2.1. Cell Culture and Zn Treatment

MC3T3-E1 subclone 4 (SC4, high osteoblast differentiation, ATCC, CRL-2593) murine pre-osteoblast was purchased from ATCC Cell Bank (Manassas, VA, USA). To test the effects of Zn on osteoblasts, the of 1 × 10⁵ of MC3T3-E1 cells were maintained in Minimum Essential Medium α (α-MEM) containing 10% FBS, 1 mM sodium pyruvate and 1% penicillin and streptomycin. The cells were incubated under a humidified atmosphere at 37 °C and 5% CO₂ to reach up to 80% confluency. Subsequently, the cells were treated with either 1 μM (low Zn) or 15 μM (high Zn) of ZnCl₂ (Sigma, St. Louis, MO, USA) along with 5 μM of intracellular Zn chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine, TPEN; Sigma) to induce the cellular Zn depletion. The normal osteogenic differentiation medium (OSM; growth medium supplemented with osteogenic growth factors of 3 mM glycerol-2-phosphate and 50 μg/mL ascorbic acid) without Zn and TPEN treatment was used as a normal control. MC3T3-E1 cells were then exposed to OSM, low Zn, or high Zn for 24 h.