In Vitro Aging of Osteoblasts

Osteoblasts were multiple-passaged as an in vitro model of age-induced bone loss, as previously described [26 (link),27 (link),28 (link)]. Upon euthanasia, primary (P0) calvarial osteoblasts were isolated from 3-month-old mice and incubated in DMEM and 10% FBS. Primary osteoblasts (5 × 10⁵/well, 6-well plates) were incubated in medium for 3 days. After 10 passages, osteoblasts were designated to the aged group (P10). In some experiments, 10⁵ cells/well (24-well plates) were incubated in 100 nM 5-Aza-2′-deoxycystidine (5-aza; A3656, Sigma-Aldrich, St Louis, MO, USA) or 10 μM N-acetylcysteine (NAC; A9165, Sigma-Aldrich, St Louis, MO, USA) for 4 h and incubated in an osteogenic medium.