Hydrogel-Dependent Angiogenic Sprouting in the Ex Vivo Aortic Ring Assay: A Comparative Functional Approach for Biomaterial Evaluation

Insufficient vascularization remains a major limitation in tissue engineering, restricting the survival and maturation of larger bioengineered constructs. While candidate hydrogels are commonly characterized with regard to physicochemical properties, gelation behavior, mechanical performance, and cytocompatibility, simple functional assays that assess their capacity to support vascular sprouting are less frequently integrated into early-stage biomaterial evaluation. Here, we investigated the established ex vivo aortic ring assay (ARA) as an exploratory functional approach for the initial comparison of selected hydrogel formulations. Murine aortic rings were embedded in collagen I (Col I), alginate (Alg), or gelatin methacryloyl (GelMA) and cultured under control conditions or with vascular endothelial growth factor A (VEGF-A) stimulation. These hydrogels were intentionally selected as a proof-of-concept panel of representative, non-equivalent material classes with distinct expected cell-interactive properties. After five days, Col I supported robust capillary-like outgrowth that was further enhanced by VEGF-A, whereas the tested GelMA formulation supported only limited cellular migration and the tested unmodified Alg formulation showed no detectable sprouting under the conditions examined. Cluster of differentiation 31 (CD31) immunostaining supported the presence of an endothelial component within the Col I-supported sprouting structures. These findings demonstrate that the ARA can detect pronounced formulation-dependent differences among the specific hydrogels tested using straightforward morphological and immunostaining readouts. Within the scope of the formulations tested, these findings support the ARA as a complementary functional readout alongside conventional biomaterial characterization before more complex tissue engineering or biofabrication studies are performed.