Development of Pre-Vascularized Tissues Containing Aligned and Perfusable Microvessels

Abstract

The single greatest restraint in tissue engineering is the inability to create and perfuse functional microvasculature in dense engineered tissues of physiological stiffness. Without active delivery of nutrients and oxygen, tissue size is diffusion-limited to thicknesses of around 400 µm, or much less for highly metabolic tissues. Thus, the creation of pre-vascularized tissues that have a high density of organized microvessels that could be perfused is a major goal of tissue engineering. The present work makes significant advances toward this goal. Tissue patches containing a high density of human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and in both cases, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. In vitro, a high-throughput assay was developed to investigate optimal conditions for angiogenic sprouting, vasculogenic microvascular network formation, and inosculation of the sprouts and microvessels in 3D fibrin gels. Samples loaded with vascular endothelial growth factor and fibroblast growth factor exhibited enhanced angiogenic sprouting, and a hybrid medium culture regimen resulted in enhanced sprouting, well-developed microvascular networks, and inosculation of the microvessels and sprouts. These results showed potential for the in vitro perfusion of larger-scale microvascular tissues. An engineering strategy was developed to perfuse endothelialized microchannels that could form sprouts into fibrin gels containing a microvascular network. An in vitro perfusion bioreactor was designed and tested that enabled these microvascular tissues to be cultured, compacted, and aligned to form a dense network of microvessels that also contained perfusable microchannels with sprouts. Different microchannel seeding regimens and perfusion regimens were applied and it was determined which conditions ultimately led to microchannel endothelialization, sprouting, perfusion, and maintenance during gel compaction. While inosculation and perfusion of the microvessels has yet to be achieved, this work presents the building blocks for a potential strategy that could ultimately enable the perfusion of a dense, aligned microvascular network through anastomoses of sprouts and microvessels. Achievement of this goal would unlock a number of tissue engineering opportunities in the development of large engineered tissues for regenerative therapies.