Applications Of Layer-by-layer Assembly Technique On Construction Of Biofunctional Interface On Titanium Substrates

As one kinds of compatible metallic biomaterials,titanium and its alloys are widely used in the preparation of various replacement devices,such as dental implants,bone fixation devices and hip replacement devices.However,the statistic data showed that the average clinical life of bone fixation devices(including taitanium,titanium alloy Ti6Al4 V,etc.)was only 12-15 years.One important reason is the poor osseointegration between the implant and surrounding bone tissue,which leads to the lack of long-term stability of the implant.Moreover,many biological response including infection,inflammation,allograft reaction,and fiber cyst around implant might induce the loosening and dislocation of implant,and then result in implant failure.To overcome the above defect of titanium and its alloys,this study employed layer-by-layer assembly technique(LBL technique)to construct functional microenvironment on the surface of titanium substrates to endow them with biological functions and regulate the cell behavior around implant,including adhesion,migration as well as differentation.The main research contents and conclusions are listed as follows:(1)Construction of different multilayer films on the surface of titanium substrates and their inflammatory effectsGelatin(Gel)/ chitosan(Chi),sodium polystyrene sulfonate(PSS)/ chitosan(Chi)and dextran sulfate(DS)/ chitosan(Chi)multilayer films were individually constructed onto the surface of titanium substrates via LBL technique,and the construction of different multilayer films were verified by the contact angle and scanning electron microscopy measurement.Then the inflammatory response of macrophage(RAW 264.7)on different multilayered titanium surface were studied,including the cell morphology,cell activity,acid phosphatase(ACP)activity,nitric oxide(NO)production,as well as inflammatory cytokines(TNF-?and IL-1?)expression.The results demonstrated that compared with PSS/Chi and DS/Chi multilayer films the activation and inflammatory response of macrophages on the surface of Gel/Chi multilayers modified titanium substrates were lower,indicating the multilayer structure could reduce the inflammatory response of the implant in vivo.(2)Preparation of silver nanoparticles embeded multilayer films on titanium substrates and their antibacterial effectsFirstly,sulfhydrylated chitosan(Chi-SH)/ gelatin(Gel)multilayers were fabricated onto the surface of titainium substrates via spin-assisted LBL technique under ultraviolet irradiation.Ag ions would be enriched in the multilayer films via the specific interactions between Ag ions and –HS groups in Chi-HS,thus leading to the formation of Ag nanoparticles in situ by photo-catalytic reaction(ultraviolet irradiation),without any other reducing agents treatment.And the results of the contact angle detection and scanning electron microscope observation demonstrated that the silver nanoparticles embeded multilayer films were successfully constructed onto the surface of titanium substrates.Meanwhile,the functional multilayered films on titanium substrates(Ti/PEI/(Gel/Chi-SH/Ag)n/Gel)were found to effectively inhibit the growth and activity of both Escherichia coli and Bacillus subtilies with low cytotoxicity via Kirby-Bauer test,bacterial adhesion assay,and cell biocompatibility test.(3)Construction of functional poly-amidoamine dendrimers and human BMP2 gene multilayer films on titanium substrates and their effects on bone formationThe multilayers comprising cationic poly(amidoamine)(PAMAM)dendrimer/ EGFP-hBMP2 plasmid DNA complex(d-DNA)and anionic naked plasmid were deposited on titanium substrates via LBL technique,and the successful construction were observed by the contact angle measurement,atomic force microscopy observation and release profiles.The expression of marker gene EGFP and functional gene hBMP2 were observed in osteoblasts.Then,the abilities of such gene actived titanium substrates on inducing the differentiation of osteoblasts in vitro and bone formation around those implants in vivo were investigated.The assays of alkaline phosphatase(AKP)activity,collagen secretion,ECM mineralization and osteogenesis-related genes expression indicated that the multilayer films containing functional gene hBMP2 improved the osteogenic differentiation.Furthermore,the histological examination and Micro-CT imaging showed that the gene actived titanium substrates demonstrated a singnificant higher level of gene expression of GFP,BMP2,OCN,OPN as well as new bone fomation around implants than those of the control group.