| ObjectiveThe seed cells, which were the co-cultured cells of chondrocyte induced and diff-erenttiate from rabbit bone marrow mesenchymal stem cells (BMSCs) and allogeneic rabbit BMSCs, were implanted to animal-derived osteochondral composite scaffold to construct cell-scaffold complex, and the complex was planted to the allogeneic rabbit knee osteochondral complex defect, observe the repair results of rabbit knee cartilage and subchondral bone defects, explore the feasibility through this method of repairing osteochondral composite defect in animals and preliminary evaluate the value of this new approach.Methods1. BMSCs were isolated and cultured from the 4-weeks-old rabbit through cell adherence culture.2. Identify the BMSCs by morphology observations and observed the surface antigen, cellcycle and cell activities by flow cytometry method.3. BMSCs were induced by using specific induction culture medium and differentiate into chondrocyte, identified them by morphology observation, stained with Toluidine Blue and typeⅡcollagen staining.4. The induced differentiation of chondrocytes and BMSCs were cocultured according to the ratio of 1:2 the co-cultured cells were act as seed cells.5. The cylindrical osteochondral complexes obtain from rabbit knee through animal experiments were de-antigened by deep frozen, degreased, decalcified, vacuum freeze dried, radiation sterilized and preparated osteochondral complex scaffold, then observe the scaffold using the scanning electron microscope.6. The co-cultured seed cells were planted to the osteochondral complex scaffold to construct the cell-scaffold complex, observe the cell-scaffold complex using the scan-ingelectron microscope.7. The cell-scaffold complexes were implanted to allogeneic rabbit knee osteochondral defects, respectively 4 weeks,8 weeks,12 weeks after surgery, evaluate the restoration of defect by gross observation and histological score.Result1. The rabbit BMSCs could be obtained through cell adherence culture and could expand in vitro.2. The morphology of BMSCs were spindle shape under optical microscope, fluor-esceence-activated cell sorter:BMSCs of cell surface antigen high expression of CD29, CD44, low expression of CD34, CD45, The cell activities was 95.27% and cells of Go-Gi phase were 96.48%.3. The cell morphology changed from spindle shape to ellipsoid and triangle shape after BMSCs were induced by specific induction cultural medium. Toluidine Blue staining and typeⅡcollagen staining are both Positive.4. Induced differentiation of chondrocytes and BMSCs can co-exist and growth main-tainning their original shape.5. Animal-derived osteochondral composite scaffold were finded porous structure by using scanning electron microscope, the porosity of osteogenic zone is 88%, pore dia-meter is 150～550μm,300μm in average, the porosity of cartilage zone is 92%, pore diameter is 25～55μm,40μm in average, cultured cells were seen better adhesion and growth and collagen adhesion in the composite scaffolds.6.4 weeks,8 weeks,12 weeks after surgery to gross observation and histological score, the experimental group form the osseous tissue and cartilage tissue, osteogenic zone and cartilage zone interface coupling is significant better than the control group and the blank group.Conclusion1. The rabbit BMSCs, which were obtained through cell adherence culture, can be cultured stably and amplified for long term in vitro and can be induced into chondrocyte by specific induction culture medium.2. The induced differentiation of chondrocytes and BMSCs can be co-cultured according to the ratio of 1:2, and they also promotes the proliferation and differentiation of each other. 3. The animal-derived osteochondral composite scaffold has preferable cytocompa-tibility, it is an ideal scaffold.4. The constructed cells-scaffold complex, which were obtained from the co-culture cell implanted to the animal-derived osteochondral composite scaffold, can repair rabbit knee cartilage and subchondral bone defects successfully.
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