Distinct matrix viscoelasticity in bone fracture hematoma steers macrophage polarization

Physical properties of the extracellular matrix (ECM) are key regulators of cellular behavior. Following injury, the formation of a hematoma establishes a provisional niche that initiates and regulates healing responses. However, the influence of hematoma viscoelastic properties on immune cell behavior remains poorly understood. Here, we show that distinct ECM viscoelastic properties of the maturing fracture hematoma steer macrophage polarization from pro-inflammatory to pro-regenerative characteristics. Tissue analyses of ex vivo human samples revealed that hematoma viscoelastic properties change with ECM remodeling during healing progression, with the late-phase stress relaxation time constant, τ2, increasing significantly with days post-injury. Using alginate hydrogels in 3D culture, we engineered extracellular microenvironments with tunable τ₂ but constant stiffness to study their role in macrophage polarization. Our data demonstrate that ECM τ₂ properties guide macrophage phenotype, characterized by high τ₂ promoting pro-inflammatory activation, while low τ₂ supported anti-inflammatory phenotypes. This regulation of macrophage polarization by ECM stress relaxation properties persists even under toll-like receptor-coactivation. Single-cell RNA sequencing revealed distinct transcriptional programs associated with different ECM τ₂ values, with many of the differentially expressed genes related to metabolic processes. The transcriptomic profiles of macrophages primed by different ECM τ₂ aligned with in vivo healing trajectories, with the gene signature score of the low ECM τ2 decreasing over time. Our findings uncover the immune-regulatory function of specific hematoma stress relaxation properties associated with healing progress after injury, and suggest τ₂ as potential mechanobiological target to be leveraged in novel biomaterials-based regenerative therapies.