Bone samples were cleaned of adherent soft tissue and osteoblasts isolated by two methods.
a. The bone was cut into small pieces (2 mm × 2 mm), washed in Ca2+- and Mg2+-free digestion’s solution (10 min), and then sequentially digested (1 mg/ml trypsin, 10 min; 2 mg/ml Dispase, 20 min; and 3 mg/ml collagenase, 2 × 30 min) in digestion’s solution at 37°C. Cells released by the collagenase digestions were washed and grown to confluence in 25 cm culture flasks (Falcon) in primary cell culture system supplemented with 10% fetal calf serum (FCS). Incubations were carried out at 37°C in a humidified atmosphere of 5% CO2/ 95% air; the medium was changed every 2-3 days.
b. Bone samples were cut into small pieces and plated into 5 cm Petri dishes containing 4 ml primary cell culture system supplemented with 10% FCS. Bone cells were allowed to grow out from the explants until confluent; the medium was changed every 2-3 days.