Linking Polysaccharide Structure, Gelation Kinetics, and Function in Dynamic Acylhydrazone Hydrogels

Abstract

Dynamic covalent hydrogels formed through reversible acylhydrazone crosslinking have emerged as promising injectable biomaterials. However, a fundamental gap remains in understanding how the macromolecular structure of oxidized polysaccharides (OxPs) governs gelation kinetics and how these kinetics pathways translate into material properties and cellular responses. We address this question by developing an acylhydrazone hydrogel library composed of alginate adipohydrazide crosslinked with oxidized alginate (OxA) or oxidized dextran (OxD), two reactive aldehyde-bearing polymers with comparable chemical functionality but fundamentally distinct backbone structure. By varying polysaccharide type, oxidation degree, and reaction pH, we decoupled the effects of chemical functionality from macromolecular structure and established quantitative structure-kinetics-property-function relationships. OxD-based hydrogels undergo rapid, largely pH-independent gelation, whereas OxA-based systems display pronounced pH-dependent kinetics with significantly delayed network formation under physiological pH. These differences in gelation kinetics and OxPs macromolecular structures lead to marked variations in hydrogel mechanics, including stiffness, stress relaxation, stability, injectability, and post-injection recovery. Importantly, differences in gelation kinetics modulate cell-matrix interactions in three-dimensional culture. Slowly forming OxA hydrogels maintained rounded chondrocyte shape, while rapidly gelling OxD networks induced transient cell elongation. Mesenchymal stem cells displayed similar shapes regardless of gelation kinetics, indicating cell-type-specific responses to matrix formation dynamics.