Mechanical confinement balances ECM remodeling in chondrocytes via MAPK and Hedgehog signaling.

Mechanical cues from the extracellular matrix (ECM) are critical regulators of chondrocyte behavior and cartilage homeostasis. Mechanical confinement, determined by the stress relaxation properties of the surrounding matrix, can drive anabolic ECM production or catabolic activity. However, the intracellular signaling pathways linking confinement to ECM remodeling remain poorly defined. Here, alginate hydrogels with tunable stress relaxation properties were used to investigate how confinement regulates signaling and matrix production. Low-confinement, fast-relaxing matrices promoted ECM deposition, whereas high-confinement, slow-relaxing matrices increased inflammatory and catabolic gene expression. Kinase activity profiling identified mitogen-activated protein kinase (MAPK) signaling as a key pathway modulated by confinement, with subsequent activation of Hedgehog (Hh) signaling. Pharmacological activation of Hh signaling enhanced matrix deposition and restored chondrogenic morphology in low-confinement conditions. ECM formation negatively correlated with primary cilia length, and confinement-mediated changes in chondrocyte volume occurred even without primary cilia. These findings support a model in which confinement regulates matrix production via MAPK and Hh signaling to maintain homeostasis, with alterations in confinement, such as those in osteoarthritis or aging, linked to changes in chondrocyte phenotype. This work positions confinement as a central regulator of cartilage mechanobiology and provides design principles for viscoelastic biomaterials supporting balanced ECM remodeling.