"A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling towards regeneration of healthy tissue rather than fibrocontractive tissue. The goal of this thesis was to elucidate the interactions between the boundary stiffness of three-dimensional (3D) matrix and soluble factors on valvular interstitial cell (VIC) phenotype with a quantitative approach. The first part of the work presented in this thesis was to characterize the combined effects of boundary stiffness and transforming growth factor-β1 (TGF-β1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing VICs within micro-scale fibrin gels between compliant posts (0.15-1.05 nN/nm) in chemically-defined media with TGF-β1 (0-5 ng/mL). The VICs generated 100 to 3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a 3D culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-β1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-β1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-β1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-β1 concentration. In the second part, by using the quantitative information obtained from the first part, we investigated the effects of dynamic changes in stiffness on cell phenotype in a 3D protein matrix, quantitatively. Our novel method utilizing magnetic force to constrain the motion of one of two flexible posts between which VIC-populated micro-tissues were cultured effectively doubled the boundary stiffness and resulted in a significant increase in cell-generated forces. When the magnetic force was removed, the effective boundary stiffness was halved and the tissue tension dropped to 65-87% of the peak value. Surprisingly, following release the cell-generated forces continued to increase for the next two days rather than reducing down to the homeostatic tension level of the control group with identical (but constant) boundary stiffness. The rapid release of tension with the return to baseline boundary stiffness did not result in a decrease in number of cells with α-SMA positive stress fibers or an increase in apoptosis. When samples were entirely released from the boundaries and cultured free floating (where tension is minimal but cannot be measured), the proportion of apoptotic cells in middle region of the micro-tissues increased more than five-fold to 31%. Together, these data indicate that modest temporary changes in boundary stiffness can have lasting effects on myofibroblast activation and persistence in 3D matrices, and that a large decrease in the ability of the cells to generate tension is required to trigger de-differentiation and apoptosis. "