Engineering Hierarchical Vasculature for Regenerative Medicine

Abstract

In the human body, blood flows from the heart to organs and tissues through a hierarchical vascular tree, consisting of large arteries that branch into arterioles and further into capillaries where gas and nutrient exchange occur. Engineering a complete, integrated vascular hierarchy possessing vessels large enough to suture, strong enough to withstand hemodynamic forces, and a branching structure to permit immediate perfusion of a fluidic circuit across length scales would be transformative for the field of regenerative medicine, enabling the translation of engineered tissues of clinically-relevant size, and perhaps whole organs. Within the field of tissue engineering, there has been extensive research towards engineering vessels at each caliber individually, while hierarchical vasculature has been comparatively understudied. This dissertation investigated the engineering of various two-scale hierarchical vascular tissues (HVT) which were integrated to form a complete, three-scale HVT composed of capillaries, mesovessels, and a macrovessel suitable for surgical anastomosis.

Aim 1 investigated the fabrication of meso-microvascular hierarchies and explored how stromal cell (SC) identity influenced endothelial cell (EC) morphogenesis and the formation of perfusable HVT. A microfluidic lab-on-a-chip system was adopted and modified to enable the formation of both microvascular capillary beds and mesovessels, and then used to evaluate inosculation between the two scales to support functional perfusion. The presence of supportive SC that can adopt perivascular phenotypes was essential, and lung fibroblasts (LF), dermal fibroblasts, and mesenchymal stem cells were compared and contrasted for their abilities to support EC morphogenesis and subsequent perfusion of the HVT. LF supported microvascular network morphologies with the highest vessel density, diameter, and interconnectivity, and were the only SC type to support functional perfusion of the hierarchy. By comparing three SC types and their abilities to support the formation of multiscale HVT, this study provided insights regarding the choice of cells for vascular cell-based therapies and highlighted the importance of SC identity in the regulation of tissue-specific vasculature.

Aim 2 focused on incorporating a functional macrovessel to suture the HVT into circulation. We compared and contrasted vessels of venous, arterial, thoracic, and femoral origins for their ability to sprout EC capable of inosculating with surrounding microvasculature. We identified the thoracic aorta as the vessel yielding the greatest degree of sprouting and interconnection to surrounding capillaries and the only vessel capable of cannulation. The presence of cells undergoing vascular morphogenesis in the surrounding hydrogel attenuated EC sprouting from the macrovessel, but ultimately sprouted EC interacted with capillaries in the bulk supporting an interconnected HVT. This study yielded HVT suitable for surgical anastomosis and a platform to study vascular inosculation to provide insights for cell-based therapies.

Aim 3 focused on the biomanufacturing of fibrin-based tissue engineered vascular grafts for scale-up of HVT towards translational applications avoiding the need for autologous vessel harvest. Three-layered grafts mimicking the native tunica intima, media, and adventitia composed of smooth muscle cells (media layer), LF and EC (adventitial layer), and EC only (intima layer) were successfully engineered and evaluated. Cells in the adventitial layer formed a vasa vasorum that sprouted into surrounding hydrogels that also contained microvasculature and mesovessels to support the formation of integrated HVT. Extended adventitia culture was critical for integration across length scales. Overall, this dissertation integrated top-down and bottom-up fabrication approaches towards the engineering of a complete HVT suitable for surgical anastomosis for translational regenerative medicine applications.