Engineered microvessels for improved integration with host coronary circulation and for the generation of pre-vascularized cardiac constructs
Redd, Meredith Ann
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Engineered cardiovascular tissues are at the forefront of regenerative medicine research and have the potential to redefine the treatment of heart disease in the United States and worldwide. There are many challenges associated with tissue engineering such as mimicking native structure, pre-vascularization, tissue maturation, and functional integration with the host. The following dissertation addresses many of these challenges, with a particular focus on improving vascularization methods and promoting host integration following in vivo transplantation. Pre-vascularization of engineered cardiac constructs is essential for the survival of the tissue, but requires precise control of structural and geometric features which is technically very challenging. Here, we engineered cardiac constructs with patterned vasculature by optimizing cardiomyocyte culture conditions for compatibility with our previously established method to fabricate 3D perfusable microvessels in native collagen matrix. While collagen-based cardiac constructs have previously been generated in our lab and in others, we found that human stem cell derived cardiomyocytes are unable to form functional constructs in dense collagen matrix, which is required to maintain vessel patency during fabrication. However, when co-cultured with matrix-remodeling stromal cells, we observed synchronous electrical wave propagation, force generating contractions, and markedly improved cellular maturation as well as uniaxial alignment. With these co-culture conditions in dense collagen, we were able to incorporate patterned endothelialized channels and generated pre-vascularized cardiac tissue constructs. This work represented an important achievement, but looking forward, tissue engineering strategies will ultimately require efficient host integration to have therapeutic success. Additionally, the ability to generate all of the required cell types (i.e. cardiomyocytes, stromal cells, and endothelial cells) from a single cell source would be more suitable for clinical applications by enabling patient-specific or autologous construct formation with human induced pluripotent stem cell technology. Towards this end, we generated human stem cell derived endothelial cells (hESC-ECs) and characterized them both in vitro and in vivo. hESC-ECs were used to make engineered microvessels and were found to retain structural and functional characteristics of mature endothelial cells, including patent lumen formation with intact junctions and non-thrombogenic blood interactions. To determine whether our pre-patterned and perfusable hESC-EC vascular networks were able to improve host integration following in vivo implantation, we developed a novel method to image vascular perfusion ex vivo of grafts implanted on injured rat myocardium for five days. Compared to unpatterned controls, patterned vascular networks showed improved vascular perfusion dynamics with flow velocities comparable to those in healthy myocardial regions. To our knowledge, this was the first demonstration of improved graft perfusion through the use of patterned vasculature in the heart. Taken together, this research demonstrated the successful use of a patterned vascular system for the generation of pre-vascularized cardiac constructs and for improved graft perfusion dynamics. Collectively, this work has many implications in the field of cardiovascular tissue engineering with particular regard to maintaining cardiac structure during vascular incorporation, host integration strategies, and perfusion assessment methods.
- Bioengineering