Patients with an age between 18 and 80 years with a good general health were considered for the study. Specific exclusion criteria were proven osteometabolic diseases, inflammatory and/or infectious pathologic conditions at the site of surgery, genetic alterations, neoplastic disorders, kidney disease, diabetes, chronic inflammatory diseases and treatment with corticosteroids, bisphosphonates or denosumab.

Four subjects heterogeneous as for age and sex but homogeneous as for anatomical site of intervention and sampling, were selected. Three of them underwent extraction of wisdom teeth whereas the fourth underwent implant fixture insertion (Table 1).

For this study, waste fragments of bone tissue released during the osteotomy procedure (functional to the basic surgery) were used. They were used in accordance with the Declaration of Helsinki and its later amendments, and the study was approved by the local Ethical Committee (Rif. 7447 – Prot. 0141/2024). Osteotomy was performed with rotary instruments, under irrigation. Using a surgical excavator, the fragments of bone tissue released during the intervention were collected from the surgical site, placed into a tube containing Modified Essential Medium with α modification (αMEM, Merck, Darmstadt, Germany) supplemented with 1% solution of Penicillin and Streptomycin (P/S) and immediately sent to the laboratory under temperature-controlled conditions.

To isolate bone marrow stromal cells (BMSCs) and osteoblast precursors, jaw trabecular bone fragments were minced in small pieces and incubated in 10 ml of a 2 mg/ml Collagenase II solution (240 U/mg, Gibco) in Hanks’ Balanced Salt Solution (HBSS) for 45 min at 37 °C on a shaking plate. After digestion, 5 ml of αMEM supplemented with 20% Fetal Bovine Serum (FBS), 1% P/S and 1% L-glutamine (L-gln) (complete growth medium) were added and the resulting suspension was filtered through a 100 μm nylon mesh in order to separate bone fragments from dissociated cells. The filtered suspension was then centrifuged at 1300 rcf (relative centrifugal force) for 5 min. The resulting pellet was resuspended in 10 ml of complete growth medium and cells were plated in a 100 mm-diameter dish. Medium was changed twice a week; when the cells reached 80% of confluency were lifted by adding 0,25% Trypsin for 5 min and plated in 24-well plates at the density of 2⋅104 cells per well. Six wells per plate were used and the remaining wells were filled with a Phenol Red-containing αMEM to shield the cell cultures and to avoid a secondary radiation during the laser treatment.

Cells were induced to osteogenic differentiation by incubation for 14 days with Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, USA) with no Phenol Red, supplemented with 10% FBS, 1% P/S, 1% L-gln, 10− 8 M Dexamethasone, 4 mM β-glycerophosphate and 50 μg/ml of Ascorbic Acid (Merck). During osteogenic differentiation, cells underwent laser treatment according to different schedules (Table 2). At the end of differentiation, cells were either collected for gene expression analyses or fixed for 10 min with 4% formaldehyde solution for von Kossa and alkaline phosphatase (ALP) stainings.

A double diode red (650 nm) and NIR (910 nm) superpulsed laser was used (Lumix 2 diode laser, Fisioline LTD, Verduno CN, Italy). The 650 nm emitting diode was set at 100mW in continuous wave, while the 910 nm emitted at 500mW, at 9 kHz. Each irradiation had a duration of 58 s to release a total amount of 4 Joules/well, chosen according to the settings described by many studies [10]. Three different modes of laser administration were performed (Table 2): a single dose on the 1st day of culture (LT 1); repeated dose on the 1st day of culture and then every other day up to 2 weeks (LT 2); a single dose on the 12th day of culture (LT 3).

In order to expose all cells to the same environmental conditions throughout the study both treated and untreated cultures were kept at room temperature during laser irradiation.

The ability of cells to produce matrix mineralization (mineralized nodules) was analyzed by von Kossa stain. Cells were incubated with 1% Silver Nitrate solution for 20 min under ultraviolet light, washed with distilled water and then incubated with 5% sodium thiosulfate solution for 5 min. Pictures of mineralized nodules were taken with an inverted microscope and quantified with Adobe Photoshop. At least 4 fields from each well were photographed, and at least 3 wells for each experimental point were considered.

Alkaline phosphatase (ALP) is an important enzyme required to enhance the concentration of inorganic phosphates therefore facilitating mineralization [11]. For this reason, it is used as marker of osteogenic differentiation.

ALP cytochemistry was performed using Sigma Aldrich kit reagents (Merck) as previously described [12]. Briefly, 30 mg of Naphtol AS Phosphate were dissolved in 0,5 mL N, N-dimethylformamide and mixed with 100 mL borate buffer with 100 mg of AS blue BB salt. The solution was added to the wells and incubated for 5–10 min at 37 °C. Pictures from at least 4 fields each well were taken and analyzed by measuring the optical density using ImageJ software.

Total RNA was isolated using the TRI Reagent® following manufacturer’s instructions (Merck). Reverse transcription was performed by using PrimeScript RT Reagent Kit (Takara, Kusatsu, Japan). cDNA samples were used as templates for quantitative PCR (qPCR) analysis on a 7500 Fast Real-Time PCR System (Applied Biosystem, Waltham, MA, USA), performed using PowerUP Sybr Green (Thermo Fisher Scientific) and specific primers (Table 3) recognizing genes involved in osteogenic differentiation (CBFA1, SP7, ALPL, BGLAP), matrix deposition and remodelling (COL1A1, COL3A1, COL10A1, MMP1, MMP8, MMP13), osteoclastogenesis (TNFSF11, TNFRSF11B, CSF1) and inflammation (IL6, IL6R, IL1B). The expression level of each gene was normalized to GAPDH expression.

From each patient, at least 4 technical replicates for each analysis were generated. The mean from the 4 replicates was then used to represent each donor in the sample size. In order to show the general trend of the laser treatments on the cells from each donor, results of each biological replicate are shown as relative to each control, which is therefore reported as 1.

A repeated measure one-way ANOVA, corrected for multiple comparisons using Dunnet method was used to compare the effect of the treatment on gene expression analyses, Von Kossa and ALP staining. In all experiments a p-value < 0.05 was considered statistically significant. All graphs and statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software, La Jolla, CA, USA).