A series of poly(NaSS-co-DMAAm) hydrogels with ζ potentials (ζ) in the range −8.8–−20.5 mV and a constant Young's modulus (E) of ca. 200 kPa were designed for studying the effect of the charge density of hydrogels on dynamic cell behavior. The ζ was adjusted by tuning the molar friction of negatively charged sodium p-styrene sulfonate (NaSS) contained in the monomer mixture [NaSS and N, N-dimethyl acrylamide (DMAAm)] for gelation. A critical ζ potential, which controls cell behavior, ζcrit = −14.0 mV, was observed. When ζ > ζcrit, the cells exhibited a small spreading area, fast migration velocity, and a large migration distance, with concurrent deficiency in actin fibers and less prominent focal adhesions. Conversely, when ζ ≤ ζcrit, the cells exhibited a large spreading area, slow migration velocity, and a short migration distance, with concurrent well-developed actin fibers and prominent focal adhesions. Furthermore, cells repeatedly oscillated in a stick–slip mode from a spreading shape to a round shape on the hydrogels with ζ > ζcrit, although were unable to proliferate. The cell behavior is well correlated with the adsorbed fibronectin on the hydrogel surfaces.
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