| ObjectiveTo isolate and cultivate BMSCs from New Zealand rabbits, fabricate three dimensional PPC/CSNF composite porous scaffolds, and then evaluate the compatibility of the PPC/CSNF composite porous scaffolds and the BMSCs of rabbits in vitro and in vivo. To plant cell-scaffold compound fabricated in vivo to repair bone defect of rabbits, evaluate the effect and conduct a preliminary study on this new tissue engineering method.Methods1. BMSCs were isolated from New Zealand rabbits by the whole bone marrow adherence method and cultured.2. The surface antigen, cell cycle and viability of BMSCs were observed with morphology and flow cytometry methods.3. BMSCs were induced directionally and differentiated into osteoblast by using the induction culture medium. Their capability of osteogenic differentiation is worked out through morphologic observation, ALP staining, mineralized nodule staining, type I collagen staining, etc.4. PPC porous scaffold was firstly fabricated by combining a solution-casting and a porogen-leaching technique, followed by in situ phase separation of chitosan solution within the PPC scaffold to form PPC/CSNF composite porous scaffold. The scaffolds were observed by a scaning electron microscope (SEM). And the porosity of the scaffolds were characterized.5. The two tpyes of scaffolds were co-cultured with BMSCs. They were observed by scanning electron microscope.6. PPC/CSNF scaffolds were planted into the subcutaneous tissue of rabbits, and were taken after 1,2 and 4 weeks later separately. General observation and HE staining were conducted to analyze the histocompatibility of the materials.7. The rabbits'femoral condyle defects were modeled and experimented in four groups. PPC/CSNF/BMSCs compound was planted into the defected position in the experimental group; autogenous bone in the criterion group; PPC/CSNF scaffolds in matched group; and the control group was left without any treatment.4,8 and 12 weeks after the operations, general observation, radiology examination and HE staining were made to evaluate the status of defect repairing.Result1. BMSCs of rabbit can be obtained through whole bone marrow adherence method and can be cultured in vitro.2. BMSCs expressed CD29 and CD44 at 89.23% and 90.57% respectively while expressed CD34 and CD45 at 0.53% and 0.61% respectively. The cell cells of G0-G1 phase were 92.32%.3. The cells morphology changed from spindle to ellipsoid and triangle after been induced. Positive reactions were showed in ALP and collagen typeâ… staining. Red dense nodules were seen in the mineralized nodules staining.4. The pores of PPC scaffolds feature diameters varying from 200 to 500μm and have good connectivity. The diameter of chitosan nanofibers in the PPC/CSNF scaffold varied from 50nm to 500nm. All the scaffolds showed a high porosity of 92%.5. Observed with the scanning electron microscope, the BMSCs had good adhesion with the scaffold.6. No tissue rejection was seen after the planting of the materials, and no distinct inflammation and rejection was found in the HE staining.7. The following results were observed after the operation: in experimental group, bone tissue was formed in the defected position, the scaffolds degraded and the effect of repairing was similar to that of the criterion group; in matched group, some bone tissue was formed and the scaffold degraded partly; in control group, the defect was mainly filled with fibrillar connective tissueConclusion1. The BMSCs of rabbit can be obtained through the whole bone marrow adherence method.. They can be cultured stably for long term and be induced into osteoblast. They are the ideal choice for tissue engineering. 2. PPC/CSNF porous scaffold is characterized with good adhesion and tissue compatibility and can degrade gradually in vivo.3. PPC/CSNF porous scaffold composited with BMSCs of rabbit have a good abliity to accelerate the formation of new bone, and repair the bone defect finally。... |
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