Osteoclasts are important for angiogenesis in bone explants. (A) Osteoclast inhibition decreases angiogenesis in metatarsal explants. Metatarsal explants stained for endothelial cells (red, CD31); 17.5 days postcoitum outbred fetal mouse metatarsals were cultured with indicated treatments for 15 days before fixation. indicates area of osteoclast resorption that is prominent in the control bones and decreases with increasing OPG. (B) Quantification of angiogenic outgrowth and osteoclast number and activity in metatarsal explants. Number of branches and other angiogenesis tube formation parameters quantified at the end of the assay period (15 days). CTX (RatLaps) assayed from metatarsal explant-conditioned media collected from days 7 to 9 of culture. TRAP activity extracted by homogenization of bones after 15 days of culture and assayed by color development using a TRAP substrate. Data are mean ± SEM. *P < .05. Images acquired as whole mounts in water with an Olympus IX71 microscope with a UPlanFLN objective, numeric aperture (NA) of 0.13, and a Spot RTKE camera with Spot Advanced software (original magnification ×4). (C) Correlation of osteoclast formation and angiogenesis. The TRAP activity extracted from bone explants and angiogenesis (branch points) was correlated for all samples treated with various concentrations of OPG-Fc and analyzed by linear regression. r indicates Pearson correlation coefficient; P, significance from 1-sample F test for linear regression. Results are from 1 representative experiment of at least 2 performed.