Identification of cell cycle-arrested quiescent osteoclast precursors in vivo.

How are sites suitable for osteoclastogenesis determined? We addressed this issue using in vivo and in vitro experimental systems. We first examined the formation of osteoclasts in ectopic bone induced by BMP-2. When collagen disks which contained BMP-2 (BMP-2-disks) or vehicle (control-disks) were implanted into wild-type mice, osteoclasts and osteoblasts appeared in the BMP-2-disks, but not in the control disks. RANKL-deficient (RANKL(-/-)) mice exhibited osteopetrosis, with an absence of osteoclasts. BMP-2 and control disks were implanted into RANKL(-/-) mice, which were intraperitoneally injected with RANKL. Osteoclasts formed in the BMP-2-disks, but not in the control disks. In the BMP-2-disks, osteoclasts were observed in the vicinity of osteoblasts. Cell cycle-arrested quiescent osteoclast precursors (QOP) were identified as the committed osteoclast precursors in vitro. Experiments in vivo showed that QOPs survived for several weeks, and differentiated into osteoclasts in response to M-CSF and RANKL. QOPs were identified as RANK and c-Fms double-positive cells, and detected along bone surfaces in the vicinity of osteoblasts in RANKL(-/-) mice. QOPs were also observed in the ectopic bone induced by BMP-2 implanted into RANKL(-/-) mice, suggesting that QOPs were circulating. These results imply that osteoblasts support the homing of QOPs to bone tissues. In response to bone-resorbing stimuli, QOPs promptly differentiate into osteoclasts. Therefore, the distribution of QOPs appears to determine the correct site of osteoclastic development.