A four-dimensional spatial atlas of spinal cord injury reveals predominant Spp1-integrin signaling driving microglia-fibroblast crosstalk in fibrotic niches.

BACKGROUND: Spinal cord injury (SCI) leads to fibrotic scarring that blocks axonal regeneration, yet the molecular mechanisms controlling scar formation remain incompletely understood. Microglia and fibroblasts accumulate at lesion sites, but their intercellular communication, spatial organization and therapeutic tractability have not been systematically mapped. METHODS: We integrated spatial transcriptomics, single-cell transcriptomics, bulk RNA sequencing, and microarray profiling across multiple post-injury time points in mouse and rat SCI models. A four-dimensional spatial atlas spanning three spatial dimensions and time was constructed to map microglia-fibroblast interactions. Fibroblast differentiation trajectories were used to establish molecular subtypes and a risk prediction model, validated in independent cohorts and human peripheral blood. NT3-chitosan was tested in a complete transection model with functional and molecular assessments. RESULTS: The four-dimensional spatial atlas identified Spp1-Itgav/Itgb1 as the dominant microglia-to-fibroblast signaling axis during peak scar consolidation, spatially colocalized with NF-κB and TGF-β transcriptional programs. Fibroblast differentiation-based classification stratified SCI into three subtypes associated with injury severity and immune remodeling. A risk model based on fibroblast differentiation-associated signatures in peripheral blood predicted ASIA grade A with AUCs of 0.883, 0.939, and 0.807 in the total, training, and test sets, respectively. NT3-chitosan treatment was associated with reduced Spp1-Itgav/Itgb1 signaling, diminished fibrotic scarring, and sustained locomotor recovery over 52 weeks. CONCLUSION: Microglia-fibroblast crosstalk via Spp1-Itgav/Itgb1 is enriched in regions of fibrotic scar maturation, alongside NF-κB and TGF-β transcriptional activation. The fibroblast differentiation-based framework enables risk stratification and guides scar-targeted interventions, providing a clinically actionable pathway for SCI therapy.