Figure 1

RANKL attenuates the effect of bisphosphonates on osteoclast number, apoptosis, and bone resorption in vitro. Cultures of mature osteoclasts from rabbit bones were treated with 100 μM ALN or CLO, ± 50 ng/mL RANKL for 48 hours. Cells were then fixed, stained for tartrate-resistant acid phosphatase (TRAP), and counterstained with DAPI. (a) Results are the mean number of TRAP-positive multinucleated osteoclasts (more than three nuclei per cell) ± standard error of the mean (SEM) (n = 3) or (b) the percentage of non-apoptotic and apoptotic osteoclasts. Data are expressed as the mean ± SEM (n = 3 replicates). **P = 0.01, ***P = 0.001 compared with ALN or CLO alone (analysis of variance). ^#Treatment with ALN or CLO alone caused a significant decrease in osteoclast number compared with control (CTL) cultures (P = 0.01) and a significant increase in osteoclast apoptosis compared with control cultures (P = 0.001). (c) Values of resorption area are the mean resorbed area (mm²) per slice ± SEM (n = 6 slices). ***P = 0.001 compared with CLO alone and **P = 0.01 compared with ALN alone (analysis of variance). ^#Treatment with ALN or CLO alone caused a significant decrease in osteoclastic bone resorption compared with control cultures (P = 0.001). The data shown are representative of three independent experiments. ALN, 4-amino-1-hydroxy-butylidene-1,1-bisphosphonate (alendronate); CLO, dichloromethylene-1,1-bisphosphonate (clodronate); DAPI, 4,6-diamidino-2-phenylindole; RANKL, receptor activator of nuclear factor-kappa-B ligand.