Osteoclasts Derive Predominantly from Bone Marrow–Resident CX₃CR1⁺ Precursor Cells in Homeostasis, whereas Circulating CX₃CR1⁺ Cells Contribute to Osteoclast Development during Fracture Repair

Abstract

Osteoclasts (OC) originate from either bone marrow (BM)–resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX₃CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX₃CR1-EGFP/TRAP tdTomato mice, we found CX₃CR1 expression in mononuclear cells, but not in multinucleated TRAP⁺ OC. However, CX₃CR1-expressing cells generated TRAP⁺ OC on bone within 5 d in CX₃CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato⁺ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX₃CR1⁺ cells migrated to bone, lost expression of CX₃CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX₃CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX₃CR1⁺ cells can become OC.