comparison of chemically and pharmaceutically modified … · dental implants overall, 6 types of...

YIJOM-1445; No of Pages 8

Research PaperDental Implants

Int. J. Oral Maxillofac. Surg. 2008; xxx: xxx–xxxdoi:10.1016/j.ijom.2008.09.008, available online at http://www.sciencedirect.com

Comparison of chemically andpharmaceutically modifiedtitanium and zirconia implantsurfaces in dentistry: a study insheepJ. D. Langhoff, K. Voelter, D. Scharnweber, M. Schnabelrauch, F. Schlottig, T. Hefti,K. Kalchofner, K. Nuss, B. von Rechenberg: Comparison of chemically andpharmaceutically modified titanium and zirconia implant surfaces in dentistry: a studyin sheep. Int. J. Oral Maxillofac. Surg. 2008; xxx: xxx–xxx. # 2008 InternationalAssociation of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rightsreserved.

Abstract. Advanced surface modifications and materials were tested on the sameimplant geometry. Six types of dental implants were tested for osseointegrationafter 2, 4 and 8 weeks in a sheep pelvis model. Four titanium implant types weretreated with newly developed surface modifications, of which two were chemicallyand two were pharmacologically modified. One implant was made of zirconia. Asandblasted and acid-etched titanium surface was used as reference. The chemicallymodified implants were plasma-anodized or coated with calcium phosphate. Thepharmacological coatings contained either bisphosphonate or collagen type I withchondroitin sulphate. The implants were evaluated using macroscopic, radiographicand histomorphometric methods.

All implants were well osseointegrated at the time of death. All titanium implantshad similar bone implant contact (BIC) at 2 weeks (57–61%); only zirconia wasbetter (77%). The main BIC increase was between 2 and 4 weeks. Thepharmacologically coated implants (78–79%) and the calcium phosphate coating(83%) showed similar results compared with the reference implant (80%) at 8weeks. There were no significant differences in BIC. Compared with previousstudies the results of all implants were comparatively good.

Please cite this article in press as: Langhoff JD, et al., Comparison of chemically and pharm

implant surfaces in dentistry: a study in sheep, Int J Oral Maxillofac Surg (2008), doi:10

0901-5027/000001+08 $30.00/0 # 2008 International Association of Oral and Maxillofacial Surgeon

J. D. Langhoff1,*, K. Voelter1,*,D. Scharnweber2,M. Schnabelrauch3, F. Schlottig4,T. Hefti4, K. Kalchofner5, K. Nuss1,B. von Rechenberg1

1Musculoskeletal Research Unit, EquineHospital, Vetsuisse Faculty ZH, University ofZurich, Winterthurerstr. 260, CH-8057 Zurich,Switzerland; 2Max Bergmann Center forBiomaterials, Institute of Materials Science,Dresden University of Technology,Budapester Str. 27, 01069 Dresden,Germany; 3Biomaterials Department,INNOVENT e. V., Pruessingstrasse 27B, D-07745 Jena, Germany; 4Thommen Medical,Hauptstrasse 26d, CH 4437 Waldenburg,Switzerland; 5Veterinary Anesthesiology,Equine Hospital, Vetsuisse Faculty ZH,University of Zurich, Winterthurerstr. 260, CH-8057 Zurich, Switzerland

* The first two authors contributed equallyto this work.

Over the last decades, titanium or its alloyshas become a gold standard as a base fortooth reconstruction in dental implantol-ogy, because of its mechanical strength,chemical stability and excellent biocom-

patibility22. These properties ensure goodanchorage within the mandible or maxil-lary bone. The aim is to achieve shorterhealing periods for implants, in order toload them as soon as possible after sur-

Keywords: dental implants; zirconia; titanium;surface modification; histomorphometry;sheep.

Accepted for publication 3 September 2008

gery, while allowing efficient osseointe-gration. Apart from good implant design

aceutically modified titanium and zirconia

.1016/j.ijom.2008.09.008

s. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ijom.2008.09.008


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Table 1. Implant groups, their abbreviations and sample size.

Implant type Abbreviation Weeks Sample size

Chemical Sandblasted and acid etched* Ref 2 94 68 6

Calcium Phosphate CaP 2 64 68 6

Plasma Anodized APC 2 64 68 6

Pharmacological Collagen I + Chondroitin Sulfate Coll+ 2 64 68 6

Bisphosphonate BisP 2 64 68 6

Chemical Zirconia Zr 2 64 68 6

* SPI ELEMENT, Thommen Medical.

related to mechanical anchorage, theserequirements may be met by modifyingthe implant surface, bearing in mind thatthe most important surface properties for(metallic) implants are topography, chem-istry, surface charge and wettability5.

To improve surface properties twomain approaches were used either opti-mizing the micro-roughness (e.g. sand-blasting and acid-etching) or applyingbioactive coatings (e.g. calcium phos-phate, bisphosphonate, collagen). Opti-mizing the micro-roughness results inenlarged surfaces providing improvedconditions for osteogenic cell attachmentand proliferation. In recent studies, histo-mophometric and biomechanical compar-isons of such optimized implant surfacesto machined implants showed bettervalues for short time osseointegration6.These surfaces were also optimized fortheir wettability for potentially enhancedimplant–tissue interaction and betterosseointegration, achieved by rinsingunder an N2 atmosphere and submersionin an isotonic NaCl solution followingacid-etching7. The new generation of thincalcium phosphate based coatings pro-vide high wettability and were describedas highly potential24.

Another approach is to add bioactivecomponents to titanium surfaces. Onemethod uses extracellular matrix ligands,the RGD-peptide sequence, for betterosteoblast attachment and enhanced boneremodelling18. SCHULER et al.19 used afunctionalized coating (poly(L-lysine)-graft-poly(ethylene glycol)) to presentbioligands for interaction with osteoblastsin vitro. Faster colonization of the implantsurface by osteoblasts also inhibits bacter-ial growth.

A new method uses nucleic acid, singlestrands, fixed electrochemically via theirtermini by anodically growing an oxidelayer on Ti6Al7Nb as anchor structures toload surfaces with bioactive moleculeslinked to complementary strands12.

The bioactivity of surfaces can beenhanced using drug eluting coatings,which are supposed to influence bonehealing, for example by activating osteo-blasts, suppressing osteoclasts or stimulat-ing the production and distribution ofgrowth factors such as BMP-2.

An increase in the mechanical fixationof implants has been achieved with localdelivery of bisphosphonates15. Other stu-dies showed the high potential of growthfactors such as BMP-28.

Zirconia has gained attention as animplant material because of its white col-our, which makes it aesthetically attrac-tive14. Apical bone loss and gingival

Please cite this article in press as: Langhoff J

implant surfaces in dentistry: a study in she

degeneration associated with implantsoften uncover parts of the metal implantshowing a bluish discoloration of the over-lying gingiva. The use of zirconia implantsavoids this complication and accedes tothe request of many patients for metal-freeimplants. The material also provides highstrength, fracture toughness and biocom-patibility16. Osseointegration is approxi-mately the same as with titanium9.

The authors hypothesize that chemicaland pharmacological surface modifica-tions to titanium initiate a stronger boneresponse than an advanced sandblastedand acid-etched surface alone. They testedwhether a surface-treated zirconia cancompete with sophisticated titanium sur-faces. The bone response to the implantmodifications was tested on the identicalestablished implant geometry using histo-morphometry.

Material and methods

Dental implants

Overall, 6 types of implants with identicalimplant geometry were tested (Table 1).All titanium and zirconia implants weresandblasted and partially etched prior tothe surface treatments, similar to the refer-ence. The surfaces of the chemically mod-ified implants were either plasma anodizedor coated with calcium phosphate. Thepharmacologically modified implantswere either coated with bisphosphonateor collagen type I. An acid-etched andsandblasted implant made of titanium(grade 4, SPI1ELEMENT, ThommenMedical AG, Waldenburg, Switzerland)served as the reference and control forthe surface modifications.

D, et al., Comparison of chemically and pharm

ep, Int J Oral Maxillofac Surg (2008), doi:10

Surface details

The calcium phosphate surface was coatedusing electrochemical assistance in anaqueous solution containing calcium andphosphate ions. The coating consists of thetwo calcium phosphate phases, hydroxya-patite and brushite, and is commerciallyavailable. The anodic plasma chemicalsurface modification method is anadvanced anodization method, whichallows anodic oxide layer formation andincorporation of calcium phosphatephases in a single process step. Themethod exploits the dielectric breakdownof anodic oxide films to produce a porousoxide layer that contains significantamounts of electrolyte components. Theelectrolyte contained calcium and phos-phate ions, leading to a porous surfacecontaining calcium phosphate.

The collagen coating was based on anextracellular matrix containing chondroi-tin sulphate, prepared by fibrillogenesis ofthe collagen in the presence of CS, andperformed as dip coating in a collagen/chondroitin sulphate solution. The bispho-sphonate coated implants were immobi-lized with an alendronate solution, to afinal concentration of 10 mg/cm2.

The zirconia implants were manufac-tured from yttrium partially stabilized zir-conia, medical grade. The zirconiaimplants were sandblasted and etched inan alkaline bath.

Animal model and study design

A total of 15 sheep underwent surgery. Allsheep were full-grown, aged 2–3 years,not gestating females and 49–87 kg (aver-age 68 kg). General guidelines for care


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Comparison of chemically and pharmaceutically modified titanium and zirconia implant surfaces in dentistry 3


Fig. 1. Implant locations in the iliac bone of a sheep in a dorso-ventral view.

and use of animals in research have beenfollowed and all experiments wereapproved by the local veterinary authori-ties (approval no.159/2005). The sheepwere kept in groups containing a maxi-mum of 4 animals. Their general conditionwas checked three times a day to accom-plish pain monitoring, to detect variationsin wellbeing and injuries of the musculos-keletal system. All implants (totaln = 110) were placed in the iliac bonesof the pelvis. Bone structure was predo-minantly of cancellous quality in the cra-nial part with increasing cortical thickness(up to 3 mm) toward the caudal part. Animplantation scheme was worked out todistribute all implant types homogenouslyto 7 implantation sites per iliac bone(Fig. 1). The study design aimed toachieve the statistical minimum of 6 sam-ples per implant group (one implant typewas not evaluated for the present study)for each healing period of 2, 4 and 8weeks, with 5 animals per time point.Additional implants were placed for aconcurrent biomechanical analysis (thetopic of a separate study). Animals werekilled in the University’s slaughterhouseaccording to ethical standards.

Animal surgery

After sedation with medetomidine (5 mg/kg, DomitorTM, Orion Pharma AnimalHealth, Finland) anaesthesia was inducedusing ketamine (2 mg/kg, Narketan1 10,Chassot GmbH, Germany) in combinationwith diazepam (0.01 mg/kg, Valium1,Roche, Switzerland). After intubationanaesthesia was maintained with 0.8Vol% isoflurane (Forene1, Abbot AG,Switzerland) in O2 and an infusion ofRinger’s solution with 60 mg/l ketamine(NarketanTM 10, Chassot GmbH, Ger-many) at a rate of 10 ml/kg/h. As a pro-phylaxis against infection all animalsreceived 30,000 IU/kg penicillin (HoechstAG, Germany) and 6 mg/kg gentamicin(Streuli & Co AG, Switzerland) intrave-



nously thre times a day. In addition, theyreceived 500 Units of equine tetanusserum as a single subcutaneous applica-tion (Tetanus Serum Veterinaria AG, Zur-ich, Switzerland).

The animals were placed in lateralrecumbency and access to the pelviswas achieved using a standard operationprocedure. A 20 cm long cut was made inthe skin in the longitudinal direction of theiliac bone at the mid-pelvis line. Thefascia was cut and the middle glutealmuscle and tensor fasciae latae were sepa-rated by blunt dissection. In the distal halfof the iliac bone the tendinous insertion ofthe deep and middle gluteal muscles wassevered from the iliac crest with a scalpeland the muscles were bluntly removedfrom the iliac bone shaft. A Finocchiettoretractor was used to expose the entireiliac wing. Holes were drilled using theSPI1VECTOdrillTM-System (ThommenMedical AG, Waldenburg, Switzerland)with a 2.0 mm pilot drill, widened witha 2.8 mm and finally with a 3.5 mm drill.A drill sleeve was used to ensure thedesignated drill depth according to theimplant design and the depth was con-firmed with a depth gauge (ThommenMedical AG, Waldenburg, Switzerland).The self-tapping implants (SPI1ELE-MENT, Thommen Medical AG, Walden-burg, Switzerland) were placed accordingto the implantation scheme and using thespecific instruments supplied with theimplant system. Healing caps were placedto prevent tissue ingrowth in the abutmentconnection area of the implant head.Implant setting was documented with digi-tal photographs. The muscles were reposi-tioned and the tendinous insertionresutured to its origin using a cross patternof single (at the edges) and continuoussutures. Fascia and subcutis were closedwith the same synthetic resorbable suture(Polyglactin; Vicryl1 2-0, Johnson&-Johnson Intl.) while the skin was closedwith staples (Davis + Geck ApposeULC1). Gauze was applied as protection



for the wound before the animal wasturned over to the other side. The contral-ateral pelvis was operated on in an iden-tical manner.

Postoperative treatment consisted of anantiphlogistic and analgesic, as well asantibiotic medication for 4 days (bupre-norphin 0.01 mg/kg i.m. t.i.d. during thefirst 24 h, benzylpenicillin (30000 I.U./kgi.v. b.i.d.), gentamycin (4 mg/kg i.v. s.i.d.)and carprofen (4 mg/kg i.v. s.i.d.).

Fluorochrome labelling

Bone healing and remodelling was fol-lowed by labelling new bone appositionwith fluorochrome dyes17 at defined pointsof time. The first labelling with calceingreen (10 mg/kg s.c.) was performed 2weeks after implantation. In the 8 week-group, a second label was injected at 6weeks using xylenol orange (90 mg/kgs.c.).

Preparation and evaluation of bonesamples

The bones were harvested after killing theanimals. They were freed of all soft tissue,revealing the implants in the iliac bone.The firm seat of the implants within thebone was tested qualitatively by manualpressure and the caps were removed.Thereafter, the intact pelvis bone wasradiographed using a faxitron machine(Cabinet X-ray-faxitron series, model43855A, Hewlett Packard1, USA) fordocumentation of implant placement andverification of proper seat. Then the bonewas cut into 1.5 � 1.5 cm cubes with aband saw (K 410, Kolbe GmbH, Elchin-gen, Germany), containing one implant.Samples were fixed in 40% alcoholic solu-tion for 14 days and were routinely pro-cessed for non-decalcified bonehistology11. They were submitted to adehydration process in an ascending seriesof ethanol solutions (50, 70, 96, 100%),before degreasing in xylene undervacuum. Samples were infiltrated inpMMA solution (poly methacrylic acid-methylester; dibuthylphtalate and perka-dox in a proportion 89.5: 10: 0.5) for 7days, embedded and polymerized inTeflon containers. Samples were posi-tioned to assure that implants were cutparallel to the longitudinal axis. Twoground sections were cut at the maximumdiameter of the implant using a low speeddiamond saw (Leica1 SP 1600, Leica1

Instruments GmbH, Nussloch, Germany).One section of 200 mm was used for nor-mal bone histology, applying a surfacestaining with toluidine blue. The thinner,


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4 Langhoff et al.


Fig. 2. Illustration of the thread wise evaluation of the bone implant contact (BIC). Estimationof the percentage was supported with a 10% step grid.

Fig. 3. Calcification of new bone formation (arrows) at the implant was proved by matchingareas of radiodense (microradiograph, on the left) and stained structures (toluidine blue dye, onthe right) in histological thick sections. Overview picture (5.8�) of a bisphosphonate-coatedtitanium implant at 4 weeks.

native section (150 mm) was used forfluorescence microscopy (Leica, DMR,UV light source and Filter I3 for calceingreen and xylenol orange, Glattbrugg,Switzerland). Before the 200 mm sectionswere glued to the opal, acrylic Plexiglasslides (Wachendorf, Perspex GS, Acrylic-glas Opal 1013) microradiographs, using ahigh-resolution analogue film (KodakOncology Film, Eastman Kodak Com-pany, Rochester, NY), were taken tovisualize the stage of calcification of thebone samples adjacent to the metallicimplants.

Evaluation

Using the toluidine-stained thick sections,a semi-quantitative evaluation of the boneimplant contact (BIC) was made. For this,the percentage of direct contact betweenmineralized bone and the titanium surfacewas determined by intersection countingwithin the thread area. Six thread pitcheswere counted per sample. The evaluationwas performed at calibrated digital pic-tures at 10x magnification (Leica macro-scope M420, Leica DFC320, 3088x2550pixels, Leica Microsystems, Germany).Two pictures covered the full threadedpart in high resolution. The percentageof BIC was estimated in steps of 10%(Fig. 2). Means of thread counts perimplant were calculated.

Statistical analysis

In a first step, factors as individual differ-ence and position of the implant could beexcluded as not significant. In a secondstep, comparison of implant types at eachpoint of time was performed. The analysisof variance (ANOVA) was used to iden-



tify significant differences, mean valuesand standard deviations using a specificsoft ware (SPSS 13.0 for Macintosh). Sig-nificance level was set at p < 0.05.

Results

Surgery and postoperative period

All surgery was uneventful and the ani-mals recovered from anaesthesia quickly.The sheep were able to walk immediatelyafter recovery, but showed signs of mildmuscle soreness for 1–2 days after sur-gery. Thereafter, no signs of lameness orother discomfort were seen. Insertion ofall implants proceeded smoothly, but dur-



ing implantation it was noticed, that thezirconia implants required slightly moreforce for insertion compared with the tita-nium implants.

Macroscopic and radiological evaluation

After preparation of the muscle above theimplants, the tissue layer directly at thebone–implant surface was gel- and fat-likeafter 2 weeks. A soft tissue layer hadformed after 4 weeks, which developedinto a periosteum-like layer with callusformation later. At 2 and 4 weeks, hae-matoma were rarely visible around theimplants. Overall, no signs of inflamma-tion or infection could be found, indicatedthrough swelling, reddening or otherdegradation of surrounding tissue. Allimplants were firmly seated.

Radiographs demonstrated all implantsto be still in place. No fractures or zones ofbone resorption could be found.

Microradiographic evaluation

Radiographs of the thick sections con-firmed the macroradiographic results ofabsence of bone resorption. Radiodensestructures were visible in detail and couldbe clearly identified as bone (Fig. 3).Radiodense structures and bone tissuestained with toluidine blue matchedexactly. Except for a small seam of osteoidall of the new bone formation was calci-fied at all time points. The microradio-graphs were not evaluated additionallybesides the stained histologies.


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Fig. 4. A matrix of representative histological pictures of all implant types and time points (2, 4 and 8 weeks) at 10�magnification. Surface wereeither sandblasted and acid etched (Ref), anodic plasma treated (APC), calcium phosphate (CaP), bisphosphonate (BisP), collagen withchondroitin sulfate (Coll+) coated or of zirconia (Zr).

Evaluation of histology samples

All sections were cut precisely in themiddle axis, capturing the entire implant,enabling standardized evaluation.

Qualitative microscopic evaluationrevealed that implants were generallywell seated within the bone. New boneformation, visible as dark-bluish stain,was present around all implants in thecancellous bone by 2 weeks and builtup steadily until 8 weeks (Fig. 4). Bonydebris was found in the remaining cavityof the implant tip, where new bone wasfound by 2 weeks.



Remodeling in the cortical bone startedby 4 weeks in some samples and wasprominent at all implant sites at 8 weeks.There were no signs of pathological boneresorption indicative of excessive mechan-ical instability or issues of bioincompat-ibility (accumulation of inflammatorycells) in any implant. Striking differencesbetween the implant types were notobserved in the qualitative evaluation.

Evaluation of BIC

Results of the BIC measurements (Fig. 5)demonstrated clear trends between surface



types and time points. All titanium typeswere nearly similar at 2 weeks (59–62%BIC) and increased with time (78–83%),except the plasma anodized surface (58%).

The two chemical surface modificationsperformed very differently. The calciumphosphate surface showed similar values,with the main increase at 2–4 weeks, likethe reference, and a slight increasetowards week 8. In contrast, the plasmaanodized surface lost 2% bone contactinitially and did not improve after 4 weeks.

Pharmacologically modified surfacesperformed close to the reference. The


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6 Langhoff et al.


Fig. 5. Results of the bone-implant contact (BIC) measurements are given according to the three groups of implant types: Chemical (a) andpharmacological (b) titanium surface modifications and a zirconia implant (c) were evaluated for bone response and referenced by a sandblastedand acid etched implant (SPI1ELEMENT). Significant differences were not found between the groups of 6 samples per implant and time point.

collagen with chondroitin sulphate surfaceshowed slightly higher values than thereference implant at 2 weeks and contin-ued nearly equally, whereas the bispho-sphonate coated surface was higher at 2and 4 weeks.

The zirconia implant presented 20%more bone contact than the titaniumimplants at 2 weeks, improved toward 4weeks, then reduced at 8 weeks to belowthe level of the reference surface.

The overall performance of the newsurfaces, except the plasma anodized,was better than the reference. Statisticallysignificant differences for BIC were notfound.

Evaluation of fluorochrome labeling

At 4 weeks, calcein green fluorescent dyewas exclusively visible in the new bonedirectly at the implant, while in the 8 weeksections xylenol orange was found directlyat the implant surface. In those specimenscalcein green was found at a greater dis-tance from the implant surface. Differ-ences in signals or deposition offluorochrome dyes could not be foundbetween the implant types and, therefore,further histomorphometrical evaluationswere not performed.

Discussion

In this study the osseointegration of mar-ket standard dental implants (titaniumgrade 4, sandblasted, acid etched) wascompared with surface-treated implantsthat were either chemically (plasma ano-dized, calcium phosphate coated) or phar-macologically modified (bisphosphonate,collagen type 1 containing chondroitinsulphate) or to zirconia implants. Anexperimental sheep pelvis model wasused, where all implants showed goodbiocompatibility and osseointegration.Although statistically not significant, therewas a clear tendency for the chemicallyand pharmacologically modified implants



to show better BIC values at 8 weekscompared to the anodic plasma treatedsurface or zirconia implants.

Finding an appropriate animal modelfor testing dental implants is difficult,mainly because the morphology of teethin animals is different from that ofhumans. Pigs and dogs are commonlyused as experimental animals, if dentalimplants are applied intra-orally1. Apartfrom different root systems and the formof the incisor and molar teeth, mouthhygiene is a problem in those animalsafter setting dental implants and, thus,healing without infection may pose a pro-blem.

Osseointegration is often tested in otherlocations, such as the femoral condyle13.Even though the risk of infection may beexcluded, the cancellous bone of thefemoral condyle is more compact andstronger compared to the mandible. Theauthors’ group has developed an animalmodel in the iliac shaft of sheep, where thestructure of the bone is similar to that ofthe human mandible, as described by theLekholm and Zarb index10. Sheep is awell-established animal for orthopedicresearch, because of the similar remodel-ing rate, bone structure and bone propor-tions as humans3. The pelvis model allowsthe implantation of a relatively high num-ber of implants in one sheep; by operatingon both sides intra- and inter-individualcomparisons can be made. The animalmodel serves well from an ethical stand-point considering animal welfare and pro-tection, because surgery does not interferesignificantly with normal ambulation ofthe sheep, housing can be easily providedappropriate to the species and handlingdoes not cause excessive stress.

The study design achieved the statisticalminimum within the limitations of a jus-tifiable use of animals. The implantationscheme reduced the influences of the indi-vidual and the implantation site using arotation system of sample distribution.The iliac bone as implantation site could



be established as a standard with a zerofailure rate of operation and implantation.Standard dental equipment (SPI1 -Sys-tem, Thommen Medical) could be usedwithout limitations for predrilling andimplant placement. In this manner, clin-ical standard procedures and precisioncould be applied. Also sample preparationfor histology did not involve complica-tions or loss of samples. The sample pre-paration proved to be a very effective andreliable method for longitudinal sections.All sections were cut in the centre of theimplants with very little variation, so his-tological evaluation of osseointegrationcould be well standardized. The presentstudy was mainly focused on the morpho-logical aspects of osseointegration, as thehistological picture did not show anyabnormalities on the cellular level. As acommon tool in dental research, BIC wasregarded as an appropriate method to mea-sure the performance of an implant23. Theestimation of bone contact in 10% stepsper screw thread was regarded as ade-quate. Calculation of means for eachimplant was close to the accuracy of aquantitative method, since the fullthreaded part of the implant was evalu-ated.

Although good standardization of sur-gery and sample preparation procedurescould be achieved, differences betweengroups did not reach statistical signifi-cance. The two main reasons for this werethe relatively small sample size and thegood material properties of all the testedimplants. The minimal sample size forstatistical evaluation was chosen consider-ing animal welfare issues and ethical con-cerns related to the use of animals inexperimental research. Since the sand-blasted and acid-etched implants (Thom-men Medical SPI1-System) used as areference show good performance owingto their original titanium properties6, dif-ferences to the modified implants wereexpected to be relatively small. Tenden-cies for improved osseointegration follow-


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ing implant modification were clearlyshown and allow further research andtesting to be focused on the implantsshowing superior performance.

The improved values of the pharmaco-logically modified surfaces may be attrib-uted to the enhanced adhesion ofosteoblasts and the suppression of osteo-clasts, or a combination of both. The earlyattachment of the old bone to the implant,including the inhibition of function ofosteoclasts (bisphosphonates) can hamperresorption and lead to a better anchorage atthe very early stage of bone healing20. Thereduction of micromotion at a very earlystage after implantation is therefore con-sidered responsible for good osseointegra-tion of bisphosphonate-coated implants,since initially mainly bone formationoccurs. Resorption of the old bone matrixmay take place later when the bispho-sphonates are resorbed and the implanthas gained a certain stability25. This dif-ference was not confirmed for BIC in thisstudy.

Collagen containing chondroitin sul-phate surfaces increased cell proliferationand activated osteoblasts in cell cultures asdemonstrated through higher values ofbone markers (osteopontin, alkaline phos-phatase) and larger cell size4. Adhesionmolecules such as vinculin, actin andintegrins were up-regulated in vitro. Inhi-bition of osteoclasts does not occur inparallel. Recruitment and activation ofosteoclasts and subsequent bone resorp-tion at the surface of the bone lesion is notinhibited and takes its normal course. Asbone resorption normally precedes newbone formation and deposition, it mayresult in temporary microinstability atthe bone–implant interface and thus, lessstability of implants in the immediate andearly postoperative phase21.

Cell proliferation, cell size and regula-tion of adhesion molecules were not inves-tigated in the current study, whereosseointegration was assessed using thehistology of non-decalcified bone samplescontaining the implants alone. Although itwould be interesting to understand theexact mechanism of osseointegration ona molecular level, it would not change thepractical and clinical results, where histol-ogy demonstrated a sound performancefor bisphosphonate-coated implants.

Zirconia implants showed goodosseointegration in histology. The addi-tional etching process and the roughnessachieved was good for cell attachment andbone apposition and seemed to make adifference in the early postoperative phaseat 2 and 4 weeks. Later the BIC valueswere lower compared with the chemically



or pharmacologically treated implants andthe reference titanium implants. Whetherthis is due to the surface of the implant isunknown and will be investigated.

Calcium phosphate coated implantsshowed similar BIC rates as the pharma-cologically treated surfaces, with a BICrate of approximately 80% after 8 weeks,comparable with those of other advancedbioactive surfaces2,5.

In conclusion, the hypothesis ofimproved surfaces could not be acceptedeven though there were trends for betterperformance for some surface modifica-tions. All tested implant types demon-strated good biocompatibility andosseointegration, with only small differ-ences compared with the referenceimplant surface.

Further studies will refine the concen-tration of bioactive substances used in thisstudy and explain the reactions on a cel-lular level as well as prove those conceptsin clinical conditions.

Acknowledgements. Implants were pro-vided by Thommen Medical AG, Walden-burg, Switzerland. The excellent work byS. Bierbaum (Biomaterials Department,INNOVENT e. V) for providing the col-lagen containing chondroitin sulfate coat-ing, by V.M.Frauchiger (Dr RobertMathys Foundation, Bischmattstrasse 12,2544 Bettlach, Switzerland) for providingthe plasma anodized coating, by A.Kautz(Biomaterials Department, INNOVENT e.V) for providing the bisphosphonate coat-ing and P.Zeggel (DOT GmbH, Charles-Darwin-Ring 1a, 18059 Rostock, Ger-many) for providing the calcium phos-phate coating is highly appreciated.

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Address:Jens D. LanghoffMusculoskeletal Research UnitEquine HospitalVetsuisse Faculty ZHUniversity of ZurichWinterthurerstr. 2608057 ZurichSwitzerlandTel: +41 76 432 27 38Fax: +41 44 635 8905E-mail: [email protected]


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