MC3T3-E1 cells (1.0 × 105 cells/well) were cultured onto 48-well plates, and treated with DMEM (Wako) supplemented with 10% FBS (Invitrogen), antibiotics (0.2 mg/ml gentamicin) (Gibco), 50 mg/L ascorbic acid, hydrocortisone, and 10 mM β-glycerophosphate (Takara Bio, Shiga, Japan) in the presence and absence of TER (0.1–10 μM) and recombinant mouse bone morphogenetic protein (BMP)-2 (50 ng/ml) (BioLegend, San Diego, CA, United States) for 21 days. Prior to staining, all cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Then, the cells were stained with an Alizarin red staining kit (Cosmo Bio Co., Ltd., Tokyo, Japan) and an ALP Staining Kit (Fuji Films Co., Tokyo, Japan), following the manufacturer’s instructions (Aaron et al., 2010).

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