Oxidized Xanthan Gum Cross-Linked N−O Carboxymethyl Chitosan Hydrogel Promotes Spheroid Formation of Murine Fibroblast by Increasing Cell−Cell Interaction and Integrin αv Expression

Naturally derived Schiff-based hydrogels are widely fabricated for tissue engineering applications. However, limited studies have explored how the physicochemical and functional groups on polymer chains affect cell behavior in three dimensions. To address this limitation, we fabricated cytocompatible N-O carboxymethyl chitosan (NOCC) cross-linked with oxidized xanthan gum (OXG), incorporating various aldehyde (-CHO) contents (NO1, NO2, and NO3) while maintaining a constant concentration of NOCC, resulting in hydrogels with diverse viscoelastic and aldehyde content properties. The results demonstrated significant differences in storage modulus (<i>G</i>') and loss modulus (<i>G</i>″), attributed to cross-linking density through imine bonds (-C═<i>N</i>-). These differences influenced murine fibroblast aggregation, spheroid formation, and cell migration, proliferation, and viability over time. Both NO1 and NO2 exhibited good cell viability, with slight differences in spheroid morphology compared to those of NO3 and Matrigel samples. To further explore cell behaviors, integrin αV (CD51) expression was assessed using fluorescence-activated cell sorting (FACS) and immunofluorescence. The results aligned with prior observations, with the quantitative analysis of integrin αV expression, normalized to 4',6-diamidino-2-phenylindole (DAPI) fluorescence, revealing a notable 2.1-fold increase in fluorescence intensity for the NO2 hydrogel in comparison to NO1 (<i>p</i> < 0.0001). These findings indicate that the hydrogel composed of 2% (w/v) NOCC cross-linked with 2% (w/v) OXG in a 1:1 (v/v) ratio represents the optimal condition for promoting murine fibroblast growth and spheroid formation. These results provide a robust foundation for future research aimed at modulating cell behavior through precise adjustments of scaffold properties, thereby advancing the potential for translational applications from laboratory research to clinical settings.