Alkaline Ozonation-Induced TiO₂ Nanoscaffold on Titanium Alloy for Surface-Mediated Osteogenic Guidance

Numerous surface modification strategies, particularly nanoengineering approaches, have been explored to tailor the physicochemical and topographical properties of titanium surfaces in order to enhance osteogenic responses at the implant interface. In this study, we propose an alkaline ozonation strategy as a novel approach to generate nanostructured TiO₂ layers on Ti-6Al-4V alloy surfaces. Titanium discs were treated in a 6 M KOH solution under continuous bubbling of ozone, allowing the formation of reactive oxygen species (ROS) responsible for oxidative surface restructuring. Scanning electron microscopy (SEM) revealed the formation of a homogeneous three-dimensional TiO₂ nanonetwork composed of intertwined nanofibers. X-ray photoelectron spectroscopy (XPS) confirmed the oxidative reconstruction of the Ti alloy surface. The fraction of Ti⁴⁺ species characteristic of TiO₂ increased markedly from 44.2 at% to 92.2 at%, accompanied by a strong reduction in Ti⁰ (from 40.2 at% to 5.8 at%) and Ti³⁺ (from 15.7 at% to 2.1 at%). Concomitantly, lattice oxygen associated with Ti-O-Ti bonding increased from 48 at% to 78 at% as deduced from the O 1s signal, while the surface carbon content decreased from 48 at% to 18 at%. The modification induced a pronounced increase in surface hydrophilicity, with the water contact angle decreasing from 85° to 32° and the surface free energy increasing from 40.8 mJ/m² to 69.8 mJ/m². In vitro studies demonstrated good cytocompatibility and enhanced osteogenic differentiation of human mesenchymal stem cells, with twice as much alkaline phosphatase activity after 14 days and mineralization of the extracellular matrix after 28 days than those on TCPS, and also significantly higher than those on the nonmodified Ti alloy control. These findings indicate that the generated three-dimensional TiO₂ nanonetwork acts as a surface-confined nanoscaffold providing nanoscale cues that promote osteogenic cell responses on titanium implant surfaces.