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The interactions between substrate materials and cells usually play an important role in the hydrogel-based 3D cell cultures. However, the hydrogels that are usually used could not be parametrically regulated, especially for quantitatively regulating the spatial distribution of the adhesion sites for cells in 3D. Here, we employed the semisynthetic hydrogel consisting of maleimide-dextran, Arg-Gly-Asp (RGD) peptides, and cell degradable crosslinkers to biochemically characterize the evolutionary behaviors of NIH-3T3 fibroblasts and C2C12 cells in 3D. Moreover, by comparing the cell-adhesive efficacy of 3D dextran hydrogels with four different RGD clustering rates, we explored the underlying regulation law of C2C12 connections and 3T3 aggregations. The results showed that mal-dextran hydrogel could promise cells stable viability and continuous proliferation, and induce more self-organized multicellular structures relative to 2D culture. More importantly, we found that RGD-clustered mal-dextran hydrogel has the advantage of enhancing C2C12 cell elongation and the breadthwise-aggregated connection, and promoting the 3T3 cell aggregating degree compared to that with homogenous RGD. Further, the advantages of RGD clustering hydrogel could be amplified by appropriately reducing RGD concentration. Such RGD-composition controllable mal-dextran hydrogel can function as a regulator of the collective cellular behaviors, which provides useful information for quantitatively designing the tailored hydrogel system and exploiting advanced biomaterials.

Characterization and Analysis of Collective Cellular Behaviors in 3D Dextran Hydrogels with Homogenous and Clustered RGD Compositions