Abstract: Objective: To study the surface properties of 3D-printed titanium alloy (Ti₆Al₄V) with hydrogenated TiO₂ nanotubes (TNTs) prepared by selective laser melting (SLM) technology, and its influence on the biological behavior of human gingival fibroblasts (HGFs). Methods: 3D-printed titanium alloy specimens (3D-Ti) were prepared by SLM technology and subjected to electrochemical anodic oxidation. 3D-TNTs were formed, which were further subjected to high-temperature hydrogenation treatment to obtain 3D-H₂-TNTs. Mimic polished titanium alloy (MP-Ti) were used as the control group. The surface morphology, hydrophilicity, and roughness of MP-Ti, 3D-Ti, 3D-TNTs, and 3D-H₂-TNTs were measured and analyzed by field emission scanning electron microscopy (SEM), contact angle measurement instrument, surface roughness measurement instrument, and atomic force microscope. HGFs were cultured on the surfaces of the four groups materials, and the cell morphology was observed by SEM. The early adhesion and proliferation of cells were detected by fluorescence staining (DAPI) and cell counting kit-8 (CCK-8). Real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of adhesion-related genes and fibronectin (FN). Results: The surface of the nano-titanium spheres of 3D-Ti was anodized to obtain a radial, regular, and ordered nanotube structure. After further hydrogenation, a superhydrophilic surface with a contact angle less than 5° was obtained. Cell experiments showed that HGFs on the surface of 3D-H₂-TNTs were the most spreading, with more and longer pseudopodia, and the number of early cell adhesion was significantly higher than the other three groups (P<0.05) in the early 4 hours of culture. The proliferation of HGFs on 3D-Ti, MP-Ti, 3D-H₂-TNTs, and 3D-TNTs decreased gradually when the culture time was extended to 1, 3, 5 days. Compared to the early stage of cell culture, the dominance of 3D-H₂-TNTs in promoting cell growth disappeared. The expression of integrin-beta 1 (ITG-β1), focal adhesion kinase (FAK), vinculin (VCL), and FN secretion of HGFs on the 3D-H₂-TNTs were significantly increased compared with the control group (P<0.05) at 4 hours and 1 day. Conclusion: The superhydrophilic micro/nano composite morphology can be formed on the surface of 3D printed titanium alloy by electrochemical anodization and high temperature hydrogenation, which can promote the early adhesion and proliferation of HGFs and enhance the expression of adhesion-related genes and fibronectin.

Key words: 3D-printed titanium alloy, TiO₂ nanotubes, hydrogenation treatment, human gingival fibroblasts