The low in vitro stability of dynamic covalent hydrogels with fast exchanging bonds prevents their long-term applications despite their benefits, like fast self-healing and stress relaxation. To circumvent this while maintaining the fast dynamics, Schiff base crosslinked alginate hydrogels were developed with different crosslinking chemistry and structure. Alginate dialdehyde (ADA) was synthesized and used to prepare hydrazone and oxime crosslinked hydrogels. Two crosslinking strategies were followed, one group was prepared by ADA and difunctional small molecule crosslinkers having hydrazide and oxyamine functionalities. The other group was prepared by hydrazide-modified alginate and ADA to study the effects of changing the crosslinker structure while maintaining the crosslinking chemistry on the hydrogel dynamics. The effects of the polymer concentration, crosslinking chemistry, and structure on the hydrogel properties, self-healing ability, and injectability were evaluated. Mitigating the low in vitro stability, the fast exchange of dynamic bonds was exploited to control the delivery of L-Arginine (L-Arg). The release of L-Arg was assessed at different pH to investigate the stimuli-responsiveness of the hydrogels and was compared with the release from ionically crosslinked hydrogels. Furthermore, the effects of incorporating L-Arg as a competitor in forming imine bonds on the mechanical properties of the hydrogels were investigated.

The dynamic interplay between cells and their native extracellular matrix (ECM) influences cellular behavior, imposing a challenge in biomaterial design. Dynamic covalent hydrogels are viscoelastic and show self-healing ability, making them a potential scaffold for recapitulating native ECM properties. We aimed to implement kinetically and thermodynamically distinct crosslinkers to prepare self-healing dynamic hydrogels to explore the arising properties and their effects on cellular behavior. To do so, aldehyde-substituted hyaluronic acid (HA) was synthesized to generate imine, hydrazone, and oxime crosslinked dynamic covalent hydrogels. Differences in equilibrium constants of these bonds yielded distinct properties including stiffness, stress relaxation, and self-healing ability. The effects of degree of substitution (DS), polymer concentration, crosslinker to aldehyde ratio, and crosslinker functionality on hydrogel properties were evaluated. The self-healing ability of hydrogels was investigated on samples of the same and different crosslinkers and DS to obtain hydrogels with gradient properties. Subsequently, human dermal fibroblasts were cultured in 2D and 3D to assess the cellular response considering the dynamic properties of the hydrogels. Moreover, assessing cell spreading and morphology on hydrogels having similar modulus but different stress relaxation rates showed the effects of matrix viscoelasticity with higher cell spreading in slower relaxing hydrogels.