Unveiling the Effect of Aqueous-Phase Dynamics on Chitosan Hydrogel Film Mechanical Properties Through AFM Nanoindentation and Tensile Testing.

The mechanical properties of cell scaffolds are strongly influenced by their hydration state. In this study, we investigated the effect of the aqueous phase on the elastic modulus of chitosan hydrogel films using two complementary techniques: uniaxial tensile testing and atomic force microscopy (AFM) nanoindentation. Our results demonstrate that hydration markedly reduced the elastic modulus, decreasing from approximately 2 GPa in dry films to 120 kPa in swollen films, primarily due to the plasticizing effect of water. Moreover, hydrogel films in equilibrium with the aqueous phase exhibited a Young's modulus three times lower than that of swollen films not in equilibrium. Raman spectroscopy further reveals a solvent "squeeze-out" phenomenon, as evidenced by an increased signal intensity in the 850-1200 cm region for stretched films that were out of swelling equilibrium, whereas equilibrated films showed stable spectral features. These findings highlight the crucial role of hydration dynamics in determining the mechanical behavior of chitosan hydrogel films, offering valuable insights for tailoring their properties in biomedical scaffold applications.