Osteoblast differentiation and osteoclastogenesis assay in vitro

Human mesenchymal stem cells (HMSCs) were purchased from ScienCell (Carlsbad, CA, USA), plated in cell culture flasks and expanded in mesenchymal stem cell growth medium (MSCM; ScienCell) at 37°C in a 5% CO₂ atmosphere for 7–10 days. Subsequent to having grown to an adequate density (70–80% convergence), the adherent cells were trypsinized and seeded at a density of 5,000 cells/cm² on a 24-well plastic plate. After 24 h, MSCM was removed, and osteogenic induction medium (ScienCell) was added to induce osteoblast differentiation (day 0). On the seventh day, the osteoblast cells were plated at 1×10⁶ cells per well in 24-well plates, at a 1:1 ratio of basal culture medium/filtered CM (harvested from 24 h incubation of confluent tumor cells). The medium was replaced every 2 days. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7 using a Leukocyte Acid Phosphatase kit from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) (14). TRAP-positive multinucleated cells were considered mature osteoclasts and were included in the number of osteoclasts per well.