Transparent titanium dioxide nanotubes: Processing, characterization, and application in establishing cellular response mechanisms

The therapeutic applications of titanium dioxide nanotubes as osteogenic surface treatments for titanium-based implants are largely due to the finely tunable physical characteristics of these nanostructures. As these characteristics change, so does the cellular response, yet the exact mechanisms for this relationship remains largely undefined. We present a novel TiO₂ NT imaging platform that is suitable for use with live-cell imaging techniques, thereby enabling, for the first time, dynamic investigation of those mechanisms. In this work, fabrication methods for producing transparent TiO₂ NTs with diameters of 56 ± 6 nm, 75 ± 7 nm, 92 ± 9 nm, and 116 ± 10 nm are described. To demonstrate the diagnostic potential of these TiO₂ NT imaging platforms, the focal adhesion protein vinculin and actin cytoskeletal filaments were fluorescently tagged in osteoblasts and real-time, high-resolution fluorescent microscopy of live-cell interactions with TiO₂ NT substrates were observed. The scope of such a platform is expected to extend far beyond the current proof-of-concept, with great potential for addressing the dynamic response of cells interacting with nanostructured substrates. Statement of significance Titanium dioxide (TiO₂) nanotubes are known to strongly enhance bone/mesenchymal stem cell behavior and, consequently, have gained attention as potential osteogenic surface treatments for titanium-bone implants. The exact mechanism by which TiO₂ nanotubes influence cellular function remains controversial, partly due to limitations in existing cellular imaging methods with opaque substrates. This work identifies fabrication conditions for the successful production of transparent TiO₂ nanotube arrays with tailorable diameters, as well as their functionality with pre-osteoblast mouse cells (MC3T3-E1) transfected with fluorescent focal adhesion protein vinculin and cytoskeletal filament actin. We demonstrate a means of recording live-cell, cell-substrate interaction mechanisms via high-resolution fluorescent microscopy and customizable, transparent TiO₂ nanotubes to begin defining the relationship between TiO₂ nanotube features and cell function.