Csf1r⁻ macrophages govern the second wave of neutrophils for bone repair

Macrophages play a pivotal role in tissue regeneration, including bone healing; however, the mechanisms and cell types through which they establish regenerative niches are not yet fully understood. Here, we identified a distinct subset of osteodirective macrophages (OdMacs), characterized by the expression profile F4/80⁺ Csf1r⁻ Cx3cr1hi Ccr2hi, which contribute to the formation of bone regenerative niches following injury. Histological analysis and FACS analysis, combined with transcriptomic profiling (RNA-seq and scRNA-seq), revealed that OdMacs facilitate the recruitment of activin A-expressing preneutrophils (preNeu) to sites of bone injury. This recruitment enhances the proliferation and osteogenic differentiation of LepR⁺ bone marrow stromal cells (BMSCs), thereby promoting bone regeneration. Furthermore, genetic studies using Axin2-Cre; tdTomato, Inhba, and Wls conditional knockout mice underscored the essential roles of Wnt signaling in BMSCs and preNeu-derived activin A in orchestrating this regenerative process. The OdMac-regulated regenerative niche represents a promising therapeutic target for regenerative medicine.