CD301b+ tissue-resident macrophages drive bone repair as an early-responding subpopulation.

Bone healing is a complex immune-regulatory process. Specifically, bone tissue-resident macrophages (TRMs) play a critical role in this process. Furthermore, TRMs exhibit niche-specific functional heterogeneity within the skeletal environment. However, the functional contributions and molecular mechanisms of specific TRM subsets during bone repair remain poorly understood. Using single-cell RNA sequencing integrated with in vivo functional assays, we identified a unique CD301b+TRMs subpopulation that expands rapidly post-injury. This subset is molecularly defined by the high co-expression of CD301b and the canonical resident marker Timd4. Pseudotime and RNA velocity analyses confirmed that CD301b+TRMs act as key transcriptional precursors within a parallel evolution model. These cells orchestrate the remodeling of early signaling networks via the Ptn and Gas6 axes, recruiting macrophages and driving their transition toward pro-repair and osteogenic states. Functional studies demonstrated that the specific depletion of this subset impairs bone regeneration in mice. This impairment is characterized by a significant reduction in mineralized bone volume, disordered collagen deposition, and the loss of key osteogenic factors including Sp7. This study establishes CD301b as a reliable biomarker for identifying pro-regenerative TRMs. Our findings highlight CD301b+TRMs as critical regulators of the osteo-immune microenvironment and suggest their potential as a therapeutic target for addressing various orthopedic regeneration.