| Titanium and titanium alloys have been applied for hard tissue alternatives extensively such as orthopedics and dental devices based on their excellent mechanical properties,high corrosion resistance and benign biocompatibility.However,the intrinsic inert nature of titanium leads to insufficient bioactivity,and their non-antibacterial property may cause infection,looseness and implant failure finally.Many studies indicated that the osseointegration property of titanium can be improved significantly by micro-nano hierarchical structure,due to combining merits of mechanical locking capability and improving adhesion,proliferation and differentiation performance which derive from micro-roughness surface and nano-morphology on titanium respectively.Thus,inspired by the precise assembly of organic/inorganic materials across scales of natural bone tissue,we constructed a series of ordered multiscale micro-submicro-nano structures on titanium implants from bionics point of view.The effects of surface characteristics,such as micro-nano scale,the order of nano morphology,wettability and crystallographic form etc.,on adhesion,proliferation and osteogenic differentiation of pre-osteoblasts were analyzed systematically.Meanwhile the molecular mechanism of micro/nano multilevel structure influencing biological behaviors of cells was investigated thoroughly.Additionally,in order to develop high efficiency and safety of antibacterial coatings on implant materials for clinical application,we firstly introduced bioactive peptide with low cytotoxicity and broad-spectrum antibacterial characteristics on titanium implants by assembling the composite films.And the corresponding mechanism of antibacterial and osteogenic activity of the prepared composite films was discussed as well.The main research results and progress are as follows:1.The hierarchical structures on titanium with micro-pits,sub-micro-protuberances and varying scaled nanotubes were constructed successfully via electrochemical self-organizing etching,chemical etching and anodizing.And the diameter of nanotubes was positively correlated with the anodic oxidation voltage.The morphology of micro-pits maintained and wettability was enhanced greatly by further construction of nano-morphology.In vitro,micro/sub-micro(EE)and micro/sub-micro/nano(EE+CE)morphology imparted superior cell behaviors to titanium materials compared to micro-pits structure alone.The smaller diameters of nanotubes and roughness would be beneficial to early adhesion and propagation for pre-osteoblasts MC3T3-E1,while the higher ALP activity,collagen secretion,ECM mineralization and osteogenic specific genes expression were discovered on 20V samples which possessed larger diameter of nanotubes and roughness.The production of osteogenic differentiation indicators would be suppressed by IWP-2,a Wnt/β-catenin pathway inhibitor,which indicated that this signaling pathway played an important role in promoting osteogenic differentiation on ordered micro-submicro-nano multilevel titanium.2.The micro/nano multilevel structure of varying sized micro-pits,identical submicro and nano morphology were fabricated on titanium by adjusting secondary etching voltages during electrochemical self-organizing etching.The size of micro-pits was enlarged gradually with the increase of secondary etching voltage.Similarly,the morphology and size of micro-pits were not changed after sub-micro and nano structure introduction.In vitro,good performance of cell adhesion,spreading and proliferation were proved on micro-nano hierarchical titanium,and there was no obvious influence on cell early adhesion although different micro pits size,which manifested the vital role of nano-morphology on cell adhesion.The 6V and 8V samples with smaller micron size and roughness exhibited faster cell proliferation,while the amount of cells on 10V samples was relatively lower.The results of ALP activity and bone formation related genes expression demonstrated that multilevel structure titanium with larger size of micro-pits and roughness might be prone to promote cell differentiate towards osteogenesis.Importantly,micro-nano surface on titanium would stimulate ALP activity,collagen secretion and extracellular matrix mineralization by activating Wnt/β-catenin pathway,moreover enhancing the osseointegration of implant materials.3.By altering the electrochemical etching parameters,the ordered micro pits and ordered nanotubes array or disordered nano-sponge structure with identical micro morphology and similar surface chemistry,wettability and roughness were prepared on titanium successfully.The anatase phase and anatase/rutile mixture phase was detected after annealing under 450℃ and 550℃ respectively,meanwhile mineralization capacity of micro-nano multilevel structure titanium in SBF could be strengthened by crystallization process.And there was no significant effect on the formation of bone-like apatite whether for order or disorder nano-morphology.It was found that the cell adhesion and proliferation could be elevated on anatase/rutile surface,and the disordered nano-morphology played an active role on cell adhesion.The ALP activity,collagen secretion,extracellular matrix mineralization and osteogenic genes expression have been enhanced by ordered micro-pits/nanotubes hierarchical structure on titanium compared to the ordered micro-pits/disordered nano-sponge structure,especially for that made by 20V-450℃.The Wnt/β-catenin pathway of MC3T3-E1 could be activated by ordered micro/nano structure via increased phosphorylation of GSK-3β and suppressing degradation of β-catenin,then promoted osseointegration property of titanium implants.4.The ε-polylysine(ε-PL)and gum Arabic(GA)multilayer composite films were immobilized combining covalent grafting and layer by layer(LBL)on anodized titanium(TNTs)with the assisstance of polydopamine for the first time.The antibacterial properties against both Staphylococcus aureus and Escherichia coli were enhanced with an increase in the loading amount of ε-PL.And the samples with multilayer films(PL2 and PL3)possessed long-term antibacterial ability last for 21 days via synergistic effects of inhibiting cell adhesion and contact killing.Meanwhile,the chemical characteristics were improved greatly by ε-PL and GA on multilayer composite films decorated TNTs,thus the cell proliferation and osteogenic differentiation of BMSCs including ALP activity,collagen secretion etc.on LBL TNTs were significantly raised.Remarkably,a facile method to optimize anti-infective and osteogenic properties of medical titanium has been developed,and it was demonstrated that the ε-PL and GA multilayer composite films with assistance of polydopamine were able to endow the orthopedic implant materials both improved antibacterial property and excellent biocompatibility,which is of profound significance for enhancing the osseointegration of implants. |
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