Tissues with Patterned Vessels or Protein Release Induce Vascular Chemotaxis in an In Vitro Platform

Engineered tissues designed for translational applications in regenerative medicine require vascular networks to deliver oxygen and nutrients rapidly to the implanted cells. A limiting factor of in vivo translation is the rapid and successful inosculation, or connection, of host and implanted vascular networks and subsequent perfusion of the implant. An approach gaining favor in vascular tissue engineering is to provide instructive cues from the engineered tissue to enhance host vascular penetration and connection with the implant. Here, we use a novel in vitro platform based on the aortic ring assay to evaluate the impact of patterned, endothelialized vessels or growth factor release from engineered constructs on preinosculative vascular cell outgrowth from surrogate host tissue in a controlled, defined environment, and introduce robust tools for evaluating vascular morphogenesis and chemotaxis. We demonstrate the creation of engineered vessels at the arteriole scale, which develop basement membrane, exhibit tight junctions, and actively sprout into the surrounding bulk hydrogel. Vessel-containing constructs are co-cultured adjacent to rodent aortic rings, and the resulting heterocellular outgrowth is quantified. Cells originating from the aortic ring migrate preferentially toward constructs containing engineered vessels with 1.5-fold faster outgrowth kinetics, 2.5-fold increased cellular density, and 1.6-fold greater network formation versus control (no endothelial cells and growth factor-reduced culture medium). Growth factor release from constructs with nonendothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in endothelial growth medium 2 (EGM-2) medium. In conclusion, we show that three-dimensional endothelialized patterned vessels or growth factor release stimulate a robust, host-derived vascular cell chemotactic response at early time points critical for instructive angiogenic cues. Further, we developed robust, unbiased tools to quantify metrics of vascular morphogenesis and preinosculative heterocellular outgrowth from rat aortic rings and demonstrated the utility of our complex, controlled environment, heterocellular in vitro platform. Impact statement Using a novel in vitro platform, we show that engineered constructs with patterned vessels or angiogenic growth factor release, two methods of instructing host revascularization responses, equivalently improve early host-derived vascular outgrowth. Our platform leverages the aortic ring assay in a tissue engineering context to study preinosculative vascular cell chemotaxis from surrogate host vascular cells in response to paracrine cues from co-cultured engineered tissues using robust, open-source quantification tools. Our accessible and flexible platform enables translationally focused studies in revascularization using implantable therapeutics containing prepatterned vessels with greater environmental control than in vivo studies to advance vascular tissue engineering.