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 pre-inosculative 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 towards 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 non-endothelialized channels and in reduced factor medium equivalently stimulates sustained vascular outgrowth distance, cellular density, and network formation, akin to engineered vessels in EGM-2 medium. In conclusion, we show that 3D endothelialized patterned vessels or growth factor release both 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 pre-inosculative heterocellular outgrowth from rat aortic rings and demonstrate the utility of our complex, controlled environment, heterocellular in vitro platform.