Tissue-engineered heart valve replacements have the potential to overcome the limitations of existing surgical therapies for aortic valve disease; however, these cell-based therapies require careful manipulation of cellular function and phenotype. Valvular interstitial cells (VICs), the main cell-type of the cardiac leaflet, are a heterogeneous population of cells which can become myofibroblasts, activated mesenchymal cells hypothesized to have wound healing responsibilities. This thesis proposes that the unique contractile, synthetic, and secretory functions of the myofibroblast should be considered in the development of a heart valve substitute that has the capacity to grow, repair, and remodel. The goal of this thesis was to identify the essential chemical signals within the extracellular environment that regulate valvular interstitial cell (VIC) secretory and myofibroblast properties, and utilize this knowledge towards the design of biomaterial niches for VIC culture and tissue regeneration. First, changes in fundamental cellular properties were assessed in response to defined components of varying complexity from within the extracellular matrix. Second, soluble factors inhibiting myofibroblast activation were identified and their intracellular signaling pathways were explored. Lastly, a biomaterial niche was created by functionalizing poly(ethylene glycol) hydrogels with selected chemical signals, allowing facile promotion or suppression of myofibroblast activity. This research provides the foundation for regulating the myofibroblast properties of VICs for applications in heart valve tissue regeneration.