3689

Abstract. – OBJECTIVE: The purpose of this study is to evaluate the histocompatibility and feasibility of structure and function of bone mar-row mesenchymal stem cells (BMSCs) and silk fibroin meniscus porous scaffolds in repairing meniscus injury in rabbits.

MATERIALS AND METHODS: BMSCs were cultured and identified in vitro, silk fibroin me-niscus porous scaffolds were prepared and the histocompatibility of porous scaffolds was eval-uated via in vitro culture. Silk fibroin-BMSCs po-rous scaffolds were implanted into the meniscus defect area of New Zealand white rabbits as the experimental group, the blank scaffold group without BMSCs, pure BMSCs group and blank control group were set up with 10 rabbits in each group. Joint capsules were opened for general observation at 6 weeks and 12 weeks after oper-ation. The specimens received the HE staining, alkaline toluidine blue staining, and immunohis-tochemical staining of Type I and Type II colla-gen and S100 protein.

RESULTS: In vitro HE staining and SEM ob-servation showed that the scaffolds showed the sponge-like structure with abundant mi-croporous structure on the surface and inside, which had a good histocompatibility with BM-SCs. At 6 and 12 weeks after operation in ex-perimental group, meniscus-like tissues were found in the meniscus defect area. HE staining showed the fibrous cartilage-like structure and obvious collagenous fiber structure around ar-ranged in an orderly manner. Alkaline toluidine blue staining showed the cartilage-specific gly-cosaminoglycan (GAG); Type I and Type II col-lagen and S100 protein staining were strongly positive. However, the above changes were not seen in other groups.

CONCLUSIONS: BMSCs and silk fibroin me-niscus porous scaffolds have a better histo-

compatibility and feasibility of structure and function in repairing meniscus injury in rab-bits.

Key Words:Bone marrow mesenchymal stem cell (BMSC), Silk fi-

broin meniscus porous scaffold, Histocompatibility.

Introduction

Meniscus is an important crescent-shaped structure of knee joint cavity, which functioning in load transfer, joints lubrication, stress expan-sion, stability and consistency1. However, the-re is about 20-35% occurrence rate of meniscus injury in population with heavy load exercises, and 30-40% in the elderly patients with osteopo-rosis2. As a commonly occurred injury in knee structure, meniscus injury often leads to the se-condary osteoarthritis, osteophyte formation, ar-ticular cartilage degeneration, etc.3,4. A variety of meniscus prostheses were used previously, such as polytetrafluoroethylene, silicon resin, carbon fiber and absorbable polylactic acid. However, the flexibility, creep property and physiological function cannot meet the requirements of human meniscus5,6. It has been found that silk fibroin has a good biocompatibility and mechanical property, which may be an ideal alternative of meniscus tis-sue7,8. This study aimed to evaluate the feasibility of bone marrow mesenchymal stem cells (BM-SCs) and silk fibroin meniscus porous scaffolds in repairing meniscus injury in rabbits.

European Review for Medical and Pharmacological Sciences 2018; 22: 3689-3693

X.-Z. YING2, J.-J. QIAN1, L. PENG3, Q. ZHENG2, B. ZHU2, Y.-H. JIN2

1Department of Ultrasound, The Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang, China2Department of Orthopedics, Integrated Chinese and Western Medicine Hospital of Zhejiang Province, Hangzhou, Zhejiang, China3Traumatology Department, Affiliated Hospital of Hainan Medical University, Haikou, Hainan, China

Xiaozhang Ying and Jingjing Qian contributed equally as first authors

Corresponding Author: Jingjing Qian, MD; e-mail: Yxz99010@163.com

Model research on repairing meniscus injury in rabbits using bone marrow mesenchymal stem cells and silk fibroin meniscus porous scaffold

X.-Z. Ying, J.-J. Qian, L. Peng, Q. Zheng, B. Zhu, Y.-H. Jin

3690

Materials and Methods

MaterialsNew Zealand white rabbits of either gender

aged 3-5 months weighing (2.5±0.6) kg were pur-chased from Shanghai Sangon Animal Co., Ltd. (Shanghai, China). The rabbits were fed normal-ly to adapt to the environment for 1 week. Dried silkworm cocoons were purchased from Animal Breeding Center of Beijing Agricultural Universi-ty (Beijing, China). Heraeus 5060/BB16 automa-tic temperature control CO2 incubator (Hamburg, Germany) was obtained from Shanghai Lichuang Scientific Instrument Co., Ltd. (Shanghai, Chi-na); scanning electron microscope was purchased from Nikon (Tokyo, Japan); FACS Caliber flow cytometer was obtained from BD (Lake Franklin, NJ, USA); liquid DMEM was purchased from Gibco BRL (Waltham, MA, USA); HE staining and alkaline toluidine blue staining reagents were obtained from Jiangsu Beyotime Technology Co., Ltd. (Nanjing, Jiangsu, China); Type I, Type II collagen, S100 protein immunohistochemi-cal staining kits, and supporting reagents, were purchased from Sigma-Aldrich (St. Louis, MO, USA). This research was approved by the Ethi-cal Committee of Zhejiang University School of Medicine.

Experimental Methods

In vitro Culture and Identification of BMSCs About 5.0 mL bone marrow was extracted

from the posterior superior spine of New Zealand rabbits. Bone marrow was separated using Hi-stopaque-1077 density gradient method to obtain mononuclear cells. Then, mononuclear cells were added into DMEM culture solution contai-ning 10% FCS to resuspend into cell density of 2.0×107 /mL. Next, cells were incubated with 5% CO2 with saturated humidity at 37°C. After 72 h, non-adherent cells were abandoned. The re-maining cells continued to be cultured for sub-culture until the fifth generation, and after they were co-cultured with OEC. The expressions of markers in BMSCs, such as SH2, CD90 and CD4, were detected by flow cytometry.

Preparation of Silk Fibroin Meniscus Porous Scaffold

Dried silkworm cocoon with good living con-ditions was soaked in 2% neutral soap solution and repeatedly refined in a water bath kettle at 98°C. Next, the silkworm cocoon was soaked in

0.05% Na2CO3 solution for about 20 min, and repeatedly washed and dehydrated to obtain the refined silk fibroin. The silk fibroin was dissol-ved and filtered in 100 mL CaCl2 and C2H5OH mixed solution, and the filtrate was dialyzed to re-move the small-molecule hydrolyzed silk fibroin. Finally, the silk fibroin solution in concentration of 10% was obtained. Silk fibroin solution and NaCl granules were mixed and placed into the si-licone tube of medial meniscus of knee joint in rabbits to prepare the porous scaffold. The solu-tion was evaporated at room temperature for 48 h, and NaCl granules were dissolved by distilled water. Next, the scaffold was frozen at -80°C for 1 d, and then stored.

Evaluation of Histocompatibility of Porous Scaffolds via in vitro Culture

The third-generation BMSCs were taken and inoculated on a 6-well culture plate. 2 cm×2 cm silk fibroin scaffolds were added into each well. High-glucose DMEM culture solution was added and changed every other day. After 2 weeks, the cells were taken out, followed by HE staining and SEM observation.

Animal Model ResearchThe medial collateral ligament was separated

from the femoral attachment layer by layer via the medial parapatellar approach in New Zealand rabbits. The fully-exposed medial meniscus was completely removed. The silk fibroin-BMSCs po-rous scaffold was implanted into the meniscus de-fect area of New Zealand rabbits. The soft tissues in medial collateral ligament and femoral attach-ment were tightly sutured to close the incision as the experimental group. The blank scaffold group without BMSCs, pure BMSCs group and blank control group (direct incision suture) were set up with 10 rabbits in each group. The rabbits were fed in separate cages after operation and lower limbs were not immobilized. The rats were sa-crificed via excessive anesthesia at the 6 and 12 weeks after operation. Joint capsules were opened for general observation, and the specimens recei-ved the HE staining, alkaline toluidine blue stai-ning, and immunohistochemical staining of Type I and Type II collagen and S100 protein according to the instructions.

Statistical AnalysisStatistical analysis was performed using

SPSS20.0 software (SPSS Inc., Chicago, IL, USA). The data were presented as mean ± stan-

Silk fibroin-BMSCs porous scaffolds and repair of meniscus injury in rabbits

3691

dard deviation. One-way ANOVA was used for comparison among groups. Enumeration data were presented as case or percentage (%), and

c2 test was used for comparison among groups. p<0.05 suggested that the difference was statisti-cally significant.

Results

In vitro Histocompatibility AnalysisIn vitro HE staining and SEM observation

showed that the scaffolds showed the sponge-like structure with abundant microporous structure both on the surface and inside, which had a good histocompatibility with BMSCs (Figure 1).

General Observation of Animal ModelAt 6 weeks and 12 weeks after operation in

experimental group, meniscus-like tissues were found in the meniscus defect area. The above change was not seen in other groups (Figure 2).

HE Staining and Alkaline Toluidine Blue Staining

At 6 weeks and 12 weeks after operation in expe-rimental group, HE staining showed the fibrous car-tilage-like structure and obvious collagenous fiber structure around arranged in an orderly manner. Alkaline toluidine blue staining showed the carti-lage-specific glycosaminoglycan (GAG). The above changes were not seen in other groups (Figure 3).

Immunohistochemical StainingAt 6 weeks and 12 weeks after operation in

experimental group, Type I and Type II collagen

Figure 1. In vitro histocompatibility analysis Left: (HE staining, 100×). It could be seen that more cartilage-like cells adhered and grew in the loose porous scaffold structure, and cell viability was good; Right: (SEM, 500×). It could be seen that scaffolds showed the sponge-like structure with abundant microporous structure on the surface and inside, collagenous fiber bundles crossed forming the three-dimensional network structure, and cells connected each other through neurite or adhered to the pore wall of scaffold via pseudopodium.

Figure 2. General observation of repaired meniscus in experimental group at 12 weeks after operation. The defect area was healed well in experimental group at 6 weeks after operation, and meniscus-like tissues replaced the defect area at 12 weeks without significant difference from the surroun-ding normal meniscus in texture, elasticity and glossiness, and the corresponding femoral and tibial articular surfaces were smooth. Fibroid neoplasm replaced most defect area in blank scaffold group at 12 weeks after operation, defect area became smaller, a little meniscus-like tissue could be seen in the border between defect area and normal meniscus, and the corresponding femoral and tibial articular surfaces were rough. Defect areas still existed in the other two groups wi-thout significant reduction covered by fibroid neoplasm, and the corresponding femoral and tibial articular surfaces were significantly worn.

X.-Z. Ying, J.-J. Qian, L. Peng, Q. Zheng, B. Zhu, Y.-H. Jin

3692

and S100 protein staining were strongly positive. The above changes were not seen in other groups (Figure 4).

Discussion

The porosity, pore diameter and mechanical properties of silk fibroin porous scaffolds were detected in vitro at early stage. This study further investigated its histocompatibility and feasibility of structure and function in rabbit model. In vitro HE staining and SEM observation showed that BMSCs in silk fibroin meniscus porous scaffold could differentiate into cartilage-like cells, parti-cipate in repairing meniscus injury and adhere to the scaffold closely, and the cell viability was good,

indicating that the scaffold is stable with a better histocompatibility9,10. At 6 weeks and 12 weeks after operation in experimental group, general observation, HE staining, alkaline toluidine blue staining and Type I and Type II collagen and S100 protein immunohistochemical staining showed that silk fibroin porous scaffold combined with BMSCs could induce the meniscus repair with a better feasibility in the structure and function. At present, the allogeneic meniscus transplantation is still the main choice for the treatment of joint pain and osteoarthritis after total meniscectomy11. However, its disadvantages include the late tran-splantation failure caused by the slow immuno-logical rejection12, hepatitis and other related di-seases, and changes in graft structure, etc.13. The development of tissue engineering technology

Figure 3. HE staining and alkaline toluidine blue staining in experimental group at 12 week after operation. Left: (HE stai-ning, 100×). At 6 weeks after operation, a little cellular infiltration could be seen in the repaired tissues, and new collagenous fibers were arranged in a disorderly manner, at 12 weeks after operation, meniscus fibrous cartilage-like structure could be seen, chondrocytes showed the oval, shuttle and polygonal shapes, etc., and the surrounding collagenous fiber structure was obvious, arranged in an orderly manner. Right: (alkaline toluidine blue staining, 100×). At 6 weeks after operation, some areas showed the metachromasia, suggesting that BMSCs differentiate into chondrocytes and secrete cartilage-specific glycosami-noglycan. At 12 weeks, the blue metachromatic area was significantly expanded with darker color.

Figure 4. Immunohistochemical staining in experimental group at 12 weeks after operation. From left to right: Type I colla-gen, Type II collagen and S100 protein, 100×. At 6 weeks and 12 weeks after operation, the staining scope and depth of Type I and Type II collagen and S100 protein were gradually increased, but the above changes were not seen in other groups)

Silk fibroin-BMSCs porous scaffolds and repair of meniscus injury in rabbits

3693

and material science provides the possibility for the application of meniscus scaffold. Altman et al14 found that silk fibroin is a kind of naturally large biological molecule without physiological activity, whose structure is based on the stable an-tiparallel β-sheet conformation, so its strength is high in the long axis direction; also, it has certain ductility with significantly better comprehensive mechanical properties than ordinary synthetic fibers. Silk fibroin has been widely used as the repair material in bone, cartilage, intervertebral disc and tendon tissue engineering15,16.

Conclusions

The significance of this study is to provide an important reference for the clinical application of silk fibroin porous scaffold combined with BM-SCs in meniscus injury, and provide important methods of preparing silk fibroin meniscus po-rous scaffold in vitro and scaffold combined with BMSCs, which has important scientific values and real economic significance.

Conflict of InterestThe Authors declare that they have no conflict of interest.

References

1) Naimark mB, kegel g, O’DONNell T, lavigNe S, He-veraN C, CrawfOrD DC. knee function assessment in patients with meniscus injury: a preliminary study of reproducibility, response to treatment, and correlation with patient-reported question-naire outcomes. Orthop J Sports Med 2014; 2: 2325967114550987.

2) Yu m, TakeSHi m, kuNikazu T, HiDeYuki k, iCHirO S. mesenchymal stem cells in synovial fluid increase after meniscus injury. Clin Orthop Relat Res 2014; 472: 1357-1364.

3) TakaTOmO m, kOiCHirO i, HirOYuki k, rYO D, kazuki C. Unusual appearance of an osteochondral lesion accompanying medial meniscus injury. Arthrosc Tech 2014; 3: e111-e114.

4) SHeN wl, CHeN Jl, zHu T, YiN z, CHeN X, CHeN lk, faNg z, HeNg BC, Ji Jf, CHeN wS, OuYaNg Hw. Osteoarthritis prevention through meniscal re-generation induced by intra-articular injection of meniscus stem cells. Stem Cells Dev 2013; 22: 2071-2082.

5) Di maTTeO B, PerDiSa f, gOSTYNSka N, kON e, filar-DO g, marCaCCi m. Meniscal scaffolds – preclinical evidence to support their use: a systematic re-view. Open Orthop J 2015; 9: 143-156.

6) guO wm, liu SY, zHu Y, Yu Cl, SHiBi lu, YuaN m, gaO Y, HuaNg JX, YuaN zg, PeNg J, waNg aY, waNg Y, CHeN Jf, zHaNg l, Sui X, Xu wJ, guO QY. advan-ces and prospects in tissue-engineered meniscal scaffolds for meniscus regeneration. Stem Cells Int 2015; 2015: 517520.

7) gruCHeNBerg k, igNaTiuS a, friemerT B, vON lüBkeN f, Skaer N, gellYNCk k, keSSler O, DürSeleN l. In vivo performance of a novel silk fibroin scaffold for partial meniscal replacement in a sheep model. Knee Surg Sports Traumatol Arthrosc 2015; 23: 2218-2229.

8) rONgeN JJ, vaN TieNeN Tg, vaN BOCHOve B, griJPma Dw, Buma P. Biomaterials in search of a meniscus substitute. Biomaterials 2014; 35: 3527-3540.

9) SeO Yk, YOON HH, SONg kY, kwON SY, lee HS, Park YS, Park Jk. Increase in cell migration and an-giogenesis in a composite silk scaffold for tis-sue-engineered ligaments. J Orthop Res 2009; 27: 495-503.

10) meiNel l, HOfmaNN S, karageOrgiOu v, kirker-HeaD C, mCCOOl J, grONOwiCz g, ziCHNer l, laNger r, vuNJak-NOvakOviC g, kaPlaN Dl. The inflammatory responses to silk films in vitro and in vivo. Bioma-terials 2005; 26: 147-155.

11) SiHvONeN r, PaavOla m, malmivaara a, JärviNeN Tl. Finnish Degenerative Meniscal Lesion Study (FIDELITY): a protocol for a randomised, place-bo surgery controlled trial on the efficacy of ar-throscopic partial meniscectomy for patients with degenerative meniscus injury with a novel ‘RCT within-a-cohort’ study design. BMJ Open 2013; 3. pii:e002510.

12) BulgHerONi e, graSSi a, CamPagNOlO m, BulgHerONi P, muDHigere a, gOBBi a. Comparative study of col-lagen versus synthetic-based meniscal scaffolds in treating meniscal deficiency in young active po-pulation. Cartilage 2016; 7: 29-38.

13) mOrONi l, lamBerS fm, wilSON w, vaN DONkela-ar CC, De wiJN Jr, HuiSkeSB r, vaN BliTTerSwiJk Ca. Finite element analysis of meniscal anatomical 3D scaffolds: implications for tissue engineering. Open Biomed Eng J 2007; 1: 23-34.

14) alTmaN gH, Diaz f, JakuBa C, CalaBrO T, HOraN rl, CHeN J, lu H, riCHmOND J, kaPlaN Dl. Silk-based biomaterials. Biomaterials 2003; 24: 401-416.

15) alTmaN gH, HOraN rl, lu HH, mOreau J, marTiN i, riCHmOND JC, kaPlaN Dl. Silk matrix for tissue en-gineered anterior cruciate ligaments. Biomaterials 2002; 23: 4131-4141.

16) maCiNTOSH aC, kearNS vr, CrawfOrD a, HaTTON Pv. Skeletal tissue engineering using silk biomate-rials. J Tissue Eng Regen Med 2008; 2: 71-80.