Preparation Of TiO₂ Nanorods Array On Ti Substrate And The Study Of Morphology Of Osteoblasts Adhered On Different Nanostructured TiO₂ Surfaces

Cell adhesion to surface of implants is the key and early period for bone tissues formation after implanting, which determine following cell proliferate, differentiation, functional expression and the eventual success or failure of the bone implants. Cell adhesion is influenced by many factors, such as surface chemical composition, surface topology, surface roughness and hydrophilicity et al. Therefore, cell adhesion may be modulated by reforming implants surface[1-3]. Nanostructured materials with properties distinctly different from ordinary materials were widely used to reform materials surface, and it has been reported that the same material with smooth surface and nanostructured surface showed great differences for cell adhesion[4-5]. Currently TiO2 nanostructures which have been used to study cell adhesion on mainly include TiO2 nanotubes array[6-11], TiO2 nanoparticles film[12] and TiO2 nano-network[13,14]. However, there is still no report on the interaction of cells between with TiO2 nanorods array.Nowadays, SEM, immunofluorescence staining, MTT assay and LDH assay et al are the main methods to evaluate cell adhesion on biomaterials surface. Actually, all of these methods require high vacuum or offline conditions and can't investigate entire process of cell adhesion on line, so it is very necessary to develop a new method to monitor the cell adhesion on real-time.In the above research background, four different kinds of TiO2 nanorods were successfully synthesized on titanium substrates by a hydrothermal method. The adhesion, spreading and viability of osteoblasts to these TiO2 nanorods surfaces were investigated. In addition, cell adhesion to nanostructured surface is monitored by QCM technique combining with nanoparticle self-assembly technique. The abstract of the content of the work is as followed:(1) Hydrothermal method was used to synthesize TiO2 nanorods array on the titanium foil. The influence of experimental conditions including the reaction time, the reaction temperature and NaCl concentration to TiO2 nanorods morphology was studied. The results showed that the higher NaCl concentration, the better TiO2 nanorods orientation. With reaction time prolonging, TiO2 nanorods array increase in length, but no significant change in diameter. The higher reaction temperature, the greater diameter and length of TiO2 nanorods and the smaller diameter of about 20nm of TiO2 nanorod array is formed at 100℃and the diameter of about 100nm is formed at 160℃. Finally, we synthesized four kinds of useful TiO2 nanorods in appropriate experimental condition, respectively mentioned as TNR1, TNR2, TNR3 and TNR4. The hydrophilicity of TNR1 and TNR2 is better than titanium, however TNR3 and TNR4 is hydrophobic materials and hydrophilic is worse than titanium.(2) The responses of osteoblasts to the four kinds of different TiO2 nanorods surfaces were investigated by SEM, immunofluorescence staining and LDH activity assay. The results showed that cell amount on TiO2 nanorods array surface is more than on titanium at the same culture time. The rate of cell adhesion and spreading on TiO2 nanorods array surface is faster than on titanium and the activity of cells on the surface of TiO2 nanorod is higher than it on titanium. In these four materials, TNR1 and TNR2 mostly benefit the cell adhesion, and the cell activity of TNR2 surface is the highest.(3) 1,2,3 and 4 layers of titanium dioxide(TiO2) nanoparticle thin films were successfully synthesized via layer-by-layer (LbL) nano-assembly technique on the gold-coated quartz crystal resonator. Relationship between resonator frequency and the assembled layers was studied by QCM technique and morphology, thickness and hydrophilicity of TiO2 nanoparticles thin films were tested by AFM, SEM, stylus profiler and contact angle tester. We observed that surface roughness, hydrophilicity and thickness were increasing with increasing number of layer-by-layer assembled TiO2 nanoparticles thin films. The response of osteoblasts to the assembled gold-coated quartz crystal resonator were investigated through SEM, MTT assay, LDH activity assay and immunofluorescence staining, the results showed that the more layers of the film surface, the better cell adhesion and spreading. Initially, cell activity on TiO2 nanoparticle films is higher than titanium foil. However, when time extended the cell activity on 4 layers of TiO2 nanoparticle film layer surface is lower than titanium foil. This maybe because TiO2 nanoparticles fall off 4 layers of TiO2 nanoparticle films were phagocytosed by cells to cause cell death. From dynamic process of cell adhesion, we found that proteins adhesion is the first period as biomaterials and cells are co-cultured. Cells begin to adhere to biomaterial surface after proteins adhesion reach to balance. We can monitor the initial process of cell adhesion by this method.