MC3T3-E1 cells (ATCC, Manassas, VA, United States) were used as pre-osteoblasts. MC3T3-E1 cells were cultured for 3 days in culture dishes containing DMEM (Wako) supplemented with 10% FBS (Invitrogen) and antibiotics (0.2 mg/ml gentamicin) (Gibco). Osteogenic differentiation was investigated by supplementing media with 50 mg/L ascorbic acid, 10 μM hydrocortisone, and 10 mM β-glycerophosphate (Takara Bio, Shiga, Japan) (Aaron et al., 2010).

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