Osteoarthritic chondrocytes exert higher contractile forces and exhibit enhanced protrusive activity when cultured in...

Osteoarthritis (OA) induces phenotypic changes in chondrocytes as well as alterations in matrix composition and mechanics. Yet, its impact on active cell-generated forces, a key indicator of cell-matrix interaction, remains poorly characterized. In this study, we systematically compared the force generation capacity and associated proteins of interest between human OA and non-OA articular chondrocytes and how they are affected by cell culture dimensionality (2D versus 3D) and matrix degradability. Using traction force microscopy (TFM) combined with high-resolution immunostainings we show that OA alters the expression and organization of proteins involved in force exertion and transmission across both 2D and 3D cultures, but only in a 3D degradable hydrogel environment do these changes translate into higher cell-generated contractile forces This increased force generation correlates with elevated protrusive activity, higher actomyosin content and engagement, as well as altered localization of adhesion and matrix proteins, all of which could contribute to increased cell-matrix interaction in OA chondrocytes. In contrast, OA chondrocytes display no increase in cell tractions when cultured on 2D hydrogel substrates. These findings demonstrate that the detection and interpretation of OA-related alterations in chondrocyte mechanobiology are strongly dependent on the dimensionality and degradable properties of the culture system. Our results highlight the critical role of 3D degradable environments in revealing disease-associated changes in chondrocyte force generation and emphasize the necessity of carefully selecting model systems when investigating OA mechanobiology. STATEMENT OF SIGNIFICANCE: Chondrocytes, cells essential for cartilage maintenance, are disrupted in osteoarthritis (OA) through mechanisms that remain poorly understood. We developed an in vitro 3D degradable hydrogel system that mimics chondrocyte physiological environment better than traditional 2D cultures, allowing to study OA-driven changes in cell-matrix interactions. Using this system, we applied computational techniques to compare the ability of patient-derived non-OA and OA articular chondrocytes to generate forces on their environment. Our results revealed that OA chondrocytes exert higher contractile forces and exhibit enhanced protrusive activity in 3D, but not in 2D. This combination of techniques provides new insight into OA-driven changes in cell-matrix interactions and facilitates the design of more predictive in vitro models for cartilage research.