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Abstract Background The repair of critical-sized bone defect represents a challenging problem in bone tissue engineering. To address the most important problem in bone defect repair, namely insufficient blood supply, this study aimed to find a method that can promote the formation of vascularized bone tissue. Method The phenotypes of ASCs and EPCs were identified respectively, and ASCs/EPCs were co-cultured in vitro to detect the expression of osteogenic and angiogenic genes. Furthermore, the co-culture system combined with scaffold material was used to repair the critical-sized bone defects of the cranial bone in rats. Results The co-culture of ASCs/EPCs could increase osteogenesis and angiogenesis-related gene expression in vitro. The results of in vivo animal experiments demonstrated that the ASC/EPC group could promote bone regeneration and vascularization in the meantime and then significantly accelerate the repair of critical-sized bone defects. Conclusion It is feasible to replace traditional single seed cells with ASC/EPC co-culture system for vascularized bone regeneration. This system could ultimately enable clinicians to better repair the defect of craniofacial bone and avoid donor site morbidity.