MALADAPTIVE IMMUNE-FIBROTIC AXIS DRIVES IMPAIRED LONG BONE REGENERATION UNDER MECHANICAL INSTABILITY.

Abstract

Delayed and nonhealing fractures, affecting 5-10% of incidents, result in prolonged disability and reduced quality of life for the patient. While acute inflammation initiates healing, dysregulated immune responses exacerbate bone resorption and fibrosis. Current animal models fail to replicate mechanical instability, the primary driver of clinical hypertrophic nonunion, limiting translational insights. Here, we engineer a murine delayed-healing model using tunable intramedullary nails to impose controlled interfragmentary strains, mimicking human hypertrophic nonunion. High-strain fractures (15-30%) generated larger calluses with delayed ossification, 2.9-fold increase in fibrotic tissue ( p = 0.0099), and inferior biomechanical strength (stiffness: 1.6-fold lower, p = 0.024) compared to low-strain controls (<5%). Spatial transcriptomics revealed persistent fibrotic niches in high-strain calluses enriched with fibroblast-associated genes ( Pdgfrb, Lgals3 ) alongside dysregulated macrophage-osteoclast signaling ( Spp1, Mmp9 ). Systemic immune profiling revealed CD206+ macrophages and CD25+ T-regulatory cells as predictive biomarkers, with early polarization determining long-term outcomes ( R = 0.72, p = 0.004). Multivariate modeling linked delayed healing to persistent CD8+ T cells and deficient Treg recruitment, perpetuating inflammation. These findings establish mechanical instability as a catalyst for pathological immune-stromal crosstalk and provide a platform for mechano-informed immunotherapies. Our work redefines hypertrophic nonunion as a disorder of mechano-immunology, offering novel diagnostic and therapeutic strategies to mitigate fibrosis and restore regeneration.

Teaser: Mechanical strain hijacks immune signaling to induce fibrosis and block bone regeneration in unstable fractures.