Protein Adsorption On TiO₂ Nanotube Surfaces And Its Influence On Osteoblasts

Titanium and its alloys have been extensivly applied in orthopedics for their mechanical, physical and biocompatibility properties. However, the titanium implants cannot meet specific biocompatibility requirements for the biomaterials because of their sample mechanical conformity with bones. Surface modification of biomaterials was often ultilized to improve their biocompatibility. In this study, Titanium dioxide (TiO2) nanotube arrays were fabricated by anodic oxidation of a pure titanium plate. Nanotubes of different diameters and lengths could be fabricated by changing applied voltages and oxidation time. Titanium plate was modified by micro-arc-oxidation method; a connected microporous layer was formed on titanium surface. The surface morphology and crystal structure of the nanotube arrays were analyzed by scanning electron microscope (SEM), X-ray diffractometer (XRD). The hydrophilicity and surface energy of the nanotube arrays were analyzed by a standard goniometer instrument. Adsorption of protein onto the TiO2 nanotube arrays was used to investigate the kinetics, the thermodynamics mechanisms and the mode of the protein adsorption. The effect of the protein adsorption on cellular behavior of osteoblast were evaluated in vitro before and after adsorbed on TiO2 nanotube arrays. The protein release mechanism was explored by the in vitro release experiment.The voltages were adjusted to 5V,10V,20V and two-step anodic oxidation (20V and then 25V) to prepare nanotubes with about 20nm,50nm, 100nm and 200nm diameters respectively. TiO2 nanotube arrays with the diameter of 100nm and the lengths of-500nm and-1000nm were fabricated by anodic oxidation under the constant voltage of 20V for 1 hour and 6 hours. Microporous film with depth of 200-400nm was attained by micro-arc-oxidation on titanium plate surface. After heat treatment at 450℃for 3 hours, amorphous TiO2 crystal transformed into the anatase crystal. The crystal of the samples treated by micro-arc-oxidation was anatase and rutile crystal after the heat treatment. The tubular morphology of the TiO2 arrays did not apparently altered by the heat treatment at 450℃.When the implants were placed into the human body, the reaction would happen between the specimens and adsorbed biomacromolecules. Thus, we selected different proteins with different initial concentrations to explore the mechanism of the protein adsorption on the titanium and TiO2 nanotube arrays. The results suggested that TiO2 nanotube arrays have a higher capacity of protein adsorption than the flat titanium plate and the titanium with microporous layer. The protein adsorption quantity increased with the increasing of the nanotube diameter and the decrecent of the nanotube length.But the protein adsorbance showed decreased trends with increasing of the nanotube length. The nanotube samples had the best bioactivity among three types of samples was attributed to its high specific surface area which could provide more adsorption sites for protein. The amount of protein adsorbed on the nanotublar surfaces increased with increasing of the protein initial concentrations. When different proteins were chosen to study, the maximal adsorption amount was calculated through the Langumir isotherm model. XPS and FTIR analyses indicated that BSA adsorbed onto the nanotublar surfaces by chemical adsorption, but FN adsorbed onto the nanotublar surfaces by physical adsorption. The adsorption capacity of FN was better than BSA. However, fluorescence and SDS-PAGE analyses indicated that a protein multilayer which was most composed of BSA molecules will be formed on nanotublar surface when different proteins coexisted.The release experiment results suggested that 98 percents proteins adsorbed on flat titanium and microporous layer were released back into solution with the same release speeds. While a few of proteins were released from the nanobular surfaces. The release behaviors of BSA and FN on the nanotube arrays involved both of the bust release and the gradual release, the release mechanism was consistent with Fickian diffusion.The osteoblast culture test in vitro indicated that osteoblast on the titanium surface modified by the nanotube arrays have the higher cell activity. The samples with protein adsorption are good for cell adhesion and proliferation. The sample with the different protein on it had the different effects to the cell adhesion and proliferation. FN was helpful to cell proliferation and differentiation than other two proteins.