Preparation And In Vitro Performance Of Silk Fibroin GBR Membrane Loaded With Phlorotannins

Purposes: A novel bilayer silk fibroin membrane was constructed with silk fibroin and plorotannins(PT)as raw materials,and its mechanical properties under wet conditions,cell adhesion ability,biocompatibility and osteogenesis were tested,so as to explore its possibility as a barrier membrane for guided bone regeneration.Methods: Silk fibron(SF)solution treated with different degumming time was prepared into SF bilayer membranes by solution pouring,directional freezing and freeze-drying techniques.The tensile strength of each group of membrane samples(n=5)was measured by mechanical universal experimental machine.The SF bilayer membranes group with the highest tensile strength corresponding to SF solution with a long degassing time was further re-prepared for Microscope observation of its microstructure.PT-SF bilayer membranes are prepared by loading Phlorotannins on the rough surface.The interaction between PT and SF were analyzed by Fourier Transform Infrared Spectrometer(FTIR).Human alveolar bone marrow mesenchymal stem cells(h ABMSCs)were extracted and identified by flow cytometry.h ABMSCs were inoculated on the rough surface of PT-SF bilayer membranes,and their adhesion on the rough surface was observed by SEM.The biocompatibility of PT-SF bilayer membranes was investigated by CCK-8 and the staining of live and dead cells.q RT-PCR was performed to detect the expression of osteogenic genes including: The expression of Alkaline phosphatase(ALP),RUNX family transcription factor 2(RUNX2)and osteoblast-specific transcription factor(OSX),The effect of PT-SF bilayer membranes on osteogenic differentiation of h ABMSCs was investigated by ALP staining and alizarin red staining.Results: The tensile strength of the SF bilayer membranes decreased with the increasing degumming time.The highest wet tensile strength of the SF bilayer membrane was obtained from the SF solution with the degumming time of 30 min,which is(3.293±0.276)MPa.Under SEM,the smooth surface of the SF bilayer membrane appeared dense and smooth,while the rough surface was longitudinally ordered loose porous structure.FTIR results showed that PT was loaded onto the rough surface of SF bilayer membrane through hydrogen bonding and hydrophobic force.h ABMSCs were able to attach to the rough surface of PT-SF bilayer membranes,and the staining results of CCK-8 and live cells showed no significant difference between the blank group,SF group and PT-SF group.q RT-PCR results showed up-regulation of ALP,RUNX2,and OSX genes in the PT-SF group compared with the SF group without PT.Additionally,ALP staining and alizarin red staining results also showed that,compared with the blank group and SF group,the positive results of ALP were more obvious in the PT-SF group7 days after osteogenesis induction,and greater calcium salt deposition occurred in the PT-SF group 14 days after osteogenesis induction.These results suggest that PT-SF bilayer membranes can promote osteogenic differentiation of h ABMSCs.Conclusion: The dense layer of PT-SF bilayer membranes exhibits good mechanical properties under wet conditions,while the rough surface of its porous layer can provide adhesion sites for h ABMSCs.In vitro experiments confirmed that the PT-SF bilayer has excellent biocompatibility and can promote the osteogenic differentiation of h ABMSCs to a certain extent.These results indicate that PT-SF bilayer membranes has been successfully fabricated,providing a solid foundation for future in vivo experiments.