Kollert, M. R. | Search bioRxiv

bioRxiv2025

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, {tau}2, increasing significantly with days post-injury. Using alginate hydrogels in 3D culture, we engineered extracellular microenvironments with tunable {tau}2 but constant stiffness to study their role in macrophage polarization. Our data demonstrate that ECM {tau}2 properties guide macrophage phenotype, characterized by high {tau}2 promoting pro-inflammatory activation, while low {tau}2 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 {tau}2 values, with many of the differentially expressed genes related to metabolic processes. The transcriptomic profiles of macrophages primed by different ECM {tau}2 aligned with in vivo healing trajectories, with the gene signature score of the low ECM {tau}2 decreasing over time. Our findings uncover the immune-regulatory function of specific hematoma stress relaxation properties associated with healing progress after injury, and suggest {tau}2 as potential mechanobiological target to be leveraged in novel biomaterials-based regenerative therapies.

bioengineering↗

bioRxiv2025

Osmotic niche changes as multifaceted trigger of cellular regenerative processes in organ injury

Cell niches are organ-specific and characterized by a variety of distinct biophysical cues, including mechanics and osmolality. Injury disrupts this cellular environment and marks the start of regenerative processes. It remains unclear whether bone fracture alters the osmolality of bone marrow, and how associated changes in the extracellular matrix (ECM) affect marrow-resident cells in the onset of regeneration. Here we present analyses of human tissue samples indicating that osmolality differences among tissue types lead to a sudden drop in bone marrow osmolality upon fracture, which in turn enhances ECM viscoelasticity. We reveal that a sudden osmolality drop, mimicked in vitro by lowering ion concentrations, triggers bone regenerative processes in mesenchymal stromal cells (MSCs), markedly enhancing their spreading, proliferation, and osteogenic differentiation while residing in osmolality- responsive viscoelastic ECM. Conversely, in non-physiologically elastic ECM, similarly increased osmolality augments MSC osteogenic differentiation, suggesting that ECM viscous dissipation redirects cellular responses to osmotic changes. Mechanistically, the regenerative function of the osmolality drop depends on the matrix providing cell-adhesion ligands and physiological viscoelastic properties. Sequencing data show altered gene expression already two hours after differentiation start, with distinct characteristics related to chromatin structural changes specifically associated with hypoosmolality. Our results suggest that the osmolality drop serves as fast-acting regenerative stimulus for MSCs by extracellularly enhancing matrix viscoelasticity, while altering chromatin structure intracellularly. This stimulus upon injury potentially orchestrates the individual responses of multiple cell types within a niche, facilitating a collective action towards regeneration. Learning how to leverage osmotic cues to induce regenerative cascades may eventually advance local and personalized therapeutic strategies for patients with impaired healing capacity. We anticipate that the integration of osmotic and mechanical ECM properties, as demonstrated in our assay, will catalyze advanced 3D cell culture systems and offer new perspectives on material design in tissue engineering, disease modeling, and mechanobiology.

bioengineering↗