Collagenase dispase enzyme solution

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)].

Primary osteoblasts were isolated from calvariae of 1-day-old ICR mice after dissected aseptically and treated with 0.2% collagenase-dispase enzyme solution (Sigma-Aldrich, St. Louis, MO, USA). Cells (passage 0) were collected by centrifuge after repeated digestions and cultured in α-minimum essential medium without L-ascorbic acid supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. The cells were detached and reseeded at ~70–80% confluence, and then the cells (passage 1) were used for the experiments reported here. To test the effects of TMDQ on osteoblast differentiation, osteoblastic differentiation was induced by changing OS medium containing 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate when the cells are ~80% confluent. The recombinant noggin and DKK1 were purchased from Invitrogen (Carlsbad, CA, USA; Catalog Number: PHC1506) and PeproTech (Rocky Hill, NJ, USA; Catalog Number: 120–30), respectively. The medium was replaced every 2 days during the incubation period. TMDQ was dissolved in 100% DMSO and then diluted it (1 : 1000) directly into the medium. Final concentration of 0.1% DMSO was used as the vehicle control. For siRNA transfection, cells were transfected using Lipofectamine RNAiMAX according to the manufacturer's specification (Invitrogen).

Primary calvarial osteoblasts were isolated from calvariae of 1-day-old ICR mice using 0.2% collagenase-dispase enzyme solution (Sigma-Aldrich, St. Louis, MO). The cells isolated from the last four to six digests were cultured separately in α-modified minimum essential medium (α-MEM) (Gibco Laboratories, Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco Laboratories) and antibiotics (100 mg/mL penicillin G and 100 IU/mL streptomycin). After reaching a subconfluent state, the cells were removed from each flask and combined together as osteoblasts. The cells were used for all experiments at second passage, as described below. Cells were cultured in α-MEM containing 10% FBS, 5 mmol/L β-glycerophosphate, ascorbic acid (50 μg/mL), and antibiotics. For siRNA transfection, cells were transfected using Lipofectamine RNAiMAX according to the manufacturer’s specification (Invitrogen, Carlsbad, CA, USA).