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

Abstract

are missing extensive pericyte coverage. In addition, lymphatics exhibit an irregularly shaped or collapsed lumen lacking red blood cells (RBCs). [1][2][3] Lymphatic vascular structures are generally classified as either capillaries (diameter 10-60 µm) or collecting lymphatic vessels (diameter 50-200 µm). [4] Similarly, blood vessels can be categorized into either capillaries (4-10 µm) or larger structures such as arterioles and venules (10-100 µm). [5] Despite their structural differences, the blood and lymphatic vasculature are closely interconnected on a functional level to ensure nutritional supply of cells and tissue homeostasis. [6][7][8] The absence of a functional vascular network in vitro is one of the main drawbacks of many bioengineered tissues and a frequent cause of necrosis in vivo. [9] Whereas most previous studies focused on the blood vasculature, recent discoveries have brought increased attention to the role of lymphatics and their role in tissue regeneration. [7,8] Yet only a few studies have so far attempted to co-engineer blood and lymphatic microcapillaries. Marino et al. described a simple co-culturing system of blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) based on native fibrin and collagen hydrogels. [10] The resulting microcapillaries remained in their early developmental stages, with phenotypes exhibiting mostly short capillary fragments 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.

Digital Light Processing 3D Bioprinting of Gelatin‐Norbornene Hydrogel for Enhanced Vascularization

“…However, in many instances, hydrogels with higher moduli may be preferred, but they would not support a high degree or rapid vascularization. In such instances, stiffer hydrogels may be conjugated with vasculogenic peptides, such as VEGF‐mimetic QK peptide, which has been demonstrated to enhance angiogenesis in vitro [ 30,63,64 ] and in vivo. [ 65 ] To this end, we tethered a cysteine‐terminated QK peptide in the DLP‐printed GelNB hydrogels during thiol‐norbornene hydrogel cross‐linking.…”

The Use of Collagen-Based Materials in Bone Tissue Engineering

“…Currently, collagens directly extracted from animal tissues, or produced as recombinant proteins, with or without further modifications according to crosslinking, polymerization, or fibrillization, have been widely applied in vitro as standardized 3D materials to investigate the influence of microstructural and mechanical features on cell behaviors, such as cell attachment, cell contraction, cell motility, and related gene expressions [ 108 , 109 ]. Collagen-based materials in various forms, including membranes, sponges or matrices, hydrogel, and composite scaffolds, are also widely used in vivo to support bone tissue regeneration in different clinical applications [ 18 , 110 ] ( Figure 4 ).…”

Injectable‐Hydrogel‐Based Tissue Sealant for Hemostasis, Bacteria Inhibition, and Pro‐Angiogenesis in Organ Bleeding Wounds and Therapeutic Outcome Monitoring Via NIR‐II Optical Imaging

“…Some PAPs (such as the vascular endothelial growth factor (VEGF) mimetic peptide QK) can also address the limitations associated with growth factors. [ 42 ] Therefore, combining AMPs and PAPs within a biocompatible injectable hydrogel may afford a feasible approach to enhance wound healing; while, effectively safeguarding against any potential bacterial infections.…”