Micro/nanofibrous Membrane As Bionic Periosteum For Bone Regeneration Inspired By Periosteal Development

Objective:Prepare bionic periosteum which possesses micro/nano structure and VEGF based on micro-sol electrospinning and collagen self-assembly techniques.Verify the assumption that the bionic periosteum can complete the periosteum and bone regeneration through an exogenous-endogenous combined pathway to replace periosteal fibrous layer,promote early revascularization and mesenchymal cell proliferation,differentiation.Methods:The core-sheath structure was synthesized by micro-sol(MS)electrospinning technique,with PLLA(L-polylactic acid)as the outer sheath and HA(hyaluronic acid)as the core,in which exogenous VEGF was encapsulated.Collagen(Col)self-assembly technique was applied to finally form a bionic periosteum with micro/nano structure.Scanning electron microscopy(SEM),transmission electron microscopy(TEM),infrared spectroscopy(FTIR)and mechanical testing machine were used to characterize different fibrous membranes(PLLA,MS,PLLA-Col,MS-Col).The distribution of the drug in single fiber was traced by fluorescent labeling method,and the sustained release effect of VEGF was detected by ELISA.Rat mesenchymal stem cells(BMSCs)were cultured on different fibrous membranes to evaluate the effect of bionic periosteum on cell activity,adhesion,spreading,proliferation and differentiation by live/dead staining,CCK-8 assay,immunofluorescence staining,and alkaline phosphatase activity and calcium nodule assay.Different membranes and human umbilical vein endothelial cells(HUVECs)were cultured through Trans-well plates to verify the effect of released VEGF on endothelial cell angiogenesis.Finally,the rat skull defect model was used to evaluate the ability of bionic periosteal to promote periosteal regeneration and bone defect repair in vivo.Micro-CT,H&E staining,masson staining,periostin and CD31 immunohistochemical staining were used for qualitative and quantitative analysis.Results:Fluorescently labeled drug tracer experiment showed that the drug was relatively evenly distributed among each fiber.The final cumulative release of VEGF was about 80.7%±1.4%,and VEGF could be sustained released for more than 4 weeks.In the cell viability and proliferation experiments,live/dead staining and CCK-8 tests indicated good biocompatibility and ability to promote cell proliferation on the bionic periosteum.Integrin and vinculin immunofluorescent staining showed the ability of micro/nanostructures to promote cell adhesion and spreading.In the osteogenic differentiation test,the ALP activity and calcium nodules staining were significantly increased compared with the control group.In endothelial cell tube assay,the junctions and total length of tubes were significantly higher than those of control group.In animal experiments,the group covered with bionic periosteum indicated obvious periosteal and bone regeneration.Finally,the linear arrangement of periostin was observed by tissue section analysis.Conclusion:Micro/nanofibrous periosteum has good biocompatibility and biological activity,and can effectively promote osteogenic differentiation of mesenchymal stem cells and angiogenesis of endothelial cells in vitro.Bionic periosteum has good abilities to prevent scar tissue proliferation and promote blood vessel and bone regeneration in vivo.It can complete the repair of periosteum through an exogenous-endogenous combined strategy in vivo,and heal bone defect through the inherent osteogenic mechanism of periosteum.