2.2. Cell culture

MC3T3-E1 cells (Riken, Hirosaka, Japan) were inoculated in T-75 flasks and cultured in α-MEM medium containing 10% (v/v) FBS and 1% (v/v) double antibodies in an incubator at 37 °C, 95% relative humidity, 5% CO₂, and changed twice a week. The cells were digested with 0.25% trypsin in the culture flask for 3 min to remove the cells from the wall. Then, the digestion was terminated by adding a fresh cell culture medium. The cells were counted under a hemocytometer, and the cell culture medium matched the cell density to the required value. The cell suspensions were added to the 24-well plates with samples prepared as described above. The wells without samples were used as blank controls, then placed in the incubator and cultured separately to start the following assays.