Sandwich-like Nano-composite Electrospun Membrane For Both Promotion Of Osteogenesis And Antibacterial Activities

Backgroud:Barrier membranes have broad prospects in guided bone regeneration(GBR),but they are severely challenged by a germ-laden microenvironment and soft-hard tissue hierarchy in orthopedic and dental applications.Objective:To fabricate a GBR membrane(GBRM)with improved osteogenic and antibacterial properties and a hierarchical structure in accordance with the physiological and anatomical structure of the oral alveolar bone defect through electrospinning technology.Then,evaluating the potential application of the membrane in GBR technology by studying the physical and chemical properties,osteogenic properties in vitro and in vivo,and antibacterial activities of the membrane.Methods:First,Silk fibroin(SF)was extracted from Bombyx mori silkworm cocoons;then,a novel sandwich-like nano-composite electrospun silk fibroin membrane(nZnO/SF/nHA)with both enhanced osteogenic properties and antimicrobial activities was fabricated by sequential electrospinning of zinc oxide nanoparticle-doped SF(nZnO/SF),pure SF,and hydroxyapatite nanoparticle-doped SF(nHA/SF).Second,the physicochemical characterization of the membrane was detected by scanning electronmicroscope(SEM),fourier transform infrared spectrometer(FTIR),X-ray diffractometer(XRD),and contact angle(CA),and the mechanical properties of the membrane was assessed via a uniaxial tensile test;Third,the biocompatibility of the membrane was examined by CCK8 and live-dead staining and the osteogenic effect of the membrane in vitro was assayed by PCR,alkaline phosphatase(ALP)staining and alizarin red staining(ARS).Four,the antimicrobial activities of the membrane were tested against Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli).Furthermore,the prepared membrane was implanted into the calvaria bone defect of Sprague Dawley rats and newly formed bone tissue was analyzed by micro-CT and histological analysis.Results : SEM images demonstrated a well-developed three-dimensional sandwich-like hierarchical structure with randomly oriented nanofibers and highly interconnected pores.While FTIR,XRD and EDX confirmed that nZnO and nHA were successfully incorporated into SF nanofibers.The water contact angle test found that the membrane exhibited satisfactory hydrophilicity after integrated with nZnO and nHA.In vitro biological experiments proved that the nZnO/SF/nHA membrane had good cytocompatibility to MC3T3-E1 and L929 cells and promoted the differentiation and mineralization of MC3T3-E1 cells.In addition,antibacterial assays suggested that the nZnO/SF/nHA membrane had noticeable antimicrobial activities against S.aureus and E.coli.Moreover,the nZnO/SF/nHA membrane induced more bone formation in rat calvaria defect models within 12 weeks.Conclusion: Consequently,the sandwich-like hierarchical multifunctional nanofibrous nZnO/SF/nHA membrane,which shows good biocompatibility and enhanced osteogenic and antimicrobial properties simultaneously,is promising for GBR applications.