Experimental Study On Rabbit Articular Cartilage Full-thickness Defects Repaired With Homograft Of Mesenchymal Stem Cells Seeded Onto Novel Scaffold Composite HA/CPP/PLLA

OBJECTIVE: 1. To investigate the methods of isolation, culturing, purification and identifying mesenchymal stem cells(MSCs) in vitro. 2. To examine the physical mechanics properties and biocompatibility of hydroxyapatite(HA)/calcium Polyphosphate fibers (CPPf) and poly-(L-lactic acid)(PLLA)cartilage tissue engineering scaffold composite. 3. To investigate the curative effects of autograft MSCs seeded onto HA/CPP/PLLA to repair articular cartilage defects. 4. To investigate the possibilities of composite material HA/CPP/PLLA used as tissue engeering scaffold.METHODS: 1. Mesenchymal stem cells derived from bone marrow of Qingzilan rabbits aged 4-6 months and weighted 2.5-3.5kg were cultured in vitro. Morphoginic characteristics were identified using mouse anti-rabbit antibody CD44, CD34, CD29, CD105 and CD166. 2. The degradation rate of HA/CPP/PLLA was tested in vitro by soaking it into phosphate-buffered saline (PBS) solution. 3. The holing rate of HA/CPP/PLLA was tested by liquid replacement method. 4. The regenerative rate and multiplying time of different passage of MSCs were detected and the regenerative capacity of MSCs in different gap was compared. 5. The adhesion rate of MSCs onto HA/CPP/PLLA was also tested. 6. MSCs were seeded onto scaffold composite material HA/CPP/PLLA. The MSCs/HA/CPP/PLLA complex was co-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 bonemarrow mesenchymal stem cells and HA/CPP/PLLA complexes(MSCs/HA/CPP/PLLA) in group A(management A), with only HA/CPP/PLLA in group B(management B),and with nothing in group C(management Q.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: L The cultured cells were positively reacted to antigen CD44, CD29,CD105, and antigen CD 166, but negative to antigen CD34 . 2.The degradation rate of HA/CPP//PLLA was accelerated with the prolonging of time. The degrading time was about 12-15 weeks. 3. The holing rate tested was 78.15. 4. The second passage cells have a higher regenerative rate than first and third passage ones. 5. The cells of 1 X105 have a higher adhesive rate to HA/CPP/PLLA. 6. The defects filled with MSCs/HA/CPP/PLLA complex group formed 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 MSCs/HA/CPP/PLLA group excelled HA/CPP/PLLA group and control group significantly (/)<0.05) , HA/CPP/PLLA group excelled control group (/)<0.05) .CONCLUSION: L_The cells isolated from bone marrow have a morphoginic characteristic of MSCs. They could be used as the source of seeds cells for cartilage tissue engineering. 2/The degradation graph of HA/CPP/PLLA was matched to the graph of MSCs' regenerative ones. The second passage cells were best cells in repairing of cartilage defect. 3. The concentration of 1 X 105 has a higher adhesion rate onto HA/CPP/PLLA. 4. The full-thickness cartilage defects of rabbits were repaired with autograft of mesenchymal stem cells seeded onto scaffold composite material HA/CPP/PLLA, which is a promising way fortreatment cartilage defects.