Osteogenic differentiation. Results of osteogenic differentiation after 3 weeks. (A) Representative micrographs showing Alizarin Red stained mineral deposits on cultured BMSCs from each experimental group. Magnification bars represent 100 μm in all images. (B) Percentage of total area viewed that was positively stained with Alizarin Red. (C) Quantified calcium cation (mM) determined using a colorimetric calcium assay. For B and C, The '#' sign indicates a significant difference between passage 1 and passage 6 cultures from similarly aged donors, '‡' indicates a significant difference between the indicated group and any passage 1 groups from younger donors, and '¥' indicates a significant difference between the indicated group and any passage 6 groups from younger donors (p < 0.05). Age-related decreases in staining and calcium were significant across all age groups, and significant passage related decreases were only observed in BMSCs from 6-day-old donors. Error bars designate means ± standard deviation (n = 3).