Quantitatively Designed Cross-Linker-Clustered Maleimide-Dextran Hydrogels for Rationally Regulating the Behaviors of...

Biomaterials can serve as the cornerstone of extracellular matrices (ECMs) in three-dimensional (3D) cell culture. Designing of diverse ECM materials with adjustable inner structures exhibits growing superiority in inducing varied cellular behaviors, such as spreading, proliferation, migration, differentiation, and apoptosis. However, a rational and easy-to-implement strategy for regulating cell behaviors during cellular self-organization in 3D hydrogels is still lacking. In this study, we propose that maleimide-functionalized dextran hydrogels are cross-linked with quantitatively predesigned nanoscaled heterogeneity. The heterogeneity is obtained via mixing two parts of a dextran polymer with different amounts of available cross-linking sites. We experimentally demonstrate that this rational strategy has a noteworthy impact on cell morphology and fate. The cross-linker-clustered hydrogels are found to contain more microscopic heterogeneous pore structures and show larger heterogeneity in local stiffness, compared to the homogeneously cross-linked hydrogels. Further, 3D cell culture results show that myoblast cells exhibit better spreading, lower circularity, and perceptible changes in the expression of a differentiation marker in cross-linker-clustered hydrogels, which can also be tuned with the cross-linker-clustering degree. The possible mechanisms are discussed by considering the cellular durotaxis in nanoscales, mechanical cues, and biological cues in extracellular and intracellular networks. It is inferred that the effect of nanoscaled durotaxis is triggered during the tentative protrusion of the C2C12 cells, which thus repeatedly and continuously enhances the cellular elongation during their oriented polarization. In conclusion, the predesigned 3D cross-linker-clustered maleimide-dextran hydrogels are able to affect the cell morphology and fate in a rational way by modulating physical, chemical, and biological cues related to the cell-hydrogel interactions. This study provides a promising strategy for quantitative designing and fabrication of functional biomaterials, and theoretical study of mechanobiological mechanisms underlying the cellular behavior.