Research Paper Volume 11, Issue 19 pp 8103—8119

Figure 1. Ethanol increases RANKL-induced OC formation and bone resorption in vitro. (A) BMMs were stimulated with RANKL at different concentrations of ethanol and histochemically stained for TRAP detection. TRAP-positive cells with ≥3 nuclei were considered as OCs. (B) Numbers and areas of TRAP-positive multinucleated (>3 nuclei) cells formed in the presence of increasing concentrations of ethanol (n = 3). (C) Equal number of pro-osteoclasts were cultured on bone slices in the presence of different concentrations of ethanol. After 5 days, bone resorption lacunae were observed by scanning electron microscopy. (D) Area of bone resorption was measured using ImageJ software. (E) Disulfiram dramatically inhibited RANKL-induced osteoclastogenesis. TRAP-positive cells with ≥3 nuclei were considered OCs (magnification 100×; scale bar = 200 μm). (F) Analysis of the numbers and areas of TRAP-positive multinucleated (>3 nuclei) cells (n = 3). (G) Equal number of pro-osteoclasts were cultured on bone slices treated with indicated condition. After 5 days, bone resorption lacunae were observed by scanning electron microscopy. (H) Area of bone resorption was measured using ImageJ software. Data are the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the respective controls.