Isolation and culture of murine primary osteoblasts

Murine primary osteoblasts were obtained as described in previous research [24 (link), 25 (link)]. Briefly, the calvarial bone was carefully dissected from one-day-old neonatal Wistar rats and washed with PBS solutions containing 100 U/ml penicillin and 100 µg/ml streptomycin thrice. The semi-transparent calvarial bone was incubated with 0.25% trypsin at 37 °C for thirty minutes, then sliced and incubated with 0.1% collagenase I at 37 °C overnight. The isolated osteoblasts were collected from the final digests and resuspended in the complete medium consisting of α-MEM, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured in the humidified atmosphere of 5% CO₂.