(315a) 3D Bioprinting of Dynamic Covalent Hydrogels Using a Small Molecule Competitor and Catalyst

Extrusion-based three-dimensional (3D) bioprinting is a promising technique for fabricating tissue-like structures by spatially patterning bioinks composed of cells and biomaterials. However, the number of materials that are both printable and cell compatible remains small, and currently available bioinks offer limited tunability in regards to matrix mechanical and biochemical properties. Since it is well-established that cell growth, phenotype, and differentiation are all dependent on these 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 functional printed structures. Most extracellular matrices within the body are viscoelastic and stress-relaxing, allowing them to be dynamically remodeled by cell-generated forces, yet few bioinks are able to replicate this behavior. Here, we present a family of bioinks crosslinked by dynamic covalent bonds to enable stress relaxation within printed constructs. To prepare these inks, we modified various backbone polymers with either an aldehyde or hydrazine functional group. When mixed, these two components form hydrazone bonds, which can break and reform at physiological conditions, creating a material that is shear-thinning, self-healing, and stress-relaxing. We further modulate the printability of these inks by introducing two small molecules: a competitor and a catalyst. The competitor binds to free aldehydes present in the bioink 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 easily extrudable 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 this method can be used to create bioinks with either gelatin, hyaluronic acid (HA), elastin-like protein (ELP), or polyethylene glycol (PEG) as the backbone polymer. In all cases, addition of the competitor reduced the stiffness of the materials 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. Importantly, diffusion of the competitor and catalyst out of the printed structure occurred within 24 hours, allowing the structure to be readily extracted from the support bath and stabilized for longer-term culture. Additionally, we demonstrate that the small molecule competitor and catalyst are cell compatible, and that cell viability remains high within the printed constructs. Altogether, we present a bioink system based on dynamic covalent crosslinking in which we are able to modulate printability and long-term stability based on the addition of a small molecule competitor and catalyst.