Effects of Stiffness and Degradability on Cardiac Fibroblast Contractility and Extracellular Matrix Secretion in Three-Dimensional Hydrogel Scaffolds

Cardiac fibrosis results from persistent cardiac fibroblast activation and is heavily dependent on the interplay of extracellular matrix mechanics and proinflammatory cytokines. Studying this interplay using <i>in vitro</i> disease models is of interest for developing strategies to treat cardiac fibrosis. However, current metrics for quantifying myofibroblast activation rely heavily on the presence of α-SMA stress fibers, which works well for two-dimensional (2D) culture systems but not 3D cell scaffolds. Here, we investigate how contractility and extracellular matrix secretions, which are two phenotypic markers of cardiac myofibroblasts, correlate with 3D matrix stiffness and TGF-β concentration (a proinflammatory cytokine). Cardiac fibroblasts encapsulated in soft, degradable hydrogels were larger and more contractile and secreted more extracellular matrix than cells encapsulated in stiff, degradable hydrogels or nondegradable hydrogels. The addition of TGF-β to the soft, degradable hydrogels increased the volume, contractility, and extracellular matrix secretions, indicating myofibroblast activation. In addition, the presence of α-SMA increased, but α-SMA stress fibers were not detected. These results highlight the importance of local degradability in 3D hydrogels for cellular contractility and remodeling of the extracellular matrix. They also suggest the use of additional phenotypic markers to probe myofibroblast activation in 3D cellular scaffolds.