Bioinks with varying densities of physical and chemical crosslinks modulate cellular responses in 3D by altering the viscoelasticity of the cell microenvironment

Abstract

The biological function and clinical translation of bioprinted cell-laden constructs largely depend on the bioink printability and biomechanical cues presented to embedded cells. Despite multiple biomaterials and crosslinking reactions have been explored for bioink design, how the type and density of crosslinks used in bioink development determine the relationship between bioink printability and viscoelasticity, and how in turn the resulting alterations in viscoelasticity regulate cell behavior within 3D bioprinted constructs remain largely unknown. Here, we developed double crosslinked bioinks with controllable printability and time-dependent mechanical properties by varying the density of reversible (ionic) to static (thioether) crosslinks in the gel network. We utilized these bioinks to investigate how the altered density of physical and chemical crosslinks affects the viscoelasticity of bioprinted cell constructs and how they regulate fundamental cellular responses in 3D. From our results, it was evident that increased density of reversible bonds in bioprinted constructs significantly promotes not only rapid cell spreading, but also the formation of interconnected cellular networks by enhancing matrix viscoelasticity and stress relaxation. By co-printing bioinks whose viscoelasticity can be adjusted independently from its cell-adhesiveness by varying the degree of covalent crosslinking via photoclick thiol-ene reaction, we showed that cell spreading and morphology are spatially regulated in step-gradient hydrogels by the viscoelasticity of their surrounding environment. Our findings reveal that bioinks with similar printability elicit distinct cell responses in bioprinted 3D constructs via altered matrix viscoelasticity, which is determined by the type and density of crosslinks employed for bioink crosslinking. Taken together, these results underscore matrix viscoelasticity as a key parameter in the rational design of mechano-instructive bioinks for bioprinting applications in tissue repair and in vitro tissue modelling.