*2.7. Cell Culture*

Osteoblast MC3T3-E1 cells derived from mouse bones were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Osteoblast MC3T3-E1 cells were prepared in α-minimum essential medium (α-MEM, Gibco, Grand Island, NE, USA) with 10% fetal bovine serum (FBS; Gibco, Grand Island, NE, USA), 100 U/mL penicillin (CO2 incubator (MCO-230AIC-PK, Panasonic, Kadoma, Japan) using a culture medium supplemented with Gibco (Grand Island, NE, USA)), and 100 U/mL streptomycin (Gibco, Grand Island, NE, USA) at 37 ◦C, 5% CO2, and 95% air. The cells were washed with phosphate-buffered saline (PBS; pH 7.4; Gibco, Grand Island, NE, USA) and 0.25% trypsin-2.65 mM EDTA (Gibco, Grand Island, NE, USA), and sub-cultured by changing the culture medium every two days.
