Studyonthe Constructionand Osteogenic Propertyof Silk Biomimetic Bone Composite Scaffoldswith Anisotropy

Purpose:Based on the different responsiveness of amorphous and beta silk nanofibers to chemical cross-linking and electric field driving,the behavior of the two nanofibers was separately regulated in the same system.Then,the silk protein bone material was constructed with the anisotropic structure of cortical bone in the outer layer and the random porous structure of cancellous bone in the inner layer from the view of bionic the structure of cortical bone and cancellous bone in natural bone tissue.Through the optimization design of bionic bone structure,the osteoinduction and immune regulation ability of materials are improved,and the performance of scaffolds in promoting bone tissue regeneration is improved.Methods:1.The raw silk is degummed,dissolved in lithium bromide,dialyzed,centrifuged and concentrated to obtain amorphous and beta silk nanofibers.Then the two nanofibers are mixed and poured into a prefabricated annular graphite mold in a certain proportion,and the orientation of beta silk nanofibers and the freezing cross-linking of amorphous nanofibers were adjusting by electric field and chemical cross-linking,respectively 2.Cut the inner and outer layers of the scaffold,and set the outer,inner and combined groups.Mechanical properties,secondary structure,microscopic morphology and degradation behavior of silk protein scaffolds were investigated by compressive modulus,infrared absorption spectroscopy,scanning electron microscopy,pore size and orientation,water retention and degradation experiments.3.Using immunofluorescence and DNA content detection to evaluate the biocompatibility of silk protein scaffolds.4.By establishing a co-culture system of macrophages and bone marrow mesenchymal stem cells,using immunofluorescence,q RT-PCR,Western Blot,ELISA,alizarin red and alkaline phosphatase staining were used to evaluate the regulation of macrophage polarization and osteogenic performance of scaffolds.5.By establishing a rat femoral defect model,the ability of the material to promote bone regeneration in vivo and its regulation of macrophage polarization were evaluated by Micro-CT and histopathological section staining.Results:1.A circular silk protein biomimetic bone scaffold with an anisotropic outer layer and an inner random porous structure was prepared.2.Infrared spectroscopy showed that the main secondary structure of the silk protein scaffold wasβ-sheet.The pore size,porosity,and water retention rate of the outer scaffold are smaller than those of the inner scaffold.The outer scaffold has higher mechanical properties than the inner scaffold.In vitro degradation experiments show that the scaffold can be slowly degraded and absorbed,and play a role in long-term intervention.3.The results of laser confocal observation and DNA content detection of cells showed that the scaffold has good biocompatibility.In addition,the inner layer and the composite layer showed a better proliferation trend than the outer layer because of their larger pore size,which is more conducive to the proliferation of cells.4.The co-culture system of macrophages and bone marrow mesenchymal stem cells suggests that the expressions of IL-10,CD 206,BMP-2,RUNX2,alizarin red and alkaline phosphatase of the scaffolds with anisotropic structures were higher than that of the inner layer group.And the composite group had better proliferation performance,and the expressions of each were higher than that of the outer layer group.However,the expression of IL-6 and i NOS in the inner layer group was higher,and the other indexes were lower,indicating that the existence of anisotropic structure can induce macrophages to polarize to M₂type and have better osteogenic properties.5.The animal experiments’results of Micro-CT and histopathological section staining were consistent with cell experiments.The composite group had the best ability to induce macrophage polarization to M₂type and promote bone tissue repair.Conclusion:The formed scaffold can simulate the structural characteristics of cortex and cancellous bone in physiological state to the greatest extent,and improve the overall ability of the scaffold to promote bone tissue repair by regulating the polarization of macrophage.The integration of multiple factors in the same system is realized,which provides ideas and possibilities for the construction of biomimetic bone models and organoids in vitro in the future.