In vitro behaviour of human gingival fibroblasts cultured on 3D-printed titanium alloy with hydrogenated TiO nanotubes.

Selective laser melting (SLM), as one of the most common 3D-printed technologies, can form personalized implants, which after further surface modification can obtain excellent osseointegration. To study the surface properties of SLM titanium alloy (Ti6Al4V) with hydrogenated titanium dioxide (TiO)nanotubes (TNTs) and its influence on the biological behaviour of human gingival fibroblasts (HGFs), we used SLM to prepare 3D-printed titanium alloy samples (3D-Ti), which were electrochemically anodizing to fabricate 3D-TNTs and then further hydrogenated at high temperature to obtain 3D-H-TNTs. Polished cast titanium alloy (MP-Ti) was used as the control group. The surface morphology, hydrophilicity and roughness of MP-Ti, 3D-Ti, 3D-TNTs and 3D-H-TNTs were measured and analysed by scanning electron microscopy (SEM), contact angle metre, surface roughness measuring instrument and atomic force microscope, respectively. HGFs were cultured on the four groups of samples, and the cell morphology was observed by SEM. Fluorescence staining (DAPI) was used to observe the number of adhered cell nuclei, while a cell counting kit (CCK-8) was used to detect the early adhesion and proliferation of HGFs. Fluorescence quantitative real time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of adhesion-related genes and fibronectin (FN), respectively. The results of this in vitro comparison study indicated that electrochemical anodic oxidation and high-temperature hydrogenation can form a superhydrophilic micro-nano composite morphology on the surface of SLM titanium alloy, which can promote both the early adhesion and proliferation of human gingival fibroblasts and improve the expression of cell adhesion-related genes and fibronectin. Graphical abstract.