(25e) Vascularization of Engineered Tissues Via Synthetic Regulation of Cell Signaling

The vascularization of engineered tissues remains a significant challenge for the biomedical community. While numerous biomaterial platforms have demonstrated that spatiotemporal control of growth factor presentation can direct vascularization, clinical translation of these approaches has been limited due to the high cost and short shelf-life of growth factors. In contrast, clinical trials have explored the use of gene or cell therapies to induce vascularization, but the inability to control gene expression or cell behavior results in vasculature that is short-lived and non-functional. Inspired by the process of vascular morphogenesis, in which tight regulation of paracrine signals controls the formation of functional vasculature, we hypothesize that the ability to precisely regulate gene expression and paracrine signaling in engineered cells will lead to improved control over vessel formation and maturation for applications in ischemic disease and regenerative medicine. Here, we demonstrate that the magnitude and timing of VEGF production can be controlled by engineering fibroblasts with an inducible genetic circuit, which enables VEGF secretion in a manner controlled by the concentration of chemical inducer (e.g., grazoprevir). Titration of VEGF secretion yields dose-dependent differences in vessel density and architecture in a variety of synthetic and natural biomaterials, such as fibrin and methacrylate-functionalized dextran. Using a microfluidic model of vascular morphogenesis, we find that controlling VEGF signaling alone is insufficient to produce perfused vascular beds with tunable density. Instead, temporal coordination of VEGF and extracellular matrix production is necessary to establish highly-perfused vascular beds with variable architecture. Engineering fibroblasts with dual-channel inducible switches enables orthogonal control of VEGF secretion and extracellular matrix gene expression, thereby enabling the formation of stable and perfused vascular beds with tunable density.

Overall, these results provide insight into how paracrine and extracellular matrix signaling are regulated during vascular morphogenesis to give rise to functional vascular networks with varied architecture. More broadly, the toolbox developed in this work for controlling gene expression and signaling offers a new generalizable approach to investigate and uncover a wide variety of tissue morphogenetic programs that could be used to direct the assembly of artificial tissues. Ongoing efforts are focused on employing engineered fibroblasts to vascularize biomaterials in vivo and develop vascularized parenchymal tissue implants.