Preparation And Characterization Of Nanofiber-reinforced Bilayer GBR Membrane In Vitro And In Vivo

Objective Constructing a new type of bilayer composite membrane by utilized sodium alginate（SA）,bacterial cellulose（BC）,chitosan（CS）and nanohydroxyapatite（n HAP）via a combination of self-evaporation technique and ice-templating method.The mechanical properties under dry and wet state,the cytocompatibility,the bacteriostasis,the ability of cell adhesion,the osteogenesis in vitro and the histocompatibility in vivo of the composite membrane were investigated to explore the potential application of the bilayer composite membrane used as guided bone regeneration（GBR）membrane.Methods SA and BC were mixed in a certain proportion and were dispersed evenly by ultrasonic.After self-evaporation,the membrane was form,and then the loose and porous structure containing CS and n HAP was engineered through ice-templating procedure,and finally a complete double layer composite membrane was form.The microstructures of the as-fabricated membrane were characterized by scanning electron microscope（SEM）and transmission electron microscope（TEM）.The mechanical properties of the membrane were measured by mechanical universal testing machine.To characterized the raw materials of the composite membrane,X-ray diffraction（XRD）and fourier transform infrared（FTIR）spectrometer were used.The biocompatibility in vitro was detected by cell counting kit-8（CCK-8）.Staphylococcus aureus and Escherichia coli were selected to study the antibacterial properties of the bilayer membrane.Rat bone marrow stem cells（RBMSCs）and NIH3T3 were selected to evaluate the cell adhesion ability.The osteogenic activity in vitro was assessed by alkaline phosphatase（ALP）activity detection and ALP staining.Subcutaneous implantation of the membrane was performed to investigate the histocompatibility of the membrane in vivo.Results SEM shows that the cross-section of the composite membrane presents tightly bonded double layer structures,the lower layer is dense lamellar structure while the upper one is loose and porous structure.In the dense layer of the composite membrane,with the introduction of BC into SA,the tensile strength increased first and then decreased with the increase of BC ratio.When the ratio of SA:BC is 10:4,the tensile strength of the membrane reaches the highest（～160 MPa）,and the corresponding tensile strength in wet state are about 40 MPa（in aqueous）and 12 MPa in phosphate buffer solution（PBS）,far beyond than that of the Bio-Gide membrane in dry and wet state.After the membrane was cocultured with rat bone marrow stem cells（RBMSCs）for 1,4 and 7 days,there was no statistically significant difference compared with the blank control group.After incubating with Staphylococcus aureus and Escherichia coli for 24 h,the optical density（OD）value of the membrane group was lower than that of the control group.RBMSCs and NIH3T3 were cultured on the porous surface and smooth surface of the composite membrane for 4 days.The results of SEM,cytoskeleton and nuclear staining showed that the number of RBMSCs adhered to the porous surface was significantly higher than that of NIH3T3 adhered to the smooth surface;After coculture of RBMSCs with porous layer of the composite membrane for 7 and 14 days,ALP activity increased dramatically compared with blank group and dense layer group,and the ALP staining was more obvious;The material was implanted into the subcutaneous of rats and the results of H＆E staining showed that there were no obvious inflammatory cells around the composite membrane.Conclusion The dense layer of the double-layer membrane serves as the mechanical support.The tensile strength reaches the maximum when SA:BC is 10:4,and also has high strength in H₂O and PBS.In vitro assays reveal that the composite membrane possesses good cytocompatibility,bacteriostatic ability and osteogenic activity.In vivo experiments show that the membrane has good histocompatibility in vivo.The novel bilayer composite membrane prepared here has great prospects in the field of GBR.