Innovative thermosensitive alginate bioink combining cations for enhanced 3D extrusion bioprinting for tissue engineering

Sodium alginate (SA) hydrogels are widely used in 3D extrusion bioprinting, but their isolated use does not meet all the requirements for this application. To overcome this problem, crosslinking with divalent cations and combinations with other polymers, such as gelatin (Gel), are employed to improve their mechanical performance and bioactivity. In this study, we proposed a new concept of pre-crosslinking SA and SA/Gel inks with divalent cations Ca²⁺, Co²⁺, and Zn²⁺ and their binary mixtures. These inks were successfully formulated and characterized, and it was observed that different ion ratios can impart essential characteristics and properties for 3D extrusion bioprinting. To evaluate the thermosensitive response of these inks, it was included gelatin in a dispersed phase, giving the 3D-printed system a 4D character. The hydrogel with the best mechanical and biological performance was the pre-crosslinked composition with mixtures of divalent Ca²⁺/Co²⁺ ions, whereas it was observed through the live/dead assay that the presence of Zn²⁺ ions in the hydrogels on day 3 reduced the cell viability. This composition was used to develop a bioink for 4D printing using cell spheroid or single cells, with spheroids presenting better viability after 7 days than single cells. These results emphasize the importance of obtaining a pre-crosslinked bioink with modulated properties by employing divalent ions for 4D biofabrication and that 3D cell culture ensures superior resistance to 3D extrusion bioprinting when compared to single cells. Those characteristics give us an interesting bioink with high potential to be used in regenerative medicine of soft tissues.