Study On Antifouling And Osteogenic Performance Mechanism Of Choline Phosphate Functionalized Titanium Surface

Titanium（Ti）and its alloys have been widely used in orthopedic implants and cardiovascular stents owing to their excellent tissue compatibility and corrosion resistance.However,the bare Ti surface promotes unnecessary protein adsorption by activating the coagulation pathway in blood-contacting devices,and the stable oxide layer structure naturally formed on the surface of titanium limits cell adhesion at a physiological p H,which inhibits biological activity.Therefore,there is potential value in developing Ti implant surfaces that inhibit protein adsorption to prevent undesirable responses and promote cell adhesion conducive to organizational integration in the blood-device microenvironment for applications in the biomedical field.Surface-initiated atom transfer radical polymerization（SI-ATRP）is a universal and gentle surface modification technology,which is widely used in the surface modification of biological materials.In recent years,the rapid development of polymer chemistry has made it possible to modify metal substrates with multifunctional polymer brushes.Zwitterionic polymers can serve as antifouling and blood-compatible surface coatings,and Ti and other biomedical metals have been functionalized with these molecules to inhibit protein adsorption,blood platelet adhesion and subsequent thrombus formation,Moreover,phospholipid polymers,such as 2-methacryloyloxyethyl phosphorylcholine（MPC）,have improved biocompatibility and can form a zwitterion structure that mimics the outer membrane of eukaryotic cell membranes at the water interface.Although these studies used immobilization of zwitterions onto Ti to achieve a hemocompatible surface,in most cases,this also inhibits cell adhesion as a result of suppressed protein adsorption.Therefore,there is a need to modify the Ti surface to resist protein adsorption and simultaneously promote cell adhesion to overcome the limitations of Ti.PC is a well-known hydrophilic molecule in eukaryotic cell membranes,and choline phosphate（CP）is an interesting molecule that contains ammonium and phosphate groups in reverse order to that in PC.There is an electrostatic interaction between PC and CP,and multivalent CP molecules can quickly adhere to the surface of a variety of cell membranes.Furthermore,2-（methacryloyloxy）ethyl choline phosphate（MCP）is designed to resist protein adsorption because of its zwitterionic properties and is widely used to construct non-biofouling surfaces for biomedical applications.In our previous study,we modified the surface of materials with a monolayer or multilayer of CP molecules,and the results indicated that the CP-decorated surfaces can improve the adhesion and proliferation of human umbilical vein endothelial cells,and prevent the protein adsorption.However,to the best of our knowledge,there are few reports on the introduction of CP groups onto Ti surfaces,and further investigation on its more remarkable cell-adhesive and protein-resistant properties is required.In this study,we used a two-step method to synthesize MCP monomer with double bonds,and characterized the structure of MCP by proton nuclear magnetic resonance spectroscopy.Then we modified the Ti sheet surface with poly-MCP（PMCP）and MCP monomer was grafted by combining surface-initiated atom transfer radical polymerization（SI-ATRP）with mussel-inspired polydopamine（PDA）chemistry,and different amounts of MCP monomers（2,4,8,and 10 mmol）were polymerized onto the surface of Ti to form the PMCP polymer.X-ray photoelectron spectroscopy（XPS）revealed that PMCP was successfully modified on the Ti surface.Fibrinogen and bovine serum albumin（BSA）were used as model proteins to study the antifouling properties of Ti-PMCP surfaces,and the results showed that the titanium surface modified by PMCP can effectively resist the adhesion of proteins,inhibit the adhesion of bacteria,and had good blood compatibility.In addition,the cell adhesion,proliferation,and osteogenic differentiation properties of Ti-PMCP surfaces were investigated in vitro.In order to further explore the role of Ti-PMCP surface and osteoblast-related cells,the ability of osteoblast-related cells（MC3T3-E1 and BMSCs）on the surface of adhesion,proliferation and osteogenic differentiation was observed.Cell viability assay experiments show that Ti-PMCP surface can effectively promote the adhesion and proliferation of MC3T3-E1 cells.The osteogenic differentiation morphology of BMSCs on the surface of Ti-PMCP was observed by fluorescence microscopy.The staining of calcium nodules indicated that the surface of Ti-PMCP had good osteoinductive activity.The gene expression of osteogenic related genes Alp,Ocn,Col-I and Run×2 was detected by q RT-PCR,and the results showed that the osteoinductive activity of the Ti-PMCP₈group was the highest.In summary,this study successfully grafted MCP to the surface of Ti sheet surface through the SI-ATRP method,and the Ti-PMCP surface resisted the adsorption of proteins,promoted cell adhesion,and improved osteogenic differentiation.Moreover,as more MCP monomers were grafted to the surface,the Ti-PMCP exhibited higher hydrophilicity and improved resistance to protein adherence.Furthermore,the decorated Ti surface promoted MC3T3-E1 cells and bone marrow mesenchymal cells（BMSCs）adhesion and proliferation via the specific CP–PC interaction,and the Ti-PMCP surface was effective in promoting the osteogenic differentiation of MC3T3-E1 cells and BMSCs because the phosphate group in MCP can stimulate osteogenic signaling pathways.Therefore,these findings may provide a general concept for biomaterial surface modification with MCP molecules to improve specific cell adhesion and resist unnecessary protein adsorption and has great potential in bone tissue engineering.