The Experimental Research On The Repair Of Rabbit Articular Cartilage Defects With Allogenic Marrow Mesenchymal Stem Cells Derived Chondrocytes

Objective To investigate the effect of repairing rabbit articular cartilage defects with tissue engineered cartilage constructed in vivo by the composite of chondrocyte precursor cells induced by allogenic marrow mesenchymal stem cells(MSC_s) in vitro and autologous "two-phase" bone matrix gelatin(BMG).To compare the ability of the chondrocyte precursor cells induced by autologous and allogenic MSC_s seeded onto "two-phase" allogeneic BMG to construct tissue engineering cartilage in repairing articular cartilage defects,and observing the effect of repairing articular cartilage defects with the different tissue engineering cartilage ,to provide the basis for the clinical experiment of tissue engineering cartilage for further.Methods 1.The bone marrow which was aspirated from the rabbit's shinbone,high glucose Dulbeco,s modified Eagle medium(H-DMEM) was added into the marrow, after blowing, beating, and centrifuging, cells suspension was inoculated in the culture bottle to preceed the primary culture, which changed the culture medium gradually to take away cells which didn't stick the wall to obtain the MSC_s.2. It was easy to get enough quantity of the MSC_s through passaging and proliferating in vitro. The immunohistochemistries of CD44 and CD34 were used to identify MSC_s cultured in vitro.Meanwhile,we observed its morphology and drew the growth curve.3.When the P3 MSC_s gathered together,the growth factor: transforming growth factor-beta1 (TGF-β₁), insulin-like growth factor- I (IGF- I ),vitamin C were added in and induced the MSC_s to convert to cartilage cells, and provided the seed cells for repairing the cartilage defects in tissue engineering.4.Getting out the part of the rabbit's ilium, according to the method of modified Urist, the "two-phase" BMG was acquired from degreasing, taking off the calcium and getting out the protein, and was cut as the certain size and shape as needed. It could be conserved after sterilizing within a low temperature.5.Taked three rabbits to divide into three groups: autologous BMG was implanted the acral muscles; Allogenic BMG was implanted the acral muscles;The composite of allogenic MSC_s and autologous BMG was implanted the acral muscles.The specimen was observed roughly in the twelfth week by morphological examination,HE staining was applied.6.Inoculated the chondrocyte precursor cells into autologous BMG to set up a composite of the cell-carrier, observed it through the electric-microscope after culturing three days.7.Made the model of articular cartilage defects of rabbits, divided it into three groups,the experimental group was implanted the composite of allogenic cell-carrier,the positive control group was implanted the composite of autologous cell-carrier,the blank group was implanted nothing.In the eighth week and the twelfth week,rabbits were killed respectively,and then the specimens were obtained.The specimens were observed by morphological examination,histochemistry and immunohistochemistry,and then graded score with Wakitani histological grading system.Results 1.MSC_s could be isolated,cultured and proliferated by marrow gentrifugalism and adherence to plastic flask culture.The results of immunohistochemistry demonstrated that in vitro expanded MSC_s expressed mesenchymal cell marker,CD44.However,they didn' t express CD34. We acquired the large quantity of MSC_s through culture in vitro.2.The MSC_s were successfully induced into cartilage cells, which were verified by the positive results of collagen II through immunohistochemistry and toluidine blue staining.3.Observed the BMG and the composite of the cell-carrier by the electric-microscope, the surface of the BMG was rough and had many small openings.In cancellous part,the pore size ranged was 100um-800um,in which the cells proliferated;4.After the decalcify,degreasing and escaped antigenprocessing,the antigenicity of BMG was low.To compare with autologous BMG, there were little immunological rejection with implanting allogenic BMG.But the host could tolerate absolutely and the implanted BMG was observed totally absorbed in the twelfth week. 5.In the fifth week and the eighth week,cartilage defects were repaired by hyaline-like cartilage in the experimental group and the positive control group,but the blank group was fiber repair at the same time.There was no statistical difference according to Wakitani histological grading system between the experimental group and the positive control group in the eighth week and the twelfth week (P>0.05).But there was signigicant difference in the specimens from the experimental group and the positive control group between 8 weeks and 12 weeks (P<0.05).Meanwhile there was statistical difference between the autologous or allogenic MSC_s group and the blank group (P<0.05).Conclusion The composite of chondrocyte precursor cells induced by allogenic MSC_s and autologous BMG can construct the tissue engineering cartilage in vivo which can repair defects of articular cartilage.It is no obvious difference with that of autologous cell-carrier transplanted,but it is better than that of nothing transplanted.