An Experimental Study On Rabbit Joints'Cartilageinjury Treatment Through The Cartilageinduced By ADSCs With Combined Frame

It is an important task to promote cartilage regeneration after injury or defect in surgery. Recent technical progress of molecular biology, cell biology and microsurgery results in better outcome, but not all patients have satisfactory results. It is a nodus how to improve the recovery after cartilage injury in surgery. Adipose-derived stem cells (ADSCs) is one kind of adult stem cells that have self-renewal and multilineage differentiation potential. It has recently been reported that ADSCs can differentiate into catilage cells, opening new frontiers in therapy for cartilage injury. In this study, we repaired of rabbit cartilage injury by transplantation of ADSCs, and examined the mechanism of ADSCs transplantation for promoting regeneration of cartilage cells. We wanted to offer the experimental data for clarifying the mechanism and clinical application. Our experimental study were divied into three parts:Part I: ADCs (adipose-derived stem cells) derived from rabbits'fat were isolated and culture expanded efficiently in vitro and its biological features are detected. We selected suitable culture medium and FBS concentration, separated and purified the ADSCs with adherent method. The morphology of ADSCs was observed with invert-microscope constantly. ADSCs' curve of growth was depicted and cell cycle was analyzed by flowing cytometry. ADSCs were induced to differentiate into osteoblasts and adipocytes in induction medium containing desamethasone ,β-Glycerophosphate and horse serum. Then differentiated cells were identificated by AKP and oil O stain. Results showed ADSCs could be isolated and proliferated by adherent method in vitro. The appearance of ADSCs were fibroblast-like cells and its growth curves was like shape of S. Cell cycle analysis showed that most of ADSCs were in G₁/G₀ phase. ADSCs can be differentiated into osteoblasts and adipocytes in vitro.Part II: Investigate the cartilage cell differentiation potential and expression mucopolysaccharide and typeâ…¡-collagen in ADSCs. The 5^th generation ADSCs were put into the centrifugate tubes, induced with hTGF-β2 in L-DMEM condition medium. After 14d, the cell mass were digested by trypsogen, dispersed into single cell suspebsion, and seeded on the cover slips. 24h later, we did immunohistochemistry and RT-PCR. Then expression of proteoglycans and typeâ…¡-collagen was confirmed. The RT-PCR had a bright strap,which shows that the gene trascripition level of the typeâ…¡-collagen and proteoglycans raise up after ADSCs are induced.Part III: Study on construction and transplantation of engineered cartilage tissue. Group A: ADSCs and PLGAs are transplanted into the deep layer of PLGAs. The induced cartilage cells are seeded into the superficial layer of PLGAs. Then the two layers are tightly pressed together. Group B Positive control group only ADSCs with PLGA are transplanted into the defects. Group C Negative control group .Only the PLGA is put into the defects. A 5mm deep hole is made by a drill with a 4mm-diameter into rabbits'femoral condyles, which causes joint cartilage defects. The above three groups of frame are separately transplanted into the holes. The test rabbits are killed after 12 weeks and 24 weeks. The femoral condyles are takens out to observe the quality of the transplantation.It shows that resuls of Group A and Group B are better than Group C and the Group A is the best.Conclusion:(1) Rabbit ADSCs have been successfully isolated and culture-expanded in vitro with adherent method; (2) ADSCs can be differentiated into cartilage postive cells in vitro; (3) ADSCs and PLGA can form the cartilage frame compomers; (4) The cartilage frame compomers can be transplanted into the carilage defect and well sound grow and repair the defect.