TiO ₂ Nanotubes Co‐Functionalized With Hydroxyapatite, Collagen, and Nisin for Bioactive Titanium Surfaces: A Preliminary In Vitro Study

ABSTRACT

One of the main causes of clinical failure related to implants is the low stability of the implant in the bone, frequently associated with the formation of bacterial biofilms on the surface of the devices. In this context, modifying the titanium surface to obtain bioactive and antibacterial surfaces represents a potential strategy for further investigation in implant applications. In this preliminary in vitro study, TiO ₂ nanotubes were obtained by pulsed anodization and functionalized with hydroxyapatite, type I collagen, and nisin, aiming at applications in metallic implants. The nanotubes presented a well‐organized morphology, with average internal diameters of approximately 50 nm and predominantly anatase crystalline phases. Surface functionalization was confirmed by physicochemical analyses, which detected carbon and nitrogen by XPS on the collagen‐ and nisin‐functionalized surfaces, with calcium and phosphorus found at low levels, mainly beneath the outermost surface layer, in addition to a marked increase in hydrophilicity, with a reduction in the contact angle from 84.1° ± 2.5° (Ti) to 34.7° ± 3.9° (NT.HA.Col.Nis) after functionalization. Assays with MC3T3‐E1 pre‐osteoblastic cells showed metabolic activity above 90% relative to unmodified titanium, while collagen‐containing surfaces promoted greater cell adhesion and significantly increased metabolic activity at 72 h. Furthermore, alkaline phosphatase activity, an early marker of osteoblastic differentiation, increased significantly on collagen‐containing surfaces compared with unmodified titanium. The incorporation of nisin reduced Staphylococcus aureus colonization, as observed by SEM and LIVE/DEAD analyses, without inducing cytotoxic effects. These results indicate that TiO ₂ nanotubes functionalized with hydroxyapatite, collagen, and nisin are a promising approach for developing implant surfaces with early osteoblastic‐stimulating and antibacterial properties, warranting further validation in preclinical models.