| Part 1:The study on investigation to isolate, culture and identify rabbit adipose-derived mesenchymal stem cells and its ability to differentiate into lipocytes and osteoblasts in vitroObjective:Establish the method to isolate and culture rabbit adipose-derived mesenchymal stem cells (rADSCs) in vitro, in order to study their morphology, cell surface markers and biological properties. To investigate the method of rADSCs differentiate into lipocytes and osteoblasts in vitro.Methods:Primary rADSCs were isolated from the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits by 0.1% collagenase I digestion. The rADSCs were passaged and amplified by the trypsin digestion. The fourth passage of rADSCs was induced to differentiate after exposure to adipogenic or osteogenic medium. The Oil Red 0 staining, alkaline phosphatase (ALP) and Alizarin red staining were used to detect the results. The growth curve of the fourth passage of rADSCs was drawn by optical density value (OD value) using the CCK-8 method. Cell surface markers CD29, CD34, CD44, CD45 and CD90 were examined using flow cytometry.Results:The rADSCs isolated from the subcutaneous adipose tissue of rabbits had a strong capability of proliferation with a fusiform adherent growth observed under inverted microscope, primary rADSCs 5-6hours after inoculation began to stick wall, about 7d cells can be single fusion up to 80%-90%, cells were fusiform after wedding, beam shaped and vortex pattern. After being cultured in the adipogenic differentiation medium, the fourth passage of rADSCs were Oil Red 0 staining positive. After being cultured in the osteogenic differentiation medium, the cells expressed positive to ALP and Alizarin red staining. CD29, CD90, CD44, CD45 and CD34 markers flow cytomerty instrument detection, visible rADSCs CD29, CD90 and CD44 in the positive rate is high, CD45 and CD34 negative expression.Conclusion:The rADSCs were successfully isolated and cultured from the subcutaneous adipose tissue of rabbits. These cells are pluripotent with the potential to differentiate into lipocytes and osteoblasts.Part 2:The investigation of the establishment of rADSCs/β-TCP bone tissue engineering composite and osteogenic differentiation in vitroObjective:To establish rADSCs and β-TCP bone tissue engineering composite in order to observe the differentiation of rADSCs into osteoblasts in scaffolds.Methods:The fourth passage rADSCs in the β-TCP was inoculated in β-TCP scaffold,cell suspension was added. The growth of the cell was monitored by optical density value (OD value) using CCK-8 method every day. On day 7 the expression of ALP was determined. The fourth passage rADSCs inoculated in 12-well plate for culture was used as control group in the mean while proliferation rate and ALP activation was investigated. The results were analyzed statically. The cell morphology was observed by scanning electron microscope (SEM).Results:CCK-8 method was used to investigate cell proliferation in the scaffold, OD value of each group gradually increased with time and no significant difference was observed in cell cultured on the scaffold. Moreover the scaffold had no toxic effect on the cell and acquired normal growth similar to the control group. ALP expression of both cell groups had statically significant differences, the scaffold induces osteoblastic differentiation in rADSCs.Conclusion: β-TCP scaffold had good biocompatibility, rADSCs does not have a co-culture cytotoxicity, but is also possess strong ability of cell proliferation and osteogenic differentiation.Part 3:The study of the use of rADSCs/β-TCP Bone tissue engineering complex for repairing of large segmental radial bone defect in rabbitObjective:To investigate the possibility of repairing large segmental radial bone defect in rabbit by using rADSCs/β-TCP composite bone tissue engineering.Methods:In vitro cultured rADSCs was inoculated in β-TCP, rADSCs/β-TCP Bone tissue engineering complex was co-culture in vitro, Treatment of bone defects were divided into three groups:control group with no implant (group A), control β-TCP (group B), rADSCs/β-TCP composite bone tissue as experimental group(group C). Each group had 8 rabbits with 16 bone defect. Radiographic examination with Lane-Sandhu X ray semi-quantitative analysis was done 2w,4w,6w and 8w after surgery. From each group,2 rabbits were sacrificed, specimens were harvested for gross observation, hematoxylin-eosin staining at the same time period mentioned above. The stained samples were observed for bone repair and allograft rejection under Light microscopy (LM).Result:Lane-Sandhu X ray semi-quantitative analysis was used for Statistical Analysis. The repair effect in group C was prominent compared to group B and was statically significant. In Both of these group Lane-Sandhu X ray score was significantly higher compared to group A. In Histological observation group C at 4 weeks after surgery had new bone growth around the defect and there was a gradual increase in bone mass with increase postoperative period.6 weeks after surgery there was a gradual degradation of the material, and new bone growth was observed in Pores within the material. Material degradation was significant at 8 weeks after surgery, pores disappeared, and recanalization of the bone marrow was complete. But at 8 weeks after surgery, two cases of group B and four cases in group A impaired bone was not completely healed, bone marrow was not recanalized. There were significant differences when compared to the experimental group. The allografts showed no histological signs of acute rejection.Conclusion:rADSCs/β-TCP Bone tissue engineering complex had healing property and the large segmental radial defect in rabbit was successfully repaired. Thus supported the fact that ADSCs is expected to become the ideal repair substrate of bone defects, which can be used as Seed cells for bone tissue engineering. After implantation, ADSCs do not provoke an immune response. |
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