Fig. 4.
ACTH promotes chondrogenic mineralized nodule formation in rat BMSC enriched for mesenchymal progenitors in a dose-dependent manner. γ2–MSH and α-MSH do not exhibit this effect. Cultures were stained for mineral (a–d), stained for alkaline phosphatase (e–h) or stained for proteoglycan matrix (i–k) after 14 days of osteogenic culture in the presence or absence of ACTH at a range of doses (10−8 to 10−6 M). Photomicrographs were taken using bright field under scanning power (40X total magnification) to emphasize differences in nodule numbers with ACTH treatment, Bar = 500 µM. Photomicrographs in m–o were taken using bright field (200X total magnification) to emphasize the multi-cellular alkaline phosphatase (m), proteoglycan rich (n), mineralizing nodules (o) as well as the condensed cells in the periphery. Bar = 100 µM. Arrowheads indicate condensed cells in the periphery of the nodules as well as chondrocytes in lacunae. In p–r, the number of mineralized nodules per well in ACTH treated cultures as well as α-MSH and γ2-MSH cultures are presented as % of untreated control (0) ± SEM. Data are representative of 3 separate experiments with each treatment group having an n = 3–5 wells. Individual comparisons were made after a significant one-way ANOVA using the Bonferroni correction. a-significantly different from 0, 10−8 M, b-significantly different from 0, 10−8, and 10−7 M.