Light microscopy

Non-differentiated and differentiated cells were cultured on 12-well plates. After 15 days of incubation, the cells were washed with DPBS and fixed with 4% paraformaldehyde (EMS, Hatfield, PA, USA) for 30 min, in the dark, at room temperature. Fixed cells were washed twice with DPBS, and mineral deposits were stained with 2% Alizarin Red S (Merck, Darmstadt, Germany) for 3 min. After staining, cells were washed twice with water and analyzed by light microscopy (Optika, Ponteranica, Italy).

The human commercial cell line, DPSCs (Lonza, Basel, Switzerland) was used. For all studies examining the molecular mechanism of osteogenic differentiation, cells were seeded in 25 cm² flasks and grown under a humidified atmosphere of 5% CO₂ at 37 °C. Cells were precultured in DPSCs growth medium consisted of α-MEM (Merck Sigma-Aldrich, Darmstadt, Germany) supplemented with 2 mM GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA), 0.2 mM ascorbic acid 2-phosphate (Sigma-Aldrich), 10% fetal bovine serum (Biosera, Nuaille, France), 100 U/mL penicillin, and 100 µg/mL streptomycin (Biosera). The medium was replaced every three days. During preculture, cells remained in an undifferentiated stage. After reaching 80–90% confluence, cells were harvested using TrypLE Express Enzyme (Thermo Fisher Scientific) and then seeded at 5000 cells/cm². When DPSCs reached 50% confluence (day 0), the growth medium was replaced with osteoinductive medium OsteoMAX-XF (Merck Sigma-Aldrich) for osteogenic differentiation and mineralization. The cells were grown in this specific medium for 24 days. In parallel, undifferentiated DPSCs (control) were cultured in the DPSCs growth medium. Media were changed every 3 days. Cell morphology was monitored during the experiment by light microscopy (Optika, Via Rigla, Italy).