Osteoblast-like cells were isolated from the calvariae of 3–5-day-old Wistar rats (Sankyo Labo Service Corporation, Tokyo, Japan) as described previously (Yokose et al., 1996; Gu et al., 2006). Calvariae without periosteums were aseptically dissected and processed by serial enzymatic digestion. Briefly, the calvariae were cut into pieces using scissors, which were suspended in 3 mL enzyme mixture and incubated in a water bath shaker at 37°C for 20 min. After the incubation, the supernatant containing released cells were collected in a new tube and mixed with an equal volume of growth medium. The growth medium was alpha minimal essential medium (α-MEM; Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, United States) and 1% antibiotic-antimycotic mixture (Invitrogen, Carlsbad, CA, United States). This enzymatic digestion was repeated four times; the cells isolated from the last three fractions, which are abundant in osteoblast-like cells (Gu et al., 2006), were used in all experiments. All protocols for animal use and euthanasia were approved by the Animal Care Committee of the Experimental Animal Center at Tokyo Medical and Dental University (A2019-098C3).

Cells were precultured in 10-cm culture dishes in growth medium. When the cells reached 80% confluency, they were seeded in 35-mm dishes for cell proliferation assay, calcification assay, and evaluation of gene expression. All cultures were maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The medium was changed every 3 days.

An Er:YAG laser apparatus (DELight; HOYA ConBio, Fremont, CA, United States) emitting at a wavelength of 2.94 μm was employed in this study. Laser irradiation was performed perpendicularly to the bottom of the culture dish at a distance of 25 cm, with the handpiece fixed using a stand as described previously (Aleksic et al., 2010). To completely irradiate the 35-mm dish, neither cover sleeve nor contact tip was mounted with the handpiece. The medium was removed immediately before irradiation and all irradiations were performed in the absence of culture medium. The output energy settings were 35, 55, 70 mJ/pulse and 20 Hz on the panel, with an irradiation time of 60 s. The actual energy levels at the dish surface were 17.6, 26.4, 34.5 mJ/pulse, and the actual energy densities were 1.8, 2.7, 3.6 mJ/pulse/cm², resulting in total energy densities of 2.2, 3.3, 4.3 J/cm², respectively. Immediately after irradiation, the appropriate medium was added to the dish, according to the subsequent experiments.

Cell surface temperatures were measured before, during, and after laser treatment using a non-contact infrared laser thermometer (wavelength 630–670 nm, output <1 mW; MiniTemp, RayTek Corporation, Santa Cruz, CA, United States) on monolayer MC3T3-E1 cells at room temperature (22–23°C).

Osteoblast-like cells seeded at 1 × 10³ per dish were cultured for 48 h with growth medium, then serum-starved by replacing with starvation medium (α-MEM containing 0.5% FBS and 1% antibiotic-antimycotic mixture) for an additional 24 h to induce cell-cycle synchronization. Prior to laser irradiation, the cells were labeled with 0.5 μL of 5 μM CellTrace Violet (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s protocol. At 24 h following irradiation, the cells were analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific). As an initial generation control, cells were labeled with CellTrace Violet immediately before cytometry. Non-labeled cells were used as a negative control.

We also evaluated cell proliferation using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) at 1, 2, and 3 days after laser irradiation as described previously (Aleksic et al., 2010; Kong et al., 2018). Laser irradiation was performed in the absence of medium after a 24 h-starvation. Immediately after irradiation, dishes were refilled with growth medium. CCK-8 was applied to non-irradiated control and irradiated cells at 1, 2, and 3 days after laser irradiation. A 1:10 mixture of kit solution and α-MEM (750 μL) was added to the cultured osteoblast-like cells and the dishes were incubated at 37°C for 30 min. The optical absorbance of samples was measured using a fluorescence microplate reader at a wavelength of 450 nm (FMAX, Molecular Devices, Sunnyvale, CA, United States). Relative cell proliferation was expressed as a ratio of proliferation activity of irradiated cells to that of non-irradiated cells.

Osteoblast-like cells were seeded at 5 × 10⁴ cells per dish. When cells reached confluency, laser irradiations were performed in the absence of the medium. Immediately after irradiation, the dishes were re-filled with osteoinduction medium [growth medium containing 50 μg/mL ascorbic acid (Wako, Osaka, Japan), 10 mM β-glycerophosphate (Sigma-Aldrich), and 10 nM dexamethasone (Sigma-Aldrich)] for osteoinduction (Ozawa et al., 1998). At 7, 14, and 21 days following irradiation, dishes were stained with 1% Alizarin red S to assess calcified matrix production. Relative areas of staining of irradiated and non-irradiated cells were measured using Photoshop CC software (Adobe Systems Inc., San Jose, CA, United States).

Quantitative evaluation of Alizarin red S staining by solubilization was performed and absorbance measured as described previously (Gregory et al., 2004). Briefly, dye from stained monolayers was extracted by 10% acetic acid and neutralized with 10% ammonium hydroxide, and absorbance of the supernatants was detected at 405 nm. Relative cell calcification is expressed as absorbance relative to that of non-irradiated cells.

Osteoblast-like cells were seeded at 5 × 10⁴ per dish. When cells reached confluency, laser irradiations were performed in the absence of medium. Immediately after irradiation, the dishes were re-filled with osteoinduction medium as described above. Cells were collected for RNA extraction at 3, 6, and 12 h, as well as 1, 3, 7, and 14 days, after irradiation.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States) with NucleoSpin RNA (TaKaRa Bio, Shiga, Japan). Complementary DNA was synthesized using a PrimeScript RT Master Mix (TaKaRa Bio) according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq II (TaKaRa Bio) and Thermal Cycler Dice Real Time System II (TaKaRa Bio). The 18s rRNA expression level was used as an internal control. The results are shown as a ratio of the gene expression level relative to the non-irradiated control samples from the same time point. PCR primers are listed in Supplementary Table S1.

The Agilent Low Input Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA, United States) was used to generate cRNA from an input of 200 ng total RNA for single-color (Cy3) microarray analysis. Then, cRNAs were analyzed by hybridization onto an Agilent SurePrint G3 Unrestricted Gene Expression 8 × 60 K Microarray (Agilent Technologies). Fluorescence signals were detected using the Agilent Microarray Scanner System (Agilent Technologies). Raw microarray data were extracted using Feature Extraction Software (ver. 11.0.1.1; Agilent Technologies).

Data are presented as means ± standard error (SE). Experiments were repeated six times unless stated otherwise. Data distributions were assessed with the Shapiro–Wilk test. Paired t-tests with Bonferroni corrections were performed for multiple-group comparisons in parametric data. For non-parametric data, the Steel method was applied for comparisons with control samples. Data were analyzed using JMP v.9 software (SAS Institute Inc., Cary, NC, United States). P < 0.05 was considered statistically significant.

Microarray data were quantile normalized and log₂-transformed (Shippy et al., 2006) using R (ver. 3.6.0). The Limma Bioconductor package (ver. 3.40.6) (Ritchie et al., 2015) was used to identify differentially expressed genes. Gene set enrichment analysis (GSEA¹; Subramanian et al., 2005) was carried out with hallmark gene sets (Liberzon et al., 2015).

Irradiation at 2.2, 3.3, and 4.3 J/cm² increased the surface temperatures of cell monolayers from 22.8 ± 0.1°C before irradiation to 25.2 ± 0.1, 26.2 ± 0.1, and 27.4 ± 0.2°C, respectively, with an average increase of 2.4, 3.4, and 4.6°C, respectively (Figure 1A). The area under the curve clearly showed a significant difference in thermogenesis between groups (Figure 1B).

The fluorescence intensity distributions of cells labeled by CellTrace Violet at 24 h following irradiation are shown in Figure 2A. Because CellTrace Violet dye molecules are sequentially halved by cell division, cell proliferation measurements can be derived from the sequential reduction of its fluorescence (Kumar et al., 2013). Cells irradiated at 2.2, 3.3, and 4.3 J/cm² and non-irradiated control cells showed a shift of the histogram peak toward the left relative to initial-generation control cells, indicating proliferation during the 24-h period following irradiation. However, there was no significant difference in average fluorescence values between non-irradiated control and irradiated cells (Figure 2B).

Cell proliferation evaluated by CCK-8 are shown in Figures 2C–E. Laser irradiation at any fluence showed no significant differences in proliferation between non-irradiated and irradiated cells at 1, 2, and 3 days after laser irradiation (n = 6).

Osteoblast-like cells showed calcification after osteoinduction (Supplementary Figures S1A–C). Calcification data from 7 days after Er:YAG laser irradiation at 2.2, 3.3, and 4.3 J/cm² is shown in Figure 3A. Calcification was significantly increased in cells irradiated at 3.3 J/cm² compared to non-irradiated control cells. However, there was no significant difference in Alizarin red S staining area between cells irradiated at 2.2 or 4.3 J/cm² and non-irradiated control cells (Figure 3B). The relative absorbances of solutions extracted from stained cells irradiated at 3.3 J/cm² were significantly higher than those of non-irradiated control cells (Figure 3C).

Both non-irradiated and irradiated cells showed considerable mineral deposits at 14 and 21 days after irradiation (Figures 3D,G). The relative areas stained by Alizarin red S for all fluences showed no significant differences relative to those of non-irradiated control cells on days 14 and 21 (Figures 3E,H). There were no significant differences in relative absorbances of solutions extracted from stained cells between irradiated and control cells at 14 and 21 days after irradiation (Figures 3F,I).

Since calcification of osteoblast-like cells was significantly increased after 3.3 J/cm² Er:YAG laser irradiation, we evaluated Runx2, Sp7, Alpl, Bglap, Msx1, Msx2, and Dlx5 mRNA expression (Figures 4A1–G3). Bglap expression was dramatically increased after osteoinduction (Supplementary Figure S1D). Irradiation produced no significant changes at 3 h in Runx2 expression (Figure 4A1). Runx2 expression tended to increase in cells irradiated at 3.3 J/cm² after 6 h, but the difference was not significant (Figure 4A2). Runx2 expression in cells irradiated at 4.3 J/cm² significantly decreased at 12 h after irradiation (Figure 4A3). Meanwhile, the relative expression of Sp7 in cells irradiated at 2.2 and 3.3 J/cm² tended to increase at 3 and 6 h, but the increase did not reach statistical significance (Figures 4B1,B2). At 6 h after laser irradiation, Sp7 expression in cells irradiated at 4.3 J/cm² was significantly increased over control levels (Figure 4B2). No significant differences in Sp7 expression were observed at 12 h (Figure 4B3). Alpl expression at 6 h after irradiation at 4.3 J/cm² was significantly increased over control levels. Relative expression of Alpl in cells irradiated at 3.3 J/cm² tended to increase at 3 and 6 h after laser irradiation, but differences were not significant (Figures 4C1,C2). Also, no significant difference was observed in Alpl expression between cells irradiated at 2.2 J/cm² and control cells (Figures 4C1–C3). Bglap expression was significantly increased in cells irradiated at 3.3 J/cm² after 6 h (Figure 4D2). When cells were irradiated at 4.3 J/cm², the expression of Bglap at 3 and 12 h tended to increase, although the increase was not statistically significant. There was no significant difference in Bglap expression between cells irradiated at 2.2 J/cm² and control cells (Figures 4D1–D3). No significant changes were observed in Msx1 expression at 3, 6, and 12 h after laser irradiation at any fluences (Figures 4E1–E3). However, Er:YAG laser-irradiated cells tended to be increased Msx1 expression at 3 and 6 h (Figures 4E1,E2). Cells irradiated at 2.2 J/cm² had significantly decreased Msx2 expression at 6 h, although no significant differences were observed at 3 and 12 h (Figures 4F1–F3). Dlx5 expression in laser-irradiated cells at 3.3 and 4.3 J/cm² tended to increase at 3 and 6 h, but there was no significant difference (Figures 4G1,G2). In particular, cells irradiated at 3.3 J/cm² showed a strong increase at 6 h (Figure 4G2). Relative expression of Dlx5 in cells irradiated at 2.2 J/cm² was significantly decreased at 12 h after irradiation (Figure 4G3).

Interestingly, cells irradiated at 4.3 J/cm² showed significantly higher Hspa1a expression compared to control cells at 6 h after irradiation. There was no significant increase in Hspa1a expression after irradiation at 2.2 and 3.3 J/cm² (Figures 5A–C).

The relative expression levels of Runx2, Sp7, Alpl, Bglap, Msx1, Msx2, and Dlx5 at 1, 3, 7, and 14 days after laser-irradiation are shown in Figures 6A1–G4. Runx2 expression in cells irradiated at 3.3 J/cm² was significantly increased at 3 days after irradiation, although there were no significant differences between irradiated and non-irradiated control cells at 1, 7, and 14 days after irradiation (Figures 6A1–A4). In addition, no significant differences were observed in Sp7, Alpl, Bglap, Msx1, Msx2, and Dlx5 expression between irradiated and non-irradiated control cells after 1, 3, 7, and 14 days (Figures 6B1–G4). However, Sp7 expression at 3 days (Figure 6B2) and Msx1 expression at 7 and 14 days (Figures 6E3,E4) tended to be increased in cells irradiated at 3.3 J/cm².

Since calcification and Bglap expression at 6 h following irradiation were significantly increased in cells irradiated at 3.3 J/cm², microarray analysis was performed to obtain a comprehensive overview of the gene expression profile (control, n = 4; cells at 6 h after 3.3 J/cm² irradiation, n = 4). All microarray data are shown in Supplementary Table S2. No genes showed |fold change| >2 and q < 0.1. However, 33 genes showed changes for which P < 0.01 (Figure 7A). We subsequently focused on genes related to inflammation and Wisp2, validating the microarray data by qPCR. Wisp2 expression showed no significant difference, however, it tended to decrease in irradiated cells at 6 h after irradiation at 3.3 J/cm² (Figure 7B). The expression of Il1rl1 and Cxcl1 on cells irradiated at 3.3 J/cm² did not differ significantly at any time point after irradiation (Figures 7C,D). There were significant increases in Cxcl3 and Mmp3 expression at 6 h after irradiation (Figures 7E,F). Microarray data from this study are available in the Gene Expression Omnibus database² as GSE146098.

GSEA was performed using hallmark gene sets to evaluate the gene expression changes caused by 3.3 J/cm² laser irradiation at 6 h. The gene sets enriched in irradiated cells were identified with FDR-q < 0.25 (Table 1). The details for the Notch Signaling gene set [normalized enrichment score (NES) = 1.43, q = 0.138] are shown in Figure 8.