| Bone marrow mesenchymal stem cells(BMSCs),a multipotent adult stem cell,can differentiate into neurons,endothelium,muscle,cartilage and bone,and become an important seed cell in tissue engineering.Besides biological factors,some biophysical factors,such as mechanical properties,microstructures and so on,influence cell behaviors,such as adhesion,spreading,proliferation,migration and differentiation in cellular microenvironment.In the field of tissue engineering,it is still a challenging task to design bioactive materials that can match different tissues and simulate structures and functions.Silk hydrogel has high water content and good biocompatibility,and its structure and properties are closer to natural tissue.However,how to maintain its excellent comprehensive properties,such as micromorphology,degradability and biocompatibility,has been a difficult problem for silk fibroin researchers.In this study,the silk fibroin with nanofiber structure is used as the base,and the mechanical properties of silk hydrogel are further adjusted and improved by enzyme crosslinking.A substrate material that can simulate different tissues and extracellular matrix is constructed.First,the silk hydrogel was prepared by enzymatic crosslinking method based on the amorphous silk nanofiber solution prepared by our research group.By optimizing the ratio of silk protein to crosslinking agent horseradish peroxidase(HRP)and hydrogen peroxide(H2O2),the mechanical properties of hydrogels were adjusted.It is found that in the low concentration of silk fibroin solution(2%),the hydrogel mechanics range of the silk nanoparticles solution is narrow(0.41 k Pa-1.19 k Pa),while the hydrogel mechanics range of the silk nanofiber solution is relatively wide(1.26 k Pa-11.5 k Pa).Secondly,the optimal ratio of silk fibroin and crosslinker was selected,and the mechanical properties of the hydrogel were regulated by adjusting the concentration of silk nanofibers(1%-4%)(3.7k Pa-171 k Pa).By optimizing the hydrogel system,it not only simulates the structure of the nanofiber of the extracellular matrix,but also regulates the wide rage mechanical properties,and lays a foundation for matching a variety of tissues.In vitro experimentsshow that the silk hydrogel prepared by the enzyme crosslinking method has good biodegradability,biocompatibility,and the cells are more easily spread on the hydrogel of nanofiber structure than nanoparticles.without any inducer,the nanofiber hydrogel with low mechanical properties is beneficial to the differentiation of BMSCs to the nerve.In order to better simulate the tissue of the anisotropic structure of human body,such as bone,muscle and ligaments,the cross-linking of amorphous nanofibers and the electric field of high crystalline nanofibers were combined to prepare the silk hydrogel with morphology and mechanical anisotropy.From the proportion of amorphous and high crystalline nanofibers,the time of enzyme crosslinking and the concentration of silk fibroin,the effective regulation of the structure and properties of anisotropic hydrogel is achieved.The vitro studies demonstrated that the anisotropic hydrogel had good cellular compatibility and laid a foundation for the design of bioactive materials that simulated the anisotropic tissue in the later period.To sum up,two kinds of isotropic and anisotropic silk protein hydrogels were prepared by HRP mediated enzyme crosslinking by using the amorphous silk fibroin nanofibers and high crystalline silk nanofibers as the matrix materials.It can control the mechanical properties of silk hydrogel in a wide range. |
PDF Full Text Request