Dpscs

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).

DPSCs (Lonza, Walkersville, USA) were characterized by surface marker profiling (negative: CD34, CD45, and CD133; positive: CD105, CD166, CD29, CD90, and CD73) performed by the manufacturer. DPSCs at passage 6–8 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, Saint Louis, USA) supplemented with 10% fetal bovine serum (Bovogen, Melbourne, Australia) and 1% penicillin–streptomycin solution (Fujifilm Wako, Osaka, Japan) at 37 °C in 5% CO₂ until 80% confluency. Subsequently, these cells were washed with phosphate-buffered saline (PBS; Fujifilm Wako, Osaka, Japan), and the culture medium was replaced with the vehicle medium (serum-free DMEM). After 48 h of incubation in 21% and 1% O₂ (normoxic [N-] and hypoxic [H-] conditions, respectively), the cells were collected and centrifuged at 400 × g and 4 °C for 3 min. The supernatants were collected and centrifuged at 1700 × g and 4 °C for 3 min. Following this, the supernatants resulting from this centrifugation were collected and defined as N-CM or H-CM. CM was stored at − 80 °C before being used in the subsequent experiments.