Click method preserves but EDC method compromises the therapeutic activities of the peptide-activated hydrogels for critical ischemic vessel regeneration

Peptide-functionalized hydrogel is one of commonly used biomaterials to introduce hydrogel-induced vessel regeneration. Despite many reports about the discoveries of high-active peptides (or ligands) for regeneration, the study on the conjugating methods for the hydrogel functionalization with peptides is limited. Here, we compared the vasculogenic efficacy of the peptide-functionalized hydrogels prepared by two commonly used conjugating methods, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and Click methods, through cell models, organ-on-chips models, animal models, and RNA sequencing analysis. Two vascular-related cell types, the human umbilical vein endothelial cells (HUVECs) and the adipose-derived stem cells (ADSCs), have been cultured on the hydrogel surfaces prepared by EDC/Click methods. It showed that the hydrogels prepared by Click method supported the higher vasculogenic activities while the ones made by EDC method compromised the peptide activities on hydrogels. The vasculogenesis assays further revealed that hydrogels prepared by Click method promoted a better vascular network formation. In a critical ischemic hindlimb model, only the peptide-functionalized hydrogels prepared by Click method successfully salvaged the ischemic limb, significantly improved blood perfusion, and enhanced the functional recoveries (through gait analysis and animal behavior studies). RNA sequencing studies revealed that the hydrogels prepared by Click method significantly promoted the PI3K-AKT pathway activation compared to the hydrogels prepared by EDC method. All the results suggested that EDC method compromised the functions of the peptides, while Click method preserved the vascular regenerating capacities of the peptides on the hydrogels, illustrating the importance of the conjugating method during the preparation of the peptide-functionalized hydrogels.