CCL2/CCR2 Signalling in Mesenchymal Stem/Progenitor Cell Recruitment and Fracture Healing in Mice

Macrophage efferocytosis (clearance of apoptotic cells) is crucial for tissue homeostasis and wound repair, where macrophages secrete factors that promote resolution of inflammation and regenerative signalling. This study examined the role of efferocytic macrophage-associated CCL2 secretion, its influence on mesenchymal stem/progenitor cell (MSPC) chemotaxis, and in vivo cell recruitment using Ccr2^-/- (KO) mice with disrupted CCL2 receptor signalling in two regenerative models: ossicle implants and ulnar stress fractures. Single cell RNA sequencing and PCR validation indicated that efferocytosis of various apoptotic cells at bone injury sites (osteoblasts, pre-osteoblasts, MSPC) upregulated CCL2. CCL2 gradients enhanced MSPC migration through type I collagen matrices. In vivo, MSPC (LepR⁺) infiltration was significantly reduced while macrophage (F4/80⁺) infiltration increased in KO ossicle implants versus WT. In ulnar stress fractures, micro-CT revealed increased mineralized callus incidence in CCR2 KO male mice 5 days post injury (dpi) versus WT. By 7-dpi callus fractional bone volume, trabecular thickness, and bone mineral density were increased versus WT. Immunohistochemistry of mice 5-dpi confirmed an increase in callus area (including soft tissue); however, the percent of osteoprogenitors (%Osx⁺) within the callus was not different. These findings suggest that CCL2 differentially impacts MSPC recruitment depending on bone wound healing model.