Osteogenic differentiation

Cells (1×10⁵/well) were cultured were cultured until they reached 80% of the culture flask. Then, media were changed with osteogenic medium (100 nM dexamethasone, 50 mg/ml ascorbic acid, and 5 mM β-glycerophosphate; Sigma-Aldrich; Merck KGaA) cells were cultured for 7 or 21 days. The alkaline phosphatase (ALP) activity assay was performed following osteogenic induction for 7 days using an ALP kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). ALP staining was performed using a BCIP/NBT ALP Color Development kit according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Haimen, China). Following inducing in osteogenic medium for 21 days, Alizarin Red staining was performed. Cells were washed with 10% FBS in PBS twice. Then cells were fixed with 60% isopropanol for 1 min. Subsequently, cells were washed with distilled water for 3 min and stained using 1% Alizarin Red (Sigma-Aldrich; Merck KGaA) at room temperature for 30 min. The Alizarin Red-stained nodules were visualized under an Olympus BX51 light microscope equipped with an Olympus DP70 camera (Olympus, Co., Tokyo, Japan). To quantify Alizarin staining, mineralized nodules were dissolved in 0.5 N HCl with 0.5 ml 5% SDS for 30 min.

To quantify Alizarin Red-stained nodules, the stain was solubilized with 0.5 ml 5% SDS in 0.5 N HCl for 30 min at room temperature. Subsequently, 0.15 ml of the liquid was transferred to a 96-well plates and absorbance value were measured at 405 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT,, USA). All assays were repeated three times.