACs derived from OA and RA were same. 2. The proliferation of ACs was not affected by infliximab. Then, the proliferation of ACs treated by TNF-α 20 ng/ml was neither affected by infliximab statistically. 3. MMP-3 secretion from ACs was not affected by treating infliximab. On the other hand, MMP-3 secretion was shown an increase in ACs treated by TNF-α, and infliximab inhibited this effect dose dependently. Conclusions: In this reports, we demonstrated that infliximab did not affect ACs proliferation in vitro, but infliximab inhibited MMP-3 secretion induced by TNF-α in ACs dose dependently in vitro. In present study demonstrate that infliximab could inhibit MMP-3 secretion and inhibit cartilage degeneration under inflammatory conditions.

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Methods and Materials: Cartilage was harvested by arthroscopic knee surgery and cultivated in 3 stages. After in vitro expansion of the chondrocytes, which takes place in stages P2 (5 flasks of 75cm²) and P3 (10 flasks of 150cm²), the final number of cultivated cells was harvested for implantation. Collected data of the mean cell growth factor was correlated to age and gender, and analyzed with PASW v18. Results: The gender distribution of 180 patients was 88 female and 92 male. Age ranged from 18 to 63 (mean 41). Duration of the culturing stages ranged from 6 to 20 days for P2 (mean 10) and from 13 to 28 days for P3 (mean 19). Mean harvesting resulted in 10,6x106 cells at the end of P2 (range 4.2-21, SD 2.9) and 42.5x106 cells at the end of P3 (range 16.6-87, SD 11.8). The average growth factor per day was 1.151 in P2 (SD 0.069) and 1.076 in P3 (SD 0.026). No differences were seen with the independent samples test in respect to the genders, both in P2 and P3. Comparing the ages over and under 45 years revealed no statistical significance.Conclusions: The culturing technique for chondrocytes is suitable for a wide range of patients. The ability to grow the required cells in vitro is independent of gender or age up to 60-65 years. No adaptations of the current cultivating process is required.

P93Identification and selection of human hyaline chondrocytes with appropriate levels of differentiation and cell viability for clinical useI. Garzón, V. Carriel, A. Morales, M.S. Alaminos, M. Lobo, M. Alaminos, A. CamposGranada/Spain

Purpose: Recent reports demonstrated a lack of scientific evidence supporting the effectiveness of autologous chondrocyte implantation. This could be due to low cell viability or differentiation levels of the chondrocyte cultures that are implanted in the patient. In this work, we have performed a study of seven successive cell passages of human hyaline chondrocytes to determine the expression levels of several genes associated to cell death and chondral differentiation using expression microarrays. Methods and Materials: Human hyaline chondrocytes were isolated from the knee joint of healthy donors using collagenase II digestion, and 7 cell passages were done. Total RNA was extracted from each cell passage and the RNA expression level was quantified by using Affymetrix Human-Genome U133 plus 2.0 arrays. Expression of the gene encoding for caspase 2 and two extracellular matrix-related genes was analyzed and compared among all the cell passages analyzed here. Results: Our results showed that CASP2 levels decreased until the 2nd cell passage and then progressively increased until the 7th passage (Figure 1). In contrast, the extracellular matrix-related genes matrilin 1 (MATN1, cartilage matrix protein) tended to increase up to the 3rd passage, and decreased thereafter, with a correlation with CASP2 of r=-0.7737. Similarly, the extracellular matrix gene CHST3, encoding for carbohydrate-chondroitin 6-sulfotransferase 3, showed a sequential increase until the 5th passage and then tended to progressively decrease until the 7th passage (r=-0.6987). Conclusions: The equilibrium between cell viability as determined by CASP2 gene expression and the extracellular matrix differentiation genes as determined by MATN1 and CHST3 genes expression should be considered for selection of the most appropriate chondrocyte cell population for clinical purposes. Since the highest levels of cell differentiation and the lowest levels of caspase expression are found in the 3rd passage, we suggest that this passage should be preferentially selected for therapeutic use. Supported by PAI CTS-115 (Tissue Engineering Group).

P94The influence of anaesthetics on in vitro cultured cartilage cells; a reflection of the effects while performing Autologous Chondrocyte Implantation (ACI) in the knee?C. Pronk-Admiraal1, B. Ellermeijer2, R. Benink2 1den helder/Netherlands, 2Den Helder/Netherlands

Purpose: To repair cartilage lesions the use of autologous chondrocytes is an upcoming technique. When implantating new grown cartilage cells back into a knee of the patient, Ropivacaine and Bupivacaine are sometimes used by wound infiltration. This study will show which influence these anaesthetics and the povidon-iodine used as a desinfectant, would have on the growth-rate or further development of cartilage cells, in case the wound is not properly closed and the anaesthetic comes into contact with the cells.

P90Validation of chondrocytes loading on type I/III collagen membrane for autologous chondrocyte implantationZ. Lin1, Q. Zheng1, J. Xu2, M.H. Zheng3 1Guangzhou/China, 2Perth/Australia, 3City Beach/Australia

Purpose: Matrix Induced Autologous Chondrocyte Implantation (MACI) relies on the seeding of in vitro expanded cell on type I/III collagen membrane (Bio-Gide®) as a cell deliver scaffold prior shipping for clinical implantation. It is always impossible to standardize the cell loading time on collagen membrane due to the variation in clinical and cellular manufacture requirement. We have observed that long term incubation of chondrocyte on collagen membrane did not favor the maintenance of the cellular phenotype and function. In this study, we investigated the gene expression profile and the time-dependent adhesion rate of the chondrocytes on type I/III collagen membrane, aiming to optimize the pre-surgical time of cell incubation on Bio-Gide®. Methods and Materials: Thirty chondrocyte-associated genes were measured on the chondrocytes loading on Bio-Gide® and compared to monolayer cultured cells by real-time PCR. Cell adhesion assay was performed to determine the adhesion rate of the cells on collagen membrane.Results: Real-time PCR revealed that human chondrocytes after seeded on Bio-Gide® for 5 days, the cells display less capability for the production of cartilage-specific matrix proteins, as demonstrated by the downregulation of aggrecan, col2a1, link protein and MMPs. In cell adhesion assay, 80% of the cell attached to the collagen membrane within 7 mins after loading, and the adhesion rate raised to 90% after 15 mins. At 40 mins, approximately 99% of the chondrocytes were attached to the membrane.Conclusions: The study concluded that the pre-surgical time of cell loading could be optimized to less than 40 mins without loss the majority of the cell population and proper cellular function.

P91Effect of the mechanical properties of the substrate on the expression of cartilage markers in cultured chondrocyteA. Zabalza-Baranguá1, P. Sanz-Ramos1, P. Ripalda Cemboráin1, C. Alcaine2, S. Santander2, I. Ochoa2, G. Mora1, I. Izal Azcárate1 1Pamplona/Spain, 2Zaragoza/Spain

Purpose: Mechanical properties of the substrate may modify the behaviour and shape of cells in culture. In chondrocytes it have been studied the effect of mechanical stress, but little is known about the capacity of cartilage cells to sense the stiffness of the substrate.Methods and Materials: Sheep chondrocytes were cultured at same density using standard plastic dishes (1 GPa) and Elastic Supported Surface dishes (28 kPa). Morphology, adherence, doubling rate and the expression of cartilage markers (aggrecan and collagens type I and II) were evaluated alter 3 days in culture. Finaly, the presence of focal adhesion points was studied by immunofluorescence.Results: Results did not show any difference according to the morphology and adherence of cells. The number of doublings suffered by cells was also similar in both type of plates. RT-PCR analysis of gene expression showed a significant decrease in the quantity of RNA for collagen type II and aggrecan, while a significant increase in type I collagen. Interestingly the addition of blebbistatin (inhibitor of Non Muscle Myosin II) to the culture prevents for this change, returning values obtained to those of plastic dishes. The number of focal adhesion points resulted different according to the stiffness of the substrate.Conclusions: Chondrocytes have the capacity to sense the stiffness of the substrate on which they are cultured, and to modify their phenotype accordingly. Mechanisms for the sensing include the action of NMMII.

P92Cell growth of chondrocytes is independent of age and gender.C. Pronk-Admiraal, B. Ellermeijer, R. Benink Den Helder/Netherlands

Purpose: Autologous chondrocytes implantation is a very effective technique for repairing cartilage lesions. The development of new cartilage is triggered by cells cultivated in vitro. For large surface defects, more cells are needed, which is especially important for older patients with more severe defects. We investigated whether in vitro cell growth depends on age or gender.

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enzymatically released. The cells were cultured in alginate beads, exposed to IL-1β, and treated with the unspecific COX inhibitors ibuprofene, paracetamol, and diclofenac. Proliferation, bFGF-secretion, rate of apoptosis, and mRNA-expression of chondrogenic markers were analyzed. Results: Exposure of chondrocytes to IL-1β for 14 days resulted in a 4fold increase of metabolic cell activity, and an amplification of DNA-content by 51% (p<0.05). The addition of ibuprofene induced a further enhancement by 20% and 24%, respectively (p<0.05). In contrast, metabolic activity of chondrocytes co-cultured with LPS-stimulated PBMCs was significantly reduced by 23% (p<0.05). Despite enhanced proliferation, rate of apoptotic cells increased following IL-1β exposure by 5%; this effect could significantly be reduced by COX-inhibition in 2D and 3D chondrocyte cultures (p<0.05). Correlating with augmented proliferation, bFGF concentrations measured in the culture supernatants treated with IL-1β increased 2.85fold at day 3, elevated levels were maintained up to day 14. Addition of COX-inhibitors caused a further induction of bFGF between 84% and 245% (p<0.05). Real time PCR showed the complete suppression of the chondrogenic marker collagen type II by IL-1β, expression of aggrecan was significantly reduced by 70% (p<0.05), this was not influenced by COX inhibition. Conclusions: IL-1β induces cell death of primary human articular chondrocytes, releasing activating intracellular cytokines as bFGF, which is associated with an enhanced cell metabolism. Whereas apoptosis may be reduced by COX inhibition, downregulation of specific cartilage markers remained unchanged.

P97Effect of mechanical stress or Interleukin-4 on cartilage-specific gene expressions of rat chondrocytes in 3-D scaffoldS. Shioji1, S. Imai1, K. Mori1, K. Ando1, K. Uenaka2, K. Nishizawa1, S. Araki1, Y. Takemura1, Y. Matsusue1 1Otsu/Japan, 2Otsu City/Japan

Purpose: Interleukin-4 (IL-4) has been suggested to protect the articular cartilage. Yet, it remains long unclear whether IL-4 acts on the chondrocytes. In turn, mechanical stress has been known to influence the chondrocyte function. The aim of this study was to investigate the role of IL-4 in chondrogenesis and to research whether IL-4 plays any role in regulating the molecular function of chondrocyte in response to the mechanical stress. The present study used the 3-dimentionally (3-D) embedded chondrocytes to examine the effect of mechanical stress or IL-4 on the expression of type â…¡ collagen (Col.2) and aggrecan (AGC) mRNA.Methods and Materials: Chondrocytes were isolated from rat articular cartilage. On reaching confluence, the cells were 3-D embedded in type I collagen scaffold. The cell-seeded scaffold was cultured either under mechanical stress (MS group) or with IL-4 (IL-4 group). The mechanical stress was a cyclic compression at 5% compression, 0.33Hz for 1hours, and the IL-4 concentrations were 10ng/ml. The 3-D embedded chondrocytes with neither mechanical stress nor IL-4 served as non-stressed (NS) cells. These groups aside, before applying the mechanical stress, IL-4 soluble receptor (sIL-4R) was added to medium at different concentration. Real-time PCR was performed for Col.2, AGC, and GAPDH at 1, 7, 13, and 25 hours after the application of the mechanical stress or IL-4.Results: Expression of AGC and Col.2 was significantly upregulated in MS group as well as in IL-4 group when compared with that of the NS group. The mechanical stress-induced upregulation of the matrix synthesis was attenuated when IL-4 inhibitor was applied.Conclusions: The present results show that IL-4 and MS influence the matrix synthesis of the 3-D embedded chondrocytes. In turn, the inhibition of the mechanical stress-related enhancement of matrix synthesis by the IL-4 inhibitor strongly suggests that the mechanical stress regulates the matrix synthesis via IL-4.

P9817β-estradiol inhibits the activation of volume-sensitive Cl- current by doxorubicin in isolated rabbit articular chondrocytesK. Kumagai, F. Toyoda, S. Imai, N. Okumura, E. Isoya, Y. Matsusue, H. Matsuura Otsu/Japan

Purpose: Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis. It has been suggested that activation of volume-sensitive Cl- current (ICl,vol) mediates cell shrinkage triggering apoptosis (apoptotic volume decrease: AVD) in several cell types. We examined in vitro effects of 17β-estradiol

Orthopaedists can adapt their methods, when performing ACI into the knee, to the most optimum with respect to the results of this study. Methods and Materials: Aside from the regular cultures, culture flasks have been used to grow cells added with Ropivacaine, Bupivacaine or Povidon-iodine. All these cells grow under the same circumstances as the regular culture. The cell growth-rate and cell aspect are evaluated every three days in all culture flasks. Results: At a high concentration (1.5 mg/ml Ropivacaine and 0.83 mg/ml Bupivacaine) the cells died immediately and lysated. At lower concentrations the cells remained alive but they duplicated very chaotically with respect to the blanco. These cells also formed very large cores.When Povidon-iodine was added, the cells could not get attached to the flask. If the Povidon-iodine was added after the cells had time to get attached, they had difficulty with duplicating and had a low growth-rate. (Table 1) Conclusions: In this study we show strong effects of anaesthetics and povidon-iodine on the in vitro growth-rate of cartilage cells. With respect to this results we would advise not to use any form of local anaesthetics or povidon-iodine, since its use can damage the cells and lower the growth rate. Further research will evaluate which effect will remain when no anaesthetics would be added after three days of culture.

P95Localization of the Optimal Site for Articular Cartilage Biopsy for Autologous Chondrocyte ImplantationM. Gibson, J. Elisseeff, M. Trice Baltimore/United States of America

Purpose: We hypothesized that the quality of cartilage repair tissue in autologous chondrocyte implantation (ACI) is dictated by the quality of the articular cartilage biopsy material and have proposed that the progenitor cells obtained at biopsy for ACI are major determinants of chondrogenic capacity of redifferentiated chondrocytes. In this study, we assess the qualitative features, anatomical location, and quantitative features of articular cartilage biopsy specimens to determine their relationship to postexpansion chondrogenic capacity.Methods and Materials: We completed histologic, biochemical, live-dead, and collagen-type analyses in 10 human articular cartilage biopsy specimens. Chondrocytes were expended in three-dimensional peg culture for four to five passages and then studied for cell viability with WST-I reagent. We assessed chondrogenic capacity by measuring DNA content through fluorophotometry, glycosaminoglycan (GAG) content by using a dimethylene blue spectrophotometric assay, and gene expression by using reverse transcription-polymerase chain reaction. We analyzed all data with the Microsoft Office Excel Statistical Package and used one-way single factor analysis of variance and post hoc Tukey tests. Significance was set at p < 0.05.Results: A statistically significant correlation (p = 0.001173) was found between initial GAG concentration in biopsy specimens and the chondrogenic capacity of redifferentiated chondrocytes. When the anatomic locations of biopsy site were compared, no difference was found in cell viability, metabolic activity, histology, biochemical content, or gene expression before or after in vitro expansion.Conclusions: Anatomic biopsy location was not a factor in chondrogenicity of redifferentiated chondrocytes. However, chondrocytes from locations with higher GAG concentrations reproducibly produced chondrocytes with higher chondrogenic capacity as measured by GAG production and collagen type-II markers in gene expression analysis. A newly proposed measure, GAG to cell ratio per unit weight of biopsy specimens, proved predictive of the same ratio in redifferentiated culture. These data have relevance in choosing optimal biopsy tissue for ACI.

P96The influence of COX inhibition on primary, human articular chondrocytes following exposure to IL-1betaH. Schmal, A.T. Mehlhorn, P. Niemeyer, B. Dirhold, N.P. Suedkamp Freiburg/Germany

Purpose: COX inhibiting drugs are applied in orthopedics as a standard analgetic medication for osteoarthritis and posttraumatic pain. The effects of COX inhibition on differentiation and metabolism of human articular chondrocytes following cartilage damage are not yet completely understood. Methods and Materials: Human femoral heads were obtained during hip arthroplasties following femoral neck fractures and chondrocytes

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effects of NSAIDs on OA treatment, we evaluated the effect of the selective cyclooxygenase-2 (COX2) inhibitors aceclofenac, celebrex and tiaprofenic acid, and non-selective COX inhibitor indomethacin on extracellular matrix of human chondrocytes in vitro.Methods and Materials: Human chondrocytes were isolated from surgical specimen of OA patients. The chondrocytes were cultured in the normal medium (as a control group) or in the presence of aceclofenac, celecoxib, tiaprofenic acid and indomethacin for 7 days to examine the change in cell proliferation. The components of extracellular matrix of chondrocytes such as aggrecan and collagens were measured after 3-day treatment. The expressions of type I, II, and X collagens and aggrecan were determined by quantitative RT-PCR. The amount of total glycosaminoglycan (GAG) was measured by 1,9-dimethylmethylene blue (DMMB) binding.Results: There is no significant difference in the cell proliferation rates in the presence or absence of NSAIDs for 7 days. The expressions of type I, II, and X collagens are reduced in the presence of the tested NSAIDs. However, the expression of aggrecan are significantly increased in the presence of aceclofenac, celecoxib and tiaprofenic acid. In addition, aceclofenac significantly increased total GAG production compared to those of celecoxib, indomethacin, tiaprofenic acid and control.Conclusions: These results suggest that NSAIDs could affect the expression and production of the extracellular matrix of human chondrocytes, including increasing glycosaminoglycan and aggrecan, and decreasing collagens. Aceclofenac has the most prominent effect in increasing total GAG production. The effects of these changes in extracellular matrix components on physical property of chondrocytes need further evaluation.

P102TGFβ-1 administration on serum free-expanded chondrocyte enhances the expression of osteogenic markers and induces SMADs transcript regulation: in-vitro and in-vivo studies.R. Narcisi, R. Quarto, P. Giannoni Genova/Italy

Purpose: Culturing articular chondrocytes is required to perform cartilage resurfacing in tissue engineering-based approaches the use of serum-free medium (SF) can opportunely mimic the physiological (avascular) autocrine environment. Previously data showed that TGFdministration during the expansion phase induces loss of matrix components, positive immunodetection for type-X Collagen and apoptosis once in 3D culture system and, after osteogenic induction, TGF-expanded cells strongly mineralized with respect to SF-expanded cells. Thus we evaluated if TGF&beta-1 administration influence the expression of osteogenic markers and SMADs, signalling proteins involved in the chondrogenic development and differentiation. Moreover we evaluated the behaviour of TGF-expanded cells in an in-vivo ectopic model. Methods and Materials: Human articular chondrocytes were expanded in SF, with or without TGFβ-1. Subsequently cells were collected and seeded statically in hydroxyapatite (HA) scaffold and after 48h in osteogenic induction medium, implanted subcutaneously in nude mice for the following four weeks. Decalcified sections were assessed by mean of haematoxylin-eosin immunocitostaining. Microarray and Real time-PCR analysis were performed after the expansion phase. Results: TGF-expanded cells displaying an increased transcripts levels for Osteopontin (OP; 4.76±2.24, [mean±SD]), Bone Sialoprotein (BSP; 2.76±0.03) and Collagenase-3 (MMP13; 6.25±3.18), while Osteocalcin (OC) wasn’t influenced by TGFβ-1 (1.02±0.28 fold-increase). Moreover, microarray analysis showed that TGFβ-1 administration influenced the SMADs expression. Qualitative-PCR analysis confirmed that treated cells displayed increased level of SMAD-1 mRNA (1,77±0.26) and down-regulated SMAD-3 transcript (0.35±0.28). Interestingly, after four weeks in-vivo TGF-chondrocyte/HA implant showed nascent deposition of bone matrix and numerous blood-vessels. Conclusions: TGFβ-1 directs chondrocytes to acquire a different phenotype, firstly regulating the expression of SMADs signalling pathways. These cells showed high level of OP, BSP and MMP-13 mRNA indicating the predisposition to undertake the endochondral ossification-like progression and in-vivo analysis seem to validate this hypothesis.

on the doxorubicin-induced activation of ICl,vol in rabbit articular chondrocytes using whole-cell patch-clamp technique. Methods and Materials: Rabbit cartilages were collected from joints of male animals weighing 2.5 to 3.0 kg. The cartilage was dissected into slices and cultured in DMEM for 1-3 days. On the day of experiments, chondrocytes were isolated by enzymatic digestion. Whole-cell membrane current was recorded under conditions where Na+, K+ and Ca2+ currents were minimized. Real-time change in cell size was monitored using a CCD digital camera and the cross-sectional area of cell image was measured. Results: Exposure of isolated chondrocytes to doxorubicin (1µM) resulted in an obvious increase in the membrane Cl- conductance without any appreciable change in cell size. The doxorubicin-evoked Cl- current exhibited many properties almost identical with ICl,vol phenotype, including outward rectification, prominent inactivation at large positive potential, inhibition by hyperosmotic cell shrinkage, and sensitivity to ICl,vol blockers, arachidonic acid or DCPIB . Pretreatment of cells with 17β-estradiol inhibited the ICl,vol activation by doxorubicin as well as subsequent apoptotic events such as AVD and elevation of caspase 3 activity. It was unlikely that 17β-estradiol produced a direct action on ICl,vol, because it had little effect on ICl,vol activated by hyposmotic cell swelling. On the other hand, the effect of 17β-estradiol was significantly attenuated by an estrogen receptor blocker.Conclusions: These results suggested that 17β-estradiol may prevent the doxorubicin-induced apoptosis by interfering the activation of ICl,vol in rabbit articular chondrocytes.

P100Chondrocyte micro-aggregates enhances neo-cartilage formationL. Moreira Teixeira1, J. Leijten1, J. Sobral2, R. Jin1, P. Dijkstra1, J. Feijen1, C. van Blitterswijk1, M. Karperien1 1Enschede/Netherlands, 2Cambridge/United Kingdom

Purpose: We recently developed a novel injectable in situ crosslinkable Dextran-Tyramine 14kDa-DS=15 (Dex-TA) hydrogel, which has shown high potential for cartilage regeneration. However, cartilage repair strategies based on autologous chondrocyte implantation still rely on in vitro expansion to obtain sufficient cells with all inherent drawbacks such as dedifferentiation. Cartilage repair using mixtures of a limited number of chondrocytes with Mesenchymal Stem Cells (MSCs) can potentially overcome this hurdle. It remains unclear whether the hydrogels should be seeded with single-cell suspension or with preformed micro-aggregates of defined size. To test, this we have seeded hydrogels with microaggregates of chondrocytes, MSCs or a mixture of both and compared their performance with single-cell seeded hydrogels.Methods and Materials: High throughput formation of micro-aggregates of 50, 100 and 200 cells was achieved in micromolds. Micro-aggregates were prepared of chondrocytes, MSCs or a mixture of both. Morphology, stability and chondrogenic capacity was evaluated. Aggregates with the optimal cell density of 100 cells were incorporated into Dex-TA hydrogels, cultured in vitro and in vivo and compared to single-cell seeded hydrogels.Results: Micro-aggregates were formed in a very controlled manner and successfully incorporated into Dex-TA hydrogels. Aggregates formed by 100 cells showed a superior balance between stability and gene expression profile, with higher collagen type2 and Aggrecan expression. After incorporation of micro-aggregates into Dex-TA, long term stability and survival was observed, as well as enhanced matrix production, when compared to single-cell seeded hydrogels. More cartilage was formed in micro-aggregates consisting of a 50%/50% mixture of chondrocytes and MSCs, compared to both micro-aggregates of pure cell populations and to single-cell mixtures.Conclusions: We conclude that neocartilage formation is greatly improved by seeding hydrogels with micro-aggregates instead of single-cell suspensions. In addition, this system provided preliminary information about the effect of micro-cocultures, showing enhancement of neo-cartilage formation when using 50% chondrocytes mixed with 50% MSCs.

P101The effects of non-steroidal anti-inflammatory drugs on chondrocytesC. Hsieh, J. Tsai-Wu, S. chen, H. Chiang, C. Jiang Taipei/Taiwan

Purpose: Osteoarthritis (OA) is a painful disease with degenerating cartilage matrix components. For OA treatment, non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequently prescribed medications to relieve pain and reduce inflammation. The effects of NSAIDs on cartilage matrix remain controversial. To elucidate the

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P105Tissue engineering cartilage tissue using elderly human articular chondrocytesX. Zhao1, D.A. Bichara1, F. Ballyns2, M.A. Randolph1, L. Bonassar2, T.J. Gill1 1Boston/United States of America, 2Ithaca/United States of America

Purpose: There are many unknown aspects using human chondrocytes from middle aged or elderly patients for neo-cartilage formation and lesion repair. We hypothesized that human articular cartilage can be engineered using human chondrocytes from these patient populations that would be similar morphologically, biochemically and biomechanically to juvenile animal neo-cartilage. Methods and Materials: Articular chondrocytes from elder healthy human donors (age range: 50-60 years) and young swine (~3 months) were isolated and expanded in culture. Human and swine Cells (40-60 x 106 cells/ml) were encapsulated in fibrin gel nodules (n=30) to determine engineered cartilage formation. To evaluate cartilage integration, 100ul chondrocyte/fibrin hydrogel mixture was sandwiched between two 6mm discs of native cartilage (n=36). All constructs were implanted into nude mice subcutaneously for 12, 18, and 24 weeks. The specimens were evaluated and compared histologically, immunohistochemically, biochemically and biomechanically. Results: Similar to the swine cell constructs, the nodules with human cells showed continuous neo-cartilage matrix formation as evidenced in specimens stained with H&E, Safranin-O, Toluidine Blue, and immunohistochemical stain (type II collagen). Also neo-cartilage formed between cartilage discs in the sandwich model with human articular disks and human chondrocytes showed tight bonding and equivalent integration strength (stress was 90±26 kPa) with existing matrix. Biochemical and biomechanical data showed a similar performance between human and swine chodrocytes in engineering cartilage tissue. Conclusions: These results demonstrate that chondrogenesis and integration using articular chondrocytes from older aged people can be achieved in a predictable and reliable manner similar to that from juvenile animals. Since most patients with articular cartilage lesions are quite often middle aged or older, these results move us closer to possible clinical application.

P106Inhibition of TGF-ß prevents dedifferentiation in monolayer cultured sheep chondrocytesP. Ripalda Cemboráin, A. Zabalza-Baranguá, M. Vicente-Pascual, J. Dotor, I. Monreal, F. Borrás-Cuesta, G. Mora, I. Izal Azcárate Pamplona/Spain

Purpose: Expansion of chondrocytes in vitro can yield a great number of cells but with a depleted capacity of chondrogenesis. TGF-beta is a strong inductor of chondrocyte phenotype, but its role in cartilage is not clear. In this work we analyzed the effect of two peptides (P17 and P144) with capacity to inhibit the activity of TGF-beta in cartilage cells.Methods and Materials: Sheep chondrocytes were cultured in the presence of peptides for up to 4 passages. Apoptosis and proliferation rate were assayed after a 24 hours treatment, while RT-PCR for cartilage markers were assayed at passages 2 and 4. Chondrogenic capacity was determined by placing cells in a 3 dimensional environment for 7 additional days. Pellets formed were analyzed by immunofluorescence and collagen II content determined by ELISA.Results: Results show a significant reduction of the proliferation rate exerted by P144 but not by P17. None of them results toxic. Only P144 is able to reduce the TGF-beta induced phosphorilation of SMAD2, and a better expression of cartilage markers during the first passages. P144 increased the size of the micromass structure used for the analysis of chondrogenesis, as well as the type II collagen content measured by ELISA. The histological characterization of the pellets showed an increase in the proportion of type II collagen after the expansion with P17 and an increase in the detection of type II collagen and aggrecan after the expansion with P144.Conclusions: Inhibition of TGF-beta using peptides P17 and P144 in the monolayer culture preserves the chondrogenic capacity of expanded chondrocytes.

P103Salvage of Contaminated Osteochondral Allografts: The Effects of Chlorhexidine on Human Articular Chondrocyte ViabilityJ. Campbell1, G. Filardo2, S. Bajaj1, N.A. Friel1, A.A. Hakimiyan1, R. Grumet3, S. Chubinskaya1, B.J. Cole1 1Chicago/United States of America, 2Bologna/Italy, 3Orange/United States of America

Purpose: Osteochondral allograft (OA) transplantation procedures carry the risk of graft contamination. Because chondrocyte viability is imperative for successful transplantation, graft sterilization techniques must provide antimicrobial effects with minimal to no cartilage toxicity. Chlorhexidine is an effective disinfectant, but its use with human articular cartilage requires further investigation. The purpose of this study was to determine the maximal chlorhexidine concentration that does not affect chondrocyte viability in allografts. Methods and Materials: 6mm osteochondral plugs were harvested using OATS technique (Arthrex,Inc) from five femoral condyles obtained from AlloSource. Plugs were subjected to pulse lavage using 1L solutions of 0.002%, 0.01%, 0.05%, and 0.25% chlorhexidine gluconate (CHG) preceded and followed by 1L saline pulse lavage. Treated plugs were cultured for 0,1,2 and 7 days in media containing 10% fetal-bovine-serum and antibiotics. Two controls, an osteochondral plug immediately cultured after harvest and a plug subjected to saline pulse lavage before culture, were included. Chondrocyte viability was determined using LIVE/DEAD Assay.Results: Both controls and 0.002% CHG group showed similar cell viability ranging from 67±4% to 81±22% at all time points. In 0.01% CHG group, cell viability was reduced in comparison to control by 2-fold at Day 2 and remained at this level till Day 7 (p<0.01). 0.05% and 0.25% CHG groups showed a 2-fold reduction in cell viability already at Day 1 (p<0.01). At Day 7, cell viability was reduced to 15±18% (4-fold) for the 0.05% CHG group and 10±19% (6-fold) for the 0.25% CHG group, (p<0.01). Conclusions: 0.002% CHG pulse lavage does not cause significant cell death within 7 days following exposure, while CHG at concentrations greater than 0.002% significantly decreases chondrocyte viability within 1-2 days after exposure and therefore, should not be used for disinfection of OA. The antimicrobial properties of 0.002% CHG and its effect on OA metabolism remain to be investigated.

P104Cartilage damage treated with Chondron™ methodR. Benink, C. Pronk-Admiraal Den Helder/Netherlands

Purpose: Several treatment methods are described for post-traumatic and chronic degenerative cartilage lesions of the knee, for example arthroscopic debridement, microfracturing, Becks drilling, osteochondral transplantation (OATS) and 1st generation autologous chondrocyte implantation (ACI) and 2nd generation MACI. For the short term results and safety of the Chondron™ (3rd generation cultivated chondrocyte-gel technology) method a prospective cohort study was evaluated by ICRS criteria and KOOS scoring lists were used.Methods and Materials: Data from the first 160 consecutive patients were prospectively collected. Among them were 45 patients with a solitary medial condyle lesion(17 men, 28 women). Average age was 41 years (range: 18-60, SD ± 10), mean BMI was 26 (range: 20-37) and the average size of the chondral defect of the medial condyle was 6.5 cm2 (range: 2-16cm2; SD ± 3.5). Average number of cells per vial cultivated was 13.213 x 106 cells. Patients are under supervision of a physiotherapist for 6 to 12 months. Data was analyzed with PASW v18.Results: Clinical evaluations using the IKDC and KOOS score were processed at preoperative, 3, 6 and 12 months. Table 1 and graphic 1 shows the mean scores of the patients. The Wilcoxon signed ranks test showed statistical significant improvement at 3, 6 and 12 months. *= statistically significant p <0.05.Conclusions: These first results of the Chondron™ method for the treatment of articular cartilage defects of the knee are promising. The medial condyle lesions showed a significant improvement at 1 year, although we realize that this is only short term results.

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P109TGF-beta protects OA chondrocyte mitochondria from experimentally induced oxidative stress.V. Grishko1, A.W. Pearsall, IV2, G.L. Wilson2 136688/United States of America, 2Mobile/United States of America

Purpose: TGF- beta is a powerful anabolic factor for chondrocytes and plays an important role by enhancing cartilage repair. Oxidative stress and mitochondrial damage have been found to promote cell death, functional failure and degeneration. There is a growing body of evidence that mitochondrial dysfunction is present during the progression of osteoarthritis. The purpose of the present study was to evaluate the mitoprotective potential of TGF-beta for human OA chondrocytes in the conditions of oxidative stress. Methods and Materials: Primary OA chondrocytes cultures, generated from cartilage from patients undergoing total knee replacement, were exposed for 30 min to reactive oxygen species (ROS) generators xanthine oxidase/ hypoxanthine or peroxynitrite. Some cells were pre-incubated with 5 ng/ml of TGF-beta for 24 h prior to treatment. In some experiments cells were incubated for 48 h with IL-1 beta or TNF-alpha alone or in combination with TGF-beta. Following exposure, cells were immediately lysed for DNA or protein isolation or ATP analysis or replenished with normal growth media and left for recovery and then analyzed. Mitochondrial dysfunction was assessed in terms of mitochondrial DNA damage (Quantitative Southern blot analysis), ATP content (Bioluminescence kit), mitochondrial proteins levels (Western blot). Cell viability and apoptosis were evaluated by flow cytometry. Results: When OA chondrocytes were exposed to ROS generators, mitochondrial dysfunction and apoptosis were accumulated as a consequence. Pretreatment with TGF- beta preserved mitochondrial DNA integrity and ATP levels, restored drop in mitochondrial protein levels. Moreover, TGF-beta enhanced chondrocytes viability and diminished the appearance of apoptosis. The similar results were obtained for IL-1 beta and TNF-alpha. Conclusions: The present results demonstrate for the first time the mitoprotective properties of TGF-beta and offer some explanation of the mechanisms through which TGF-beta enhances cartilage repair. More studies required evaluating the precise mechanisms of observed effects.

P111The effects of osteogenic protein (OP)-1 on microfracture-treated cartilage defects in a goat modelL. Jeng1, H. Hsu2, R.F. Padera2, M. Spector2 1Cambridge/United States of America, 2Boston/United States of America

Purpose: The benefits of osteogenic protein (OP)-1 in promoting chondrocyte metabolism in vitro commend it for cartilage repair. The objective was to evaluate the effects of OP-1 on the amount and composition of the reparative tissue in microfracture-treated defects in a goat model.Methods and Materials: Two 4-mm chondral defects were created in the trochlear groove of right knees and treated with microfracture, and immobilized for 8 days. Treated knees (Group II) were injected with 1 mg of OP-1 in 1 ml of vehicle 3 times: at defect creation, and at 8 and 20-21 days, post-op. Control knees received the vehicle alone (Group I). After sacrifice, the joints were radiographed. Formalin-fixed, paraffin-embedded sections were evaluated histomorphometrically for the percentage fill and types of tissues filling the defect.Results: Radiographs and histology revealed the presence of ossicles in the synovial capsules of three of six 16-week Group II goats as well as in one of four 16-week Group I animals (no significant difference by Fisher’s Exact test). Histomorphometric analysis (Table 1) indicated that the percentage of the defect site that was filled with reparative tissue was much higher for the OP-1 treated group (62 ± 17%, mean ± SEM) than for the control group (19 ± 5%) 4-5 weeks postoperatively. However, by the 16-week timepoint, the total fill was similar between the 2 groups (75 ± 6% for Group I and 80 ± 10% for Group II). Two-factor ANOVA of the 16-week defects indicated that OP-1 treatment had no significant effect on the percentages of hyaline cartilage, fibrocartilage, fibrous tissue, bone ingrowth, or total fill. Conclusions: This study suggested that high dose treatment with OP-1 may be beneficial during early stages of cartilage repair but found no significant advantage of the OP-1 treatment on microfracture-treated cartilage defects in a caprine model by the 16-week timepoint.

P107Identification of hyaline and fibrous chondrocytes using a novel genetic markerA. Campos, I. Garzón, J. Garrido, V. Carriel, C. Martínez, M..S. Alaminos, M. AlaminosGranada/Spain

Purpose: Autologous chondrocyte cultures are currently used for the treatment of several conditions affecting the human cartilage. It is well known that chondrocytes kept in culture for long periods of time may tend to lose differentiation capabilities, and no specific chondrocyte markers have been described to the date. In this work, we have evaluated primary cultures of hyaline and fibrous human chondrocytes using genome-wide microarrays to identify the gene expression of a novel chondrocyte marker in both cell types. Methods and Materials: Primary cell cultures of human hyaline chondrocytes were obtained from small biopsies of the knee joint using collagenase II digestion and of human fibroblasts from oral mucosa biopsies. Then, RNA was extracted from the cell cultures and the RNA expression of the gene SUSD2 (sushi domain-containing 2) was quantified by using Affymetrix Human-Genome U133 plus 2.0 arrays. Results: Our analysis revealed that the gene SUSD2 was highly expressed by both hyaline and fibrous human chondrocytes cultures, but not by other cells of mesenchymal origin (p<0.001 for both comparisons) (Figure 1). In addition, the expression of this gene was significantly higher in hyaline chondrocytes than in fibrous chondrocytes (p<0.001). Conclusions: These results suggest that expression of the gene SUSD2 is highly specific of human chondrocytes and could be used as a novel marker of cartilage cells. Additionally, this marker allows for an efficient ex vivo distinction of hyaline versus fibrous chondrocytes to be used in cell and tissue engineering protocols. Supported by G.I. PAI CTS-115 (Tissue Engineering Group).

P108Age-related change of collagen in patellar cartilage of normal rabbits: FTIR and histologyH. Kuroki, M. Kobayashi-Miura, K. Tsuchimoto, A. Ito, H. Inoue, M. Kobayashi, K. Nishitani, T. Shirai, T. Satake, Y. Nakagawa, T. Nakamura Kyoto/Japan

Purpose: Fourier transform infrared spectrometer (FTIR) microscope has been used for a novel tool to assess cartilage matrix content. The purpose of this study is to determine whether FTIR could evaluate age-related change of patellar cartilage from surface to deep layer, especially more detailed distribution of collagen in combination with histological information.Methods and Materials: Five groups of rabbits of various ages (3-week, 8-week, 6-month, 1-year, 2.5-year) consisting of three rabbits per group were examined. Patellar cartilage samples were sectioned and analyzed by FTIR. Collagen (amide-I peak; 1710-1590 cm-1) was evaluated with collagen II immunostaining. The amide-I peak was measured with five depths of cartilage, from surface to 100-, 200-, 300-, 400-, 500-micrometer, respectively.Results: Mean intensity of amide-I peak in the depth from surface to 100-micrometer of 3-week, 8-week, 6-month, 1-year and 2.5-year samples were 27.5, 45.1, 42.1, 47.0 and 49.5 (arbitrary unit), respectively. Those were 31.3, 43.0, 40.9, 46.4 and 45.8, respectively, in the depth from surface to 200-micrometer; 33.6, 43.4, 41.2, 44.1 and 42.6, respectively, in the depth to 300-micrometer; 33.4, 43,4, 40.9, 42.5 and 41.5, respectively, in the depth to 400-micrometer; and were 30.9, 43.8, 41.4, 42.0 and 41.0, respectively, in the depth to 500-micrometer. Amide-I peak distribution was consistent with the localization of collagen-II.Conclusions: Mean intensity of amide-I peak in the 3-week specimens differed from those of the others. We suppose that, combining FTIR with collagen II immunostaining, it is feasible to determine the age-related change of collagen of patellar cartilage from surface to deep layer.

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P114Osteocyte-derived HB-GAM is associated with bone formation and mechanical loadingY. Takemura, S. Imai, Y. Matsusue Otsu/Japan

Purpose: HB-GAM is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro.Methods and Materials: We used the mice lacking HB-GAM in C57BL/6 background and the C57BL/6 mice for control. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and µ-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. Results: The skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed between 3weeks and 2 months of age. This suggests a link to physical activity. Adult HB-GAM knockout mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo. Osteocytic HB-GAM expression was up regulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro.Conclusions: Our results suggest that HB-GAM could be involved in mediating the osteogenic effects of mechanical loading on bone.

P115Effect of intraarticular pH change on structure and metabolism of cartilage tissueS. Ergün, B. Kocaoğlu, U. Akgün, R. Nuran, O. Başçı, M. Karahan Istanbul/Turkey

Purpose: Hyalin cartilage is an avascular tissue and it is only nourished from synovial fluid by diffusion. Decrease in pH of synovial fluid in the presence of osteoarthritis and trauma has been reported in previous investigations. We hereby aimed to show the negative effects of low synovial fluid pH on cartilage metabolism and synthesis of extracellular matrix components by Real Time Polymerase Chain Reaction method.Methods and Materials: Cartilage tissue obtained from bovine femoral condyles were incubated in 3 different medias; acidic (pH 7.2), physiologic (pH 7.4) and basic (pH 7.6), for four days. At the end of incubation, mRNA isolation and quantitative gene expression analysis were done. Type 2 collagen, aggrecan and hypoxia inducible factor 1 alpha were the genes investigated. Normalizing was done with beta actin housekeeping gene by 2-ddCt method.Results: According to results, expression levels of all genes in physiologic pH media was higher compared to acidic and basic media. Gene expression quantity of type 2 collagen in acidic media was 0.739, in basic media 0.755 when compared to physiologic pH media gene expression quantity which is 1. This ratio was 0.615 in acidic and 0.681 in basic media of gene aggrecan and 0.695 in acidic and 0.652 in basic of gene Hif 1 alpha.Conclusions: Conclusion, decrease in synovial fluid pH as a consequence of osteoarthritis, trauma or intraarticular hyaluronic acid injection, negatively effects chondrocyte metabolism and production of extracellular matrix components.

P116Recombinant human fibroblast growth factor-18 combined with a biphasic collagen/GAG scaffold produces superior articular cartilage compared with BMP-7 in an ovine osteochondral defect modelA. Getgood1, F.M. Henson1, R. Brooks1, A.K. Lynn1, H. Guehring2, N. Rushton1 1Cambridge/United Kingdom, 2Darmstadt/Germany

Purpose: The aim of this study was to investigate the effect of combining rhFGF18 with a biphasic collagen/GAG osteochondral scaffold (Chondromimetic), on the treatment of osteochondral defects in sheep.

P112IL-1ra solution blocks IL-1β and TNFα-induced MMP-13 production from human articular chondrocytesJ. Woodell-May, A. Matuska, J. Hoeppner Warsaw/United States of America

Purpose: Inflammatory cytokines such as IL-1β and TNFα can induce chondrocytes to produce matrix metalloproteinases (MMPs) which are responsible for degradation of cartilage matrix (1). Autologous protein solution (APS) rich in IL-1ra can be prepared in less than 30 minutes from blood collected during a draw (2). The purpose of this study is to determine if incubation with APS, derived from human blood and rich in IL-1ra, can decrease the production of MMP-13 from IL-1β and TNFα stimulated chondrocytes. Methods and Materials: APS was prepared from 10 consented human donors. Human knee articular chondrocytes (NHAC, Lonza Inc.) were seeded in 12 well plates. For the positive controls and treatments, 5ng/ml rhIL-1β (Sigma) and 100 ng/ml rhTNFα (Prospec) were added to the wells. APS, rhIL-1ra, or rhsTNF-RI were added 2 hours prior to the addition of IL-1β and TNFα. Wells without IL-1β, TNFα, or APS were left as negative controls. After 24 hours, the supernatant was removed and frozen at -50° C. The supernatant was assayed for MMP-13 by ELISA (R&D Systems). Results: Chondrocytes stimulated with TNFα, IL-1β, and the combination of TNFα and IL-1β, produced 104, 403, and 565 fold more MMP-13, respectively, than negative control chondrocytes. Recombinant IL-1ra and sTNF-RI blocked MMP-13 production in stimulated cells. Treatment with APS reduced MMP-13 production of the respective samples by 90.9±9.7%, 92.9±3.3%, and 89.7±4.4%. Conclusions: Articular chondrocytes dosed with known inflammatory cytokines and treated with APS demonstrated a reduction in MMP-13 production when compared to untreated samples. The results emphasize the role of IL-1β and TNFα in the breakdown of cartilage matrix seen in osteoarthritis and warrant further studies to investigate a potential treatment for patients suffering from OA. 1.) Goldring MB et al., Arthritus Rheum, 2000; 43(9); 1916-26. 2.) Vangsness, T. et al., ORS, 2008.

P113Gene profiles of the regenerated cartilage tissue induced by implantation of a novel double-network hydrogelR. Imabuchi, H.J. Kwon, N. Kitamura, T. Kurokawa, J.P. Gong, Y. Ohmiya, K. Yasuda2 Sapporo/Japan

Purpose: We have developed an innovative method to induce spontaneous hyaline cartilage regeneration in vivo by implanting a double-network (DN) hydrogel composed of poly-(2-Acrylamido-2-methylpropanesulfonic acid) and poly-(N,N´-Dimetyl acrylamide). The purpose of this study is to investigate the gene expression profiles in the regenerated cartilage tissue induced by this DN gel in comparison with normal cartilage. Methods and Materials: Ten mature Japanese white rabbits were used. An osteochondral defect having a 4.5-mm diameter was created in the femoral groove. A cylindrical DN gel plug was implanted into the defect having 2-mm depth remained after surgery. Five rabbits were sacrificed at 2 (Group-2W) and 4 (Group-4W) weeks after surgery, respectively. Regenerated cartilage in the defect were collected and mRNAs were isolated. The gene expression profiles were analyzed with use of custom-made DNA microarray. The normal knee cartilage of the same age of Group-2W and -4W was used as control.Results: The number of the probe sets with an expression ratio greater than 2, 5, 10 between 2W and control was 2,423, 452, and 174, respectively and that between 4W and control was 2,122, 391, and 164, respectively. We defined a probe with the ratio greater than 5 as differentially expressed gene for further analysis; with respected to cartilage-related genes, Col2, Col5, Col10, Agc1, Crtl1, Dcn, Comp, Prg4, Cilp, Ctgf, Pthr1 were upregulated in both 2W and 4W. The functional classification of the differentially expressed gene was also performed. A similar distribution of categories was observed in 2W and 4W when comparing to control (Fig)..Conclusions: We first reported the gene expression profiles of the regenerated cartilage tissue using a rabbit model. We conclude that the gene expression profiles of the regenerated cartilage tissue in both 2W and 4W were different from control and that of 2W and 4W were similar to each other.

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and healthy cartilage –CTR-). At the same time, they were subdivided in two subgroups based on the time of study (5 postsurgical weeks -16s- and 8 postsurgical weeks -19s-). The PRGF and PCB rabbits were subjected to a complete thickness chondral defect in the medial femoral condyle of both knees. After this, the therapeutic infiltrations were begun according to the protocol established for each group. The presence of type II collagen in the reparative tissues was examined immunohistochemycally using a mouse monoclonal antibody against human type II collagen (Fuji Chemical, Takaoda, Japan) that specifically crossreact with rabbit type II collagen. The sections were counterstained with hematoxylin. The repaired areas were microscopically assessed according to a semi-quantitative scale. Statistical significance among groups was estimated using a Kruskall-Wallis Test, and a Mann-Whitney’s U test for the difference between times. Results: The statistical study showed no significant differences, either among groups or between the stages of evolution, for the collagen values.Conclusions: The application of plasma rich in growth factors has not produced significant differences in the synthesis of collagen type II in the articular cartilage tissue repair, comparing with the healthy cartilage and its physiological repair.

P121Repeated sub-threshold dosing of TGF-β1 is ineffective in stimulating human bone marrow cells: implications for cartilage repair strategiesC. Chu, V. Yao, K.A. Payne Pittsburgh/United States of America

Purpose: Since the articular cartilage has a limited capability to heal, improving cartilage repair is an important strategy to reducing pain and disability. Transforming growth factor-beta 1 (TGF-β1) induces chondrogenesis of human bone marrow cells (hBMC), an important cell source for cartilage repair. This study tests the hypothesis that (1) TGF-β1 signaling can be induced and maintained by either single or repeated administrations in vitro, and (2) hBMC mediated cartilage repair cells remain responsive to sustained exposure to TGF-β1 in vivo.Methods and Materials: To test hypothesis 1, hBMC were stimulated with single administration of 5, 10 and 15ng/mL of TGF-β1, and the expression of TβRII and phospho-SMAD3 compared with multiple administrations of TGF-β1. Protein was isolated at 0, 2h, 4h, 1d, 3d and 7d after the last administration of TGF-β1. To test hypothesis 2, TβRII expression were assessed in athymic rat osteochondral defects at 4 weeks following implantation of hBMC transduced with AAV-GFP or AAV-TGF-β1.Results: In vitro, TβRII expression increased significantly at 2h post-stimulation with 15ng/mL of TGF-β1, and at 4h and 1d post-stimulation with 10ng/mL of TGF-β1. Phospho-SMAD3 expression increased at 2h post-stimulation with 10ng/mL of TGF-β1. However, the expression of both proteins declined thereafter. In addition, multiple administrations of 10ng/mL of TGF-β1 increased TβRII and phospho-SMAD3 expression up to 7 days post-stimulation.In vivo, TβRII is strongly expressed throughout the repair tissue of osteochondral defects receiving hBMC expressing TGF-β1, but not those receiving hBMC expressing GFP.Conclusions: The data show that the effects of single administration of TGF-β1 are transient suggesting that repetitive administration of 10ng/mL of TGF-β1 may be required to sustain cellular responsiveness. In addition, cartilage repair cells remain responsive to repeated exposure to TGF-β1 in vivo. The implication for in vivo repair strategies are that sustained exposure of hBMC to TGF-β1 is critical and can be achieved using AAV.

P122The effect of formalin fixation on Equilibrium Partitioning of an Ionic Contrast with microcomputed tomography (EPIC-µCT) imaging of osteochondral samplesK.E.M. Benders1, J. Malda1, D.B. Saris1, W.J.A. Dhert1, R. Steck2, D.W. Hutmacher3, T.J. Klein2 1Utrecht/Netherlands, 2Kelvin Grove/Australia, 3Brisbane/Australia

Purpose: EPIC-µCT is a non-invasive technique to quantify and visualize the three-dimensional distribution of glycosaminoglycans (GAGs) in cartilage. While this technique provides valuable high-resolution 3D data for fresh tissues, sample preparation and imaging are time-intensive and limit the number of samples that can be analysed at one time-point. Fixation may help overcome these

Methods and Materials: Osteochondral defects (5.8x6mm) were created in the medial femoral condyle (MFC) and the lateral trochlea sulcus (LTS) of the stifle joint of 24 sheep. Sheep were randomly assigned to four groups in the study (n=6); 1) empty defect, 2) scaffold only, 3) scaffold + rhFGF-18 (30μg) and 4) scaffld + BMP-7 (100µg). At 6 months the defects underwent mechanical testing, gross assessment of the repair tissue (ICRS score) and histological analysis (Modified O’Driscoll score). Results: ICRS gross repair score (Fig.1): Defects treated with rhFGF18 (mean 9.83, 95% CI 8.43-11.23) and BMP-7 (10, 9.06-10.94) in the MFC had significantly improved ICRS repair scores compared to empty defects (4.2, 0-8.80) (p=0.002). Mechanical properties: BMP-7 treated defects (mean 64.35, 95% CI 56.88-71.82) were significantly less stiff than both the rhFGF18 (mean 84.1, 95% CI 76.8-91.4) and empty defects in the LTS, compared to both contralateral limb (p=0.003), and the perilesional articular cartilage (p<0.001). Histology (Fig.2): Statistically significant improvements in the modified O’Driscoll score were observed in the rhFGF18 treated group (mean 16.83, 95% CI 13.65-20.61) compared to the empty defects (mean 9, 95% CI 4.88-13.12) (p=0.039). Excellent tissue fill, lateral integration and proteoglycan staining was observed. Only the rhFGF18 defects showed pericellular type VI collagen staining with positive type II collagen and reduced positive type I collagen staining indicative of hyaline like repair tissue. The majority of defects in the control and BMP-7 groups’ demonstrated fibrocartilagenous repair tissue. Conclusions: Statistically significant improvements in gross repair, mechanical properties and histological score were found over empty osteochondral defects when Chondromimetic was combined with rhFGF18. These results suggest that rhFGF18 may play a significant role in articular cartilage repair applications.

P119Inhibition of TGFß to repair articular cartilage in a sheep modelR. Escribano, P. Ripalda Cemboráin, P. Sanz-Ramos, A. Zabalza-Baranguá, J. Dotor, I. Izal Azcárate, G. Mora Pamplona/Spain

Purpose: Repairing of lesions in cartilage is an issue of interest for orthopaedic surgeons, as the fully regeneration of the tissue has not been yet acomplished. TGF-beta is a growth factor with a well described anabolic activity in cartilage. Interestingly, results in our laboratory have shown that inhibition of TGF-beta using peptide P144 brakes the loosing of the chondrogenic capacity of in vitro expanded chondrocytes. The aim of this study is to evaluate the effect of the inhibition of TGF-beta has in an in vivo model of cartilage repairing using a commercially available system.Methods and Materials: A model of osteochondral lesions was performed in the load bearing area of internal femoral condyle of healthy sheep. The peptide was loaded in TruFit cylinders (Smith & Nephew) by centrifugation, resuspended in PBS and implanted in the right knee according to the guidelines of the manufacturer. Left knee was used as a control and implanted with the cylinder embedded in PBS. After 3 months animals were sacrificed and samples obtained analyzed by gross appearance and by histomorphometry.Results: The gross aspect of the lesioned cartilage show an improvement of the tissue covering the lesioned area in lesions treated with the peptide. Histological analysis confirms these results and showed an improvement of the repairing tissue in knees that received P144 with respect to untreated knees.Conclusions: Inhibition of TGF-beta unsing peptide P144 must be taken into account for the development of new strategies for the repairing of cartilage lesions.

P120Collagen type II evaluation of the articular tissue repair treated with autologous plasma rich in growth factors (PRGF).C.I. Serra1, C. Soler2, J.I. Redondo1, J.M. Carrillo1, J.J. Sopena1, R. Cugat2 1Valencia/Spain, 2Barcelona/Spain

Purpose: To value the autologous plasma rich in platelets application, in the type II collagen formation of tissue repair in chondral deffects.Methods and Materials: 36 Californian rabbits were divided in 3 groups based on the treatment that they would receive (physiologic saline serum –PCB-, activated plasma rich in growth factors –PRGF-

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with sterile operation. Divided into control group and 0.2mmol / L lidocaine group, Cell density of 2×106/ml,vitro culture. Detect the apoptosis of chondrocytes at 24h and 48h. The GAG and Collagen type II concentrations of supernatants were measured at 3th,6th and9th day.Results: 0.2mmol/L Lidocaine has no effect on apoptosis of chondrocytes (p>0.05). The secretion of GAG was inhibited by Lidocaine at the 3th day(p<0.05) but was accelerated at the 9th day(p<0.05); Compared with the control groupe, the secretion of Collagen type II of Lidocaine group has reduce trend but no Statistical differences(p>0.05).Conclusions: 0.2mmol/L Lidocaine has no effect on apoptosis of chondrocytes.The secretion of GAG was inhibited by Lidocaine at Short-term culture,but accelerated at long-term culture; Lidocaine also has no effect on the secretion of Collagen type II.

P126The Potential for the End Stage Osteoarthritic Sclerotic Lesion to Participate in Cartilage RepairL.L. Johnson1, C. Verioti1, J. Gelber1, D.D. D’Lima1, M. Spector2, A. Pittsley3 1La Jolla, CA/United States of America, 2Boston/United States of America, 3Okemos/United States of America

Purpose: The use of arthroscopic debridement procedures in the treatment of degenerative arthritis of the knee remains controversial. Reports in the literature on the end stage osteoarthritic sclerotic lesion of the knee undergoing cartilage repair following unloading from high tibial osteotomy indicate an intrinsic potential for regeneration. The purpose of this study was to examine in depth the nature of the pathology of such a lesion and explore whether there was any potential for this lesion to participate in cartilage repair. Methods and Materials: Specimens harvested following total knee surgery were examined for gross pathology including staining with Safranin O. Multiple explants of the lesion were placed in tissue culture for three and six weeks. Gross examination and histological examination was made after simulated arthroscopic surgery and following each interval in culture. Results: The pathology of the end stage osteoarthritic lesion showed sclerotic bone, dead osteons, hypervascularity and scattered cartilaginous aggregates. Cartilaginous aggregates Additional observations showed multiple pitting on the sclerotic surface which histologically was related to three events; fragmentation of dead bone, ruptured blood vessels, and eroded aggregates. Vascular pit There were no pathological or biological changes following the time in tissue culture, and no outgrowth of chondrocytes from the cartilaginous aggregates. Conclusions: The in-depth pathological evaluation showed the end stage osteoarthritic lesion to have certain features with potential to facilitate cartilage repair. The cartilaginous aggregates remained unchanged in tissue culture absent the normal synovial joint environment and therefore further testing with a different experimental model would be necessary to establish these aggregates as a source of cartilage regeneration. The clinical relevance of this pathological study is that the cartilaginous aggregates remain following arthroscopic abrasion arthroplasty and microfracture and therefore may be participants in cartilage repair following those procedures. The small depressions in the lesion may be a “home” for various arthroscopic delivered cellular therapeutics.

P127Interaction Of Human Osteoarthritic Cartilage And SynoviumM. Beekhuizen1, L.B. Creemers1, Y.M. Bastiaansen-Jenniskens2, D.B. Saris1, W.J.A. Dhert1, G.J.V.M. Van Osch2 1Utrecht/Netherlands, 2Rotterdam/Netherlands

Purpose: Cartilage and synovium are both affected in osteoarthritis (OA) and are known to interact. However, a long term co-culture model with synovium and cartilage explants have not been described before. Such a co-culture may provide a suitable model to study these interactions and mimic the OA joint more closely as well as to screen new therapies for cartilage degeneration in OA. Therefore, the aim of the study is to develop an in vitro model that includes both synovium and cartilage.Methods and Materials: Osteoarthritic cartilage and synovium were cultured together or alone for 21 days. To assess viability of the synovium immunohistochemistry, a live/dead assay, and the release of lactate dehydrogenase (LDH) were used. For functionality of the cultured synovium a multiplex ELISA was used. Di-methylmethylene-blue assay was used to determine glycosaminoglycan (GAG) release and content.

issues; however, the effects of fixation on EPIC-μCT outcomes are unknown. This study aimed to determine whether formalin fixation of bovine cartilage affects x-ray attenuation, and therefore the interpretation of EPIC-µCT data. Methods and Materials: Osteochondral samples from bovine trochlear grooves were incubated with ioxaglate, an anionic contrast agent, for 22 hours prior to µCT scanning. Samples were scanned in both fresh and formalin-fixed conditions, and samples scanned fresh were also re-scanned after fixation (n = 8 per group). Wet weight, dry weight, and GAG content were determined for fresh and fixed samples (n = 5). Data were analyzed by ANOVA, with significance determined by p < 0.05. Results: The expected zonal distribution of contrast agent/GAGs was observed for both fixed and fresh cartilage specimens. However, the output range required for visualization of this distribution varied between fresh and fixed specimens (A). Correspondingly, average attenuation levels of formalin-fixed cartilage were significantly lower (14.3%) than in fresh samples (B, p < 0.001). Despite the difference in attenuation, there were no significant differences in wet weight or dry weight (C), or GAG concentration (D,E) between fixed and fresh samples. Conclusions: Formalin fixation reduces the attenuation of EPIC-µCT imaged cartilage. This reduction in attenuation is not due to changes in volume or GAG concentration following fixation. EPIC-μCT remains useful for studying GAG distributions in fixed tissues, such as archival samples, but the reduced attenuation should be taken into account when quantifying GAG by using standard curves specific for fixed cartilage.

P124Omega-3 (n-3) Polyunsaturated Fatty Acids in Regulating Disease in a Spontaneous Model of OsteoarthritisJ. Tarlton, L. Knott Bristol/United Kingdom

Purpose: The aim of this study was to examine effects of a high omega-3 (n-3) poly-unsaturated fatty acid (PUFA) diet on development of osteoarthritis (OA) in a spontaneous guinea pig model, and to further characterise the model in terms of its pathogenesis. Modern diets low in n-3 PUFAs have been linked with increases in inflammatory disorders. It may be that n-3 is beneficial in osteoarthritis, as this too has an inflammatory component. However, n-3 also increases bone density, and this may be a contributing factor in osteoarthritis. Therefore net effects of n-3 on OA are not known. Methods and Materials: The OA-prone Dunkin Hartley (DH) Guinea pig was compared with the OA-resistant Bristol Strain-2 (BS2), each fed standard or n-3 diets (10-30wks). We examined parameters of cartilage and subchondral bone pathology and biochemistry, including histological scoring, collagen crosslinks, matrix metalloproteinases (MMPs), alkaline phosphatase, glycosaminoglycan, and total and denatured type II collagen. Results: Most cartilage parameters in the DH strain were modified by n-3 diet towards those seen in the non-pathological BS2 strain. Those reaching significance included cartilage pathology, pro- and active MMP-2, collagen hydroxylation and lysyl-pyridinoline. The only exception to this trend was proMMP-9 which increased with n-3 but was lower in the BS2. GAG content was higher in the n-3 group, but not significantly so in the BS2s. There was no difference in total or denatured type II collagen. All subchondral bone parameters in the DH n-3 group also changed towards those seen in the non-pathological strain, of these alkaline phosphatase, proMMP-9 and calcium:phosphate ratios were significant. Conclusions: We confirmed reduced pathology in the BS2 strain by comparison with DH. Dietary n-3 PUFA reduced pathology in the OA-prone strain, and changes in biochemical markers were consistent with those associated with the non-OA strain. Omega-3 did not increase markers of pathology in either strain.

P125Effects of Na+ Ion channel blocker Lidocaine on metabolism of cultured rabbit articular chondrocytesW. Qi1, X. Wei2 1030001/China, 2Taiyuan/China

Purpose: To study the effect of Na+ Ion channel blocker Lidocaine on apoptosis and metabolism of cultured rabbit articular chondrocytes.Methods and Materials: Chondrocytes were isolated from the knee joints of five two-month New Zealand White Rabbit knees

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the defect to represent the desired repair surface. The algorithm then arranged a set of grafts on the defect site. Each graft was chosen from the potential donor regions. The algorithm optimized the position and orientation of the grafts to best fit the desired repair surface. The RMS errors between the planned repair surfaces and the original, uninjured surfaces were computed. Results: The algorithm had mean RMS error of 0.25 mm (95% CI: 0.21-0.30, min 0.12, max 0.45) and took five minutes. The expert human achieved mean RMS error of 0.30 mm (95% CI: 0.16-0.44, min 0.08, max 1.04) and took twenty minutes. Conclusions: No statistically significant difference in RMS error between the algorithm and the expert was found. The algorithm was faster and produced surfaces with less variance.

P130The degree of osteoarthritic alterations modulates the effects of drugs on human articular cartilageJ. Steinmeyer, J. Kordelle, H. Stuerz Giessen/Germany

Purpose: Several studies have implicated that tetracyclines, in addition to their antimicrobial properties, can slow down the progression of cartilage damage both in animal models of osteoarthritis (OA) as well as in humans. In search for the underlying mechansims the purpose of this in vitro study was to determine systematically (1) whether tetracyclines influence the synthesis and release of proteoglycans (PGs), MMP-1, -8, -13 and PGE2 from human OA cartilage, and (2) whether these pharmacological effects are affected by the degree of OA alterations.Methods and Materials: Full-thickness cartilage explants of the lateral compartment of the femoral condyles were taken from OA patients undergoing knee replacement surgery. Explants from mild (Collins grade 0-1.5) or moderately (Collins grade >1.5-3) affected human OA condyles were cultured separately in supplemented Ham s F12 media with media changes every 3-4 days. Explants were treated with 1, 10, 50 or 100 µM minocycline, doxycycline or tetracycline in the presence or absence of rhIL-1ß. PG synthesis was determined by the incorporation of radioactive sulfate during the final 18 h of the 11 days experiments whereas the content of PGs were quantitated with the DMMB-assay. MMP-1, -8, and -13 as well as PGE2 were determined with ELISAs. Results were compared to untreated explants removed from the same condyles (N=6).Results: The degree of OA alterations of explants can significantly modulate the pharmacological effects of tetracylines on cartilage metabolism. Furthermore, doxycycline was less effective on cartilage metabolism than minocycline, whereas tetracycline displayed no effect.Conclusions: Our study indicate that the pharmacological efficacy of drugs can be dependent on the clinical stage of OA.

P131Pleiotropic Mechanism of Action for Flavocoxid: Enzymatic Inhibition, Antioxidant Activity and Down-regulation of Inflammatory Gene ExpressionB. Burnett, L. Pillai, R. Levy Scottsdale/United States of America

Purpose: Flavocoxid, a USFDA-regulated, prescription medical food for the clinical dietary management of osteoarthritis (OA) under the supervision of a physician, shows equivalent efficacy to NSAIDs in clinical studies with fewer side effects. Its mechanism of action, however, for cyclooxygenase-1 (COX-1) and COX-2 as well as 5-lipoxygenase (5-LOX) inhibition is poorly understood. The antioxidant capacity of flavocoxid and effect on inducible gene expression is also not well-defined. Experiments were undertaken to clarify flavocoxid’s overall mechanism of action. Methods and Materials: Purified enzymes were used to assess flavocoxid’s anti-peroxidase and anti-cyclooxygenase activity on COX-1 and COX-2 as well as inhibitory activity on 5-LOX. Multiple standard antioxidant assay assays were utilized to judge flavocoxid’s antioxidant capacity and cell co-culture of LPS-stimulated human peripheral blood monocytes (PBMCs) with flavocoxid for its effect on inducible inflammatory gene expression of COX-2, IL-1β, IL-6 and TNFα.Results: Flavocoxid showed balanced inhibition of COX-1 and COX-2 peroxidase activities with inhibitory concentrations (IC50s) of 12.3 and 11.3µg/ml, respectively, while the 5-LOX IC50 was 110µg/ml. No detectable 5-LOX inhibition was found for rofecoxib, celecoxib, valdecoxib, diclofenac, meloxicam, naproxen, ibuprofen or aspirin. Flavocoxid showed minimal inhibition of cyclooxygenase activity for COX-1 (IC50=25µg/ml) compared to indomethacin

Results: Throughout the entire culture synovium showed viable cells by a LDH assay, a live/dead assay and immunohistochemistry demonstrated the presence of macrophages and T-cells. Several cytokines, which were previously demonstrated in synovial fluid of osteoarthritic patients, were secreted by synovium tissue in culture during the 21 days of culture. Co-culture of cartilage and synovium enhanced GAG release and reduced GAG content after 21 days of culture compared to cartilage alone (Fig. 1). Addition of Triamcinolone to the co-culture inhibited these catabolic effects of synovium on cartilage.Conclusions: From the present data, we can conclude that it is possible to culture synovium explants for at least 21 days. A co-culture with synovium explants enhances GAG release by the cartilage explants, indicating the catabolic environment that is present in OA, may be mimicked more closely in co-culture than in single explant culture. This study indicates the applicability of this co-culture model for use in testing new therapies for cartilage degeneration in OA.

P128Selection of Chondroprogenitors from Bone Marrow by Adhesion to Chondroitin SulfateJ. O’Sullivan1, C. Coleman2, M. Murphy1, F. Barry1 1Galway/Ireland, 2Galway City/Ireland

Purpose: Osteoarthritis is a disease of the joints characterized by progressive destruction of articular cartilage. As cartilage is composed primarily of extracellular matrix (ECM) with limited capacity for self-repair, the therapeutic application of mesenchymal stromal cells (MSCs) offers potential to regenerate cartilage. It was therefore hypothesized that a chondrogenic population of MSCs within the bone marrow (BM) can be isolated by preferential adhesion to cartilaginous ECM proteins. Methods and Materials: Proceeding MSC isolation, culture flasks were coated with 1mg/ml hyaluronan (HA) or chondrotin sulphate (CS) at 4°C overnight (Figure 1). BM from the iliac crest of healthy consenting human donors was exposed to uncoated, HA or CS coated plates for 24hrs or 5 days before non-adherent cell removal as per traditional methods. Confluent MSCs were passaged and expanded on uncoated plastic. At P2, MSCs were induced towards chondrogenesis, adipogenesis and osteogenesis.1 Results: All isolated cells retained a fibroblastic morphology with comparable expansion characteristics. Adipogenic differentiation was significantly increased in all ECM isolated MSCs, especially in the early adherent HA and CS isolated cells. Osteogenic potential was enhanced in all ECM selected MSCs especially in early adherent CS cells. Chondrogenic differentiation was enhanced in all ECM isolated groups, particularly in CS isolated cells (Figure 2). Conclusions: Cartilaginous ECM molecules (HA and CS) were utilized to isolate MSC subpopulations from BM. Cells adhering to CS had the greatest capacity for chondrogenic differentiation, suggesting cells expressing receptors for CS in the bone marrow may be more potent chondroprogenitors. Specific isolation of these subpopulations for clinical application will enable a reduction in the number of MSCs required for clinical efficacy and tissue regeneration.

P129Automated Planning of Computer-Guided Mosaic ArthroplastyJ. Inoue1, M. Kunz1, S. Waldman1, M.B. Hurtig2, J. Stewart1 1Kingston/Canada, 2Guelph/Canada

Purpose: Computer-guided mosaic arthroplasty requires a time consuming planningprocess to choose and place several osteochondral grafts on a computermodel of the joint, in order to reconstruct the original curvature of the articular surface over a defect. We investigated whether a computer algorithm can achieve reconstruction plans as good as those of an expert human.Methods and Materials: Surface mesh models from 14 sheep knees in original condition and three months after an impact-induced cartilage defect were created. For each knee, two plans were made (one by an expert human, the other by a computer algorithm) to reconstruct the articular cartilage over the three-month defect. Each plan consisted of donor and placement sites for three or four osteochondral grafts. The expert human operator manually selected and placed cartilage grafts using a computer interface. Grafts could be positioned and oriented and had their cartilage surface tilted according to the donor site. With the computer algorithm, a human operator outlined the defect, outlined potential donor regions, and placed a spline surface over

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Results: Standard debridement generally did not violate the tide mark line in 87.5% of the cases (35/40 specimens), with only occasional minor openings of approximately 20 µm in diameter with a smooth edge. Twenty-eight specimens (70%) showed at least remnants of uncalcified cartilage, 5 samples (12.5%) one large area with a missing bone plate and an open bone marrow space. Conclusions: Because of the fine structure of the minor openings, they are likely due to increased vascular penetration through the tide mark in the pathologically altered bone-cartilage interface in OA. Standard debridement only occasionally violates the subchondral bone plate in OA knee joints under in vivo conditions. The exact consequences of limited hemorrhage through minor openings or selected large defects are still unkown. Therefore, the traditional surgical preparation technique appears suitable for cartilage regeneration even in cases of osteoarthritic defects.

P134Therapeutic Effects of Intra-articular Administration of Ultra-purified Low Endotoxin Alginate on the Progression of Experimental Osteoarthritis in RabbitsT. Igarashi, N. Iwasaki, D. Kawamura, N. Tsukuda, Y. Kasahara, A. Minami 1Sapporo/Japan

Purpose: We have developed an ultra-purified low-endotoxin alginate (UPLE-alginate), which can drastically reduce endotoxin levels. Our hypothesis was that the UPLE-alginate could promote anti-arthritic activity in experimental osteoarthritis (OA). To test this hypothesis, we examined the activity of intra-articular administration of the UPLE-alginate using a rabbit OA model. Our aims were to clarify the effects of the UPLE-alginate administration on OA progression, and to determine the adequate molecular weight of the UPLE-alginate for therapeutic effects. Methods and Materials: To induce knee OA, 35 Japanese white rabbits underwent anterior cruciate ligament transection. Intra-articular injections of a 0.3 ml solution of each material started at 4 weeks postoperatively for a total of 5 weekly injections. Seventy knees were randomly divided into 5 treatment groups as follows; AL20 (430 kDa molecular weight UPLE-alginate), AL100 (1,000 kDa), AL500 (1,700 kDa), HA (hyaluronan), and NS (normal saline). At 9 weeks postoperatively, all knees were assessed macroscopically, histologically, and mechanically. Mechanical testing determined the friction coefficient as a joint lubrication. Results: Gross morphology. The NS and HA groups showed extensive cartilage erosion. The focal deep erosions were smaller in the treatment groups than the NS group. The UPLE-alginate groups exhibited milder cartilage degradation compared to that of the NS and HA groups. Histological findings. The NS and HA groups showed loss of the superficial layer and fibrillation. An obvious reduction in the severerity of OA was found in the UPLE-alginate groups. The scores described by Kikuchi et al. were superior in the AL100 group compared to other groups. Mechanical test. The friction coefficient of samples obtained from the AL20 and AL100 groups was significantly lower than that of the NS group. Conclusions: The current study indicates that our novel UPLE-alginates, especially AL100 (1,000 kDa molecular weight), have promising potential as an effective agent in preventing OA progression.

P135Effect of Light-Emitting Diode (LED) Therapy on the Development of Osteoarthritis (OA) in a Rabbit ModelY. Oshima1, R.D. Coutts1, N.M. Badlani1, R. Healey1, T. Kubo2, D. Amiel1 1La Jolla/United States of America, 2Kyoto/Japan

Purpose: Light-emitting diode (LED) therapy has proven beneficial for pain relief and wound healing. Our objective was to evaluate the effect of LED therapy on osteoarthritic (OA) knee joints using the anterior cruciate ligament transection (ACLT) model in vivo.Methods and Materials: Four weeks after ACLT of mature New Zealand rabbits, LED therapy began at intervals of 10 min/day, 5 days/week for 5 weeks (n=7). Therapy on the ACLT knees used a custom-designed brace that fit over the knee using red and IR (wavelength: 630 nm and 870 nm, respectively). The amount of energy delivered to the skin was calculated to be 2.4J/cm2. In the control group (n=7), the rabbits were sacrificed 9 weeks after ACLT without treatment. The femoral surface was evaluated with

(IC50=0.012µg/ml) and no cyclooxygenase inhibition of COX-2 compared to NS-398 (IC50=0.095μg/ml). Flavocoxid also demonstrated a strong antioxidant capacity against a variety of reactive oxygen species: ORAChydro=3700 µmolTE/g; ORAClipo= 19µmolTE/g; FRAP=1145µmolTE/g; HORAC= 1326µmolCAE/g; NORAC=1936μmolTE/g; SORAC= 27kunitSODeq/g; TEAC=2456µmolTE/g; and DPPH=767µmolTE/g. In LPS-stimulated PBMCs, flavocoxid strongly reduced gene expression of COX-2 (80-fold), TNFα (11-fold), IL-1β (10-fold) and IL-6 (40-fold) and, to a lesser extent, COX-1 (2.8-fold). Expression of NF-κB gene was also reduced (2.2-fold).Conclusions: These results suggest that the clinically favourable effects and equivalency to NSAIDs in the management of OA are achieved by simultaneous modification at multiple points in the inflammatory process making flavocoxid a unique anti-inflammatory acting via an antioxidant mechanism.

P132Joint distraction in canine experimentally induced osteoarthritis leads to cartilage repair accompanied by sustained relieve of pain.S.C. Mastbergen, F. Intema, P.M. van Roermund, H.A.W. Hazewinkel, F.P.J.G. Lafeber Utrecht/Netherlands

Purpose: Joint distraction in the treatment of osteoarthritis in humans results in long-term clinical benefit. The mechanism responsible for this benefit is unclear. Tissue structure modification was suggested to be involved. Therefore, joint distraction was applied in a canine experimental model of osteoarthritis to study the involvement of cartilage repair. Methods and Materials: Osteoarthritis was induced in the right knee joint according to the Groove model (condylar surgically applied damage) in 16 dogs. Ten weeks post-induction, the right knee joint was distracted for 3-5 mm by use of a hinged external fixator for 8 weeks in 9 dogs (distraction group). Seven dogs were left untreated (OA group). Pain was studied by (un)loading of the joint using force-plate-analysis every 5-10 weeks. Twenty-five weeks after removal of the external fixators, cartilage integrity of the tibial plateau was analysed. Results: Cartilage showed a decreased PG-content (-18%, p<0.01), an increased PG-release (+20%, p<0.03), and an increased histological grade of cartilage damage (+5; p<0.05). This loss of cartilage integrity was accompanied by decreased loading of the affected joint. The loss of PG-content was significantly less in the distraction group (-7%; p<0.02) compared to the OA group. PG-release (5%) was also less as was the histological grade of cartilage damage (-3, p<0.05). This relative improvement of cartilage integrity was accompanied by a persistent increase of loading of the osteoarthritic joint. Conclusions: Joint distraction resulted in less cartilage damage and normalization of loading of the affected knee. This study corroborates the clinical finding that cartilage repair is induced by joint distraction in treatment of osteoarthritis and that this may underlie the clinical benefit.

P133Surgical preparation for articular cartilage regeneration in patients with osteoarthritisJ. Mika1, T.O. Clanton2, R.W. Kinne3, C.G. Ambrose2 1Jena/Germany, 2Houston/United States of America, 3Jena/Eisenberg/Germany

Purpose: The autologous chondrocyte implantation/transplantation (ACI/ACT) has been described as a treatment option even in patients with osteoarthritis (OA). To prevent hemorrhage, fibrin clot formation, and subsequent activation of the inflammatory response, surgical preparation for articular cartilage regeneration should avoid penetration of the subchondral bone plate. We examined whether current surgical procedures with ring curettes preserve the integrity of the subchondral bone plate in patients with OA. Methods and Materials: The subchondral bone plates of femoral condyles (n = 40) in OA human knees undergoing total knee arthroplasty (TKA) were prepared in vivo using traditional debridement for ACI/ACT. The force on the ring curette was 15.2 ± 0.4 N. In order to approximate regular wear, only areas with maximally grade 3A (ICRS-score) cartilage were prepared. Effects were analyzed by light microscopy.

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As a control, PLGA gel without EP2 agonist was placed in one side (Control group). Rabbits were sacrificed either 2 or 12 weeks after the operation, and articular cartilages were examined histologically by HE and Safranin O staining and evaluated by Mankin ‘s score. Results: In the control group, significant cartilage degeneration was observed in both knee joints at 2 weeks. Among treated groups, the Mankin’s score of distal femur was significantly lower in knee joints treated with 80ug of EP2 agonist. At 12 weeks, the joint degeneration was accelerated in the control group, whereas no significant effect of EP2 agonist was observed in either 80ug or 400ug-treated group. Conclusions: We found that the administration of low dose (80ug) EP2 agonist was effective to prevent joint degeneration for short term, which may be due to the limitation of current drug delivery system. Continuous stimulation of EP2 agonist by new drug delivery system may prevent the degeneration for long term. EP2 agonist could be a promising drug for osteoarthritis.

P138Hyperbaric oxygen treatment enhances the expression of heat shock protein 70 and prevents nitric oxide-induced apoptosis in chondrocyteL. Yuan, S. Lin, C. Niu, C. Yang, S.W.N. Ueng, W. Chen Kweishan, Taoyuan/Taiwan

Purpose: We investigated the effect of hyperbaric oxygen (HBO) on (1) NO-induced apoptosis of rabbit chondrocytes and (2) the healing of rabbit articular cartilage defects.Methods and Materials: In vitro, interleukin-1β treatment induced NO in chondrocytes, and all hyperoxic cells were exposed to 100% oxygen at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting to detect mRNA and protein expression of HSP 70, inducible NO synthase (iNOS), and caspase 3. In vivo, the HBO group was exposed to 100% oxygen at 2.5 ATA for 2 h, 5 days a week for 10 weeks. After sacrifice, specimen sections were sent for histological and histochemical examination using a standardized scoring system. In situ analysis of iNOS, HSP 70, and caspase 3 expression were performed by immunohistostainingResults: Our in vitro study demonstrated that HBO treatment up-regulated mRNA and protein expression of HSP 70 and suppressed iNOS and caspase 3 expression. In vivo, the histological and histochemical scores showed that HBO treatment significantly enhanced the cartilage repair. Moreover, immunohistostaining showed that HBO treatment enhanced HSP 70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes.Conclusions: HBO treatment enhances the expression of HSP 70 and prevents NO-induced apoptosis in articular cartilage injury.

P139Tissue Engineering Osteochondral Graft for Cartilage RepairJ. Davies1, S. Wilshaw2, D. Shaw1, Z. Jin2, E. Ingham2, J. Fisher2 1Bradford/United Kingdom, 2Leeds/United Kingdom

Purpose: A novel acellular xenogenic graft is proposed as a biological cartilage replacement, for repair of osteochondral defects. Acellular porcine cartilage has been produced using repeated freeze thaw cycles and washing using hypotonic buffers and sodium dodecyl sulphate solution (SDS; Keir, 2008). DNA content was reduced by 93.3% compared to native cartilage, with corresponding reduction in glycosaminoglycan (GAG) content. It was hypothesised that penetration of decellularisation solutions into native tissue could be improved through deformation of cartilage under confined compression, and then allowing the osteochondral pin to recover in solution, allowing greater retention of the GAGs Methods and Materials: A perforated indenter applied a ramped load in a confined chamber, using a tensile testing machine (Instron). Osteochondral pins (9.8 mm diameter; n = 3) were loaded to 5 MPa contact stress for 20 minutes, and then placed in SDS 0.1% (v/v) in hypotonic Tris buffer; pH 8.0 and then treated according to the established protocol. For comparison, osteochondral pins (n = 3) were treated using the unmodified standard protocol. Histological and biochemical analyses were performed.Results: There was no difference in the reduction in DNA content of loading modified versus standard protocol decellularisation cartilage (0.017 mcg.mg-1 +/- 0.006 mcg.mg-1 (95% CI), loaded, 0.018 mcg.mg-1 +/- 0.011 mcg.mg-1 (95 % CI), standard). There was no difference in the GAG content (19.769 mcg.mg-1 +/- 33.581

a modified Outerbridge OA grading: Grades I, II, III and IV. mRNA expressions of various markers from cartilage of the femoral condyle and synovial tissue were evaluated by RT-PCR. Statistical analysis was performed. Results: The control group showed 4 Grade II, 2 Grade III and 1 Grade IV (average was 2.6) macroscopically. In the experimental group after the 5-week treatment with the light brace there were no remarkable side effects seen on the knee joints. There were 2 Grade I and 5 Grade II (average 1.7), however no severe OA joints. Aggrecan expression in the cartilage showed no differences between the groups; however, type II collagen expression increased in the experimental group compared to the control (Table 1). Expression of TNF-α in both cartilage and synovial tissues was significantly decreased in the experimental group compared to the control (Table 2). Conclusions: This is the first trial of LEDs in the orthopaedic area, and results showed that cartilage was preserved and type II collagen increased in the experimental group. Inflammation seemed decreased in both cartilage and synovium.

P136Effects of potassium and chloride channel blockers on the matrix anabolism of rabbit articular chondrocytes in vitroL. Ren, X. Wei Taiyuan/China

Purpose: To investgate the effects of potassium and chloride channel blockers on The glycosaminoglycan(GAG) and collagen type 2 synthesis of rabbit articular chondrocytes in vitro. Methods and Materials: 2 months New Zealand rabbits were killed and their knee joint were taken out under aseptic condition. The chondrocytes were isolated by enzymolysis method and cultivated in 24-well plates (seeded 5×104 cells per well).Then the chondrocytes were divided into three groups randomly after cultured 2 days when the cells were adherent. The control group was cultured by DMEM/F12 while the potassium channel blockon group(4-AP group)was cultured in the media contained 1mmol/L 4-aminopyridine, which can lead to the blockon of the potassium channel,and the chloride channel blockon group(SITS group)was cultured in the media contained 1mmol/L 4-acetamido-4 -isothiocyano-2,2 -disulfonic acid stilbene(SITS), which can lead to the blockon of the chloride channel.The GAG synthesis were measured by Alician Blue method and collagen type 2 synthesis were estimated by the ELISA in the media when medium was change after3-, 6-and 9-day. Results: Compared with the control group,the GAG content of 4-AP group has significant decrease at 3th day,but has no significant difference at 6th and 9th day,while the collagen type 2 content has significant increased at 3th and 6th day, and has decrease tendency at 9th day, but have no significant difference.The GAG content of SITS group has significant increased at 3th, 6th and 9th day,while the collagen type 2 content has significant increased at 3th and 6th day,and has increased tendency at 9th day, but have no significant difference.Conclusions: The blockon of potassium and chloride channel can increase the GAG and collagen type2 synthesis of chondrocytes in vitro.Especially when block chloride channel,the GAG synthesis was significant.

P137Prostaglandin E2 prevents the degeneration of articular cartilage via EP2 receptor in rabbit osteoarthritis modelH. Mitsui1, T. Aoyama2, M. Furu2, K. Ito3, Y. Jin2, T. Kasahara4, K. Hayakawa2, K. kobayashi2, T. Maruyama4, T. Kanaji4, S. Fujimura4, H. Sugihara4, T. Otsuka3, T. Nakamura2, J. Toguchida2 1Kyoto City/Japan, 2Kyoto/Japan, 3Nagoya/Japan, 4Osaka/Japan

Purpose: We have reported that a selective agonist for EP2 receptor of prostaglandin E2 (EP2 agonist) induces the proliferation of articular chondrocytes and promotes the regeneration of the osteochondral defect of rabbits. The purpose of the current study is to analyze the effect of EP2 agonist on articular cartilage regeneration in traumatic joint degeneration model of rabbits.Methods and Materials: Female Japanese white rabbits (3-months-old) were used for this study. Traumatic degeneration model was created by transection of anterior cruciate ligament and partial menisectomy of medial meniscus in both knee joints. Immediately after the operation, poly lactic-co-glycolic acid gel (PLGA) containing EP2 agonist (80ug or 400ug) was placed on one side (Treated groups), and no treatment was performed on contralateral side.

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media. Cell death remained localized to the site of injury immediately following injury; at 21 days post injury, injured regions were void of dead cells, suggesting that cell death had not spread through apoptotic pathways, as previously reported in the literature for in vitro explant injury models.Conclusions: Tissue culture storage in CM protects native and mature engineered cartilage by confining cell death to the injury site. In engineered cartilage, biochemical and mechanical properties are maintained at control levels after injury. Future work will look to utilize CM to enhance explant survival after harvesting. These findings may lead to development of better strategies for engineered cartilage as well as osteochondral harvest/storage that optimize the use of allograft tissues for clinical repair of cartilage.

P143Arthroscopic Mosaicplasty for Osteochondral Lesions of the Knee: Computer-Assisted Navigation vs. Freehand TechniqueD. Koulalis1, P. Di Benedetto2, M. Citak3, P.F. O’Loughlin4, C. Cranchi4, A. Pearle4, D. Kendoff4 1Athens/Greece, 2Udine/Italy, 3Hannover/Germany, 4New York/United States of America

Purpose: The purpose of this study was to compare a freehand arthroscopic approach to mosaicplasty for treatment of osteochondral lesions of the knee with a navigated arthroscopic approach.Methods and Materials: Four whole cadaveric legs were used. A conventional navigation system was used in combination with an autologous ostechondral graft transplantation system. A customized reference tracker was fixed to both the OATS recipient and donor guides. Results: Mean angle of graft harvest was 3.4° (range 0° to 10°, SD +/- 3.10°) in navigated group versus 14.8° (range 6° to 26°, SD +/- 7.53°) in freehand group (p < 0.0003) Mean angle for recipient site coring was 1.50° (range 0° to 5°, SD +/- 1.65°) in the navigated group versus 12.60° (range 4° to 17°, SD +/- 3.98°) in the freehand group (Fig. 2) (p < 0.0003). Mean angle of graft placement was 2.0° (range 1° to 5°, SD 1.25°) in the navigated group versus 10.8° (range 5° to 15°, SD +/- 3.23°) in the freehand group (Fig. 3) (p = 0.0002). Mean difference between angle of graft harvest and graft placement was 2.40° (range 0° to 8°, SD +/- 2.46°) in the navigated group versus 7° (range 1° to 16°, SD +/- 5.19°) (p = 0.0207) in the freehand group. Mean difference between the angle of recipient site coring and graft placement was 0.90° (range 0° to 2°, SD +/- 0.74°) in the navigated group versus 9.2° (range 6° to 14°, SD 2.57°) in the freehand group (p = 0.002). Conclusions: This study demonstrates in a cadaveric model that improved accuracy and reproducibility may be achieved with a navigated arthroscopic mosaicplasty versus a freehand arthroscopic technique.

P144A systematic assessment of hierarchical clustering methods for flow cytometry data analysis: application to the study of chondrosarcomasJ. Diaz-Romero1, S. Romeo2, J.V.M.G. Bovée3, P.C.W. Hogendoorn3, D. Nesic1, P. Mainil-Varlet1 1Bern/Switzerland, 2Treviso/Italy, 3Leiden/Netherlands

Purpose: We wanted to establish a method to categorize different cell types of mesenchymal origin based on their pattern of surface marker expression combining flow cytometry data with hierarchical clustering. Cell types for which an independent knowledge of cluster membership was available were used to (a) assess cluster accuracy and guide the choice of the different clustering approaches tested, and (b) establish similarities with chondrosarcoma cells that could provide clues concerning the cellular origin of these tumors. Methods and Materials: Human primary conventional central chondrosarcoma cells, articular chondrocytes, mesenchymal stem cells, fibroblasts, and a panel of tumor cell lines of chondrocytic or epithelial origin were clustered based on the expression profile of eleven surface markers, previously shown to be differentially expressed on the cell types analyzed (Fig.1). Eight hierarchical clustering algorithms, three distance metrics, as well as several approaches for data preprocessing, including multivariate outlier detection, logarithmic transformation, and z-score normalization, were systematically evaluated. Results: Careful selection of clustering approaches allowed separation of chondrocytes, mesenchymal stem cells, fibroblasts,

mcg.mg-1 (95% CI), loaded, and 13.530 mcg.mg-1 +/- 21.207 mcg.mg-1 (95 % CI), standard). DAPI fluorescence demonstrated residual DNA in deep zones and at the cartilage bone interface in the standard protocol, which was to a lesser degree with loading modified cartilage.Conclusions: Loading modification did not improve DNA reduction or GAG retention in acellular porcine cartilage.

P140Repair of rabbit osteochondral defects using a novel bioabsorbable bone-originated apatite with body fluid permeabitityY. Kasahara1, N. Iwasaki1, M. Murata2, T. Akazawa1, T. Igarashi1, D. Kawamura1, Y. Tsukuda1, A. Minami1 1Sapporo/Japan, 2Tobetsu-Chou/Japan

Purpose: We successfully developed a bioabsorbable bone-originated apatite with body fluid permeability (functionally graded apatite, fg-HAp). The purpose of this study was to determine the efficacy of this material for the treatment of osteochondral defects using a rabbit model.Methods and Materials: Preparation and physicochemical characterization of the fg-HAp blocks were described previously. As a control, commercially manufactured interconnected porous hydroxyapatite ceramic (IP-CHA; Toshiba Ceramics, Tokyo) was used in this study. Twenty skeletally mature female rabbits were used for further analysis. A 4.5 mm diameter osteochondral defect was created in the femoral trochlea of each rabbit. In both treatment groups, the HAp blocks were press-fit implanted into the defects without periosteum coverage. At 4 and 12 weeks postoperatively, the macroscopic and histological appearance of the samples was evaluated in each group (n=5) using Wayne and modified O’Driscoll scoring systems, respectively.Results: Table 1 summarizes macroscopic and histological evaluations of both treatment groups. From 4 to 12 weeks postoperatively, the implanted apatites of the fg-HAp group were indistinguishable from the reparative tissue and the surface became smooth. The macroscopic score of the fgHAp group was statistically higher than that of the IP-CHA group at 4 weeks. The histological findings of the fg-HAp group demonstrated that the reparative tissue at the articular surface was complete integration between native and reparative tissue (Fig. 1). Although the fg-HAp bulk was stained by HE, the IP-CHA bulk was not stained by HE, suggesting that body fluid hardly permeated into the bulk structure. The histological score of the fgHAp group was statistically higher than that of the IP-CHA group at 12 weeks.Conclusions: We succeeded in osteochondral repair with the newly developed fg-HAp alone. Our study indicates the efficacy of the fg-HAp as a bioabsorbable scaffold for the treatment of osoteochondral defects without growth factors nor cultured cells.

P142Chondrogenic media (CM) protects native and engineered cartilage constructs from cell death after injuryA.R. Tan, J.P. Andry, G.A. Ateshian, C.T. Hung New York/United States of America

Purpose: In cartilage explant injury models cell death has been characterized as progressing away from the site of injury and persisting long after the initial insult. Here, we look at protecting both native cartilage explants and mature engineered cartilage after mechanical insult by storage in a serum-free, chondrogenic media (CM).Methods and Materials: Explants and chondrocytes were harvested from juvenile bovine wrists. After 35 days in culture, chondrocyte-seeded agarose constructs attained native Young’s modulus and glycosaminoglycan content. Two orthogonal cuts were made in each construct by pushing a razor blade to 50% of the specimen thickness. Cuts were similarly made in fresh cartilage explants. Controls were handled similarly but without insult. Samples were returned to culture following injury and maintained in CM (hgDMEM plus 1X PSF, 0.1uM dexamethasone, 50ug/mL ascorbate 2-phosphate, 40ug/mL L-proline, 100ug/mL sodium pyruvate, and 1X ITS+ premix (Becton Dickinson, Franklin Lakes, NJ). Cell viability was assessed by Live/Dead cytotoxicity assay at days 1, 7 and 21 post-trauma.Results: Cartilage injury and cell death were mitigated in cartilage explants and engineered cartilage when cultured in chondrogenic

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of morphology but not in terms of gene expression; the intermediate and external areas are composed of cells that present fibroblast-like morphology and phenotype. Interestingly, there is no expression of type II collagen throughout the whole young meniscus. In the adult meniscus, however, the three analyzed areas did not show the differences in cell morphology noted for the young tissue. Moreover, all cell populations were positive for type II and type I collagen presenting a gradient of type II versus type I: from the inner to the outer area, type II collagen decreases while type I increases.Conclusions: These results lead to the conclusion that meniscus maturation, from young to adult, is accompanied by changes in cell phenotype: in the early stages of life the cells from the intermediate and outer part are still immature and far from a chondocyte-like phenotype; in adult life all meniscus cells show an intermediate phenotype between chondrocytes and fibroblasts.

P147Integration of Repair Cartilage: an in vitro modelR. Kandel, J.A. DeCroos, J. Theodoropoulos Toronto/Canada

Purpose: In previous studies, we developed methodology to generate biphasic constructs composed of hyaline cartilage formed in vitro and anchored to the top surface of a porous bone-interfacing component. These constructs are suitable to use for cartilage repair as shown in an ovine model. In contrast to osteochondral transfer (OATS), the in vitro formed cartilage layer integrates laterally with the surrounding native tissue. The aim of this investigation was to develop this as an in vitro model in order to be able to study the mechanisms regulating tissue engineered cartilage integration to native cartilage.Methods and Materials: A single biphasic construct (5.7 mm diameter) was placed into the center of doughnut-shaped 1cm diameter osteochondral cylinder, which had been harvested from bovine joints. These were maintained in culture for up to eight weeks. Autologous osteochondral plugs served as controls. Results: Histological evaluation by light and electron microscopy showed that bioengineered cartilage exhibited increasing integration over time. Collagen fibers were seen crossing the interface ultrastructurally in regions of integration. In contrast autologous osteochondral implants did not integrate. Biochemical evaluation demonstrated a significant increase in collagen content in the cartilage of the biphasic implant over time but no change in proteoglycan content. The strength of repair–native cartilage integration increased significantly between 4 and 8 weeks. When carboxy-fluorescein diacetate-labeled chondrocytes were used to generate cartilage tissue, fluorescent cells were detected in the adjacent native cartilage up to 1.5mm from the integration site.Conclusions: This study demonstrates development of an in vitro model to study repair cartilage integration. The presence of cells from the repair tissue in the adjacent cartilage suggests that the integration to the surrounding native tissue may be a result of these migratory cells. Further study is required to evaluate the role of these cells in cartilage integration. This work was supported by a grant from CIHR (RK).

P148Development of In Vitro Articular Cartilage Injury: Novel Strategy Utilizing Impaction and Bioreactor to Induce and Treat LesionsD. Wilensky1, L.D. Kaplan1, C. Wong2, C.C. Huang1 1Miami/United States of America, 2Coral Gables/United States of America

Purpose: To induce an articular cartilage injury and recreate the native environment in vitro to facilitate the assessment of early hyaluronic acid delivery in a human articular cartilage model.Methods and Materials: Human full-thickness articular cartilage plugs were harvested from the femoral condyles of tissue donors upon cessation. Cartilage injuries were induced with a drop tower and treated at 1 hr or 24 hrs post impaction. The articular cartilage plugs were then subjected to displacement-controlled cyclic compressive loading at a frequency of 1 Hz and magnitude of 10% strain in the presence of hyaluronic acid. The effectiveness of the hyaluronic acid treatment was then assessed utilizing an ethidium homodimer-1 assay for cell viability determination and a TUNEL assay for apoptotic activity. GAG content was determined using a DMMB assay while safranin O staining was employed to visualize proteoglycan distribution in the extracellular matrix. Morphologic analysis was performed with a Hematoxylin and Eosin stain.

and tumor cell lines in four well differentiated clusters (Fig.2). Primary chondrosacoma cells were grouped in two main clusters with distinctive marker signatures: one group clustered together with mesenchymal stem cells (CD49b-high/CD10-low/CD221-high) and a second group formed an independent cluster close to fibroblasts (CD49b-low/CD10-high/CD221-low). Substantial differences between primary chondrosarcoma cells and established chondrosarcoma cell lines were revealed, with the latter not only segregating apart from primary tumor and normal tissue cells, but clustering with cell lines from epithelial origin. Conclusions: Hierarchical clustering of flow cytometry data is a powerful tool to classify samples according to surface marker expression patterns. It distinguishes between closely related cell types, such as fibroblasts from chondrocytes, allows tumor classification according to similarity with normal cell types, and could be used to validate the use of cell lines mimicking normal chondrocytes or primary tumors.

P145Phenotypic gradient from the avascular to the vascular zone of the young meniscusD. Deponti, C. De Palma, A. Pozzi, R. Ballis, A. Di Giancamillo, C. Domeneghini, L. Mangiavini, G.F. Fraschini, G.M. Peretti Milan/Italy

Purpose: The meniscus plays an important role in the biomechanics of the knee joint. This tissue has a poor healing potential, partly due to the absence of vasculature: blood vessels are present only in the outer 10-30% of the meniscal body. The term fibrochondrocytes has been introduced to identify the typical characteristics of the meniscus cells, but a clear description of the phenotype of these cells is still missing. This work was aimed to study the characteristics of the meniscus cells by focusing on three areas of the meniscus, the inner avascular zone, the intermediate, and the external vascular zone.Methods and Materials: Meniscus cells proliferation and differentiation were compared in these three different areas by FACS analysis, moreover the expression of cartilage specific genes was confirmed by real time PCR; additionally, cell morphology was analyzed both in the native meniscus by histology and by microscopy analysis after enzymatic isolation from the tissue. Meniscus cells were compared to isolated articular chondrocytes and tendon fibroblasts. Results: The data show that the inner region of the meniscus is composed of cells that are similar to articular chondrocytes in terms of morphology and proliferation rate, but they express lower amount of cartilage specific genes, such as collagen type II and aggrecan; the intermediate and external areas are composed of cells that showed a gradient in morphology and cartilage specific gene expression compared to the cells of the inner area; however, interestingly, they show a lower proliferation rate. Conclusions: These results suggest the presence of an “intermediate phenotype” located in the middle and external meniscal areas having morphological and functional characteristics intermediate between the fibrochondrocytes of the inner region and the tendon fibroblasts; these evidences could lead to new options in the choice of the optimal cell source for meniscus tissue engineering.

P146Meniscus cells phenotype: new insights in the use of the term “fibrochondrocytes”D. Deponti, C. De Palma, A. Pozzi, R. Ballis, M. Melato, A. Di Giancamillo, C. Domeneghini, G.F. Fraschini, G.M. Peretti Milan/Italy

Purpose: The meniscus plays an important role in the biomechanics of the knee joint. This tissue has a poor healing potential, partly due to the absence of vasculature. Due to the presence of both collagen I and II, the meniscus has properties of fibrous and cartilaginous tissue. The term fibrochondrocytes has been introduced to identify the typical characteristics of the meniscus cells, but a clear description of the phenotype of these cells is still missing.Methods and Materials: This work was aimed to study the characteristics of swine meniscus cells by analyzing three areas of the meniscus: the inner avascular zone, the intermediate and the external vascular zone. Meniscus cells morphology, proliferation, differentiation and gene expression were evaluated.Results: The data showed that in the young meniscus the inner region is composed of cells that are similar to chondrocytes in terms

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Conclusions: Longitudinal in vivo results show persistent and stable AAV-mediated gene expression following a single intra-articular injection. The use of inducible AAV-TRE-Luciferase demonstrates the feasibility to control transgene expression in the rat knee joint. These results suggest that AAV is a potential therapeutic approach to deliver growth factors to the knee joint. The rare occurrence of GFP+ cells in intact articular cartilage indicates that more studies on AAV delivery to damaged articular cartilage, as seen in cartilage injuries, are needed. This continued characterization of the length, location and ability to control transgene expression following intra-articular injection of AAV will help to better tailor gene therapy for articular cartilage repair.

P151Imaging of osteochondral samples using delayed Gadolinium-Enhanced MR Imaging of Cartilage (dGEMRIC) is not affected by formalin fixationK.E.M. Benders, J.E.J. Bekkers, W. Schuurman, L.B. Creemers, W.J.A. Dhert, D.B. Saris, J. Malda Utrecht/Netherlands

Purpose: Delayed Gadolinium-Enhanced MR Imaging of Cartilage (dGEMRIC) is a non-destructive method that could facilitate non-destructive ex vivo visualization of glycosaminoglycans (GAGs) in experimental settings. This study aimed at investigating whether formalin fixation affects dGEMRIC outcomes. Methods and Materials: Osteochondral plugs (n=72) were harvested from the medial and lateral condyles of equine stifle joints (n=3). Samples were divided into three groups: no digestion (samples were kept in PBS (n=24)), digest 1 (0.25% trypsin for 20 minutes (n=24), and digest 2 (0.25% trypsin for 40 minutes (n=24). Per group half of the plugs were fixed using 10% formalin for 22 hours (n=36) while the other half was kept in PBS. Samples were incubated for 22 hours in 1mM Magnevist (Gadolinium). A 3-dimensional T1 MRI measurement protocol, at 1.5T, with 5 different inversion times (50, 150, 350, 650 and 1650ms) was used. The T1gd (dGEMRIC index) was calculated by averaging the T1gd across the region of interest (ROI) using in-house developed software. The ROI was defined as the full-thickness cartilage in all the sagittal slices. After scanning, the cartilage was cut from the samples and GAGs were determined biochemically using a DMMB assay. Correlations and differences in GAG/mg wet weight were statistically analyzed using Pearson’s correlations and one-way ANOVAs with post-hoc LSD tests. Results: Digestion of the samples resulted in a GAG content that ranged from approximately 20 to 75 mg GAG per mg tissue. Good correlations were found between the T1gd and total GAG/mg cartilage (R=0.763, p<0.01). No significant differences were found in T1gd and GAG content per mg between fixed and non-fixed osteochondral samples (p>0.1).Conclusions: Formalin fixation of osteochondral plugs does not alter the T1gd-time. Therefore, the results suggest that dGEMRIC can be used on both fixed and non-fixed osteochondral ex vivo samples.

P152Construction of a bio-active meniscus via bio-plottingE. Berneel, J. Schelfhout, H. Declercq, P. Dubruel, M. Cornelissen Ghent/Belgium

Purpose: Injuries to the meniscus, particularly those in the a-vascular region, pose a complex problem. Tissue engineering of a replacement tissue might be the solution. Rapid proto-typing is an innovating technology that allows to construct tailor-made scaffolds. Layer-by layer disposition enables to fabricate a 3D-scaffold with a variable micro-architecture. Another advantage is the possibility to plot cells together with the material so that plotted scaffolds contain a homogenous dispersed cell population. Moreover different cell types can be used during plotting. We started with the construction of the gelatinous core of the meniscus. A porcine meniscus functions as a reference because of its analogous structure and cell distribution as the human meniscus: fibrochondrocytes in the inner and middle part of the meniscus and fibroblast-like cells in the outer part of the meniscus. Our aim is to encapsulate and plot these cells with a suitable material and to evaluate their cell viability and phenotype. A polyester such a polycaprolactone will be used to give more strength to the construct.Methods and Materials: Fibrochondrocytes derived from porcine menisci were isolated, cultured and identified with following markers: collagen I, collagen II and aggrecan. MC3T3 cells were

Results: Injured groups subjected to mechanical loading and hyaluronic acid treatment showed increased viability mainly in the superficial tangential and middle zones. The injured group with no treatment revealed lower overall proteoglycan content with reduced distribution in the more superficial areas. Fluorescent microscopy demonstrated that there was a strong trend for reduced chondrocyte death in the mechanically loaded groups challenged with hyaluronic acid.Conclusions: This study suggested that early hyaluronic acid delivery coupled with cyclic mechanical loading, to acutely injured cartilage, reduces cell death and significantly improves cell metabolism. With further experimentation, the use of hyaluronic acid shortly following injury may prove to be beneficial for clinical use in humans to minimize cartilage death and improve healing after traumatic injury.

P149T2 relaxation in healthy neutral, varus and valgus knee joints: does malalignment provoke cartilage deteriorationM. Sauerschnig1, G.M. Salzmann2, L. Kohn1, J.S. Bauer1, K. Wörtler1, S. Landwehr1, S. Hinterwimmer1, A.B. Imhoff1 1Munich/Germany, 2Freiburg/Germany

Purpose: We hypothesize that static knee joint malalignment affects the articular cartilage in asymptomatic subjects who display evidence of an early chronic joint impairment. Methods and Materials: The study had institutional review board approval and was HIPAA compliant; all subjects provided informed consent prior to testing. In 4 groups of active, age-matched (26.1 ± 1.8 years) patients [neutral (0.44° ± 0.29), mild varus (2.95° ± 0.52), severe varus (5.25° ± 1.45), or valgus leg deformity (2.95° ± 0.75) both knee joints were clinically (Lysholm, Tegner) and MR-radiographically (hip-knee-ankle angle, T2 mapping) evaluated. Patients were without knee complaints (no history of major knee trauma, knee surgical intervention) with either to define 4 different groups (n=6 subjects/group; n=12 knees/group). Regions of interest for T2 assessment were within full-thickness cartilage across the joint surface and were divided with respect to compartmental as well as functional joint anatomy. Results: Leg deformity was significantly different between groups, while no difference emerged from the clinical measures. When compared to neutral knee angle subjects, prolonged cartilage T2 was found at the medial tibia plateau and medial posterior femoral condyle in varus aligned subjects, while no such differences emerged among valgus knees. Conclusions: There are early alterations within the articular surface of asymptomatic subjects that have noteable varus knee malalignment. These changes can be detected by quantitative MR-imaging.

P150Single intra-articular injection of AAV results in stable and controllable in vivo transgene expressionC. Chu1, K.A. Payne1, H.H. Lee1, A.M. Haleem1, C. Martins1, X. Xiao2 1Pittsburgh/United States of America, 2Chapel Hill/United States of America

Purpose: This study characterized the localization, persistence, and ability to externally control transgene expression in the rat knee joint after a single intra-articular injection of adeno-associated virus (AAV2). Methods and Materials: Nine male Sprague-Dawley rats received AAV2-CMV-GFP and AAV2-CMV-Luciferase into their right and left knees, respectively. Luciferase expression was evaluated over a 1-year period using the IVIS® Imaging System (Xenogen). After sacrifice, tissues within the joint were analyzed by fluorescence stereomicroscopy to detect GFP+ cells. To study the ability to control AAV transgene expression in the rat knee joint, five male Sprague-Dawley rats received AAV2-tetracycline response element (TRE)-Luciferase and AAV2-CMV-transcriptional transactivator into the right knee. To induce expression of Luciferase, doxycycline (Dox; 2 mg/ml) was added to the drinking water, and rats were imaged twice a week as described above. Results: Luciferase expression was detectable in all rats by 7 days post-injection, and showed continued and stable expression through 1 year. GFP+ cells were mainly found in soft tissues surrounding articular cartilage, with rare GFP+ chondrocytes. With the use of inducible AAV-TRE-Luciferase, gene expression could be upregulated with Dox, and could then be downregulated by its removal from the water. This was repeated in multiple cycles.

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P154Manipulative structural re-organization of superficial zone extracellular matrix: articular chondron density increases in response to non-ablation radiofrequency energyK. Ganguly1, I.D. McRury2, P.M. Goodwin1, R.E. Morgan2, W.K. Augé, II3 1Los Alamos/United States of America, 2Fall River/United States of America, 3Santa Fe/United States of America

Purpose: Chondron density within the Superficial Zone has been shown to decreased with age, disease, injury, and in response to some interventions and may predispose articular cartilage to extracellular matrix-based failure through an inability to support the mechanotransductive demands of physiologic loading (1). Since chondron shape and orientation reflect inter-territorial extracellular matrix architecture, chondron density is an important descriptor for functional cartilage (2, 3). Interventions that alter chondron density may provide insight into the treatment outcome of focal lesions. This study evaluates radiofrequency energy effects on native Superficial Zone chondron density in articular cartilage demonstrating early lacunar emptying without significant surface fibrillation.Methods and Materials: Radiofrequency energy was delivered by two methods to ex-vivo femoral condyle osteochondral specimens obtained from patients undergoing total joint replacement; Ablation and Non-ablation. Untreated control and treated specimens were sectioned, prepared with Live/Dead cell viability stain, and assessed by confocal laser microscopy. Chondron density was determined by quantifying live chondrocyte populations per square millimeter in two-dimensional section images.Results: The mean total Superficial Zone cell number in control sections was 1480 per mm2. The Ablation method fully corrupted the Superficial Zone by volumetric loss or near complete cellular necrosis. The Non-Ablation method retained the Superficial Zone with a mean total number of cells of 1468 per mm2 (0.8% difference from control) with increased live chondron density of 12% over control. Chondron image character was not altered by treatment; whereas, the increased live chondron density originated from preferential extracellular matrix volume contraction.Conclusions: This report suggests that non-ablative radiofrequency energy can increase articular cartilage Superficial Zone live chondron density. The Superficial Zone extracellular matrix, because of its distinctive composition, is uniquely suited to manipulative structural reorganization. Resetting functional chondron density patterns may have the potential to create a more chondro-supportive environment for articular cartilage as it inherently responds to focal disease (4).

P156Mechanical Forces can change the microstructure of cartilage using a joint mimicking bioreactor systemW.H. Choi1, Y.J. Kim1, B.H. Choi2, S.R. Park2, B. Min1 1Suwon/Korea, Democratic People’s Republic of, 2Incheon/Korea

Purpose: Articular cartilage consists of 4 horizontal layers separated by cell arrangement and macromolecular (proteoglycan and collagen) distribution and organization. The zonation is important to maintain cartilage function such as lubrication and cushion. In this study, we hypothesized the complex stimuli (combination of shear, hydrostatic pressure and compression) during the joint loading movement effects on zonal differentiation by applying bioreactor system that imitated human joint loading condition. We examined the change of cell arrangement and macromolecular organization on porcine cartilage explants. Methods and Materials: To mimic the joint loading condition, we manufactured the bioreactor system that has a device similar to the shape of a femoral and tibial condyle in human knee. This device is designed to produce the shear and compression forces. Cylinder shaped cartilage plugs (D X H = 5mm x 4mm) were taken from a young porcine (2w) femoral head and were placed between both condylar mold of the device of bioreactor. The cartilage plugs were stimulated with bioreactor for 1 hour per day over the course of 4 weeks. Control group was static cultured without stimuli. After 4 weeks, the tissues were cut into 2 pieces (the top and bottom layers) for chemical analysis (glycosaminoglycan (GAG), collagen, water). Cell arrangement and macromolecular organization were analyzed through histology.Results: All groups have a quantitative difference between the upper and lower layers on chemical contents. The amount of GAG and collagen was larger in the lower layer, but water content was not. In histology data, larger amounts of GAG were expressed

encapsulated in and seeded on methacylamide modified gelatin (DS 61%). The hydrogels were cross-linked using uv-irradiation in the presence of a photo-initiator Irgacure® 2959. Sol-gel fractions of the hydrogels were determined in aqua dest, PBS and culture medium. The gels (0,4 ml) were casted in a 24 well plate and cross-linked with uv-irradiation. Cell survival was evaluated with MTT en live-dead assay. Polycaprolactone scaffolds were plotted with the 3D-bioplotter device and were modified to enhance cell adhesion and improve biocompatibility of PCL. Cell viability and adhesion of HFF (human foreskin fibroblast) cells was tested on thin modified polycaprolactone films (2D) and 3D scaffolds with MTT and live-dead assay.Results: First results showed a good cell survival of MC3T3 cells seeded on the hydrogel constructs but a low cell survival of encapsulated MC3T3 cells in methacrylamide modified gelatin. The effect of uv-irradiation on MC3T3 cells was also evaluated and the preliminary results are promising. This might indicate that the low cell viability in the hydrogel may be due to radical formation during uv-irradiation but further research is needed. The HFF cells demonstrated a good cell survival on the modified 2D PCL films and 3D plotted constructs which indicates that modified PCL is a good choice of material to construct the outer part of the meniscus.Conclusions: First results showed a good cell survival of MC3T3 cells seeded on the hydrogel constructs but a low cell survival of encapsulated MC3T3 cells in methacrylamide modified gelatin. The effect of uv-irradiation on MC3T3 cells was also evaluated and the preliminary results are promising. This might indicate that the low cell viability in the hydrogel may be due to radical formation during uv-irradiation but further research is needed. The HFF cells demonstrated a good cell survival on the modified 2D PCL films and 3D plotted constructs which indicates that modified PCL is a good choice of material to construct the outer part of the meniscus.

P153Coefficient of Friction Measurement for Early Performance Screening of Artificial Materials in Articulation Against Joint CartilageN. Stark1, B. Thomas2, R. Klabunde1, M. Bürgi1, T. Schwenke1 1Winterthur/Switzerland, 2Warsaw/United States of America

Purpose: Cartilage replacement with artificial materials may pose as a successful early intervention procedure in patients with confined areas of cartilage lesion or degradation. Cartilage – cartilage articulations have shown a very low coefficient of friction (COF) in the range of 0.0181. Therefore we hypothesized that the coefficient of friction measurement of cartilage articulating against artificial material can provide early information on the general mechanical performance of a material for this application. Methods and Materials: COF testing was performed on a pin-on-rotating-disk wear testing device (Figure 1). Cartilage pins (ø: 6 mm), harvested from the femoral condyle of bovine calves, were articulated against flat discs of different artificial materials. Constant sliding velocity (25 mm / s) and a constant vertical load (5 N -> 0.18 MPa) were applied. The contact was submersed in phosphate-buffered saline at room temperature (20±2°C). Friction forces were constantly measured for 60 minutes. The artificial materials were: cobalt-chrome alloy (CoCr, ISO 5832-12), polyethylene (PE, ISO 5834-2), and PCU (Bionate 55D). Three samples were tested per material, using a fresh cartilage pin for each test. Results: Two materials (CoCr, PE) experienced an increasing COF with increasing testing time (Figure 2). The COF of the PCU discs in articulation against cartilage pins remained constant at a level of µ = 0.16. Conclusions: The three different artificial materials, when tested against bovine cartilage pins, developed different behavior with respect to their coefficient of friction over time. Along with our hypothesis, this testing approach may be useful as an early and quick assessment parameter for the performance of the artificial material. 1Schmidt, Sah RL. Effect of synovial fluid on boundary lubrication of articular cartilage. Osteoarthritis and Cartilage 2007

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Results: Based on data from literature concerning the preferential targets of the respective proteinases, results were expressed as ratios of aggrecan and Col 2 to MMP-3 (MI3) and MMP-13 (MI13), respectively. Both, MI3 as well as MI13, significantly increased by mechanostimulation compared to control indicating an “anabolic shift”. Moreover, chondrocyte morphology proved to be drastically altered by appearing round rather than spindle-shaped in histological slices of stimulated compared to control matrices.Conclusions: We got strong indications of a shift from catabolic towards anabolic transcription activity. Our findings further suggest the importance of mechanical stress for metabolism and function of chondrocytes and indicate that the supposed catabolic phenotype of matrix-embedded osteoarthritic chondrocytes might be reversible by mechanostimulation.

P160Change in cartilage quality following opening wedge valgus tibial osteotomy, measured using dGEMRICM. Rutgers1, W. Bartels1, R.V. Heerwaarden2, L.B. Creemers1, R. Castelein1, W.J.A. Dhert1, K.L. Vincken1, D.B. Saris1 1Utrecht/Netherlands, 2Woerden/Netherlands

Purpose: Opening wedge valgus tibial osteotomy (VTO) is clinically effective for the treatment of knee pain caused by medial compartment osteoarthritis with varus of the knee. Although its effectiveness in pain reduction has been repeatedly shown, little is known about the effects of the change in alignment on knee cartilage quality. This study aims to evaluate the effects of such an osteotomy on knee cartilage quality, by means of dGEMRIC non-invasive imaging.Methods and Materials: 10 patients with unicompartmental osteoarthritis of the knee were treated with opening wedge VTO. Before and 9 months after surgery, cartilage quality of medial and lateral compartments were compared using dGEMRIC (3D TFE T1-mapping). In order to reduce artifacts, hardware was removed before the post-operative MRI. T1 measurements were made in selected anterior and posterior regions of interest (area size: 10 pixels) of medial and lateral condylar cartilage. Changes in clinical scores (VAS for pain, KOOS, KSCRS) were evaluated using paired T-testing and related to cartilage quality improvement.Results: Before osteotomy, average T1 was greater in the lateral compartment than in the medial compartment (p < 0.01). Clinical scores of patients undergoing osteotomy improved during follow-up (VAS p=0.02, KOOS pain p=0.05). dGEMRIC cartilage quality changed in varying degrees post-operatively, from improvement of medial cartilage quality to further deterioration of both medial and lateral cartilage quality.Conclusions: dGEMRIC confirms that patients with unicompartmental knee osteoarthritis have higher cartilage quality in the lateral than in the medial compartment. While clinical scores improved following VTO, changes in medial cartilage were accompanied by either improvement or worsening of lateral cartilage quality. Axis alignment alteration alone does not prevent further deterioration of medial cartilage quality. Moreover, clinical improvement does not seem to depend on cartilage quality. Although dGEMRIC seems valuable as research tool, its value for patient selection and clinical decision making should be further evaluated.

P161Anti-angiogenic properties of articular cartilage glycosaminoglycansJ.J. Bara1, W.E.B. Johnson2, B. Caterson3, S. Roberts4 1Oswestry/United Kingdom, 2Birmingham/United Kingdom, 3Cardiff/United Kingdom, 4Shropshire/United Kingdom

Purpose: Mature articular cartilage is normally avascular, but vascularisation can occur in diseased cartilage (e.g. osteoarthritis and degenerative disc disease). This in-growth of blood vessels has been associated with a loss of proteoglycan from the tissue. Human aggrecan is known to inhibit the adhesion and migration of endothelial cells in monolayer culture depending on the presence of its glycosaminoglycan (GAG) sidechains. We have developed an in vitro 3D model to investigate whether GAG depletion of cartilage explants increased the susceptibility of the tissue to invasion by endothelial cells. Methods and Materials: Bovine cartilage explants were treated for 24 hours with hyaluronidase. Explants were seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was quantified 4 hours and 7 days after cell seeding. GAG content was quantified using the DMMB assay and chondrocyte viability was assessed using live/dead scoring. Biochemical

on the bioreactor group, while it degraded on the control group (static cultivation). In the bioreactor group, the cells were arranged horizontally paralleled to the surface, but control group was not. The result of this study suggests that complex stimuli could affect the cartilage zonal differentiation.Conclusions: Our bioreactor loading condition that imitated joint loading movement could be a useful system in manufacturing the native mimic artificial cartilage using cells/scaffold.

P158Prostaglandin E2 up-regulation by cyclic compressive loading on 3-D tissue of human synovium-derived stem cells and inhibitory effects of COX2 selective inhibitor or dexamethasoneK. Shimomura1, T. Kanamoto1, N. Nakamura2, T. Mae1, Y. Take1, H. Kohda1, S. Kuroda1, Y. Akamine1, K. Ota1, H. Yoshikawa1, K. Nakata1 1Suita/Japan, 2Osaka/Japan

Purpose: Excessive mechanical stress on synovial joint causes osteoarthritis along with production of prostaglandin E2 (PGE2), a key molecule of arthritis, by synovial fibroblasts or chondrocytes. However, the molecular mechanism of PGE2 production by mechanical stress or the effects of NSAIDs or steroid is still unclear. The purpose of this study was to examine the expression of PGE2 by cyclic compressive loading on human synovium-derived mesenchymal stem cells (MSCs) and clarify the effects of NSAIDs or steroid.Methods and Materials: Human synovium-derived MSCs were cultured onto collagen scaffolds to produce three-dimensional constructs. Cyclic compressive loading (40kPa, 0.5Hz, 1hr) was applied to the constructs with or without the administration of COX2 selective inhibitor or dexamethasone. After 6 hours incubation, the concentrations of PGE2, IL-1beta, TNF-alpha, IL-6, IL-8 in supernatant were measured by HTRF and the mRNA expressions for COX2 and mPGES-1 genes were examined by RT-PCR.Results: The concentrations of PGE2, IL-6, and IL-8 in supernatant were significantly higher by mechanical stress, but those of IL-1beta and TNF-alpha unchanged. By administering COX2 selective inhibitor, the increased concentration of PGE2 by mechanical stress was impeded but those of IL-6 and IL-8 remained high. mRNA levels of COX2 and mPGES-1 genes were up-regulated by mechanical stress and this up-regulation of COX2 was not suppressed by COX2 selective inhibitor, while that of mPGES-1 was suppressed. With dexamethasone, up-regulation of PGE2, IL-6, and IL-8 was suppressed, and mRNA levels of COX2 and mPGES-1 were also suppressed.Conclusions: Mechanical stress on the constructs up-regulated PGE2 production, while pro-inflammatory cytokines such as IL-1beta and TNF-alpha did not change. These results indicate that PGE2 up-regulation by mechanical stress is controlled not via these cytokines but via another signal pathway. COX2 selective inhibitor suppressed PGE2 production without changing COX2 mRNAl level, while dexamethasone did by suppressing COX2 gene expression.

P159Train the chondrocytes: Mechanostimulation reverses the catabolic phenotype of human matrix-embedded chondrocytes - a preliminary reportF. Halbwirth, E. Niculescu-Morzsa, H. Zwickl, S. Nehrer 1Krems and der Donau/Austria

Purpose: The goal of this study was to determine the effect of cyclic mechanical stimulation on matrix-embedded human chondrocytes derived from osteoarthritic cartilage. For that purpose expression of genes known to be affected in diseased cartilage (MMP-3, MMP-13, collagen II, aggrecan) and its alteration upon mechanical stress was investigated via RT-PCR. In addition, matrix-embedded chondrocytes were histologically evaluated concerning a potential effect of mechanostimulation on cell morphology. Methods and Materials: Human articular cartilage was obtained from osteoarthritis patients subjected to total knee arthroplasty. After isolation of the chondrocytes from cartilage tissue and an expansion under standard cell culture conditions for 14 to 20 days they were embedded in a collagen I-matrix. Following cultivation for 12 to 16 days, cell-seeded matrices were either mechanically stimulated (cyclic sinusoid compression regime for 4 days) or further cultivated without stimulus. For measurement of gene expression, chondrocytes were isolated and mRNA levels of the genes of interest were gathered via RT-PCR. Moreover, morphology and distribution of chondrocytes in the differentially treated matrices were histologically determined.

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Results: The cells were monitored every 24h until day 5, when was performed the analysis of Alkaline Phosphatase expression and the quantification of total protein. These evaluations were also realized at day 10, when was determinate the initial cell differentiation. The proliferation results shows decrease of RFU (Relative Fluorescence Units) after 24h due to environment changing and cell adaptation on the morphologies of each collagen membrane. The results of resasurin increases after 72h and 96h by the proliferation into the porous surfaces (see Fluorescence Microscopy day 3) and decrease after 120h. The ends of these evaluations are the comparison between Alcaline phosphatase expression and amount of protein for 5 and 10 days.Conclusions: The biodegradable collagen membranes Bilayer and Hydroxyapatite coated showed excellent morphology for cell growing. These biomaterials have presented good properties for tissue engineering as scaffold for cells culture from human mesenchymal stem cells.The authors are grateful to FAPEMIG for the financial support.

P165TGF-beta1 release from hyaluronan-coated poly-e-caprolactone nanofibrous and microfibrous scaffolds promotes expression of collagens type I and II in human mesenchymal stem cellsJ.C. Schagemann1, S. Paul1, M.E. Casper2, J. Rohwedel1, J. Kramer1, J.S. Fitzsimmons2, C. Kaps3, M. Fehr4, M. Russlies1, S. O’Driscoll2, G.G. Reinholz2 1Luebeck/Germany, 2Rochester/United States of America, 3Berlin/Germany, 4Hannover/Germany

Purpose: The objective was to develop a scaffold that presents cartilage-like matrix to bmdMSCs together with a release of growth factors for cell recruitment, proliferation and chondrogenic differentiation, which would make supply of inductive factors redundant.Methods and Materials: Nanofibrous (~400 nm) and microfibrous (~10 um) PCL scaffolds were combined with 1% HMW sodium hyaluronate (NHA/MHA), 1% sodium hyaluronate and 200 ng TGF-beta1 (NTGF/MTGF), or 0.1% BSA (N/M). Scaffolds were seeded with bmdMSCs and cultured without growth factors in vitro. Cultures with chondrogenic medium served as controls (ScInd). Proliferation, migration, and release of TGF-beta1 were investigated. Cell differentiation was evaluated by rtPCR.Results: MTGF released TGF-beta1 for 72 hrs, NTGF for 12 hrs. None of the scaffolds recruited bmdMSCs. Composites containing HA demonstrated elevated seeding efficiency. TGF-beta1 had no proliferative effect. There was an increase in COL2 expression with TGF-beta1 containing scaffolds promoting higher yields. This was independent of scaffold composition or fiber size. Significantly higher values were found for ScInd. All scaffolds allowed for COL1 expression with TGF-beta1 containing scaffolds showing highest yields. COL1 expression decreased in NTGF/MTGF and NHA/MHA after 21 days, whereas scaffolds supplied with TGF-beta1 in the medium promoted an increase until day 21. PCR identified also bone-specific markers, yet not as striking as for bmdMSCs supplied with osteogenic medium. Varying alkaline phosphatase levels were found in N/M and NHA/MHA, and a decline in NTGF/MTGF after 21 days. Osteopontin decreased regardless of scaffold composition after 21 days.Conclusions: Combining HA and TGF-beta1 with PCL scaffolds fosters cell seeding and proliferation. TGF-beta1 released from MTGF promotes COL2 expression, although not as effective as TGF-beta1 supplied with the medium. TGF-beta1 induces also COL1 followed by a decline after 21 days when released from NTGF/MTGF. The presence of nanofibrous PCL and HA suppresses COL1 compared to a TGF-beta1 containing milieu. No significant impact of fiber size was seen in our model.

P166The assembly of platelet lysate-loaded chitosan-chondroitin sulfate nanoparticles as a three dimensional hydrogel construct for encapsulation of adipose derived stem cells for cartilage regenerationV. Espírito Santo, M.E. Gomes, J.F. Mano, R.L. Reis Caldas das Taipas, Guimarães/Portugal

Purpose: Platelet lysates (PL) are an outstanding natural source of growth factors (GFs) that can play an enhancement role over the proliferation and differentiation ability of mesenchymal stem cells. Moreover, their autologous nature is an advantage for regenerative medicine applications as they can be easily obtained

composition of the ECM was investigated immunohistochemically with antibodies against aggrecan core protein, chondroitin-6-sulphate, chondroitin-4-sulphate, keratan sulphate and lubricin. Results: Hyaluronidase treatment reduced explant GAG content by 79±3% compared to controls. GAG depletion was associated with significantly more HMEC-1 adherence (11±1 cells/mm) compared to controls (3±0.4 cells/mm) at 4 hours (p<0.0001). HMEC-1 cells adhered to the surface and deeper/calcified zones of the cartilage, but did not appear to migrate into the tissue during culture. Chondrocyte viability was significantly lower in GAG-depleted explants compared to controls, i.e. 66±2% vs. 87±1% respectively (p<0.0001); however further experiments confirmed that reduced cell viability alone did not increase HMEC-1 adhesion. Hyaluronidase treatment altered chondroitin sulphate patterning, but not lubricin. Conclusions: This work supports the hypothesis that GAGs in mature articular cartilage have anti-angiogenic properties. Hence, the loss of proteoglycan that occurs in OA may make the tissue more susceptible to pathological vascularisation. Furthermore, manipulating GAG content may be a useful technique to control vascularisation in osteochondral tissue engineering.

P163The redifferentiation potential of human articular chondrocytes can be modulated by the pore size of poly(urethane urea) scaffoldsH. Stenhamre1, U. Nannmark2, A. Lindahl2, P. Gatenholm2, M. Brittberg3 1Gäteborg/Sweden, 2Göteborg/Sweden, 3Kungsbacka/Sweden

Purpose: It is suggested that the chemical and physical properties of scaffolds affect cellular behavior. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold (ArtelonÒ) could be a suitable material for cartilage tissue engineering. In addition we investigated whether the pore sizes of the scaffold could modulate the redifferentiation capacity of in vitro expanded articular chondrocytes. Methods and Materials: Scaffolds with different pore sizes, <150µm, 150-300µm and 300-500µm, were seeded with chondrocytes and cultured in chondrogenic media up to 28 days in vitro. Results: The chondrocytes redifferentiated and produced cartilage specific ECM in the poly(urtehane urea) scaffolds. The scaffolds with the smaller pore size favoured redifferentiation and thus enhanced the hyaline-like extracellular matrix production. Conclusions: In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. The chondrogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes the performance of the tissue engineered cartilage constructs is modulated and thus also the clinical outcome in the long run.

P164Human stem cells culture into biodegradable collagen scaffolds for guided tissue regeneration.R.F.C. Marques1, H. Tavares2, A.F. Fraga3, H.M.G. Tómas4, J.L. Santos4 1Poços de Caldas/Brazil, 2São carlos/Brazil, 3São Carlos/Brazil, 4Funchal - Ilha da Madeira/Portugal

Purpose: Biomaterials like collagen barriers are current employed in guided tissue regeneration (GTR) for plastic and reconstructive surgery, dental implants and bone graft procedures. The actual challenges of sportive medicine are trauma in muscle, tendon and cartilage structures where clinical and surgical treatment shows many controversies. The aim of this preliminary study was to evaluate the behavior of human stem cells and proliferation after the initial stage of cell adhesion among collagen matrices, with different compositions, nanostructures and mechanical properties.Methods and Materials: After local ethical committee approbation, human mesenchimal stem cell (hMSC) from fourth passage with density of 5x104 cells/sample were seeds onto collagen membranes manufactured by PROCELL BIOLOGICS LTDA. The experiments were realized with (n=6) for all experimental groups. The cell adhesion and proliferation was analyzed by fluorescent method using Alamar Blue assay. In order to evaluate morphological aspects, fluorescent labeling by Alexafluor were performed.

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MW of either 25kDa or 50kDa. Based on a large sweep, two water-dioxane (0.0105 and 0.0405) ratios were used producing four different scaffolds. The cells were cultured for 1, 3 and 6 days. Using quantitative RT-PCR expression of chodrogenic markers sox9, collagen type 1 and 2 and aggrecan as well as cytoskeletal- and adhesion markes beta-actin, fibronectin, vinculin and integrin 10aplha were investigated. Reference genes were B2M and RPL13A. Possible differences were investigated using three-way ANOVA measurements.Results: Scaffolds with a lyophilized PCL compound of 25kDa resulted in a significantly higher expression of collagen type 2, aggrecan and vinculin (pConclusions: We successfully constructed a structurally graded scaffold for cartilage repair. There was a significant positive effect of the structure made by 25kDa-lyophilized material compared to 50kDa. The lower MW might lead to a combination of faster degradation time and increased chondrogenic viability for scaffold-aided cartilage repair techniques.

P169In situ gelling Tetronic depots for sustained delivery of growth factorsJ. Couceiro, A. Rey-Rico, M. Silva, A. Concheiro, C. Alvarez-Lorenzo Santiago de Compostela/Spain

Purpose: Local delivery of growth factors using minimally invasive in situ gelling solutions is an efficient and patient-friendly alternative to surgical grafts. The aim of this work was to evaluate the potential of X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers, named as Tetronics, as components of liquid and easily syringeable solutions that undergo a transition to the gel state at the body temperatureMethods and Materials: A cytoxicity screening of a wide number of Tetronic varieties, the in situ gelling capability of Tetronic 908, 1107, 1301 and 1307 solutions was rheologically characterized. The release profile of BSA and BMP-2 was recorded and the ALP activity measured using mesenchymal stem cells.Results: The gelling temperature of 20% w/w solutions of 908 was 33ºC and of 1107, 1301 and 1307 solutions around 25ºC; being highly viscoelastic at 37ºC. Such solutions evidenced a remarkable capability to sustain the delivery of proteins under physiological-like conditions. Particularly, formulation of BMP-2 led to relevant differentiation of mesenchymal cells to osteoblasts, quantified as alkaline phosphatase activity.Conclusions: Tetronic varieties (908, 1107, 1301 and 1307) combine good cytocompatibility with the ability to undergo sol to gel transitions at body temperature. Autoclaving does not alter the rheological properties, which is an important aspect for the development of injectable formulations. Tetronic gels can modulate the release of proteins depending on hydrophilicity and molecular weight of the copolymer. Initially rapid delivery of an important amount of BMP-2 followed by slower release enables the Tetronic gels to exhibit a marked ALP activity at day 7 and matrix mineralization at day 14. Thus, Tetronic is a very promising material for syringeable in situ gelling formulations with osteogenic performance, acting as a suitable BMP carrier. Similar application could be used for cartilage cells differentiation by means of delivery of proper growth factors.

P170Development of a structurally graded polycaprolactone (SG-PCL) scaffolds for hyaline cartilage repairB.B. Christensen, C.B. Foldager, A. Kristiansen, D. Le, J.V. Nygaard, C. Bünger, M. LindAarhus/Denmark

Purpose: Rapid prototyping is a very precise scaffold manufacturing technique where scaffolds can be constructed from MRI or CT scans to fit into the individual tissue defects. The aim of this study is to develop a novel Structurally Graded Poly-Capro-Lactone scaffold using rapid prototyping for cartilage tissue engineering.Methods and Materials: A novel SG-PCL scaffold was constructed using rapid prototyping (Ø 4 mm, height 2 mm). PCL fibers (MW 50 kDa) with a diameter of 120 µm were plotted producing a 3-dimensional web. The scaffold was subsequently submerged into a mixture of dioxane, PCL and water, and lyophilized at -32°C, creating an extremely porous graded structure. By shifting the water-dioxane ratio, 16 batches of scaffolds with different pore

from a simple blood sample from the own patient in treatment. Natural based chitosan/chondroitin sulfate nanoparticles (CH/CS NPs) were developed and characterized with the ultimate goal of encapsulating bioactive agents to promote and enhance cartilage regeneration. Previous studies performed in our group reported the successful incorporation of PL in these NPs, which were then released in a controlled manner in two and three dimensional (2D and 3D) in vitro cultures of human adipose derived stem cells (hASCs), enhancing the proliferation rate of hASCs while they are differentiating into the chondrogenic phenotype. When used at determined concentrations, the PL-releasing NPs can assemble in simple and quick mode and form a 3D stable hydrogel while in suspension with hASCs, following a mild centrifugation, allowing the cells to be entrapped in this enriched 3D environment. Methods and Materials: The hydrogels were cultured in vitro in chondrogenic medium and basal medium up to 28 days and were characterized for cell viability, proliferation, glycosaminoglycans production, histology, immunohistochemistry and gene expression (by polymerase chain reaction).Results: The presence of high levels of GFs such as Transforming Growth Factor β1 (TGF-β1) influence the biological response of the entrapped cells, stimulating their viability, proliferation and the production of a cartilage extracellular matrix throughout the culture time.Conclusions: PL is a strengthening factor in the morphological stability of the assembled NPs as they enhance the interactions between the individual natural-based complexes and, in combination with ASCs, it is obtained an innovative system with a high potential of application in cartilage tissue regeneration.

P167Long-term clinical and radiological follow-up of collagen meniscal implants.P.C. Verdonk1, J. van der Maas2, T. Tampere2, K.F. Almqvist2, R. Verdonk2 1Gent-Zwijnaarde/Belgium, 2Gent/Belgium

Purpose: There is growing evidence in literature that meniscal substitution in the post-meniscectomized knee results in significant pain relief and functional improvement of the involved joint. The collagen matrix implant (CMI) is a bioabsorbable scaffold to substitute a partial medial meniscus defect. Long-term data on clinical and radiological outcome of this CMI implant are however scarce.Methods and Materials: We evaluated 15 transplants with a mean time of follow-up of 9,9 ± 1,9 years, mean age at surgery was 31,6 years (range 16-49). Clinically, the patients were evaluated using a KOOS, SF-36, HSS, VAS, Tegner and Lysholm score. Each patient received radiographs (AP, profile and Rosenberg view). Radiological outcome parameters were joint space width narrowing and Fairbank changes and were scored according to IKDC. Failures were defined as graft removal or conversion to an arthroplasty.Results: HSS-scores at long-term follow-up were 184 ± 21. Lysholm-score was 84 ± 19, which was defined as a good result. Mean VAS-score was 1,7 ± 2,7, mean Tegner was 4 ± 2. There were no significant differences between following subgroups: left or right knee and male or female. Five (33%) of the fifteen implants failed after a mean of 3,8 years. Conclusions: Transplantation of a collagen matrix implant scaffold can significantly relieve pain and improve function of the knee joint. Survival analysis showed that this beneficial clinical effect remained in approximately only 70% of the patients at ten years.

P168Chondrocyte viability is affected by PCL molecular weight in structurally graded polycaprolactone (SG-PCL) scaffoldsC.B. Foldager, B.B. Christensen, A. Kristiansen, D. Le, J.V. Nygaard, C. Bünger, M. Lind 1Aarhus/Denmark

Purpose: The aim of the study is to investigate cellular effects of the physical structure in a novel SG-PCL scaffold on human chondrocytes, when the scaffold is produced with different molecular weights (MW) and water-dioxane ratios.Methods and Materials: Rapid prototyping was used to make a porous PCL backbone macro-structure (Ø=4mm, height=2, MW=50kDa). Micro and nano-structure in the scaffold porosity was made by lyophilization of a PCL, water and dioxane mixture. Two different lyophilized structures was made using PCL with a

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Results: By postoperative week 1, the osteochondral defects of both groups were filled with hematoma. At week 2, fibrous membrane covered the surface and the border between the defect and surrounding bone became unclear at the deep area in both groups. At week 4, cartilage regeneration was found at the whole surface of the scaffold. In the control group, the regenerated tissue was concaved and cartilage regeneration was observed only at the peripheral area of the defect even at week 4. VEGF was detected in the whole area of defect by week 2. At week 4, in the scaffold group, localization of VEGF decreased and type II collagen was observed at the surface of the scaffold. VEGF was still remained at the concaved surface in the control group. Conclusions: In the present study, VEGF was localized at the whole osteochondral defect and decreased time dependently at the surface of regenerated tissue, and type II collagen was observed at week 4. The angiogenesis during chondrogenic differentiation is one of the important issues affecting the application of stem cells for cartilage regeneration.

P173Osteochondral defect repair using a bilayer scaffold implant containing BMP-7 and IGF-1M. Ast1, P. Razzano2, D. Grande2 1New Hyde Park/United States of America, 2Manhasset/United States of America

Purpose: Articular cartilage injuries in the knee are painful and disabling, predisposing the patient to early arthritis. In this study we use a bilayer scaffold implanted with cells engineered to overexpress BMP-7 and IGF-1 in an attempt to improve the quality of the repair tissue. It is our hypothesis that gene-modified tissue engineering using this bilayer implant will regenerate a durable, physiologic articular surface.Methods and Materials: BMP-7 and IGF-1 were isolated from human cells using RT-PCR and retroviral expression plasmids were generated for each based on the LNCX series of vectors. Northern blot analysis was then used to demonstrate effective gene transfer and ELISA was performed to confirm BMP-7 and IGF-1 secretion from primary rabbit periosteal cells. 4 x 106 gene-enhanced periosteal cells were seeded onto PGA scaffolds prior to implantation. Animals were randomly assigned to one of four experimental groups which included: 1) Empty Defect, 2) PGA Alone, 3) LNCX/PGA, 4) BMP-1/IGF-1. The BMP-7 cell laden scaffold was placed in the bony defect and the IGF-1 containing scaffold layer was placed congruent with the cartilage layer. Animals were then sacrificed at 6, 10, 12, and 24 post operative weeks. The rabbit knees were fixed and evaluated for histology. Histological sections were graded using a modified O’Driscoll scoring system.Results: The experimental defects treated with a bilayer BMP-7 and IGF-1 scaffold showed histologically superior repair tissue when compared to each of the other groups.Conclusions: Gene-modified tissue engineering using a bilayer implant containing both BMP-7 and IGF-1 to repair an osteochondral defect can regenerate a histologically superior repair tissue when compared to no intervention, or implantation of an PGA scaffold alone.

P174Growth Factor Release from Platelet-rich Plasma (PRP) Loaded TRUFIT(TM) Scaffolds without the Use of ThrombinA. Au Andover/United States of America

Purpose: To assess the loading of PRP in TRUFIT scaffolds, determine the in vitro growth factor (GF) release kinetics from thrombin-free PRP-loaded TRUFIT scaffolds, and the activation state of the platelets. Methods and Materials: PRP was produced using Caption PRC and loaded into TRUFIT scaffolds. No thrombin was used in any part of the process. To measure GF release, PRP-loaded scaffolds (n=3) were placed in DMEM media at 37ºC. During the course of 10 days, media was collected at each time point and replenished with fresh DMEM. GF concentration in the collected media was measured by ELISA. Platelet activation in PRP and whole blood was quantified by microparticle count, percentage of P-selectin+ platelets and PAC-1+ platelets using flow cytometry (n=3). Results: PRP filled up (95.0±7.3)% of the voids in TRUFIT scaffolds after loading. PRP gelled in TRUFIT scaffolds in ~5 minutes, faster than PRP alone and likely owing to the calcium sulphate in the

sizes were made. Using scanning electron microscopy, two scaffolds were selected, based on porous structure. The two scaffolds were then constructed with a graded structure of either 25 kDa or 50 kDa, giving a total of four different scaffolds. They were cultured with human chondrocytes and the viability was analyzed using confocal microscopy after 1, 3 and 6 days. The scaffolds were rated based on cell migration, cell shape and distribution of viable cells.Results: The scaffolds contained macro-, micro-, and nano-pores. A large difference in investigated parameters was observed and a water-dioxane ratio of 0.0415 provided the most viable environment for chondrocytes.Conclusions: We successfully constructed a SG-PCL scaffold that can be used in future in vivo experiments, and has the potential of subsequent functionalization with nanoparticles and growth- and differentiation factors.

P171A Hybrid Scaffold For The Functional Repair Of Articular Cartilage DefectsK.W. Ng1, H. Hsu2, K. Joh1, P.A. Torzilli1, R.F. Warren1, S.A. Maher1 1New York/United States of America, 2Taichung/Taiwan

Purpose: Clinical success in the use of scaffolds to treat focal cartilage defects depends on the scaffold’s ability to bear mechanical loads and integrate with surrounding tissue. In this study, we hypothesized that a hybrid scaffold with a solid polymer core and a porous periphery would improve the load-bearing ability of the scaffold while maintaining the ability to facilitate chondrocyte migration. Methods and Materials: Scaffold Fabrication: Cylindrical gelatin sponges (Ø7x2mm) were impregnated with 10% polyvinyl-alcohol (PVA) solution and freeze-thawed 6 times. In half of the sponges, a hybrid scaffold was creating by excising a Ø5mm central core and filling the hole with 10% PVA solution. All sponges were freeze-thawed an additional 6 times and digested with collagenase. Scaffold morphology was evaluated using environmental scanning electron microscopy. Mechanical properties were determined using unconfined compression tests. In vitro cartilage defect model: Disks of middle-zone, calf cartilage were cored to create annuli. These annuli were treated briefly with collagenase and then cell-free porous scaffolds were press-fit to fill the central defects of the annuli. These scaffold-cartilage constructs were then cultured and histologically analyzed. Results: Macropores were present in the porous scaffolds and the porous periphery of the hybrid scaffolds (Figure 1), with no significant differences in average pore diameter (~15μm) or apparently porosity (~80%). Compressive modulus was ~12kPa for porous scaffolds and ~55kPa for hybrid scaffolds. In the defect model (Figure 2), chondrocytes infiltrated the scaffolds and produced a proteoglycan rich matrix, with cell number and migration distance increasing significantly over time to ~500 μm after 28 days. Conclusions: This hybrid scaffold presents a modular design where the compressive modulus of the polymer core can be controlled and the porous periphery can facilitate cell infiltration towards tissue integration. Varying the polymer concentration of the solid core to improve load bearing of the scaffold will be explored in future work.

P172Localization of VEGF in cartilage repair using bioabsorbable synthetic polymer scaffoldR. Sakata1, H. Fujioka2, K. Makino2, T. kokubu1, I. Nagura1, N. Toyokawa1, A. Inui1, M. Satake3, H. Kaneko3, M. Kurosaka1 1Kobe/Japan, 2Nishinomiya/Japan, 3Tokyo/Japan

Purpose: The purpose of the present study is to investigate the localization of VEGF at the early stage of cartilage repair using bioabsorbable synthetic scaffold in a rabbit model. Methods and Materials: Forty-eight female Japanese white rabbits were used in this study. Osteochondral defect (5 mm diameter, 5 mm depth) was created on the patellar groove of the right knee under general anesthesia using the Osteochondral Autograft Transfer System. Then, rabbits were divided into two groups. The defect was treated with PLG scaffold in the scaffold group and the defect was left untreated in the control group. At postoperative 1, 3, 5 days and 1, 2, 4 weeks, the specimens are examined macroscopically and histologically. Immunolocalization of VEGF and type II collagen were also analyzed.

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compressive stress for 10h followed by a 10h relaxation period under 0.05MPa. Integration strength testing was performed on the three layered constructs using plexiglass rods mounted in a mechanical test frame and pulled to failure in tension at a displacement rate of 10 µm/s, with resultant loads recorded at 5Hz. Results: All constructs had a similar appearance to native cartilage shown by Safranin-O (Fig. 1B) and toluidine blue stains (Fig. 1C). Specimens stained positive for COL II (Fig. 1D). Creep strain (%) after 20 h of loading showed the PVA-FG chondrocyte group performing similarly to native cartilage (Fig. 1E). The presence of cells and FG significantly enhanced the integration strength of layered constructs (p<0.05 for both parameters by 2 way ANOVA). The combination of cells and fibrin glue increased integration strength more than 11 fold from 12 kPa to 133 kPa (Fig. 1F). Conclusions: Bio-synthetic cartilage was engineered using articular chondrocytes in vivo with a novel porous PVA-gel hybrid.

P177Mechanical properties of PLLA scaffolds for cartilage tissue engineering depending on their micro and macrostructureV. Acosta1, H. Deplaine2, I. Ochoa1, G. Gallego Ferrer2, M. Doblaré1, J.L. Gómez-Ribelles2, J.M. García-Aznar1 1Zaragoza/Spain, 2Valencia/Spain

Purpose: Actually, the application of biodegradable polymeric materials with three-dimensional structure to facilitate the adhesion, diffusion and proliferation of cells for tissue regeneration represents an important field of investigation. The structural scaffolds not only permit passive mechanical support but also perform an interactive physical-chemical role in the tissue regeneration. This work presents a characterization of Poly,L-lactic acid (PLLA) scaffolds, comparing experimental mechanical properties (Young and Aggregate Modulus), permeability, porosity and pore size. This information allows a better understanding of how the mechanical properties of PLLA scaffolds change depending on the micro and macrostructure. Different concentrations of PLLA/Dioxan and PLLA/Porogen could be used in order to achieve a correct structural scaffold’s design. Methods and Materials: Uniaxial Compression (Unconfined (UC) and Confined (CC)) static tests were performed to characterize the mechanical properties of the scaffolds. To determine a relation between interconnected porosity and pore size, a permeability test was carried out.The microstruture was obtained by micro CT and SEM.Results: Regarding the PLLA/Porogen concentrations, a higher amount of Porogen causes lost of stiffness and raise of the permeability and porosity values (figure 1). macropore distribution and micropore sizes are associated with an augment of structure stiffness (figure 2). The results show that an increase of the wt% PLLA augments the uniformity and the distribution of scaffold’s pores and trabeculae, granting higher mechanical properties illustrated by the Modulus values. When the wt% PLLA increases the permeability value decreases. All this results are independent from the changes in porogen relation. Conclusions: It’s important to underline that if scaffolds are designed for confined application, Aggregate Modulus should be taken as critical factor, whereas for scaffolds subject to a high lateral deformation, the Young Modulus represents the most relevant design parameter. A correct scaffold design should take into account the microstrututre as a key factor to control the mechanical properties.

P178Ultrasonic Noninvasive Evaluation of Preconditioned Scaffolds in Articular CartilageS.R. Ghorayeb1, T. Awad1, P. Razzano2, D. Grande2 1Hempstead/United States of America, 2Manhasset/United States of America

Purpose: While several imaging modalities have been utilized to observe articular cartilage injuries in the knee, ultrasonography has not played a major role in this area. This study demonstrates that ultrasound may be used to non-invasively monitor the healing process of osteochondral defects that are implanted with preconditioned bioactive scaffolds.Methods and Materials: Rabbit marrow stromal cells were retrovirally transduced with either BMP-7 or IGF-1 genes and altered for 9 weeks in non-woven PLLA scaffolds, then frozen and lyophilized. A total of 16 adult male White New Zealand rabbits

implant. The significantly higher percentage of microparticles, P-selectin positive platelets and PAC-1 positive platelets in PRP than whole blood indicates the platelets in PRP were activated, even without thrombin. SEM showed activated platelets of spiculated shape and mesh-like fibrin network within the scaffold. The gradual release of GFs from PRP-loaded TRUFIT scaffolds continued out to 10 days in vitro. The 10-day cumulative amount of VEGF, TGF-β1 and PDGF-AB released was (2.7±0.9), (4.0±1.8) and (0.8±0.0) times the GF content of lysed whole blood of the same volume respectively. The low enrichment of PDGF-AB is expected as the concentration of PDGF-AB in PRP generated by Caption was less than 1.5 times the concentration in whole blood. Conclusions: PRP-loaded TRUFIT scaffolds released GFs gradually for up to 10 days without thrombin. TRUFIT scaffolds with PRP can be used to deliver autologous GFs to repair sites.

P175In vitro 3-D scaffold-based cultures for potential human meniscus repairU. Freymann, D. Paape, L. Litau, L. Morawietz, K. Neumann, M. Endres, C. Kaps Berlin/Germany

Purpose: Treatment options for meniscal lesions are rarely developed. Recent approaches use scaffold-based techniques based on the long experience with matrix-induced treatment options for hyaline cartilage.The aim of the study was the investigation of meniscal matrix formation of in vitro expanded human meniscal cells in a 3-dimensional bioresorbable scaffold for potential meniscal repair approaches.Methods and Materials: Cultivation of the human meniscus cells was performed in a resorbable scaffold material made of polyglycolic acid (PGA) with the application of hyaluronic acid (HA). To investigate the impact of human serum (HS) and HA as potential inducing factors for meniscus matrix formation, high-density micromass cultures supplemented with human serum (HS) or HA were additionally examined in comparison to an non-stimulated control. Biocompatibility and cell vitality of human meniscus cell seeded PGA scaffolds were evaluated by fluorescein diacetate (FDA) and propidium iodide (PI) staining. Verification of typical meniscal markers like type I collagen and other extracellular matrix molecules was performed histologically, immunohistochemically and by gen expression analysis in meniscus cell seeded PGA scaffolds and in high-density micromass cultures. Results: In results, 3-D scaffold-based meniscus cultures showed an excellent cell vitality and cell expansion over an observational period of 21 days. On the protein level type I collagen and proteoglycan expression was documented. Gen expression analysis confirmed the expression of meniscus-specific markers. Supplementation of HS or HA in high-density cultures was shown to stimulate meniscal matrix formation. Conclusions: In this study we demonstrated, that in vitro expanded human meniscus cells allow for formation of meniscal matrix components when cultured in PGA scaffolds supplemented with HS and HA. This results encourage scaffold-based approaches for the treatment of meniscal lesions.

P176Porous Poly(vinyl Alcohol) Hydrogel Matrix-Engineered Bio-Synthetic CartilageD.A. Bichara1, X. Zhao1, H. Bodugoz-Senturk1, F. Ballyns2, L. Bonasser2, M.A. Randolph1, O.K. Muratoglu1 1Boston/United States of America, 2Ithaca/United States of America

Purpose: Poly(vinyl alcohol) (PVA) gel is a non-degradable material that can be tailored to be viscoelastic and have a high water content. We hypothesized that porous PVA combined with articular chondrocytes suspended in fibrin gel (FG) creating a PVA-FG hybrid construct could mimic the properties of native cartilage.Methods and Materials: PVA hydrogel was prepared by theta gelation by dissolving PVA, polyacrylamide-co-acrylic acid and poly(ethylene glycol) in 90°C deionized water. The solution was molded and cooled down for gelation. Hydrogel was imaged by SEM (Fig. 1A). Chondrocytes from swine knees were isolated, expanded and mixed with thrombin and fibrinogen to create FG that was injected into PVA discs (6mm x 2.1mm) and a three layered construct model (Peretti, Tissue Eng. 1999). Constructs were implanted for 12w into nude mice and subjected to histological and immunohistochemical analysis. PVA discs underwent creep strain by applying a 0.5MPa

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Methods and Materials: Multipotent mesenchyaml stem cell-like cells (MSC-hESCs) were derived from the hESC line, KCL-2, by plating 7-day embryoid bodies (EBs) in serum containing medium with FGF-2. The cells were characterized for surface markers by FACS and were seeded onto PGA or HYAFF-11 scaffolds with or without fibronectin coating. The constructs were incubated with different combinations of growth factors for 35 days on a rotating platform and markers of chondrogenic differentiation were analyzed by quantitative RT-PCR and immunoassays.Results: MSC-hESCs had fibroblastic morphology and expanded for more than 60 days. They expressed several makers found on adult MSCs including CD105, CD73, CD117, BMPR1 and VCAM and were multipotent upon adipogenic, osteogenic and chondrogenic stimulation in monolayer. The best chondrogenic stimulation on 3D scaffolds was found with fibronectin-coated scaffolds in the presence of TGF-β1 and BMP-7 but not with BMP-2, BMP-7 or TGF-β1 alone. Quantitative analysis of dry weight, aggrecan and type II collagen mRNA and protein in cartilage engineered constructs revealed levels that were 10-30% of constructs engineered using adults MSCs stimulated with TGF-β3. The MSC-hESC constructs had double the hydroxyproline content when compared to adult MSC constructs.Conclusions: The data highlights the importance for quantitative analysis in assessing the quality of cartilage tissue engineering using hESCs and the need for optimal matrix and soluble signals to drive their chondrogenic differentiation.

P182The influence of culture media and supplements on phenotype and differentiation properties of human bone-marrow derived mesenchymal stromal cellsS. Hagmann, B. Moradi, W. Richter, T. Gotterbarm Heidelberg/Germany

Purpose: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal cells (BM-MSCs) is of major importance in modern tissue engineering, including novel cartilage repair procedures. Different cell isolation and expansion conditions have been described, with distinct variations of culture media, growth factors and other supplements. The aim of this study was to analyse the influence of culture media and supplements on immunophenotype and chondrogenic differentiation potential of human BM-MSCs. Methods and Materials: Bone marrow derived mononuclear cells (BM-MNCs) were isolated with gradient centrifugation and expanded in different cell culture media (alphaMEM, DMEM-LG, DMEM-HG, DMEM-F12, n=6), with different supplements described earlier. Additionally, cells from 6 donors were expanded in alpha-MEM and DMEM with or without addition of bFGF. In P4, immunophenotyping was performed by FACS analysis for fluorochrome labelled CD10, CD13, CD14, CD34, CD44, CD45, CD49, CD73, CD90, CD105, CD117, CD133, CD 140b, CD 166, CD271, CD340, Stro-1 and HLA-ABC. Also, chondrogenic and osteogenic differentiation were induced using standard protocols. Results: Cell proliferation differed substantially between the media with significantly lower growth index values for DMEM-LG with 10% FCS. “Negative markers” for MSCs (e.g. CD14, CD34, CD45) showed a high reliability in all donors and media, while expression of certain surface markers (e.g. CD10, CD49) was strongly donor-dependent. The expression of CD10 and CD146 showed significant differences depending on the media used for expansion. Supplementing bFGF caused significant changes of CD10, CD49 (Fig.1) and CD146 expression while the expression of CD13, CD73 and CD133 appeared to be independent of bFGF addition. “Negative” surface markers showed to be independent of bFGF addition. The different cell culture media had a significant influence on the differentiation ability of MSCs, with the highest chondrogenic potential obtained after expansion in DMEM-LG with 10% FCS. Conclusions: Our results show that distinct culture media have a determining influence on both immunophenotype and therapeutic potential of MSCs.

underwent bilateral knee arthrotomies to create 3.7 mm diameter osteochondral defects. 24 defects were randomly implanted with preconditioned PLLA scaffolds and 8 defects were left empty as controls. The knees were then harvested at 3, 6, and 12 weeks post-surgery and evaluated using scanning acoustic microscopy. B-scans were obtained and compared to histological results.Results: The osteochondral defects were clearly observed in each ultrasound signature. Images containing an empty defect were characterized as having a gap between the grooves of the femur, while those containing a scaffold displayed a filling within the area. There were no significant differences between images of scaffolds treated with IGF-1 or BMP-7. A defect depth of 4 mm and scaffold thickness of 2.5 mm were measured. Also a gradual increase of healing bordering the defects for the 3, 6 and 12 week samples was observed. Histological results revealed similar outcomes where the scaffold was still intact along with the cartilage and minimal bone growth within the defect.Conclusions: Ultrasound can aid in monitoring implantation of preconditioned scaffolds in osteochondral defects and thus assessing the healing process and cartilage/bone quality. This method was successful at distinguishing the physical border between the defect and the scaffold. Lateral edge integration was observed in the B-scans and correlates well with histological results.

P179Cell proliferation and differentiation capacity of magnetically labeled mesenchymal stem cells – the effect of an external magnetic forceG. Kamei, N. Adachi, H. Shibuya, Y. Nagata, S. Ohkawa, M. Ochi Hiroshima/Japan

Purpose: Many studies on regenerative medicine are currently focused on tissue engineering. Tissue engineering usually requires technically demanding procedures with proper scaffolds or growth factors. Therefore, intravenous or intraarticular cell transplantation without scaffolds and growth factors is a more attractive option. We developed a novel stem cell delivery system for regenerated medicine using MSCs with superparamagnetic iron oxide (magnetically labeled MSCs:m-MSCs) and an external magnetic device to accumulate a relatively small number of MSCs to a desired area. But, it had not been approved whether m-MSCs that were exposed to external magnetic force could proliferate and differentiate. The purpose of this study was to evaluate cell proliferation, differentiation into chondrogenesis, osteogenesis and adipogenesis of m- MSCs that was exposed to external magnetic force, and the effect of the magnetic force strength and exposure time. Methods and Materials: MSCs were labeled overnight with ferucarbotran and protamine as a transfection agent. We evaluated cell proliferation using cell-counting kit and compared the absorbance of day 0 with that of day 7 about non-magnetically labeled MSCs (non m-MSCs), m-MSCs and m-MSCs that were exposed to magnetic force with various condition (Magnetic force: 0.6T, 1.5T, and 3.0T. Exposure time: 10 minutes, 30minutes and 60 minutes). Also we evaluated chondrogenesis, osteogenesis and adipogenesis in their conditions.Results: Cell proliferation of m- MSCs that were exposed to magnetic force was significantly larger than that of m-MSCs and non m-MSCs. And magnetic labeling and exposure to magnetic force do not have any adverse effects to chondrogenesis, osteogenesis and adipogenesis.Conclusions: This study showed that magnetically labeling of MSCs had no advertise effect on cell proliferation and differentiation, and we could use them safety. We believe that we can apply this novel stem cell delivery system using m-MSCs and an external magnetic device for regenerated medicine.

P180Cartilage tissue engineering by human embryonic stem cellsK. Ridgway1, H. Jia1, S.C. Dickinson1, T.J. Sims1, S. Minger2, A.P. Hollander1, W. Kafienah1 1Bristol/United Kingdom, 2London/United Kingdom

Purpose: The use of human embryonic stem cells (hESCs) as a source for chondrogenic stem cells remains to be established. Previous attempts were qualitative and limited to using H1 and H9 cell lines in pellet cultures or hydrogels. Our aim was to investigate quantitatively the optimal factors that drive cartilage tissue engineering by hESCs using therapeutic biodegradable scaffolds.

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Conclusions: Our data reveal that in young and elderly patients alike, synovial explants can be induced to undergo chondrogenesis in vitro, even if the tissue is derived from differently diseased joints.

P185Tendon cells from rotator cuff differentiate into chondrogenic lineageI. Nagura, T. kokubu, T. Makino, H. Nishimoto, R. Sakata, A. Inui, M. Kurosaka Kobe/Japan

Purpose: Entheses serve to decrease stress between the bone and the tendon and reconstruction of these interfaces is an issue of considerable importance. We targeted degenerated cells from human rotator cuff and investigated whether human rotator cuff derived cells have the capacity for chondrogenic differentiation.Methods and Materials: The edges of the rotator cuff were harvested from patients who had sustained a rotator cuff tear and underwent arthroscopic rotator cuff repair. Cells were cultured in monolayer culture with α-MEM containing 10%FBS, 2mM L-glutamine and antibiotics on 100 mm diameter culture dish. The cultures were incubated at 37oC with 5% humidified CO2. Flow cytometric analyses have also performed using monoclonal antibodies for the following antigens: CD14, CD29, CD34, CD44, CD45, CD105, CD133, CD166. To induce chondrogenesis, a pellet culture was performed for three-dimensional culture. About 2.5x105 cells were spun in serum free ITS-medium containing dexamethasone, ascorbate, proline, recombinant BMP-6 and recombinant TGF-β3. Results: Flow cytometric analyses showed positive immunoreactivities for CD29, 44, 105, 166. The other tested markers were negative. After 21 days of differentiation culture the cells showed a chondrogenic differentiation potential as evidenced by the histological immunohistochemistryies and RT-PCR analyses. In contrast, the tissue specific markers of the control cells without supplements did not increased.Conclusions: The results of this study showed that degenerated cells derived from human rotator cuff have the capacity for chondrogenic differentiation. The ability to generate bone-ligament interface is specifically of great importance, as it can be useful in healing torn tendons that require reattachment to bone. This study suggests that degenerated human rotator cuff derived cells have a chondrogenic differentiation potential. Further investigation was needs to analysis the best condition to chodrogenic differentiation.

P186Enhanced in vitro cartilage formation by co-culture of human primary chondrocytes and mesenchymal stromal cellsA. Barbero1, C. Acharya1, A. Adesida2, P. Zajac1, S. Schaeren1, M. Jakob1, J. Riesle3, I. Martin1 1Basel/Switzerland, 2Basel/Canada, 3Bilthoven/Netherlands

Purpose: Co-culture of mesenchymal stromal cells (MSC) with chondrocytes (Ch) has been reported to improve cartilaginous matrix accumulation (phenomenon here named chondro-induction, CI). In this study, we investigate the type(s) of communication between the two cell types responsible for CI. Methods and Materials: Expanded bone marrow-MSC or fibroblast (as control cells) and freshly isolated Ch were cultured in pellets alone (pure pellets) or after being mixed (co-culture pellets, Ch:MSC ratio 25%:75%). Selected pellets were generated combining: human MSC with bovine Ch, and MSC from HLA-A2+ with Ch from HLA-A2- donors. MSC and Ch were also cultured in transwells, with the two cell types physically separated. Pellets were assessed biochemically [to quantify CI as a ratio GAGmeasured/GAGexpected (GAGexpected=75%GAGpure_MSC+25%GAGpure_Ch)], by RT-PCR using human and bovine specific primers and probes for collagen-II, and cytofluorimetrically. Tissues formed in the inserts of the transwells were assessed histologically and biochemically. Results: CI was higher when Ch were co-cultured with MSC (1.6±0.1) than with fibroblasts (1.3±0.1). RT-PCR of pellets generated by bovine Ch and human MSC showed an increase in the expression of human collagen-II following co-culture. FACS quantification with antibodies specific for HLA-A2 indicated that: Ch number increased (4.2-fold) in the co-culture pellets while remaining constant in pure pellets, MSC number decreased in the co-culture and pure pellets to a similar extent (5.0-fold). GAG content of tissues formed by MSC or Ch in the inserts of transwells were not modulated by the presence of either cell types in the bottom layer of the same transwells.

P183Bone marrow-derived culture-expanded mesenchymal stem cells to augment healing of chondral lesions treated with microfractureW. McIlwraith1, D.D. Frisbie1, W.G. Rodkey2, J.R. Steadman2, J. Kisiday3, C. Kawcak1 1Fort Collins/United States of America, 2Vail/United States of America

Purpose: We tested bone marrow-derived culture-expanded mesenchymal stem cells (BMSC) to augment healing after full-thickness cartilage defects were microfractured. We hypothesized that intraarticular injection of autogenous BMSC with microfracture would enhance healing in full-thickness defects compared to microfracture alone.Methods and Materials: At index surgery, ten skeletally mature horses had 1cm2 defects arthroscopically made on medial femoral condyles of both stifles (femorotibial joints). Defects were debrided to subchondral bone followed by routine microfracture. One month later, one medial femorotibial joint in each horse randomly received intraarticular injection of 20x106 BMSC with hyaluronan or hyaluronan alone. A routine strenuous postoperative rehabilitation protocol was followed. Throughout the study, horses underwent routine musculoskeletal and radiographic examinations bimonthly and re-look arthroscopy at six months post index surgery. All examinations were done with evaluators unaware of treatment. Horses were euthanized 12 months post index surgery, and study joints underwent MRI. Gross, histologic, histomorphometric, immunohistochemical and biochemical examinations were performed.Results: Subjective arthroscopic and gross evaluation of study joints confirmed significantly increased repair tissue firmness in BMSC-treated joints. There also was a trend for better overall repair tissue quality (cumulative score of all arthroscopic and gross grading criteria) in BMSC-treated joints. Immunohistochemical analysis demonstrated significantly greater levels of aggrecan in repair tissue associated with BMSC treatment although no difference in type II collagen was noted. There were no significant effects between treatment groups on clinical (musculoskeletal, radiographic or MRI) parameters. Likewise, histologic analysis of synovial membrane and articular cartilage including repair tissue showed no differences between treatments.Conclusions: Long-term improvement was enhanced in repair tissue firmness and aggrecan staining following intraarticular administration of BMSC. These findings persisted even with strenuous exercise and may be of long-term clinical benefits in other species. These positive findings support further in vivo research, including human clinical trials to use BMSC to enhance outcomes of microfracture.

P184The chondrogenic potential of human synovial tissue derived from young adults with femoral acetabular impingement and from elderly osteoarthritic patientsY. Himeda, N. Shintani, E. Hunziker Bern/Switzerland

Purpose: The synovium – like other mesenchymal tissues – contains sub-populations of multipotent stem cells that are capable of undergoing chondrogenesis, as well as osteogenesis, adipogenesis and myogenesis. We wished to ascertain whether synovial explants that were derived from young adults with femoral acetabular impingement (FAI) and from elderly osteoarthritic patients could be induced by exogenous stimulation to undergo chondrogenesis in vitro.Methods and Materials: Synovial biopsies were derived from the hip joints of five 20- to 28-year-old individuals with FAI and from the knee joints of six 67- to 81-year-old osteoarthritic patients. Agarose-cultured synovial explants were exposed for two weeks under serum-free conditions to a combination of BMP-2 (2000ng/ml) and TGF-β1 (10ng/ml). The gene-expression levels of a panel of cartilaginous markers and of catabolic ones were quantified by the real-time PCR-method.Results: In both groups of patients, stimulation with BMP-2/TGF-β1 induced the most profound increases (>10-fold) in the gene-expression levels of the chondrogenic markers collagen types II, IX, X and XI, aggrecan, COMP and alkaline phosphatase; those of lubricin and type-I collagen were lower (<10-fold increases), and that of osteocalcin remained unchanged. Amongst the catabolic markers, the levels of MMP-13, Cox-2 and iNOS were elevated (<10-fold), those of IL-1, IL-6 and ADAMTS-4 remained unchanged, and those of IL-4, matrilin-1 and TNF-α lay below the detection level. Although the trends were similar for the two groups of patients, the magnitude of the increases for collagen types II and XI, aggrecan, COMP, Cox-2 and iNOS were greater in the FAI- than in the osteoarthritic patients.

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P189Cartilage tissue engineering with acellular cartilage matrix and synovial stem cellsY. Hsu, C.H. Liao, H.W. Fang, C.H. Chang Taipei/Taiwan

Purpose: Cartilage tissue engineering has been a new strategy for treatment of cartilage injury. However, there are some limitations from the clinical perspective, such as cell source or type, ideal scaffold, and optimal culture conditions. The aim of this study was to investigate the potential of the discard cartilage and synovium from total knee replacement (TKR) surgery to develop decellularized cartilage ECM powder as chondrogenic scaffold and to isolate synovial stem cells for replacement of autogenous chondrocytes.Methods and Materials: The cartilage slices were harvested from the patients who underwent TKR surgery. After decellularization process, the DNA, GAG, and collagen contents of acellular cartilage matrix (ACM) were determined and its morphology was observed. The cytotoxicity of ACM was analyzed as well. Finally, the chondrogenic properties of isolated synovial stem cells in cell-ACM construct were characterized.Results: The DNA assay showed that the amount of DNA of the cartilage powder after the decellularization procedure was decreased, and the GAG and collagen assays showed that the amounts of GAG and collagen were retained from the untreated powder. The cytotoxicity assay indicated that the ACM was toxic free for cell adhesion and growth. After induction, the results from qPCR of synovial stem cell and ACM co-culture showed that the chondrogenesis was slightly promoted with ACM existence.Conclusions: In this study, we utilized human, natural, and discard materials from the TKR surgery to develop a method for cartilage tissue engineering with ACM and synovial stem cells. The results of this study may provide an alternative option for clinical treatment on articular cartilage injury.

P190Iron oxide labeling does not affect the chondrogenic differentiation ‎capacity of mesenchymal stem cellsH. Nejadnik1, S. Wang2, J.H.P. Hui1 1Singapore/Singapore, 2Sydney/Australia

Purpose: Cell based therapy is one of the most promising treatments for cartilage injuries. Monitoring cells’ fate by live tracking of the cells is essential to understand the homing mechanism and for the improvement of cell therapy. Magnetic resonance imaging (MRI) is a repeatable, non invasive way to track the cells in the living animal; by labeling the stem cells with super para magnetic iron oxide (SPIO) nanoparticle, monitoring of the cells with MRI will be possible.Methods and Materials: Bone marrow derived mesenchymal stem cells (BMSCs) were extracted and expanded till passage 2 from iliac crest of 3 different porcines. Labeling was done using five different concentration of Resovist® (Ferucarbotran) (25, 50, 75, 100, 125 µg/ml in culture media). To optimize labeling concentration and to evaluate effects of labeling on the cells, proliferation study (MTS assay), atomic absorption spectrometry (AAS), Prussian blue staining, transmission electron microscopy (TEM), adipogenic, osteogenic, and chondrogenic differentiation studies were done.Results: Prussian blue staining showed that the efficiency of labeling was more than 90 percent of the cells. Transmission electron microscopy confirmed that the endocytosed particles were in the cytoplasm. The average iron content of the BMSCs was increased according to the increase of the particle concentration in the culture media. Labeling of the BMSCs with different concentration of Resovist did not affect the proliferation rate of the cells. The detection capability of the labeled cells was assessed with clinical MR imaging at 1.5T and 3T. Moreover, labeling of the BMSCs did not affect the adipogenic, osteogenic and especially the chondrogenic differentiation capacity of the cells.Conclusions: This study showed that labeling of BMSCs by SPIO does not affect cellular function of the BMSCs especially the chondrogenic differentiation capacity. Therefore, it can be used for tracking the cells and to study their homing in the articular cartilage defect.

Conclusions: Mutual communication between Ch and MSC occurs in co-culture: Ch stimulate MSC for higher collagen-II expression and, in turn, MSC stimulate Ch to proliferate. These effects are not mediated by soluble factors alone but require cell-cell contacts. In vivo studies are necessary to assess the clinical relevance of our findings in the context of cartilage repair.

P187A Strategy of Using Cartilage Fragments for Chondrogenesis of MSC.C.C. Chen, C.H. Chang, H.W. Fang, C.H. Liao Taipei/Taiwan

Purpose: Human mesenchymal stem cells (hMSC) can differentiate into cells of connective tissue lineages, including cartilage. To overcome the limiting autogenous chondrocyte populations available in cartilage repair, various methods have been developed to maximize chondrogenesis of hMSCs in vitro. In this study, we investigated the effects of cartilage fragments for chondrogenesis of MSCs in fibrin glue and developed a favor method for cartilage repair.Methods and Materials: Human cartilage fragments obtained from TKR surgery and immortalized human bone marrow-derived MSC were mixed well and embedded into fibrin glue. The cartilage-glue-BMMSC constructs were implanted into subcutaneous tissue of nude mice. After 2 and 4 weeks, gene expression, histological and immunohistochemical stain of the constructs were analyzed. The fibrin glue-MSC constructs without minced cartilage fragments were as a control group.Results: The results of the cartilage fragments-fibrin glue-MSC constructs presented round and elongated chondrocyte-like appearance with positive stain of GAG which inducted into cartilage tissue. Moreover, RT-PCR analysis showed the gene expression of aggrecan, type II, X collagen, and sox-9 increased over time in the cartilage fragments-fibrin glue-MSC constructs. However, there was no gene expression of type II collagen in control group, and suggesting the MSCs in fibrin glue without cartilage fragments did not differentiate into chondrocytes. Conclusions: According to our results in this study, we have suggested that cartilage fragments own the potential to help chondrogenic induction of MSCs and develop a favor method for cartilage repair.

P188Relative percentage and zonal distribution of mesenchymal progenitor cells in adult human articular cartilageD. Pretzel, S. Linss, S. Rochler, R. Kinne Eisenberg/Germany

Purpose: Combined expression of the surface molecules CD105 and CD166 unequivocally identifies mesenchymal progenitor cells (MPC) in adult human articular cartilage. Therefore, these marker molecules were employed to quantify and analyze the zonal distribution of resident MPC in cartilage. Methods and Materials: Specimens of human osteoarthritic cartilage (n = 8) were either used for cell isolation or for immunohistochemistry in paraffin sections. Isolated cells were analyzed by flow cytometry (FACS) or immunofluorescence in chamber slides for the expression of CD105 and CD166. Following separation of CD166+/- cells with magnetobead-coupled antibodies, multi-lineage differentiation assays were performed. In addition, the zonal distribution of CD166+ cells within the cartilage matrix was analyzed by immunohistochemistry in paraffin-embedded tissue sections. Results: FACS analysis showed that 16.2 ± 2.1% (mean ± SEM) of the chondrocytes were CD105+/CD166+ and thus carried the established MPC marker combination. Similar results (approx. 12% CD105+/CD166+ cells) were observed by immunofluorescence in adherent cells. The CD166+-enriched cell population showed a stronger induction of the chondrogenic phenotype in differentiation assays than the CD166--enriched cell population and thereby underlined the chondrogenic potential of the MPC. Interestingly, the CD166+ cells were mainly located in the superficial and middle zone of articular cartilage and only sporadically in the deep zone. Conclusions: CD 166 appears to be a novel suitable biomarker to characterize the zonal distribution of resident MPC with a high chondrogenic potential in human articular cartilage. Strikingly, the percentage of MPC in osteoarthritic cartilage was substantially higher than previously reported, supporting the concept of a yet unexplored reserve capacity for regeneration.

Posters 235

P193Correlation of human bone marrow mesenchymal stem cell characteristics with donor ageV. Dexheimer, S. Müller, F. Braatz, W. Richter Heidelberg/Germany

Purpose: Due to their self-renewing and multilineage differentiation capacity, mesenchymal stem cells (MSC) are an attractive source for cell-based cartilage repair strategies. Therefore the question arises if donor age has an influence on MSC properties, in particular their number, clonal expandability and chondrogenic differentiation capacity.Methods and Materials: MSC from 28 donors of three age groups were analyzed for their ability to form Colony-forming unit fibroblasts (CFU-F). During expansion single cell cloning efficiency (SSCE) was assessed at passage 0 (P0) and generation determined at P0-P4. Chondrogenic differentiation was induced in a high density pellet culture at P3 for 6 weeks in chondrogenic induction medium with TGF-β1 and pellets were analyzed for proteoglycan and collagen type II content.Results: The number of MSC obtained per 106 mononuclear cells was highly variable between donors but independent of donor age. Clonal expandability was reduced in the old age group compared to the young and middle aged groups. Cells proliferated slower with increasing passages. Generation time in passage two and three was however significantly higher in the old compared to the young and middle aged group. After chondrogenesis the collagen type II and proteoglycan content of pellets was similar in all age groups. Generation time correlated with collagen type II deposition in every age group with the fastest cells producing the best differentiating results in each age group.Conclusions: The cells from aged donors show a reduced speed of proliferation. The in vitro chondrogenic differentiation potential is highly variable between donors but donor age would not be a criterion to exclude patients from future applications of MSC. In the context of cartilage repair similar numbers of MSC are obtained from bone marrow of young and old donors.

P194PTHrP - Indian Hedgehog autoregulation during chondrogenic differentiation of human mesenchymal stem cells – a PTH-receptor-independent mechanismA. Brauckhoff, J. Fischer, E. Steck, W. Richter Heidelberg/Germany

Purpose: Bone marrow-derived mesenchymal stem cells (BMSC) are an attractive alternative for cartilage tissue engineering. However, long-term phenotypic stability along with functional suitability in-vivo and the adoption of a non-hypertrophic chondrocyte phenotype are imperative for cartilage repair and still represent a challenge for current in vitro chondrogenic protocols. Parathyroid hormone-like peptide (PTHrP) is a negative regulator of chondrocyte differentiation and MSC chondrogenesis, and a known inhibitor of chondrocyte hypertrophy. The aim of this study was to evaluate whether modulation of PTHrP-signalling is a means to improve chondrogenic in vitro differentiation of human MSC.Methods and Materials: Chondrogenic differentiation of human BMSC was induced in pellet culture for 6 weeks in chondrogenic medium containing TGFß. Expression of PTHrP, PTH-receptor 1 (PTHR1) and Indian Hedgehog (IHH) mRNA was assessed over time. Part of the cultures were supplemented with PTHrP(1-34), its antagonist PTHrP(7-34) or a combination of both from day 0 or 21 on. Differentiation was assessed by histology, real-time PCR and detection of ALP activity. Results: PTHrP was expressed up to 2-3 weeks and downregulated in favour of IHH and PTH1R up-regulation. PTHrP(1-34) inhibited the TGF-β-driven collagen type II and collagen type X expression and alkaline phosphatase (ALP) induction, while the antagonist alone showed no evident effects. PTHrP action could surprisingly not be antagonised by PTHrP(7-34).Conclusions: Similar to observations in immature chondrocytes in the growth plate, early chondrogenesis of MSC was characterized by PTHrP expression which declined spontaneously in favour of IHH up-regulation allowing further maturation of cells towards hypertrophy. PTHrP exposure from day 21 on suppressed IHH up-regulation and reduced hypertrophy and surprisingly, this occurred independent of PTH1R action. Future steps will be to develop alternate differentiation protocols in which the here discovered PTHrP/IHH autoregulation is modulated in favour of a sustained nonhypertrophic chondrocyte phenotype desired for articular cartilage repair studies.

P191Comparative labelling of mesenchymal stem cells with magnetic iron oxide nanoparticles for MR imaging in vitroH. Jülke, C. Geißler, I. Ribitsch, W. Brehm, E. Ludewig, U. Delling Leipzig/Germany

Purpose: In vitro and magnetic resonance imaging (MRI) trials were employed to investigate 3 commercial supraparamagnetic iron oxide nanoparticles (SPIO) products for labelling bone marrow derived mesenchymal stem cells (MSC). Labelling efficiency, longitudinal cellular detectability by MRI and biologic effects on MSC were evaluated. Results of this study will help to define the most appropriate SPIO for in vivo cell tracking of locally applied MSC in degenerative joint disease.Methods and Materials: Ovine and equine MSC were routinely obtained from 5 donors, expanded until p4 and labelled with one of the following SPIO products: Molday ION Rhodamine B (Biopal, USA), Endorem (Guerbet, France), or Resovist (Bayer HealthCare, Germany). Labelling efficiency and cellular SPIO retention until p7 were graded by a semiquantitative histological scoring system based on Prussian blue staining. Cellular detection was evaluated by a 0.5T MRI system using GRE 3DT2*w sequences for up to 3 weeks after labelling (p4 to p7). Proliferation and differentiation capacities were assessed by in vitro assays.Results: MSC were successfully labelled by all 3 SPIO products. High iron uptake and selective intracellular iron presence was achieved by Molday ION Rhodamine B, only. Labelling with Resovist led to prominent extracellular iron presence, labelling with Endorem was less intense. In MRI, all labelled cells showed strong hypointense signals contrary to unlabelled controls. Resovist caused most hypointense signals, followed by Molday ION Rhodamine B and Endorem. MRI detectability decreased proportionally from p4 to p6. In p7, 3 weeks after labelling, only Resovist labelled cells were detectable. Proliferation, adipogenic and osteogenic differentiation were similar between labelled and unlabelled cells. Chondrogenic differentiation decreased proportional to increasing intracellular iron quantities.Conclusions: Out of the 3 products, Molday ION Rhodamine B is the most promising labelling agent for in vivo cell tracking. Advantages are reliable intracellular iron uptake without extracellular SPIO and consistent hypointense signals in MRI.

P192Heterogeneity in the chondrogenic capacity of bone marrow stromal cellsS.C. Dickinson, C.A. Sutton, W. Kafienah, A.P. Hollander Bristol/United Kingdom

Purpose: There is great potential for the use of cell-based therapies for the treatment of cartilage lesions caused by osteoarthritis (OA). Bone marrow stromal cells (BMSCs) are a heterogeneous population of multipotent stem cells which can differentiate into chondrocytes and produce three-dimensional hyaline cartilage. The aim of our study is to determine whether sub-populations of BMSCs are able to create cartilage of superior quality to the whole population and to identify markers of the most chondrogenic cells.Methods and Materials: BMSCs were isolated from patients with OA. Stem cell clones were prepared by using a sterile cell sorter to deliver individual cells into wells of a 96-well plate. Each clone was proliferated in the presence of FGF-2 to generate a sufficient number of cells to assess multipotential. Chondrogenesis was assessed by TGF-β3 stimulated cartilage tissue engineering. The quality of the cartilage produced by each individual clone was measured using specific biochemical assays.Results: A total of 21 individual stem cell clones were proliferated, with negligible signs of senescence, to generate sufficient cells to perform cartilage tissue engineering. Both the macroscopic appearance (Figures 1A&B) and the extracellular matrix protein content of the resulting cartilage were highly variable between clones. Five stem cell clones were highly chondrogenic, generating cartilage containing more type II collagen than cartilage produced from the whole BMSC population (Figure 1C). There was no correlation between the chondrogenic capacity of different clones and their adipogenic and osteogenic potential.Conclusions: We have demonstrated that the BMSC population is highly heterogeneous in terms of chondrogenic capacity. This highlights the importance of identifying and isolating the most chondrogenic cells for use in clinical therapies. We are currently performing a gene array analysis to compare gene expression in highly and poorly chondrogenic clones to identify unique markers for the isolation of the most effective cells for therapeutic use.

Posters236

P197Retinoic acid signalling is important for chondrogenic differentiation of human mesenchymal stem cellsS. Diederichs, W. RichterHeidelberg/Germany

Purpose: Retinoid signalling has long been known to play a major role in early skeletal development. On the one hand, retinoic acid (RA) – the active vitamin A metabolite – inhibited in vitro chondrogenesis of primary mouse and chick limb bud mesenchymal cultures and stimulated hypertrophy of chicken sternal chondrocytes. On the other hand, RA receptor antagonists promoted chondrogenic differentiation of mouse limb bud mesenchymal cultures and prevented chondrocyte hypertrophy in developing chick limbs, suggesting that they are attractive factors to improve chondrogenic differentiation of mesenchymal stromal cell (MSC) cultures. Indeed, the RA receptor-β selective antagonist (LE135) was recently reported to drive chondrogenesis of human MSC in PGA scaffolds. Aim of this study was to unravel whether LE135 can drive and improve in vitro chondrogenesis of human MSCs in pellet culture in the presence or absence of TGF-β.Methods and Materials: Expanded MSCs from five human donors were cultured as micromasses (500,000 cells each) in serum free chondrogenic medium supplemented either with TGF-β (positive control), LE135 (0.1 µM / 1 µM) or a combination of both. Chondrogenic differentiation was assessed after six weeks based on proteoglycan and collagen II detection (histology, quantitative PCR).Results: While TGF-β treated MSCs were strongly positive for proteoglycans and collagen-type II, LE135 was not sufficient to promote chondrogenesis of human MSCs. In combination with TGF-β, LE135 even inhibited chondrogenesis.Conclusions: While prevention of RA signalling by RA receptor-β inhibition improved chondrogenesis of in vivo committed chondroprogenitors, such an inhibition did not create a sufficiently positive signal to drive non-committed human MSCs into the chondrogenic lineage. In the presence of the potent inducer TGF-β, RA receptor-β inhibition blocked differentiation suggesting that RA signalling is, however, a crucial part also of in vitro chondrogenesis of human MSCs. LE135 is, thus, currently not attractive to improve chondrogenesis in this model.

P198Inhibition of chondrogenesis by sex hormonesP. Angele, Z. Jenei-Lanzl, T. Dienstknecht, R. Kujat Regensburg/Germany

Purpose: Objective Articular cartilage is a sex-hormone sensitive tissue. estrogens have been shown to affect cartilage under physiological and pathological conditions. Estrogens have different mechanisms of action via classical (ER α/β) or membrane receptors such as GPR30. The purpose of the present study was to investigate the effect and the possible mechanism of estradiol treatment during chondrogenesis of bone marrow derived MSCs with respect to repair of chondral defects and for the prevention of trauma-dependent osteoarthritis. Methods and Materials: Methods Bone marrow was obtained from the iliac crest of young male humans. Ficoll-separated hMSCs proliferated as a monolayer in serum-containing medium. After achieving confluence, aggregates were created and cultured in a serum-free differentiation medium. 17β-estradiol (E2) with or without the specific estrogen receptor inhibitor ICI 182.780, membrane impermeable E2-BSA, ICI 182.780 alone, a GPR30 agonist G-1, and a GPR30 antagonist G15, were added. After 21 days, the aggregates were analyzed histologically and immunohistochemically; synthesized type II collagen, DNA content, glycosaminoglycan concentrations, and type X collagen and MMP-13 expression were quantified. Results: Results The existence of intracellular and membrane-associated E2 receptors was shown at various stages of chondrogenesis. Smaller aggregates, significant lower type II collagen, and sGAG content but enhanced type X collagen and MMP-13 expression were detected after E2 and E2-BSA treatment in a dose-dependent manner. The co-incubation of ICI 182.780 with estradiol enhanced suppression of chondrogenesis compared to estradiol alone. The treatment with specific GPR30 agonists alone (G-1 and ICI 182.780) resulted in a considerable inhibition of chondrogenesis and enhancement of hypertrophy. Conclusions: Conclusion The experiments revealed a suppression of chondrogenesis by estradiol via membrane receptors (GPR30). The study opens new perspectives to influence chondrogenesis on the basis of classical and non-classical estradiol signalling.

P195Effects of coculture conditions on chondrogenic commitment of human wharton jelly stem cellsR.C. Pereira, A.R. Pinto, A.M. Frias, N.M. Neves, H.S. Azevedo, R.L. Reis Caldas das Taipas – Guimarães/Portugal

Purpose: Cell-based therapies have shown great promise for the repair of articular cartilage lesions.Autologous chondrocyte implantation(ACI)proposed by Britteberg is a clear example of a cell-based therapy with excellent clinical results.The use of ACI is associated with several limitations involving lengthy and costly cell isolation and expansion steps of human articular chondrocytes(hACs)which have low cellular mitosis and are prone to dedifferentiation.Potential improvement in ACI procedure is the application of mesenchymal stem cells(MSCs),recognized for the potential to differentiate into the chondrogenic lineage,instead of hACs.Coculture systems using relevant cells can be used as valuable tools to offer therapeutic possibilities in cartilage regeneration.Realizing that MSCs present a limited degree of proliferation with maintenance of their multipotency capacity of differentiation,we used human wharton jelly stem cells(hWJSCs)to evaluate their chondrogenic differentiation capability.This study was undertaken to test if hWJSCs,in non direct coculture with hACs,would be able to present a chondrogenic commitment compared to those not exposed to chondrocytes metabolic products. Methods and Materials: Cell differential potential was assessed by qRT-PCR,and by micromass pellet culture.Immunohistochemistry was also performed.Results: Immunolocalization of collagen type I and II of hWJSCs cocultured with hACs in micromass pellets shows a promising trend towards chondrogenic phenotype.Moreover,hWJSCs in coculture presented a homogeneous cell distribution and, for at least 20 cell duplications, higher levels of collagen type II, aggrecan, Sox9, TGF-β and COMP expression compared to those cultured alone, suggesting a potential commitment toward the chondrogenic lineage.Conclusions: These findings suggest that coculture of hWJSCs with hACs may provide a suitable environment for their chondrogenic commitment and could significantly improve the development of cell-based therapies for treating cartilage lesions.

P196Articular chondrocytes secrete PTHrP and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesisJ. Fischer, A. Dickhut, M. Rickert, W. RichterHeidelberg/Germany

Purpose: Bone marrow-derived mesenchymal stem cells (MSC) are promising for cell-based cartilage regeneration. A yet unsolved problem is, however, the unwanted upregulation of hypertrophic markers like alkaline phosphatase (ALP) and collagen-type X during in vitro chondrogenesis and formation of instable calcifying cartilage at heterotopic sites. In contrast, stable non-mineralizing cartilage is obtained from articular chondrocytes. Aim of this study was to address whether coculture with human articular chondrocytes (HAC) has the capacity to suppress undesired hypertrophy in differentiating MSC.Methods and Materials: MSC were differentiated in chondrogenic medium which had or had not been conditioned by parallel chondrocyte pellet cultures, or were mixed in the same pellet with chondrocytes (1:1, 1:2) and cultured for six weeks. Following in vitro differentiation, pellets were transplanted into SCID mice.Results: The gene expression ratio of COL10A1/COL2A1 and IHH/COL2A1 was significantly reduced by HAC-conditioned medium and less collagen-type X protein was deposited relative to collagen-type II. ALP-activity was significantly lower (p<0.05) in the conditioned-medium-group and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of ALP was dose-dependent and in vivo calcification was fully inhibited. Chondrocytes secreted PTHrP throughout culture while PTHrP was downregulated in favour of IHH upregulation in control MSC after 2-3 weeks of chondrogenesis. Main inhibitory effects seen with HAC-conditioned medium could be reproduced by PTHrP supplementation of unconditioned medium.Conclusions: HAC-derived soluble factors and direct coculture are potent means to improve chondrogenesis and suppress hypertrophic development of MSC. PTHrP is one important soluble candidate factor involved in this effect.

Posters 237

P202Repair of chondral defects and meniscus using autologous mesenchymal stem cells: a preliminary study in sheepJ. Vives1, F. García Arnás1, J. Barrachina2, J. García2, R. Soler Rich2, L. Orozco2 1Bellaterra/Spain, 2Barcelona/Spain

Purpose: We herein present our recent findings, in an ovine model, on the use of autologous mesenchymal cells expanded from bone marrow aspirates for the repair of damaged cartilage, as a model, of degenerative effects of osteoarthritis or osteochondral lesions. Such animal model also permitted us to study the regeneration of meniscal lesions in the same knee. Methods and Materials: Ten 2-year old ewes were divided in two groups (for analysis at 6 and 12 month). Arthroscopically guided chondral lesions of approximately 60 mm2 were created in the femoral medial condyles and the anterior horn of the medial meniscus of the two posterior legs. Cellular treatments were applied at a max dosage of 50x106 MSCs 30 days later and the animals were monitored by MRI, echographic and X ray at 6 and 12 months. A full necropsy was performed at 12 months to evaluate possible adverse effects of the treatment. Results: Although MRI, ecographic and X ray control were not extremely informative to assess the progression of the reparative process, the histological and immunohystochemical analysis (at 6 and 12 months) confirmed the hyaline quality of the regenerated cartilage of the condyles of the knee of treated animals. Meniscal lesions were also partially repaired in some cases. Most importantly, the intra-joint application of autologous mesenchymal stem cells was demonstrated to be safe at 12 months post-treatment. Conclusions: We demonstrated that autologous mesenchymal stem cells are safe and capable to regenerate both hyaline cartilage and meniscal fibrocartilage with the advantage of using a straightforward infiltration approach.

P204Microarray analysis reveals signature clusters of gene expression during in vitro chondrogenesis of human bone marrow-derived mesenchymal stem cellsS.R. Herlofsen, A.M. Küchler, J.E. Brinchmann Oslo/Norway

Purpose: Chondrogenic differentiation of hBM-MSCs in alginate discs was performed to develop clinically useful cartilage implants, and to facilitate the kinetic analysis of gene and protein expression.Methods and Materials: After monolayer expansion MSCs were set into a selfgelling alginate system forming a 1-2 mm thick disc and differentiated in chondrogenic medium containing TGF-β1 and BMP2. Samples were collected at day 0, 7, 14 and 21. Chondrogenesis was evaluated by realtime PCR and immunofluorescence histochemistry. Matrix production was additionally quantified by GAG concentration measurement. Finally, microarray technology and similarity search statistics were used to identify signature clusters of gene expression patterns.Results: COL2, COL10 and SOX5, 6 and 9 mRNA were greatly elevated already at day 7, while COL1 and versican were gradually reduced. COL2 and aggrecan were seen throughout the entire ECM. COL1 seemed to be produced by only some of the cells. COL10 was found predominantly in the cytoplasm while SOX proteins were localized in the nuclei. We detected increasing levels of GAGs in the supernatant. By similarity search of differentially expressed genes (p<0.05, >2-fold change) we identified a signature cluster of ECM genes upregulated similar to COL2 by differentiation medium, and clusters of genes involved in blood vessel development and immune responses downregulated similar to the chemokine CXCL12.Conclusions: We show that hBM-MSCs differentiated in alginate discs express genes and secrete proteins with kinetics and distribution similar to hyaline cartilage. At the same time they downregulate genes involved in non-chondrogenic functions of BM-MSCs. Analysis of the signature chondrogenic clusters identified here may provide a better understanding of how the stem cell fate could be directed to produce perfect chondrocytes for celltherapy.

P199Clinically applicable cell tracking in cartilage repair using MRIG.M. van Buul, G. Kotek, P.A. Wielopolski, E. Farrell, P.K. Bos, H. Weinans, J.A.N. Verhaar, G.P. Krestin, M.R. Bernsen, G.J.V.M. Van Osch Rotterdam/Netherlands

Purpose: Cell tracking is a useful tool to optimize the use of human bone marrow stromal cells (hBMSCs) for cartilage repair and to elucidate the relevant repair mechanisms. Cell labeling using superparamagnetic iron oxides (SPIOs) provides the possibility for non-invasive in vivo cell tracking using MRI. We investigated the safety, intra-articular MRI traceability and SPIO re-uptake of this cell tracking technique.Methods and Materials: Part 1: hBMSCs were labeled with SPIO (ferumoxides-protamine sulphate), dose range 0 - 250 µg/ml. Cell viability was assessed and cell activity was quantified up to 7 days. Part 2: SPIO-labeled hBMSCs (100,000 – 5,000,000 cells) were injected in pig knees post mortem. Furthermore, SPIO-labeled cells (30,000 - 100,000 per 75 µl) were seeded in cartilage defects in vitro. Scanning was performed on a clinical 3.0 T MRI scanner. Part 3: To show possible SPIO re-uptake by synovial cells, viable and dead GFP-SPIO double-labeled chondrocytes were co-cultured on human synovium explants for five days. Samples were analyzed using fluorescence- and light microscopy. Results: Part 1: SPIO labeling resulted in labeling efficiencies of ± 95% and did not impair cell viability or -activity at any dose. Part 2: Intra-articular injected SPIO-labeled hBMSCs were visualized by MRI in a dose dependent manner, and could be differentiated from articular anatomical structures. SPIO-labeled cells seeded in cartilage defects were visualized and quantified using a T2* mapping MRI technique Part 3: GFP+-SPIO+ cells, indicating originally seeded cells, were seen in samples containing live cells. GFP‾-SPIO+ cells, indicating SPIO re-uptake by synovial cells, were found in samples containing dead cells. Conclusions: Although possible SPIO re-uptake by host cells has to be considered, we showed promising results for the use of SPIO labeling for cell tracking in clinical cartilage repair. This approach provides the extra advantage to simultaneously track cells and evaluate cartilage repair in one MRI session.

P201Efficacy of Adeno-associated gene therapy in equine bone marrow derived mesenchymal stem cells for cartilage resurfacing applicationsL. Goodrich1, A. Mandel1, V. Choi2, J. Kisiday1, W. McIlwraith1, J. Samulski3 1Fort Collins/United States of America, 2Cambridge/United States of America, 3Chapel Hill/United States of America

Purpose: Gene therapy for joint disease relies on a non-immunogenic gene delivery vector that efficiently and persistently transduces joint specific tissues. Recombinant self-complimentary adenoassociated virus (scAAV) is a promising vector due to its potential to efficiently express therapeutic genes for long periods of time. Our goal was to investigate which serotypes of AAV would be best suited to deliver the gene encoding green fluorescent protein (GFP) into bone marrow derived mesenchymal stem cells (BMDMSC) for future gene transfer applications in the joint.Methods and Materials: Bone marrow derived mesenchymal stem cells were harvested from adult horses and humans. Cell monolayers were cultured in a mesenchymogenic media. Two days after seeding, the scAAV vector serotypes including S1-6 and S8 carrying a GFP expression cassette were used to transduce cells. The cells were suspended into 1.6% alginate for 6 weeks. Flow cytometry was performed every two weeks to measure efficacy of gene modification of these cells and realtime PCR was performed to detect any increases in inflammatory molecules as a result of gene modification.Results: Excellent gene modification of these cells was accomplished for scAAV serotype 2 and 3. Moderate transduction was achieved for scAAV5 and 6. Intense fluorescence was achieved for a period of at least 6 weeks for these cells when transduced by scAAV 2 and 3 as measured by flow cytometry. No significant elevations were noted in inflammatory molecules as measured by PCR.Conclusions: We established the use of specific AAV serotypes for efficient gene modification of BMDMSC. Transgene expression continued past 60 days and no apparent inflammation or reduction in cell viability appears to be associated with gene modification of these cells. Gene therapy using BMDMSC for gene therapy in joint resurfacing applications appear to be clinically applicable with scAAV serotype 2 and 3 being the most efficacious means of vector transfer.

Posters238

P209Development of the scaffold free cell processing robot for osteochodral regenerationK. Nakayama, T. Shimoto, S. Akieda, X.Y. Zhang, S. Matsuda, Y. Iwamoto Fukuoka/Japan

Purpose: We’ve been working for developing scaffold free cell delivery system, and show good regeneration of rabbit osteochondral defect until more than three years by implantation of molded mesenchymal stem cells (MSC) construct without use of exogenous factors. The entire procedure is simple, yet, it takes time and labor to build a single construct even experienced hands. In this study, we developed a cell processing robot for building Scaffold free HD MACs (High-density mesenchymal stem cell autologus constructs).Methods and Materials: We customized ready-made-scalar robot and the electric cylinder for cell handling under sterilized and GMP-ready condition. The entire system are placed in clean bench. The system has automatic 96-well plate changer. When the isolated cells and empty 96-well plates in a magazine rack were set, the system automatically dispense the cell suspension into each well. After 24 hours in normal incubator with the magazine rack,the system will also automatically collect cell aggregate into the HD-MACs molding chamber. Another 2 to 3 days in the molding chamber under incubator, the HD-MACs were ready to implant to the knee.Results: The obtained scaffold free HD MACs as same as manual procedure without contamination. The processing times are almost same compared with experienced human,while the robot system may able to work much faster with some optimization.Conclusions: In this study, we developed the cell processing robot for cartilage regeneration. The entire system is almost GMP ready. We are preparing clinical trials in Kyushu university hospital for autologous MSC implantation to cartilage defect with this robot system.

P210hMSC TNAP Expression is a Marker of Differentiation PotentialS. D’Arcy1, C. Coleman2, M. Murphy1, F. Barry1 1Galway/Ireland, 2Galway City/Ireland

Purpose: Due to their regenerative, tri-lineage potential, mesenchymal stromal cells (hMSC) offer new prospects for clinical treatments over current therapies. Although hMSCs are CD90+, CD105+, CD73+, CD44+ and CD45-, no standardized marker has been identified. Recently, expression of the cell surface protein Tissue Non-specific Alkaline Phosphatase (TNAP) has been used to directly isolate hMSC from bone marrow (1, 2). We hypothesized that expression of TNAP may offer a methodology to isolate hMSC subpopulations with varying differentiation potential. Methods and Materials: hMSC subpopulations were isolated from whole BM using anti-TNAP microbeads according to manufacturer’s instructions (3). TNAP+, TNAP-, and parent (TNAP+ and TNAP-) populations were expanded in alpha-MEM with 10% FBS and 5ng/ml FGF-2. All populations were assessed for surface phenotype (flow cytometry), expansion potential, morphology, and tri-lineage differentiation potential using standard methods (4). Results: TNAP+ hMSC, unlike parent and TNAP- fractions, initiated as distinct colonies and showed enhanced proliferative ability (Fig 1). Expression of traditional hMSC markers (CD105, CD73, CD44) was maintained in all populations, however TNAP- hMSC also showed CD271 expression was increased significantly, indicative of osteogenic potential (5). Osteogenic differentiation confirmed this when TNAP- hMSC showed significantly enhanced calcium deposition compared to parent and TNAP+ populations (Fig 2a). TNAP+ hMSC demonstrated enhanced adipogenic potential, which was completely depleted in the TNAP- fraction (Fig 2c). Chondrogenic assessment (GAG deposition) indicated complete inhibition of TNAP- hMSC potential, accompanied by significant reduction of TNAP+ hMSC, to chondrify (Fig 2b). Conclusions: TNAP expression was found to be related to both hMSC proliferation and differentiation capability. TNAP absence on hMSC was indicative of osteogenic potential, and presence of TNAP suggested a more proliferative cell with enhanced adipogenic potential. Interestingly, for chondrogenic differentiation to arise, the combination of TNAP expressing and non-expressing cells was required, indicating a necessity for subpopulations to communicate for hMSC chondrogenesis.

P206Stem cell- based augmentation of meniscus repairE. Chen1, M. Ast1, M. Goldstein1, N.V. Shah2, N. Chahine2, P. Razzano2, D. Grande2 1New Hyde Park/United States of America, 2Manhasset/United States of America

Purpose: Meniscal injuries represent one of the most common musculoskeletal complaints, with partial meniscectomy being the most frequently performed orthopaedic procedure in the US. The importance of the meniscus as a weight-bearing structure has been established, with meniscectomy shown to predispose to earlier onset of articular cartilage degeneration. Yet meniscal repair is infrequently performed, in part from concerns for healing due to limited vascular supply. In this study, we attempt to enhance the meniscus repair process. It is our hypothesis that cellular augmentation with either meniscal fibrochondrocytes or stem cells will enhance the histological repair quality of meniscus tissue.Methods and Materials: Experimental defects were created in fresh bovine menisci in a radial orientation. Defects were treated with either meniscal fibrochondrocytes, adipose-derived stem cells, synovial-derived stem cells, or without cells. Cells were delivered either alone in a collagen gel, scaffold, or with hyalurinoc acid. Each of these cell lines were derived from bovine tissue, cultured and expanded, and labeled with BrdU for later tracking. The repaired meniscal samples were then implanted subcutaneously into the dorsum of nude rats and the animals sacrificed at 3, 6, and 9 weeks. The samples were then be prepared for histologic examination and stained with H & E, Mallory Trichrome, and DAPI.Results: Meniscal defects treated with any of these cell types showed a histologically superior repair when compared to controls. Cells implanted in the absence of scaffold showed a higher density of cellular repair tissue, although it is unclear whether this reflects a more structurally sound repair. Synovial-derived cells resulted in significantly higher levels of collagen matrix synthesis and better overall integrative repair.Conclusions: The addition of meniscal fibrochondrocytes, adipose-, or synovial- derived stem cells to a bovine meniscus injury model results in a histologically superior repair tissue when compared to repair without cellular augmentation.

P208GDF-5 Supplementation Enhances MSC ChondrogenesisC. Coleman1, C. Curtin2, F. Barry3 1Galway City/Ireland, 2Dublin/Ireland, 3Galway/Ireland

Purpose: Deficient secretion of growth/differentiation factor 5 (GDF-5) contributes to the induction of arthritis. As arthritic cartilage fails to self-repair, treatment with mesenchymal stem cells (MSCs) may contribute to the repair of fibrillated cartilage. The aim of this project was to assess the effects of MSC treatment with GDF-5, focusing on the deposition of matrix proteins. Methods and Materials: Bone marrow (BM) was harvested from healthy, consenting donors. MSCs were isolated and expanded from the BM by direct plating using traditional methods, followed by induction towards chondrogenesis by pellet culture in incomplete chondrogenic medium (ICM), ICM supplemented with TGF-beta3 (CCM) or CCM with 100, 150 or 200ng/ml GDF-5. Glycosaminoglycan (GAG) content was determined histologically by Safranin-O staining, and quantitatively by DMMB assay. DNA content was determined by Pico Green analysis. Immunohistological (IHC) analysis following paraffin embedding included blocking in 2.5% BSA, incubation with anti-collagen II or X antibodies and DAB development.Results: MSCs in pellet culture initiated the deposition of a collagen and GAG containing ECM. As assessed by Safranin-O and DMMB analysis, pellets treated with GDF-5 contained significantly greater quantities of GAG as compared to CCM controls (Figure 1). Although treatment with GDF-5 resulted in enhanced GAG deposition, IHC analysis for collagen II illustrated the presence of similar quantities of protein in pellets treated with CCM or GDF-5. Collagen X expression, a marker of hypertrophy, was eliminated in GDF-5 treated pellets when expression was observed in CCM pellets (Figure 2). Conclusions: Treatment of MSC chondrogenic pellets with 150ng/ml GDF-5 resulted specifically in significant enhancement of GAG deposition, but no obvious change in collagen II secretion. The presence of GDF-5, however, eliminated collagen X from the ECM, an indication of undesirable hypertrophy. Therefore, the co-administration of GDF-5 with MSCs as a therapeutic for arthritis may result in enhanced chondrogenesis as well as the prevention of hypertrophy.

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P215Gene expression in subchondral bone osteoblasts from hip osteoarthritis, rapidly destructive coxarthrosis and rheumatoid arthritisK. Honjo1, Y. Arai1, K. Takahashi1, R. Terauchi1, S. Nakagawa1, N. Hiraoka1, H. Inoue1, M. Saito1, S. Tsuchida1, T. Kubo2 1Kyoto/Japan, 2Kyoto City/Japan

Purpose: The representative hip joint diseases include osteoarthritis (OA), rapidly destructive coxarthrosis (RDC) and rheumatoid arthritis (RA). OA progresses slowly, whereas RDC and RA result in rapid joint deterioration. Recent studies suggested that an abnormal subchondral bone metabolism might be involved in the progression of joint destruction. In this study, we examined the gene expression of inflammatory cytokines, proteases and factors related to bone metabolism in the subchondral bone of these hip joint diseases. Methods and Materials: The subjects were 7 OA, 3 RDC and 3 RA patients who underwent total hip arthroplasty. Four patients with femoral neck fracture (FNF) were used as controls. Subchondral bone tissues of femoral head were harvested. The subchondral bone osteoblasts (SBOs) were isolated. The mRNA expression of IL-1β, IL-6, IL-8, TNF-α, MMP-13, ADAMTS-5, RANKL, RANK and OPG were analyzed by real time RT-PCR.Results: The expression levels of IL-β, IL-6, ADAMTS-5 and MMP-13 in the SBOs from OA patients were significantly higher than those from FNF. RANK/ RANKL/ OPG among these two groups had no significant difference. Comparing RDC with OA, IL-8 gene expression and RANKL/ OPG ratio in the RDC were higher. The ADAMTS-5 mRNA in the RA was significantly higher than OA, but the level of OPG was lower. RANKL/ OPG ratio in the RA was upregulated.Conclusions: In relation to the joint destruction, vulnerability of subchondral bone and activities of osteoclasts have been discussed. In the present study, highly expressed IL-β, IL-6, ADAMTS-5 and MMP-13 around the subchondral bone area might have an influence on the bone turnover, which contribute to the progression of OA. In addition to the highly expression of inflammatory cytokines and proteases, upregulated osteoclastic activities might accelerate the joint destruction and result in the rapid clinical course of RDC and RA.

P216Bipolar fresh osteochondral allograft in the shoulder: a possible alternative to traditional arthroplasty?S. Giannini, R. Buda, A. Ruffilli, M. Cavallo, B. Grigolo, M. Nanni, F. Vannini Bologna/Italy

Purpose: Surgical treatment of severe post-traumatic shoulder arthritis in young active patients is a challenge. Bipolar fresh osteochondral allograft (BFOA) is a fascinating option for patients without severe cuff tear arthropaty. The purpose of this study is to report the clinical and histological results of 2 cases of BFOA in the shoulder. Methods and Materials: 2 patients, mean age 48.5 ± 0.7 years, affected by post-traumatic unilateral arthritis of shoulder, received BFOA. Patients evaluation was carried out clinically and radiographically by X-Rays, CT scans and MRI. Immunosuppressive therapy was used for 6 months. Bioptic specimens were harvested during hardware removal at 12 months follow-up in both patients. Bioptic specimens were examined histochemically and immunohistochemically and by immunohistochemistry to analyze specific cellular markers. ICRS II scoring system was used to evaluate the grafted tissue. Results: At 24 months both patients obtained good clinical result with pain relief and ROM comparable with that of the controlateral healthy joint. Good radiographic consolidation of the graft was observed at 5 months. Radiographic arthritis of the BFOA was nevertheless evident at follow-up. Bioptic specimens showed cartilaginous structure with an high proteoglycan content and a normal cellular distribution, collagen Type II expression. Analysis of synovial biopsies demonstrated limited number of macrophages, without prominent perivascular inflammatory cell infiltrates or lymphoid aggregates. Conclusions: For properly selected patients without symptomatic and disabling rotator cuff deficiency, BFOA can result in improvements in pain, motion, function, and patient satisfaction. Furthermore, unknown factors still have an influence on allografts outcome. Radiographic arthritis at follow-up, was evident although immunosuppressive therapy, correct positioning and size of the graft. This finding, even if uncorrelated to the clinical results, still remain cause of concern. Further research regarding the immunological behavior of transplanted cartilage are needed in order to improve the results.

P212A novel cell therapy for cartilage regeneration based on adipose mesenchymal stem cells and a new biomaterial of natural origin.B. Castro1, V. Soto-Cerrato2, M. Nicolàs2, A. Herrero1, I. Gartzia1, M. Montes1, M. del Olmo1, A.G. Fernández2, A. Guglietta2 1Derio/Spain, 2Barcelona/Spain

Purpose: Articular cartilage damage is a common disease that may lead to severe osteoarthritis of the joint. Spontaneous healing of cartilage is difficult due to the avascular nature and low cell density of this tissue. Our purpose is to develop a new tissue engineering strategy to optimize cartilage repair by integrating cells in a scaffold material that functions as an extracellular matrix. Methods and Materials: We evaluated cell viability using live/dead and MTT assays. Gene expression profile was analyzed by qRT-PCR. Results: We first describe Histogel, a novel biomaterial of natural origin with optimal properties for its use in cartilage regeneration. Histogel behaves as a hydrogel, with viscoelastic properties yielding high adhesiveness in the injury and fluidity to be used through arthroscopic implantation. We have identified the expression profile of several key genes to comparatively study the chondrogenesis process in human adipose mesenchimal stem cells (aMSC) either conventionally cultured or introduced into the biomaterial. In the same conditions, cell viability was evaluated showing cell biocompatibility up to 96 hours in the biomaterial. Furthermore, when human cartilage explants were incubated together with this new biomaterial, chondrocytes seemed to be attracted by the hydrogel in a higher extent than when incubated with hyaluronic acid. Conclusions: These results show the potential of Histogel to be a very advantageous scaffold for cartilage regeneration owing to its biological safety and its ability to promote cell adhesion, proliferation and chondrocytic differentiation. Additionally, Histogel has chemotactic trails that will be relevant to recruit cells to the chondral defect.

P214Effects of combining chondrogenic and anti-angiogenic factors on a one-step cartilage repair approachK. Gelse, J. Gusinde, M. Blanke, A. Olk, F. Hennig Erlangen/Germany

Purpose: Microfracture (Mfx) is a simple, minimally-invasive technique that is frequently applied for the induction of cartilage repair tissue, however, this approach mostly generates inferior fibrocartilage. Furthermore, an unphysiological hypertrophy of the subchondral bone plate with vascular invasion and subsequent endochondral ossification within the repair tissue is a frequently observed phenomenon that may interfere with the long-term outcome. Therefore, this study followed a strategy to combine Mfx with the application of recombinant osteogenic protein-1 (OP1) to promote chondrogenesis, and with the application of recombinant Thrombospondin-1 (TSP-1), a protein with anti-angiogenic properties, to prevent excessive endochondral ossification within the repair matrix. Methods and Materials: In miniature pigs, cartilage defects in a total of 12 knee joints were treated by the microfracture technique (Mfx). The Mfx-treated defects either received no further treatment (Mfx), or were filled with collagen-bound OP1 (Mfx+OP1), collagen-bound TSP-1 (Mfx+TSP-1), or with a combination of the two factors (Mfx+TSP-1+OP1), respectively. Six or 26 weeks later, the outcome was assessed by the ICRS histological assessment scale. Results: Defects treated by Mfx were typically characterized by fibrocartilaginous repair tissue and hypertrophic reactions of subchondral bone tissue. The application of OP1 (Mfx+OP) significantly improved the quality of the repair tissue with a hyaline-like, proteoglycan-rich matrix, but was also associated with excessive endochondral ossification. In contrast, the application of TSP-1 (Mfx+TSP) completely prevented subchondral bone hypertrophy and matrix calcification, but did not support chondrogenesis within the repair tissue. Only the combined treatment with Mfx+TSP-1+OP1 could achieve both generation of a hyaline-like repair matrix and prevention of endochondral ossification. Conclusions: These data indicate that the induction of chondrogenesis and the prevention of terminal chondrocyte differentiation have to be addressed by different factors. Future studies may further optimize this “factor-cocktail” and release kinetics in order to yield the unique articular chondrocyte phenotype within the cartilage repair tissue.

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P219Mid-Term Functional and Radiological Outcomes of Allograft Meniscal TransplantationJ.C. Monllau, G. Gonzalez-Lucena, P.E. Gelber, X. Pelfort, M. Tey Barcelona/Spain

Purpose: The purpose of this study was to evaluate the functional and radiographic results on a mid term basis as well as the complications in a first series of meniscal allograft transplantation (MAT) performed by means of suture fixation without any bone block. Methods and Materials: A series of 33 meniscal allograft transplantation were operated on in our institution from January 2001 to October 2003. Inclusion criteria was patients with compartmental joint line pain due to a previous meniscectomy in an otherwise well aligned knee. There were 24 males and 9 females with a mean age of 38.8 years (range, 21 to 54 years). The functional outcomes were evaluated by use of Lysholm and Tegner scores at a mean follow-up of 6.5 years. A Visual Analogical Scale (VAS) for pain was also used. Radiographic assessment included joint space narrowing in Rosenberg view and MRI evaluation. Results: The Lysholm and Tegner scores significantly improved from 65.4 to 88.6 (p < 0.001) and from 3.1 to 5.5 (p < 0.001) respectively, after index surgery. Similarly, the VAS score significantly dropped from 6.4 to 1.5 (p < 0.001). The radiographic evaluation did not show any joint space narrowing (p = 0.38). Meniscal extrusion was a constant finding, averaging 36.3% of total meniscal size. According to the Van Arkel criteria, the survival rate was 87.8% at 6.5 years. The rate of complications was 33%. Conclusions: This study suggests that MAT provides significant pain relief and functional improvement in selected symptomatic individuals on a mid term basis. with a non-negligible reoperation rate.

P220Matrix associated and Autologous Chondrocyte Implantation in the Ankle: Clinical and MRI Follow-up after 2 to 14 YearsC. Hirschfeld, S. Domayer, D. Stelzeneder, R. Dorotka Vienna/Austria

Purpose: The aim of this study was to assess the clinical mid- to long-term results of autologous condrocyte transplantation in the ankle in 19 cases and to evaluate repair tissue quality with T2 mapping at 3T. Methods and Materials: 19 cases with talar cartilage defects were assessed with the AOFAS Score, a modified Cincinnati Score and a subjective Ankle-Hindfoot Score at mean 4.5 ( 2 to 14) years after surgery. Nine patients consented to an additional MRI exam including T2 mapping at 3T. ACI was carried out with a periosteal flap (4 cases), with a matrix assisted ACI technique (Hyalograft C, 13 cases) or with a matrix assisted collagen-I-gel condrocyte transplantation system (CaReS, 2 cases). At the time of surgery, the patients had a mean age of 27,8 ± 7,5 years (21-42) and the defect size measured during surgery was 1.48 ± 0,64 cm² (0.5-3).Results: Significant improvement was found in all cases. The AOFAS improved from 52.2 to 87.0 , the AHS from 45.8 to 81.4 , and the modified Cincinnati Score from 3.0 to 6.9. All cases that underwent MRI had an intact interface of the repair tissue and the adjacent talar cartilage. T2 mapping results indicated that the collagen and water content of the repair tissue was comparable to adjacent cartilage. Repair tissue (RT) T2 was 30.9 ± 7.9 (22 – 43) ms and reference cartilage (RC) T2 was 37.3 ± 7.5 (27 – 47) ms. The unpaired double tailed student t-test demonstrated no significant difference between RT and RC T2 (P=0.095). Conclusions: ACI is a valuable treatment for talar cartilage lesions that can provide excellent outcome in the long term. Matrix assisted implantation allows for a less invasive procedure and can apparently provide good defect filling as well as repair tissue with a water and collagen content similar to the adjacent cartilage.

P221Autologous Chondrocyte Implantation of the AnkleR. Ferkel, S.K. Kwak Van Nuys/United States of America

Purpose: To report long-term outcomes on patients who have had autologous chondrocyte implantation of the talusMethods and Materials: Thirty-two patients (16 male, 16 female; mean age 34) underwent ACI of the talus after previous failed

P217Isolated Osteochondral Allografting of the PatellaW. Bugbee, A. DeYoung, S. Görtz La Jolla/United States of America

Purpose: Cartilage disease of the patellofemoral joint represents a difficult clinical challenge. Although osteochondral allografting has been shown to be effective in the treatment of chondral and osteochondral lesions of the femoral condyle and tibial plateau, there are few clinical studies on osteochondral allografting of the patellofemoral joint. In this study, we report on the outcome of isolated osteochondral allografting of the patella.Methods and Materials: Twenty patients (22 knees) underwent isolated osteochondral allografting (OCA) of the patella. Patients with bipolar or multifocal allografts were excluded. The mean age was 35 years (range, 17–64 years) and 60% were female. 91% had previous surgery (range, 1–10 procedures), including seven patients who had surgery for realignment of the extensor mechanism. The majority of patients had traumatic or degenerative chondral disease or fracture malunion. Mean allograft area was 11.1 cm2 (range, 4.0–17.8 cm2) and 86% (19/22) had total patellar resurfacing. Results: Thirty-two percent had failure of the allograft (defined as revision, patellectomy, or conversion to TKA) at a mean of 19.6 months (range, 6–40 months). Seven of 22 (32%) had further surgery with retention of the allograft. Mean follow-up of the 15 non-failures was 102 months (range, 24–310 months). Mean IKDC pain scores improved from 6.8 to 3.2 (p< 0.01). Mean IKDC function scores improved from 4.3 to 6.6 (p= 0.06). 93% reported less pain, 80% reported better function, and 93% reported improvement. All patients were satisfied with their results and would choose allograft surgery again under similar circumstances. Conclusions: Patella allografting resulted in a high (64%) reoperation rate. However, patients that retained their allografts had significant improvement in pain, better function, and were satisfied with their treatment. We conclude that patella allografting is a useful salvage treatment option in carefully selected patients.

P218Fresh total shell osteochondral allograft of the knee: surgical technique, clinical and histological resultsS. Giannini, R. Buda, A. Ruffilli, M. Cavallo, G. Desando, S. Neri, B. Grigolo, F. Vannini Bologna/Italy

Purpose: Post-traumatic arthritis of the knee poses a reconstructive challenge for the young active patient. Fresh total shell osteochondral allograft (FTSOA) is a fascinating option since chondrocytes showed to be viable many years after transplantation. Nevertheless, the source of cells in transplanted cartilage has not been confirmed as being from the donor or the recipient. Purpose of this study was to examine the effect of FTSOA as an alternative to prosthesis in post-traumatic arthritis of the knee and to provide a characterization of chondrocytes DNA. Methods and Materials: 8 patients, age 42±12 years, affected by pst-traumatic knee arthritis, received FTSOA. Patients were evaluated clinically and by X-Rays, CT scans and MRI. The technique provided a complete total joint resurfacing with a continuous 1.5cm thick FTSOA, including the resurfacing of the patella and menisci. Biopsies, including cartilage, ligament, synovia and menisci were obtained during revision and evaluated by histological, immunohistochemical analyses and by allograft genetic typing by microsatellite analysis in respect to recipient and donor DNA. Results: Satisfactory allograft consolidation, ROM and weight-bearing were resumed at 6-8 months in all the cases. A severe joint laxity developed at 10-16 months in 6 patients requiring arthroplasty. Two patients are satisfied with the result. Histological evaluation showed cartilage degenerative changes with hypocellularity. Fibrocartilaginous aspect and positivity to extracellular matrix degrading enzymes were evident in the soft tissues. The genetic typing showed a mixed recipient/donor DNA profile in all the cases. Conclusions: Knee FTSOA demonstrated high rate of failure, although specifically designed jigs, excellent positioning and good consolidation rates. The integration of recipient cells into transplanted knee allograft investigated by DNA typing, resulted in persistence of donor cells in the graft together with presence of cells from the host, thus suggesting partial in growth of the transplanted allograft by host cells at different degrees.

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Results: PRP administration induced a significant decrease P<0,005 in COMP concentrations both in plasma ( tendinitis: 11,06 U/L±1,55SD); tenosynovitis 14,15 U/L±7,32SD; OA:19,98 U/L ±7,4SD), and increases in GAG concentrations although not statistically significant in the samples tested. Fluctuations in HA concentrations were seen but were not significant. . Discussion: The significant decrease in COMP concentrations may be interpreted as a sign of decrease in degradation and the increase in GAGs although not statistically significant, as a sign of synthesis. Conclusions: From the 3 mollecules tested in this study COMP was the marker that reflected both baseline lesional status and response to PRP treatment.

P224Synovial fibrinolysis activity as a potential early marker in horses with inflammatory joint disease.Thais Ribera, Luis Monreal, M. Ángeles Delgado, José Ríos, M Prades Departament de Medicina i Cirurgia Animal , Universitat Autònoma de BarcelonaM. Prades1, T. Ribera2 1Cerdanyola/Spain, 2Barcelona/Spain

Purpose: To assess synovial fibrinolysis activity in horses with joint inflammatory disease. Methods and Materials: Study Design: Prospective observational clinical study and pilot experimental study. Sample Population: 34 synovial fluid samples from 24 horses with joint disease and 35 controls. Additionally, 5 synovial fluid samples collected before and after induction of chondral lesion from 3 experimental horses. Methods: Horses with synovial disease had a diagnosis consistent with osteoarthritis (OA) or osteochondritis dissecans (OCD). D-dimer concentration was measured in synovial fluid samples. Synovial fluid also was routinely analyzed. Results: In the clinical study, synovial D-dimer concentration was significantly (P= 0.002) higher in the inflammatory group (median, interquartile range; 25,024 ng/mL, 7,914-55,104) compared to controls (10,832 ng/mL, 4,808-16,652). The logistic regression analysis showed that horses with synovial D-dimer concentration above 20,000 ng/mL were statistically (P<0.001) more consistent with inflammatory joint disease, resulting in an OR= 9.8 (95% CI= 2.9 to 33.8) for inflammatory joint disease when synovial D-dimer concentration was above 20,000 ng/mL. Conclusions: Inflammatory joint disorders in horses are associated with an increased fibrinolysis activity in synovial fluid.

P225Clinical and radiological evaluation of matrix-induced autologous chondrocyte implantation (MACI) at five years.J. Ebert, M. Fallon, W.B. Robertson, T.R. Ackland, M.H. Zheng, D.J. Wood Perth/Australia

Purpose: This study presents radiological and clinical outcome to five years post surgery, for a consecutive series of patients following matrix-induced autologous chondrocyte implantations (MACI), to evaluate whether MACI provides a suitable mid-term treatment option for articular cartilage defects in the knee.Methods and Materials: An uncontrolled, prospective study design was used to assess clinical and radiological outcome in 41 patients (44 knees; 53 grafts) to five years following MACI. Following surgery, patients underwent a structured, supervised rehabilitation program of progressive exercise and graduated load bearing to protect, and then stimulate the healing process. Clinical outcomes were measured using the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Short-Form Health Survey (SF-36), the six-minute walk test and knee range of motion. High resolution magnetic resonance imaging (MRI) was undertaken at 3, 12 and 24 months, as well as at five years post-surgery, to describe the quality and quantity of repair tissue.Results: Patients demonstrated a significant improvement (p<0.05) throughout the post-operative timeline for all five subscales of the KOOS, both subscales of the SF-36, the six-minute walk test and active knee extension. Patients also demonstrated an increased MRI composite score over time that improved significantly (p<0.05) from three months to five years post-surgery. Post-hoc analysis demonstrated the improvement occurred predominantly in the first 12 months post-operatively, though was maintained to five years.Conclusions: Both clinical and radiological outcome improved up until two years post-surgery, and were maintained to five years. Based on these data, it would appear that MACI provides a suitable mid-term treatment option for full thickness, localised articular cartilage defects in the knee.

surgical management for osteochondral lesions. There were 24 medial and 8 lateral lesions, with an average size of 18x11 mm (198 mm2). Twenty-three patients underwent ACI of the talus alone; nine had ACI with a “sandwich procedure”. Mean follow-up was 66 months (range 24-113 months). 25 of 32 patients underwent second look arthroscopy during hardware removal at 1 year.Results: Preoperatively, 25 patients rated their ankles as poor and 3 as fair, using the simplified symptomatology evaluation. At last follow-up, 8 were classified as excellent, 12 as good, 5 as fair and 1 as poor. One patient qualified as a failure having had a fusion at 4 years post-op. Tegner activity scale improved from 1.6 to 4.4. The Finsen score (modified Weber score) showed significant improvement in total score. There was also overall agreement between the Finsen score and the American Orthopaedic Foot and Ankle Society ankle hindfoot score, with significant improvement from 50.2 to 86.4. Nine of eleven previously reported patients showed continued maintanence of improvement in Tegner, Finsen and AOFAS scores.Conclusions: Autologous chondrocyte implantation of the talus yields significant functional improvement with enduring long-term results in patients who have failed previous surgery..

P222Synovial fibrinolysis activity in foals with septic joint disease: Ribera T, Monreal L, Armengou L & Prades M : Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain.M. Prades1, T. Ribera2 1Cerdanyola/Spain, 2Barcelona/Spain

Purpose: The objective of this study was to assess synovial fibrinolysis activity in foals with septic arthritis Methods and Materials: Prospective observational clinical study. Foals admitted for septic joint disease (neonates or not) were included. Synovial D-dimer concentration was determined together with routine analysis. Septic joint was diagnosed when it resulted consistent with septic disease (synovial TNCC> 30,000 cells/ïL, total protein> 4 g/dL and percentage of neutrophils> 80%, or a positive synovial culture) Results: The study included synovial fluid samples from 18 septic newborn foals with polyarthritis, 9 septic newborn foals without polyarthritis, 9 older foals with a septic joint, and 4 control foals. Synovial D-dimer concentration was markedly higher in septic/polyarthritic foals (median, interquartile range 299,104 ng/mL, 122,960-679,390) and septic joint foals (400,384 ng/mL, 256,768-632,064) when compared to septic/non-polyarthritis foals (40,640 ng/mL, 21,240-89,426) and controls (36,753 ng/mL, 30,760-43,562). A decrease towards normalization in synovial D-dimer concentration was seen in 10 septic foals after receiving treatment. A positive correlation between an increased synovial D-dimer concentration and other synovial fluid analysis variables was observed. Discussion: Septic joint disorders in foals are associated with a marked synovial fibrinolysis activation to subsequently destroy fibrin formed. This has been studied in degenerative joint diseases in humans and experimental animals, and more recently in horses, but no information is available in patients with acute septic arthritis Conclusions: Synovial fibrinolysis activity was higher in foals with septic joint disease, and may be used as a marker of inflammatory joint disease for diagnostic and follow up purposes.

P223Local PRP therapy induces fluctuations on plasmatic and synovial fluid biomarker concentrationsM. Prades1, I. Abellanet2 1Cerdanyola/Spain, 2Barcelona/Spain

Purpose: To report the effect of intralesional or synovial injection of PRP on plasmatic and synovial fluid concentrations of 2 biomarkers (COMP and GAGS) and hyaluronan. Methods and Materials: Seven horses with DJD, 8 horses with tendonitis, 4 patients with tenosynovitis, seven horses free of musculoskeletal disease (negative controls) and 2 horses with lesions and no treatment (2 positive controls) were included in the study. Baseline COMP, GAGs and HA plasmatic and sinovial fluid concentrations (in DJD affected horses) were measured initially and then after treatment with PRP and normal saline in some horses. A non parametric test was used for statystical analysis (Wilcoxon p<0.05). Biomarker concentrations and HA were measured by ELISA at the IBB (Biomedicine and Biotechnology Institute at the UAB).

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from articular cartilage inevitably dedifferente into fibroblast-like cells. Spontaneous generation of hyaline cartilage - is physiologically present only in skeletal growth centers. A unique accessible site of hyaline producing cells was found in the neonatal porcine mandibular condyle with the capacity to differentiate into stable hyaline cartilage spontaneously.Methods and Materials: Mandibular condyle (MC)-derived chondrocytes (MCDC), harvested from neonatal SPF porcine were cultured in vitro. The cultured cells create a continuous implantable cartilage film (Cartimove™), offering the possibility of simple mechanical handling. Re-culturing/implantation preserves its proliferating and differentiating activities as demonstrated by immunohistochemistry and RT-PCR. In the pre-clinical studies, Cartimove was implanted in lesions created in the medial femoral condyle of 8 goats. Three, 6 and 13 months implants were retrieved and analyzed morphologically, biochemically and biomechanicallyResults: In vitro, at 10 days, the expression of type II collagen is amplified by about 50 folds (RT-PCR), concomitantly, type I collagen expression is reducing to negligible values. Co-culturing with human cells, showed no transmittable pathogen. The preclinical studies, demonstrated tissue filling, environmental integration and hyaline characteristics. Local as well as lymphatic organ analysis, showed no detectable immune response.Conclusions: We propose that Cartimove cultured from porcine-derived MCDC cells, is capable of replenishing cartilage lesions with genuine hyaline cartilage.

P229Correlation between localisation of 4° chondral leasions in knees and the clinical result after autologous chondrocyte transplantationB. Böttenberg, P. Schaeferhoff Köln/Germany

Purpose: The method of the ACT demands to repair deep and huge size cartilage defects with hyalin respectively hyalin like cartilage. Previous studys describe only rare the effect of the ACT in knees regarding to their chondral defect region. Methods and Materials: This monocenter study presents the correlation between the localisation of 4° chondral leasions in knees and the clinical outcome after ACT treatment. The clinical evaluation was performed using the subjective and the objective part of the IKDC2000 score for 52 patients before and after treated with ACT (meanfollowup 38,6 months). Results: 52(35m/17f) patients in the age between 18 and 45 years (mean age 38 years) participate in this study. The defect size average is 6,13cm². Concerning to the cartilage defect localisation we develop five different cartilage defect region groups.Group 1 (medial femoral, n=15), Group 2 (lateral femoral, n=10), Group 3 (trochlear, n=10 ), Group 4 (retropatellar, n=8) and Group 5 (combinied defect regions, n=9). The follow up examination shows in all five groups a signifivant rise concerning to the subjective und objective IKDC2000 score after ACT treatment ( p<0,001). The subjective part of the IKDC2000 score shows significant poorly post-operatively results for the the patients in the groups 4 and 5 than in the groups 1,2,3 (p<0,001), where as there is no significant difference betwenn the 5 groups pre-operatively. Regarding to the objective part of the IKDC2000 score with a view to the chondral leasion area there are no significant differences between the pre- and postoperative evaluated IKDC2000 scoure values (p<0,001). Conclusions: The presented data indicate autologous chondrocyte transplantation as an effective and safe option for the treatment of large full thickness cartilage defects in knee joints with significant better subjective results for patients with cartilage defects in the medial or femoral condyle and trochlear region than in retropatellar or combinied cartilage leasion regions.

P230Can Joint Preservation be possible for Osteonecrosis of the Knee by Autogenous Osteochondral Graft Transplantation? -Middle-term Results-Y. Matsusue1, M. Kubo1, Y. Nakagawa2 1Otsu/Japan, 2Kyoto/Japan

Purpose: Repair of the osteochondral lesion of osteonecrosis of the knee is a difficult and controversial issue. In this paper, we present the middle-term clinical results of autogenous osteochondral grafting for osteonecrosis of the knee including steroid-induced osteonecrosis.

P226Open Autologous Osteochondral Transplantation on Posterior Aspect of Medial Femoral Chondyle with Medial EpicondylectomyY.G. Koh, S. Kim, S. Jo Seoul/Korea

Purpose: The purpose of this presentation is to introduce a novel approaching method of autologous osteochondral transplantation for treatment of chondral and osteochondral lesions on posterior aspect of the medial femoral chondyle(MFC) of the knee.Methods and Materials: Operative technique : After the arthroscopic procedure, about 8cm skin incision is made on the midline of the knee joint and arthrotomy is performed with midvastus approach. MFC is exposed and the medial epicondyle is confirmed. Medial epicondylectomy is done with including about 2cm diameter bone fragment and attached medial collateral ligament. Subsequently was carried out with the OATS technique, which allows for press-fit graft implantation. Without medial epicondylectomy, OATS devices cannot be applied perpendicularly on the osteochondral lesion of posterior aspect of the MFC then, it is impossible to achieve perpendicular graft insertion. After autologous osteochondral transplantation is performed, reattachment of the medial epicondyle is done with one washer and one 5.0 mm diameter cannulated screw.Results: Case: Patient was a 48-year old male complained pain on the right knee when he standed up from sitting or squating position, climbed a mountain and went up the stairs. There was an osteochondral lesion on posterior aspect of the MFC on the preoperatively evaluated MRI. Autologous osteochondral Transplantation was performed with medial epicondylectomy approach. Postoperative evaluation was performed when one year passed after surgery. Lysholm score improved from 56 preoperatively to 89 postoperatively. IKDC assessment was improved from abnormal(C) preoperatively to normal(A) postoperatively. VAS score were improved 8 to 2 on standing-up motion and 8 to 3 on going-up stairs motion. Congruency of the articular surface was restored on postoperatively evaluated MRI.Conclusions: An approach with medial epicondylectomy including attached medial collateral ligament is one of the recommendable alternative for treatment of chondral and osteochondral lesions on posterior aspect of the MFC of the knee with OATS technique.

P227Combined autologous chondrocyte implantation (ACI) with supra-condylar femoral varus osteotomy, following lateral growth-plate damage in the knee of an adolescentS. Vijayan1, G. Bentley2, R. Carrington1 1Stanmore/United Kingdom, 2London/United Kingdom

Purpose: We report the 8-year clinical and radiographic outcome of an adolescent patient with a large osteochondral defect of the lateral femoral condyle, and ipsilateral genu valgum secondary to an epiphyseal injury.Methods and Materials: This patient was successfully managed with Autologous Chondrocyte Implantation (ACI) and supracondylar re-alignment femoral osteotomy.Results: Long-term clinical success was achieved using this method, with the patient reporting significant improvements in Modified cincinnati, Bentley and Visual analogue scores post-operatively when compared to their pre-opartive state. Since treatment, the patient has resumed semi-professional sporting activities which was was not possible before.Conclusions: We conclude that this technique illustrates the effective use of re-alignment osteotomy in correcting mal-alignment of the knee, protecting the ACI graft site and providing the optimum environment for cartilage repair and regeneration. This is the first report of combined ACI and femoral osteotomy for such a case.

P228Xenograft chondrocytes for cartilage repair– A unique source for genuine hyaline cartilage implantG. Maor, R. Goldberg, S. Bar-Zvi, G. Nierenberg Haifa/Israel

Purpose: Since hyaline cartilage does not regenerate, biological long term solutions are based on tissue regenerative therapies. The current available cell base therapy is autologous chondrocyte implantation (ACI), which results invariably in mixed fibrous / hyaline-like tissue in various proportions. Chondrocytes separated

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P232Matrix guided Autologous Chondrocyte Transplantation (MACI) is effective as second line treatment of large cartilage defects in the knee: 2-year clinical resultsP.E. Müller, M.F. Pietschmann, A. Horng, T. Niethammer, S. Lehmann, M. Feist, I. Pagenstert, C. Glaser, V. Jansson Munich/Germany

Purpose: Over the last years Matrix guided Autologous Chondrocyte Implantation (MACI) has become an important surgical technique for treating large cartilage defects. The aim of our prospective study was to evaluate the effectiveness of MACI as first and second line treatment option for cartilage repair of large defects in the knee.Methods and Materials: 43 pts. were treated with MACI for cartilage defects of the knee between 2004 and 2008. We assessed clinical situation pre- and postoperatively using the IKDC Knee Examination Form, VAS for “pain at rest” and “pain at weight bearing”. MRI scans for evaluation of cartilage regeneration were obtained postoperatively. The modified MOCART score and T2 relaxation time were applied to assess cartilage regeneration.Results: One third of the cartilage defects were localized retropatellar the remaining on the femoral condyles. The average defect size was 5,7 cm2. After one year the clinical assessment showed a significant improvement across all patients. After two years we found an improvement as well. With regard to first or second line therapy MACI showed similar results in both groups. Etiology and age of the cartilage lesion are important factors that influence the postoperative clinical outcome. The MRI showed an significant improvement of the implanted scaffold over time as well.Conclusions: The present study confirms the benefits of MACI in young pts. with large cartilage defects of the knee. Patients with MACI as second treatment option achieved similar results as patient which had MACI done as first therapy. We expect a good long-term outcome of MACI comparable to that of classic ACI.

P233Patellar dislocation causing severe patellar-trochlear cartilage damage, treated by BioCart™II implantation. A case studyA. Friedman1, G. Chaimsky1, J. Lowe1, H. Barkay2, A. Yayon2 1Jerusalem/Israel, 2Ness Ziona/Israel

Purpose: A major challenge for matrix-assisted autologous chondrocyte implantation is the susceptibility of implants to mechanical loads and shear forces in the joint during neocartilage maturation, integration and remodeling, particularly in multiple lesions where rapid implant maturation is critical for rapid mobilization and rehabilitation. Here we describe treatment with BioCart™II, a novel fibrin-HA porous mechano-transductive scaffold, supporting autologous chondrocyte implantation in both opposing lesions in a patella-trochlear kissing lesion Methods and Materials: Right knee arthroscopy in a 24-year old policeman injured playing football revealed severe chondral damage to the medial patellar facet (1x0.8cm) and lateral trochlea (2x2cm), both ICRS grade IIIc, compatible with lateral patellar dislocation. Several loose cartilagenous bodies were removed. Pre-operative MRI revealed a “kissing” lesion. Five weeks later, the patient underwent arthrotomy. BioCart™II was implanted in both the patella and trochlea using autologous chondrocytes cultured in ProChon. A protective osteotomy (Fulkerson) with anterio-medialization of the tibial tuberosity was performed. Post-surgery, a knee immobilizer was applied and weight bearing in extension was allowed with CPM 0-40 degrees then intensive physiotherapy rehabilitation for 4 months. .Results: MRI 8 months post-surgery showed almost normal appearance of the implanted cartilage. Two years after implantation of two opposing but overlapping BioCart™II implants it the patella and trochlea, the patient is back to full service as a policeman, including sport activities. The physical examination of the knee is normal with only 10 degrees of reduction in knee flexion and very mild reduction in quadriceps muscle tone. Conclusions: BioCart™II has been scientifically designed to mimic the topography of natural hyaline cartilage. Together with the growth factor-directed culture of the chondrocytes which preserves the chondrogenic potential of the cells an accelerated rehabilitation schedule can be followed which facilitates the return to normal physical activity. There is a need for more experience before extensively prescribing BioCart™II for such complex injuries.

Methods and Materials: Thirty-two patients (36 knees) with at least 24-months follow-up periods were included in this study. The age ranged from 21 to 76 years of age, with a mean of 48. The follow-up period ranged from 24 to 165 months with a mean of 52. The cause of osteonecrosis was steroid-induced in 10 and idiopathic in 26. There was one knee with osteonecrosis of the medial tibial plateau. Correction osteotomy was performed in 22 knees at the same time of osteochondral grafting. The size of the osteochondral defect ranged from 156 to 1080 mm2 with a mean of 475. The number of transplanted grafts ranged from 1 to 6 with a mean of 2.94.Results: The clinical results by ICRS cartilage evaluation form were normal in 14, nearly normal in 20, and abnormal in 2 knees. The range of motion was normal except in one knee with a flexion of 135 degrees. Second-look arthroscopy revealed complete integration in 59% of the cases and the others showed partially incomplete integration between the grafts. MRI showed that complete healing of the necrotic lesion was observed in 14 knees (41%) and a significant lesion was remained in one knee. In steroid induced osteonecrosis with unstable but intact articular cartilage on the necrotic lesion, the lesion is treated by multiple drilling and bone grafting through cartilage windows created OATS instruments; then followed by two large osteochondral plugs transplantation into the windows (eyeplass-plasty). Abnormal results were obtained in one patient who had non-union of osteotomy and another patient with steroid-induced osteonecrosis who had avulsion of the remaining cartilage after transplantation. When a proper alignment was obtained, autogenous osteochondral grafting for osteonecrosis of the knee can provide a good knee function including full flexion and could avoid total knee arthroplasty.Conclusions: Autogenous osteochondral grafting can give satisfactory middle-term results for the osteonecrosis of the knee including steroid-induced osteonecrosis.

P231Arthroscopic matrix-induced autologous chondrocyte implantation (MACI) in combination with accelerated rehabilitationJ. Ebert1, R. Carey-Smith2, M. Fallon1, H. Davies1, S. Garett1, D.J. Wood1, G. Janes1 1Perth/Australia, 2Coventry/United Kingdom

Purpose: This study presents the clinical and radiological outcome in patients treated with arthroscopically performed matrix-induced autologous chondrocyte implantation (MACI), combined with an ‘accelerated’ return to full weight bearing (WB). Methods and Materials: Fifteen patients with full-thickness defects to the femoral or tibial condyles were treated with MACI performed arthroscopically. Following surgery, patients underwent a graduated rehabilitation program that aimed to protect the implant initially, then incrementally increase the load until full WB was attained at 8-weeks post-surgery. Clinical outcomes were measured pre-surgery and at 3, 6 and 12 months post-surgery using the KOOS, VAS, the six-minute walk test and knee range of motion. Clinical outcomes were compared with previously established clinical scores from patients who had undergone MACI performed through open arthrotomy, in combination with an identical, accelerated rehabilitation protocol. High resolution MRI was undertaken at 3 and 12 months post-surgery.Results: From pre-surgery to 12 months post-surgery, KOOS and VAS scores demonstrated significant improvement (p<0.05), though there was no difference (p>0.05) to patients who underwent open arthrotomic MACI. There was no difference (p>0.05) in six-minute walk distance, though significantly better (p<0.05) active knee flexion was observed at 3 and 6 months in the arthroscopic MACI group. Patients demonstrated an increased MRI composite score over time that improved significantly (p<0.05) from three to 12 months post-surgery, but did not differ (p>0.05) from patients following open MACI. Conclusions: Arthroscopic MACI in combination with ‘accelerated’ rehabilitation has shown encouraging early results, with good early graft infill and no graft complications. Arthroscopically performed MACI may reduce the co-morbidity associated with open arthrotomy, such as adhesions, decreased range of motion, pain and impressive scars, whilst providing faster post-operative rehabilitation due to reduced pain and muscular deficits.

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P236Therapeutic response to Autologous Cartilage Tissue Implant (ACTI), Neocart is significantly greater in comparison to microfracture (MF) arthroplasty surgery following treatment of distal femoral cartilage lesionsD.C. Crawford1, R.J. Williams, III2 1Portland/United States of America, 2New York/United States of America

Purpose: This exploratory multi-site (FDA and IRB monitored) phase II prospective randomized clinical trial evaluates the efficacy of a tissue-engineered bio-implant, Neocart, for treatment of hyaline cartilage knee injury in direct comparison to microfracture arthroplasty.Methods and Materials: Thirty patients were randomized (2:1;ACTI:MF) at arthroscopic confirmation of grade III ICRS lesion(s). Lesional microfracture arthroplasty or hyaline cartilage biopsy was performed. The ACTI (Neocart) produced by seeding collagen I matrix scaffold with autogenous chondrocytes and bioreactor treatment, was implanted 6 weeks post-arthroscopic biopsy. Standard interval evaluations applied multiple validated clinical outcomes measures. Responder analysis was applied using a dual threshold criteria based on previously reported MPCI (minimal perceptible clinical improvement) thresholds for both the KOOS pain and IKDC outcomes measures.Results: Twelve month data for all enrolled (21ACTI:9MF) and for the first ten (7ACTI:3MF) reaching 24 months is reported. Mean age (40+/-9yrs), BMI (28+/-4), injury acuity (3+/-5yrs) and lesion size (ACTI 287+/-138mm2 v. MF 247+/-127mm2) is similar. Clinical outcomes demonstrated by IKDC, KOOS, SF-36, and VAS Pain scores are shown in Table 1. In both groups SF-36 physical and IKDC improved from baseline (p<0.025) at one year. Improvement for ACTI v. baseline was significant (p<0.025) for all additional measures at twelve months and all but KOOS Sports at six months. ACTI showed greater change from baseline than MF in IKDC (p<0.025) and KOOS pain (p<0.025) at one and two years. Using ANOVA, the difference in both IKDC and KOOS Pain change from baseline between MF and ACTI was also significant (p=0.028) and (p=0.016) for each measure. Similarly, more patients (P=0.0125, Fischer’s exact test) in the ACTI arm were therapeutic responders at 6 months (48% v. 25%, data not shown) and 12 months (76% v. 22%) (Fig. 1). Conclusions: This initial prospective randomized study indicates ACTI, using the NeoCart implant treatment has greater clinical efficacy than microfracture.

P237A Novel technique for fully arthroscopically performed 3-dimensional autologous chondrocyte implantation (ACT-3D ) for retropatellar lesions. Preliminary results.S. Alevrogiannis, G. Skarpas, A. Triantafyllopoulos Athens/Greece

Purpose: To present our experience in using a novel technique for autologous 3D chondrocyte implantation (ACT-3D), performed in fully arthroscopical manner, for treatment of retropatellar cartilage defects. Methods and Materials: We treated operatively in our Dept., 5 symptomatic patients with retropatellar lesions. All patients were recreational athletes and the mean age was 32 years old (17-54 y.o.). The mean area of cartilage defect was 3.8 cm² and all the cases were classified as grade III(3) and IV(2) according to Outerbrigde scale. All of them were treated fully arthroscopically. 3 patients were male and 4 patients had the operation at the right knee. In 2 cases the defect was due to trauma, while 3 were due to osteochondritis dissecans. 3 of them had MFx procedure performed elsewhere, more than 5 years ago. We performed 5 applications of ACT3D as single procedure, with the patient in prone position. Preop. and postoperative evaluation of patients was done using the Modified Cincinatti (MC) Rating System(0-100), the VAS (visual analogue pain score) (0-10), IKDC Knee examination score and Patient Outcome Function score. Results: All the procedures progressed uneventfully. A specialized rehabilitation protocol was followed. We assessed the patient at six months and one year post-operatively; using Lysholm & Gillquist Score, VAS pain score and the Patient Outcome Function score. The follow-up using MRI showed adequate filling of the defect without significant graft-associated complications for the same period. The clinical outcome was excellent . Conclusions: Our preliminary results of this novel technique for autologous-3D chondrocyte implantation for the treatment of retropatellar cartilage defects, seems to be more than encouraging.Rehabilitation protocol is quicker due to minimal trauma. As far as we know this is the first publication in the literature regarding a novel technique for 3rd generation ACI fully arthroscopically performed, concerning retropatellar lesions.

P234Osteochondral transplantation in the therapy of osteochondral lesions in the elbowS. Vogt, A.B. Imhoff München/Germany

Purpose: Effective treatment of osteochondral lesions in the elbow is difficult. Débridement and microfracture or drilling techniques are often insufficient and provide only temporary symptomatic relief. The purpose of this study was to evaluate the treatment of these lesions with osteochondral autografts. Methods and Materials: From 1996 to 2009, 19 patients with osteochondral lesions (n=20) of the capitellum humeri, trochlea, or radial head were treated with osteochondral grafts, which were harvested from the proximal aspect of the lateral femoral condyle. The patients were evaluated preoperatively and postoperatively, with an average follow-up of 6.6 years. The Broberg/Morrey score was chosen for functional evaluation of the elbow, and the ASES score was used for the analysis of pain. All patients had imaging studies done preoperatively to evaluate the defect, postoperatively to assess the ingrowth and viability of the graft and at the final follow-up. The ipsilateral knee was examined for donor-site morbidity. Results: The Broberg and Morrey score improved significantly from preoperatively to postoperatively, and pain scores were significantly reduced (p < 0.05). At the time of the final follow-up, flexion and extension were equal bilaterally in all patients. The postoperative magnetic resonance imaging scans showed graft viability and a congruent chondral surface in all patients. At the last follow-up there were only minor degenerative changes in some patients. No donor-site morbidity was noted at one year postoperatively and at the final follow-up. Conclusions: The osteochondral autograft procedure described in the present study provides the opportunity to retain viable hyaline cartilage for the repair of osteochondral lesions in the elbow while restoring joint congruity and function. These results suggest that the risks of a two joint procedure are modest and justifiable.

P235Phenotypic modulation of chondrocytes from human knee joints with osteochondritis dissecans: Implications for chondrocyte implantation procedures.M. Aurich1, G.O. Hofmann1, B. Rolauffs2, J. Mollenhauer3 1Jena/Germany, 2Tuebingen/Germany, 3Reutlingen/Germany

Purpose: To characterize the biological quality and potential of chondrocytes from the dissected fragment. Methods and Materials: 20 patients with cartilage lesions in the knee were involved. Dissected fragments from 10 patients with typical OCD were harvested at arthroscopy. A biopsy of cartilage from the intercondylar notch was taken from the same joint and from 10 patients with a traumatic full thickness cartilage defect as part of the autologous chondrocyte implantation (ACI) procedure. Chondrocytes were isolated by enzymatic digestion and expanded by subsequent primary cell culture. Before and after cultivation, mRNA levels of collagen types I, -II, and -X were determined by semi quantitative RT-PCR. In two patients, a biopsy of the repair tissue was taken 6 and 18 months after ACI.Results: In total, 0.81 ± 0.33 x 106 cells per gram tissue could be recovered with no difference between dissecate and notch cartilages (cell viability ≥ 90 %). Compared with the notch chondrocytes, cells from the dissecate expressed similar levels of collagen type I and -II mRNA including a 100,000fold relative increase of col1 over col2 after cell expansion. Expression of collagen type X mRNA is significantly less in trauma joints compared to OCD cartilages before and after cell culture. The level of collagen type X message is approx. 50fold decreased after cell culture, indicating a loss of hypertrophic cells or expression of hypertrophic genes in chondrocytes. Post-implantation biopsies show features of hyaline-like cartilage without signs of hypertrophy or mineralization (Figure 1). Conclusions: The high viability, quality and activity of the extracted cells suggest a still preserved intrinsic repair capacity of the dissecates. The molecular analysis indicates phenotypic modulation of the isolated chondrocytes during cell culture. The similar quality of the cells from both dissecate and notch after cell culture suggests the use of either cartilages as a cell source for ACI.

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P240Characterized Chondrocyte Implantation in the Patellofemoral Joint: a 1 to 3 Year Follow-UpJ. Vanlauwe, T. Claes Leuven/Belgium

Purpose: To assess clinical outcome in patients treated with Characterized Chondrocyte Implantation (CCI) for full thickness lesions in the patellofemoral joint up to 36 months post surgery.Methods and Materials: Patients with symptomatic patellofemoral full thickness cartilage lesions were treated with ACI using characterized chondrocytes (ChondroCelect®) covered with a Chondro-gide® membrane. clinical outcome was assessed using the Knee Injury and Osteoarthritis Outcome score (KOOS) and a Visual Analogue Scale (VAS) for pain. responders were defined using 5 categories (>10 points and >20, 30, 50, 70%) based on KOOS and VAS.Results: Thirty-six patients, with a mean defect size of 4.89 cm² (1.5 to 11 cm²), were treated in the patella (n=25), trochlea (n=8) or kissing lesion (trochlea and patella; n=3). The mean follow-up period was 24.67 months . Treated patients showed statistically significant improvements in KOOS (at 6, 12, 18, 24 and 36 months) and VAS (at 12 and 24 months). Responder analysis identified approximately 75% of patients with a clinically relevant improvement greater than 10 points. Treatment failure was observed in 4 patients. The most commonly reported adverse events were joint crepitation (n=17) and arthrofibrosis (n=7).Conclusions: CCI resulted in statistically significant and clinically relevant improvement over time. these results add to the evidence demonstrating that ACI is a useful technique for the repair of patellofemoral cartilage lesions and illustrate the potential of CCI in the context.

P241Autologous chondrocyte implantation does not prevent the need for arthroplasty in patients with pre-existing osteoarthritisS. Nawaz1, P.K. Jaiswal2, K. Gallagher2, G. Bentley3, D. Park3, R. Carrington3, J. Skinner3, T. Briggs3 14LP/United Kingdom, 2Stanmore/United Kingdom, 3London/United Kingdom

Purpose: The rate of arthroplasty or osteotomy in patients who had undergone autologous chondrocyte implantation (ACI) for osteochondral defects in the knee was determined. Furthermore, we investigated whether any radiographic evidence of osteoarthritis (OA) prior to ACI was associated with poorer outcome following surgery. Methods and Materials: We retrospectively reviewed the medical notes and radiographs of 144 patients (mean age 34.9) who underwent ACI from 1998 to 2005 at our institution. Knee function was assessed according to the Modified Cincinnati Score (MCS) pre-operatively and at a mean of 64.3 months postoperatively (range 12 – 130). Radiographic changes were graded according to the Stanmore grading system. Results: Patients were divided into 2 groups; Group A were patients with no evidence of OA (n=72) and Group B were patients with OA (n=72). In group A, two patients required total knee replacement (TKR) or unicondylar knee replacement (UKR) and 3 required high tibial osteotomy (overall revision rate 6.9%). In group B, 14 patients required patello-femoral replacement, or UKR or TKR and 7 patients required osteotomy (overall revision rate 29.2%). This difference was significant (p < 0.01). At latest follow up, the mean MCS was significantly higher in Group A (72.5 versus 51.8, p < 0.01). Conclusions: Patients with early radiographic of evidence of OA are unlikely to gain maximum benefit from ACI. The results suggest that ACI does not prevent patients from progressing in their arthritic process and hence requiring joint replacement.

P242Autologous chondrocyte implantation for degenerative cartilage defects of patello - femoral joint.K.P. Slynarski, E. Kurowska Warsaw/Poland

Purpose: Autologous Chondrocyte Implantation (ACI) is typically indicated for focal lesions contained within boundaries of healthy tissue of femoral condyles. Our hypothesis was that population of patients with degenerative changes of patello-femoral joint could also benefit from this procedure and this could reverse or stop the course of osteoarthritis.

P238Simultaneous autologous chondrocyte implantation (ACI) and mosaicplasty for “Kissing” osteochondral defects of the patello-femoral joint : A 10-year follow-up of 2 casesS. Vijayan1, G. Bentley2, J. Skinner1, R. Carrington1 1Stanmore/United Kingdom, 2London/United Kingdom

Purpose: Bi-polar lesions, especially those involving the patello-femoral joint, are difficult-to-treat. The ideal approach to management of “kissing” osteochondral lesions of the patello-femoral joint is a topic of much debate.Methods and Materials: We report the 10-year clinical outcome of two patients with “kissing” osteochondral defects of the patella and trochlear groove of the knee, managed by simultaneous patellar autologous chondrocyte implantation (ACI) with type I/III collagen (chondrogide) membrane (ACI-C) and trochlear mosaicplasty. This is unreported in the Orthopaedic literature.Results: At 10 year follow-up, long-term clinical success was achieved using this method. Significant improvements in the Modified cincinnati, Bentley and Visual analogue scores post-operatively when compared with pre-operative scores were reported. Clinical and arthroscopic assessment of both patients showed excellent outcome, with adequate cartilage regeneration being shown. Conclusions: This case illustrates the effective use of trochlear mosaicplasty for re-creating the anatomical contour of the joint and therefore reducing the likelihood of disrupting the opposing patellar ACI. We conclude that such a technique, allows for the successfull treatment of “kissing” defects of the patello-femoral joint in an otherwise diffcult to treat group of patients.

P239Change over time in cartilage repair after chondrocyte transplantation with a fibrin-hyaluronan matrix - correlation of morphological MRI, biochemical T2 mapping and clinical outcome.I. Eshed1, S. Trattnig2, M. Sharon1, R. Arbel3, G. Nierenberg4, E. Konen1, A. Yayon5 1Ramat Gan/Israel, 2Vienna/Austria, 3Hod Hasharon/Israel, 4Haifa/Israel, 5Rehovot/Israel

Purpose: BioCart™II is a second generation matrix-assisted implantation system composed of autologous chondrocytes cultured in vitro, seeded on a fibrin/ hyaluronic acid based scaffold and implanted by miniarthrotomy. We aimed to evaluate change over time after implantation with BioCart™II of clinical scores, morphological MRI and quantitative T2 values, novel MR sequences that permit the assessment of ultrastructural elements in the neocartilage. Methods and Materials: Thirty-one patients (18-55 years) including those with previous knee operations (e.g. ACL, meniscal repair), were recruited 6-49 months post surgery with BioCart™II to correct a single full-thickness cartilage defect in the femoral condyle. Each subject underwent MRI (morphological (Figures 1 and 2)and T2-mapping sequences) and completed a clinical questionnaire (International Knee Documentation Committee (IKDC) knee evaluation form). MRI scans were scored using the Magnetic resonance Observation of Cartilage Repair Tissue (MOCART) system and a T2-mapping evaluation of the zones in the repair and non-operated cartilage. Analysis included correlation of IKDC scores, MOCART and T2 evaluation with each other, with implant age and with previous intervention history. Results: The IKDC score correlated significantly with implant age (r=0.35 p=0.056) and MOCART score (r=-0.39 p=0.031) and inversely correlated with previous interventions (r=-0.39 p=0.034). A slight, but not significant higher MOCART score was seen in patients with longer duration implants (p=0.199). Zonal cartilage T2 values, indicative of collagen concentration and orientation were significantly lower in longer duration implants (p<0.002) in patients without previous intervention. This trend was also seen in patients with previous interventions although absolute values were higher. Conclusions: Significant improvement with time from BioCart™II implantation was seen by IKDC scoring and MRI T2-mapping values. Patients with previous knee operations also improved, though to a lesser extent, and can benefit from this procedure.

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Results: Significant improvements were found in the Lysholm score (64.1 (±8.7) to 91.4 (±8.3) points) as well as in the Tegner s activity level (2.7 (±1.2) to 4.5 (±0.7) points, both p<0.001). On a visual analogous 10-point scale (VAS) pain decreased significantly from 5.9 (±2.1) to 1.7 (±2.0) while subjective knee function improved from 4.9 (±1.9) to 7.5 (±1.9). There were two arthroscopic revisions after 8/18 months. Both repair tissues revealed a nearly normal result with regards to surface formation, filling and integration according to Brittberg s classification (each 11 out of 12 points). MRI follow-ups showed an adequate filling of the defects, no prolonged effusion occurred. In three cases some residual bone marrow edema was detectable. There were no complications like loss of the scaffold or the bone graft. Conclusions: Combination of scaffold ACI and autologous cancellous bone grafting was shown to be an efficient therapeutic option for osteochondritis dissecans in the knee. Performed as an one step procedure invasivity can be reduced and rehabilitation fastened. Primary and secondary interventions did not show a different outcome.

P245Early results of Autologous Chondrocyte Implantation in the HipK.S. Kumar1, H.S. McCarthy1, S. Roberts1, J.C. Parker1, I.W. McCall2, V. Murakibhavi3, E. Robinson2, P. Harrison2, J. Richardson2 1Shropshire/United Kingdom, 2Oswestry/United Kingdom, 3Belgaum/India

Purpose: There are few surgical options available to treat symptomatic chondral or osteochondral lesions of the hip. Autologous chondrocyte implantation (ACI) is most commonly used to treat cartilage defects in the knee with good results and a long term durable outcome. There are only a few studies of its use in other joints, mainly the ankle. The aim of this study was to assess whether ACI could be used in the treatment of osteochondral lesions in the hip.Methods and Materials: We describe a consecutive series of 16 patients with chondral or osteochondral lesions of the femoral head that underwent ACI. Pre-operative hip function and structure was assessed by the patient-completed Harris hip score (HHS) and where possible, magnetic resonance imaging (MRI). ACI procedures were performed via an antero-lateral or posterior approach, covering the defect with collagen patches (Chondro-Gide®) in twelve patients and periosteum in four. MRI was performed pre-operatively (n=11), at 1 year (n=7) and at 2 years or later (n=8) and HHS was collected annually. Failure was defined as receiving hip arthroplasty.Results: Patients were on average 34 years old (range 14-52) when treated. They improved clinically with time with HHS improving from 55±15 (range 31-78) pre-operatively to 74±17 (range 46-100) at two years and 96±7 (range 86-100) at five years post-operatively. However, 7 patients progressed to hip arthroplasty (mean 30 months, range 13-47 months); of these 6 had cyst formation, 5 had osteophytes, 5 oedema and 5 avascular necrosis (AVN).Conclusions: These results suggest that ACI can be a viable option for treatment of either chondral or osteochondral lesions of the hip but only with careful patient selection. Young patients may benefit, at least in the short term, delaying the need for arthroplasty. However, the presence of cysts, osteophytes, oedema or the development of AVN may indicate a shorter period of benefit.

P248matrix-based autologous chondrocyte implantation for cartilage repair of knee: two-year follow-up in 15 patientsZ. Zhang, S. Hou, Z. Tang, Z. Yang, X. Zhong Beijing/China

Purpose: To evaluate the safety and efficacy of matrix-induced autologous chondrocyte implantation treatment (MACI, Genzyme America) for patients suffering from cartilage lesions of knee in two years follow-up study. Methods and Materials: MACI operation were performed in a total of 15 patients (16 knees), with a median age of 33.7 years and average defect size of 8.54 cm2 from 2004 to 2009. The procedure of MACI began with harvesting of cartilage tissue which obtained from autologous non-weight-bearing region for in vitro proliferation. The cultivated chrondrocytes were seeded onto type â… /type â…¢ collagen bilayer membrane which was glued by fibrin sealant to cartilage defect area after suitable shaped. Knee injury and Osteoarthritis Outcome Score（KOOS）and Magnetic resonance imaging (MRI) were evaluated for clinical rehabilitation at postoperative 3,6,12 and 24months. In addition, 4 arthroscopic biopsies and 2 histological examinations were performed post-surgery.

Methods and Materials: We evaluated 11 patients, aged between 36 to 55 years, with extensive degenerative changes located on patella or patellar facet of femur. Follow up ranged from 4 years to 6 months. All patients underwent chondrocyte implantation on collagen membrane and same rehabilitation process. Weight-bearing status, range of motion restrictions, muscle strengthening, proprioceptive retraining and soft tissue conditioning were the main goals of rehabilitation in those patients.All patients were prospectively clinically evaluated using the KOOS score, SF-36 score, analogue pain score and patient’s subjective assessment and MRI.Results: The KOOS score increased significantly during the observation period. For pain the mean preoperative score was 42 and the score at follow-up was 84. The corresponding scores for symptoms were 36 and 81 for activity of daily life (ADL) 38 and 85 for sport and recreation 26 and 64 and quality of life 19 and 63. The MRI evaluation well correlated with clinical outcome.Conclusions: ACI for degenerative changes of patello-femoral joint is off-label indication however our results suggest that covering exposed degenerative bone with implanted cells on collagen membrane, leading to cartilage-like tissue regeneration as confirmed on MRI, will reduce the pain, improve joint function and possibly protect from further joint destruction.

P243Treatment of cartilage defects in the knee using alginate beads containing human mature allogenic chondrocytes: Clinical results at 3 years of follow-up.A.A.M. Dhollander1, P.C. Verdonk2, R. Verdonk1, G. Verbruggen1, K.F. Almqvist1 1Ghent/Belgium, 2Gent-Zwijnaarde/Belgium

Purpose: The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects in the knee.Methods and Materials: A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of chondral and osteochondral lesions in the knee. Twenty-one patients were clinically prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9, 12, 24 and 36 months of follow-up.Results: A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 36 months of follow-up. Adverse reactions to the alginate/fibrin matrix seeded with the allogenic cartilage cells were not observed. Two of the procedures failed. One of the patients had loosening of the periosteal flap, which was attributed to a failure of the surgical procedure. The other failure case was the result of the poor quality and quantity of the repair tissue itself.Conclusions: The results of this pilot study show that the alginate-based scaffold containing human mature allogenic chondrocytes is feasible for the treatment of symptomatic cartilage defects in the knee. The described technique provides clinical outcomes equal to those of other cartilage repair techniques.

P244Scaffold ACI and cancellous bone grafting for osteochondritis dissecans in the knee – mid-term resultsS. Anders, J. Schaumburger, O. Wiech, J. Beckmann, J. Grifka Bad Abbach/Germany

Purpose: Osteochondritis dissecans (OD) of the femoral condyle in the knee joint needs extensive osseous debridement for revitalization as well as reconstruction of the joint surface. This can be achieved by autologous cancellous bone grafting combined with scaffold autologous chondrocyte implantation (ACI). Our mid-term results with this technique are presented. Methods and Materials: 20 patients (mean age 23.7 (14-42) years, mean defect size 4.4 (1.8-8.0) cm²) with 21 cases of osteochondritis dissecans of the femoral condyle (ICRS III-IV°, 19 medial, 2 lateral) were treated by scaffold ACI (MACI®) and autologous cancellous bone grafting. 14 patients have had a at least one (1-3) previous OD treatments before (6x drilling, 4x refixation, 2x cancellous bone grafting, 2x retrogarde drilling, 1x shaving, 1x removal only). 7 cases were treated primarily. All results were evaluated prospectively by functional outcome scores, subjective clinical ratings and MRI with an average follow-up of 30.2 (6-62) months.

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examination was performed in 149 patients between 06/03 and 12/07 by capturing the IKDC score pre-operatively, after 6 months, 12 months and then yearly. This investigation was focusing on clinical improvement performing covariance calculations to learn about the impact of defect size, patients age, etiology, cell-viability and more. The average follow-up here is 3 years (0,5-5). Results: The IKDC-score increased for 18 points on an average and reached a plateau after 2 years. Graft-related adverse events have been observed in 6% of all patients (CI: 2,6- 12,4%). Patients age just had impact on the postoperative joint function (p=0,004). The patella showed highly significant (p<0,001)worse results in terms of graft failure and revision surgeries.Regarding etiology, degenerative defects showed significantly (p=0,005) worse results compared to traumatic defects or OD. Whereas in the latter in IKDC-increase for 18,4 points can be expected, improvement in degenerative defects will be just 10,8 points (p=0,07) on average. Conclusions: The performed covariance-analyses allow a pretty detailed chance- and risk-profile for ACT-patients that can be used for a more sophisticated pre-operative interview and informed consent.

P251The harvest site in ACI treatment is not associated with long term joint morbidity: a study of ACI treated hips with knee harvest siteH.S. McCarthy1, J. Richardson2, E. Robinson2, J.C. Parker1, S. Roberts1 1Shropshire/United Kingdom, 2Oswestry/United Kingdom

Purpose: Autologous chondrocyte implantation (ACI) is commonly used for the treatment of cartilage defects in the knee but more recently has been used to treat similar defects in the hip. Patients who have had ACI of the hip with chondrocytes harvested from the knee provide a unique opportunity to assess the contribution of the harvest site to joint morbidity up to 7 years post-treatment.Methods and Materials: In a study of 12 patients receiving ACI treatment of the hip, chondrocytes were harvested from the knee (11 ipsilateral, 1 contralateral). Clinical knee scores (Lysholm) were obtained pre-harvest, post-harvest (range 2-12 weeks) and annually thereafter. Statistical significance was determined using the Kruskal-Wallis analysis of ranks and the Mann-Whitney U test.Results: Pre-harvest, mean Lysholm scores were 87±18 (range 59-100) indicating that the joint was generally in good health prior to cell harvest. Initially, the Lysholm scores post-harvest were reduced (but not significantly) to 72±17 (range 61-97), with an increase in pain, locking sensation and limping, a decrease in movement and an impaired ability to climb stairs. Cell harvest did not cause swelling. By 12 months post-ACI however, mean Lysholm scores had increased to just below pre-harvest scores (mean 82±25, range 44-100) with all normal functioning of the knee restored. Scores had returned to pre-harvest levels or greater from 2 years through to 7 years.Conclusions: These results indicate that that there is an initial decrease in joint function post-harvest for ACI. However, by 2 years post-harvest, joint function had returned to pre-harvest levels. Hence, there is no long term harvest site morbidity of the knee associated with harvesting cells for use in ACI treatment of the hip. Thus, the harvest site is unlikely to have contributed to the demise of the joint for those patients for whom ACI treatment of the knee has failed.

P253Novel arthroscopic matrix-encapsulated autologous chondrocyte implantation: clinical, ultrasonographic, MRI, T2-mapping, and arthroscopic results. Pilot study.E. Villalobos Jr, C. Ibarra, C. Velasquillo, V. Martinez, F. Pérez, A. Izaguirre, S. Cortes, G. Franco, D. Chavez, C. Pineda, V. Guevara, L.G. IbarraMexico City/Mexico

Purpose: The purpose of this study is to evaluate the efficacy and biosafety of a novel autologous chondrocyte implantation technique at 6 to18 months follow up. Methods and Materials: We selected patients younger than 50 years old with localized 1-2 cm² ICRS grade IV femoral condyles chondral lesions for arthroscopic chondrocyte implantation. We treated concomitant pathology of patients and took a chondral biopsy of non-weight bearing site near the femoral notch. After cell culture and proliferation, we implanted matrix encapsulated autologous chondrocytes arthroscopically and fixed the construct with a bioabsorbable mini-anchor. All the patients followed the same rehabilitation protocol with supervised physical therapy, 8

Results: There were no postoperative complications in 15 patients and no adverse events related with MACI operation. In postoperative 3 months, evaluation of KOOS in pain showed significantly improved compared with baseline(p<0.05). In postoperative 6 months, KOOS results demonstrated significantly improved (p<0.05) in pain, ymptoms, ADL, sport and recreation function and knee-related QoL. MRI score in postoperative 3 months showed the regenerated cartilage filled the defect areas nearly completely. In postoperative 6 months, MRI score exhibited that neo generated cartilage completely filled defect regions. MRI score in postoperative 12 and 24 months presented significantly improved in defect filling, integration, signal intensity and subchondral bone. Histological evaluation of postoperative 15 and 24 months presented the predominated hyaline cartilage in new generated tissue. MACI operation time was controlled in 2.0 hours with less than 100mL bleeding. Conclusions: Our clinical results of two-year follow-up study affirmed that MACI technique was a safe, reliable and valid treatment with characteristics with non-complicated technique, short operating time and small amount of surgical bleeding.

P249Clinical and radiological results in patients operated on the femoral condyles with the classical ACI after 10 yearsM. Drobnič, D. Martinčič, B. Koritnik, M. Gorensek, D. Radosavljevič Ljubljana/Slovenia

Purpose: A prospective non-randomized clinical study analyzed long-term clinical and radiological results in a cohort of patients treated (1996-2000) with the classical ACI technique due to the cartilage lesions on femoral condyles.Methods and Materials: The initial study group comprised of 30 patients with the average age of 28.6 years. 2 patients did not respond to follow-up, 1 sustained another injury of the operated knee, and 1 suffered from another handicapping illness. 3 patients had to undergo additional joint resurfacing procedure (1 osteotomy, 2 arthroplasties). Therefore only 23 patients were available for clinical and radiological examination (Lysholm-Tegner, IKDC subjective and examination, and Kellgren-Lawrence radiologic scores) at 10 years. The acquired results were compared to their follow-up scores at 2 and 5 years.Results: The average scores for the cohort of patients were: Lysholm 50 pre-operatively, 76 at 2 years, 72 at 5 years, 73 at 10 years; Tegner 2.9 pre-operatively, 4.0 at 2 years, 4.1 at 5 years, 3.8 at 10 years; IKDC 36 pre-operatively, 61 at 2 years, 65 at 5 years, 59 at 10 years. 23 operated knees were at 10 years examination classified as: normal 9, nearly normal 8, abnormal 5, and severely abnormal 1. The average Kellgren-Lawrence score at 10 years was 1.9. Kellgren-Lawrence scores of 2 or more were found in the subgroups of patients as follows: solitary lesions 28%, OCD 15%, lesion with an ACL reconstruction 67%.Conclusions: The clinical results demonstrated stability between 2 and 10 years of follow-up. Patients with low clinical scores at the 2nd post-operative year did not improve later. High radiological evidence of OA in ACL reconstructed subgroup and low OA in OCD patients indicate that classical ACI worked best for localized low-impact cartilage lesions.

P250Impact of patient’s age, etiology and site of cartilage defect in the knee on clinical outcome of autologous matrix-based chondrocyte transplantationJ. Fritz1, C. Gaissmaier2, J. Volck2, B. Schewe3 1Dusslingen/Germany, 2Reutlingen/Germany, 3Rottenburg/Germany

Purpose: The main indications for autologous chondrocyte transplantation (ACT) are circumscribed full-thickness defects in younger patients, located mainly at the femoral condyles, caused by trauma or OD. Little is known about the performance on ACT in degenerative defects or in patellar lesions. We analyzed our results of a retrospective survey and a prospective clinical observation to learn more about the impact of defect-site, etiology of defects and patients age on the clinical outcome of matrix-based ACT.Methods and Materials: 1. From 433 patients, who underwent matrix-ACT (NOVOCART 3D, TETEC AG, Germany), 422 (=97,4%) have been followed-up after 7 months (2-30) with means of a standardized questionnaire. This survey was focusing on early basic clinical outcome and mainly on adverse events. 2. A prospective

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Methods and Materials: Ten 2-3 years-old female sheep were included. A full 10 x 10 mm incision was made in the articular cartilage of the medial femoral condyle. After the induction of a chondral defect in the femoral condyle the size of the defect was measured, and a fragment of the same size was cut from the membrane. All the cell culture (1 million or 5 million cells) was deposited on the fragment and after waiting for 15 minutes it was implanted to the animal. After 12 weeks the animal were sacrificed and the implantation area was compared between both groups and also compared with a control healthy sample taken near the implantation area. Results: Control samples showed the highest score of the histological parameters recommended by the International Cartilage Repair Society to evaluate cartilage reparation. Although without statistical differences, we have found that the histological parameters scores were highest in the samples taken from the 5 million cells implantation. We have also found that the expression of agrecan and type II collagen was highest in the samples from the 5 million cells implantation. Conclusions: The implantation of 5 million of cultured autologous chondrocytes on I/III collagen membranes seems to give better histological and molecular results than 1 million cells.

P256Tissue engineering in the treatment of 41 patients with osteochondritis dissecans: results at mean 6 years follow-upG. Filardo, E. Kon, M. Berruto, A. Di Martino, S. Patella, G. Altadonna, M. MarcacciBologna/Italy

Purpose: Osteochondritis Dissecans (OCD) is a focal osteochondral separation of the weight-bearing portion of the articular surface. Autologous tissue engineered cartilage (Hyalograft C) using a biodegradable, biocompatible hyaluronian-based scaffold for cell proliferation was developed for the treatment of chondral knee lesions. The use of autologous chondrocyte transplantation in OCD presents a problem to promote only the cartilaginous but not the bone regeneration. For this reason we utilized the two-step technique: autologous bone grafting followed by autologouos condrocyte Hyalograft C transplantation. The aim of this study is to evaluate Hyalograft C transplantation with or without bone grafting for the treatment of OCD at mean 6 years follow-up. Methods and Materials: Autologous chondrocytes transplantation was performed from 1997 in 41 patients. Two-step technique consists in the first arthroscopic surgical step autologous bone harvested from omolateral tibia is utilized to fill the bone defect. In the same surgical procedure healthy cartilage is harvested from intercondylar notch in order to expand autologous chondrocytes. The second surgical procedure is performed 4/6 months later, when the integration of the autologous bone graft is achieved and consists of arthroscopic autologous chondrocyte transplantation. All the patients were clinically evaluated and analyzed according to the International Repair Cartilage Society score. Results: No complications related to the Hyalograft C implant and no serious adverse events were observed during the treatment and follow-up period. IKDC subjective score showed satisfactory clinical results, with 79/100 at the latest follow-up. Conclusions: The autologous chondrocyte transplantation provides satisfactory clinical results. The second generation of autologous tissue engineered cartilage transplantation avoids the use of periosteal flap, simplify the surgical procedure and permit to perform the arthroscopic implant. Thus, recovery time for the patient are reduced. The association of the autologous bone grafting permits to restore also a bone loss in order to recreate the articular surface.

P257Autologous chondrocyte implantation for treatment of symptomatic deep cartilage defects in the knee. 3-year follow-up study comparing clinical and MRI evaluation.W. Widuchowski1, H. Bursig1, A. Wysocka1, W. Wawrzynek2, R. Faltus2, B. Koczy2, J. Widuchowski2 1Katowice/Poland, 2Piekary Śląskie/Poland

Purpose: This prospective cohort study reviews and compares clinical and morphologic results of ACI in the treatment of deep cartilage defects in the knee. Methods and Materials: Forty patients with symptomatic single ICRS grade IV lesion localized within medial or lateral femoral condyle participated in that prospective controlled trial. Average size of defect was 3.5 cm2 (range; 2-9 cm2). Mean follow-up was

weeks of non-weight bearing, 6 weeks of CPM, and an isokinetic strengthening program at the 4th month after the surgery. We assessed prospectively clinical and structural outcomes at 3, 6, and 12 mo follow-up. Second look evaluation was done in patients at one year f/u. We conducted non parametric hypothesis tests with 2-tailed significance set at (p<0.05). Results: Mean age was 32.3 years old (23-46). All patients had clinical improvement after surgery, with mean Lysholm preop results 51.82±22.12 and postop 72.86 ± 18 .7 (p=0.028); Tegner activity scale preinjury was 7.58±1.4 and 7.63± 1.2 (p=0.61) at final f/u. Safety evaluation with ultrasonographic assessment through the study was done and permitted identify that the implant was placed correctly. A slight increase in MOCART evaluation and a decrease in relaxation time with T2-mapping was observed. Second look evaluation showed a repaired area with cartilage like tissue. No serious adverse events were observed in this study. Conclusions: Matrix encapsulated autologous chondrocyte implantation with a novel arthroscopic technique is efficacious and safe supported by clinical, macroscopic, and imaging studies at the short term.

P254Matrix associated autologous chondrocyte transplantation in the knee: Clinical results at 3 to 8 years follow-upD. Stelzeneder1, S. Domayer1, R. Dorotka1, M. Brix1, C. Chiari1, S. Nehrer2 1Vienna/Austria, 2Krems/Austria

Purpose: The aim of this study was to prospectively assess clinical outcome after matrix associated autologous chondrocyte transplantation (MACT) in the knee using Hyalograft C. Methods and Materials: Fifty-three patients were treated with Hyalograft C in the knee. Patient characteristics at baseline were as follows (mean±standard deviation): 22 female, 31 male, age 32±12 years, body mass index (BMI) 24.5±3.8 kg/m2 and defect size 4.4±1.9 cm2. Fifty patients had single defects and 3 had multiple defects (41 medial femoral condyle, 6 lateral femoral condyle, 2 patella, 1 tibia). All defects were rated Outerbridge grade III or IV. Primary inclusion criteria (stable ligaments, regular knee alignment (±3°), singular defect within otherwise healthy adjacent cartilage, age <55 years) were met by 42 patients. However 11 patients with secondary indication, not fulfilling all criteria, were included. Instability or malalignment were treated before or at the time of graft implantation. For patients with small defects (<2 cm2) microfracturing was used as first-line treatment. Full weight bearing was allowed 7-12 weeks after graft implantation. Clinical assessment was performed once a year with the following scoring systems: Subjective IKDC knee form, Lysholm score, a modified Cincinnati Knee Rating System and objective IKDC knee form. Descriptive statistics are given. Results: Clinical parameters at baseline were as follows (median and interquartile range): Lysholm 59 (47;69), subjective IKDC 40.0 (27.1;46.5), Cincinnati 3 (1;4), objective IKDC C (B;D) Results at 8 years were: Lysholm 92 (77;95), subjective IKDC 76.3 (61.1;91.7), Cincinnati 8.5 (6;10), objective IKDC B (A;B). Twelve cases (9 secondary indications) needed further surgical intervention (graft failure). Graphical results are shown in Figure 1 and 2. Conclusions: Our findings suggest that MACT with Hyalograft C is able to provide satisfactory clinical results at 8 years of follow-up. In particular young patients with singular defects benefit from this treatment.

P255Treatment of chondral defects with autologous chondrocytes on type I/III collagen membranes: comparison between 1 and 5 million cells in the sheep animal modelP. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos, M. Guillen-Vicente, F. Garcia-Gomez, E. Santos, T. Fernandez-Jaen, J.M. Lopez-Alcorocho Madrid/Spain

Purpose: Autologous chondrocyte implantation (ACI) combined with a periosteal flap was first performed in the human knee in 1987. The success of this technique as an alternative treatment for symptomatic chondral defects has motivated a continuous effort to improve it and today many modifications, that involve tissue engineering, coexist. In MACI , chondrocytes are seeded in a collagen I/III membrane functioning as cell carrier. The density in MACI is around 106 cells/ cm2. We have evaluated in an ovine model if increasing five times the cell density in a type I/III collagen membrane improves tissue repair.

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P260Our experience in the treatment of chondral defects with autologous chondrocyte implantation, chondrotissue and other scaffoldsE. Somma, G. La CavaRoma/Italy

Purpose: Introduction.We report our 3 year experience in the treatment of chondral defects caused by trauma or degeneration, where we implanted autologous chondrocytes on a 3D scaffold (BioSeed-C), chondrotissue and other scaffolds.Methods and Materials: We treated 197 cases of focal cartilage defects, single and multiple, with a 1 to 3-4 cm. surface, situated in the femoral chondilars, tibial plate, patella, femoral and astragalus trochlea in III and IV stage (Outerbridge classification); in some cases we associated a reconstruction of the cruciate ligaments of the knee. BioSeed-C was used in 150 cases, and it consisted in drawing a small sample of cartilage, through arthroscopy, from a healthy area of the knee without loading and a 120 ml. blood sample for culture of the chondrocytes in laboratory. The cells were then cultivated in vitro for approximately 3 weeks and spread out on a 3D reabsorbable polymeric scaffold. During the 2nd phase of treatment the matrix with the chondrocytes was implanted with an open technique (mini open), fixing the matrix with a reabsorbable pin or reabsorbable suture passing through the trans-bone tunnel with a Kirschner suture according to the technique of Erggelet. Particular post operative attention is required to deal with the active and passive flex-extension. Complete loading is not allowed for 3 weeks. After the 3rd week we allow partial loading of 15% with progressive loading to reach complete loading after the 6th week. br>Results: Our follow-up is presently 3 years; our French-Swiss colleagues (Prof. Rhenter, Prof. Erggelet) have an 8 year follow up with very satisfactory clinical radiographic results. As regards the use of Chondrotissue and other scaffolds this has been used in 47 cases and requires the application of a reabsorbable scaffold of 2 cm x 3 cm x 1 mm of polyclicolic acid (marked CE class 3) and ialuronic acid (marked CE class 3) in the seat of the cartilage damage prior to courettage of the lesion, Pridie perforations by means of reabasorbable pins or reabsorbable suture passed through the trans bone tunnel with Kirschner suture according to Erggelet’s technique or, in minor lesions, and particularly in the astragalar and tibial region, with fibrin glue. After the operation we used the same protocol utilized for the previous technique but we loaded earlier in percentage. Our follow-up with this technique is 1 year and 6 months with RMN controls after 3, 6 and 9 months. br>Conclusions: Considering the simple surgical procedure which foresees only one operation and very encouraging results, we are of the opinion that Chondrotissue and other scaffold can be utilized more frequently in this type of lesion especially in the over 50s. br>

P261Arthroscopic Cartilage Regeneration Facilitating Procedure (ACRFP) for OA KneeS. Lyu Chiayi/Taiwan

Purpose: A novel concept of arthroscopic procedure for the treatment of osteoarthritic (OA) knee has been developed based on the conceptualization of a possible pathogenesis process for OA knee featured by focal abrasion phenomenon and soft tissue imbalance.Methods and Materials: From January to December of 2005, 571 knees of 367 patients with OA knee have received this procedure. We used Kallgren-Lawrence classification for pre-operative staging. Pain domain of Knee Society Score (KSS-P) and Knee Injury and Osteoarthritis Outcome Score (KOOS) were used for subjective outcome study. Measurement for the roentgenographic changes of femoral-tibial angle (FTA) and joint space width (JSW) was evaluated for objective outcome study. The mean follow-up period was 43 months (range, 41 to 54). Results: There were 70 (19%) male and 297 (81%) female and the mean age was 60 years (range, 29 to 82). One hundred and thirty-four knees retured and completed the thorough outcome evaluation. Another 381 knees completed the subjective telephone questionnaires outcome evaluation. The total follow-up rate was 90.2%. The satisfactory rate for the whole series was 85.5%. FTA improved from 1.52 (95% confidence interval, 0.84~2.19) to 1.93 (1.21~2.64)(p=0.03). JSW increased from 2.03 (2.81~3.24) to 2.18 (2.97~3.38)(p=0.01). Conclusions: ACRFP is a good option of treatment for OA knee. The beneficial effects of this procedure might be the improvement of the general environment of the knee joint for cartilaginous regeneration after eradication of the possible etiologic factors.

26.2 months (range; 12-38 months). Clinical and MRI evaluation were performed preoperatively and 6, 12, 24 and 36 months postoperatively. Functional outcome scores were collected using Lysholm, Tegner and ICRS scores. Results: All clinical rating scores showed significant improvements (p<0.05) compared to pre-operative levels with ongoing sequential improvement. MRI showed overall good healing process of cartilage defects; 70-90% of defect filling, osseous or cartilagenous hypertrophy in 32% of cases, subchondral bone marrow edema in 11% of cases. There was no statistically significant correlation between clinical and MRI scores. Conclusions: In short-term observation ACI produces significant improvement in knee function in patients affected by symptomatic full-thickness isolated focal cartilage defect. There are likely to be factors other than morphology of repaired tissue that may influence the overall outcome of ACI.

P258ACI or AMIC for osteochondritis dissecans of the knee? A 3-year follow-up study.W. Widuchowski1, P. Łukasik2, H. Bursig1, W. Wawrzynek2, M. Szczęśniak2, J. Widuchowski2 1Katowice/Poland, 2Piekary Śląskie/Poland

Purpose: The aim of the study was to compare the results of Autologous Chondrocyte Implantation (ACI) and Autologous Matrix-Induced Chondrogenesis for osteochondritis dissecans treatment of the knee joint. Methods and Materials: Clinical evaluation of the results are based on established scores (Lysholm, Tegner, ICRS). MRI was performed for each patient; preoperatively and 6, 12, 24 and 36 months postoperatively. The study comprised of 31 patients (21 male, 10 female, average age at surgery 28; range 15-32) affected by single grade III and IV isolated focal lesion (3-8 cm2; average 4,1 cm2). 17 patients were treated with ACI and 14 with AMIC. Both groups were evaluated prospectively. Results: Significant (p<0.05) differences between pre- and post-operative values have been observed for all patients within both groups, but without distinction between groups. MRI evaluation revealed better defect filling in ACI patients, but difference was not statistically significant. Osseous or cartilagenous hypertrophy was observed in 30% (ACI) of cases in ACI group and in 40% of cases (AMIC). Conclusions: Clinical and MRI scores demonstrate that both ACI and AMIC techniques allow to obtain good short-term results in the treatment of OD of the knee.

P259ACT3D fully arthroscopically performed for multiple chondral defects, at the knee. Preliminary results.S. Alevrogiannis, G. Skarpas, A. Triantafyllopoulos, G. Kakavas Athens/Greece

Purpose: To present our experience in using 3rd generation autologous cartilage transplantation with 3-dimensional chondrospheres for treatment of multiple cartilage defects, at the knee. Methods and Materials: 12 symptomatic patients with multifocal cartilage lesions, were treated operatively in our Dept., between March 2007 and June 2009. All patients were recreational athletes and the mean age was 32 years old. The mean area of cartilage defect was 6.75cm² and were classified as Outerbridge grade III(7) and IV(5). All of them were treated fully arthroscopically. 8 patients were male; 6 pts had the operation at the right knee and the remaining 6 at their left knee. The cartilage lesions were located in the weight-bearing surface of the MFC+LFC in 7 patients, 2 in LFC+FT, 2 in the MFC together with retropatellar lesions and 1 in MFC+LFC+LTP. In most of the cases (9) the defect was due to trauma, while 3 of them were caused due to OCD. 3 patients had failed MFx performed elsewhere in the past. We performed 12 applications of ACT3D as a single procedure. Preop. and postoperative evaluation of patients was done using the Modified Cincinatti Rating System, the VAS, IKDC score and Patient Outcome Function score. Results: All cases followed a specialized rehabilitation protocol. We assessed the patient at six months and 1 year post-operatively using the MCK score , the VAS and the IKDC scores. No significant graft-associated complications were observed in MRI scans.Second look arthroscopy, showed hyaline like cartilage formation immunohistologically. Conclusions: Our preliminary results of biological resurfacing techniques at the knee, for multiple chondral defects, seems to be more than encouraging. A greater number of cases and further mid and long term follow-up has to be studied in order to prove the efficacy of the method.

Posters250

P264Satisfaction after arthroscopic femoral osteoplastyJ. Miquel, J.I. Erquicia, M. Tey Pons, X. Pelfort, J.C. Monllau, E. Cáceres Palou Barcelona/Spain

Purpose: Femoracetabular impingment is a well-know predisposal factor to hip cartilage damage. The objective of the present paper is to evaluate the satisfaction degree of patients in whom arthroscopic femoral osteoplasty was performed and its possible correlation with alpha angle correction and postoperative pain. Methods and Materials: A series of 20 patients operated on 2009 were prospectively studied. Inclusion criteria were patients in whom arthroscopic femoral osteoplasty was performed. The patients were clinically assessed by the Satisfaction Cincinatti Score (SCS) and Visual Analogical Scale (VAS) pre and postoperatively. The degree of alpha angle correction was radiologically calculated by means of a Dünn axial view. Femoral head correction was correlated to VAS and SCS. Spearman’s-rho test was used to statistically study correlations among variables, using the programme SPSS 15.0. The level of significance was set at p< 0.05. Results: At a mean postoperative follow-up of 9.9 months, femoral head correction averaged 21.9º ± 11.9 (range 2 to 41º), while VAS averaged 2.8 ± 2.4 (6.2 ± 2 preoperatively). Patients rated 6.9 out of 10 points in the average activity score (SCS). Most of the patients (80%) showed a moderate to excellent satisfaction level according to SCS. A significant correlation was found between patient satisfaction and alpha angle correction, as well as postoperative pain (p<0,05). Conclusions: Arthroscopic femoroacetabular osteoplasty reduces pain with a high percentatge of patient satisfaction at a short term follow-up. The procedure satisfaction seems to correlate to the alpha angle correction.

P265Further clinical improvement in a cohort of 528 patients affected by symptomatic hip osteoarthritis undergoing to repeated intra-articular ultrasound-guided administrations of hylan g-f20 (Synvisc®) during second year of followup.A. Migliore1, E. Bizzi1, U. Massafra1, S. Martin2, M. Granata1, F. Vacca1, S. Tormenta1 1Rome/Italy, 2Albano Laziale/Italy

Purpose: No data are currently available in the scientific literature reporting long followup times on patients affected by hip OA undergoing ultrasound-guided intra-articular injection of the hip with hylan G-F 20. Aim of the Study is to evaluate variability and duration of long-term benefit obtained by the ultrasound-guided intra-articular injection of the hip with hylan G-F 20 in hip OA patients and investigate if further clinical improvements are achieved during second year of therapy. Methods and Materials: Adult patients suffering from hip OA grade 1-3 according to Kellgren-Lawrence grading were considered eligible for the study. Patients were injected with one syringe (2 ml) of hylanG-F20 every 3-6 months under ultrasound guidance following Migliore-Tormenta technique. The efficacy was assessed by using the Lequesne index and VAS pain score at baseline and, then, every three months after the first injection of hylan G-F 20. NSAIDs consumption was also evaluated. Results: 528 patients were enrolled in the study; 851 injections were performed in 24 months. Scores obtained at each visit after baseline show an improvement when compared with baseline with a statistically significant difference, p<0,001 for every time point. No infectious complications were reported.Conclusions: Our data suggest that beneficial effects obtained by the ultrasound-guided intra-articular injection of the hip are present after the first injection and are maintained over two years by repeat injections. Furthermore, when comparing data obtained at 12 months followup whit data obtained at 24 months followup, a statistically significative reduction was observed in NSAID consumption (p=0.01) and mean Lequesne index value (p=0.01)(Fig.1 and Fig.2).

P262Intra-articular Ultrasonic Assessment of Cartilage IntegrityE. Kaleva1, T. Viren1, S. Saarakkala1, J. Sahlman1, J. Sirola1, J. Puhakka2, T. Paatela2, I. Kiviranta2, H. Kröger1, J. Jurvelin1, J. Töyräs1 1Kuopio/Finland, 2Helsinki/Finland

Purpose: We tested whether a minimally invasive intra-articular ultrasound (IAUS) method could be used to evaluate structural integrity of articular cartilage in human knee joints in vivo.Methods and Materials: Five patients (2 female, 3 male, age 22-69 years) coming to arthroscopic surgery of the knee were enrolled in the study. The study plan was approved by the local ethics committee. During arthroscopy, ultrasonic measurements of cartilage surfaces were conducted using a miniature rotating unfocused high-frequency (40 MHz) ultrasound transducer inside a catheter (dia.=1mm, Boston Scientific). The catheter was inserted into the knee joint though the normal arthroscopic portals and the ultrasound transducer was positioned on the desired joint location under arthroscopic control. In addition to collecting ultrasound backscatter data, an IAUS video and arthroscopic video were recorded. Cartilage surfaces at the measurement sites were classified according to the ICRS macroscopic score. Results: The IAUS assessment was able to distinguish between different characteristics of the articular surfaces (e.g., intact surface, surface fibrillation and lesions of varying depth) of human articular cartilage. In addition abnormal internal cartilage structure and subchondral bone were visible in some cases in the IAUS images.Conclusions: The IAUS method served as a minimally invasive diagnostic in vivo tool for evaluation of articular cartilage. The method provided quantitative information on cartilage integrity and thickness not available in conventional arthroscopy. The ultrasound equipment is FDA approved for intravascular use, making the use of the instrument easier for intra-articular applications. The present results suggest that also the integrity of the articular cartilage repair may be quantitatively monitored with the IAUS approach in vivo.

P263Role of Compound Real Time Sonography in Evaluation of Meniscal InjuriesM. Sandhu, K. Shanbhogue, P. Singh, K. Sodhi, R. Sen Chandigarh/India

Purpose: This study was designed to assess the utility of Real Time Spatial compound sonography ( CS ) in diagnosis of meniscal injuries of the knee. Methods and Materials: 24 patients presenting with a history of meniscal injury participated in this study. All patients underwent conventional sonography ( CNS ) and CS examination followed by MRI. Only unilateral injuries were included, contra lateral knee was used as a control. Results: CS was significantly superior with respect to the clarity of tissue plane definition, excellent contrast resolution, reduction of artifacts like speckle and refractive shadows resulting in reduced posterior shadowing from the bones. Overall, the sensitivity, specificity and accuracy was 80%, 89.3%, 85.4% for CNS,and 90%, 85.7% and 87.5% for CS respectively.Negative predictive values were 86.2% for CNS and 92.3% for CS. Conclusions: We feel that CS should be routinely used for evaluation of meniscal injuries as it has better sensitivity, making it an attractive screening tool. It has the potential to become a gatekeeper to MR especially when the clinical suspicion of a meniscal tear is not very high. Unnecessary MRI examination can safely be avoided with 90% accuracy when the clinical suspicion is not very high and when USG is normal. Although it cannot replace MRI , CS is an excellent low cost alternative when MR is not available or when waiting period for MRI can cause unnecessary delay in management.

Posters 251

P268Evaluation of post-ACI cartilage repair tissue using FT-IR imaging spectroscopyA. Hanifi1, S. Roberts2, J.B. Richardson2, N. Pleshko1 1Philadelphia/United States of America, 2Shropshire/United Kingdom

Purpose: There has been limited success in correlating the clinical outcome of cartilage repair procedures with quality of the repair tissue formed. Techniques that can assess the molecular features of repair tissue could aid in evaluating the tissue response to such therapeutic interventions. Fourier transform infrared imaging spectroscopy (FT-IRIS) is a modality based on molecular vibrations that has been used to evaluate articular cartilage degradation and repair. The aim of the current study was to investigate molecular properties of cartilage repair tissue after autologous chondrocyte implantation (ACI) procedures and to assess whether there was a correlation between the FT-IRIS derived properties and the immunohistochemical and clinical outcomes.Methods and Materials: Osteochondral biopsies were obtained from knees of ACI treated patients from 1 to 5 years post-ACI procedure (N=10). Thin sections of the biopsies were evaluated for type I and II collagen immunostaining and by FT-IRIS. Clinical outcome was assessed by Lysholm score.Results: Improvement in Lysholm score occurred in 9 out of 10 samples and correlated with the FT-IRIS determined proteoglycan (PG) content of the tissue. Increase in years post-ACI correlated with an increase in FT-IRIS determined PG content, collagen helical integrity, and collagen maturity based on crosslinking (ratio of mature: immature crosslinks). Immunohistochemical staining showed a greater amount of collagen type II near the calcified cartilage and bone, which was inversely related to the distribution of the collagen crosslinking parameter.Conclusions: We conclude that FT-IRIS derived parameters could be useful for predicting cartilage repair success.

P269Clinical utility of T2 relaxation time at proximal tibiofibular cartilage in osteoarthritis MR imagingK. Kwack, B. Min, S. Yoon, Y.M. Lee Suwon/Korea, Democratic People’s Republic of

Purpose: The proximal tibiofibular joint (PTFJ) can be considered the fourth compartment of the knee joint. However, there have been no studies of the T2 values (T2 relaxation time) of PTFJ cartilage. Purpose: To assess the T2 values of PTFJ cartilage at 3T magnetic resonance imaging (MRI), and to show the clinical utility of T2 values of PTFJ cartilage for the diagnosis of osteoarthritis (OA).Methods and Materials: 118 patients who had knee MR imaging and knee radiography were enrolled. MRI was performed using a 3T MRI scanner, and T2 maps were calculated from a sagittal multi-echo acquisition. Two regions of interest (ROIs) were positioned within PTFJ cartilage and medial femoral condyle (MFC) cartilage. The average T2 value and standard deviation (SD) of each ROI were recorded. Using PTFJ cartilage as a standard reference, the T2 index ((MFC/PTFJ)x100) and T2(SD) index ((MFC(SD)/PTFJ(SD))x100) were calculated. A paired t test was performed to compare the mean and SD of ROIs within PTFJ and MFC cartilage. Correlation analyses were performed among the parameters age, Kellgren-Lawrence (KL) score, means and SDs of ROIs within PTFJ and MFC cartilage, T2 index, and T2(SD) index.Results: PTFJ cartilage had a significantly shorter T2 value than did MFC cartilage (P<0.0001). ROIs within PTFJ cartilage showed significantly smaller SDs than did those within MFC cartilage (P<0.0001). The average T2 value and SD of MFC and the T2(SD) index showed a positive correlation to the KL score (P<0.05). The correlation coefficients for the average T2 value, SD, and T2(SD) index of MFC were R=0.203, 0.254, and 0.268, respectively. However, there was no significant correlation between T2 values of PTFJ cartilage and KL score (P=0.643).Conclusions: PTFJ cartilage may be a useful internal standard reference to diagnose OA and would be a good candidate as a standard reference for cartilage imaging.

P266PRP intra-articular ultrasound-guided injections for the treatment of hip osteoarthritisA. Timoncini, M. Battaglia, R. Buda, G. Filardo, T. Buscio, S. Giannini Bologna/Italy

Purpose: Platelet Rich Plasma (PRP) is a natural concentrate of autologous growth factors from the blood. Currently PRP is widely experimented in orthopaedic practice in order to test its potential in tissue regeneration, especially its benefits on articular cartilage degeneration of the knee. The aim of our study is to explore this novel approach to treat osteoarthritis of the hip. Methods and Materials: Nineteen patients affected by unilateral symptomatic osteoarthritis of the hip were treated with 3 PRP intra-articular ultrasound-guided injections. The procedure consisted of 150 ml of venous blood collected and twice centrifuged to produce 3 units of 5 ml each of PRP. Three ml PRP added of 0,3 ml Calcium Gluconate were biweekly injected in the hip joint under ultrasound-guide using Convex probe (1-4 Mhz) aligned with the anterior long axis of the femoral neck. Median value of follow-up was 8 months (range 6-12): patients were clinically evaluated at enrolment, and at 1, 3, 6 months and at final follow-up according to HHS and VAS. Statistical analysis (Non-parametric Mann Whitney test, Paired t-test, Pearson and Spearman correlations) was performed in order to evaluate the final outcome and find its correlation with sex, age, grade of OA, BMI and femoral-acetabular impingement. Results: A statistically significant improvement of all clinical scores was obtained from basal evaluation to 1 and 3 months follow-up (p<0.0005). The results decreased with statistical significance (p=0,016) from 3 to 6 months follow up, and remained stable (p=0,5) until final follow-up even if still significantly higher with respect to the basal level (p<0,005). OA grade and femoral-acetabular impingement showed statistical correlation with the clinical improvement at 1 and 3 months follow-up (p<0,023, p=0,049). One patient stopped the treatment at first injection. Conclusions: PRP intra-articular ultrasound-guided injections showed to reduce pain and improve articular function in hip osteoarthritis without femoral-acetabular impingement at short follow-up.

P267Primary stability in the hip of a membrane used for autologous chondrocyte transplantation. A cadaveric studyA. Fontana Monza/Italy

Purpose: The treatment of chondral defects is a challenging issue in joint surgery. Tissue engineering and chondrocyte transplantation are actually giving new prospectives to this field, especially in the knee joint. Nevertheless the use of membranes as scaffolds for chondrocytes implantation in the hip is scarcely investigated. The aim of this study is to evaluate the primary stability of a membrane used for autologous chondrocyte transplantation.Methods and Materials: The membrane was a polyglactin 910 and poly-p-dioxanone reabsorbable, which favors the three-dimensional growth of the cellular culture. The stability of the membrane was evaluated after implantation in 12 hips in 6 cadavers on a cartilage defect specifically formed on the anterior part of the acetabulum and on a cartilage defect specifically formed on the polar sector of the femoral head. All the samples were subjected to 50 complete movement cycles including coxo-femoral movements, for the acetabulum and for the femoral head, respectively. Fifty additional cycles were evaluated for the implants on the acetabulum which showed stability at the first evaluation.Results: The implanted membranes showed stability in 83.3% of the acetabular defects and in 33.3% of the defects of the femoral head. The additional 50 movement cycles carried out for evaluating the acetabulum showed stability of the implant in 80% of the cases. The results suggest the specific use of this tissue, therefore, in the treatment of cartilage defects of the acetabulum.Conclusions: The hypothesis to use membranes as scaffolds for chondrocytes implantation in the hip and their stability without addictive fixation is extremely attractive for hip surgery and hip arthroscopy.

Posters252

P274Autologous Matrix Induced Chondrogenesis (AMIC plus) for the treatment of patellar cartilage defects in the kneeA.A.M. Dhollander1, F. De Neve1, K.F. Almqvist1, R. Verdonk1, G. Verbruggen1, P.C. Verdonk2 1Gent/Belgium, 2Gent-Zwijnaarde/Belgium

Purpose: The present study was designed to evaluate the AMIC plus technique for the treatment of symptomatic patellar cartilage defects in the knee. MRI was used for the morphological analysis of cartilage repair.Methods and Materials: The AMIC plus technique (combination of the original AMIC technique and the use of platelet rich plasma) was used for the treatment of symptomatic chondral and osteochondral patellar lesions in the knee. Five patients were clinically prospectively evaluated with use of the Knee Injury and Osteoarthritis Outcome Score (KOOS) , a Visual Analogue Scale (VAS) for pain, the Tegner activity level scale and Kujala scale preoperatively and at 12 and 24 months of follow-up. All 5 patients had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) scoring system.Results: A statistically significant clinical improvement became apparent after 24 months of follow-up. The MOCART scoring system revealed no significant deterioration or improvement of the repair tissue between one and two years of follow-up. Twenty-four months after the operation hypertrophy was found in 40 %. Subchondral bone changes and intralesional osteophytes were seen in all cases (100%). Synovitis and adhesions were not observed in the study patients at that time of follow-up.Conclusions: AMIC plus resulted in clinically significant improvement in all patients. The favourable clinical outcome of the AMIC plus technique was not confirmed by the MRI findings as determined by the MOCART score. More specifically, all cases showed subchondral lamina and bone changes, including intralesional osteophytes, were observed.

P275T2 relaxation time mapping of human cartilage with variable kinds of MR pulse sequences: correlation analysis with biochemical assayK. Kwack1, B. Min1, S. Yoon1, L.H. Jin1, X. Cui2, S. Hong2, H. Kim2, Y.M. Lee1 1Suwon/Korea, Democratic People’s Republic of, 2Incheon/Korea

Purpose: T2 relaxation time mapping has been universally used to evaluate the cartilage of knee joint. Variable kinds of pulse sequences have been used to do T2 relaxation time mapping. However, there has been no study that compares variable kinds of T2 relaxation time mapping with variable MR pulse sequences. Our study aim is to compare variable kinds of T2 relaxation time mapping with Turbo Spin Echo (TSE), GRASE (hybrid echo) and Fast Field Echo (FFE for T2 star mapping). Methods and Materials: In this study, 11 osteochondral specimens were taken from TKR samples. We performed 3 different T2 relaxation time mappings with 3 kinds of pulse sequences (TSE, GRASE and FFE for T2 star mapping). After 3 kinds of MR imaging all the osteochondral specimens were analyzed with biochemical assay (glycosaminoglycans (GAG), water content and hydroxy proline (HP) for collagen content). T2 relaxation time (T2 msec) of the cartilage in the same ROI (region of interest) from 3 different pulse sequences were calculated. Correlation analyses were performed between T2 msec, GAG, HP and water contents. Results: The mean values of ROIs with each pulse sequences (TSE, GRASE and FFE) were 58.2 msec, 70.4 msec and 20.8 msec respectively. Correlation analyses revealed significant correlation between T2 relaxation values with FFE and HP (r=0.782, p < 0.01). Statistically, no other significant correlation was proven between T2 relaxation values with TSE and GRASE and biochemical contents in cartilage. Conclusions: T2 msec in T2 star mapping using FFE correlated well with HP content in the cartilage of human knee. T2 star mapping with FFE is a promising MR imaging method to detect collagen depletion in cartilage.

P270Sequentially Programmed Magnetic Field (SPMF) Therapy as an effective Treatment for Osteoarthritis, our ExperienceV.G. Vasishta Bangalore/India

Purpose: Sequentially Programmed Electromagnetic Field (SPMF) Therapy utilizes Magnetic Field Generators (MFG) that can be precisely controlled and focused onto the affected tissues. The normal physiological remodeling of cartilage occurs due to piezoelectric stimuli which are lost in osteoarthritic patients. By utilizing specific frequencies in the range of 6-30Hz based on tissue type and grade of osteoarthritis this stimulus is recreated by SPMF therapy leading to cartilage regeneration. Methods and Materials: Methods: 195 patients with bilateral osteoarthritis (OA) of knees were assessed by internationally recognized Knee Society clinical rating system; the scores computed prior to treatment, after 21 days of therapy, and at 3 months. In addition, MRI of the treated knees was done using standard protocol, before treatment and at three months, to measure objective changes in cartilage thickness. Results: Results: The results showed statistically highly significant improvement in pain scores, total knee scores, total functional scores and the range of motion, immediately after the treatment vis-à-vis pre-treatment values, and this improvement persisted when re-evaluated at three months. There was also a significant increase in cartilage thickness at three months, from 0.64mm (±0.02) baseline to 0.88 mm (±0.07) in left knee, and 0.65mm (±0.02) to 0.89mm (±0.05) in the right knee joint (p<0.001). The 9 month follow up results showed a significant improvement in the cartilage thickness, of 1.26mm (±0.02mm)in the left knee and 1.23mm (±0.03mm) in the right knee Conclusions: Conclusion: Therapeutic exposure to SPMF therapy is effective in ameliorating the signs and symptoms of OA, and inducing regeneration of chondrocytes as evidenced by increase in cartilage thickness. SPMF therapy is an effective treatment modality for osteoarthritis.

P271Anteriomedialization of tibial tubercle for patellofemoral disordersM.I. Iosifidis, D. Neophytou, I. Melas, T. Liakos, D. Alvanos, A. Kyriakidis Thessaloniki/Greece

Purpose: Patella recurrent dislocation and patellofemoral pain syndrome affect many young people. In the present study we present the results of the extension mechanism realignment through the oblique osteotomy of the tibial tubercleMethods and Materials: During the last three years 18 patients (12 men, 6 women, mean age 30.3/ range 20-41) were treated operatively for recurrent patella dislocation (12 patients) and patellofemoral pain syndrome without patella dislocation episodes (6 patients). All patients underwent knee arthroscopy for the treatment of possible chondral lesions or loose bodies removal and for lateral retinaculum release. After that, we performed oblique tibial tubercle osteotomy, which is fixed with 2 cortical screws. This oblique osteotomy allows the concomitant anteriorization and medialization of tibial tubercle. In addition, we performed also medial patella retinaculum plication. All the patients followed the same rehabilitation program. We followed up the patients a mean period of 14 months (range 9-24 months). Tegner activity and Lysholm scores were recorded pre- and postoperatively at 3, 6, 9, 12 months and 2 years. We also evaluated the patellofemoral pain through the visual analogue scale (VAS).Results: There was no new patella dislocation episode and any other early or long term complication. The improvement of both functional scores after the 6th p.o. month was statistically significant (p<0.001). There was also statistically significant improvement of pain (p<0.001). Overall, all patients during their last follow up examination had a painless knee with almost full ROM and marked improvement of the patella tracking.Conclusions: Our results support that the oblique osteotomy of tibila tubercle along with lateral release and medial retinaculum plication, is an excellent option for the treatment of recurrent patella instability and relief of the patellofemoral pain.

Posters 253

P278Correlation between the three-dimensional deformation of meniscus with horizontal tear during knee flexion and patients’ Lysholm scoreH. Amano1, K. Nakata2, T. Mae2, K. Shino2, H. Yoshikawa2 1Toyonaka/Japan, 2Suita/Japan

Purpose: The purpose of this study was to quantitatively evaluate the three-dimensional deformation of menisci with horizontal tear during knee flexion using MRI and compare the deformation of horizontal tear with patients’ Lysholm score.Methods and Materials: Fifteen patients (10 male, 5 female, mean age: 32 y/o,) with symptomatic horizontal tear in the posterior segment of medial meniscus underwent 3-D MRI evaluation at 0 and 60 degrees of knee flexion. Volume of bone, cartilage, and meniscus was semi-automatically extracted from the 3-D images by intensity threshold techniques, and 3-D models were created. The percent of circumferential length of horizontal tear against the whole circumferential length of meniscus in the 3-D meniscus model was defined as %tear. The deformation and opening of horizontal tear during knee flexion were calculated in the para-sagittal plane in the 3-D meniscus model. Patients’ Lysholm scores were compared with the degree of opening of meniscus tear with knee flexion. Results: The mean value of %tear was 36.0%. The gap of tear became wide by knee flexion and the mean vertical gap statistically significantly increased from 1.7mm at 0 degree to 3.2mm at 60 degree of knee flexion (p=0.004). There was a positive correlation (r=0.60, p<0.05) between the %tear and the opening of vertical gap. The patient with wide open tear of meniscus by knee flexion showed lower Lysholm, score. There was an inverse correlations between patients’ Lysholm score and the opening of meniscus tear with knee flexion (r= -0.7, p<0.01).Conclusions: Three-dimensional dynamic MRI evaluation of meniscus with horizontal tear has revealed that the gap of tear opened with knee flexion in accordance with the size of circumferential length of horizontal tear, and patients’ Lysholm score correlated with the opening of meniscus tear by knee flexion.

P279Use of Magnetic Resonance Imaging to Assess Tissue Regeneration Following Implantation of a Novel Meniscus ScaffoldP.C. Verdonk1, W.C.J. Huysse2, R. Verdonk2 1Gent-Zwijnaarde/Belgium, 2Gent/Belgium

Purpose: To assess tissue ingrowth, meniscal defect filling and cartilage status using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and anatomic magnetic resonance imaging (MRI) up to 24 months after implantation of a novel resorbable meniscal scaffold for the treatment of partial meniscal tears or meniscal loss.Methods and Materials: This was a prospective, non-randomised, single-arm, multicentre clinical investigation in patients with an irreparable medial or lateral meniscal tear or partial meniscus loss, with an intact rim and the presence of both horns and an International Cartilage Repair Society (ICRS) classification ≤2, and ≤3 previous surgeries on the index knee. Following implantation, DCE-MRI was performed to assess tissue ingrowth at 3 months, and anatomic MRI was performed to assess meniscal defect filling and cartilage scores at 1 week, and 3, 12 and 24 months.Results: Overall, 52 patients (34 medial: 18 lateral) were treated with the meniscal scaffold. Subjects were predominantly Caucasian (51/52; 98%), male (75%) and had previously undergone one (65%) or two (23%) surgeries on the treated meniscus. Subjects had a mean age of 30.8±9.4 years and a mean longitudinal defect length of 47.1±10.0 mm. DCE-MRI performed 3 months post-implantation revealed tissue ingrowth in 37/43 (86%) subjects. Anatomic MRI scans indicated that articular cartilage grading remained stable or improved in the majority of patients (43/46; 93%) and there was no indication that the scaffold caused cartilage damage. Filling of the meniscal defect was observed in all subjects who had data available at 3 and 12 months. Data obtained at 24 months post-implantation will also be presented.Conclusions: Tissue ingrowth and stable or improved cartilage scores were demonstrated by MRI assessments performed up to 12 months after implantation of a novel meniscus scaffold for the treatment of meniscal tears or loss. Data at 24 months post-implantation will be presented.

P276Functional and radiographic results after fixation of the unstable oeteochondritis dissecans lesions using PLLA pinsN. Adachi, M. Ochi, M. Deie, A. Okuhara Hiroshima/Japan

Purpose: There are several surgical treatments for unstable osteochondritis dissecans (OCD) lesions of the knee. If the unstable OCD lesions are left in the joint with the adequate conditions for fixation, internal fixation should be the preferred method of treatment, since it preserves the natural contour of the distal femur. The purpose of this study was to evaluate the functional and radiographic outcome of fixation of the unstable OCD lesions of the knee after minimum 2 years follow-up.Methods and Materials: 30 unstable OCD lesions in 28 patients who were treated with fixation of the OCD lesions and followed up for more than 2 years were included in this study. They were 22 males and 6 females with an average age of 14.8 years (range:11-22). Fixation of the OCD lesions was performed through arthrotomy in 22 knees and under arthroscopy in 8 knees using PLLA pins. The functional outcomes were evaluated using the Lysholm score at an average follow-up of 3.2 years after the surgery and healing of the lesions were confirmed using plain radiographs and MRI.Results: The average Lysholm score significantly improved postoperatively (from 78.4 to 96.8). 28 of 30 lesions healed after fixation of the lesion. Healing was achieved at an average of 2.4 months on plain radiographs and 4.2 months on MRI. Two lesions which did not heal required additional surgery. 22 of 24 patients who had been involved in sports activities returned to their previous activities without reduction of their activity levels within 6 months after the operation. Conclusions: This study clearly demonstrated that fixation of the lesions was an effective treatment option for patients with unstable OCD of the knee, as proved by their functional and radiographic improvement.

P277Arthroscopic pullout repair of posterior root tear of the medial meniscus: The anterior approach using medial collateral ligament partial releaseY.G. Koh Seoul/Korea

Purpose: The purpose of this study was to report the clinical outcome after arthroscopic double transosseous pullout repair of posterior root tears of the medial meniscus through the anterior approach using partial controlled medial collateral ligament release. Methods and Materials: Patients who underwent arthroscopic repair of posterior root tears of the medial meniscus between May 2008 and March 2009 were reviewed retrospectively. Fifty four cases (8 men and 46 women) were available with a mean follow-up of 15 months (range, 12 to 22 months). The age at surgery ranged from 36 to 75 years of age (average: 57 years). The posterior part of deep medial collateral ligament was partially released to expand the medial joint space with an 18 gauge needle. Two anteromedial transtibial tunnels were made into the foot print. Two PDS sutures were passed in the red-red zone approximately 5mm medial to the torn margin and pulled out to the anteromedial tibial cortex and secured by a sliding knot. After surgery, a cylinder splint was applied for 4 weeks with the knee in extension. Clinical outcome was evaluated with Lysholm score preoperatively and at the latest follow-up. Results: The mean preoperative Lysholm score significantly improved from 43 before surgery to 79 after surgery. In most patients, the pain and the tenderness on the MCL disappeared by 3 months after surgery and no clinical medial instability or stiffness of the knee joint occurred. Conclusions: This technique using partial MCL release can be a safe and easy alternative in arthroscopic repair of the posterior root tears of the medial meniscus.

Posters254

P282Clinical outcome scores following implantation of a novel meniscal scaffold for the treatment of irreparable partial meniscus tears and/or partial meniscal tissue loss, 2 year follow up.R. Verdonk1, P. beaufils2, J. Bellemans3, P. colombet4, R. Cugat5, P. djian6, H. Laprell7, P. Neyret8, H. paessler9, P.C. Verdonk10 1Gent/Belgium, 2Versailles/France, 3Leuven/Belgium, 4Bordeaux/France, 5Barcelona/Spain, 6Paris/France, 7Kiel/Germany, 8Lyon/France, 9Heidelberg/Germany, 10Gent-Zwijnaarde/Belgium

Purpose: To assess the clinical efficacy of a novel, biodegradable, polyurethane meniscal scaffold designed for the treatment of irreparable partial meniscus tears and/or partial meniscal tissue loss.Methods and Materials: This was a prospective, non-randomised, single-arm, multicentre clinical investigation in patients with an irreparable medial or lateral meniscal tear or partial meniscus loss, with an intact rim and the presence of both horns. The scaffold is designed to support the body’s own physiological pathways of tissue repair by providing a temporary three-dimensional matrix for cellular and vascular ingrowth. Clinical efficacy was assessed at up to 24 months post-surgery using the following scores: Visual Analog Scale (VAS), the International Knee Documentation Committee (IKDC), the Knee and Osteoarthritis Outcome Score (KOOS) and the Lysholm score.Results: Overall, 52 patients (34 medial: 18 lateral) were treated with the meniscal scaffold. The population recruited into the study was young (mean age, 30.8±9.4 years), predominately male (75%) and the majority (88%) had undergone 1 or 2 prior surgeries on the involved meniscus. The mean longitudinal length of the meniscal defect after surgical debridement was 47.1±10.0 mm. Clinical efficacy of the meniscal scaffold was demonstrated at 12 months by a statistically and clinically significant mean reduction in knee pain on VAS (p<0.0001), and statistically and clinically significant improvements in IKDC and Lysholm scores, and all outcome categories of the KOOS questionnaire (p≤0.0001). It is anticipated that the 24 month data will be presented here.Conclusions: Implantation of the meniscal scaffold resulted in statistically significant and clinically relevant improvements in all subjective clinical outcome scores at 12 months post-surgery demonstrating the efficacy of the scaffold in the treatment of irreparable meniscal tears. It is envisaged that 24 month data will further support the efficacy of this novel meniscal scaffold and will be presented.

P283Treatment with mesenquimal cells derived from the Hoffa’s fat pad for the reparation of chondral defects in a ovine animal modelP. Guillen, E. Rodriguez-Iñigo, I. Guillen-Vicente, R. Caballero-Santos, M. Guillen-Vicente, E. Santos, F. Garcia-Gomez, T. Fernandez-Jaen, J.M. Lopez-Alcorocho Madrid/Spain

Purpose: Current therapies, such as transplantation of healthy cartilage, mosaicplasty, microfracture of the subchondral bone and implantation of artificial polymers or metal prostheses, have many limitations for cartilage reparation. Some research have been focused in developing techniques based on cell therapy using autologous chondrocytes and mesenchymal cells (MSC). We have used an ovine model to evaluate from the histological and molecular point of view the use of Hoffa’s fat pad derived MSC for the reparation of chondral defects.Methods and Materials: Five 2-3 years-old female sheep were included. A full 10 x 10 mm incision was made in the articular cartilage of the medial femoral condyle. A second lesion of the same size was done at the trochlea. In this lesion, microperforations were done. A sample of adipose tissue from the Hoffa’s fat pad was taken to isolate MSC and 5 million of cultured MSC seeded on a collagen I/III membrane were implanted. After 12 weeks the animals were sacrificed and tissue samples in the following areas were taken: a) MSC implant area, b) perforations area, and c) healthy tissue near of perforation area. Histological study was carried-out made by hematoxilin-eosin and safranin-O staining. Relative expression of agrecan and types I and II collagens was determined by real-time polymerase chain-reaction. Results: Among the parameters recommended by International Cartilage Repair Society to study cartilage reparation we have found that most of them were similar when the samples taken from the implant area were compared to those of the perforated ones, being different to controls. The differences were statistically significant in the case of surface and cell distribution. Expression of agrecan and type II was similar in perforations and implanted samples and lower than that found in control samples. Conclusions: Microperforations and Hoffa’s fat pad derived MSC seem to have no role in the reparation of damaged cartilage.

P280The short term clinical and MRI results after meniscus transplantation ; ninety nine casesY.G. Koh Seoul/Korea

Purpose: The purpose of this study was to compare the allograft extrusion using MRI in short term period after the medial and lateral meniscal allograft transplantation and correlate the extrusion with clinical outcome.Methods and Materials: Ninety nine cases (67 men and 32 women) were available for MRI evaluations. The age at surgery ranged from 21 to 52 years of age (average: 35 years). Seventy three lateral and 26 medial meniscus allografts were evaluated with a mean follow-up of 22 months (range, 12 to 53 months). The absolute value and relative percentage of the width of extruded menisci was measured in the coronal image that showed maximal extrusion. Clinical outcome was evaluated with Lysholm score. Results: The mean extrusion was 4.5mm in lateral meniscus versus 2.7mm in medial meniscus (p=0.000) and the relative percentage of extrusion was 50.0% versus 30.8% (p=0.000). Lysholm score significantly increased from 49.0 preoperatively to 87.3 postoperatively in lateral meniscus and from 50.9 to 89.7 in medial meniscus. The difference of final score between the groups was not significant (p=0.381). The overall Lysholm score was not correlated with the degree of extrusion (p=0.202) Conclusions: The presented data shows that the transplanted lateral meniscus extrudes in the lateral direction significantly more than the medial meniscus. However, the clinical outcome after meniscus transplantation was not adversely affected by the allograft extrusion.

P281Twenty-six years of Meniscal Allograft Transplantation (MAT): Is it still experimental? Meta-analysis of 44 trials.P.C. Verdonk1, M. ElAttar2, K.F. Almqvist2, R. Verdonk2 1Gent-Zwijnaarde/Belgium, 2Gent/Belgium

Purpose: Since the first MAT procedure in 1984, thousands of patients with post-meniscectomy symptoms have been treated through allograft replacement. Nevertheless, MAT is still considered experimental surgery. AIM OF THE WORK: Collection, presentation and meta-analysis of published trials presenting outcomes of meniscal transplantation to establish safety and reliability of the MAT procedure. Methods and Materials: A pubmed search was conducted using different combinations of keywords with reviewing of the abstracts excluding all but English-language trials that presented more than 6 months clinical, radiological and/or histological outcome in human subjects. We analyzed 44 trials presenting 1136 grafts in 1068 patients.Results: The literature presented the outcomes of 678 medial and 458 lateral grafts in 613 male, 265 female and 190 non-defined patients with an average age of 34.8 years. Sixty-four percent of MATs were parts of combined procedures while only 36% were isolated. The outcomes were presented through 12 scoring systems, 4 radiographic modalities, 2nd look arthroscopy plus histological analysis. Whatever the follow-up period and the scoring system used, patients continuously showed clinical improvement. Failure rate averaged 10.6% and a total of 128 (21.3%) non-major complications, mainly observed in combined procedures, was reported.Conclusions: Continuous satisfactory outcomes with restoration of working ability in this active patients group were observed in all studies. The complication and failure rate are considered acceptable by all authors. Salvage procedures included osteotomy and arthroplasty without secondary difficulties. MAT can be considered safe and reliable for the treatment of refractory post-meniscectomy symptoms in selected patients.

Posters 255

P286Distraction arthroplasty for severe osteoarthritis of the ankle jointH. Shibuya, N. Adachi, T. Nakasa, K. Fukuhara, M. Ochi Hiroshima/Japan

Purpose: There are many reports for the treatment for osteoarthritis (OA)and rheumatoid arthritis(RA) of ankle joint. However, we have no consensus on treatment of severe stage of arthritis. Distraction arthroplasty has been proposed as one of these options for the patients in whom fusion or joint replacement is not appropriate. Distraction arthroplasty and arthroscopic bone marrow stimulating technique can be a new treatment strategy for severe OA of the ankle joint.The purpose of this study was to evaluate the results of distraction arthroplasty and arthroscopic bone marrow stimulating technique for severe OA of ankle joint.Methods and Materials: We performed drilling or microfracture extensively for cartilage defect of tibial plafound and tatar dome. Then we applied an external fixtator to distract the joint. This device allow the patients to more joint actively. arthroplasty system (Kobayashi MedicalTM). Patients began ROM exercise as soon as possible after the surgery. Weight bearing was permitted 2ï½Å¾3 months postoperatively.Between April 2002 and March 2010, 5 patients underwent distraction arthroplasty at our hospital. Of these, OA was 4 cases and RA was one case. 3 cases were male, 2 were female. The mean age was 48.6 years (range, 38 to 65) at operation time.Results: Mean AOSAF score improved significantly postoperatively in 4 patients. The other patient underwent arthrodesis 24 months after the initial operation due to residual ankle pain. Representative case: 44 years old male. He was amateur high level soccer player. He suffered from left ankle joint pain from 40 years old. After operation, his symptom was disappeared. He finally returned to soccer. Conclusions: This report indicated that this procedure is one option for patients with severe OA of the ankle joint replacement is not appropriate.

P287A Retrospective Functional and T2 MRI Comparison Study of Arthroscopic Bone Marrow Stimulation for Osteochondral Lesions of the Talus – With and Without BMACC.D. Murawski, J.G. Kennedy New York/United States of America

Purpose: Osteochondral lesions of the talus are increasingly recognized injuries and may occur in up to 50% of acute and chronic ankle sprains. Arthroscopic marrow stimulation (i.e., microfracture, drilling) has provided good results in the short to medium-term as a means of treatment for lesions up to 15 millimeters in size. Nevertheless, fibrocartilage is biomechanically inferior to native hyaline cartilage and is subject to degradation over time. In an effort to address these concerns, the current authors reserve the marrow stimulation technique for smaller sized lesions and utilize an autologous bone marrow aspirate concentrate (BMAC). The current authors hypothesize that the BMAC in conjunction with smaller sized lesions will provide durable functional outcomes and improved appearance of quantitative T2 mapping MRI by comparison to the control group.Methods and Materials: Between January 2005 and February 2009, 76 patients underwent arthroscopic microfracture surgery under the care of the senior author. Thirty-five patients underwent microfracture without BMAC injection while 31 patients did have the BMAC injection. The Harvest SmartPReP 2 BMAC device was utilized to prepare the bone marrow aspirate. No lesion treated was larger than 8 millimeters in diameter. All patients had functional AOFAS outcome scores at 1 year post-operatively in addition to quantitative T2 mapping MRI.Results: The average size lesion was 6.22 mm (range 1.52 - 8.04 mm) in the anterior to posterior direction and 6.04 mm (range 1.64 - 7.87 mm) in the medial to lateral direction. The average patient age at the time of surgery was 35.55 years (range 13-66 years). The mean AOFAS functional outcome score at 1 year post-operatively in the group not treated with BMAC was 86.51. The group treated with BMAC had a mean AOFAS score at 1 year of 88.12. These differences were not statistically significant (p=.127). Quantitative T2 mapping MRI in the group not treated with BMAC demonstrated prolongation of T2 relaxation times in 32/35 patients and poor collagen fiber architecture in 35/35 patients. By comparison, the group treated with BMAC demonstrated improved fiber orientation in 21/31 patients and good overlying repair cartilage infill in 23/31 patients.

P284The Efficacy of Combination of Microfracture and ArtifilmTM Covering for the Treatment of Cartilage Defects : Comparison with Conventional Microfracture Technique in a Prospective Randomized TrialB. Min, K. Oh, Y. Lee, J. Kim Suwon/Korea, Democratic People’s Republic of

Purpose: Microfracture has been used as a first-line treatment to repair symptomatic articular cartilage defects. In this study, a new technique using an extracelluar matrix biomembrane (ArtifilmTM) to cover cartilage lesions during microfracture healing was compared with conventional microfracture technique in a prospective randomized trial. Methods and Materials: A total of 35 patients (37 cases) without general osteoarthritis or ligament instability who had symptomatic articular cartilage lesions were randomly assigned in each group. Fourteen patients (15 cases) underwent conventional microfracture procedure (group M) and 21 patients (22 cases) received microfracture and ArtifilmTM covering concomitantly (group MA). Clinical assessment was done through 1 year postoperatively using the subjective International Knee Documentation Committee IKDC questionnaire, and visual analog scale (VAS) for pain and satisfaction. Magnetic resonance imaging (MRI) was performed at 1 year after the operation in all patients. Results: In clinical outcomes, no significant differences were observed between the groups at each visit (final outcomes of VAS for pain, p=0.413; VAS for satisfaction, p=0.579; IKDC, p=0.365). The MRI showed good to complete defect fill (67 to 100%) in 6 (group M) and 17 (group MA), respectively. In group M, 6 patients with poor defect fill (less than 33%) was observed, whereas 3 in group MA (p=0.067). Assessment of peripheral integration revealed no gap formation in 5 patients (group M) and 14 patients (group MA). While, 6 patients in group M showed fissures greater than 2mm and 4 patients in group MA (p=0.112). No serious complications or adverse effects related to the ArtifilmTM were reported.Conclusions: Although not statistically significant, the patients undergoing microfracture only had less defect fill on MRI scan. These results indicated a trend toward increased incomplete cartilage healing in patients undergoing conventional microfracture compared with those undergoing combined procedure, microfracture and ArtifilmTM covering.

P285Effect of microfracture and jamshidi needle biopsy on ex vivo symptomatic OA knee condylesC.D. Hoemann1, J. Sun2, Y. Gosselin1, M.B. Hurtig3, A. Carli1, H. Chen1, W.D. Stanish4 1Montreal/Canada, 2Laval/Canada, 3Guelph/Canada, 4Halifax/Canada

Purpose: The aim of this study was to elucidate practical aspects of microfracture and Jamshidi osteochondral biopsy of debrided symptomatic condyles and resulting subchondral bone hole structure. Methods and Materials: Lateral and medial femoral condyle (LFC and MFC) surgical waste was obtained through IRB-approved protocols from total knee arthroplasty (N=4) in consented patients (49 to 73 years, BMI 30.2 – 34.2, 8-10 pain score with 0=none to 10=extreme). 2 subjects had prior steroid treatment. Condyles were warmed to 37°C for 1 hour, biopsied in the middle with a Jamshidi 11G bone biopsy needle; the proximal region was debrided of the calcified layer then microfractured 4mm deep with 3 awls (Arthrex, Linvatec, Smith & Nephew). Samples were fixed and scanned with a SkyScan 1172 at 15 µm resolution to measure bone hole depth and diameter by 3-D Micro-computed tomography. Agarose gels impregnated with calcium-phosphate were used as tool phantoms. Results: The subchondral plate was very hard below full-thickness grade IV MFC lesions compared to LFC specimens debrided of 2.5±0.9 mm thickness cartilage. Bone was difficult to microfracture in samples from subjects with prior intra-articular steroid treatment. Arthrex, Linvatec and Smith & Nephew awls formed 2.9-3.9 mm deep holes with a 1.8, 1.3 and 1.6 mm superficial hole diameter: 0.16-0.3 mm smaller than the respective hole diameter cast in agarose. 7mm deep Jamshidi holes were 2.8 mm diameter in both agarose and bone.Conclusions: MFX holes did not always attain the 4mm target depth, potentially due to non-elastic bone material properties that preserved the contours specific to each microfracture awl. Jamshidi holes were less affected by condyle hardness than the MFX tools, probably because that the biopsy removed osseous tissue instead of compacting bone into a conical hole. Patient subchondral bone has a stiffness spectrum that tends to extreme hardness as lesions become chronically degraded.

Posters256

P291Evaluation of the structure-modifying effect of avocado-soybean unsaponifiables (ASU) in hip osteoarthritis (OA): results of the ERADIAS study, a 3-year prospective, randomized, double-blind, placebo controlled trial.E. Maheu1, C. Cadet1, M. Marty2, D. Moyse3, I. Kerloch4, P. Coste4, M. Dougados1, B. Mazières5, T. Spector6, E. Vignon7, J. Grouin8, M. Lequesne1 1Paris/France, 2Créteil/France, 3Tours/France, 4Courbevoie/France, 5Toulouse/France, 6London/United Kingdom, 7Lyon/France, 8Rouen/France

Purpose: To assess the ability of ASU to slow radiographic progression in hip OA Methods and Materials: Randomized, double-blind, placebo-controlled 3-year trial. Patients with hip OA (ACR), ≥ 45 years, symptomatic (pain ≥ 1 year, Lequesne ≥ 3) with a minimum joint space width (JSW) between 1-4 mm on a pelvic radiograph were randomly assigned to ASU 300 mg or placebo. 3 standing radiographs were taken annually: pelvis, target hip, and oblique views. JSW was measured at the narrowest point on pelvic or target hip view by manual measurement (0.1 mm-graduated magnifying glass) by the best of 2 readers. The primary outcome was based on the 3-year loss in the narrowest JSW. Patients with at least 2 radiographs of the same view composed the Full Analysis dataSet (FAS). Minimum JSW change was compared by a continuous approach (mean JS loss) and a more robust analysis: comparison of the percentage of “progressors” (loss ≥ 0.5 mm, SDD of the reader).Results: 399 patients randomized, 345 in the FAS. Demographic and hip OA characteristics were similar in both groups: age 62, 54% women, BMI= 27, symptom duration 4 years, 0-100 normalised Lequesne index 30, pain VAS 37 mm. Baseline JSW was 2.8 (0.9) mm. Mean JSW loss and % of progressors at year 3

%

progressors

Mean JSW loss

(sem)

ASU n = 166 Placebo n = 179 Cochrane

M a n t e l

Haenzel P

adjusted

o n

stratus

ASU Placebo P

Total 40% 50% 0.039 -0.64 (0.07) - 0 . 6 7

(0.06)

There was no significant intergroup difference on the mean JSW loss, but the number of progressors was 20% lower in the ASU group (p = 0.039).Conclusions: A 3-year treatment by ASU appears to reduce the % of JSW deteriorating patients compared to placebo, indicating a structure-modifying effect in hip OA. The clinical relevance of these results requires further assessment.

P292Efficacy of chondroitin sulfate on synovitis in patients with knee osteoarthritis: an ultrasound studyI. Möller Barcelona/Spain

Purpose: To assess the efficacy of chondroitin sulfate (CS) alone or combined with glucosamine sulfate (GS) on synovitis in patients with knee osteoarthritis.Methods and Materials: Retrospective study in 115 patients who received CS 800mg (N=36), CS800mg +SG1500mg (N=41) or acetaminophen (ACET) 500mg (N=38) daily for 6 months. Synovitis was measured in the suprapatellar recess using ultrasonography with high frequency linear array. Synovitis was defined as present in measures ≥ 4mm. Huskisson’s VAS, extra acetaminophen consumption, meniscal extrusions and Baker’s cysts were also assessed. Patient’s follow-up was performed after 1, 2, 3 and 6 months.Results: Among all subjects, a correlation between BMI and size of synovitis (r=0,138;p=0,002) and knee pain (r=0,136;p=0,003) was noted. A correlation between synovitis and pain (r=0,385;p=0,000) was also detected.Regarding ultrasonography assessments, all groups experienced a decrease in synovitis. Patients treated with SYSADOAs reached physiological values after 2 months. This improvement was maintained in the course of treatment with a mean synovitis (SD) of 3,2 (2,7) mm in CS group and of 2,9 (2,6) mm in CS+SG group.

Conclusions: The functional outcome scores of both groups (with/without BMAC) had good AOFAS functional outcome scores at 1 year post-operatively. Nevertheless, quantitative mapping T2 MRI demonstrated improved repair cartilage in the BMAC group. These differences may be indicative of more rapid deterioration in the group not treated with BMAC. While further investigation is warranted with longer-term follow-up, BMAC is a beneficial adjunct in cartilage repair.

P288Cartilage repair by a cell-free polymer-based cartilage implant immersed with platelet-rich plasma: histological and functional results of 52 patientsA. Siclari1, G. Mascaro1, C. Gentili2, R. Cancedda2, E. Boux1 1Biella/Italy, 2Genova/Italy

Purpose: In 52 patients, cartilage repair by a cell-free cartilage implant (chondrotissue®) immersed with autologous platelet-rich plasma was evaluated clinically. Methods and Materials: During arthroscopy, a chondrotissue® matrix and platelet-rich plasma were implanted after Pride drilling into articular cartilage defects. At 9 months after surgery, clinical outcome was assessed by second-look arthroscopy, histological staining and by evaluation of the Knee injury and Osteoarthritis Outcome Score (KOOS) compared to the pre-operative situation. Results: In results, second-look arthroscopy showed an excellent defect filling with cartilaginous repair tissue, firmly integrated to the adjacent tissue. Histologically, the formation of an homogenous matrix with hyaline-like repair tissue was evident. KOOS evaluation at 9 months after surgery showed significant improvement (P< 0,05) of the patients´ situation with increased subscores for pain (55 to 90; P = 2.75E-033), symptom (57 to 86.5; P = 2.40E-033), activities of daily living (69 to 85; P = 2.64E-033), sports and recreation (36 to 69.5; P = 1.78E-033) and quality of life (38 to 71; P = 2.39E-033). In the follow-up period, no revision surgery was indicated. No serious side effects like infection, inflammation, allergic reaction, thrombosis or symptomatic hypertrophy occurred. Conclusions: In conclusion, the chondrotissue® implant immersed with platelet-rich plasma supports cartilage repair after bone-marrow stimulation and showed significant clinical improvement in the patients´ situation and mobility. This advanced technique is suggested to be a safe and effective approach for cartilage repair.

P289Repair of full-thickness cartilage defects with human umbilical cord blood-derived mesenchymal stem cells and hyaluronate gel composite in a rabbit modelC. Ha Seoul/Korea, Democratic People’s Republic of

Purpose: This study investigated the cartilage regeneration effects of human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) plus sodium hyaluronate using an articular cartilage defect model in rabbits.Methods and Materials: Full-thickness chondral defects, 3 mm wide × 3 mm deep, were created in the trochlear groove of the right femur in 53 healthy New Zealand White (NZW) rabbits. The control group had only a chondral defect in the trochlear groove and 4 study groups administered the composite directly into the defect (0.2 ml/cm2). The MSCs concentration ranged between 0.1 and 1.5 x 107 cells/mL were applied. At 4, 8, and 16 weeks after surgery, cartilage repair was evaluated macroscopically and histologically using a semiquantitative grading scale.Results: The mean scores of the gross and histological evaluation grade in the experimental groups were significantly superior to those in the control group at 8 and 16 weeks (P <0.05).Conclusions: These findings suggested that human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) plus sodium hyaluronate can be used for the regenerative treatment of full thickness articular cartilage defect.

Posters 257

Correlation R S

IL-8 OLS open eyes/Area 0,44 0,04

IL-8 OLS open eyes/Amplitude ML 0,54 0,01

IL-12 OLS open eyes/Velocity ML 0,56 0,028

IL-12 OLS open eyes/Velocity AP 0,57 0,027

IL-12 OLS close eyes/Velocity AP 0,50 0,05

IL-6 OLS open eyes/Amplitude ML 0,43 0,04

IL-6 OLS open eyes/Area 0,55 0,009

IlL-6 OLS close eyes/Amplitude ML 0,52 0,015

IL-6 OLS close eyes/Amplitude AP 0,52 0,015

OLS = One leg stance ML = Mediolateral AP= Anteroposterior Conclusions: The biological markers were detected in SF from patients with OA in the early grades and were correlated to the functional activities that influence postural control, thus, these finds could be used as a way to measure the evolution of the functional capacity of patients with OA.

P295A Novel Implantable Load Absorber for the Treatment of Medial Compartment Knee OAN.J. London1, C.S. Waller2, D.A. Hayes3, A.G. Clifford4 1Harrogate/United Kingdom, 2Sidney/Australia, 3Brisbane/Australia, 4Hayward/United States of America

Purpose: Knee overload correlates with OA incidence, symptoms, radiographic, morphologic and biological changes. An implantable load absorber has been developed to treat the medial OA knee. The implant resides in the extra-capsular tissue of the medial knee and provides compartment unloading whilst preserving joint integrity. This study reports on the basic science of, and our early clinical observations with, this novel device. Methods and Materials: Changes in intra-articular loads provided by the implant were determined through dynamic gait simulations on cadaver knees with early OA. Preclinical safety was determined using a chronic ovine model (n=15) sacrificed at 4, 12, 26 and 52 weeks. The surgical approach used in our early clinical experience was similar to that described in preclinical studies. Post operative recovery and soft tissue healing in these patients were monitored as part of standard clinical follow-up. Results: Dynamic gait simulations in cadaveric specimens demonstrated a statistically significant reduction in medial compartment loads. Medial compartment loads were reduced by 129 ± 64 N, comparable to results reported for high tibial osteotomy. Wound healing in the chronic ovine model followed a normal course. Post implantation, a fibrous pseudocapsule, non adherent to the implant, formed around the device and matured between 12 and 26 weeks. In our clinical experience post operative recovery was rapid and medial knee tissues remodeled to accommodate the subcutaneous device. Patient’s exhibited excellent tolerance for the implant through a broad range of knee motions and activities. Conclusions: The load absorber preserves the structural tissues of the knee whilst providing clinically meaningful joint unloading. This unique surgical solution may be adopted as a primary or adjunctive surgical treatment to address medial knee degeneration. Our clinical experience suggests a major role for this treatment would be for the active patient, for whom preservation of both joint structure and active lifestyle are primary objectives.

P296A comparison of patellofemoral alignment between individuals with and without patellofemoral cartilage lesionsS. Duncan1, B. Noehren2, C. Lattermann2 10200/United States of America, 2Lexington/United States of America

Purpose: Patellofemoral malaligment is often implicated as a important factor in the development of patellofemoral osteoarthritis (PFOA). However, to date no study has directly compared patellofemoral alignment between those with symptomatic PFOA vs those without. Therefore, the purpose of this study was to determine the differences in patellofemoral alignment between those with grade IV lateral patellofemoral articular cartilage lesions versus normal controls.

A significant decrease in synovitis after SYSADOA therapy vs ACET treatment after 1 month of follow-up was observed.Statistical analysis demonstrated a faster decrease in pain with CS vs ACET (p=0,000) and in the CS+SG vs ACET (p=0,000). Analysis at each visit for pain decrease showed that differences between CS+SG vs ACET were significant after 1 month of treatment (p=0,037) and between CS vs ACET after 2 months of treatment (p=0,017).Conclusions: This study suggests that CS alone or in combination with GS decrease significantly synovitis measured by ultrasonography. This decrease after SYSADOA treatment has correlated with an improvement in knee pain. The decrease in synovitis elicited by SYSADOA is in accordance with former reduction of synovitis signs shown by CS and could contribute to explain its symptomatic and structure disease modifying effects.

P293Clinical utility of (99m)Tc-methylene diphosphonate (MDP) bone scan in osteoarthritis of knee with concomitant analysis of subchondral bone changeL.H. Jin, Y.J. Kim, K. Kwack, Y. An, B. Min Suwon/Korea, Democratic People’s Republic of

Purpose: Bone scan grade system has been used to evaluate alterations of knee cartilage in osteoarthritis (OA). The aims of this study were to evaluate the correlations between bone scan score, subchondral bone change and Osteoarthritis Research Society International (OARSI) score, and to show the clinical usefulness of the bone scan score to determine the severity of osteoarthritis. Methods and Materials: In this study, 54 human osteochondral specimens from total knee replacement (TKR) samples of 38 patients were taken after all patients were examined with 99mTc-methylene diphosphonate (MDP) bone scan. The intensity of MDP isotope uptakes was graded 0 to 3 (0, normal; 1, mild; 2, moderate; 3, severe) by 3 specialists of nuclear medicine. We measured mean values and standard deviations of subchondral Hounsfield Units (HU, CT number) values of the osteochondral specimens with a Multidetector Computed Tomography (MDCT). The histological grading by OARSI system was performed after staining with hematoxylin & eosin and Safranin-O stains. The correlations between bone scan scores, two HU values and OARSI scores were statistically analyzed. Results: Correlation analysis revealed significant correlations between bone scan and OARSI score (r=0.574, P<0.01). There were also significant positive correlations between bone scan score, OARSI score and subchondral MDCT HU (P<0.01). Interestingly, the standard deviation of subchondral bone MDCT HU correlated well with the bone scan score and OARSI score. Conclusions: In this study, we found statistically significant correlation between the bone scan score, subchondral bone MDCT HU and OARSI score. Our results suggest that the bone scan is a valuable tool in clinical research to evaluate OA of knee.

P294Correlation between the presence of cytokines in the synovial fluid and postural control in early degrees of knee osteoarthritisS.M. Mattielo-Rosa, K. Gramani-Say, P.R. Serrão, F.A. Vasilceac, G.C. Lessi, A.I. Medeiros, R. Reiff, J.A. Barela São Carlos/Brazil

Purpose: The aim of this study was to verify the correlation between cytokines detectable in synovial fluid (SF) and functional tests in patients with OA. Methods and Materials: 31 male subjects (40 to 65 years) with OA of the knee, grades I or II, was enrolled. All subjects were selected by clinical and radiographic evaluation (Kellgren & Lawrence scale). The SF knee was aspirated for analysis of cytokines (TNF-a, IL-1β, IL-6, IL-8, IL-12, IL-10, TGF-b) by ELISA method (Enzyme Linked Immuno Sorbent Assay). Later, the subjects performed one leg stance postural control on the force platform with eyes open and closed. Center of pressure (COP) variables were analyzed (area, amplitude, speed and frequency in anteroposterior and mediolateral) in anteroposterior and mediolateral directions. Pearson test correlation was used (R> 0.7 strong; p ≤ 0.05).Results: There was detected in the SF cytokines IL6, IL8 and IL12, which related to inflammatory process. According to Table 1, we found a positive correlation between cytokines detected and postural control variables. Table 1: Correlation between SF cytokines and postural control variables

Posters258

Methods and Materials: During the last two years we performed 67 knee arthroscopies (67 patients, 25 men and 42 women, mean age 51,7 years), in which we found cartilage lesions alone or along with intrarticular soft tissue injuries. We classified the lesions according to Outerbridge classification (grades I through IV). During the arthroscopy chondral lesions were shaved. We classified our patients in two groups taking care each of them to be consisted of similar cartilage lesions according to size and gradinfg. Group A (30 patients) were not treated in any additionally way. Group B (37 patients) were treated postoperatively by intrarticular injection of hyaluronan in 3-time course (after the 1st p.o. month, one per week), and took from the early p.o. period glucosamine sulfate per os, at least for 6 months.Results: We followed up all our patients using Lysholm score the 1st p.o. month, the 3rd, and the 6th and after 1 year (mean 11.1 months). There was a prevalence of group B scores when there was I to III grade of cartilage defect. For the grade IV lesions there was no significant deference between the two groups. Conclusions: We believe that the combination of arthroscopic debridment and postoperative use of intrarticular injection of hyaluronan and glucosamine sulfate per os, offers better functional results and pain relief in cartilage degenerative lesions.

P299Distraction arthroplasty for treatment of the osteoarthritic kneeA. Nakamae, M. Deie, N. Adachi, T. Nakasa, H. Shibuya, T. Niimoto, A. Okuhara, M. Ochi Hiroshima/Japan

Purpose: Joint distraction is a relatively new approach in treatment of osteoarthritis. We developed a new articulated distraction arthroplasty device for treatment of the osteoarthritic knee joint. This device allowed widening of the joint spaces and the continuation of ROM exercises. The purpose of this study was to evaluate the clinical results of a new distraction arthroplasty device when used in conjunction with a bone marrow stimulating technique for the treatment of osteoarthritis of the knee. Methods and Materials: We followed up 7 patients (age range, 40 to 63 years) who underwent distraction arthroplasty using our device with an average follow-up of 39 months (8 to 67 months). Distraction was carried out for 10 weeks (7 to 13 weeks) during which full weight bearing was allowed. Assessment included the Japanese Orthopaedic Association (JOA) knee score, range of motion, pain scale, and radiographic evaluation. Results: The JOA knee scores significantly improved from a mean of 54 points before treatment to a mean of 78 points after treatment. Scores on a visual analog pain scale and joint space values, as measured by the Rosenberg radiographic view, were also significantly improved at the latest follow-up.Conclusions: We conclude that the treatment using this new device in combination with a bone marrow stimulating method was effective for osteoarthritic knees in middle-aged patients. Until now, there has been no useful treatment for middle-aged osteoarthritis. We hope that this procedure not only will delay the requirement for total knee arthroplasty but will also encourage a change in strategy for treatment of osteoarthritic knee.

P301Autologous Matrix-Induced Chondrogenesis (AMIC) on the patella plus periosteal coverage on the trochlea combined with mechanical realignment- A New Treatment Option in Symptomatic Isolated Femoropatellar Osteoarthritis due to Subluxation of the PatellaR. Jakob, M. Jacobi Fribourg/Switzerland

Purpose: Symptomatic isolated femoropatellar osteoarthritis is reported for 8% of women and 2% of men over the age of 55 years. Surgical options include osteotomy of the tibial tubercle with patellar debridement and realignment (1), patellar hemiarthroplasty and femoropatellar arthroplasty and patellectomy. We present our results of a new biological treatment option, in which (1) is combined with the AMIC procedure (autologous matrix-induced chondrogenesis) and periosteal resurfacing of the lateral trochlea femoris.Methods and Materials: Only symptomatic isolated lateral femoropatellar osteoarthritis with an unsuccessful conservative treatment was included in the study. The surgery consisted of

Methods and Materials: 9 subjects (average age 33) diagnosed with lateral PFOA participated in this study. They were compared to 11 subjects (average age 34) with no patellofemoral lesions documented during an anterior cruciate ligament repair. Radiological measurements of interest included, the Insall-Salvati ratio, Trochlear angle, lateral patellofemoral angle and the tibial tuberosity trochlear grove distance (TTTG). Groups were compared using a two tail, independent samples, t-test. Additionally, means and effect sizes were calculated. Results: The PFOA group had a significantly higher Insall Salvati ratio (1.13 vs 0.94, p= 0.004, ES= 1.6), Troclear angle (142.2 vs 136, p= 0.04, ES= 1.8), and TTTG (15 vs 9, p= 0.02, ES= 1.81). Although there was no significant difference in lateral patellfemoral angle (12 vs 16, p= 0.09, ES= 0.8) it was associated with a large effect size. Conclusions: The results of this study clearly show that those with PFOA have alterations in their patellofemoral alignment as compared to controls.The greater patellar height, shallower trochlea, and higher Q angle seen in this group will increase shear and compression on the lateral aspect of the patella.This may over time result in the cartilage lesions seen in PFOA. These variables are easily measured with x-ray, or MRI and should be considered in surgical planning. Lastly, in light of these findings, physicians should consider concomitant realignment procedures in addition to cartilage restoration to promote an optimal outcome.

P297Intrarticular injections of hyalouronans for knee osteoarthritisC. Zidrou, M.I. Iosifidis, I. Melas, D. Neophytou, T. Liakos, N. Valanos, D. Alvanos, A. Kyriakidis Thessaloniki/Greece

Purpose: The aim of this study is to investigate the safety and efficacy of intrarticular injections with hyalouronic acid, in patients with knee osteoarthritis. Methods and Materials: Our study performed from June 2008 until December 2009 and was carried out on 412 patients (114 men and 298 women) with osteoarthritis of the knee, suffering from painful joint symptoms. The mean age of the patients was 62,5±8,4 years. Osteoarthritis was diagnosed on the basis of clinical and radiological examinations. Three intrarticular injections of hyalouronic acid, once per week, were carried out in all patients. They were excluded from the trial those patients with severe systematic disease (e.g. rheumatoid arthritis), suspected joint infection and those receiving concomitant treatments likely to interfere with the results (e.g. corticosteroids). All patients answered a questionnaire which contained questions on basic demographic information; pre- and post injection pain, mobility and the number of nights per week the patient was awakened from sleep by knee discomfort; the occurrence of side effects (e.g. pain, allergy); whether the injection allowed them to postpone or prevent joint replacement; and willingness to repeat the treatment if required. Participants were asked to rate pre-and post- therapy pain (both at rest and with weight-bearing) using a 10-point scale (1=no pain; 10=worst possible pain). A similar scale was used to access mobility (1=patient unable to move; 10=no restriction of movement). Results: The average scores for pre- and post-therapy pain were significantly improved (rest pain: before injection 5,9±2,4; after injection 3,8±2,3 – pain with weight-bearing: before injection 8,1±2,3; after injection 4,2±2,5). Average scores for mobility (before injection 4,2±2,5; after injection 6,8±2,6) and frequency of night-time awakenings (before injection 3,9±2,6; after injection 1,5±2,2) were also significantly improved. Knee replacement surgery was temporary avoided in 49% of the patients while 51% were willing to repeat the treatment. Side effects were not recorded. Conclusions: Intrarticular injections of hyalouronans are a safe and effective treatment for knee osteoarthritis and can be used to postpone or prevent joint replacement.

P298A combination of surgical and conservative treatment for knee articular cartilage degenerationM.I. Iosifidis, I. Melas, D. Neophytou, T. Liakos, E. Papantoniou, D. Metaxiotis, D. Alvanos, C. Zidrou, A. Kyriakidis Thessaloniki/Greece

Purpose: Articular cartilage degeneration is a common finding during arthroscopy. The aim of this early report is to present our results of a combination between arthroscopic debridement and postoperative use of intrarticular injections of hyaluronans and glucosamine sulfate per os.

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(Table 1). The AMF and lateral compartments consistently showed the greatest thickness (red areas on figure 1), corresponding with the thick cartilage commonly found in the PF joint. Some wear of the PF joint was seen in the knees with more advanced OA. The posterior regions of the lateral and medial condyles were intact in most cases, with relatively minor cartilage loss in some of the severe cases. The medial femoral and tibial lesions mirrored each other (Figure 1), starting with small areas in the centers of the condyles, then extending both A-P and medially as the OA progressed. On the tibia, anterior medial and posterior medial cartilage showed most loss for the majority of the patients. The medial side consistently showed more wear than the lateral side but there was no single region on the medial side which showed consistent wear for all patients.Conclusions: Our results showed that most early OA in the knee presented in the distal region of the medial femoral articular cartilage, leaving the posterior condyles intact. The medial tibia also matched the wear shown on the femoral condyle but did not present a consistent region of wear, which may be explained by the variations in ligament laxity and the condition of the meniscus. These patterns of cartilage loss are consistent with the impact forces generated at the heel strike and the toe off phases of walking. The information from this study is crucial for the design of future EI implants, as it points to the opportunity of preserving maximal viable cartilage while resurfacing only the damaged areas. Such early intervention techniques could significantly delay the progression of knee osteoarthritis and thus improve the quality of life of many patients.

P303Secondary Osteoarthritis in the Pediatric Hip Joint: Underlying ConditionsH.K. Gahunia, A.S. Doria, P.S. Babyn Toronto/Canada

Purpose: The aim of this study was to investigate the incidence of secondary OA in the pediatric hip joint.Methods and Materials: We retrospectively reviewed the imaging data of all identified pediatric patients with secondary OA of the hip (N=18, age range 8-17 years, 1:1 male:female ratio) obtained from our computerized database (1996 to 2009). Study cohort inclusion criteria for hip OA were based on two or more radiographically characteristic features of OA: joint space narrowing, subchondral bone sclerosis, cyst, and osteophyte formation. Primary diagnoses included: Dysplasia (spondyloepiphyseal-SED; Developmental Dysplasia of the Hip-DDH), rheumatologic diseases (Systemic juvenile rheumatoid arthritis-SJRA, Systemic Lupus Erythematosus-SLE), infections (Septic Arthritis-SA) as well as Slipped Capital Femoral Epiphysis (SCFE) and Legg-Calve-Perthes Disease (LCPD).Results: Unilateral hip OA (N = 12) presented more commonly in the left hip (67%) compared to the right (33%), with the OA lesions predominantly found in the acetabulum and femoral head. Unilateral hip OA was more commonly seen in patients with SCFE (25%) and LCPD (25%), followed by children with DDH (16%), SA (16%), SED (8%) and SLE (8%). In contrast, bilateral hip OA (N=6) lesions were identified in SJRA (83%) and one case of SED. In addition to the narrowing of the hip joint space, the radiographic presentation of OA lesions included subchondral sclerosis, cyst formation of the acetabulum and/or femoral head, osteophyte formation at the acetabular margins and irregular outline of the acetabulum. The magnetic resonance imaging of subchondral sclerosis showed low signal intensity on both T1- and T2-weighted images whereas subchondral cyst showed high signal intensity on T2-weighted images.Conclusions: Our series of cases suggests that pediatric secondary OA of the hip predominantly affects the left hip joint with similar incidence amongst females and males. Patients diagnosed with inflammatory arthritis, particularly systemic juvenile rheumatoid arthritis, appear to have an increased risk of developing secondary OA.

an AMIC procedure described by Behrens on the retropatellar cartilage defect and a periosteal coverage of the trochlear cartilage defect, both naked surfaces prepared by 1, 1 mm drillings combined with resection of the lateral pole of the patella with lateral release, medial reefing and a tibial tubercle medialisation and advancement. Lack of improvement and need for reoperation were defined as endpoints.Results: From 2003 to 2009 a total number of 21 patients underwent this described new procedure of which two were operated bilaterally. 1/4 of patients had a lack of improvement, thus 3/4 were satisfied. One patient who underwent bilateral surgery was satisfied at the last follow-up but died later of an unrelated cause. The other patient with bilateral surgery had persistent anterior knee pain on the one knee and a good result on the other. One patient had a reoperation with the AMIC procedure and is now satisfied. Another patient underwent partial meniscectomy. His arthroscopy showed fibrous cartilage coverage. One patient had total knee replacement. The oldest patient in this series showed radiographic signs of patellar osteolysis. Patients over the age of 75 years all were failures.Conclusions: The proposed combination of mechanical and biologic treatment modalities has proven to be a valid alternative to debridement and alignment alone or to arthroplasty in patients under the age of 75 years. It shows that there is a chondroplastic potential in osteoarthritis and advanced age.

P302MRI Analysis of the Progression of Articular Cartilage Loss in Knee Osteoarthritis PatientsG. Yildirim, S. Arno, E. Khelmenstkaya, R. Regatte, P. Walker New York/United States of America

Purpose: Magnetic Resonance Imaging (MRI) has been used to characterize the properties of articular cartilage, as well as local thickness. Medical and engineering software is now available whereby three dimensional models can be made from MRI scans, together with analyses of thickness and volume. These techniques can have a useful application in studying the progress of Osteoarthritis (OA) and in planning and designing Early Intervention (EI) treatments. While many studies have been carried out on the end-stage of OA, there have been few reports of the progress of the cartilage loss from the earliest stages. The goal of this study was to determine the locations and magnitudes of the cartilage loss of the knee joint, and the progression of OA in order to understand the mechanical factors involved. Further, from this data, our second goal was to determine what types of EI treatments, such as small implant components, could be employed to arrest the progress of the OA process.Methods and Materials: The MRI scans (3T Siemens, Malvern, PA) from 18 patients with symptomatic early OA were analyzed. The cases were taken at random from a group of 150 patients enrolled in the NIH study titled Leukocyte Gene Expression in Osteoarthritis by PI Steven B Abramson at the Department of Rheumatology, NYU-HJD. The Kellgren-Lawrence (K-L) scale was the biomarker used to classify the severity of the OA. 3D Doctor Software (Able Software Corp., Lexington, MA) was used to contour the articular cartilage layers at 0.6mm resolution. 3D models of the cartilage layers were then rendered in RapidForm software by lofting the contour slices (INUS Technology Inc., Seoul, Korea). The femoral and tibial cartilage models were subdivided into 6 regions, as follows: anterior lateral (AL), distal lateral (DL), posterior lateral (PL), anterior medial (AM), distal medial (DM), and posterior medial (PM). The anterior femoral region was defined as the anterior-most point of the cartilage to 2-3mm above the trochlear groove. The posterior region began 10mm posterior to the center of the circle formed by the condylar curvature to the posterior-most point of the femoral cartilage. Finally, the distal section lay between the anterior and posterior regions. The tibia was subdivided into 3 equal regions for each condyle. Each region then underwent thickness analysis displayed with a color-coded thickness map in RapidForm. From the 18 cases, 4 representative examples with increasing K-L grades were selected to show the progression of the cartilage loss (Figure 1). The overall cartilage volume was measured and the thickness at each point was divided by this overall volume to give a ratio for each region. This data was used to construct Table 1 showing the variations between the regions across the 18 patients, for increasing K-L grades.Results: For the majority of the 18 knees analyzed, the distal femoral regions showed the most cartilage loss; ranging between 0 and 1.5mm of remaining thickness. As expected, the medial distal femoral region showed greater thinning than the lateral region

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P307Osteochondral juvenile allografting: Is juvenile tissue a way to improve osteochondral defect repair?: A pilot horse study.S.M. Cokelaere, H. Brommer, J. Malda, P.R. van Weeren Utrecht/Netherlands

Purpose: Mature articular cartilage has developed definitive topographical biomechanical and biochemical heterogeneity and lacks adaptability and regenerative capacity. In contrast, fetal cartilage is still “blank” with regard to biomechanical and biochemical properties and has the ability to functionally adapt until maturity. We therefore explored a new approach in osteochondral repair by transplanting juvenile osteochondral plugs into freshly made defects in an equine carpal model.Methods and Materials: By means of arthroscopy / mini-arthrotomy, osteochondral plugs (6-mm diameter, 8-10 mm deep) were drilled out of the third carpal bone of a 5-month-old foal and directly press-fit implanted in a freshly made defect (6-mm diameter, 8-10 mm deep) in the proximal articular surface of the third carpal bone in 2 adult horses. At 2, 4 and 6 months arthrocenthesis, radiography, CT-scan and follow-up arthroscopy were performed. Sacrifices were at 6 months, with assessments by MRI (+ contrast), macroscopic inspection and histology.Results: Defects treated with osteochondral juvenile plugs showed better repair than the untreated donor sites. Follow-up arthroscopy showed a fibrocartilaginous surface with moderate integration into the surrounding cartilage. Indentation tests showed that the subchondral area was soft at 2 months, but became considerably firmer at 4 and 6 months. On CT and MRI, a gradual increase in subchondral bone density of the plug together with surrounding sclerosis was visualized. At the level of the plug, the articular surface was not exactly flush with the adjacent tissue on contrast MRI. Macroscopically and histologically, good integration at the bone-to-bone interface and minor cleft formation (filled with fibrous tissue) at the fibrocartilaginous articular surface was seen. Histologically, presence of type II collagen was evident and slight Safranin-O staining showed the (reduced) presence of glycosaminoglycans in the matrix.Conclusions: This equine pilot study shows that juvenile osteochondral allografting is an interesting concept that may be potentially useful in osteochondral defect repair.

P308Treatment of Osteochondral Lesions of the TalusA. Dalmau, D. Codina, J. Vega, F. Alvarez, R. Viladot Sant Cugat del Vallés/Spain

Purpose: The treatment of the osteochondral lesions in the weithgbearing articular surfaces is a common orthopaedic problem. Osteochondral ankle defects cause various symptoms including pain, swelling, and limited range of motion. Several surgical procedures, open or arthroscopic, attempt to resolve osteochondral lesions in the ankle: debridement and microfractures, osteochondral graft, or chondrocyte transplantation. The aim of this study is show our results of osteochondral lesion in the talar dome treated with osteochondral autograft technique (OATS), and present a guideline for its treatment. Methods and Materials: 34 patients with osteochondral lesions of talar dome, 5 to 27 mm in diameter, were treated using the osteochondral autograft transfer system (OATS). Osteochondral cylindrical grafts from the ipsilateral knee were delivered into the talar defect. These procedures were done by arthrotomy Results: The results have been evaluated by a clinical hindfoot score (AOFAS), radiographs, CT and MR images. An histological study has been made in any patients. Postoperative plain radiographs and CT images have been used to evaluate the congruence and integration of the graft. The AOFAS Score showed good to excellent results in most of the case, with a mean of 89 points, and all the patients returned to their activities and work.Conclusions: Restoring the normal convexity of the articular talar surface is a perplexing problem. To performance the graft correctly, malleolar osteotomy is necessary. Hystological exams demostrates that characteristics of hyaline cartilage is a normal hyaline cartilage with collagen type II, better with OATS than other techniques. Although OATS is more aggressive than arthroscopic procedures, no complications at the ankle or morbidity at the donnor site has been observed. Autogenous osteochondral mosaicoplasty is an alternative to treat a localized talar dome lesion with good functional results.

P305Use of autologous grafts for reconstruction of osteochondral defects of the knee: 18 years follow-up.E. Kon, A. Di Martino, G. Filardo, S. Patella, B. Di Matteo, S. Zaffagnini, M. MarcacciBologna/Italy

Purpose: One of the most important problems in orthopaedic surgery is reconstruction of damaged articular surface because of its limited regeneration capacity. An osteochondral defect is defined as a full-thickness, localized loss of bone and cartilage tissue of the articular surface with no spontaneous healing capacity. This study evaluates the very long results of a surgical technique for repairing osteochondral defects with autologous osteochondral grafts in young and middle-aged adult patients and evaluates the osteoarthritis progression of the treated knee with respect to the contralateral.Methods and Materials: Thirteen patients with femoral condylar osteochondral defects were treated with autologous osteochondral grafting. Mean patient age was 31 years (range: 16-52) at the time of surgery and mean follow-up was 18 years. The male:female ratio was 7:6. Defects were located on the medial condyle in 11 cases, on the lateral condyle in 1 case and on the lateral condyle associated with a tibial plate defect 1 case. For the clinical evaluation the Lysholm, International Knee Documentation Committee (IKDC) and Tegner scales were used. X-rays were used for radiological evaluation.Results: The results at long-term follow-up are very interesting with an high percentage of subjective satisfaction. Nine of thirteen patients are satisfied with their postoperative outcomes. The mean value of IKDC was 68 (range: 23-98.9). The Tegner mean value was 4.2. Knee degenerative progression was documented by X-ray.Conclusions: Autologous osteochondral transplantation is a valid therapeutic solution for treatment of deep osteochondral defects of the knee. At long follow-up evaluation the results are still good. Clinical and radiographic examinations confirmed good integrative capacity of the autologous graft and also survival of the chondral transplant.

P306Osteochondral autogenous transfer to the patello-femoral jointT. Satake1, M. Kobayashi2, S. Nakamura2, R. Arai2, Y. Nakagawa2, T. Nakamura2 1Otsu City/Japan, 2Kyoto/Japan

Purpose: The purpose of this study is to evaluate the result of treatment of chondral or osteochondral lesions of knee joints especially at the patello-femoral joint with osteochondral autogenous transfer (OAT).Methods and Materials: Seven knees of five patients were included in this study. Six knees of four patients were diagnosed as osteoarthritis, and one patient was diagnosed as osteonecrosis. Chondral or osteochondral lesions between 48 to 120 mm diameters were located at patellar surface in one knee and at patellar groove in six knees, and were treated with OAT. One or two grafts between 7 and 10 mm in diameter were harvested from non-weight bearing area of the femoral condyle and press-ï¬t into holes drilled into the defect. All patients were evaluated both preoperatively and postoperatively with the Lysholm knee score and Kellgren-Lawrence grade by sky-line view of plain radiographs of knee joint. Results: 5 patients of a mean age of 63 years old (55-74 years old) were followed-up for mean period of 39.1 months (24-57 months). The mean Lysholm score improved significantly from 64.57 preoperatively to 95.00 postoperatively (p<0.01). With radiographic evaluation, five knees in four patients did not be changed and two knees in one patient were improved.Conclusions: The results suggest that OAT is effective and safe method to treat chondral or osteochondral lesions of the patello-femoral joint. However, further long-term study is necessary to determine improvement of symptoms and the restoration of structural and functional integrity of graft over time.

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Methods and Materials: In study group 1, the CR-plugs were used to repair the femoral condylar or trochlear defects in a group of ten patients. The patients had an isolated grade III or IV articular cartilage lesion < 2.5 cm2. MRI scans as well as KOOS, Lysholm, IKDC subjective and SF-36 were collected pre-op and 6 months post-op with pending follow-up at 12 and 24 months. In study group 2, the CR-plugs were used as backfill in ten patients qualified by surgeon for OATS procedure. MRI scans at 6 months were collected with pending 12 and 24 months follow-up. Adverse events (AEs) were recorded. Results: One AE (tingling in the foot) possibly related to the study product was recorded and resolved. No other unexpected AEs related to the CR-plug were reported. At the 6 month post-op follow-up, the analyzed patients from study group 1 demonstrated an increase in KOOS, Lysholm, IKDC subjective and SF-36 when compared to baseline (Fig. 2C). The MRI scans at 6 months demonstrated initial remodeling of both cancellous and demineralized bone portion of the CR-plugs (Fig. 2A&B). Conclusions: We report the preliminary results from the 6 month follow-up of an ongoing clinical study using a novel allograft implant to manage small articular joint lesions. The up-to-date results demonstrate good record of safety, early signs of product incorporation and remodeling, and improvements in patient well-being.

P312Second-look arthroscopic and clinical evaluation of osteochondral autograft transplantation in osteoarthritic patients older than fifty years oldH.S. Seo, S.C. LeeSeoul/Korea, Democratic People’s Republic of

Purpose: The aim of this study is to assess second-look arthroscopic findings and clinical outcomes following osteochondral autograft transplantation in early osteoarthritic patients older than fifty years old.Methods and Materials: From June 2006 to October 2007, 23 consecutive patients (23 knees) underwent arthroscopic or mini-open osteochondral autograft transplantation (OATS) for osteoarthritic lesions of the knee joint. Inclusion criteria were: 1) age older than 50 years old; 2) persistent knee pain unresponsive to at least 6 months of conservative treatment; 3) radiologic grade 1 or 2 arthritis by Kellgren and Lawrence; 4) varus alignment less than 3 degrees; 5) Outerbridge grade 4 chondral lesion on weight-bearing area of the femoral condyle; 6) lesion size ranged from 1~4cm2. Among them, 20 patients (20 knees) received second-look arthroscopy at a mean of 12.7 months (range, 6-19 months) and were available for clinical evaluation with a minimum of 2 years’ follow-up. We evaluated the chondral lesion on second-look arthroscopy and assessed clinical outcomes using the WOMAC and IKDC scores, both preoperatively and at final follow-up.Results: On second-look arthroscopy, recipient sites of osteochondral autograft transplantation had healed completely showing normal cartilage (ICRS grade 0) in all knees. A radiographic evaluation at final follow-up revealed an increase by 1 grade according to Kellgren-Lawrence in only 1 case. The mean WOMAC score improved from 56.1 (range, 41 to 71) preoperatively to 73.0 (range, 59 to 81) at final follow-up (P = .033), and the average IKDC score also significantly increased from 54.1 (range, 40 to 66) to 77.4 (range, 67 to 85) (P = .028).Conclusions: Osteochondral autograft transplantation may be effective in treating single, localized osteoarthritic lesions in patients over 50 years old if the alignment of the knee is within the normal range.

P313Axial knee realignment technique for medial opening wedge osteotomy of the tibia with concomittant bioresorbable medial meniscus implant.K.P. Slynarski, E. Kurowska, T. Scinski Warsaw/Poland

Purpose: Main principle of medial open wedge high tibial osteotomies (MOWHTO) is to achieve a transfer of loading from medial - diseased, arthritic areas of the joint to lateral compartment with healthy cartilage. However, MOWHTO correct only knee alignment, but does not correct intraarticular pathologies of medial compartment. Our hypothesis was that patients with malalignment and medial meniscus loss would benefit with concomittant MOWTO and meniscus repair with bioresorbable meniscus implants.

P309Concurrent meniscal allograft transplatation and autologous osteochondral transplantation in meniscectomized PatientY.G. Koh, S. Jo Seoul/Korea

Purpose: The purpose of this study was to report the short -term clinical outcome performed concomitantly, AOTs(autologous osteochondral transplantation) and MAT(meniscal allograft transplantation) Methods and Materials: Patients who underwent MAT and AOTs between July 2008 and November 2008 were reviewed retrospectively . 8 cases were available with a mean follow-up 18 months(range 16 -21 months) The age at surgery ranged from 36 to 55 years of age (average : 42 years)Results: As a combined group, statistically significant improvements were observed in all standardized outcome scores at a mean 18months follow-up. Lysholm score significantly increased from 41.4 preoperatively to 82.0. Overall 87% of patients were classified as normal or nearly normal at their recent follow-up using Interanational Knee Documatation Committee examination score. Conclusions: Combined meniscal allofraft transplantation and autologous osteochondral transplanatation offers a safe alternative for patients with persistent symptoms after meniscetomy and focal cartilage injury. Long-term follow-up is needed to define the survivorship of these procedures.

P3102 year experience of Trufit osteochondral plugs for articular cartilage defects of the kneeN. Verghese1, S. Joshy2, M. Cronin2, M. Forster2, A. Robertson2 1Bridgend/United Kingdom, 2Cardiff/United Kingdom

Purpose: Recently biodegradable synthetic scaffolds (Trufit plug) have provided a novel approach to the management of chondral and osteochondral lesions. The aim of this study was to assess our 2 year experience with the Trufit plug system. Methods and Materials: 22 patients aged 20 to 50 years old all presenting with knee pain over a 2 year period were diagnosed either by MRI or arthroscopically with an isolated chondral or osteochondral lesion and proceeded to either arthroscopic or mini arthrotomy Trufit plug implantation. In 5 patients plug implantation was undertaken along with ACL reconstruction (3), medial meniscal repair (1) and contralateral knee OCD screw fixation (1). Pre and post operative IKDC scores were obtained to assess change in knee symptoms and function. Results: At a mean follow up of 15 months (range 2 – 24 months) improved IKDC scores were achieved with the scores improving with time. 2 patients had a poor result and have had further surgery for their chondral lesions. One patient had failure of graft incorporation at second look arthroscopy and went onto to have a good result after ACI. The second patient had good graft incorporation on second look but had progression of osteoarthritic degeneration throughout the other compartments of the knee which were not initially identified at the time of Trufit plugging. Conclusions: We conclude that Trufit plug is a viable alternative method for managing isolated chondral and osteochondral lesions of the knee which avoids harvest site morbidity or the need for staged surgery.

P311An allograft composed of cancellous and demineralized bone to manage osteochondral defectsJ. Farr1, A. Nawab2, S. Weinerman3, P. Stull3, D.C. Flanigan4, J. Dugas5, T.R. Carter6, D. May7, P. Bursac8 1Indianapolis/United States of America, 2Louisville/United States of America, 3Englewood/United States of America, 4Columbus/United States of America, 5Birmingham/United States of America, 6Phoenix/United States of America, 7Mechanicsville/United States of America, 8Alachua/United States of America

Purpose: This prospective multi-center study was designed to evaluate the use of a novel scaffold (CR-plug) composed of allograft cancellous bone and demineralized cortical bone for filling osteochondral defects (Fig.1). The implant was designed to a) closely biomechanically match the surrounding tissue, and b) utilize natural allograft material with a long history of safety and proven ability to induce cartilage and bone formation in animal models (e.g. Xang et al., 2008 v29(35) p4616-29).

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Methods and Materials: Using pre-defined criteria, we searched a number of electronic databases such as MEDLINE, EMBASE, The Cochrane Library to identify all the existing randomised control trials of any type of ACI treatment. Risk of bias was assessed according to the adequacy of sequence generation, concealment of allocation, blinding, completeness of follow-up, selective reporting and differences at baseline between the randomized groups. An analysis of the reported outcomes was performed. Information on the clinical efficacy and safety of ACI compared to other interventions was collected and presented.Results: Nine trials were identified with 626 patients. Patients ranged from 15 to 52 years and the size of treated lesions was between 1 and 22 cm2. ACI was associated with improvement in clinical outcomes compared to baseline. However, the body of evidence did not suggest any superiority of ACI over other treatments. Complications rates were comparable between interventions except from an increased rate of graft hypertrophies after ACI with periosteum.Conclusions: ACI is an effective treatment for full thickness chondral defects of the knee, providing an improvement of clinical outcomes. However, there is insufficient data to say whether ACI is superior to other treatment strategies. More, high quality, studies and harmonisation in the reported outcomes are needed before specific suggestions for practice can be made.

P316Measuring Pain and Function in Patients with Articular Cartilage RepairS. Lewis1, C. DeMuro1, M.M. Mordin1, J. Farr2, B.J. Cole3, K. Mithoefer4, L. Engelhart5 1Research Triangle Park/United States of America, 2Indianapolis/United States of America, 3Chicago/United States of America, 4Cambridge/United States of America, 5Milford/United States of America

Purpose: The US FDA draft guidance for industry entitled “Preparation of IDEs and INDs for Products Intended to Repair or Replace Knee Cartilage” recommends including improvement in pain and physical function as co-primary endpoints for confirmatory clinical studies. Insights into how patients assess pain intensity (PI) and the impact of pain on function are crucial to the proper selection of these outcomes for use in clinical trials. Therefore, we conducted in-depth individual interviews to assess the content validity of a single item of PI (Numeric Rating Scale - NRS) and the Knee injury and Osteoarthritis Outcome Score (KOOS) in patients with articular cartilage injury.Methods and Materials: Inclusion and exclusion criteria and a semi-structured interview guide were developed. Participants aged 18 to 65 who were either surgical candidates or at least 2 months post-op were identified through clinical sites. Participants were asked to describe the history of their knee injury and discuss ways that their lives had been impacted, followed by cognitive debriefing of the KOOS and NRS.Results: Fifteen patients aged 25-52 with mixed educational and ethnic background participated. NRS scores collected at screening ranged from 1-7 on affected knees and 0- 2 on unaffected knees. Most patients (13/15) indicated the NRS as an appropriate method to assess PI, noting its simplicity and patient familiarity with the scale. All participants stated that the KOOS was comprehensive and appropriate. Particularly, activities on the “Function, sports and recreational activities” subscale were often precipitating factors leading to injury and reflected the level of function to which participants wished to return.Conclusions: Results of this qualitative study support the use of the NRS and the KOOS subscales in assessing PI and function in patients with articular cartilage injury.

P317Biomechanical evaluation of the knee after autologous chondrocyte implantationP.C. Kreuz1, S. Müller2, A. Hirschmüller2, J. Mika3, C. Erggelet4 1Munich/Germany, 2Freiburg/Germany, 3Staufen/Germany, 4Zurich/Switzerland

Purpose: Autologous chondrocyte implantation is an effective clinical procedure for the regeneration of articular cartilage defects. However biomechanical loading and muscle strength tests are still missing in the postoperative follow-up. The present study presents a biomechanical evaluation of patients after second generation ACI in the knee.

Methods and Materials: Eight patients underwent medial opening wedge osteotomies using the AKRFX Surgical Instrument System and the iFX PEEK implant with concomittant arthroscopic medial meniscus repair with collagen (four patients) and polyurethane implants (four patients). Each of the eight patients was case-matched to a control patient who had undergone MOWHTO using the same AKRFX system with concomittant debridement arthroscopy. Patients were evaluated with specific quality of life (KOOS), SF 36 and radiographic assessments of union and maintenance of correction. Clinical union was measured by the patient’s ability to full weight bear and walk without the use of crutches.Results: At six months all 16 patients met the clinical criteria for osteotomy healing and showed statistically significant improvement in symptoms and in function. All KOOS score subscales showed statistically significant higher increase from baseline to 6 months in meniscus repair group, than in debridement alone arthroscopy. Statistically significant improvement in the SF-36 physical component score at 6 months in both groups was also observed, without statistically significant differences between them. There was no statistically significant differences with regard to type of meniscus implant used.Conclusions: The AKRFX osteotomy system achieved perfect rates of union and maintenance of correction. Clinical improvement in patients treated with concomittant medial meniscus repair were superior to group treated with osteotomy and debridement alone without statistical differences between types meniscus implants used.

P314The response of articular cartilage to periacetabular osteotomy as measured using the 3D dGEMRIC techniqueJ. Chan1, T.C. Mamisch2, Y. Kim1 1Boston/United States of America, 2Berne/Switzerland

Purpose: Periacetabular osteotomy (PAO) is a reconstructive surgery designed to treat symptoms and possibly delay the onset of osteoarthritis in patients with developmental dysplasia of the hip. The purpose of our study was to evaluate the effect of this procedure on the articular cartilage using a three-dimensional dGEMRIC sequence.Methods and Materials: Ten hips in ten patients treated with PAO for dysplasia were analyzed retrospectively. All patients were imaged pre- and post-PAO using a 1.5T MRI with three-dimensional isotropic fast T1 mapping dGEMRIC sequence (TR 15 msec, TE 3.27 msec, flip angles of 5 and 23 deg., matrix size 192/192, 16 cm FoV, voxel size 0.8 x 0.8 x 0.8 mm, acquisition 30 min post intravenous injection of contrast agent). The 3D data was reconstructed into seven radial slices of 2 mm thickness spaced 30 deg. apart (anterior - posterior) and oriented orthogonal to the acetabulum or parallel to the femoral neck axis. The mean T1 relaxation time was calculated separately for acetabular and femoral cartilage.Results: The mean age of the population was 22 ± 5.7 years and the follow-up scan was performed 12 ± 3.8 months after surgery. The mean overall dGEMRIC index (average of 7 slices) was 559 ± 109 ms (mean ± SD) pre-operatively and 519 ± 90 ms post-operatively, and the WOMAC pain score decreased by 4 ± 7.4. A global decrease in average T1 values was observed overall in both acetabular and femoral cartilage, with the largest decrease occurring in the superior-posterior region of the acetabulum and posterior-superior region of the femoral head.Conclusions: The three-dimensional assessment of cartilage in DDH patients suggests that the PAO has a minimal impact on the overall T1 average of cartilage. While the osteotomy appears to help relieve symptoms for patients, its short-term effect on the integrity of cartilage is not statistically significant.

P315Autologous Chondrocyte Implantation for the treatment of cartilage lesions of the knee. A systematic review or Randomized Control TrialsH.S. Vasiliadis1, J. Wasiak2, G. Salanti1, A. Georgoulis1 1Ioannina/Greece, 2Melburne/Australia,

Purpose: Autologous Chondrocyte Implantation (ACI) techniques are becoming more popular for the treatment of full thickness cartilage lesions of the knee joint. However, there is no systematic information for the efficacy of the new generation ACI techniques comparing to other treatment options. A systematic review of the existing evidence from randomized clinical trials of ACI treatment would contribute to understanding the advantages and limitations of this method and would inform the planning of future studies.

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the maximum isometric extensor peak torque (MIEPT) and the quadriceps steadiness (QS) between subjects of the control group (CG) and Osteoarthritis Group (OAG) and also verify the effectiveness of 11 weeks treatment protocol applied to the OAG subjects.Methods and Materials: This has been checked analyzing the average of the MIEPT and the QS for these subjects before, after 5 and 11 weeks of treatment. The sample was composed with 20 sedentary men, (45 to 65 years old), separated equally in 2 groups: CG (53,8 ± 7,7 years) without OA or knee injury and the OAG (53,1 ± 6,9 years) with knee OA grade I or II with diagnosis made by the Kellgreen & Lawrence radiological assessment. All volunteers were submitted to Isokinetic dynamometer evaluations of knee extension to verify their MIEPT and QS. The statistical tests used were ANOVA and Kruskal- Wallis to analyze the difference inter group. And Shapiro-Wilk, t-student and U Mann-Whitney test to analyze the OAG data before and after the treatment (α≤0,05). Results: At the variables analyzed peak torque (PT) and coefficient of variance (CV) any significant statistics difference was shown in both analyses, between the groups and inside the OAG before the 5 and 11 week of treatment.Conclusions: Despite of the results which didn’t show significant statistics values to prove your effectiveness, the importance to the clinic practice was notice since we checked that subjects with the initial grades of OA didn’t exhibit alterations in the maximum and steadiness force (PT and CV).

P321Case Study: Rehabilitation of an elite rugby player following osteochondral lesions in the knee. Train smart not hard!J.M. Stephen 6SX/United Kingdom

Purpose: A number of published case series have presented chondrolysis of the knee and its devastating consequences once it has occurred in young, male, elite sporting populations. This presentation outlines a successful, structured, progressive rehabilitation programme for returning an elite sportsman back to international rugby.Methods and Materials: A 23 year old male international rugby player presented with progressively worsening right knee pain and swelling on returning to play following a partial lateral meniscectomy 9 months previously. Clinical examination revealed a moderate knee joint effusion with loss of hyperextension and lateral joint line tenderness. Magnetic resonance imaging showed femoral and tibial bone oedema. Nine months following primary surgery, second look arthroscopy identified grade 3 and grade 2 chondral lesions of the lateral femur and tibia respectively, alongside a complex degenerative lateral meniscal tear (Figure 1). Surgical treatment involved a wash out and clean up of joint surfaces, with a meniscal repair undertaken to the remaining meniscal segment. The player in question underwent a lengthy rehabilitation period. A strong emphasis was placed on aquatic rehabilitation to offload the joint and encourage resumption of normal lower limb movement mechanics.Results: The player successfully returned to professional level rugby, however experienced ongoing symptoms of swelling and pain. He therefore continues to be carefully managed in terms of his training and playing. He presently plays one match in 2 weeks and undertakes team training every other day. From experience load managing these compromised athletes represents the most crucial aspect of enabling them to successfully return to sport with some career longevity.Conclusions: Articular surface compromised athletes need managed carefully in terms of weekly volume and load. They require career long modification of typical training patterns, but with correct management can demonstrate sustainability in the sport despite compromised joint integrity.

Methods and Materials: Biomechanical evaluation was performed on 44 patients 4 years after ACI with a second generation cell loaded scaffold. Isokinetic single-joint maximum strength measurements were investigated in different concentric and eccentric test modes with an isokinetic dynamometer. Reciprocal movements of the knee were performed for the flexion and extension of the knee. Statistical evaluation was performed in different defect locations of the knee using the Wilcoxon-and Mann Whitney U tests. Differences were considered significant at a p < 0.05. Results: The biomechanical evaluation revealed significant inferior maximum strength measurements for the extensor and flexor muscles in all defect locations of the knee after ACI compared to the untreated healthy knee (p < 0.05). Maximum strength values of patients with patellofemoral defects were significantly inferior compared to patients with defects on the femoral condyles (p < 0.05). Sportive patients with a higher KOOS-score and a continuous postoperative training revealed significant higher maximum strength measurements and a better balancing between extensor and flexor muscles of the thigh (p < 0.05).Conclusions: Biomechanical evaluation after ACI of the knee reveals reduced strength measurements compared to the healthy contralateral knee joint. This deficiency is often associated with a dysbalance to the disadvantage of the extensor muscles. The results have an outstanding importance in the postoperative treatment of ACI and underline the demand for a specific training of the operated extremity to avoid biomechanical deficiencies and muscle dysbalance.

P318Would you grow your own joint? An exploratory study into perceptions of endocultivation.R.G. Geddes1, J. Richardson2, P.H. Warnke3, A. Kerr2, S. Sivananthan2, R. Richardson1 1DE22 3HS/United Kingdom, 2Oswestry/United Kingdom, 3Queensland/Australia

Purpose: Regenerative medicine as an alternative to conventional orthopaedic surgery is gaining momentum in medicine through its promise of using your own body to heal your body. Endocultivation is a specific and innovative type of regenerative medicine, and has been used successfully to cultivate a mandible (Warnke et al 2004). It involves using autologous stem cells and culturing them in vivo, thus using the patient as the ‘bioreactor’ Our research aims to assess the public understanding and concerns regarding endocultivation and represents the first study in this area. We can summarise the aim of our study by asking: do people understand and want this kind of therapy?Methods and Materials: A survey instrument developed to assess attitudes towards different sources of stem cells, regenerative medicine and willingness to grow your own body part was distributed to members of the public in several European countries. Educational material on sources of stem cells was supplied to all participants. The process of endocultivation did always require an explanation.Results: Respondents (N=160) had a median age of 43 years and were from a variety of socio-cultural backgrounds. Support for a variety of sources of stem cells was higher than has been seen previously. Nearly 80% of participants would consider becoming a bioreactor and ‘growing their own joint’. In addition, people are willing to accept stem cell therapies in certain contexts.Conclusions: Acceptance of stem cell therapy in Europe is high. The use of autologous stem cells to regenerate an organ had the highest level of support, suggesting acceptance of stem cell therapy is dependent on personal context. We suggest more research is carried out in this area to inform ethics committees, policy makers and clinicians.

P320The influence of a physical training in the motor control -quadriceps steadiness - of patients with osteoarthritis grades I or IIS.M. Mattielo-Rosa, K. Gramani-Say, P.R. Serrão, Y.M. Kawaguchi, G.C. Lessi São Carlos/Brazil

Purpose: Osteoarthritis (OA) is a chronic and progressive degenerative disease, characterized by gradual loss of articular cartilage. A subject with OA exhibited substantially impaired motor control, balance instability and quadriceps weakness. All these factors come to a motor control deficit in the quadriceps steadiness. The aim of this study is to compare the average of

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function improvement in 86% of cases. Serial MRI’s of the knees showed progressive incorporation of the synthetic plugs and no adverse inflammatory reaction. Second-look arthroscopies showed complete and flush fill of the defects and their resurfacing with hyaline-like tissue under different stages of maturation. Recently, we have been able to check, clinically and by serial MRI’s, the first patients operated 24 months ago.Results: Despite the mantainance of clinical very good results, as showed by other authors, MRI images showed a delayed biologic process of incorporation of the plugs.Conclusions: This finding has not to be misinterpreted as an implant failure and the post-op rehabilitation has to be continued in order to give regenerating cartilage time to complete the maturation process.

P325The clinical safety and utility of an osteoconductive, bioabsorbable, scaffold for the use in treatment of full thickness chondral and osteochondral defects of the distal femurR.C. Lehman1, P.A. Davidson2 1Kirkwood/United States of America, 2Park City/United States of America

Purpose: This report describes the clinical safety and utility of an osteoconductive, bioabsorbable, scaffold consisting of bTCP, PLA and Type I collagen for the use in treatment of full thickness chondral and osteochondral defects (< 2cm²) of the distal femur. Methods and Materials: Patients enrolled in the study received 1 or 2 OsseoFit™ Porous Tissue Matrix™ implants on the femoral condyles (MFC or LFC) or trochlea. The study received IRB approval. Study endpoints are rate of complications related to the implant, change in KOOS scores and MRI findings with the MOCART Scale. The study was retrospective, conducted at a single center. MRI at approximately 6, 18 and 36 months post-operatively. 22 subjects with a mean age 37.7 ± 11.2 years were enrolled. 16 subjects received 1 implant and 6 subjects received 2 implants. Cartilage lesions were 1.2 ± 0.6 cm2 (0.64–2.64). 28 OsseoFit devices were implanted as follows: MFC=14, LFC=7 and trochlea=7. Concurrent surgical procedures included: meniscectomy in 13 subjects, ACL reconstruction in 6 subjects, debridement of other chondral surfaces in 16 subjects and other cartilage reparative techniques in 4 subjects. Results: At an average follow-up of 17.3 ± 4.5 months, post-operative KOOS Pain and ADL subscale scores increased by 33.8 ± 24.9 points (p<0.001) and 34.8 ± 25.3 points (p<0.001), respectively. There was no evidence of implant failure, implant delamination, hypertrophy, osseous necrosis, or significant reactive marrow edema. MRI evaluation was performed at 159 ± 80.5 days post-operative. Complete defect filling was seen in 19/28 implants. Complete integration to the surrounding cartilage was seen in 27/28 implants. Complications associated with the knee surgeries were minimal. Conclusions: At early follow-up, the OsseoFit device appears to be safe and contributed to improved clinical outcomes in the treatment of full thickness cartilage lesions (< 2cm²) of the knee.

P326Optimization of autologous fibrin carrier for chondrocyte transplantation.A. Wysocka1, H. Bursig1, P. Malinowska1, F. Kepski1, J. Dec2, T.S. Gaździk2 1Katowice/Poland, 2Sosnowiec/Poland

Purpose: There is an increasing number of publications concerning applications of cells in fibrin scaffold. Our Regional Blood Center has developed a method for obtaining fibrinogen from a single donor, which allows accurate identification and eliminates the risk of transmitting viral diseases.The objective of our work was to develop a method for autologous fibrin – chondrocyte graft preparation stabilized by fibrynolytic inhibitirs for transplantation into a cartilage defect.Methods and Materials: Cartilage was taken from the patient’s femoral condyle, non-load bearing area of knee joint, as well as blood, for the purpose of preparing autologous serum. Chondrocyte were cultured about 3 weeks. 450 ml patient own blood was collected prior to transplantation to produce autologous fibrinogen. Before surgery the chondrocyte suspension was mixed with fibrin, glue and applied into special form to prepare gel-like fibrograft.

P322Treatment of osteochondral defects using poly-lactic-glycolic acid PLGA scaffold and mesenchymal stem cells (MSCs)K. Uematsu1, Y. Ishimoto1, Y. Inagaki1, M. Ogawa1, Y. Tanaka1, K. Hattori2, H. Ohgushi2 1Kashihara/Japan, 2Amagasaki site/Japan

Purpose: The successful cartilage regeneration need a scaffold that can keep the stem cells in the defect and support to differentiate to hyaline cartilage. We have developed originally the biodegradable scaffold and that feature is that the cells can infiltrate into the scaffold without cell loss and lay in an uniform array at palisade. We have already reported the good results of the cartilage regeneration in an animal study. The aim of this study was to investigate the treatment of the symptomatic knee cartilage defects by the implantation of PLGA/MSCs composite.Methods and Materials: The patients were four men (age: 23, 25, 29 and 40 years) and one woman (age: 56 years), and three posttraumatic cartilage defects and osteonecrosis of the femur were treated. The average size of defect was about 1.7×1.8 cm. MSCs were harvested from in each ilium and incubated by standard method. After confluence, the cultured cells were seeded into PLGA scaffold, and the density of MSCs in the scaffold were 1×107 cells/cm3. We implanted PLGA/MSCs composite onto the cartilage defect by press-fit. Magnetic resonance imaging (MRI) and relook by the arthroscopy were performed in all patients at a mean of 36 months postoperatively. We evaluated the regenerated tissue endoscopically and conducted histologic assessment by a biopsy.Results: The inflammatory reaction was not recognized after one week postoperatively. The histologic evaluation by the biopsies showed the formation of hyaline-like cartilage.Conclusions: We conclude this scaffold was useful in the cartilage regeneration in human.

P323Articular cartilage repair using tru-fit plugsS. Tafazal, R. Shahid, R. Mansouri, M. Maqsood Lincoln/United Kingdom

Purpose: Articular cartilage damage is known to predispose to osteoarthritis. Many different techniques have been used to attempt repair of articular cartilage defects. Synthetic scaffolds can support cartilage formation in areas of cartilage defects. Study: We present our experience with a synthetic scaffold (Tru-Fit) which we are using to treat patients with chondral defects in knee. Methods and Materials: 15 patients with full thickness cartilage defects affecting the medial femoral condyle were treated with Tru-Fit plugs. They were followed up at a mean of 9.6 months (0.5-21) after surgery. At a minimum of 6 months follow-up MRI scans were obtained in all cases to asses healing of defect. International Knee Documentation Committee (IKDC) scores and Lysholm scores were also obtained. Results: The mean IKDC score was 55 (20-90) and the mean Lysholm score was 63 (20-100). Patients with isolated cartilage defects and no other knee pathology had a much better outcome with a mean IKDC score of 76 and a mean Lysholm score of 86. MRI scans showed good cartilage cover of the defects. Conclusions: Our early results of using Tru-Fit plugs to treat articular cartilage defects in knee are promising. Patients with isolated chondral defects in the knee show the most favourable outcomes.

P324Trufit bioresorbable scaffolds: clinical good results associated to delayed biological incorporationF.V. Sciarretta, C. AscaniRome/Italy

Purpose: Trufit resorbable scaffolds, made of semiporous copolymer, are press-fit introduced in chondral defects of articular surfaces in order to promote filling and regeneration of damaged bone and cartilage tissues.Methods and Materials: In another previous work, we have presented our good and promising results obtanied at 1 yy follow-up. Then, we have had the chance to go on on follow-ups and check patients through second-look arthroscopies and serial MRI’s: IKDC score showed 38 points improvement. WOMAC score showed statistically significant pain improvement in 89% of cases and

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Results: Five patients had small effusions, three of which resolved. One of the patients had a sizeable effusion with evidence of synovitis which persisted. The plugs show evidence of bone ingrowth in all patients by one year, which progressed on later scans. Similarly cartilage integration and subchondral lamina formation improve over time. Bone oedema is variable on early scans but improves on later scans. A good surface contour is demonstrated in all patients on early scanning, although it does deteriorate in a minority of patients over time.Conclusions: We demonstrate that the MRI appearances of TruFit plug repair follow a graded differentiation in to the respective layers over time - bone, subchondral lamina and articular like cartilage which appears to correspond with clinical improvement. These findings cannot be scored under the current Mocart scoring system and we present a robust modified Mocart scoring system to monitor the progress of this novel form of cartilage repair.

P329Ultrasound-guided corticosteroid injection in subacromial bursa for painful shoulder: a randomized, double-blind, placebo controlled, comparative trial of three dose regimensS. Yoon, J. Hong, K. Kwack, B. Min Suwon/Korea, Democratic People’s Republic of

Purpose: To determine whether ultrasound-guided subacromial bursa injection with high dose corticosteroid (4cc of 40mg triamcinolone acetonide and 1cc of 1% lidocaine, group 1, n=20), in patients with periarticular shoulder disorders, is better than low dose (2cc of 20mg triamcinolone acetonide and 3cc of 1% lidocaine, group 2, n=20) or placebo (5cc of 1% lidocaine, group 3, n=20) in improving pain, function, and active range of motion (AROM) at 2, 4, and 8 weeks. Methods and Materials: A randomized, placebo controlled, comparative trial with participant, injection operator, and outcome assessor blinding in which subacromial bursa injection with high dose regimen was compared with low dose and placebo. Outcome measures, assessed at 2, 4, and 8 weeks, included a visual analog scale (VAS) of the previous week’s average pain intensity, an AROM (includes flexion, extension, external rotation, internal rotation, and abduction angles) of shoulder, and a Shoulder Disability Questionnaire (SDQ). Results: (1) VAS: At 2 and 4 weeks, significantly greater improvements were found in participants in group 1 and 2 than in group 3. At 8 weeks, significantly greater improvement was found in group 1 than in group 2 and 3. (2) SDQ: At 8 weeks, significantly greater improvement was found in participants in group 1 than in group 2 and 3. (3) AROM: At 2, 4, and 8 weeks, significantly greater improvement was found in group 1 than in group 2 and 3. No improvement was found in other cases. Conclusions: Dose dependent efficacy of ultrasound-guided subacromial bursa injection with corticosteroid was demonstrated in patients with painful shoulder.

P330Quantification of intervertebral disc degeneration by parametric T2 and T2* mapping in patients with low back pain - initial results on the clinical use with 3.0 Tesla MRIG.H. Welsch1, D. Stelzeneder1, T. Paternostro-Sluga1, S. Goed1, D. Hornung2, S. Trattnig1, T.C. Mamisch3 1Vienna/Austria, 2Erlangen/Germany, 3Berne/Switzerland

Purpose: Quantitative T2 and T2* provide information about the interaction of water molecules and the collagen-network within the intervertebral disc (IVD). Aim of the study was to assess, compare and correlate quantitative T2 and T2* relaxation time measurements of IVDs in patients suffering from low back pain, with respect to the IVD degeneration as assessed by the morphological Pfirrmann Score. Special focus was set on the spatial variation of T2 and T2* in between the annulus fibrosus (AF) and the nucleus pulposus (NP).Methods and Materials: Thirty patients (38.1±9.1 years) suffering from low back pain were enclosed. Morphological (sagittal T1-FSE, sagittal and axial T2-FSE) and biochemical (sagittal T2- and T2*-mapping) MRI was performed at 3T covering IVDs L1-L2 to L5-S1. All IVDs were morphologically classified using the Pfirrmann score. Region-of-interest (ROI) analysis was performed on midsagittal T2/T2* maps at 5 ROIs from anterior to posterior to obtain information on spatial variation between the AF and the NP. Statistical analysis-of-variance and Pearson correlation was performed.

Results: Within 2 years 32 cultures of autologous chondrocytes were completed. The mean age of patients was 34 years. The average defect size 6,1cm2. The average concentration of autologous fibrinogen was 45 mg/ml. Stabilization of chondrocyte-fibrin construct was achieved by high doses of antifibrinolitic agent.Conclusions: Chondrocyte implantation in fibrin glue is a promising method for treatment of cartilage defect. Fibrinogen prepared at Regional Blood Center in combination with cells constituted highly plastic and adhesive grafts. This facilitates and accelerates implantation - arthroscopy can be applied. It was possible to set disintegration of fibrograft, by different concentration of antifibrinolitic agent. A novelty is the application of autologous fibrinogen in combination with autologous chondrocytes. Application of autologous fibrinogen is safer for the patient, as compared with allogenic fibrinogen, since it does not carry the risk of transmitting infectious diseases. The study was supported by grant NN403184034

P327New biodegradable and biocompatible synthetic scaffold for meniscal regeneration: preliminary clinical experienceS. Patella, E. Kon, A. Di Martino, G. Filardo, S. Zaffagnini, B. Di Matteo, M. Marcacci2

Bologna/Italy

Purpose: Either a lesion or the complete absence of the menisci can invalidate the physiological function of the knee causing important damages, even at long term. Meniscal tears are often found during the ordinary orthopaedic practice while menisci regenerative potential is very low and limited to its peripheral-vascularized part; this is why the surgeon is often almost forced to perform a partial, subtotal or even total meniscectomy, regardless of the well-known consequences of this kind of surgery. Methods and Materials: Recently a porous, biodegradable scaffold made of an aliphatic polyurethane (Actifit™,Orteq Ltd) has been developed for the arthroscopic treatment of partial and irreparable meniscal tears; this scaffold facilitates menisci regeneration preventing the potential cartilage damage due to its complete or partial lack. We treated 17 patients affected by a massive loss of meniscal substance with intraarticular or global knee pain and/or swelling. We analyzed the patients either clinically and by using the International Knee Document Committee’s (IKDC) Subjective and Objective Knee Evaluation Form and the Tegner score at the time of the very first visit with the presurgery and prelesional ones. A control MRI was done at 6 and 12 months after surgery.Results: Apparently, the properties of this scaffold help in vessels formation and tissue regeneration potentially allowing the restoration of the surgically removed portion and preventing, or delaying at least, both chondral and articular degeneration. We also performed some biopsy associated arthroscopic “second-looks” that reinforced the already good clinical results and confirmed the new tissue ingrowth into the biomaterial. Conclusions: Preliminary results suggest that this surgical procedure can be considered a really promising method for the treatment of both inveterate and symptomatic meniscal tears; however, other randomized studies with a longer follow-up should be done to confirm its reliability and potentialities.

P328Modified Mocart scoring system and three year MRI followup of Chondral repair using TruFit plugsM. Dhillon1, G. Collin1, R. Wellings1, P. Thompson1, T. Crane1, T. Spalding2 1Coventry/United Kingdom, 2Leamington Spa/United Kingdom

Purpose: TruFit plugs (a biodegradeable synthetic scaffold - Smith and Nephew) are a novel treatment of chondral damage in the knee. The normal MRI findings following TruFit repair have only a limited description in the literature. We present a qualitative longitudinal study of the TruFit plugs MRI changes in our cohort over a 3 year period with a qunatitative assessment with a modified Mocart scoring system.Methods and Materials: Twenty four patients with osteochondral defects in the knee have undergone TruFit plug synthetic bilayer scaffold repair. These patients have been followed up with MRI at six monthly intervals where possible. MRI findings of: effusion; synovitis; bone oedema; TruFit plug resorption; subchondral lamina formation; cartilage integration and surface contour were assessed and scored.

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by tissue engineering technique and with rich platelets growth factor, spongy bone chips or calcium triphosphate forming a biological structure. The affected joint was under arthroscopic assessment and we prepared the damaged area with all affected tissue resection. We finished the procedure with the application of biological structure in the bone defect. Results: In all cases we found improvement in the articular R.O.M. The Rx and M.R.I. showed bone or substitute bone integrated and soft tissue covered the defect in all cases. Histologically we found in all the biopsies performed the morphology of the “articular cartilage Hyaline”, homogeneous tissue with typical round cells forming lagoons and collagen type II with a normal basal integration that represent a “Tide-Mark” regeneration. Conclusions: The autologous bone marrow-derived Mesenchymal stem cell, associated with spongy bone chips or calcium triphosphate, show histological data of hyaline cartilage articular regeneration with very good mechanical behavior and clinical results after more than 13 months of follow up.

P333Depth-dependent healing strength of cartilage-like tissue to host cartilage: Application of a stem cell-based tissue engineered constructR. Nansai1, K. Adachi1, N. Nakamura2, H. Fujie1 1Tokyo/Japan, 2Osaka/Japan

Purpose: We have been developing a new tissue engineering technique for cartilage repair using a scaffold-free, tissue engineered construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). The present study was performed to determine the depth-dependent healing strength between the repaired cartilage-like tissue and surrounding host cartilage. Methods and Materials: Synovium-derived MSCs obtained from immature porcine knee joints were cultured in a monolayer in DMEM. After ascorbic acid 2-phosphate was added, the cells were allowed to undergo active contraction for 8 hours to develop a TEC. The TEC mass was allografted to a cylindrically shaped, chondro-defect created in the medial condyle of 3 month-old porcine femur. Six months after surgery, a dumbbell-shaped specimen including the boundary between the repaired tissue and host cartilage was extracted. The tensile test was performed for the specimen at a rate of 0.01 mm/s. Results: The healing strength of the TEC-treated repaired tissue was higher than that of the TEC-untreated repaired tissue, although it was lower than the tensile strength of the normal cartilage. The strength was almost negligible at the surface layer but was 1.7 and 2.8 MPa at the middle and deep layers, respectively. Histological examination indicated that, at the intermediate-to-deep area, the TEC-treated repaired tissue was hyaline cartilage-like while the TEC-untreated repaired tissue was fibrous cartilage-like. Conclusions: The present study indicated that the healing strength of cartilage-like repaired tissue to surrounding host cartilage was increased with the treatment of TEC although the strength remained lower than the strength of host cartilage. In addition, the healing strength was depth-dependently increased in the TEC-treated cartilage-like tissue in the same way as shown in normal cartilage. These results suggest that the healing process of cartilage-like tissue is promoted by the TEC in conjunction with an extrinsic factor possibly permeated from the bottom of the chondro-defect.

P334Regeneration of lumbar intervertebral disc using MSVs*. *Autologous Bone marrow expanded MSCs under GMP guidelines from IBGMC. Morera1, R. Soler Rich1, J.J. Velázquez1, A. Sánchez2, M. Alberca2, J. García Sancho2, L. Orozco1 1Barcelona/Spain, 2Valladolid/Spain

Purpose: We report the methodology and outcomes of the clinical trial phase I-II, Eudra-NCT:2008-001191-68, assessing the feasibility, safety and efficacy findings of regeneration after the percutaneous intradiscal infiltration of autologous bone marrow mesenchimal cells expanded under GMP. This study is carried out under the framework of the “Plan for Advanced Therapy and Regenerative Medicine” (Consejo de Ministros of the Spanish Government, 2007).

Results: Both, T2 and T2* were able to clearly differentiate between all grades of IVD degeneration according to the Pfirrmann score (p<0.05). The spatial variation, as an increase in T2 and T2* values from the AF to the NP, was highest at Pfirmann grade I and declined at higher Pfirmann grades II-IV (p<0.05). Where T2 was more sensitive in the description of the NP, T2* was more sensitive in the depiction of the AP. Correlation between T2 and T2* revealed a medium Pearson correlation (0.210 to 0.356 (p<0.001)).Conclusions: T2 mapping as well as the newer T2* mapping provide a fast and stable tool in the evaluation of IVDs. The clear differentiation of IVD degeneration and the possible quantification by means of T2 and fast T2* mapping may provide a new tool to follow-up therapy protocols in patients with low back pain.

P331T2 mapping of facet joints and intervertebral discs: Biochemical MRI at 3.0 Tesla in patients with low back painD. Stelzeneder1, G.H. Welsch1, S. Goed1, A. Messner1, T. Paternostro-Sluga1, V. Pflueger1, G. Scheurecker2, K. Friedrich1, S. Trattnig1 1Vienna/Austria, 2Linz/Austria

Purpose: Degenerative changes of the facet joints represent a possible source of pain. Our objective was to assess lumbar facet joint cartilage in comparison to the intervertebral discs by means of quantitative T2-mapping. Methods and Materials: Forty-nine lumbar spine segments from 25 patients (mean age 36 years) suffering from low back pain were examined by axial T2-mapping and morphological sequences at 3.0 Tesla. Regions of interest were drawn on a single slice across the facet joints, the nucleus pulposus and in both anterior and posterior annulus fibrosus. The Pfirrmann-score was used for morphological grading (“normal” vs. “abnormal” discs). A Pearson-correlation analysis was performed to compare disc and facet joint T2 values. Results: Twenty-six discs were graded as normal (Pfirrmann 2) and 23 as abnormal (Pfirrmann 3+4). Mean (±standard deviation) T2-relaxation-time-values (in ms) were: facet joints: 57.4±12.4, nucleus: 97.3±25.9, anterior annulus: 49.0±11.0, posterior annulus 61.6±11.5. There was no difference between facet joint mean T2-values in normal and abnormal discs: 57.1 vs 57.7 ms. Correlation analysis showed no association between nucleus and facet joint T2-values. Facet joint T2-values of all analyzed segments showed a weak correlation with the anterior annulus (r=0.31;p=0.03) and a moderate correlation with the posterior annulus fibrosus T2-values (r=0.53;p<0.001). These associations were not seen in the subgroup of normal discs. The correlation of facet joints with the posterior annulus was stronger in abnormal discs (r=0.66;p<0.001). For the facet joints and the anterior annulus only a trend was seen (r=0.41;p=0.053) in abnormal discs. Conclusions: Our report represents the first attempt of a quantitative analysis of lumbar facet joint cartilage via T2 mapping in comparison to morphological grading. Our evaluation may suggest that the biochemical structure of the posterior part of annulus fibrosus affects biochemical properties of facet joint cartilage in particular in degenerated discs. T2 mapping may help to detect early degeneration of facet joints.

P332Osteochondral Injuries: Application of Bone Marrow-Derived Mesenchymal Stem Cell Assisted by Arthroscopy.J.F. Urraza1, P. Vidal2 1Sant Pere de Ribes/Spain, 2Barcelona/Spain

Purpose: The successful regeneration of the “Hyaline cartilage” can be considered as one of the more important challenges in the field of Orthopaedic surgery. The purpose is to present a preliminary study using autologous bone marrow-derived mesenchymal stem cell in the treatment of osteochondral injuries, analyzing the feasibility of the process. Methods and Materials: We report the preliminary data of a prospective clinical study with five patients . The ICRS classification was used (grade IV B and C). The average patient age was 21 years old (17-30). All patients had received surgical treatment prior to this study and they had had the lesion for a year or more (1-11). The feasibility of the method was appraised with Rx., M.R.I., functional clinical assessment, and histological studies. Puncture of posterior iliac crest for bone marrow aspiration was made. The identification and isolation of mesenchimals cells was made

Posters 267

P336Pre-differentiation of mesenchymal stem cells improves cartilage regeneration for treatment of chronic osteochondral lesionsJ.S. Somerson1, B. Marquass2, P. Hepp2, R. Richter2, T. Aigner2, C. Josten2, A. Bader2, M. Zscharnack2, R.M. Schulz2 1Toledo/United States of America, 2Leipzig/Germany

Purpose: Large osteochondral lesions are a challenge to treat due to the lack of spontaneous healing. This project evaluated the use of marrow-derived autologous mesenchymal stem cells (MSC) in hydrogel constructs to treat osteochondral lesions. Repair quality in vivo with MSCs that had undergone in vitro chondrogenic pre-differentiation was compared with undifferentiated MSCs and cell-free constructs.Methods and Materials: An initial surgery was performed to create two osteochondral lesions (7mm diameter) on the medial femoral condyle of each hind leg in 19 merino sheep. Bone marrow aspirate was taken from the iliac crest at the time of surgery. The defects were allowed to degenerate in natural fashion for 6 weeks. After isolation of MSCs, 5 x 10^5 cells/ml were seeded into a collagen-I matrix. One group of the MSC-gels underwent chondrogenic pre-differentiation while the other MSC-gel group was cultivated using DMEM and 10% autologous serum. After 6 weeks in vitro, the constructs were implanted. Untreated lesions, cell-free constructs and chondrocyte-based gels were used in control groups. The animals were sacrificed after 6 or 12 months and the constructs were evaluated using histology and micro-MRI.Results: Quantitative assessment confirmed chondrogenic in vitro differentiation of the ovine MSCs in the collagen gels. After 6 months, the pre-differentiated MSC-gels showed the best results (ICRS Visual Histological Score (VHS):13.3±2.6; O’Driscoll score: 17.6±2.5). The cell-free gels showed a VHS of 8.2±4.3 points and an O’Driscoll score of 11.3±6.8. After 12 months, the pre-differentiated MSC-gels showed superior histological properties (O’Driscoll: 18.2±2.5) to undifferentiated MSC-gels (10.5±5.9). No significant difference was noted between the results for repair using pre-differentiated MSCs at 6 and 12 months.Conclusions: Implantation of collagen gels seeded with autologous MSCs results in regeneration with a hyaline-like structure. Repair using MSC-seeded gels appears to produce better results when chondrogenic pre-differentiation is performed prior to implantation.

P337AMIC technique enhanced by the use of concentrated bone marrow for the treatment of chondral lesion.L. de Girolamo1, P. Adravanti2, H. Schoenhuber1, P. Volpi1 1Milan/Italy, 2Parma/Italy

Purpose: AMICÒ (autologous matrix induced chondrogenesis) combines microfracturing with the application of Chondro-Gide®, a porcine collagen type I/III bilayer matrix. However, all marrow stimulation techniques collect only a poor number of mesenchymal stem cells and this number is variable among patients. For this reason it could be useful to enhance these techniques with progenitor cells harvested from iliac crest bone marrow, in order to provide a better and faster defect resurfarcing. Here we present the clinical outcome of 30 patients treated either with the standard AMICÒ technique or combined with the use of concentrated bone marrow (cBM) from a minimum follow up of 6 months up to 24 months. Methods and Materials: 30 patients (range 18-50 y/o) presenting focal cartilage lesions of the knee were treated either with the standard (n=11) or the modified AMICÒ technique (n=19). Clinical evaluation was based on Lysholm Knee Score, IKDC and VAS pain scale. For each patient MRI was performed preoperatively and 6, 12 month and 24 months postoperatively. Results: Significant differences between pre- and post-operative score values have been observed for all patients starting from 6 months. After 12 months significant best results were found in patients treated with the modified AMIC® technique (n=11) respect to those treated with standard AMICÒ (n=8), in particular in term of pain reduction (2.5±0.7 and 0.8±0.4, respectively, p<.05) and Lysholm score (84±1.4 and 95.4±4.4, p<.05). MRI showed good healing processes of the cartilage defects already after 6 months in both groups of patients. Conclusions: Enhancing the AMICÒ technique with concentrated bone marrow seems to be an effective and safe method. However, longer follow-up times are necessary to evaluate the long-term regeneration success.

Methods and Materials: 10 patients with degenerative disc disease were recruited . One of the inclusion criteria was proven competence of the annulus fibrosus by discography. The procedure starts harvesting bone marrow aspirate (80 ml) from iliac crest under sedation and local anesthesia. Collected samples are sent to the Cell Therapy Unit of the IBGM, where they carry out the cell extraction and expansion process for three weeks. The product generated (MSVs) is held to the same standard as medication under investigation. It was liberated after undergoing the bio-safety controls (sterilization, viability, phenotype and cariotype). MSVs are percutaneously infiltrated under RX guidance at a dosage of 15±5x106 MSCs per disc (max 2 discs, max dose 40x106 MSVs). Results: At the moment (6 months follow-up) the procedure has proven to be feasible and we have registered no adverse effects or toxicity signs. We are assessing the efficacy findings of the regenerative therapy evaluated through changes in density at the nucleous pulposus (re-hydration) assessed by semi quantitative MRI and the clinical outcomes using the Visual Analogue Score for pain, Oswestry Disability Index and the SF-36 Questionnaire for quality of life, all showing positive statistically significant difference. Conclusions: Infiltration of autologous bone marrow MSCs into the degenerated intervertebral nucleous pulposus showing clinical signs may improve symptoms and even achieve regression of the degenerative process. The final outcomes shall be communicated at the ICRS Congress 2010, upon completion of the one year follow-up period.

P335Chondrocytic differentiation of bone marrow-derived equine mesenchymal stem cells for future therapeutic applications in horsesA. Pineda1, M. Masri2, R. Gomez2, C. Landa2, C. Velasquillo2, C. Ibarra2 1México/Mexico, 2Mexico City/Mexico

Purpose: The objective of this study was to isolate and characterized chondrocytic differentiation of bone marrow-derived equine mesenchymal stem cells for possible future therapeutic applications in horses. We characterized using , CD45, CD34 and CD14 typical hematopoietic antigens. Methods and Materials: Bone marrow aspirates were obtained from sternebra of 7 horses, 3-5 years old. The aspirates were obtained by Jamshidi bone marrow aspirate needles and were collected into syringes containing heparin to a final concentration of 1,000 UI/ml. The fraction of mononuclear cells was separated with Ficoll (Amersham Bioscience Ficoll- Paquete Plus) and washed with PBS. The pelleted stromal cells were resuspended in DMEM F12 medium containing 10% fetal bovine serum and 100 UI/ml of penicillin and 100 µg/ml of streptomycin and cells were incubated. Part of the sample was utilized for flow cytometry test with human antibodies (CD34, CD14, and CD45). To induce chondrogenic differentiation the original media was replaced with an induction medium containing transforming growth factor β1 and bone morphogenetic protein-2 (100ng/ml). The chondrogenic differentiation potential was measure by alcian blue and safranin-O stains.Results: We found that cells from equine bone marrow stem cell markers negative for CD14, CD34 and CD 45. Under tridimensional culture conditions to promote chondrogenic differentiation, cell aggregates were detected positive for alcian blue and safranin-O.Conclusions: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSC’s, have the capacity to undergo chondrogenic differentiation and may be a clinically plausible source of cells for neo-chondrogenesis at damaged articular sites. As an autologous cell population, equine MSC’s can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.

Posters268

P338State of the art on cartilage tissue regenerationP. Vidal1, J.F. Urraza2 1Barcelona/Spain, 2Sant Pere de Ribes/Spain

Purpose: Successfully hyaline cartilage regeneration is one of the most significant challenges in the orthopedic field. Unfortunately cartilage injured doesn’t naturally grow back, and treatment options remain limited even innovative therapies which improved substantially over the past few years. Methods and Materials: The tissue engineering discipline pursues basic knowledge in tissue development and self regeneration instead of repair. It is critical to choose suitably cells, appropriate signaling proteins and biomaterials to mimic the natural physiological process of tissue regeneration. Although mostly composed by water any regenerated cartilage needs to contain a structure capable of bearing weight and being strong but flexible, definitely the main challenge to achieve. The cell-based therapies strategy fall into matrices where is depending the body natural capability to regenerate and conditioned by biomaterials applied, some tend to slowly degrade and other takes too long time. Thus, there is dissociation between cells growing to become specific local cell phenotype and matrix degradation, too short or long term degradation implies an incomplete chondrogenesis. Results: Our approach has been focused on structured autologous multilevel matrix supplying proteins and cytokines to the implanted cells in a proper environment niche that promote the ingrowing cells and support their sequential function of protein secretions, cellular metabolism, local cells commitment and differentiation. Conclusions: Bone marrow-derived mesenchymal cells are our preferred cells by their multipotentiality and progenity which correlated directly with regenerative capability. Cartilage tissue engineering incorporates cell transplantation, materials , and new surgical cell delivery, personnel who have mastered the procedures of cell harvest, manipulation, and graft design are essential for the successful application of this technology.

269Authors‘ Index

This author index lists the names of all authors and co-authors of the all congress abstracts (Podium Presentations, Poster Presentations & submitted Extended Abstracts by the invited faculty). Requests for changes have been considered until June 30, 2010. The numbers in the index refer to the final programme number and the letter “P” before the final programme number refers to the poster section.

A

Alaminos, M. P93, P93, P107Abe, S. P87Abellanet, I. 12.1.7, P223Abelow, S. P. 20.1.1Aberman, H. 25.3.4Acharya, C. P186Ackland, T. R. 12.2.2, P225Acosta, V. P48, P177Adachi, K. P333Adachi, N. 22.0.2, P23, P74,

P179, P276, P286, P299

Adelson, W. 9.1.4Adesida, A. P186Adravanti, P. P337Aicher, W. K. P83Aigner, T. P336Akamine, Y. P158Akazawa, T. P140Akgün, U. P115Akieda, S. P209Alberca, M. P334Alblas, J. 25.1.3Albrecht, C. 17.1.6Alcaine, C. P91Alevrogiannis, S. P237, P259Alibegovic, A. 9.1.3Alini, M. 9.4.2Allendorf, S. P58, P60Almqvist, K. F. 12.1.2, 12.4.2,

17.3.4, P167, P243, P274, P281

Altadonna, G. 17.1.5, P256Alvanos, D. P271, P297, P298Alvarez-Lorenzo, C. P169Alvarez, F. P308Alves da Silva, M. P37Amano, H. P278Ambrose, C. G. P133Amiel, D. 25.1.2, P1, P135Amler, E. 12.3.4An, Y. P293Anders, S. 9.2.8, P244Anderson, D. G. 12.3.2Ando, K. P97Ando, W. 3.2.3, 9.1.9, 24.3.3Andreas, K. P56Andry, J. P. P142Angele, P. P198Anitua, E. 20.1.2, P20Aoyama, T. P137Apostolidis, K. 17.4.6Apprich, S. 17.2.4, 25.2.2Arai, R. 25.1.7, P306Arai, Y. P215Arakaki, K. 9.4.5, P7, P31, P35Araki, S. P12, P17, P59,

P68, P97Arbel, R. 2.1.1, 9.4.9, P239Archer, C. 9.2.1Arno, S. P49, P50, P302

Arnold, M. P38Årøen, A. 3.1.1Ascani, C. P324Ast, M. P173, P206Aszodi, A. 9.2.9, 17.4.3Ateshian, G. A. P142Athanasiou, K. 2.3.1Au, A. P174Augé, II, W. K. P154Aurich, M. P235Awad, T. P178Azevedo, H. S. P195Azofra, J. P20

B

Babyn, P. S. P303Bach, Jr, B. R. P2Bader, A. P336Badke, A. P83Badlani, N. M. P135Bajaj, S. 12.2.7, 17.1.7, P103Balakrishnan, S. 9.2.8Balboni, F. 25.4.4Ball, S. T. 25.1.2, P1Ballis, R. P145, P146Ballyns, F. P105, P176Bar-Zvi, S. P228Bara, J. P161Barbero, A. 19.3.3, P30, P186Bardana, D. 25.4.6Barela, J. A. P294Barkay, H. P233Barnouin, L. 17.3.5, 17.3.8, 25.4.3Barr, L. 9.2.6Barrachina, J. P15, P202Barreto, R. B. P6Barry, F. 19.3.1, P128, P208,

P210Bartels, W. P160Bartl, C. 9.3.7Bartl, R. 9.3.7Basad, E. 25.1.4Bassett, E. 12.3.2Bassit, A. F. P6Bastiaansen-Jenniskens, Y. M. P127Bateman, J. F. 11.2.1Battacharya, M. P37Battaglia, M. 25.2.6, P266Bauer, J. S. P149Baum, P. 12.4.3Bascı, O. P115Beaufils, P. P282Beaulieu, A. 17.4.5Becher, C. 10.2.2Beckmann, J. P244Bédouet, L. P27Beekhuizen, M. P127Behrens, P. 17.1.3Bekkers, J. E. 12.2.8, 17.3.7, P70,

P84, P151Bell, A. 9.4.4, P58, P60Bellemans, J. 12.2.4, 12.4.2, P282Benders, K. E. 25.3.5, P122, P151Benetti, D. P9Benetti, V. P9Benink, R. P92, P94, P104Bentley, G. 12.2.6, P227, P238,

P241Berbig, R. 17.3.6Berger, M. 12.1.3Berneel, E. P152Berninger, M. 9.3.2, 12.1.5

Bernsen, M. R. P199Berruto, M. P256Bessette, L. 17.4.5Bhullar, T. P65Biant, L. C. 12.2.6Bichara, D. A. 12.3.2, P75, P105,

P176Bielecki, T. 25.4.7Bijlsma, J. W. 9.3.6Bird, J. 8.1.2, 17.1.4Bittencourt, C. 25.4.5Bittencourt, P. 25.4.5Bizzi, E. P9, P265Bizzini, M. 1.0.1Blagus, R. 9.1.3Blanke, M. P214Blasiak, A. 25.4.7Boatwright, P. P55Bobic, V. 22.0.1Bodugoz-Senturk, H. P176Bonasia, D. 12.3.5, P64Bonassar, L. 2.3.2, P105, P176Bonneau, M. P27Borrás-Cuesta, F. P106Bos, P. P199Boux, E. P288Bovée, J. P144Braatz, F. P193Bradica, G. 25.1.6Brantsing, C. 9.2.7, P21Brauckhoff, A. P194Brehm, W. P11, P191Brenner, O. 12.2.9Briggs, T. P241Brinchmann, J. E. P204Brinkerhuff, H. P34Brittberg, M. 3.1.2, 9.2.7, 10.4.1,

17.2.2, 18.4.1, P21, P163

Brix, M. P254Brochhausen, C. 16.2.1Brommer, H. P307Brooks, R. P116Brune, T. 17.3.5, 17.3.8, 25.4.3Bruzzone, M. 12.3.5, P64Brzoska, R. 25.4.7Buda, R. 9.1.5, 9.3.4, 17.2.8,

25.2.6, 25.2.9, P66, P216, P218, P266

Bugbee, W. 9.1.2, 9.1.6, 25.1.2, P1, P217

Bulgheroni, P. 17.3.6Burnett, B. P131Bursac, P. P60, P311Bursig, H. P257, P258, P326Buschmann, M. D. 9.4.3, 12.4.7, 12.4.8,

15.0.1, 17.1.2, 25.3.8, 25.4.1

Buscio, T. P266Bussiere, C. 12.4.9Byers - Kraus, V. 8.2.2Böttenberg, B. P229Bünger, C. 9.2.2, 9.4.8, P168,

P170Bürgi, M. P153

C

Carriel, V. P93, P107Caballero-Santos, R. 17.3.3, P255, P283Caborn, D. 9.2.6Cadet, C. P291Cadossi, M. 11.1.1Cairó, J. J. P15

269

Authors‘ Index270

Calabrese, G. 17.4.7Caminal, M. P15Campbell, J. P103Campos, A. P93, P107Cancedda, R. P288Carey-Smith, R. P231Carli, A. P54, P285Carnelli, D. 9.4.6Carrillo, J. M. P120Carrington, R. P227, P238, P241Carter, T. R. P311Casino, D. P43Casper, M. E. P165Castelein, R. P160Castiglione, E. 25.1.6Castoldi, F. 12.3.5, P64Castro, B. P212Caterson, B. P161Catt, C. J. 9.2.3Cavallo, C. 17.2.8, P66Cavallo, M. 9.1.5, 17.2.8, 25.2.6,

25.2.9, P66, P216, P218

Chabane, N. P78, P79, P82Chahine, N. P206Chaimsky, G. P233Chan, E. F. 25.3.4Chan, J. P314Chang, C. H. P187, P189Changoor, A. 12.4.8Chase, D. P1Chaudhary, M. P49Chavez, D. P253Chen, A. C. 25.1.2, 25.3.4, P1Chen, C. C. P187Chen, E. P206Chen, G. 12.4.7Chen, H. 25.3.8, P285Chen, S. P101Chen, W. P62, P138Cheney, M. 12.3.2Cheverud, J. 11.2.3Chevrier, A. 9.4.3, 25.3.8Chiang, H. 17.2.7, P86, P101Chiari, C. 25.3.1, P13, P254Choi, B. P16, P156Choi, V. P201Choi, W. P156Christensen, B. B. P168, P170Chu, C. 24.2.1, P121, P150Chubinskaya, S. 25.1.9, P103Citak, M. P143Claassen, B. 17.3.7Claes, T. P240Clanton, T. O. P133Clifford, A. G. P295Codina, D. P15, P308Cokelaere, S. M. P307Cole, B. J. 12.2.7, 17.1.7, 17.2.5,

25.1.9, P46, P103, P316

Coleman, C. P128, P208, P210Collett, A. 25.3.7Collin, G. P328Collo, G. P64Colombet, P. P282Concaro, C. P21Concaro, S. 9.2.7, 10.4.3, P61Concheiro, A. P169Condello, V. 2.1.1Cornelissen, M. P152Correlo, V. M. P37Cortes, S. P253

Costa-Pinto, A. P37Coste, P. P291Cottino, U. 12.3.5Couceiro, J. 19.2.1, P169Coutts, R. D. P135Cranchi, C. P143Crane, T. P328Crawford, D. C. P236Crawford, R. 9.2.5Creemers, L. 12.2.8, 17.3.7, P70,

P84, P127, P151, P160Crespo, R. 17.3.6Cronin, M. P310Cugat, R. P120, P282Cui, X. P275Curtin, C. P208Cáceres Palou, E. P3, P264

D

D’Arcy, S. P210D’Lima, D. D. P126D’Orazio, L. 17.1.5, 19.0.2, 25.1.8,

25.4.4Da Fonseca, C. F. P15Dalemans, W. 8.3.1Daley, E. 12.2.7, 17.1.7Dalmau, A. P308Daniels, D. P38Danielson, B. 25.2.7Davidson, P. A. 18.3.2, P325Davies, H. P231Davies, J. P139De Biase, C. F. 12.3.8De Boer, T. N. 9.3.6DeCroos, J. A. P147DeMuro, C. P316De Neve, F. P274De Palma, C. P145, P146DeYoung, A. 9.1.2, 9.1.6, P217Dec, J. P326Declercq, H. P152Dediu, V. P43Deforce, D. 12.1.2Dehne, T. P21Deie, M. P23, P74, P276, P299Dekker, E. 12.1.6Delattre, O. P26Delcogliano, A. 12.3.8Delcogliano, M. 9.3.4, 12.3.8Della Villa, S. 1.0.1Delling, U. P191Deplaine, H. P177Deponti, D. P145, P146DesJardins, J. P55Desando, G. P66, P218Desnoyers, J. 12.4.7, 12.4.8, 17.1.2,

25.4.1Devlin, S. 25.4.6Devreese, K. P76Dexheimer, V. P193de Girolamo, L. P337de Grauw, J. C. 25.3.5del Olmo, M. P212Dhert, W. 9.2.3, 12.2.8, 16.2.3,

17.3.7, 25.1.3, 25.3.5, P70, P84, P122, P127, P151, P160

Dhillon, M. 10.1.3, 17.1.4, P328Dhollander, A. A. P76, P243, P274Di Benedetto, P. P143Di Caprio, F. 25.2.9Di Giancamillo, A. P145, P146

Di Martino, A. 9.3.4, 17.1.5, 19.0.2, 25.1.8, 25.4.4, P256, P305, P327

Di Matteo, B. 19.0.2, 25.1.8, P305, P327

DiMicco, M. P63Diaz-Romero, J. P144Dickhut, A. P196Dickinson, S. C. P72, P180, P192Diederichs, S. P197Dienstknecht, T. P198Djian, P. P282Dijkstra, P. P100Dionigi, C. P43Dirhold, B. P96Dishkin-Paset, J. P46Doblaré, M. P48, P177Docheva, D. P69Domayer, S. 12.3-9, 12.4.4, 25.2.4,

P220, P254Domeneghini, C. P145, P146Dono, D. P77Dorais, M. 17.4.5Doria, A. S. P303Dorotka, R. 12.3-9, 25.2.4, P13,

P14, P220, P254Dos Santos, G. B. P6Dotor, J. P106, P119Dougados, M. P291Doukas, S. 9.2.8Dragomir, L. 25.3.8Drobnic, M. 9.1.3, 19.1.1, P249Drosse, I. 9.2.9Dubrana, F. 12.4.9Dubruel, P. P152Dueren, F. 17.2.9Dugas, J. P311Duguay, S. J. P63, P77Duncan, S. P296Dvorak, J. 1.0.1, 5.0.2

E

Ebert, J. 12.2.2, P225, P231El Attar, M. P281El Mansouri, F. P78, P79, P82Elewaut, D. 12.1.2, P76Eliaz, N. P41Elisseeff, J. 9.4.7, 12.3.1, 16.2.2,

P95Eliyahu, E. P88Ellermeijer, B. P92, P94Ellä, V. P25Elsner, J. 2.1.1, 9.4.9, 12.2.9,

P41Emons, J. 12.1.6Endres, M. P56, P175Engelhart, L. 17.2.5, P316Erggelet, C. 3.1.3, 15.0.2, P317Ergün, S. P115Erquicia, J. P3, P264Escribano, R. P119Eshed, I. P239Esparza, R. 12.1.9Espírito Santo, V. P166Eyre, D. R. 8.2.1

F

Facchini, A. P66Fahmi, H. P78, P79, P82Fallon, M. 12.2.2, P225, P231Faltus, R. 12.4.6, P257Fang, H. W. P187, P189

Authors‘ Index 271

Faria, S. P37Farr, J. 9.1.7, 9.1.8, 16.1.1,

17.2.5, P311, P316Farrell, E. P199Fedorovich, N. E. 25.1.3Fehr, M. P165Feijen, J. P100Feist, M. P232Feller, J. 17.2.3Ferkel, R. P221Fernandes Dias, M. 25.4.5Fernandez-Jaen, T. 17.3.3, P255, P283Fernández, A. G. P212Filardo, G. 9.3.4, 17.1.5, 19.0.2,

25.1.8, 25.1.9, 25.4.4, P103, P256, P266, P305, P327

Filova, E. 12.3.4Fischer, J. P194, P196Fisher, J. P44, P52, P139Fitzsimmons, J. S. P165Fiz, N. P20Flanigan, D. C. P311Flannery, C. R. 3.2.1Flory, C. 25.3.7Foldager, C. 9.2.2, 9.4.8, P168,

P170Fontana, A. 24.1.1, P267Foo, L. F. 12.3.7Forman, R. P49Forriol Campos, F. 12.1.9, 19.2.4Forsten, M. P25Forster, M. P310Forsyth, R. 25.4.9Fortier, L. A. 11.2.2, 18.3.1, 23.0.1,

25.1.6Fraga, A. F. P164Francin, P. 17.4.4Franco, G. P253Frankewycz, B. P69Fraschini, G. P145, P146Freire, M. F. 12.3.3Freymann, U. P175Frias, A. M. P195Friederich, N. F. P38Friedman, A. P233Friedrich, K. P331Friel, N. A. P103Frisbie, D. D. P183Fritz, J. P250Fujie, H. 9.1.9, 24.3.3, P333Fujimura, S. P137Fujioka, H. P172Fujita, N. 9.3.8, P89Fukuhara, K. P286Furu, M. P137

G

Garrido, J. P107Garzón, I. P93, P107Gahunia, H. K. P303Gaissmaier, C. P250Gallagher, K. P241Gallego Ferrer, G. P177Ganey, T. P19Ganguly, K. P154Garcia-Campillo, H. P5Garcia-Gomez, F. 17.3.3, P255, P283Garcia, Z. P5, P28García-Aznar, J. P48, P177García Arnás, F. P202García, F. P15

García, J. P15, P202García Sancho, J. P334Garett, S. P231Garnica, M. I. P28Gartzia, I. P212Gatenholm, P. P163Gavenis, K. P45Gaździk, T. S. P326Ge, Y. P88Geddes, R. G. P318Geißler, C. P191Gelber, J. P126Gelber, P. E. P219Gellissen, J. 17.1.3Gelse, K. P214Gentili, C. P288Georgi, N. P81Georgoulis, A. P315Gerner, C. 12.1.3Gersoff, W. 20.2.1Getgood, A. 9.2.6, 25.1.1, P65,

P116Ghermandi, R. 9.1.5, 17.2.8Ghodadra, N. P2Ghorayeb, S. R. P178Giacomi, R. 12.3.8Giannini, S. 9.1.5, 9.3.4, 17.2.8,

25.2.6, 25.2.9, P66, P216, P218, P266

Giannoni, P. P102Gibson, M. P95Gigante, A. 18.1.3Gill, T. J. P75, P105Gille, J. 17.1.3Gillet, P. P26Giza, E. 18.1.2Glaser, C. P232Glassner, P. P49Goad, E. 25.3.7Gobbi, A. W. 16.1.2, 18.4.2Goed, S. 12.3-9, P330, P331Goldberg, R. P228Goldring, M. 9.3.1Goldstein, M. J. 9.3.5, P206Golub, L. 9.3.5Gomes, M. E. P166Gomez-Garcia, R. P5Gomez, R. P335Gong, J. P. 9.4.5, P7, P31, P35,

P38, P113Gonzalez-Lucena, G. P219Gonzalez, G. P5Goodrich, L. P201Goodwin, P. M. P154Goranov, V. P43Gorensek, M. P249Gosselin, Y. P285Gottardi, R. 9.4.6Gotterbarm, T. P182Goyal, A. D. 25.4.8Goyal, D. 12.4.1, 25.4.8Grad, S. 9.4.2Gramani-Say, K. P294, P320Granata, M. P265Grande, D. 9.3.5, P173, P178,

P206Greenwald, R. 9.3.5Griffin, D. R. 24.1.2Grifka, J. 9.2.8, P244Grigolo, B. 9.1.5, 17.2.8, 25.2.9,

P66, P216, P218Grishko, V. 17.4.2, P109Grodzinsky, A. 16.3.1

Groeneboer, S. P76Grouin, J. P291Gruber, M. P13, P14Grumet, R. P103Guadilla, J. P20Guehring, H. P116Guevara, V. P253Guglietta, A. P212Guilak, F. 2.1.1, 9.4.9, 12.2.9,

P51Guillaume, C. 17.4.4Guillen-Vicente, I. 17.3.3, 20.1.1, P255,

P283Guillen-Vicente, M. 17.3.3, 20.1.1, 20.1.3,

P255, P283Guillen, P. 17.3.3, 20.1.1, P255,

P283Gusinde, J. P214Guzman-Morales, J. P54, P57Gòdia, F. P15Gómez-Ribelles, J. L. P177Görtz, S. 9.1.2, 9.1.6, 17.3.1,

19.1.2, 25.1.2, P1, P217

H

Ha, C. 9.3.9, 25.4.2, P289Hadlock, T. 12.3.2Haeupl, T. P56Hagmann, S. P182Hakimiyan, A. A. 25.1.9, P103Halbwirth, F. 12.1.3, 15.0.3, P159Haleem, A. M. P150Hambly, K. 12.2.1Hangody, L. 10.3.1Hanifi, A. P268Hanypsiak, B. T. 17.4.7Harris, A. P54Harrison, P. P245Hart, D. A. 9.1.9Hashimoto, S. 11.2.3Haskell, M. 25.3.7Haspl, M. P72Hattori, K. P322Haudek, V. 12.1.3Haupt, O. P30Hayakawa, K. P137Hayes, D. A. P295Hazewinkel, H. A. P132He, X. P88Healey, R. P1, P135Hedrick, M. P19Heerwaarden, R. V. P160Helder, M. 17.3.9, P53Henderson, J. P54Hennig, F. 12.4.4, P214Henson, F. M. 9.2.6, P65, P116Hepp, P. P336Herlofsen, S. R. P204Hermida, E. 17.2.6Hermida, L. F. 17.2.6Herrenbruck, T. 17.4.7Herrero, A. P212Hershman, E. 2.1.1, 12.2.9Himeda, Y. P184Hinterwimmer, S. P149Hirakura, Y. P12Hiraoka, N. P215Hirschfeld, C. P220Hirschmüller, A. P317Hoch, J. M. 12.1.8, 12.2.3, 12.2.5,

12.4.5

Authors‘ Index272

Hoechsmann, N. 17.2.9Hoemann, C. D. 9.4.1, 9.4.3, 9.4.4,

12.4.7, 16.1.3, 17.1.2, 25.3.8, P54, P57, P60, P285

Hoeppner, J. P112Hofmann, G. O. P235Hogendoorn, P. P144Hollander, A. P. 5.0.1, 10.4.4, 17.3.7,

P180, P192Holsten, D. G. 17.3.6Hong, J. P329Hong, S. P275Honjo, K. P215Honold, S. P39Horibe, S. 25.2.8Horng, A. P232Hornung, D. P330Hoshi, K. 7.2, P80Hosiner, S. 17.1.6Hou, S. P248Howard, J. S. 12.1.8, 12.2.3, 12.2.5,

12.4.5Hoyle, M. P55Hsieh, C. P86, P101Hsu, H. P111, P171Hsu, Y. P189Hu, J. 2.3.1Huang, C. C. P148Huang, Y. 17.2.7Hudetz, D. P72Hui, J. H. 7.1, 25.3.9, P190Huisman, A. M. 9.3.6Hung, C. T. P142Hunziker, E. 9.1.1, 9.2.9, P184Hurtig, M. B. 9.4.4, 24.3.1, 25.3.3,

25.3.8, 25.4.6, P58, P60, P129, P285

Hussam, A. 18.5.2Hutmacher, D. 9.2.5, 25.3.5, P122Hutton, W. P19Huysse, W. C. 25.4.9, P279Hwang, J. H. P71Hwang, N. 12.3.2

I

Ibarra, C. P5, P28, P253, P335Ibarra, L. P253Igarashi, T. P134, P140Imabuchi, R. P113Imai, S. P12, P17, P59, P67,

P68, P97, P98, P114Imhoff, A. 9.3.2, 9.3.7, 12.1.5,

P149, P234Inagaki, Y. P322Ingham, E. P44, P139Inoue, H. P108, P215Inoue, J. P129Intema, F. P132Inui, A. P172, P185Iosifidis, M. I. 17.4.6, P271, P297,

P298Ishaque, B. 25.1.4Ishida, K. 9.3.8, P89Ishii, T. 11.3.1Ishimoto, Y. P322Isoya, E. P98Ito, A. P108Ito, K. P137Itoman, M. 25.3.6, P10Ivkovic, A. P72Iwamoto, Y. P209Iwasaki, N. P134, P140

Izaguirre, A. P5, P253Izaguirre, F. P5Izal Azcárate, I. P91, P106, P119

J

Jacobi, M. 17.3.2, 17.4.8, P301Jacques, P. P76Jaiswal, P. P241Jakob, M. P186Jakob, R. 17.3.2, 17.4.8, P301Janes, G. P231Jansen, N. W. 9.3.3Jansson, V. P69, P232Jarry, C. 12.4.7Jelic, M. 19.1.3, P72Jenei-Lanzl, Z. P198Jeng, L. P111Jia, H. P180Jiang, C. 17.2.7, P86, P101Jin, L. P275, P293Jin, R. P100Jin, Y. P137Jin, Z. P44, P52, P139Jo, S. P226, P309Joh, K. P171John, D. P50Johnson, L. L. P126Johnson, W. E. P161Johnstone, B. 19.3.2Joshy, S. P310Josten, C. P336Junge, A. 1.0.1Juras, V. 25.2.4Jurvelin, J. 25.3.2, P262Järvinen, E. P25Jülke, H. P191

K

Kafienah, W. P180, P192Kakavas, G. P259Kaleva, E. P262Kalwitz, G. P56Kamei, G. P74, P179Kanaji, T. P137Kanamoto, T. 9.1.9, P158Kanaya, F. P31, P35Kanazawa, S. P80Kandel, R. 16.3.2, P147Kaneko, H. P172Kannan, K. 25.3.9Kaplan, L. D. P148Kaps, C. P56, P165, P175Karahan, M. P115Karperien, M. 12.1.6, P81, P100Kasahara, T. P137Kasahara, Y. P134, P140Kautzky-Willer, A. 12.4.4Kawaguchi, Y. M. P320Kawamura, D. P134, P140Kawcak, C. P183Kellomäki, M. P25Kendoff, D. P143Kennedy, J. G. P287Kepski, F. P326Kercher, J. 9.1.7, 12.2.7, 17.1.7,

P46Kerloch, I. P291Kerr, A. P318Khelmenstkaya, E. P302Kik, M. 25.3.5Kili, S. P65

Kim, B. P16Kim, H. P275Kim, J. P284Kim, M. P29Kim, S. P226Kim, Y. 24.1.3, P29, P156,

P293, P314Kinne, R. W. P133, P188Kirk, S. 25.1.9Kirkpatrick, C. J. 16.2.1Kisiday, J. P183, P201Kita, K. 3.2.3, 24.3.3Kitamura, H. P12Kitamura, N. 9.4.5, P7, P31, P35,

P113Kiviranta, I. 19.0.1, 25.3.2, P25,

P262Klabunde, R. P153Klein, T. 9.2.5, 25.3.5, P122Kleinschmidt, K. P11Knott, L. P124Knutsen, G. 3.3.1Kobayashi-Miura, M. P108Kobayashi, M. 25.1.7, P108, P306Kobayashi, T. P74Kobayashi, K. P137Kokubu, T. P172, P185Kocaoğlu, B. P115Koch, C. P24Koczy, B. P257Koh, Y. P226, P277, P280,

P309Kohda, H. P158Kohn, L. P149Kon, E. 9.3.4, 10.4.2, 17.1.5,

18.2.5, 18.4.3, 19.0.2, 25.1.8, 25.4.4, P256, P305, P327

Kondo, E. P7Konen, E. P239Kordelle, J. P130Koritnik, B. P249Koroglu, A. 25.1.5Kotek, G. P199Koulalis, D. P143Kramer, J. P165Krestin, G. P. P199Kreuz, P. P317Kristiansen, A. P168, P170Kröger, H. P262Kubo, M. P12, P59, P68, P230Kubo, S. 9.3.8, P89Kubo, T. P135, P215Kujat, R. P198Kulig, K. M. 12.3.2Kumagai, K. P98Kumar, K. P245Kunz, M. 25.4.6, P129Kuo, L. P50Kuroda, R. 9.3.8, P89Kuroda, S. P158Kurokawa, T. 9.4.5, P7, P31, P35,

P38, P113Kuroki, H. P108Kurosaka, M. 9.3.8, P89, P172,

P185Kurowska, E. P242, P313Kurz, A. 25.1.9Kusano, T. 17.4.8Kwack, K. P269, P275, P293,

P329Kwak, S. K. P221Kwiatkowski, G. 12.4.6Kwon, H. P113

Authors‘ Index 273

Kyriakidis, A. 17.4.6, P271, P297, P298

Kyriakidis, T. 17.4.6Küchler, A. M. P204

L

Lobo, M. P93La Cava, G. P260Labarre, D. P27Ladenburger, A. P45Lafantaisie Favreau, C. P54, P57Lafeber, F. 9.3.3, 9.3.6, 11.3.2,

P8, P132Lagae, K. 17.3.6Laganier, L. 25.4.3Lambrecht, S. 12.1.2, P76Lammi, M. 25.3.2Landa, C. P335Landini, E. 9.4.6Landwehr, S. P149Langer, R. 12.3.2Laprell, H. 18.2.4, P282Lascau-Coman, V. 9.4.4, 12.4.7, 25.3.8Lattermann, C. 12.1.8, 12.2.3, 12.2.5,

12.4.5, P296Laurent, A. P27Le, D. P168, P170Leander, M. P21Lechler, P. 9.2.8Lecona, H. P5Lee, D. 11.1.2Lee, E. H. 25.3.9, P71Lee, H. H. P150Lee, J. P71, P71Lee, S. P312Lee, W. P86Lee, Y. P269, P275, P284Lehman, R. C. 25.1.6, P325Lehmann, S. P232Leijten, J. 12.1.6, P81, P100Lequesne, M. P291Lessi, G. C. P294, P320Levy, R. P131Lewis, P. B. 12.2.7Lewis, S. P316Li, K. 17.4.9, P4Liakos, T. 17.4.6, P271, P297,

P298Liao, C. 17.2.7, P187, P189Lichtenberg, D. P11Lickorish, D. 25.3.7Lin, S. P62, P138Lin, Z. P90Lind, M. 9.2.2, 9.4.8, 17.2.3,

P168, P170Lindahl, A. 9.2.7, 17.2.2, 25.2.7,

P21, P61, P163Linder-Ganz, E. 2.1.1, 9.4.9, 12.2.9,

P41, P51Linss, S. P188Litau, L. P175Little, C. B. 24.3.2Liu, I. 25.3.4Lizhang, J. P52Ljungberg, M. 25.2.7Lloyd, D. G. 12.2.2Lohmander, S. L. 8.2.3London, N. J. P295Loosli, Y. P39Loparic, M. P72Lopez-Alcorocho, J. 17.3.3, 19.2.3, P255,

P283Lopez-Olivia, F. 12.4.7, 12.4.8, 17.1.2,

25.4.1Lopez, E. P26Lowe, J. P233Lowerison, M. 9.4.4, P58, P60Ludewig, E. P191Luginbuehl, R. P39Lukasik, P. P258Luksch, T. 12.3-9Luna-Barcenas, G. P5, P28Luyten, F. 12.2.4, 12.4.2Lynn, A. 10.3.1, P116Lytvynets, A. 12.3.4Lyu, S. P261

M

Martínez, C. P107Morales, A. P93MacDonald, P. 12.4.7, 12.4.8, 17.1.2,

25.4.1MacMull, S. 12.2.6Macule, F. 12.4.7, 12.4.8, 17.1.2,

25.4.1Madry, H. 18.4.4, 22.0.3Mae, T. 9.1.9, P158, P278Maher, S. A. P171Maheu, E. P291Maiello, A. P64Mainard, D. 17.4.4Mainil-Varlet, P. 17.3.2, P144Makino, K. P172Makino, T. P185Malda, J. 9.2.3, 9.2.4, 16.2.3,

25.1.3, 25.3.5, P122, P151, P307

Malinowska, P. P326Malo, M. 12.4.7, 12.4.8, 17.1.2,

25.4.1Mamisch, T. C. 12.3-9, 12.4.4, 25.2.2,

25.2.3, 25.2.4, P314, P330

Mandel, A. P201Mandelbaum, B. 1.0.2, 9.3.4Mangiavini, L. P145Manili, M. 12.3.8Mano, J. F. P166Mansouri, R. P323Maor, G. P228Maqsood, M. P323Marcacci, M. 9.3.4, 17.1.5, 17.3.6,

19.0.2, 25.1.8, 25.4.4, P43, P256, P305, P327

Marchand, C. 12.4.7Mardones, R. M. 17.2.1Marks, P. 12.4.7, 12.4.8, 17.1.2,

25.3.3, 25.4.1, P58Marlovits, S. 12.4.4, 17.1.6, 18.1.1,

25.2.1, 25.2.2, 25.2.3, 25.2.5

Marmotti, A. 12.3.5, P64Marotta, D. 12.3.8Marquass, B. P336Marques, R. F. P164Martel-Pelletier, J. 17.4.5, P78, P79, P82Marti, C. 17.4.8Martin, C. 17.3.5, 17.3.8Martin, I. 19.3.3, P30, P186Martin, S. P265Martinez-Lopez, V. P28Martinez, I. 12.1.4Martinez, V. P253Martinez de Albornoz, P. 12.1.9Martinova, L. 12.3.4

Martins, A. P37Martins, C. P150Martinčič, D. P249Marty, M. P291Maruyama, T. P137Mascaro, G. P288Masri, M. P335Massafra, U. P9, P265Mastbergen, S. 9.3.6, P8, P132Maticic, D. P72Matsuda, S. P209Matsumoto, T. 9.3.8, P89Matsuno, T. P87Matsushita, T. 9.3.8, P89Matsusue, Y. P12, P17, P59, P67,

P68, P97, P98, P114, P230

Matsuura, H. P98Mattacola, C. G. 12.1.8, 12.2.3, 12.2.5,

12.4.5Matthews, G. P63Mattielo-Rosa, S. M. P294, P320Matuska, A. P112Mauerer, A. 25.2.2, 25.2.3Mauthe von Degerfeld, M. 12.3.5May, D. P311Mayer, S. 17.2.9Mazières, B. P291McArthur, S. P46McCall, I. W. P245McCarrel, T. M. 25.1.6McCarthy, H. S. P245, P251McCormack, R. 12.4.7, 12.4.8, 17.1.2,

25.4.1McDonough, M. P55McGill, K. P2McIlwraith, W. 12.1.1, P183, P201McKee, M. D. 25.3.8McKeon, B. 25.2.7McLeod, L. D. 17.2.5McRury, I. D. P154Mcclelland, J. 17.2.3Mcnamara, I. P65Medeiros, A. I. P294Mehlhorn, A. T. P96Meininger, A. 25.1.9Meisel, H. P19Melas, I. 17.4.6, P271, P297,

P298Melato, M. P146Melcher, C. 17.2.9Memisoglu, K. 25.1.5Messner, A. 17.2.4, P331Metaxiotis, D. P298Methot, S. 12.4.7, 12.4.8Mezape, Y. P41, P51Midura, R. 12.3.3Migliore, A. P9, P265Mika, J. P133, P317Mimnaugh, B. P34Mimura, T. P12, P59, P68Min, B. P16, P29, P156, P269,

P275, P284, P293, P329Minami, A. P134, P140Minas, T. 17.1.1, 20.2.2Minger, S. P180Minh, D. P16Miquel, J. P3, P264Mithoefer, K. 1.0.3, P316Mitsui, H. P137Mohd Hassan, A. 25.3.9Mohtadi, N. 12.4.7, 12.4.8, 17.1.2,

25.4.1

Authors‘ Index274

Moine, L. P27Moll, X. P15Mollenhauer, J. P235Monllau, J. C. 17.3.6, P3, P219,

P264Monreal, I. P106Montaperto, C. 25.4.4Montes, M. P212Monti, C. 25.2.6Mora, G. P91, P106, P119Moradi, B. P182Morawietz, L. P175Mordin, M. M. 17.2.5, P316Moreira Teixeira, L. P100Morera, C. P334Morgan, R. E. P154Mori, K. P97Morin, F. 17.4.5Morist, A. P15Moseley, B. 3.3.2Motlík, J. 12.3.4Moya-Angeler, J. 12.1.9Moyse, D. P291Mroczka, A. 12.4.6Muezzinoglu, B. 25.1.5Muezzinoglu, S. U. 25.1.5Muhonen, V. P25Mukai, S. 25.1.7Mullender, M. 17.3.9, P53Munir, S. 9.4.8Murakibhavi, V. P245Murata, M. P140Muratoglu, O. K. P176Murawski, C. D. P287Murphy, M. P128, P210Muruzabal, F. P20Méthot, S. 17.1.2Míčková, A. 12.3.4Möller, I. P292Müller-Rath, R. P45Müller, P. E. 17.2.9, P69, P232Müller, S. P193, P317

N

Nagata, Y. P179Nagura, I. P172, P185Nakagawa, S. P215Nakagawa, Y. 25.1.7, P108, P230,

P306Nakamae, A. P299Nakamura, N. 3.2.3, 9.1.9, 24.3.3,

P158, P333Nakamura, S. P306Nakamura, T. 25.1.7, P108, P137,

P306Nakasa, T. P23, P286, P299Nakata, K. 3.2.3, 9.1.9, 24.3.3,

P158, P278Nakayama, K. P209Nanni, M. P216Nannmark, U. P163Nansai, R. 9.1.9, P333Narcisi, R. P102Naruse, K. 25.3.6, P10Nawab, A. P311Nawaz, S. P241Nebelung, S. P45Negrin, L. 25.2.4Nehrer, S. 12.1.3, 12.3-9, 15.0.3,

P13, P14, P159, P254Nejadnik, H. P190Nelea, M. 12.4.8

Nelson, L. 17.2.5Neophytou, D. 17.4.6, P271, P297,

P298Neri, S. 9.1.5, P218Nesic, D. 17.3.2, P144Netter, P. P26Neumann, K. P56, P175Neves, N. M. P37, P195Neyret, P. P282Ng, K. W. P171Nguyen, V. P27Nicolàs, M. P212Niculescu-Morzsa, E. 12.1.3, P159Nielsen, A. 9.4.8Niemeyer, P. P96Nierenberg, G. P228, P239Niethammer, T. P232Niimoto, T. P23, P299Nishimoto, H. P185Nishitani, K. P108Nishizawa, K. P17, P59, P68, P97Nishizawa, S. P80Nishuzawa, K. P12Niu, C. P62, P138Nochi, H. P87Noda, K. P12Noehren, B. P296Notbohm, H. P24Nuran, R. P115Nygaard, J. V. P168, P170Nöth, U. 17.1.8

O

O’Driscoll, S. P165O’Loughlin, P. F. P143O’Sullivan, J. P128Ochi, M. 11.3.3, P23, P74,

P179, P276, P286, P299

Ochoa, I. P48, P91, P177Ode, G. E. P46Ogawa, M. 9.4.5, P322Oh, K. P284Ohgushi, H. P322Ohkawa, S. P74, P179Ohmiya, Y. P113Okuhara, A. P23, P276, P299Okumura, N. P98Olk, A. P214Onodera, S. 9.4.5, P7Orive, G. P20Orozco, L. P202, P334Oshima, Y. P135Ota, K. P158Otsuka, T. P137Ouyang, W. 25.3.8

P

Paape, D. P175Paatela, T. P262Padera, R. F. P111Paessler, H. 12.4.3, P282Pagenstert, I. P232Pallante, A. L. 25.1.2, P1Pallu, S. 17.4.4Panseri, S. P43Papantoniou, E. P298Park, D. P241Park, S. P16, P29, P156Parker, J. C. P245, P251Parker, R. 17.4.7

Pascale, F. P27Pascher, A. P72Patella, S. 9.3.4, 17.1.5, 19.0.2,

25.1.8, 25.4.4, P256, P305, P327

Paternostro-Sluga, T. P330, P331Patsch, I. 24.2.2Paul, C. 17.3.9, P53Paul, S. P165Payne, K. A. P121, P150Pearle, A. P143Pearsall, IV, A. W. 17.4.2, P109Pecina, M. P72Pedersen, M. 9.2.2Pelet, S. 12.4.7, 12.4.8, 17.1.2,

25.4.1Pelfort, X. P3, P219, P264Pelletier, J. 17.4.5, P78, P79, P82Pelsis, J. R. 9.1.4Pereira, R. C. P195Peretti, G. M. P145, P146Peris, D. P15Peterson, L. 17.2.2, 25.2.7, P61Petrigliano, F. A. 12.3.7Peyrone, B. 12.3.5Pezzillo, F. 12.3.8Pflueger, V. P331Pietschmann, M. F. P69, P232Pillai, L. P131Pineda, A. P335Pineda, C. P5, P253Pinto, A. R. P195Pittsley, A. P126Pla, A. P15Please, C. P. 9.2.3Pleshko, N. P268Plummer, O. 18.5.1Polacek, M. 12.1.4Polak, A. A. 9.3.6Polzella, M. P9Pomerantseva, I. 12.3.2Portnoy, S. P51Potel, J. 12.4.9Potter, H. G. 12.3.7Pottie, P. 17.4.4Pozzi, A. P145, P146Prades, M. 12.1.7, P222, P223,

P224Prado, R. P20Prendergast, P. 11.1.3Presle, N. 17.4.4Pretzel, D. P188Prins, H. 25.1.3Pronk-Admiraal, C. P92, P94, P104Prosecká, E. 12.3.4Provencher, M. T. P2Puhakka, J. P262Pulkkinen, H. J. 25.3.2Pérez, F. P253

Q

Qi, W. P125Quarto, R. P102

R

Rabanal, R. P15Radosavljevič, D. P249Raducanu, A. 9.2.9Rai, M. 11.2.3Raiteri, R. 9.4.6Rampichová, M. 12.3.4

Authors‘ Index 275

Randolph, M. A. 12.3.2, P75, P105, P176

Rapko, S. P77Rappold, G. 12.1.6Rappoport, L. 25.1.9Raynauld, J. 17.4.5Razzano, P. 9.3.5, P173, P178,

P206Realmuto, C. 12.3.5, P64Reddi, H. A. 2.2.1Redondo, J. I. P120Regatte, R. P49, P302Reiff, R. P294Reinholz, G. G. P165Reis, R. L. P37, P166, P195Ren, L. P136Renger, J. P24Renkawitz, T. 9.2.8Responte, D. 2.3.1Restrepo, A. 12.4.7, 12.4.8, 17.1.2,

25.4.1Rey-Rico, A. P169Rezende, M. U. P6Ribera, T. P222, P224Ribitsch, I. P191Richardson, J. P245, P251, P268,

P318Richardson, R. P318Richmond, B. 17.4.7Richter, R. P336Richter, W. 7.3, 10.2.1, P11, P182,

P193, P194, P196, P197

Rickert, M. P196Ridgway, K. P180Riek, J. K. P22Riesle, J. P186Rijen, M. P70Ringe, J. P21Ringgard, S. 9.2.2Ripalda Cemboráin, P. P91, P106, P119Ristiluoma, M. 17.4.3Rivard, G. P57Robert, H. 12.4.9Roberts, S. P161, P245, P251,

P268Robertson, A. P310Robertson, W. B. 12.2.2, P225Robinson, E. P245, P251Rochler, S. P188Rodeo, S. 12.3.7Rodkey, W. G. P183Rodrigo, J. P55Rodriguez-Iñigo, E. 17.3.3, P255, P283Roessing, S. 12.4.3Roessler, N. P14Rohwedel, J. P165Rolauffs, B. P83, P235Romeo, A. A. P2Romeo, S. P144Ronken, S. P38Roosendaal, G. 9.3.3Rossi, P. 12.3.5, P64Rossi, R. 12.3.5, P64Rossomacha, E. 12.4.7, 17.1.2Rothdiener, M. P83Rothrock, C. 17.4.7Rousseau, M. P26Royen van, B. 17.3.9, P53Rudan, J. 25.4.6Ruffilli, A. 9.1.5, 17.2.8, 25.2.6,

25.2.9, P216, P218Ruike, T. P87

Rushton, N. 9.2.6, P116Russlies, M. P165Russo, A. P43Ruta, D. 25.1.9Rutgers, M. P160

S

Saarakkala, S. 25.3.2, P262Sachot, S. P88Sadigursky, D. P6Sadlik, B. 25.4.7Sague Doimeadios, J. L. P39Sah, R. L. 2.3.3, 19.0.3, 25.1.2,

25.3.4, P1Sahlman, J. P262Saito, M. P215Sakamoto, F. A. 12.3.3Sakata, R. P172, P185Salanti, G. P315Salata, M. 9.1.7, 17.1.7Salzmann, G. M. 9.3.2, 9.3.7, 9.4.2,

12.1.5, P149Samulski, J. P201Samuoh, L. P13Sanchez Hidalgo, R. 12.1.9Sanchez, M. 19.2.2, P20Sandell, L. J. 11.2.3Sandhu, M. P263Sandri, M. P43Santander, S. P91Santoro, R. 19.3.3Santos, E. 17.3.3, P255, P283Santos, J. L. P164Sanz-Ramos, P. P91, P119Saris, D. B. 3.3.3, 10.1.1, 10.3.2,

12.2.4, 12.2.8, 12.4.2, 17.3.7, 20.3.2, 23.0.2, 25.3.5, P70, P84, P122, P127, P151, P160

Sasaki, H. 9.3.8, P89Saska, R. 25.1.6Satake, M. P172Satake, T. P108, P306Sauerschnig, M. 9.3.2, 12.1.5, P149Schaeferhoff, P. P229Schaeren, S. P186Schagemann, J. C. P165Schaumburger, J. 9.2.8, P244Schelfhout, J. P152Scheurecker, G. 17.2.4, P331Schewe, B. P250Schieker, M. P69Schinhan, M. P13, P14Schirlin, A. 17.3.8Schmal, H. P96Schmitz, P. P69Schneider, E. 12.3.3Schoenhuber, H. P337Schreyer, T. 12.4.3, 17.1.9Schrier, D. 25.3.7Schrobback, K. 9.2.5Schuchman, E. H. P88Schulz, R. M. P336Schuseil, E. 17.1.3Schuurman, W. 9.2.3, 16.2.3, 25.1.3,

P151Schwenke, T. P153Schönfelder, M. 9.3.2, 12.1.5Schöttle, P. B. 9.3.2, 12.1.5Sciarretta, F. P324Scinski, T. P313Scopp, J. M. 8.1.1

Semler, E. 25.3.4Sen, R. P263Sengers, B. 9.2.3Seo, H. P312Serra, C. P120Serrão, P. R. P294, P320Servien, E. 12.4.9Sgaglione, N. A. 2.1.2Shah, N. V. 9.3.5, P206Shahid, R. P323Shanbhogue, K. P263Shani, J. 12.2.9Sharon, M. P239Shasha, N. P51Shaw, D. P139Shelyakova, T. P43Shen, C. 17.2.7Shewman, E. P46Shibuya, H. P23, P74, P179, P286,

P299Shieh, C. 17.2.7Shikano, M. 8.3.2Shimizu, T. 25.2.8Shimomura, K. 9.1.9, 24.3.3, P158Shimoto, T. P209Shino, K. 9.1.9, 24.3.3, P278Shintani, N. P184Shioji, S. P59, P97Shiozaki, Y. 25.2.8Shirai, T. P108Shive, M. S. 12.4.7, 12.4.8, 17.1.2,

25.3.8, 25.4.1Shterling, A. 2.1.1, 9.4.9, 12.2.9,

P41, P51Siclari, A. P288Sievers, B. 17.2.9Silva, M. P169Simon, T. 25.3.4Simonaro, C. M. P88Sims, T. J. P180Simson, J. A. 9.4.7Singh, P. P263Singleton, S. B. P55Siren, E. P25Sirola, J. P262Sittinger, M. P56Sivananthan, S. P318Skarpas, G. P237, P259Skinner, J. P238, P241Slynarski, K. P. 18.2.2, P242, P313Smit, T. 17.3.9, P53Smith, R. 16.3.3Smith, T. D. P54Sobral, J. P100Sodhi, K. P263Sol, P. P37Solecki, A. 25.4.7Soler, C. P120Soler Rich, R. P202, P334Solis-Arrieta, L. P5Solsky, I. 12.3.7Somerson, J. S. P336Somma, E. P260Son, Y. P71Song, B. P29Sopena, J. J. P120Soto-Cerrato, V. P212Spalding, T. 8.1.2, 10.1.2, 17.1.4,

18.2.3, P328Spector, M. P111, P126Spector, T. P291Spindler, K. 17.4.7Spruijt, S. 20.2.3

Authors‘ Index276

Vaquero, J. 12.4.7, 12.4.8, 17.1.2, 19.2.5, 25.4.1

Vasanji, A. 12.3.3Vasilceac, F. A. P294Vasiliadis, H. S. 17.2.2, 25.2.7, P315Vasishta, V. G. P270Vavken, P. P13Vecsei, V. 17.1.6Veen van der, A. 17.3.9, P53Vega, J. P308Velasquillo, C. P5, P28, P253, P335Velázquez, J. P334Vena, P. 9.4.6Verbruggen, G. 12.1.2, P76, P243,

P274Verdonk, P. C. 2.1.3, 12.1.2, 17.3.4,

18.2.1, 25.4.9, P167, P243, P274, P279, P281, P282

Verdonk, R. 12.4.2, 17.3.4, 18.2.1, 25.4.9, P167, P243, P274, P279, P281, P282

Verghese, N. P310Verhaar, J. A. P199Verioti, C. P126Verma, N. N. P2Verra, W. P84Vicente-Pascual, M. P106Victor, J. 12.4.2Vignon, E. P291Vijayan, S. 12.2.6, P227, P238Viladot, R. P308Villalobos Cordova, F. P28Villalobos Jr, E. P253Villegas, H. P5Vincken, K. L. P160Viren, T. 25.3.2, P262Vives, J. P15, P202Vlychou, M. 17.2.4Vogt, J. P39Vogt, S. 9.3.2, 18.4.5, P234Volck, J. P250Volpi, P. P337Vonwil, D. P30

W

Wakitani, S. 25.2.8Waldman, S. 25.4.6, P129Walgenbach, A. 9.1.4Walker, P. P49, P50, P302Waller, C. S. P295Wallstabe, S. 17.1.3Wang, N. 9.4.2Wang, Q. P83Wang, S. P190Wang, V. M. P46Warnke, P. H. P318Warren, R. F. 12.3.7, P171Wasiak, J. P315Wassef, M. P27Watzer, B. 16.2.1Wawrzynek, W. P257, P258Webster, K. 17.2.3Wei, L. 17.4.9Wei, X. 17.4.9, P4, P73, P125,

P136Weinans, H. P199Weinerman, S. P311Wellings, R. P328Welsch, G. H. 12.3-9, 12.4.4, 17.2.4,

25.2.2, 25.2.3, 25.2.4, 25.2.5, P330,

Thomas, B. P34, P153Thompson, P. 17.1.4, P328Tichy, B. 17.1.6Tiitu, V. 25.3.2Timoncini, A. 9.3.4, P266Toguchida, J. P137Tonnarelli, B. 19.3.3Tormenta, S. P265Torzilli, P. A. P171Toyama, M. 25.3.6Toyoda, F. P98Toyokawa, N. P172Tran-Khanh, N. 12.4.7, 12.4.8, 25.3.8Trattnig, S. 12.3-9, 12.4.4, 17.2.4,

24.2.3, 25.2.2, 25.2.3, 25.2.4, 25.2.5, P239, P330, P331

Triantafyllopoulos, A. P237, P259Trice, M. P95Truncale, K. 25.3.4Trüssel, A. P30Tsai-Wu, J. P86, P101Tsiridis, E. P44Tsuchida, A. I. P84Tsuchida, S. P215Tsuchimoto, K. P108Tsukuda, N. P134Tsukuda, Y. P140Tuan, R. 2.2.2Turek, T. 9.1.4Tómas, H. M. P164Töyräs, J. 25.3.2, P262

U

Uchida, K. 25.3.6, P10Uddin, S. 17.2.5Ueba, H. P17, P68Uematsu, K. P322Uenaka, K. P67, P97Ueng, S. P138Uhlík, J. 12.3.4Ulrich-Vinther, M. 9.4.8Urabe, K. 25.3.6, P10Urraza, J. P332, P338Usvald, D. 12.3.4

V

Vidal, P. P332, P338Vacanti, J. 12.3.2Vacca, F. P265Vajner, L. 12.3.4Valanos, N. P297Valderrabano, V. 10.2.3Valonen, P. 25.3.2Van Assche, D. 12.2.4Van Blitterswijk, C. 12.1.6, P81, P100Van Caspel, D. 12.2.4Van Osch, G. J. 2.2.3, P127, P199Van Thiel, G. S. P46van Buul, G. M. P199van Meegeren, M. E. 9.3.3van Roermund, P. M. P132van Weeren, P. 16.2.3, 25.1.3, 25.3.5,

P307van der Lee, J. P21van der Maas, J. 17.3.4, P167Vandercruyssen, B. P76Vanlauwe, J. 12.2.4, 12.4.2, P240Vannini, F. 9.1.5, 17.2.8, 25.2.6,

25.2.9, P66, P216, P218

Staes, F. 12.2.4Stanish, W. D. 12.4.7, 12.4.8, 17.1.2,

25.4.1, P54, P285Stark, N. P153Steadman, J. P183Steck, E. P11, P194Steck, R. P122Steinmeyer, J. 3.2.2, P130Steinwachs, M. 17.4.1Stelzeneder, D. 12.3-9, 12.4.4, 17.2.4,

25.2.4, P220, P254, P330, P331

Stenhamre, H. 9.2.7, P61, P163Stephen, J. M. P321Sterner, H. P21Stewart, J. 25.4.6, P129Sticht, C. 12.1.6Stindel, E. 12.4.9Stoddart, M. 9.4.2Stoffel, M. P45Stolz, M. 9.2.9Stone, K. R. 9.1.4Straub, R. H. 9.2.8Strauss, E. 12.2.7Strehin, I. 9.4.7Strem, B. P19Stroebel, S. 9.2.4Stuerz, H. P130Stull, P. P311Stürz, H. 25.1.4Su, H. 12.3.7Suedkamp, N. P. P96Sugihara, H. P137Sun, J. 9.4.3, 9.4.4, 25.3.8,

P54, P57, P285Sundback, C. A. 12.3.2Suri, M. P55Sutton, C. A. P192Szczęśniak, M. P258Szomolanyi, P. 25.2.4Sánchez, A. P334Søballe, K. 9.4.8Südkamp, N. P. 9.4.2Sütfels, T. 9.3.2, 12.1.5

T

Tabet, S. K. 12.3.6Tafazal, S. P323Taffetani, M. 9.4.6Takahashi, K. P215Takakura, Y. 9.4.5Takato, T. P80Takayama, K. 9.3.8, P89Take, Y. P158Takemura, Y. P59, P97, P114Tampere, T. 17.3.4, P167Tampieri, A. P43Tan, A. R. P142Tan, N. 25.4.3Tanaka, Y. 9.4.5, 25.2.8, P322Tang, Z. P248Tarella, C. P64Tarlton, J. P124Tateishi, K. 3.2.3, 9.1.9Tavares, H. P164Taylor, S. D. P44Terauchi, R. P215Tey, M. P219Tey Pons, M. P3, P264Thakkar, V. 25.4.8Theodoropoulos, J. P147Thermann, H. 12.4.3

Authors‘ Index 277

Zaslav, K. R. 8.3.3, 20.3.1Zayed, N. P78, P79, P82Zbyn, S. 25.2.4Zehbe, R. 16.2.1Zhang, Z. P248Zhang, X. P209Zhao, X. 12.3.2, P75, P105,

P176Zheng, M. H. 12.2.2, P90, P225Zheng, Q. P90Zhong, X. P248Zhou, L. 12.3.2Zidrou, C. P297, P298Zivkovic, S. 11.2.1Zorzi, C. 2.1.1Zscharnack, M. P336Zuiderbaan, H. 17.3.9, P53Zur, G. 2.1.1, 9.4.9, 12.2.9,

P41, P51Zwickl, H. 12.1.3, P159

P331Wendlandt, R. P24Wendt, D. 19.3.3Werner, S. 12.4.4White, L. M. P58Whitehead, T. 17.2.3Whiteside, R. 25.3.3, P58Wickiewicz, T. L. 12.3.7Wicks, J. P. P63Widuchowski, J. 12.4.6, P257, P258Widuchowski, W. 12.4.6, P257, P258Wiech, O. P244Wielopolski, P. A. P199Wiewiorski, M. 8.1.3Wijnberg, H. M. 25.1.3Wildi, L. 17.4.5Wilensky, D. P148Williams, III, R. J. 12.3.7, P236Williams, S. P44, P52Wilshaw, S. P139Wilson, G. L. 17.4.2, P109Wilson, R. 11.2.1Wimmer, J. 17.1.3Winalski, C. S. 12.3.3Windhager, R. P72Wirz, D. P38Wit, J. 12.1.6Wittwer, J. 17.2.3Wong, C. P148Wood, D. J. 12.2.2, P225, P231Wood, S. 25.3.7Woodell-May, J. P112Woodfield, T. B. 9.2.4, 9.2.5Wu, L. P81Wyland, D. P55Wysocka, A. P257, P326Wörtler, K. P149

X

Xiafei, R. 25.3.9Xiao, X. P150Xu, J. P90

Y

Yoo, J. P75Yang, C. P62, P138Yang, Z. P73, P248Yao, J. 9.1.7, 9.1.8Yao, V. P121Yasuda, K. 9.4.5, P7, P31, P35,

P113Yayon, A. P233, P239Yildirim, G. P302Yokota, M. P7Yonetani, Y. 25.2.8Yoon, J. P29Yoon, S. P269, P275, P329Yoshikawa, H. 3.2.3, 9.1.9, 24.3.3,

P158, P278Yuan, L. P62, P138

Z

Zabalza-Baranguá, A. P91, P106, P119Zaffagnini, S. 17.1.5, 17.3.6, P305,

P327Zajac, P. P186Zak, L. 12.4.4, 17.1.6, 25.2.2,

25.2.3, 25.2.5Zandieh Doulabi, B. 17.3.9, P53Zapero, I. 12.1.9