| Objective:To investigate the efficacy of tissue engineered bone and cartilages in repairing the defects of articular cartilage in vivo by the composites of allogeneic "two-phase" bone matrix gelatin (BMG) with chondrocytes and osteoblast induced by autologous marrow mesenchymal stem cells (MSC) in vitro。Methods:Thirty-six experimental animals were Japan big ear rabbits(2.5-3.5 g) aged 2-3 monthes and despite the gender.Experiment in vitro:1.The separation and induction of MSC:Bone marrow was separated from femur bones of 30 rabbits by sterilized myeloid puncture needle. The MSC were separated from bone marrow by density gradient centrifugation and adherent screening method, and then induced with cartilage differentiation inductor (HG-DMEM, 0.1μmol/L decaesadril,50mg/L vitamin C, 10ng/L TGF-β1,1% ITS-3146) and osteoblasts differentiation inductor (15%FBS, LG-DMEM, 0.1μmol/L decaesadril,50mg/L vitamin C, 10mmol/L sodium glycerophosphate) to express chondrocytic or osteoblastic phenotype.2. BMG preparation:"Two-phase" allogeneic BMG scaffold (one side of decalcificated bone and the other side of semi-decalcificated bone) was prepared from condylus medialis femoris, condylus lateralis femoris and upper edge of tibia bone of 6 rabbits.3. preparation of composites of chondrocytes and osteoblasts:The induced MSCs expressing chondrocytic phenotype were seeded onto the side of decalcificated bone and the MSCs expressing osteoblastic phenotype were seeded onto another side of semi-decalcificated bone. The BMG composites was cultured in vitro and tested with toluidine blue staining, alizarin red staining, ALP staining and Col-Ⅱstaining in lw,2w and 3w, respectively.Experiment in vivo:On upper and the inferior articular facets of both sides of knee joints, the similar defects of the articular cartilage were made in 30 rabbits. Autologous bone and cartilage tissue from left knee joint was planted into the upper defect on the right side of the knee joint (Group A); autologous tissue engineered composites was planted into the inferior defect of the right side (Group B); simple scaffold was planted into the upper defect of the left side (Group C); and no treatment was performed on the inferior defect of the right side (Group D:control group). Samples were collected in 4,8 and 12w by operation respectively and investigated by macroscopic observation, toluidine blue staining, alizarin red staining, ALP staining and collagen typeⅡstaining. The effects of each group were assessed using modified Wakitani scoring.Results:Experiment in vitro:As a seed cell type, MSC have the ability to differentiate into chondrocytes and osteoblasts. BMG has a satisfactory biocompatibility and could provide a three dimensional enviroment for MSC to proliferate and differentiate as a scaffold. Tissue engineered composites of cartilage and bone, which is made up of scaffold and seeded cells, could format the cartilage-like tissue and has the characteristics of living tissues.Experiment in vivo:Group A:After 4w, defect was almost completely repaired without obvious boundary between normal tissue and defect, and the elastcity of neogenetic tissue was satisfied. After 12w, the defect was completely repaired and fused together with normal tissues. Group B:after 4w, defect was basicaly repaired and have a smooth surface and elasticity; after 12w, the elasticity of neogenetic tissue was similar as normal tissue, the neogenetic tissue had a smooth sureface and similar color as normal tissue and did not have any difference between group A. GroupC:after 4w, neogenetic tissue had poor elasticity and irregular surface, and the color of neogenetic tissue was different from normal tissue; after 12w, the surface became smooth and the elasticity was improved but still not equal to the normal one. Group D: the defect was not repaired. The surface of defect was lower than normal tissue in altitude, no cartilage-like tissue was found and the neogenetic tissue has no elasticity.Modified Wakitani scoring showed that, the scores of group A and B were significantly higher than the scores of group C and D (P<0.01). The result of collagen typeⅡstaining showed that the positive cell rates of group A and B were also higher than group C and D (P<0.01)Conclusion:1. The chondrocytes and osteoblasts differentiated from MSC can proliferate on the "two-phase" allogeneic bone matrix gelatin (BMG).2. Autologous composition of bone and cartilage can repair the defect of articular cartilage.3. The tissue engineered composites made up of two cell types on the BMG could resolve the problem of poor coalescence between cartilage and underlayer of cartilage. The tissue engineered composites is a favorable scaffold material.
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