Cell isolation and culture

We obtained DPSCs from third molars extracted from three healthy volunteers between 18 and 40 years old. The study was approved by the ethics committee (ID: 3729/2021), and all participants gave written informed consent. Several vials of cells were frozen as a reserve prior to testing. Cell culture passages 3–6 were used in the experiments.

Dental pulp was cut into small pieces with a scalpel and subsequently disaggregated in a collagenase (3 mg/mL) and dispase (4 mg/mL) solution for 1 h at 37 °C. The digested extracts were strained (through a 70-μm filter) and cultured on a 100-mm cell culture plate. Cell cultures were expanded in complete cell culture medium (alpha-MEM medium supplemented with 10% FBS, 400 mM of penicillin/streptomycin, and 2.5 μg/ml of amphotericin B) and incubated at 37 °C in a 95% oxygen and 5% carbon dioxide mixture.

For alkaline phosphatase activity assays and Alizarin red S staining, we used an osteogenic medium (complete medium supplemented with 50 μl/ml ascorbic acid, 7.5 mM β-glycerol phosphate, and 1 μM dexamethasone) to induce cell differentiation.

DPSC characterization

The mesenchymal phenotype of the cells isolated was confirmed by analyzing the expression of mesenchymal markers using flow cytometry (Human Mesenchymal Stem Cell Multi-Color Flow Kit- FMC002- R&D SYSTEMS).

The cell samples were washed with 2 ml of staining buffer and centrifuged at 300 g for 5 min. Subsequently, 1.5 × 106 cells in 1 ml of staining buffer were transferred into a 5 ml cytometry tube. Then, 10 μl of four fluorochrome-conjugated antibodies (CD105, CD46, CD90 and CD45) and their corresponding isotype controls, were added to the cells. The mixture was then incubated for 45 min at room temperature in the dark. After incubation, the cells were washed with 2 ml of staining buffer, centrifuged, and resuspended in 400 μl of staining buffer for flow cytometry analysis.

Over 95% of the cells were positive for the mesenchymal markers CD105, CD46, and CD90, and negative for CD45 (Fig. 1).

Fig. 1

Characterization of dental pulp stem cells

Preparation of bone substitute

Bio-Oss® (BO) (Geistlich Pharma AG., Wolhusen, Switzerland) 0.25–1 mm particles (Batch: 82,001,136) were weighed with a precision balance in a laminar flow chamber and used as a dilution (5 mg/ml, incubated for 24 h in complete medium).

Photobiomodulation

For PBM, we used an Epic™ 10 diode laser (Biolase, CA, USA), emitting at 940 nm in continuous mode and with an output power of 0.2 W, applying an energy density of 5 J/cm2 for 25 s, 7 J/cm2 for 35 s, or 10 J/cm2 for 50 s (hereon referred to PBM5, PBM7, and PBM10, respectively). The DPSCs seeded with a bone substitute were irradiated once using the “surgical handpiece” (diameter 0.5 cm) at a standard distance of 1 mm above the top of the well plates under dark conditions.

Evaluation of DPSC viability/proliferation – the MTT assay

Cells were cultured in 96-well plates seeded at 5000 cells/cm2 with 200 μl of complete culture medium, and 24 h after incubation, they were treated with PBM5, PBM7, PBM10 and BO alone or in combination with PBM5, PBM7, or PBM10. The results were quantified at two different incubation times (48 and 72 h). After the incubation period, the medium was replaced with a 1 mg/ml solution of MTT ((3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide), and cells were incubated for 4 h. Then, MTT was removed from the well and the formazan, a product of cell metabolism, was dissolved in 100 μl of dimethyl sulfoxide. Lastly, absorbance was measured with a spectrophotometer at a wavelength of 570 nm and a reference wavelength of 690 nm.

Evaluation of DPSC viability – Viability staining assay

DPSCs were cultured as for the MTT assay (in 96-well plates, seeded at 5000 cells/cm2, and with 200 μl of complete culture medium). After 24 h of incubation, cells were treated with PBM5, PBM7, PBM10 and BO alone or in combination with PBM5, PBM7, or PBM10. The results were assessed at two different incubation times (48 and 72 h), using a Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (Biotium, Fremont, CA, USA), according to the instructions of the manufacturer. After the incubation period, cells were rinsed with PBS to remove serum esterase activity, and the solutions of calcein AM and ethidium homodimer III (Eth-DII) contained in the kit were added. After being held for 30 to 45 min at room temperature, the stains were replaced by PBS, and cells were observed using an inverted fluorescence microscope.

Evaluation of cell migration

DPSCs were cultured in 48-well plates (5000 cells/cm2 in 400 μl of complete culture medium) and incubated until monolayer confluence. Next, some areas of the cell monolayer were “wounded” (scraped away) with the sterile tip of a micropipette, the medium was replaced and the cultures were treated with PBM5 and BO alone or in combination with PBM5. Cell migration was quantified by taking photos of the damaged areas at 0 and 48 h of incubation using an inverted microscope with a digital camera (Nikon Eclipse TE2000-U, Nikon, Japan). The distance between the edges of the damaged area in the cell monolayer was measured by pixel counting using the ImageJ digital image processing software (National Institutes of Health [NIH], Maryland, USA), and the following equation was applied: migration distance = cell-free initial distance—cell-free distance after 24 h.

Evaluation of alkaline phosphatase enzyme activity

Cells were cultured as for the cell migration assay (in 48-well plates seeded at 5000 cells/cm2 with 400 μl of complete culture medium) and incubated for 72 h. Subsequently, the medium was replaced by an osteogenic medium, and cells were treated with PBM5 and BO alone or in combination with PBM5.

The activity of alkaline phosphatase was assessed using a commercial kit (Alkaline Phosphatase Activity Fluorimetric Assay Kit; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. In this kit, ALP cleaves the phosphate group of the non-fluorescent substrate of the disodium salt of 4-methylumbelliferyl phosphate (MUP), producing a strong fluorescent signal. The ALP activity is measured by the amount of fluorescence emitted in each treatment.

After 7 days of culture, cells were lysed in a 1% Triton X-100 solution (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. The cells from each treatment were transferred to Eppendorf tubes and centrifuged at 13,000 G for 3 min. Subsequently, 5 μL of the supernatant from each treatment and 105 μL of the ALP Buffer solution containing the kit were added to a 96-well fluorescence plate. Four replicates were placed for each treatment (S) and a duplicate of each one as a control (SBC). Then, 20 μL of stop solution was added to the SBC wells to stop alkaline phosphatase activity. Additionally, 20μL of the MUP substrate at 0.5 mM was added to all S and SBC wells.

A standard curve (ST) was also prepared according to the manufacturer's instructions, using concentrations of 0, 0.1, 0.2, 0.3, 0.4, and 0.5 nmol per well.

All preparations (S, SBC, and ST) were then incubated for 30 min at 25 °C, protected from light and fluorescence (relative fluorescence units, RFU) was measured in a FLUOstar Omega plate reader (BMG Lab. Technologies, USA), with an excitation wavelength of 360 nm and an emission wavelength of 440 nm.

Evaluation of in vitro biomineralization potential - Alizarin red S staining

DPSCs were cultured as for the cell migration and ATP activity assays (in 48-well plates seeded at 5000 cells/cm2 with a 400 μl of complete culture medium) and incubated for 3 days. Next, the medium was replaced by an osteogenic medium, and cells were treated with PBM5 and BO alone or in combination with PBM5. For this assay, as well as the positive control (untreated cells cultured in osteogenic medium), there was a negative control (untreated cells cultured in complete control medium). The medium was replaced every 3 days throughout the experimental study (21 days).

After the culture period, cells were rinsed with PBS, fixed with 4% formaldehyde, and treated with Alizarin red S (VWR International, Barcelona, Spain) at 2% to stain the nodules of calcium formed in each of the samples yellowish orange. Subsequently, photographs were taken of all the wells using an inverted microscope equipped with a digital camera (Nikon Eclipse TE2000-U).

Statistical analysis

Data were examined by performing analysis of variance and Tukey's multiple comparison tests using GraphPad Prism v7.0 (GraphPad Software Inc, San Diego, USA). The threshold for statistical significance was set at p = 0.05 (95% confidence interval).