Isolation and Differentiation of Primary Mouse Osteoblasts

Primary osteoblasts were isolated from calvariae of 1-day-old ICR mice after aseptic dissection and treated with 0.2% collagenase-dispase enzyme solution (Sigma-Aldrich, St. Louis, MO, USA). Cells from digestions 6–8 (10–25 × 10⁶ cells) were pooled and seeded at a density of 2 × 10⁶ cells/175 cm² in culture flasks containing α-minimum essential medium (α-MEM) supplemented with 10% FBS and antibiotics (bensylpenicillin, gentamycin sulfate, and streptomycin). The cells were cultured for 4–6 days, with a change of medium every 2 or 3 days, at 37°C in a humidified atmosphere containing 5% CO2 in air. The experiments were approved by the Ethical Committee for Animal Experiments at Kyung Hee University (Seoul, Korea). To induce differentiation, cells were cultured with sulfuretin or rh-BMP2 (Calbiochem Co., La Jolla, CA, USA) and osteogenic supplement (OS; 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate) as described previously [17 (link)].