Construction And Modification Of Micro/Nano Structures On Titanium Surfaces And Their Biological Evaluations

Surface topology and chemical composition of a biomaterial plays an importantrole in the regulation of biological behaviors including protein adsorption, cell adhesion,migration, spreading, proliferation and differentiation. With the rapid development ofmicro/nano-technology, to regulate the biological behaviors by construction of micro/nano-structures on a biomaterial surface was widely investigated. Meanwhile, from theperspective of bionics, the construction of hierarchical micro/nano structures on abiomedical material could achieve the purpose of mechanical interlocking withsurrounding bone tissue.Titanium and titanium alloys have been widely used as orthopedic, dental implantsand cardiovascular stents owing to their superior physical properties. However, titaniumsurface is inherently bio-inert, thus could not form efficient osseointegration withsurrounding bone tissue. Therefore, to improve the surface property of titanium implantis significantly important in clinical application. A desired surface modification couldnot only retain the good mechanical property of a titanium implant, but also improve itsproperty in a specific clinical application, such as biocompatibility, osseointegration,antibacterial and anticancer properties. In this study, we constructed and modifiedmicro/nano structures on titanium surfaces. Their biological responses were evaluatedas well. It provides a theoretical basis for the application of titanium in clinicalorthopedic application. Main contents and conclusion of this study are listed as follows:1. Fabrication and mechanism of TiO₂nanotubes on titanium substrates byanodizationIn this study, TiO₂nanotubes with different diameters were fabricated onto titaniumsubstrates in situ via anodization and the formation mechanism of TiO₂nanotubes wasalso investigated. Firstly, we investigated the influence of different fluorinatedelectrolytes on the formation of TiO₂nanotubes. The result demonstrated that highlyordered TiO₂nanotubes could be formed in a mixture electrolyte containing0.27MNH4F in H2O/glycerol （1:1v/v） solution; Secondly, we investigated the effect of thewater content in a mixture electrolyte of0.27M NH4F in glycerol/H2O system on theformation of TiO₂nanotubes. The result indicated that the water content has a greatinfluence on the formation of highly ordered TiO₂nanotubes; Finally, the effect ofanodization potentials （10-35V） on the diameter of TiO₂nanotubes was investigated by using the electrolyte containing0.27M NH4F in H2O/glycerol （1:1v/v）. The resultsshowed that the diameters of TiO₂nanotubes increased with increasing anodizationpotentials. We thus fabricated a series of TiO₂nanotubes with diameters ranging from27nm to110nm. Highly ordered TiO₂nanotubes could not be formed when increasingthe anodization potential to35V.2. Effects of hierarchical micro/nano-structured titanium on the differentiation ofmesenchymal stem cellsTo investigate the effect of hierarchical micro/nano-structured titanium substrateson the differentiation of mesenchymal stem cells （MSCs）, we fabricated a series ofmicro-structured titanium, nano-structured titanium with TiO₂nanotubes andmicro/nano-structured titanium substrates via dual acid etching and/or anodic oxidation.Field-emission scanning electron microscopy （FE-SEM） and contact anglemeasurement were employed to characterize those substrates, respectively. Theproliferation and differentiation of mesenchymal stem cells cultured onto thosesubstrates were investigated in vitro. The results indicated that micro/nano-structuredtitanium and nano-structured titanium substrates led to the improvement of cellfunctions such as alkaline phosphatase （ALP） and mineralization activity compared tonative titanium. More importantly, micro/nano-structured titanium substratesdemonstrated great potential to induce the differentiation of mesenchymal stem cells,which were revealed by the expressions of osteocalcin （OCN） and osteopontin （OPN）.The study provides a potential alternative to generate hierarchical micro/nano-structuredtitanium implants for enhanced osseointegration.3. Construction of nano-structured titanium with long lasting antimicrobial propertyand good biocompatibilityTo explore nano-structured titanium implant with long-lasting antibacterialproperty, we conjugated3-（trimethoxysilyl）-propyldimethyloctadecylammoniumchloride （QAS） onto Ag deposited TiO₂nanotubes. Various characterization techniques,including field-emission scanning electron microscopy （FE-SEM） equipped for energydispersive X-ray spectroscopy （EDS）, fourier transform infrared spectroscopy （FTIR）,thin-film X-ray diffraction （XRD） and contact angle monitor were used to characterizethe materials, respectively. The results indicated that Ag deposited and QAS modifiedTiO₂nanotubes substrate （TiO₂nanotubes-Ag-QAS） has been successfully fabricated.The antibacterial property of the prepared substrates was evaluated by using zone ofinhibition （ZoI） and antibacterial rates tests with Escherichia coli （E. coli）. The cytotoxicity of the substrates was evaluated in vitro as well. The results suggested thatAg deposited and QAS modified TiO₂nanotubes substrates （TiO₂nanotubes-Ag-QAS）demonstrated long-lasting antibacterial property. Moreover, TiO₂nanotubes-Ag-QASsubstrates displayed good biocompatibility. This study presented a promising approachto fabricate antibacterial titanium-based implants for future medical application.4. Fabrication of nano-structured titanium with anticancer and antibacterial propertyTo develop titanium implant with anticancer and antibacterial properties, we firstlyfabricated TiO₂nanotubes onto titanium substrates via an anodization method. Then,selenium was formed in situ within TiO₂nanotubes by electrodeposition, and finallychitosan layer was coated onto the substrate via a spin-coating technique.Field-emission scanning electron microscopy （FE-SEM） equipped for energy dispersiveX-ray spectroscopy （EDS） and contact angle measurement were employed tocharacterize the materials, respectively. The results demonstrated that Se deposited andchitosan modified TiO₂nanotubes substrate （TiO₂nanotubes-Se-Chi） was beensuccessfully constructed. Subsquently, the influence of the substrate on the growth ofboth healthy osteoblasts and cancerous osteoblasts was investigated in vitro. Theselenium loaded and chitosan coated TiO₂nanotubes substrates （TiO₂nanotubes-Se-Chi）demonstrated great potential for promoting the biological functions of healthyosteoblasts growth and inhibiting the growth of cancerous osteoblasts. The antibacterialproperty of the prepared substrates was investigated by using antibacterial rates testswith Escherichia coli （E. coli）. The results demonstrated that TiO₂nanotubes-Se-Chisubstrates had good antibacterial property. This study provides an alternative tofabricate anticancer and antibacterial titanium-based implants for clinical application.5. Fabrication of diamond-like carbon films on polished titanium substrates withvarious methods and their cytocompatibility and blood compatibilityTo improve the cytocompatibility and blood compatibility of a titanium implant,titanium substrates were treated with mechanical polishing, chemical polishing andelectrochemical polishing, respectively. Then, diamond-like carbon （DLC） films weredeposited onto those titanium substrates via magnetron sputtering. Surface topographiesand surface roughness of those titanium substrates were characterized by field emissionscanning electron microscopy （FE-SEM） and atomic force microscope （AFM）,respectively. Raman spectroscopy measurement was employed to investigate thestructure of the DLC films. The results showed that DLC deposited titanium substrateswith different surfaces roughness were successfully fabricated. The cell viability and proliferation of osteoblasts cultured onto those substrates were evaluated in vitro. Andhemolysis ratio （HR） and blood platelet adhesion test were perfomed to reflect the bloodcompatibility of those substrates. The results indicated that the relatively rough titaniumsubstrates （Chemical polished Ti, Chemical polished Ti-DLC, Electrochemical polishedTi and Electrochemical polished Ti-DLC） led to improve the cell functions, such as cellviability and cell proliferation. More importantly, the DLC coated titanium substratesdemonstrated better blood compatibility than those of non-DLC coated ones. Takentogether, the chemical polished Ti-DLC and electrochemical polished Ti-DLC substratessimultaneously displayed good cytocompatibility and blood compatibility.