(119f) Combined Cellular and Tissue Engineering Approaches for Functional Vascularization of Biomaterials

Over the past several decades, the field of tissue engineering has advanced the development of biomaterials as scaffolds for regenerative medicine. However, clinical translation continues to be hampered by the inability form functional vasculature within these materials to ensure survival and function post-transplantation. While several biomaterial platforms have been designed to regulate the delivery of angiogenic growth factors spatially and temporally, their utilization in clinical trials has been limited, potentially due to the high cost of short shelf-life of growth factors. In contrast, multiple clinical trials have investigated the delivery of cells or genes to stimulate therapeutic angiogenesis, but the inability to control cell behavior or the magnitude and timing of gene expression results in vasculature that is non-perfused and eventually regresses. Furthermore, no approach thus far has demonstrated the ability to control the extent of vascularization, which is critical for meeting the needs of different tissues and patients.

During development, the human body uses the process of vascular morphogenesis to create functional vasculature. Temporally regulated and concentration-dependent cascades of paracrine signaling between endothelial and stromal cells orchestrate the initial organization of endothelial cells into cords and direct their assembly into perfused vascular networks. Inspired by the process of vascular morphogenesis, we hypothesize that biomaterials can be functionally vascularized by the incorporation of endothelial and stromal cells that are engineered with user-controlled genetic circuits to recapitulate the paracrine signals necessary for vascularization.

In this work, we demonstrate the functional vascularization of fibrin hydrogels by encapsulation of genetically engineered endothelial and stromal cells. The engineered cells express inducible synthetic zinc finger transcription regulators (synZiFTRs) to enable user-controlled expression of paracrine factors responsible for vascular morphogenesis, such as VEGF. Using a library of small molecules (e.g. gravoprevir, 4-hydroxytamoxifen, etc.), we report orthogonal and dose-dependent expression of paracrine factors across a wide concentration of small molecule induction. Using microfluidic devices to model vascular morphogenesis, we show that vascular density can be tuned by varying the extent of paracrine signaling and explore the impact of varied ratios of paracrine factors on vascular architecture (e.g. network length and branching). Finally, we demonstrate that early- and late-stage paracrine factors can be sequentially expressed, and that the timing of late-stage paracrine factor induction influences vascular architecture and perfusion. Overall, we describe a technology for achieving dose- and time-dependent control over gene expression, which can be combined with biomaterials to achieve vascularized artificial tissues. Broadly, we envision that the combination of cellular and tissue engineering can be used not only for vascularization, but for directing tissue assembly in general.