Vibration-Induced Osteoblast Differentiation

POB cells were seeded at a density of 5 × 10⁵/well in a 6-well plate (n = 3) and cultured until confluence. Cells were then cultured in calcification medium, and the experimental groups were subjected to vibration (30 and 300 Hz) once every 2 days for 30 min. On day 14, total RNA was extracted from the experimental and control groups using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) and chloroform. After precipitating RNA using isopropanol and washing with 70% ethanol. The extracted total RNA was dissolved in RNase-free water and quantified, and its purity was measured using an ultra-trace spectrometer (Nanodrop 2000, Thermo Fisher Scientific). The ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) was used to synthesize cDNA. The THUNDERBIRD SYBR qPCR Mix (TOYOBO) was used for each cDNA for real-time PCR (n = 3) in accordance with the SYBR Green method. The PCR conditions were as follows: initial thermal denaturation at 95 °C for 15 min, cycling with thermal denaturation at 95 °C for 3 s, annealing at 62 °C for 5 s, and elongation at 72 °C for 45 s for 40 cycles. Targets of mRNA expression analysis were runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 1 chain (Col1a1), osteopontin (OPN), osteocalcin (OCN), and sclerostin, all being osteoblast differentiation markers. We also targeted p53, p21, and p16, aging-related markers, and c-fos, a cytotoxicity marker. Primers are shown in Table 1. With beta-actin as the internal control, relative mRNA expression was quantified using the double delta method.