| Objective: To establish a new cultured cell model of rabbit osteoblasts in vitro and study it's biological characterstic,to provide a new method of repairing the bone defection employing biomedical engineering,as well as to investigate the effects of insulin-like growth factor Ⅱ (IGF-Ⅱ) basic fibroblast growth factor (bFGF) and transforming growth factor β(TGF-β) on rabbit osteoblasts proliferation. Methods: We harvestd the rabbit stromal cells of bone marrow from the trochanteric regoin of femur of rabbit and cultured the cells with special cultural fluid in vitro. In about two weeks cells lined up in one layer. Then cells were generated and their biological characteristic was examined by phase-contrast microscope, scanning electron microscope, transmission electron microscope ,H-E stain, ALP stain, type I collagen stain and calvaria stain.The cells PLA compound colls-ePTFe compound were cultured for one week and planted into the rabbit muscle. The planted materials were obtained at 4,8 weeks after operation.The specimens were assessed by histology method in order to assess the bone-fanning ability of the cultured cells.Then the cultured cells of the fi>fth generation were incubated with various growth factors( IGF- Ⅱ bFGF TGF-β) concentions and combinatins.Dose-depencedence and thue-depencedence were studied by MTT colorimetric method. Result: Morphologically,the cultured cells grew well in primary culture.they lined up in one layer and appeared as flat,multi-morhologioal cells with long or short protrusions. Furthermore,cells grew in congregution and when the cells reached confluence,their cellular outline had a fibroblast-lilce appearance,with mosaic regions.Then the cells continued to crowd,and multilayer regions were fanned. With transmission electron microscope examination,the cytoplasm is rich in ribosomo, endoplasmic reticulum and Golgi bodies,demenstrated well symtlietic and secretive fundion in accord with the characteristics of osteoblast. The biochemistiy and hlstochemical stains showed that cultured cells were rich in ALP,which was often used as biochemical and histochemical marker fat their identificatIon and for the evaluation of osteogenesis.Type I collagen stain were also positive. The calcification in vitro was seen when cells were cultured with special cultural fluid.The passaged cells had the same morphological and functional features.Afler compounded with PLA and ePTFe, cells grew well. At 4 weeks after operation, there were still many osteoblast around the newly-formed bone matrix .At weeks after operation, cells reduced and the newly-fanned bou looked like typical bone tissue. In the same condition, IGF-Ⅱ bFGF TGF-β all could promoted the proliferation of osteoblast.With the dose increasing,the promotion effects of the two growth factors IGF-Ⅱ bFGF became more and more obvious with grade concentration between 0.1 ng/ml and 10 ng/ml. Over 1 Ong/ml,the stimulation effect waned. The best concentration of TGF-β was 0.1ng/ml Over 0.1ng/ml,the stimulation effect waned. AU three growth factors sigincantly increased the proliferation of osteoblasts after day 5 when treated with the best concentration. After 7 days,the effect waned. The synergetic effecte were achived when they were used in combinatlon,with bFGF+IGF- β and bFGF+IGF-Ⅱ+TGF-β being the most significant. Conclusion :1.The cultured cells derived from the rabbit bone mar... |
PDF Full Text Request