Surface Modification And Biological Evaluation Of Titanium Implants With Osteogenic And Antibacterial Properties

Due to their good mechanical property,chemical stability and biocompatibility,titanium and its alloys were widely used in clinical orthopedic and dental fields.However,the surface biological inertness of titanium implants affects the adhesion,proliferation,and differentiation of osteoblast-related cells on their surface to some extends.It would limit the early osteogenesis ability of implants with native surrounding tissues,even leading to aseptic loosening and implant failure.Furthermore,numerous studies demonstrated that bacterial infection was another major cause of implant revision and failure.Therefore,the surface modification of medical titanium alloys implants so as to endow them with excellent bone integration and antibacterial properties,has become one of the research hotspot in the field of orthopedics.Considering that,from the perspective of the simulation of extracellular matrix,we have developed a series of new strategies for the fabrication of bioactive coatings on the surface of medical titanium implants through layer-by-layer self-assembly technique(LBL),anodizing technology,and cathode electrophoresis deposition(EPD)technology.These studies would endow the titanium implants with good osseointegration and anti-bacterial infection property simultaneously,in turn to improve the long-term survival of the implants.The main research contents and conclusions of this study are listed as follows:1.Surface modification of titanium substrates with N-halamine-chitosan based multilayers for antibacterial applicationTo improve the antibacterial property of titanium(Ti)substrates,we employed layerby-layer(LBL)assembly technique to fabricate a bioactive coating composed of chitosan-1-(hydroxymethyl)-5,5-dimethylhydantoin(Chi–HDH-Cl)and gelatin(Gel).The results of Fourier transform infrared spectroscopy(FTIR),nuclear magnetic resonance(¹HNMR),and X-ray photoelectron spectroscopy(XPS)showed that Chi-HHD-Cl conjugate was successfully synthesized.Scanning electron microscopy(SEM),atomic force microscope(AFM)and water contact angle measurements were employed to monitor the morphology,roughness changes and surface wettability of Ti substrates,which proved the successful fabrication of multilayers coating.Antibacterial assay against Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus)revealed that the Gel/Chi–HDH-Cl modified Ti substrates most efficiently inhibited the adhesion and growth of bacteria.Meanwhile,in vitro cellular tests confirmed that Gel/Chi–HDH-Cl multilayers had no obvious cytotoxicity to osteoblasts.Additionally,the LBL(Cl)samples could enhence the ALP expression and up-regulate the ostegeninc-relative m RNA expressioon,including runt-related protein 2(Runx2)and alkaline phosphatase(ALP).The study thus provides a promising method to fabricate antibacterial Ti-based substrates for potential orthopedic application.2.The fabrication of pH-responsive multilayers coating on titania nanotubes arrays for bacterial infections inhibition and osteogenic activity improvementTo improve the anti-bacterial property and cytocompatibility of titanium substrates,in this work,titania nanotubes(TNTs)were employed as bone morphogenetic protein 2(BMP2)carrier.A pH-responsive multilayer film composed of alginate dialdehyde-gentamicin(ADA-Gen)and chitosan(Chi)was constructed onto BMP2-loaded TNTs substrates through layer-by-layer(LBL)assembly technique,resulting in TNT-BMP2-LBLg samples.The FTIR result revealed that the ADA-Gen conjugate was successfully prepared.Meanwhile,the LBL films were measured with AFM,XPS,surface Zeta potential test,spectroscopic ellipsometer,and nanomechanical testing system,which proved the successful fabrication of the multilayered film.The release experiments revealed that acidic environment(mimicking the bacterial infectious microenvironment)could trigger the degradation of ADA-Gen from the multilayer films and in turn accelerate the release of BMP2 from TNTs.Moreover,antibacterial assay against E.coli and S.aureus confirmed that the TNT-BMP2-LBLg had excellent antibacterial capacity both in early(6 h)and in long-term(72 h).Meanwhile,in vitro cellular tests demonstrated that TNT-BMP2-LBLg had good cytocompatibility toward osteoblasts even co-culturing with S.aureus,while with good anti-bacterial effects Importantly,the obtained TNT-BMP2-LBLg promoted differentiation of osteoblasts,including enhanced alkaline phosphatase activity(ALP),improved mineralization capability and stimulated osteogenic-relative gene expression,including runt-related protein 2(Runx2),alkaline phosphatase(ALP),collagen type I(Col I),alkaline phosphatase(ALP),osteopontin(OPN),and osteocalcin(OCN).This study provides a promising strategy to develop pH-responsive antibacterial and enhance bone integrative Ti-based implants for potential orthopedic application.3.Construction of Zn-incorporated graphene oxide on Ti substrates surface to improve osteogenic activity and inhibit bacterial adhesionMethacryloyl modified graphene oxide(GOMA)as zinc ions(Zn²⁺)reservoir and release platform was fabricated on the Ti substrates with cathode electrophoresis deposition(EPD).Afterward,phenylboronic acid(PBA)functionalized methacryloyl-gelatin(Gel MA-PBA)was reacted with GOMA through in situ free-radical polymerization to prepare GO-Zn/Gel MA-PBA coating.AFM and FTIR characterizations confirmed the successful fabrication of GO and GOMA.The obtained coating was proved by SEM,XPS,and Zn ions release tests,respectively.The results of in vitro cellular experiments including cell activity,alkaline phosphatase,collagen secretion(Col I),extracellular matrix(ECM)mineralization,osteogenic genes and proteins,revealed that GO-Zn/Gel MA-PBA coating was beneficial for enhancing the adhesion,proliferation,and differentiation of osteoblasts.The positive results were related to the existence of gelatin,formation of boronic ester between PBA groups and carbohydrates of osteoblasts surface,which was beneficial for the initial adhesion of osteoblasts on titanium substrates.The sustained release of Zn²⁺improved the proliferation and differentiation of osteoblasts.In addition,antibacterial assay against S.aureus and Pseudomonas aeruginosa(P.aeruginosa)confirmed that GO-Zn/Gel MA-PBA coating on Ti substrates had superior antibacterial capacity,availably inhibited the bacterial adhesion and prevented the formation of biofilm.Hence,this study provides a novel strategy for designing pro-osteogenesis and antibacterial coating on Ti substrates for orthopedic applications.4.Construction of ZIF-8@Levo/LBL coating on titanium implants for inhibition of bacterial-associated infection and enhancement of in vivo bone formationIn this study,we developed a strategy for the fabrication of an antibacterial coating on titanium(Ti)implants with pH-response to combat bacteria-mediated local acidic microenvironment.It includes three steps:firstly,levofloxacin(Levo)-loaded zeolitic imidazolate framework-8(ZIF-8@Levo)nanoparticles were synthesized.Secondly,the nanoparticles were loaded onto the collagen-modified Ti substrates by a cathode electrophoresis deposition(EPD)method.Finally,gelatin(Gel)and chitosan(Chi)multilayers were further spin-coated onto the modified Ti substrates,since the chelating effect of Gel and Chi would reduce the hydrolysis of ZIF-8@Levo for a sustained release of Levo and Zn²⁺.Transmission electron microscope(TEM),X-ray diffraction(XRD),FTIR,dynamic light scattering,and UV-Vis spectrophotometry affirmed that the ZIF-8@Levo nanoparticles were successfully obtained.Meanwhile,SEM measurement confirmed that ZIF-8Levo nanoparticles have been successfully fixed to the titanium surface.The MOF@Levo/LBL could maintain weak alkaline microenvironment even in the presence of bacteria.Additionally,the degradation of MOF@Levo/LBL nanoparticles demonstrated that the obtained samples presented pH responsive property in PBS with different pH values.In vitro cellular experiments including CCK-8 assay,lactate dehydrogenase(LDH),live/dead staining,skeleton staining,alkaline phosphatase staining,Sirius red staining,Alizarin Red staining,and osteogenic genes expression,demonstrated that MOF@Levo/LBL coating was beneficial for enhancing the adhesion,proliferation,and differentiation of osteoblasts.Moreover,the MOF@Levo/LBL samples exhibited strong antibacterial ability against E.coli and S.aureus through hydrolysis of ZIF-8 nanoparticles,thereby creating a marginally alkaline microenvironment.Furthermore,in vivo implantation in a femur-infected rat model revealed that MOF@Levo/LBL implants not only effectively inhibited bacterial adhesion,but also significantly improved osseointegration of the Ti implants.This study provides a promising approach for fabrication of multifunctional Ti-based implants with strong antibacterial capacity and enhanced bone formation for potential orthopedic application.