In vitro behaviour of human gingival fibroblasts cultured on 3D-printed titanium alloy with hydrogenated TiO2 nanotubes

Abstract

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 (TiO₂)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-H₂-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-H₂-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.

Graphical abstract

Fig. 1

(A) Design sketch of samples (B) MP-Ti sample (C) 3D-Ti sample

Fig. 2

Sample characterization. SEM images of (A) the MP-Ti substrate at low magnification (×5000), (a) the MP-Ti substrate at high magnification (×50,000); (B) the 3D-Ti at low magnification (×500), (b) the 3D-Ti at high magnification (×2000); (C) the 3D-TNTs at low magnification (×500), (c) the 3D-TNTs at high magnification (×50,000); (D) the 3D-H2-TNTs at low magnification (×500), (d) the 3D-H2-TNTs at high magnification (×50,000)

Fig. 3

a Digital pictures of water contact angles for the MP-Ti, 3D-Ti, 3D-TNTs and 3D-H2-TNTs at high magnification (×30,000). b Water contact angles of the samples (*p < 0.05)

Fig. 4

Cell morphology. SEM morphology of the HGFs adhered on the MP-Ti, 3D-Ti, 3D-TNTs and 3D-H2-TNTs after incubation for 1 h, 4 h, and 1 day

Fig. 5

Nuclei observation with DAPI fluorescence staining. The HGFs were cultured on the MP-Ti, 3D-Ti, 3D-TNTs and 3D-H₂-TNTs for 0.5 h, 1 h, and 4 h. The cells were labelled for nuclei (blue)

Fig. 6

Cell adhesion and proliferation assays by CCK-8. a HGFs adhesion assays on the samples after incubation for 0.5 h, 1 h and 4 h (*p < 0.05 when compared with the other groups). b HGFs proliferation assays on the samples after incubation for 1 day, 3 days, 5 days and 7 days (*p < 0.05 when compared with the other groups)

Fig. 7

ELISA and RT–qPCR results. a Concentration of FN synthesized by HGFs on the four groups samples after 4 h and 1 day. b RT–qPCR detection of the adhesion-related gene expression of HGFs cultured on the four groups of samples for 4 h and 1 day with statistical significance by *p < 0.05. Expression of ITG-β1, FAK and VCL was presented by the relative amount of mRNA with the Formula 2^{(−△△Ct)}

Fig. 8

CCK-8 assays for detecting the material toxicity to HGFs. HGFs growth assays on the 3D-TNTs, 3D-TNT_1/2, 3D-TNT₁, 3D-TNT₂ and 3D-TNT₅ after incubation for 4 h and 1day

Fig. 9

Intracellular ROS. Flow cytometry detection of the intracellular ROS of HGFs cultured on the four groups of samples for 4 h and 1 day with statistical significance at *p < 0.05