Spatial multi-omics decoding of the fracture nonunion niche: from immune–vascular–skeletal crosstalk to precision regeneration

Fracture nonunion — the failure of bone to heal without surgical intervention — affects 5%–10% of all fractures and represents a substantial clinical and economic burden. Despite decades of research, the cellular and molecular mechanisms that distinguish successful repair from nonunion remain incompletely understood, largely because the fracture niche is a spatially organized, multicellular ecosystem whose crosstalk cannot be captured by bulk analyses. The recent convergence of spatial transcriptomics, single-cell multi-omics, spatial proteomics, and computational integration methods has opened a new era in which the fracture nonunion niche can be decoded at unprecedented resolution. Here we synthesize evidence from 2022 to 2025 that implicates three interdependent axes of niche dysfunction: (i) arrested macrophage polarization and chronic inflammation driven by dysregulated CSF1R, CD163, and T-cell signaling; (ii) uncoupled angiogenesis–osteogenesis despite preserved or elevated VEGF expression, mediated by impaired type H vessel formation and disrupted PDGF-BB/SLIT3/Notch signaling; and (iii) skewed skeletal stem and progenitor cell (SSPC) fate decisions toward fibrosis and adipogenesis at the expense of osteochondrogenic differentiation, governed by impaired BMP, Wnt, and IHH pathway activation. We then review how spatial multi-omics — including Visium, Xenium, MERFISH, imaging mass cytometry, and MALDI-mass spectrometry imaging — has mapped these axes in situ , enabling the discovery of spatially restricted therapeutic targets. Finally, we examine how these mechanistic insights are being translated into precision regeneration strategies, including bone-targeted nanoparticles that reprogram macrophage polarization, controlled-release scaffolds that restore angiogenic–osteogenic coupling, and biomaterials that reactivate endogenous SSPCs. We propose that the future of nonunion treatment lies in spatially informed, patient-specific immunomodulatory and regenerative therapies guided by multi-omic profiling of the individual fracture niche.