The Compared Research On Repairment Of Articular Cartilage Defects With Tissue Engineered Cartilage Constructed By "Two-Phase" Bone Matrix Gelatin With Autologous/Allogeneic Marrow Mesenchymal Stem Cells And Chondrocyte Cells

Objective To investigate and compare the effects of tissue engineered cartilages constructed in vivo by the composites of "two phase" bone matrix gelatin(BMG) with autologous/ allogeneic chondrocyte precursor cells induced by marrow mesenchymal stem cells(MSCs)and chondrocyte cells in vitro in repairing articular cartilage defects.Methods Seventy-two Japan white rabbits were involved in the experiment."two phase" BMG scaffold(one side of porous cancellous bone and the other side of cortical bone;3 mm both in diameter and in thickness)was prepared from iliac bone by sequentially chemical method with modified Urist.The MSCs were separated from 20 rabbits and induced with growth factor:transforming growth factor-betal(TGF-β₁), insulin-like growth factor-â… (IGF-â… )and vitamin C to express chondrocytic phenotype.The chondrocytes were separated from knee joint articular cartilage of 24 rabbits by digestion method with 0.2%typeâ…¡collagenase.Cells were identified through immunohistochemistry and Mallory,Toluidine blue stainin.6 rabbits were observed the bio-activity effect of autologous,allogeneic"two phase" BMG and allogeneic MSCs composed with BMG,which seperatively grafting into limb muscle of rabbits.The chondrocyte and chondrocyte precursor cells were seeded onto autologous "two phase" BMG to construct tissue engineered cartilages.Scanning elect microscopic observation was carried out at 3 days.The defects of full thickness articular cartilage(3 mm both in diameter and in depth)were made at both sides of femoral medial condyles in 62 rabbits.The defects were repaired with the tissue engineered cartilage constructed by the composites of autologous "two phase" BMG with autologous MSCs(group A,n=10),and allogeneic MSCs(group B,n=10),and autologous chondrocyte(group C,n=18),and allogeneic chondrocyte(group D,n=10),and no cells(group B,n=10).Group F without any implant at both sides in the remaining 10 rabbits as a control(,n=10).Group G in the remaining 2 rabbits as a false-operation control.8 and 12 weeks postoperation, specimens of femoral condyles werc harvested in those groups,Respectively,cross observation,Masson's trichrome and Alcian blue staining,O'driscoll scoring and modified Wakitani scoring and in situ Hybridization of collagen typeâ…¡were carried out to assess the repair efficacy of tissue engineered cartilage.Results MSC_s could be isolated,cultured and proliferated by marrow gentrifugalism. Through passaging and proliferating in vitro,high quantity MSC_s were successfully obtained and identified by immunohistochemistries of CD44 and CD34..Meanwhile, morphology was observed and growth curve were drew.MSC_s was successfully induced to convert to cartilage precursor,the positive results were verified by immunohistochemistry collagenâ…¡mRNA,Mallory,Toluidine blue staining.The "two phase" BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part,the pore size ranged 100—800 um,in which the chondrocyte and the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage.In cortical part,the pore size ranged 10-40 um,on which the cells arranged in a layer and formed the hard part of subchondral bone.The autologous/allogeneic "two phase" BMG which implanted into limb muscle were absolutely absorbed without any new tissue reconstruction and immunological response.After 8 weeks of transplantation,the cartilage and subchondral bone were regenerated in group A,B,C,D;Especially at 12 week, defect was repaired and the structure of the articular surface and subchondral bone was in integrity.In groups E and F,defects were not repaired and the surrounding cartilage of defect was abrased.According to the modified Wakitani scoring,the indexes in group A,B,C,D were significantly higher than those in group E and F(P＜0.01)at 8 week and 12 week.There were no significant difference on that between group A,B,C,D.The positive cell rate of in situ hybridization for collagen typeâ…¡in group A B,C,D.was also higher than those in groups E and F(P＜0.01).Conclusion "Two-phase" allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous/ allogeneic chondrocyte or chondrocyte precursor cells induced from MSCs to construct the tissue engineering cartilage.The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.