Surface Biofunctionalization Of Biomedical Titanium/titanium Alloy And Biological Responses

Biomedical titanium and titanium alloys were widely used as dental implants and bone rehabilitation implants.However,many previous studies revealed that the bioinert TiO₂ layer on titanium-based implants limited the osseointegration between the implants and surrounding natural bone,which might further result in the loose/shift of implants.Such drawback becomes to be even severe for bone repair under bacterial infection or osteoporosis condition.Therefore,researches on surface engineering of titanium-based implants have become to be one of the hot topics in the related fields.How to surface modify titanium based implants so as to maintain good osseointegration at the bone-implant interface is the key problem to be resolved.On considering that,we designed and fabricated a series of osteogenesis-promoting,anti-osteoporosis or antibacterial surface biofuncitonalized titanium materials via sol-gel and layer-by-layer assembly?LBL?techniques.The biological responses of those materials were further evaluated in vitro and/or in vivo.The main research contents and conclusions of the paper were listed as follows:1.Effects of micro/nano hierarchical structures of titanium substrates on biological behavior of mesenchymal stem cellsTo investigate the effects of surface topography of titanium?Ti?substrates on osteogenesis differentiation of mesenchymal stem cells?MSCs?,a series of micro/nano hierarchical structures were fabricated onto micro-structured titanium substrates via a sol-gel technique.The results of scanning electron microscopy?SEM?,surface profiler,X-ray diffraction?XRD?,X-ray photoelectron spectroscopy?XPS?and water contact angle measurement revealed that Ti substrates displayed “valley-ridge” micro-structure with presence of various TiO₂ grains?20 nm,40 nm and 80 nm,respectively?.The formation mechanism of the micro/nano hierarchical structures was proposed.The in vitro cells tests confirmed that micro/nano hierarchical structures with large grains?80 nm?greatly promoted the proliferation and differentiation of MSCs comparing with other small grains?20 nm and 40 nm?.2.Regulation of the biological functions of osteoblasts and bone formation by Zn-containing titanium micro/nano hierarchical structuresTo improve the biological performance of titanium implants,a series of Zn-incorporated coatings were fabricated on the microrough titanium?Micro-Ti?via sol-gel and spin-coating techniques.The successful fabrication of the coating was verified by combined techniques of SEM,surface profiler,XRD,XPS,and water contact angle measurements.The incorporated zinc existed as ZnO.The Zn-incorporated samples?Ti-Zn0.08,Ti-Zn0.16,and Ti-Zn0.24?efficiently inhibited the initial adhesion of both Staphylococcus aureus?Gram-positive?and Pseudomonas aeruginosa?Gram-negative?.The in vitro evaluations including cell activity?CCK-8?,alkaline phosphatase activity?ALP?,mineralization,osteogenic genes expressions?Runx2,ALP,OPG,Col I,OPN,and OC?and tartrate resistant acid phosphatase?TRAP?,confirmed that Ti-Zn0.16 sample was the optimal one to promote the proliferation or differentiation of osteoblasts and inhibit the growth of osteoclasts.More importantly,in vivo evaluations including Micro-CT analysis,push-out test,and histological observations further verified that Ti-Zn0.16 implants could efficiently promote new bone formation.3.Titania nanotubes on titanium surfaces adsorbing alendronate for antiosteoporosis implantationThe implantation of bone implants into osteoporosis patients is a common clinic challenge.The success rate of implantation would be highly improved if the implant itself has strong anti-osteoporotic property.To endow a titanium-based implant with desirable local anti-osteoporosis property for enhancing its early osseointegration,alendronate-loaded hydroxyapatite-TiO₂ nanotube?TNT-HA-Aln?substrates were fabricated in this study.The release curves of Aln/Ca2+ verified that the release of Aln was significantly accelerated along with the acidity rise caused by osteoclast differentiation.The in vitro tests including CCK-8,ALP,mineralization,gene expressions?Runx2,Osterix,ALP,ColI,OPN,OC,OPG and RANKL?,protein production?OPG and RANKL?and TRAP,proved that TNT-HA-Aln substrates had great potential for improving osteoblast proliferation/differentiation and inhibiting osteoclast differentiation.Moreover,the in vivo tests?push-out test,micro-CT and H&E staining?proved that TNT-HA-Aln implants could efficiently improve new bone formation surrounding the implants in an osteoporotic rabbit model.4.Titanium surfaces with multilayed intercalation structures for anti-osteoporosis implantationTo endow Ti6Al7 Nb implants with local anti-osteoporosis property,functional hyaluronan-alendronate/BMP-2?HA-Aln/BMP-2?nanoparticles were embedded into the Gel/Chi multilayers on Ti6Al7 Nb surfaces?Ti6Al7Nb/LBL/NP?via a LBL technique.The release test showed that the loaded BMP-2 nanoparticles slowly released along with the degradation of multilayers.The in vitro cell experiments?CCK-8,ALP activity,mineralization and TRAP etc.?demonstrated that Ti6Al7Nb/LBL/NP implants improved the proliferation and differentiation of osteoblasts and inhibited the maturity of osteoclasts simultaneously.Moreover,the in vivo tests of push-out test,micro-CT and histological observations verified that Ti6Al7Nb/LBL/NP implants enhanced new bone formation surrounding the implants under osteoporotic condition.5.Enzyme reaponsive TiO₂ nanotubes anti-bacterial materials and anti-bacterial property evaluationBacterial infection is the second dominant factor resulting in implantation failure.Previous researches verified that adhered bacteria could secrect a large amount of hyaluronidase at the infected local site.Based on this knowledge,we fabricated hyaluronidase responsive titanium based anti-bacterial materials by employing TiO₂ nanotubes?TNTs?to load cecropin B,and then covered with hyaluronidase sensitive multilayers of chitosan/hyaluronic sodium-cecropin B [?Chi/SH-CecB?₅] via LBL technique?TNT-CecB-LBLc?.FITR and 1H-NMR characterizations proved the successful synthesis of SH-CecB.SEM,AFM,fluorescence staining and water contact angle measurements verified the successful formation of multilayers onto CecB loaded titanium substrates.The release test confirmed that the presence of S.aureus and/or exogenous hyaluronidase could effectively trigger the degradation of multilayers,thus facilitating the release of CecB from TNTs.In addition,the anti-bacterial evaluation proved that TNT-CecB-LBLc substrates had good early?4 h?and long term?72 h?antibactericidal capacity against both S.aureus and Staphylococcus epidermidis?S.epidermidis?.Moreover,the cells tests displayed that TNT-CecB-LBLc substrates had relatively good cytocompatibility for osteoblasts,even co-culture with S.aureus.6.Titanium anti-bacterial material with multilayered structure and anti-bacterial property evaluationAlthough we developed hyaluronidase-responsive titanium material above using CecB,the high cost and mild antibacterial capacity of CecB limited its clinical application to some degrees.Considering that,we further employed antibiotics?gentamicin and vancomycin?to modify sodium hyaluronate with relatively low cost.The in vitro anti-bacterial and cell tests showed that SH-Gen had comprehensive advantages over SH and SH-Van samples regarding the adhesion inhibition of S.aureus and Escherichia coli?E.coli?,as well as good cytocompatibility for osteoblasts.