Vasculogenesis is the de novo formation of a vascular network from individual endothelial progenitor cells occurring during embryonic development, organogenesis, and adult neovascularization. Vasculogenesis can be mimicked and studied in vitro using network formation assays, in which endothelial cells (ECs) spontaneously form capillary-like structures when seeded in the appropriate microenvironment. While the biochemical regulators of network formation have been well studied using these assays, the role of mechanical and topographical properties of the extracellular matrix (ECM) is less understood. Here, we utilized both natural and synthetic fibrous materials to better understand how physical attributes of the ECM influence the assembly of EC networks. Our results reveal that active cell-mediated matrix recruitment through actomyosin force generation occurs concurrently with network formation on Matrigel, a reconstituted basement membrane matrix regularly used to promote EC networks, and on synthetic matrices composed of electrospun dextran methacrylate (DexMA) fibers. Furthermore, modulating physical attributes of DexMA matrices that impair matrix recruitment consequently inhibited the formation of cellular networks. These results suggest an iterative process in which dynamic cell-induced changes to the physical microenvironment reciprocally modulate cell behavior to guide the formation and stabilization of multicellular networks.

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Mechanical interactions between cells and their surrounding extracellular matrix (ECM) guide many fundamental cell behaviors. Native connective tissue consists of highly organized, 3D networks of ECM fibers with complex, nonlinear mechanical properties. The most abundant stromal matrix component is fibrillar type I collagen, which often possesses a wavy, crimped morphology that confers strain- and load-dependent nonlinear mechanical behavior. Here, we established a new and simple method for engineering electrospun fibrous matrices composed of dextran vinyl sulfone (DexVS) with controllable crimped structure. A hydrophilic peptide was functionalized to DexVS matrices to trigger swelling of individual hydrogel fibers, resulting in crimped microstructure due to the fixed anchorage of fibers. Mechanical characterization of these matrices under tension confirmed orthogonal control over nonlinear stress-strain responses and matrix stiffness. We next examined ECM mechanosensing of individual endothelial cells (ECs), finding that fiber crimp promoted physical matrix remodeling alongside decreases in cell spreading, focal adhesion area, and nuclear localization of Yes-associated protein (YAP). These changes corresponded to an increase in migration speed along with evidence for long-range interactions between neighboring cells in crimped matrices. Interestingly, when ECs were seeded at high density in crimped matrices, capillary-like networks rapidly assembled containing tube-like cellular structures wrapped around bundles of synthetic matrix fibers due to increased physical reorganization of matrix fibers. Our work provides an additional level of mechanical and architectural tunability to synthetic fibrous matrices and implicates a critical role for mechanical nonlinearity in EC mechanosensing and network formation.

Intercellular communication is critical to the development and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, we utilized synthetic, electrospun fibrous matrices in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during the assembly of microvascular networks. We found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional migration of neighboring ECs towards each other prior to the formation of stable cell-cell connections. We also identified a critical role for intracellular calcium signaling mediated by focal adhesion kinase and TRPV4 during MIC that extends to multicellular assembly of vessel-like networks in 3D fibrin hydrogels. The results presented here are critical to the design of biomaterials that support cellular self-assembly for tissue engineering applications.