| OBJECTIVE: 1. To investigate the methods of division, culturing, and identifying bone mesenchymal stem cells(BMSC) in vitro; Make a basic for whole experimentation. 2. To research in vitro the degradation rate and holing rate of PLLA/gelatin. 3.To evaluate the regenerative and multiplying capability of BMSC. The adhesion rate of BMSC onto PLLA/gelatin is to be investigated too. 4. To investigate the curative effects of autogeneous BMSC seeded onto allogenic PLLA/gelatin on articular cartilage defects.METHODS: 1. Bone mesenchymal stem cells derived from Qingzilan rabbits aged 4 to 6 months and weighted 2.5-3.5kg were cultured in vitro. Morphoginic characteristics were identified using mouse anti-rabbit antibody CD44, CD34 and Laminin. 2. The degradation rate of PLLA/gelatin was tested in vitro by soaking it into phosphate-buffered saline (PBS) solution. 3. The holing rate of PLLA/gelatin was tested by liquid replacement method. 4.The regenerative rate and multiplying time of different passage of BMSC were detected and the regenerative capacity of BMSC in different gap was compared. 5.The adhesion rate of BMSC onto PLLA/gelatin was also tested. 6.BMSC were seeded onto PLLA/gelatin. The BMSC/PLLA/gelatin complex was cultured in vitro and transplanted into full-thickness defects on intercondylar fossa. Thirty-six healthy Qingzilan rabbits were divided into three groups randomly. Each group had 12 rabbits. The cartilage defects in the intercondylar fossa were filled with autogeneous bone mesenchymal stem cells and PLLA/gelatin complexes(BMSC/PLLA/gelatin) in group A(management A), with only PLLA/gelatin in group B(management B),and with nothing in group C(management C).Four rabbits were killed at three time points, which were 4,8 and 12 weeks after operation in each group. The reparative tissue samples were evaluated grossly,histologically,immunohistochemically and graded according to gross and histological scale. Inputted the scores to SPSS 10.0 software to proceed statistical analysis to find out if the differences between each group had statistical significance.RESULTS: 1. The cultured cells were positively reacted to antigen CD44, but negative to antigen CD34 and antigen laminin. 2.The degradation rate of PLLA/gelatin was accelerated with the prolonging of time. The degrading time was about 10-12 weeks. 3. The holing rate tested was 67.15. 4. The second passage cells have a higher regenerative rate than first and third passage ones. 5. The cells of 1×10~5 have a higher adhesive rate onto PLLA/gelatin. 6. The defects of BMSC/PLLA/gelatin transplantation were repaired by hyaline-like tissue; the other defects were repaired by fibrous tissue. Gross and histological grading scales were made on 12th week postoperatively. The data were completely random designed, and then analysis of variance (one-way-ANOVA) and student-newman-keuls (SNK-q) test were used. The results showed that BMSC/PLLA/gelatin group excelled PLLA/gelatin group and control group significantly (P< 0.05) , PLLA/gelatin group excelled control group (P<0.05) .CONCLUSION: 1.The cells isolated from bone marrow have a morphoginic characteristic of BMSC. They could be used as the source of seeds cells for cartilage tissue engineering.2. The graph of PLLA/gelatin 's degradation was corresponded to the graph of BMSC regenerative ones. The second passage cells were best cells in repairing of cartilage defect.3.The concentration of 1×10~5 has a higher adhesion rate onto PLLA/gelatin. 4;_The full-thickness cartilage defects of rabbits were repaired with autogeneous of bone mesenchymal stem cells seeded onto PLLA/gelatin, which is a promising way for the treatment of cartilage defects.
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