Effect of hydrogel properties on fibroblast morphodynamics

Cellular morphodynamics are tightly regulated by the mechanical and biochemical properties of the extracellular matrix (ECM), yet independently investigating these cues in 3D hydrogel models remains challenging. Here, we present a modular hyaluronic acid (HA)-based hydrogel system that enables systematic modulation of stiffness, degradability, and viscoelasticity to examine the regulation of fibroblast behavior. Using strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry, we engineered nanoporous HA hydrogels of defined stiffness while maintaining constant polymer density and ligand presentation. Fibroblast morphology showed a non-monotonic dependence on hydrogel formulation, with the most pronounced spreading observed in the ∼400 Pa formulation. Cells in softer and stiffer hydrogels remained mostly rounded. Protease-degradable crosslinkers accelerated, but were not strictly required for, morphological transitions, and comparison with alginate hydrogels of similar stiffness suggested a role for HA-specific remodeling mechanisms. Incorporation of xanthan gum introduced viscoelastic and viscoplastic behavior into the predominantly elastic HA hydrogels, which supported pronounced fibroblast spreading. These findings demonstrate that fibroblast morphodynamics in 3D are governed by an interplay between stiffness, enzymatic degradability, matrix identity, and time-dependent mechanical behavior. The presented hydrogel system provides a versatile platform for interrogating cell-matrix interactions and for engineering instructive microenvironments in tissue models and regenerative medicine.