Construction Of Micro-nano Biomimetic Composite Coating On Titanium And Study On Its Biological Activity In Vitro

Objective:To improve the bioactivity of titanium implant surface and promote the efficiency of early osteogenesis,the construction of biomolecule coating with bone induction ability on the implant surface has an efficient and direct effect.In this study,In this study,silk fibroin（Silk Fibroin,SF）,silk fibroin derived peptide（Silk fibroin-derived peptide,Cs）and hydroxyapatite（Hydroxyapatite,HA）were organically combined,and a composite coating simulating the composition of human bone was prepared by covalent cross-linking and electrodeposition.The effects of different preparation methods on the morphology,physical and chemical properties,self-mineralization ability and binding ability of the composite coatings were studied,and the regulatory effects of different components of coatings on bone marrow mesenchymal stem cells（BMSCs）and osteogenic differentiation were studied by cell culture and osteogenic induction in vitro,which provided a new strategy for surface modification of titanium implants.Methods:The polydopamine layer（PDA）was used as the reactive adhesion layer to covalently fix SF and Cs on the surface of titanium to construct a SF/Cs coating rich in organic active substances.The composition,morphology and hydrophilicity of the composite coating were tested by X-ray photoelectron spectroscopy（XPS）,scanning electron microscope（SEM）,atomic force microscope（AFM）and contact angle;The secondary structure of the composite coating was tested by X-ray diffraction（XRD）and Fourier transform infrared spectroscopy（FT-IR）;The protein adsorption and in vitro mineralization ability of the coating were tested by BSA-FITC solution and 1.5×simulated body fluid;In order to evaluate the cytocompatibility of the coating in vitro,bone marrow mesenchymal stem cells（BMSCs）were inoculated on the composite coating.The distribution and morphology of the cells were observed by confocal laser scanning microscope and SEM,and the cell proliferation was detected by CCK-8 method;Finally,in order to verify the effect of the coating on the differentiation of cells,BMSCs was inoculated on each coating for osteogenic induction,and the alkaline phosphatase（ALP）activity and calcium deposition were measured during the culture cycle.Secondly,through the method of electrodeposition,HA was uniformly deposited on the surface of SF/Cs coating,and an organic-inorganic biomimetic composite coating with micro-nano structure was constructed.The formation and structure of the composite coating were tested by XPS,FE-SEM,AFM,TEM and contact angle;The secondary structure of protein and the crystal form of hydroxyapatite were measured by XRD and FT-IR;The protein adsorption and in vitro mineralization ability of the coating were tested by BSA-FITC solution and 1.5×SBF.In order to evaluate the cytocompatibility of the coating in vitro,bone marrow mesenchymal stem cells（BMSCs）were inoculated on the composite coating.The distribution and morphology of the cells were observed by confocal laser scanning microscope and SEM,and the cell proliferation was detected by CCK-8 method.Finally,in order to evaluate the osteogenic activity of the coating in vitro,BMSCs were seeded on each coating for osteogenic induction.Alkaline phosphatase（ALP）activity,collagen secretion,alizarin red staining and calcium(Ca²⁺)semi-quantitative analysis were performed.Quantitative PCR（RT-PCR）technique was used to detect the expression of osteogenesis-related genes（ALP、ColⅠ、Runx2 and OC）in different sample groups cultured for 7 and 14 days.Results:1.Using polydopamine as the adhesive layer,a composite coating based on Cs peptide was successfully constructed on the surface of chemical titanium oxide.Cs was observed to bind uniformly and densely to the polydopamine coating.After chemical oxidation,the surface shows an irregular pit structure,which prevents the inactivation of Cs peptides under the action of external mechanical force and surrounding environment,thus maintaining the initial stability of Cs.Due to the addition of Cs peptides,the surface structure of SF-based coating is prevented from changing to Silk II,and the surface becomes hydrophilic,which is conducive to the adhesion of proteins.Due to the existence of Cs,it was observed that the protein adsorption,surface potential and mineralization ability in vitro were all improved.In addition,the addition of Cs peptides significantly improved the proliferation activity and mineralization ability of BMSCs in vitro,and BMSCs could adhere closely to the surface of the material,thus obtaining the best cytocompatibility and osteogenic activity.To sum up,the composite coating based on Cs peptides enhanced the bioactivity and osteogenic activity of titanium surface.2.Based on the composite coating of silk fibroin and its derived peptide,a biomimetic composite coating of silk fibroin/derived peptide/hydroxyapatite with nano-micron morphology was reconstructed by electrodeposition.Composite structures with different morphologies were formed on Cs coatings with different contents by electrodeposition.The hydrophilicity,protein adsorption capacity,surface potential and mineralization ability of the biomimetic composite coating were further improved.The bonding ability of the coating is also significantly improved.The coating can significantly improve the proliferation activity and adhesion ability of BMSCs,and the ability of osteogenic differentiation is also good.Conclusion:In this study,using PDA coating as adhesion medium,SF/Cs/HA organic-inorganic composite coating with micro-nano structure was prepared by covalent cross-linking and electrodeposition,and the biomimetic bone-like structure was constructed on titanium surface.The experimental results show that the composite coating has special micro-nano structure and stable physical and chemical properties,and can closely adhere to the Ti substrate,and the introduction of Cs and HA can effectively enhance the bioactivity of titanium surface,showing good cell compatibility and strong osteogenic induction ability in vitro,and has a wide range of clinical application potential.In addition,the preparation method used in this study is relatively simple,does not need complex processes and instruments,and has a good value of application and promotion.