5.1 Titanium plates (TPs)
Commercially available plates made of pure titanium (15 mm in diameter and 1.5 mm in thickness) were used (Fig.5). TPs were divided randomly into two groups: control (Cont) and Ca (hydrothermal treatment with CaCl2 solution (20 mmol/L) for 10 h at 160°C).
5.2 Scanning electron microscopy (SEM) of the TP surface
After the two groups of TPs had dried naturally, the surface was sprayed with gold. We observed the morphology of each TP surface by SEM using an acceleration voltage of 15 kV and magnification of ×800.
5.3 Energy-dispersive spectroscopy (EDS) of the TP surface
After TPs in the two groups had dried naturally, the surface was sprayed with gold. We analyzed the elemental composition on the surface of each TP by EDS.
5.4 Culture of hGF-1 cells
TPs in both groups were cleaned ultrasonically with pure acetone for 20 min, anhydrous ethanol for 10 min, deionized water for 10 min, and then sterilized with ultraviolet light. hGF-1 cells (2×104 cells/mL, CL-0356, Procell Life Science&Technology Co.,Ltd.) were inoculated on the surface of TPs and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum was added. Culture was undertaken in a sterile environment at constant temperature (37°C) in an incubator containing 5%CO2.
5.5 Adhesion of hGF-1 cells
Cell Counting Kit (CCK)-8 diluent solution (500 μL) was added to each TP, and the latter placed in an incubator at 37°C for 1 h away from light. The optical density of each well at 450 nm was measured, and the reference wavelength was 450 nm. The adhesion and proliferation of hGF-1 cells on the TP surface were detected at 1, 8, and 12 h, as well as at 1, 3 and 5 d.
5.6 Immunofluorescence staining for adherent proteins
TPs in both groups were cultured with hGF-1 cells for 3 days. Then, TPs were rinsed twice with phosphate-buffered saline, followed by addition with 4% paraformaldehyde, and fixed for 30 min at room temperature. Integrin-β1 (1:250 dilution) was added in a wet box at 4℃ and the TPs left overnight. TPs were rinsed thrice with PBS (5-min each time). A goat anti-rabbit secondary antibody solution (1:200 dilution) was added dropwise, and incubation allowed for 2 h at 37°C. A phalloidin solution (1:200 dilution) was added and allowed to act for 30 min at 37°C. TPs were placed in an oven at 60℃ for drying. After the tablets were sealed with anti-fluorescence quenching sealing agent, observed by fluorescence microscopy.
5.7 Statistical analyses
SPSS 23.0 (IBM, Armonk, NY, USA) was used for data analyses. All experiments were repeated independently three times. Data are the mean ± standard deviation. The experimental data of each group had a normal distribution and homogeneity of variance. One-way analysis with T-test significant difference test were also used for statistical analyses. P < 0.05 was considered significant.