Preparation of the nano-TiO2 coating

SLM 3D-printed titanium alloy (Ti6Al4V) sheets with a diameter of 10 mm and a thickness of 1 mm were printed with a metal 3D printer (AM 400, Renishaw, UK) (processing parameters: power, 200 W; point spacing, 55 μm; exposure time, 50 s). The titanium alloy sheets were cleaned in ultrapure water for 15 min and pickled, the surface melted powder was removed, and the samples were sonicated with absolute ethanol and ultrapure water for 15 min before drying naturally. Then, the sample was placed in a 30% hydrogen peroxide solution, heated at 80 °C for 1 h, removed, cleaned with ultrapure water, and allowed to dry naturally. The titanium alloy sheet was placed into a Teflon-lined stainless-steel autoclave, and a precursor solution containing hexafluorotitanic acid (H2TiF6, 0.885 mM, Aladine), hydrochloric acid (HCl, 13 mM, Yonghua), isopropanol (C3H8O, 33 mM, Sinopharm), and hydrogen peroxide (H2O2, 13 mM, Yonghua) was added. After the reaction, the titanium alloy sheets were removed and cleaned with ultrapure water for 5 min; the experimental group was labelled Ti6Al4V@TiO2, and the control group did not undergo a hydrothermal reaction and was labelled Ti6Al4V. Both samples were placed in ultrapure water at 80 °C overnight and autoclaved at 120 °C for backup use.

Materials characterization

The sample surface was observed via field-emission scanning electron microscopy (FE-SEM, SU 8000, Hitachi, Japan), and the surface elements were quantified by using energy dispersive X-ray spectrometry (EDS). The water contact angle was measured at room temperature with an optical contact angle metre (OCA 20, Dataphysic, Germany) to assess its hydrophilicity, and ultrapure water was used as the liquid (n = 5 per group). The crystal structure was identified via X-ray diffraction (XRD, X’Pert PRO, PANalytical) with a scan rate of 20°/min and a Cu-Kα source operated at 40 kV-40 mA.

In vitro photothermal propertiesPhotothermal conversion capacity

The sample was immersed in 1 mL of phosphate-buffered saline (PBS) and irradiated with 808 nm NIR light (LSR808NL, Ningbo Yuanming, China) at a power of 0.8 W/cm2 for 15 min. The sample temperature was measured every 1 min with an infrared thermal imager, and the heating curve was plotted (n = 3 per group).

Photothermal stability

The sample was immersed in 1 mL of PBS and irradiated with 808 nm NIR light at a power of 0.8 W/cm2 for 15 min. Then, the light source was turned off, and the sample was naturally cooled for 15 min; this was considered one on‒off cycle. The sample temperature was measured every 1 min with an infrared thermal imager, and the temperature changes were repeated for three cycles.

In vitro antibacterial propertiesViable plate count experiment

The experimental materials were divided into four groups and labelled Ti6Al4V, Ti6Al4V + NIR, Ti6Al4V@TiO2, and Ti6Al4V@TiO2 + NIR according to the presence or absence of illumination. The sterilized samples were placed in a 24-well plate, submerged with 1 mL S. aureus (ATCC 25923, People’s Hospital of Quzhou) / E. coli (ATCC 25922, People’s Hospital of Quzhou) suspension (1 × 106 CFU/mL) and incubated at 37 °C for 4 h. The samples were subsequently transferred to a new 24-well plate, 1 mL of PBS was added, and the illumination group was irradiated with NIR light at a wavelength of 808 nm at 0.8 W/cm2 for 15 min and rinsed with PBS to remove surface floating bacteria. The sample was placed in a centrifuge tube containing 5 mL of sterile PBS and shaken on a vortex oscillator for 5 min to shed bacteria from the sample surface into PBS. After the same multiple of dilutions for each group, 10 µL of bacterial suspension was evenly coated on agar plates and incubated at 37 °C for 24 h, the number of bacterial colonies was recorded, and the antibacterial rate of each group was calculated. The antibacterial rate = [(λ0 - λt)/λ0] × 100%, where λ0 is the average number of viable bacteria in the Ti6Al4V group and λt is the average number of viable bacteria in the Ti6Al4V + NIR, Ti6Al4V@TiO2, and Ti6Al4V@TiO2 + NIR groups (n = 5 per group).

Live/dead bacterial staining

The sterilized samples were placed in a 24-well plate, submerged with 1 mL bacterial solution of 1 × 106 CFU/mL, and incubated at 37 °C for 4 h. The samples were subsequently transferred to a new 24-well plate, 1 mL of PBS was added, and the illumination group was irradiated with NIR light at a wavelength of 808 nm at 0.8 W/cm2 for 15 min and rinsed with PBS to remove surface floating bacteria. In accordance with the instructions of the Live/Dead Bacteria Staining Kit (L13152, Thermo Fisher, USA), a mixture of SYTO 9 and propidium iodide (PI) was added to the sample surface, incubated for 15 min in a light-free environment. The samples were subsequently observed and photographed under a fluorescence inverted microscope. Images were fitted with ImageJ software (n = 5 per group).

In vitro biocompatibility assessment

The Ti6Al4V group and the Ti6Al4V@TiO2 + NIR group were selected for the in vitro biocompatibility experiments based on the in vitro antimicrobial experimental results.

Culture of the osteoblasts

Mouse preosteoblasts (MC3T3-E1 Subclone 14, Propsy, China) were cultured in MC3T3-E1 Subclone 14 cell-specific medium (Propsy, China) at 37 °C and 5% CO2. The cells were digested with 0.25% trypsin (Yuanye, China) when they reached 90% confluence.

Cell adhesion

The sterilized samples were placed into a 24-well plate, the digested cells were inoculated onto the sample surface at a density of 2 × 104 cells per well, and the illumination groups were irradiated with near-infrared light for 15 min. The samples were removed after 30, 60 and 120 min of culture, rinsed with PBS and placed in a new 24-well plate; next, 50 µL of CCK-8 solution (Solarbio, China) and 500 µL of cell culture medium were added to each well, and the samples were incubated in a 37 °C incubator for 2 h. 100 µL of the reaction mixture were added to a 96-well plate, and the OD at 450 nm was measured via a microplate reader (n = 5 per group).

Cell proliferation

The sterilized samples were placed into a 24-well plate, the digested cells were inoculated onto the sample surface at a density of 8000 cells per well, and the illumination groups were irradiated with near-infrared light for 15 min. Samples were removed after 1, 3, and 7 d of culture, rinsed with PBS and placed into a new 24-well plate, and 50 µL of CCK-8 solution and 500 µL of cell medium were added to each well; the well-plate was placed in a 37 °C incubator for 2 h. 100 µL of the reaction mixture were added to a 96-well plate, and the OD at 450 nm was measured via a microplate reader (n = 5 per group).

Cell morphology analysis

The sterilized samples were placed into a 24-well plate, the digested cells were inoculated onto the sample surface at a density of 8000 cells per well, and the illumination groups were irradiated with near-infrared light for 15 min. The samples were removed after 1, 3, and 7 d of culture; next, 2.5% glutaraldehyde (Yuanye, China) was added to the samples, and then they were placed in a 4 °C refrigerator overnight. The samples were rinsed with PBS and dehydrated with a gradient of 50%, 70%, 80%, 90%, and 100% ethanol. Afterward, tert-butanol was added to the samples, which were incubated at 4 °C for 10 min. The adhesion morphology of the cells on the sample surface was observed via SEM after drying and metal spraying (n = 5 per group).

Actin staining

The sterilized samples were placed into a 24-well plate, the digested cells were inoculated onto the sample surface at a density of 2 × 104 cells per well, and the illumination groups were irradiated with near-infrared light for 15 min. The samples were removed after 2 and 6 h of incubation, rinsed with PBS, and fixed in 4% paraformaldehyde (Yuanye, China) for 20 min at room temperature. Then, the samples were treated with 0.5% Triton X-100 (Yuanye, China) for 5 min and rinsed with PBS. FITC phalloidine (Solarbio, China) was added, the sample was incubated for 30 min in a light-free environment. The cell nuclei were counterstained with DAPI dye solution for 3 min. Cell morphology and actin were observed under a fluorescence inverted microscope (n = 5 per group).

Statistical analysis

Experimental data analysis was performed using SPSS 25.0 software. The results are presented as the mean ± standard deviation (SD). The Shapiro-Wilk test was used to assess the normality of variables and the Levene’s test was performed to assess the equality of variances. The results showed normal distribution and of the measurement values, and the datasets meet the assumptions of homoscedasticity. The differences between the groups were evaluated via t tests and one-way ANOVA followed by a least significant difference test. Statistical significance was accepted at *p < 0.05.