Macrophage reprogramming nodes for bone repair identified by single-cell and spatial omics.

Early fracture repairs are characterized by dynamic immune-skeletal interactions. While immune cells are known to be critical, how macrophage polarization (M1 to M2) and metabolism jointly shape the microenvironment repairs remains unclear. Here, we integrated three mouse long bone fracture sc/snRNA-seq datasets with multi-algorithm consensus annotation. Fracture expanded and rewired intercellular communication, with redistribution of incoming signaling toward immune populations, especially macrophage subsets, and increased relative flow through TGF-β, BMP, and FN1 pathways. From days 1 to 7 post-injury, macrophages followed a graded M1-to-M2 continuum, while M1-like cells remained prevalent across this interval. Distinct transcriptional programs were associated with M1-like and M2-like macrophages, with Creb3l2/Fos enriched in M1-like cells and Maf/Mafb enriched in M2-like cells alongside differential metabolic features. Data-driven prioritization across integrated public mouse omics datasets nominated Pbx3, Creb3l2, Nfix, Maf, and Mafb as candidate regulators associated with macrophage polarization, with spatial enrichment in macrophage-associated niches. A fracture-associated repair module comprised skeletal stem/progenitor cells (SSPCs), fibroblasts, macrophages, and osteoclasts, and was accompanied by predicted metabolite-mediated communication, with communication involving glutamine, sterol/cholesterol, and GABA prioritized as relatively increased and communication involving heme and 27-hydroxycholesterol as relatively reduced. SSPC lineage tracing revealed Taco1 as an early dynamic marker and branch-specific drivers, Runx2/Egfr (osteogenesis), Ebf1 (chondrogenesis), and Stat5a (adipogenesis). Collectively, these findings provide a computational atlas of early fracture healing, suggest that macrophages may play an important coordinating role during this stage, and prioritize transcriptional and metabolic candidates for future experimental validation.