| Objective: To explore the method of tissue-engineered bone construction in vitro and evaluate it's effect on bone defect healing. For elucidating the mechanism of regulation, NO and eNOS were investigated. Methods: After the cell-scaffold composites were immediate constructed or cultured in RCCS, the effect of bone healing in rabbit were observed. The mRNA expressions of eNOS and NO concentrations in cell culture supernatants were tested, whose effect on bone healing were investigated. Finally eNOS gene modified tissue-engineered bone was used to test the effect of ectopic osteogenesis in nude mice. Results: In experiment of rabbit cranial defect, immediate construction of tissue-engineered bone led to poor bone formation, but application of GTR membrane increased the bone formation. Three-dimension tissue-engineered bone was successfully constructed in RCCS in vitro whose osteogenetic potential was better than those constructed in static environment in rabbit body. Moreover there is no significant difference between 24 hours groups and 72 hours groups. The mRNA level of eNOS and the concentration of NO in RCCS were higher than those in static environment. L-argine can accelerate NO generation which further promote bone formation in rabbit mandibular defect experiment. MSCs transferred with pEGFP-N3-eNOS were implanted in nude mice, of which osteogenesis and vasculogenesis were active. Conclusion: Immediate implantation of cell-scaffold composite combined with GTR membrane can prevent cell lost and increase bone formation. RCCS may be an effective method for rapid construction of tissue engineered bone in vitro, 24 hours in which is enough for cell-scaffold composite to possess a good osteogenetic ability. RCCS enhance the expression of eNOS and production of NO, thereby promote bone healing. eNOS gene modified tissue-engineered bone might be an ideal alternative for the repair of bone.
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