Osteoblast Differentiation of MC3T3-E1 Cells

Pre-osteoblast MC3T3E-1 cells (#CRL-2593, American-type culture collection, Manassas, VA, USA) were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air using α-minimum essential medium (α-MEM) (WELGEME, Inc., Gyeonggido, Korea) without L-ascorbic acid (L-AA) (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 10% fetal bovine serum and 1 X Gibco ^® Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA), as previously described [48 (link)]. The osteoblast differentiation of MC3T3E-1 cells was induced by changing to an osteogenic supplement medium (OS) containing 50 μg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP), as previously described [48 (link)]. The OS was replaced every 2 days during the incubation period. One hundred percent DMSO was used for the dissolution of SoyB, and the vehicle control was used at a final concentration of 0.1% DMSO.