Osteoclast and Osteoblast Generation

OCs were generated from healthy donor peripheral blood mononuclear cells (HD-PBMCs) as previously described.15 (link) HD-PBMCs were seeded into 6-well or 96-well plates at the density of 2×10⁶ cells/cm². After 2 hours, non-adherent cells were removed to enrich for OC progenitors, which were then cultured in α-MEM containing 10% FBS, 1% penicillin-streptomycin, RANKL 50 ng/mL, and M-CSF 50 ng/mL. OBs were differentiated from healthy donor mesenchymal stem cells (hMSCs, LONZA, Switzerland) as previously described.16 (link) Briefly, hMSCs were seeded in 96-well plates at a density of 6×10³ cells/cm² and cultured in osteogenic media, consisting of α-MEM with 20% FBS, 1% penicillin/streptomycin, 2,5% L-glutamine, 2.16 mg/mL β-glycerol phosphate, 0.05 mg/mL ascorbic acid, and 10 nM dexamethasone.