UC Irvine

Biophysical properties of the fibrous extracellular matrix (ECM) are critical regulators of cell behavior. Bulk measurements at single time points have often been used to investigate matrix stiffness as an instructive cue. However, our lab has demonstrated that local pericellular stiffness can be heterogeneous, anisotropic, and vary greatly from the measured bulk properties. Thus, we developed a multi-axis optical tweezers-based active microrheology (AMR) system which is capable of measuring ECM properties on a length scale relevant to cells.The fibrillar architecture of the native ECMs is well known to give rise to nonlinear mechanical properties that govern force transmission and long-range cell-cell communication. Despite this, direct comparisons of pericellular stiffening in fibrous and amorphous hydrogels remain limited. In this thesis work, I used multi-axis AMR to investigate how ECM architecture regulates the emergence of long-range stiffening and stiffness anisotropy.In an initial study in collaboration with Professor Andrew Putnam, we used AMR in a fibrin hydrogel vascular self-assembly model to assess how different supportive stromal cells (SCs), in co-culture with endothelial cells (ECs), differentially stiffen the ECM and mediate vascular morphogenesis. We found that lung fibroblasts (LFs) lead to enhanced vascular development, concurrent with significant anisotropic stiffening of the fibrous ECM in the direction of capillary growth, as well as extensive ECM remodeling compared to bone marrow-derived mesenchymal stem cells (MSCs). Notably, this stiffening extended far beyond the immediate pericellular region, consistent with long-range force transmission mediated by strain-hardening in fibrous matrices.To further explore the direct impact of a fibrillar ECM, we used AMR to map pericellular stiffness around LFs and MSCs in stiffness-matched fibrin and amorphous gelatin methacryloyl (GelMA) hydrogels. While both cells stiffened fibrin relative to cell-free controls, they reduced stiffness in GelMA. Inhibitor studies implicated cell contractility as a predominant mediator of pericellular stiffness changes. Additionally, fibrin supported significant stiffness anisotropy around cells while GelMA remained relatively isotropic. Together, these data highlight the potential link between fibrous ECM structure and the development of stiffness anisotropy and long-range stiffening, which may be important mediators of complex biological processes like vascular morphogenesis.