| Part Ⅰ Preparation and characterization silk porous scaffold with biomimetic structure Objective: Organic protein component(collagen)is one of the main components of bone tissue.Nanofibrillar collagen provides the necessary three-dimensional structure template for tissue mineralization,and provides a good microenvironment for osteoblast adhesion,proliferation and osteogenesis.Therefore,the ideal bone tissue engineering scaffold should have protein composition and nanofiber structure characteristics.To this end,this study intends to replace the collagen with silk fibroin,combined with modern processing technology,to construct a silk fibroin porous scaffold with biomimetic nanostructure.Methods: A new solvent system is used to dissolved silk into silk nanofiber solution.On the basis of obtaining silk fibroin nanofibers solution,a biomimetic silk fibroin porous scaffold was prepared by salt leaching method.The biomimeic silk scaffold shows macroscopic porous structure and a microscopic nanofibrou structure.The morphology,structure and properties of the scaffold were characterized and analyzed by scanning electron microscopy,infrared spectroscopy,X-ray diffraction and mechanical testing.Results: The novel dissolution system(salt/formic acid complex solution)can dissolve microsized silk into nanosized silk fibrils,these silk fibrils can be used to form nanofibrous structure in silk scaffolds.After dissolving silk in organic solution,sodium chloride is used as porogen to prepare silk scaffold with pore diameter in the range of 200-500 μm.Closer examination of these pore wall in the scaffold indicates a nanofibrou morphology.The structural characterization showed that the biomimetic silk fibroin porous scaffold was mainly composed of β-sheet structure,thus had water stability.Mechanical tests showed that the scaffold had good mechanical compression performance.Three different desalting and treatment processes of methanol,ethanol and water were used in the preparation of the scaffolds.The different treatment methods did not affect the nanofiber structure and secondary structure,but influnced the pore size,porosity and compression properties.Conclusion: The biomimetic silk fibroin scaffold with macroscopic three-dimensional porous structure and microscopic nanofiber structure was prepared by dissolving silk in Ca Cl2-formica acid solution combined with porogen.The porous structure,porosity and compression properties of the scaffold can be micro-regulated by different post-treatment processes.Part Ⅱ Biocompatibility study of biomimetic silk fibroin scaffoldObjective: In order to verify the biocompatibility of the biomimetic silk fibroin porous scaffold,especially the effect of different solvent post-treatment on the biocompatibility of the scaffold,cell was cultured on the scaffold to study its adhesion,proliferation and function,thus optimizing the preparation process.Methods: The cells were culture on the scaffolds post-treated with methanol,ethanol and water.After a certain period of time,the morphology and growth state of the cells were observed by laser confocal microscopy and scanning electron microscopy;the cell proliferation behavior was detected by CCK-8;the cell-related gene expression was detected by RT-PCR;and the relevant protein content in cell culture medium was detected by ELISA.Results: The confocal microscopy and scanning electron microscopy results showed that the silk fibroin scaffolds obtained by different post-treatment processes all supported cell adhesion,spreading and proliferation.The results of CCK-8 showed that the scaffold obtained by water treatment was more conducive to rapid cell proliferation compared with the organic solvent of methanol and ethanol treated scaffolds.The results of RT-PCR and ELISA showed that the scaffolds obtained by organic solvent treatment had lower expression levels of related genes and proteins.Differences in cell proliferation,gene and protein expression levels may be related to organic solvent residues and may be related to the different pore structure and mechanical property of the scaffolds.In summary,the relevant test results show that the scaffold obtained by water treatment is more conducive to maintaining the normal phenotype and function of the cells.Conclusion: The biomimetic silk fibroin scaffold supports the adhesion,spreading and proliferation of OECs,thus displayed good biocompatibility.The SF scaffold obtained by water treatment is more favorable for the proliferation of OECs than the organic solvent(methanol,ethanol)treated scaffolds,which may be due to residual cytotoxicity caused by residual organic solvent.The solvent treated groups further affects cell function,such as the expression of OECs functional genes and proteins.Therefore,the biomimetic SF scaffold obtained by water treatment is a more excellent tissue repair material.Part Ⅲ Preparation and characterization of biomimetic hydroxyapatite/silk composite scaffoldsObjective: Pure silk fibroin scaffold lacks the necessary osteoinductive and osteoconductive properties.In this respect,hydroxyapatite is not only one of the main components of bone tissue,but also has been applied to clinical bone defect regeneration,showing excellent osteoinductivity,osteoconductive and biocompatible.Therefore,the scaffold formed by organically combining silk fibroin and hydroxyapatite has a composition and structure similar to bone tissue,and is an ideal bone tissue engineering composite scaffold.In this part of the research,based on the above construction of biomimetic silk fibroin scaffolds,the introduction of hydroxyapatite into silk fibroin scaffolds was designed to construct a uniform composite scaffold for bone defect repair.Methods: Different from the conventional direct mixing method or mineralization method,this experiment directly dissolves hydroxyapatite into the silk dissolving solution,and then dissolves the silk fibroin to obtain a silk fibroin solution.On the basis of obtaining a silk fibroin solution containing hydroxyapatite,a hydroxyapatite/silk fibroin composite bionic scaffold was obtained by using sodium chloride as porogen and a water treatment process.The morphology,structure and properties of the scaffold were characterized and analyzed by scanning electron microscopy,energy spectrum,infrared spectroscopy and mechanical testing.Results: Hydroxyapatite/silk fibroin composite scaffolds with different ratios can be constructed easily.The highest content of hydroxyapatite in the composite scaffold is 40%.Scanning electron microscopy(SEM)results show that the composite scaffold has a good porous structure,and the hydroxyapatite is uniformly dispersed and forms a network structure together with silk fibroin.The presence of calcium and phosphorus in the composite scaffold was detected by energy spectrum analysis,and it was confirmed that hydroxyapatite was present in the composite scaffold.Infrared spectroscopy results indicated that the composite scaffold silk fibroin was mainly β-sheet structure with good stability.The mechanical properties test showed that the addition of hydroxyapatite improved the mechanical properties of the composite scaffold,and the higher the hydroxyapatite content,the higher the mechanical properties of the composite scaffold.Conclusion: This study successfully constructed a hydroxyapatite/silk fibroin composite scaffold with different composition ratios.The composite scaffold has a complete morphological structure,a uniform porous structure,and a biomimetic nano-network structure with typical silk II structure and improved mechanical compression performance.Part Ⅳ In vitro and in vivo osteogenesis of biomimetic hydroxyapatite/silk composite scaffoldsObjective: The design and preparation of biomimetic hydroxyapatite/silk fibroin composite scaffold is to meet the requirements of bone defect repair.Therefore,the osteogenesis of the composite scaffold is an important part of its application value and prospect.Therefore,based on the construction of biomimetic hydroxyapatite/silk fibroin composite scaffold,this part will study the osteogenic characteristics of the composite scaffold by in vitro cell differentiation experiments and in vivo bone defect regeneration experiments.Methods: The human umbilical cord blood mesenchymal stem cells were inoculated on the composite scaffold,the morphology and growth state of the cells were observed by confocal microscopy,and the proliferation behavior of the cells was quantitatively detected by CCK-8;the stem cells cultured on the composite scaffold were induced to differentiate into osteoblasts.The alkaline phosphatase activity was detected and RT-PCR was used to detect the expression of type I collagen,RUNX2 and calcitonin gene.The rat skull defect model was constructed.The preferred hydroxyapatite/silk fibroin composite scaffold and clinical bone powder were implanted into the rat skull defect model.Micro-CT was used to evaluate the bone defect repair after 1 and 2 month.Results: The hydroxyapatite/silk fibroin composite scaffold supports the early adhesion and spreading of human umbilical cord blood mesenchymal stem cells,and cell proliferation during the culture time.The scaffold has been completely covered by cell layer at 10 days.CCK-8 results showed that there was no significant difference in cell proliferation between scaffolds with different composite ratios.Compared with the pure silk fibroin scaffold,the hydroxyapatite/silk fibroin composite scaffold can significantly promote the alkaline phosphatase activity and up-regulate the expression level of osteogenic differentiation genes,.Compared with clinical bone powder,the composite scaffold with 40% hydroxyapatite content is more effective in promoting bone defect regeneration,which may be related to its biomimetic nanostructures and components.Conclusion: In conclusion,we demonstrated the good biocompatibility of hydroxyapatite/silk fibroin composite scaffolds with human umbilical cord blood mesenchymal stem cells,supporting cell adhesion,spreading and proliferation.It was found that the composite scaffold is more conducive to osteoblast differentiation of stem cells,and the higher the content of hydroxyapatite,the better the osteogenic differentiation effect.Bone repair experiments in animals preliminarily confirmed that hydroxyapatite/silk fibroin composite scaffolds can promote bone defect regeneration,which is better than clinical bone powder.Based on the above results,we believe that the biomimetic hydroxyapatite/silk fibroin composite scaffold prepared in this study is an ideal bone tissue engineering scaffold and is expected to be used for clinical bone defect regeneration. |
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