Mc3t3 e1 pre osteoblasts

MC3T3‐E1 pre‐osteoblasts (#CRL‐2593) were obtained from the ATCC (Manassas, VA). Pre‐osteoblasts were grown at 37°C, 5% CO₂ and 95% air under a humidified atmosphere in α‐MEM without L‐AA (WELGEME, Inc.) containing 10% foetal bovine serum (FBS) and 1× Gibco antibiotic‐antimycotic (Thermo Fisher Scientific). OS containing 10% FBS, 1× Gibco antibiotic‐antimycotic, 50 μg/ml L‐AA (Sigma‐Aldrich) and 10 mM β‐GP (Sigma‐Aldrich) with SCOP was used to stimulate osteoblast differentiation. 0.1, 1, 5, 10, 20, 30, 40, 50 and 100 mM SCOP stocks were prepared with 100% dimethyl sulfoxide (DMSO) and diluted to a final concentration (1:1000 dilution, 0.1% DMSO); 0.1% DMSO was treated as a control. During osteoblast differentiation, OS was changed every 2 days as described previously.¹⁵

MC3T3-E1 pre-osteoblasts were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in α-minimum essential medium without L-ascorbic acid (L-AA) (WELGEME, Inc., Gyeonggido, Korea), containing 10% fetal bovine serum and 1× antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA, USA), in a CO₂ incubator MCO-18AC (Panasonic, Osaka, Japan). Osteoblast differentiation was initiated by using an osteogenic supplement medium OS containing 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/mL L-AA (Sigma-Aldrich) in a CO₂ incubator. The OS was changed every 2 days for differentiation.

MC3T3E-1 preosteoblasts (#CRL-2593) purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) were kindly provided by Bioevaluation Center (Korea Research Institute of Bioscience and Biotechnology, Republic of Korea). The cells were cultured in α-minimum essential medium (α-MEM) without L-ascorbic acid (WELGEME, Inc., Seoul, Republic of Korea) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Osteoblast differentiation was induced by changing to osteogenic supplement medium (OS) containing 50 μg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP) (Sigma-Aldrich, St. Louis, MO, USA). The medium was replaced every two days during the incubation period as previously described [50 (link)].

MC3T3-E1 pre-osteoblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). For experiments, cells of passage 12–25 were used. Cells were grown and maintained in 75 cm² culture flasks (Greiner, Bio-one, Alphen a/d Rijn, The Netherlands) containing α-MEM supplemented with 10% FBS and antibiotics in a humidified atmosphere with 5% CO₂ in air at 37 °C. The medium was changed every 3 days. After reaching ~80% confluency, cells were detached using 0.25% trypsin (Gibco, Invitrogen, Waltham, MA, USA) and 0.1% ethylenediaminetetraacetic acid (EDTA; Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS; Gibco) at 37 °C. Cells were then resuspended in α-MEM with 10% FBS and antibiotics, seeded in 96, 48, or 24-well culture plates (Greiner), and cultured for different periods, from 1 h up to 28 days, dependent on the outcome parameter measured (see below). For determination of alkaline phosphatase (ALP) activity and matrix mineralization, 0.1 mg/mL ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (phosphate donor; Sigma-Aldrich) were added to the culture medium.

Sodium phosphate monobasic dihydrate (NaH₂PO₄·2H₂O) (99.0%), sodium hydroxide (NaOH) (98%), and dimethyl sulfoxide (99.0%) were purchased from Samchun (Republic of Korea). Calcium nitrate tetrahydrate (Ca (NO₃)₂·4H₂O) (98.0%), urea (99.0%), and phosphoric acid (H₃PO₄) (85.0%) were purchased from Junsei (Japan). Calcium hydroxide (Ca (OH)₂) (98.0%) was purchased from Acros Organics (USA). Deionized (DI) water (18.2 MΩ cm) was prepared using a Sartorius Arium®Pro Ultrapure water system and was used for all experiments. All reagents were used without further purification. All glassware was cleaned using aqua regia before use. As necessary, special care was employed in handling aqua regia. Mouse MC3T3-E1 preosteoblasts were used in this study were purchased from American Type Culture Collection (USA). Alpha minimum essential medium (α-MEM; Gibco, USA) was used to grow the cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphate (ALP) assay kits used to evaluate the proliferation and ALP activity of the cells were purchased from Sigma-Aldrich (USA). All materials were used without further purification.