3D Bioprinting of Dynamic Covalent Hydrogels Enabled by Small Molecule Competitor and Catalyst

Extrusion-based three-dimensional (3D) bioprinting is an emerging biofabrication technique in which bioinks composed of a mixture of cells and polymer are spatially patterned to create tissue-like constructs. While advances have been made to enable printing of more geometrically complex architectures, the technique remains limited by a lack of biomaterials suitable for both printing and subsequent cell culture. Since it is well-established that cell behaviors such as growth, migration, and differentiation are dependent on matrix properties, it is becoming increasingly important to tailor a bioink’s matrix cues to the cell type of interest in order to create more biomimetic and biofunctional structures. In particular, recent work has demonstrated the importance of a material’s viscoelastic, time-dependent properties in regulating cell behavior. Most extracellular matrices within the body are viscoelastic and stress-relaxing, which allows for dynamic remodeling of the matrix through cell-generated forces. However, few currently available bioinks are able to replicate such dynamic behavior. Here, we present a bioink crosslinked by dynamic covalent bonds which enable cellular remodeling within printed constructs, and demonstrate how tuning the kinetics of these dynamic bonds impacts bioink printability. To prepare these inks, we modified hyaluronan (HA) with aldehyde functional groups and a recombinant elastin-like protein (ELP) with hydrazine functional groups to create a hyaluronan elastin-like protein (HELP) matrix. When mixed, these two components form hydrazone bonds, which can break and reform at physiological conditions, creating a material that is stress-relaxing, but difficult to extrude. We modulate the printability of these inks by introducing two small molecules: a competitor and a catalyst. The competitor binds to aldehydes present on the functionalized HA, which disrupts crosslink formation and thus reduces the overall stiffness of the ink. The catalyst increases the rate of hydrazone bond exchange and thus increases the ink’s ability to shear-thin. Together, these two molecules enable the ink to be more readily extruded during printing. When printed into a gel support bath, these small molecules can then freely diffuse away from the printed structure while the ink remains in place, stiffening and stabilizing the final structure. We demonstrate that addition of the competitor reduced the stiffness of the HELP material up to three orders of magnitude in a dose-dependent manner. Addition of the catalyst was shown to improve the ink’s ability to shear-thin without changing the overall stiffness of the material. Sufficient diffusion of the competitor and catalyst out of the printed structure occurred within hours, allowing stabilized printed structures to be removed from the support bath and cultured for one week. Finally, we utilize these inks to print a 3D model of breast cancer invasion, and show that incorporating dynamic crosslinks into the ink increases growth factor-induced cell invasion as compared to a statically crosslinked control. Altogether, we present a dynamic bioink material in which we are able to modulate printability and long-term stability based on the addition of a small molecule competitor and catalyst to create 3D <i>in vitro</i> models of the tumor microenvironment that better recapitulate native physiology.