The thermo-responsive behavior of Poly(N-isopropylacrylamide) makes it an ideal candidate to easily embed cells and allows the polymer mixture to be injected. However, P(NiPAAm) hydrogels possess minor mechanical properties. To increase the mechanical properties, a covalent bond is introduced into the P(NIPAAm) network through a biocompatible thiol-ene click-reaction by mixing two polymer solutions. Co-polymers with variable thiol or acrylate groups to thermo-responsive co-monomer ratios, ranging from 1% to 10%, were synthesized. Precise control of the crosslink density allowed customization of the hydrogel’s mechanical properties to match different tissue stiffness levels. Increasing the temperature of the hydrogel above its transition temperature of 31 °C induced the formation of additional physical interactions. These additional interactions both further increased the stiffness of the material and impacted its relaxation behavior. The developed optimized hydrogels reach stiffnesses more than ten times higher compared to the state of the art using similar polymers. Furthermore, when adding cells to the precursor polymer solutions, homogeneous thermo-responsive hydrogels with good cell viability were created upon mixing. In future work, the influence of the mechanical micro-environment on the cell’s behavior can be studied in vitro in a continuous manner by changing the incubation temperature.

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.