Enzymatically Crosslinked Collagen as a Versatile Matrix for In Vitro and In Vivo Co‐Engineering of Blood and Lymphatic Vasculature

Abstract Adequate vascularization is required for the successful translation of many in vitro engineered tissues. This study presents a novel collagen derivative that harbors multiple recognition peptides for orthogonal enzymatic crosslinking based on sortase A (SrtA) and Factor XIII (FXIII). SrtA‐mediated crosslinking enables the rapid co‐engineering of human blood and lymphatic microcapillaries and mesoscale capillaries in bulk hydrogels. Whereas tuning of gel stiffness determines the extent of neovascularization, the relative number of blood and lymphatic capillaries recapitulates the ratio of blood and lymphatic endothelial cells originally seeded into the hydrogel. Bioengineered capillaries readily form luminal structures and exhibit typical maturation markers both in vitro and in vivo. The secondary crosslinking enzyme Factor XIII is used for in situ tethering of the VEGF mimetic QK peptide to collagen. This approach supports the formation of blood and lymphatic capillaries in the absence of exogenous VEGF. Orthogonal enzymatic crosslinking is further used to bioengineer hydrogels with spatially defined polymer compositions with pro‐ and anti‐angiogenic properties. Finally, macroporous scaffolds based on secondary crosslinking of microgels enable vascularization independent from supporting fibroblasts. Overall, this work demonstrates for the first time the co‐engineering of mature micro‐ and meso‐sized blood and lymphatic capillaries using a highly versatile collagen derivative.