Zheng, YingMurry, CharlesZeinstra, Nicole2022-07-142022-07-142022Zeinstra_washington_0250E_24165.pdfhttp://hdl.handle.net/1773/48825Thesis (Ph.D.)--University of Washington, 2022Implantable engineered cardiac patches have emerged as a promising approach to improve heart function after myocardial infarction. However, inadequate vascularization has prevented the generation and application of large-scale cardiac tissues. Improved methods for engineering perfusable, three-dimensional vasculature are therefore needed to support tissue survival prior to implantation as well as promote rapid host vascular integration post-implantation. The following dissertation reports advances in vascularization of implantable tissues and tools for assessing perfusion in the coronary vasculature. Here, we first established non-invasive, depth-resolved optical coherence tomography-based imaging tools to assess vascular structure and perfusion dynamics of the coronary vasculature and to visualize the fiber orientation of the heart, comparing healthy and infarcted ex vivo fixed rat hearts to demonstrate the capabilities of this imaging platform. We utilized optical microangiography (OMAG) to create three-dimensional maps of the microvasculature and developed an automated processing framework to quantify differences between the vascular morphology of healthy and infarcted hearts. We further utilized OMAG-based velocimetry (OMAG-V) to characterize perfusion and polarization sensitive optical coherence tomography (PSOCT) to visualized fiber orientation within healthy and infarcted tissue. These studies demonstrate the ability of optical coherence tomography-based tools to quantitatively assess remodeling in the heart after infarction without need for tissue processing. The established platform can further be utilized to aid in development of cardiac therapies, including assessing perfusion of implanted cardiac patches. Next, we generated engineered tissues containing perfusable, endothelialized microvessels in a collagen matrix for application in cardiac repair. We created stem cell-derived endothelial cell-lined microvessels with planar geometry and found that these tissues are angiogenic, minimally thrombogenic when perfused with blood, and have gene expression associated with vascular and tissue development. We additionally generated cardiac tissues with perfusable microvessels and showed that when implanted onto infarcted rat hearts, the perfusable microvessel grafts integrate with the coronary vasculature to a greater degree and have higher cardiomyocyte density than non-perfusable self-assembled constructs at 5 days post-implantation. Towards generation of large-scale tissues, we further adapted this platform to engineer thick, perfusable, highly vascularized constructs by stacking multiple layers of patterned collagen membranes, creating a three-dimensional vascular network within the collagen matrix. We combined confocal microscopy, scanning electron microscopy, optical microangiography, and transcriptional profiling to present the in vitro structure and perfusion of the thick vascularized networks. We then implanted these tissues onto infarcted rat hearts and found that multilayer grafts had greater in vivo perfusion and perfused vascular density than non-perfusable self-assembled controls at 5-days post-implantation as indicated by optical microangiography and histological analysis. Together, these data suggest the feasibility of generating large-scale cardiac tissues that achieve timely perfusion upon implantation. This work introduces critical advances in vascular imaging and tissue vascularization, facilitating progress towards therapeutics for cardiac repair.application/pdfen-USCC BYBioengineeringBioengineeringThesis