Design of Universal, Bioorthogonally Crosslinked Inks to Enable 3D Bioprinting

Three-dimensional (3D) bioprinting has emerged as a promising technology for producing complex, functional constructs containing precisely patterned cells. However, it remains limited by the number of materials that can be used as bioinks, especially in comparison to the vast array of biomaterials developed for non-printed tissue engineering scaffolds. Because cells are exquisitely sensitive to the materials in which they are grown, different cell types will require different inks. Ideally each cell type would be printed in its own customizable bioink so that the bioink is tuned to fit the cellular and structural needs of the desired tissue application, but many widely used bioinks do not currently have this flexibility. We present a new family of bioinks that use a universal, bioorthogonal crosslinking mechanism that is completely cell compatible and works with any type of biopolymer. To prepare these inks, we modified the biopolymer backbone with either an azide or a bicyclononyne (BCN) group. We then mixed the functionalized polymer with cells to create a bioink, and extruded it into a gel-based support bath containing multifunctional crosslinkers modified with the partner bioorthogonal functional group. Diffusion of the crosslinkers into the printed structure, followed by rapid bioorthogonal click chemistry between the BCN and azide functional groups, results in the formation of triazole linkages that stabilize the printed structure post-extrusion. We demonstrate that this universal crosslinking technique can be used to create bioinks with either gelatin, hyaluronic acid (HA), elastin-like protein (ELP), or polyethylene glycol (PEG) as the backbone polymer. The bioink mechanics can be tuned by changing the polymer weight percentage to create inks with stiffnesses ranging from 10¹ to 10³ Pa. Since the bioinks are crosslinked by a universal mechanism, we show that multiple materials can be printed at once and that the approach is compatible with different gel-based support baths so long as the crosslinker can diffuse through the bath and into the bioink materials. Multiple sensitive cell types, including corneal stromal cells and iPSC derived neural progenitor spheroids, could be printed in these bioink materials. The cells within the printed constructs maintained high cell viability and expressed characteristic phenotypic markers immediately after the construct was removed from the support bath and after one week in culture. Together, these results show that our bioinks are tunable, customizable, and highly cell compatible, thus expanding the toolkit of bioinks that can be utilized for 3D bioprinting.