β glycerol phosphate disodium salt

BMSCs were seeded at a density of 50 cells/cm² and cultured until they reached 70% confluency. The CCM was then changed to OM (fresh complete medium with 15% FBS) supplemented with 50 µg/ml ascorbic acid (Sigma Aldrich; Merck KGaA, Darmstadt, Germany), 10 mM β-glycerol phosphate disodium salt (Sigma Aldrich; Merck KGaA), and 100 nM dexamethasone (Sigma Aldrich; Merck KGaA). Cells cultured in osteogenic medium only comprised the OM group; while in the curcumin group (CR group), 15 µM curcumin was added to the OM. This concentration was selected based on previous studies that used curcumin in cell culture to induce osteogenic differentiation with minimal cytotoxicity (53 (link),78 (link),79 (link)). curcumin was prepared according to the method previously described by Gu et al (53 (link)). Briefly, curcumin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was dissolved in dimethyl sulfoxide and stored at −20°C. In the ATRA group, 1 µM ATRA (Sigma-Aldrich; Merck KGaA) was added to the OM. This concentration has repeatedly been used to study the effects of ATRA on in vitro osteogenic differentiation; since the normal physiological level of ATRA is ≤0.01 µM and the effective pharmacological concentration is >0.1 µM, the 1 µM concentration was selected to induce osteogenic differentiation (27 (link),80 (link)–82 (link)).