Rigidity Sensing of Inclusions Directs Differentiated Cell Elongation and Force Generation across Phenotypes

Fibrosis is driven in part by the transition of healthy fibroblasts to a contractile phenotype called myofibroblasts. The mechanics of the extracellular matrix play a crucial role in regulating cell fates and behaviors during this transition. However, most studies to date focus on cells grown on 2D surfaces and matrices with homogeneous properties. This leaves open how local rigidity differentially regulates the behaviors of both phenotypes in 3D environments, including polarization, contraction, and maintenance of phenotypes, during remodeling. Here, we engineer 3D microgel-in-collagen composites by embedding low-volume fractions of cell-scale microgels with two levels of rigidity, mimicking healthy and pathological tissues that are stiffer than the surrounding collagen but do not significantly change the bulk modulus. We find that microgels serve as mechanical centers: both phenotypes polarize toward microgel inclusions. The polarization response decays as a power-law with distance ∼<i>r</i>^-<i>n</i>, decreasing more slowly for myofibroblasts (<i>n</i> ≈ 0.35) than fibroblasts (<i>n</i> ≈ 0.81), indicating that myofibroblasts are more sensitive to small mechanical variations. In situ measurements finds that forces are highest for myofibroblasts near stiff microgels and lowest for fibroblasts near soft microgels. Local rigidity also stabilizes the myofibroblast phenotype: Both the ordering of the proinflammatory marker α-smooth muscle actin and nuclear Yes-associated protein localization persist for cells cultured with stiff microgels over several days but diminish quickly for those cultured with soft microgels and in pure collagen. Together, these results reveal a rigidity- and phenotype-dependent feedback loop: stiff inclusions induce cell polarization and collagen remodeling via a contractile force, which in turn maintain the myofibroblast phenotype. Our study positions mechanical heterogeneity as a useful and sensitive handle to probe and potentially modulate early fibrotic progressions.