Rapid mineralization of hydrothermally induced TiO₂ coating on titanium and early osteogenic responses in vitro

This study evaluated hydrothermally induced nanostructured TiO₂ coatings (HT-TiO₂) on Grade 1 titanium (Ti) with a focus on surface mineralization behavior and early cellular responses. Rapid mineralization (RM) was achieved by immersion in modified simulated body fluid (m-SBF). Ti discs were subjected to hydrothermal treatment (HT) to produce a TiO₂ coating, then mineralized in m-SBF for 2, 4, or 6 h. Surface characterization was conducted using SEM, EDX, XRD, and FTIR. Cellular responses were assessed using pre-osteoblastic cells and mesenchymal stem cell (MSC)‑like cells in vitro. Cell proliferation on Ti substrates was evaluated over 7 days, and alkaline phosphatase (ALP) activity in MSC-like cells was measured at days 7 and 14 across four groups: NC (non-coated), HT-TiO₂, RM4 (HT-TiO₂ with 4-h RM), and RM6 (HT-TiO₂ with 6-h RM) discs. Cell attachment and spreading were evaluated by fluorescence microscopy at days 3 and 7. The intensity of the Ca-P layer and the molar composition increased with mineralization time (2-6 h). All surfaces supported pre-osteoblast proliferation without significant differences between groups. ALP activity was significantly elevated in the HT-TiO₂ and RM6 groups compared to controls, indicating early osteogenic activity in a subset of cells. Fluorescence imaging showed flattened cell morphology on HT-TiO₂-discs and more elongated morphology on RM discs at day 3, while comparable cell confluency was observed on all surfaces by day 7. Overall, hydrothermally-induced nanostructured TiO₂ coatings on Ti surfaces can be rapidly mineralized, and the resulting Ca-P layer supports cell attachment, proliferation, and early osteogenic activity in vitro. These findings highlight the potential of surface‑driven mineralization strategies for modulating early cellular responses to titanium implants.