Tuning scaffold degradation with non-natural peptidomimetics to control human umbilical vein endothelial cell morphology and vessel formation

The vascularization of 3D tissue constructs, such as hydrogels, remains a paramount challenge in tissue engineering. Extracellular matrix degradation and remodeling are key parts of the vascularization process; however, it is difficult to isolate the effects of degradability in both natural and synthetic matrix models. Naturally-derived matrices typically couple degradability to other material properties, whereas synthetic matrices rely on short peptide sequences to impart degradability, which may exhibit substrate overlap to many proteases and confound degradability trends in vivo. Here, we present a method to systematically tune 3D hydrogel degradation across multiple proteases using crosslinkers with non-natural peptoid (N-substituted glycine) substitutions. Increased peptoid substitutions reduced hydrogel degradability to proteases without altering hydrogel modulus, swelling ratio, or crosslinker length. Using this approach, human umbilical vein endothelial cells (HUVECs) encapsulated in more degradable hydrogels proliferated more, formed more vessels, exhibited higher metabolic activity, and secreted more extracellular matrix than HUVECs encapsulated in less degradable or non-degradable hydrogels. Interestingly, HUVECs encapsulated in the least degradable hydrogels secreted significantly higher matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) than HUVECs encapsulated in the most degradable hydrogels, suggesting higher MMP secretion to compensate for the reduced matrix degradability. Overall, this work highlights the importance of protease-mediated remodeling on vascularization and suggests that peptoid substitutions are effective for tuning hydrogel degradability for a variety of 3D cell applications. STATEMENT OF SIGNIFICANCE: Vascularization of 3D tissue constructs relies on scaffold degradability to accommodate matrix remodeling. Many strategies for proteolytic degradability have been developed; however, these strategies typically couple degradability to other material properties or do not predictably decrease degradation to a complex protease profile. This work presents a strategy to tune hydrogel degradability using peptide crosslinkers with non-natural peptoid substitutions. Increased peptoid substitutions decrease hydrogel proteolytic degradability without altering chemical composition, crosslinker length, or other material properties of the scaffold. The degradability of the developed hydrogels significantly impacted human umbilical vein endothelial cell vessel formation, metabolic activity, morphology, and protease secretion, indicating this strategy is effective for decoupling degradability from other scaffold properties for a variety of biological applications.