Figure 5.

Effect of MDA-1833-conditioned medium on osteoclast differentiation and resorption activity. (a) Timeline of the osteoclast differentiation assay. Pre-osteoclasts are seeded after five days of culturing and stimulated with the MDA-1833 cell secretome for four more days, refreshing the media at day 7. (b) Representative micrographs of differentiated osteoclasts stained with HCS CellMask Deep Red Stain. Scale bar—100 μM. (c) Osteoclast number and (d) area after four days of MDA-1833 secretome stimulation. Osteoclasts with three or more nuclei were included in the analysis. Data are expressed as the median of individual data points from six independent experiments (Kruskal–Wallis test). One data point from the CTRL condition was excluded due to a technical error. (e) Timeline of the resorption assay. Osteoclasts are differentiated for nine days and seeded on top of bone slices, followed by incubation with MDA-1833 cell secretome for another three days. (f) Representative micrographs of the surface of bone slices after three days of resorption. Resorption events are highlighted by black arrows. Scale bar—200 μM. (g) Estimated percentage of eroded surface, (h) trench percentage per total eroded surface and (i) TRAcP activity quantified at the end of the experiment. Data are expressed as the median of individual data points from five independent experiments (Friedman’s test followed by Dunn’s multiple comparison test, * p ≤ 0.05).