Fig. 1. Dynamics of osteoclastogenesis.

(A,B) Human osteoclast precursors where treated with MCSF (33 ng/ml) and RANKL (33 ng/ml) and representative images were obtained. (A) Live osteoclast differentiation cultures at different time after plating. (B) Fixed, TRAP stained cells cultured for 5 days without (left) or with RANKL (right). Scale bars: 50 µm (applies to all images). (C) Human precursors were cultured with MCSF (33 ng/ml) and RANKL (15, 33 and 66 ng/ml) for 0–6 days, and osteoclasts were manually counted. Data are means ± SEM, n = 3 independent experiments. (D,E) Human precursors were cultured with MCSF (33 ng/ml) and RANKL (0–132 ng/ml) for 5–6 days, and osteoclasts were manually counted (D) or osteoclast planar area was measured (E). For panel D, data are means ± SEM, n = 2–10 independent experiments. For panel E, the box-plots indicate the minimal and maximal values (whiskers), the 25th and 75th percentiles (the limits of the box), and the median values (the line within the box), n for each concentration is shown in italics. (F) Human precursors were cultured on osteoassay bone plate with MCSF (33 ng/ml) and RANKL (0–264 ng/ml) for 6 days, then the osteoclast numbers were counted (plotted on the x-axis) and the amount of degraded collagen peptides (CTX) in conditioned medium was measured (plotted on the y-axis). Data are means ± SEM, n = 2 independent experiments. (G,H) Human precursors were cultured with MCSF (33 ng/ml) and RANKL (0–66 ng/ml) for 1–6 days and the expression of cathepsin K (CtsK, scale on the right), calcitonin receptor (CTR), MMP-9, RANK and TRAP was assessed, normalized to endogenous control GAPDH and expressed relative to samples cultured without RANKL. Data are means ± SEM, n = 2–4 independent experiments. (G) Time course of changes in gene expression of osteoclast marker genes in cultures treated with 33–66 ng/ml RANKL. (H) RANKL concentration dependence of changes in gene expression of osteoclast marker genes on day 5–6.