The Molecular Conformation Of Silk Fibroin Regulates Osteogenic Cell Behavior By Modulating The Stability Of The Material Interface

Background: Silk fibroin is a natural biological macromolecular protein extracted from B.mori silk.SF has been widely used in production of bone-related biomaterials for its robust mechanical properties and hypoallergenic features,etc.An essential step during the preparation progress is to partially transform the SF molecules from random coil to stable β-sheet conformation to render the materials water-insoluble.However,the efficiency of β-sheet transformation highly depends on the applied processing methods,which is totally different in various experiments.The influences of SF conformation on in vitro osteogenic cell behavior at the material surface is still not fully understood.Objective: Exploring the possible mechanism of the influence of silk fibroin conformation on the osteogenic behavior of stem cells.Methods: By crosslinking silk fibroin films with water vapor at different temperatures,the different proportions of molecular conformations were transformed from random coils to β-sheets,and accordingly three groups of silk fibroin materials with low β,medium β and high β were induced.The physical and chemical properties of the materials were tested by scanning electron microscope(SEM)and atomic force microscope(AFM).The surface stability of the material was detected under external ultrasonic stimulation,and the adsorption capacity and stability of fibronectin on the material surface were detected by immunofluorescence,ELISA and other methods.Bone marrow mesenchymal stem cells were incubated on the surface of the material,and the corresponding changes of intracellular mechanical signal transduction pathway were detected by immunofluorescence and q RTPCR.Finally,the osteogenic ability of BMSCs was detected by ALP detection,alizarin red staining,immunofluorescence and q RT-PCR.Results: Three kinds of stiff silk fibroin materials with high,medium and low β-sheet content were successfully prepared by changing the temperature of water vapor-crosslinking.It was found that the three groups of SF membranes had comparable chemical composition,surface morphology and wettability.When fibronectin was used as the adsorbed model protein,it was found that there was no significant difference in the initial adhesion of the protein.However,when certain external stimulation was applied,the results showed that the stability of the material-interface proteins increased with the increase of the SF β-sheet content.In addition,when bone marrow mesenchymal stem cells were cultured on different conformation silk fibroin substrates,immunofluorescence staining showed that the high β group compared with the low β: i)the area of cytoskeleton-related focal adhesions was larger,ii)the cytoskeleton was arranged more orderly,and iii)the cells stretched slenderer.In addition,the m RNA expression level of Vinculin activator was higher in SFH group,and the nuclear transport trend of YAP/TAZ and RUNX2 increased with the increase of β-sheets.Finally,osteogenic assay showed that the osteogenic differentiation ability of cells in high β group surface was better than that in low β group.Conclusion: The β-sheet content influences osteogenic differentiation of BMSCs on SF materials in vitro by modulating the stability of adsorbed protein-material interface,which proceeds via protein-focal adhesion-cytoskeleton links and subsequent intracellular mechanotransduction.The higher the content of β-sheets,the more stable the surface of silk fibroin membrane and the better the osteogenic property.