Effect of melatonin and vitamin D on calcium accumulation in ADSCs during differentiation. (A) calcium accumulation, after 21 days of treatment, in cells cultured in basic medium (BM) and ADSCs exposed to differentiation medium (ADM) or ADM together Melatonin (Melatonin+ADM) or both Melatonin and vitamin D (Melatonin+VitaminD+ADM). Positive control (CTRL+) are ADSCs cultured in osteogenic conditioned medium. Scale bar=100 µm. The percentage of mineralization (B) was calculated using ImageJ, with ADSCs cultured for 21 days in osteogenic medium as positive control (black bar), considered as 1, and ADSCs cultured in basic medium (white bar) as negative control for calcium accumulation. ADSCs were exposed for 21 days in the presence of ADM only (Blue bar), or ADM with Melatonin (yellow bar) or with both Melatonin and Vitamin D (red bar). Data are expressed as mean ± SD and are representative of 6 different experiments. An average was made from three technical replicates.