Titanium surface is usually modified using a spark anodizing technique for biomedical application, and the resultant anodic TiO
2 layer on the surface plays a crucial role in promoting the favorable biologic response. However, despite the fundamental importance of anodic TiO
2 layer, little is known about its long-term stability or changes in vivo. In this study, as a first step to elucidate the surface changes of anodized titanium in vivo, changes in surface characteristics of anodic TiO
2 layer arising from water immersion at 37°C were investigated. It was found that after water immersion for 1 week, numerous nanoparticles emerged on the surface of anodized titanium fabricated at a voltage of 200 V, and with the further increase of immersion time, more nanopartiles formed and interconnected with each other, resulting in a nanostructured surface layer. Transmission electron microscopy investigation of anodic TiO
2 layer before and after water immersion indicated that water immersion transformed the originally amorphous TiO
2 layer into crystalline anatase gradually, and the lap-shear test of oxide layers demonstrated the decreased shear strength with water immersion. In addition, it was found that the increased anodization voltage led to the anodic TiO
2 layer with better stability.
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