Interconnected Porous Hydrogels with Tunable Anisotropy Through Aqueous Emulsion Bioprinting

Abstract Interconnected microporous hydrogels have the potential to overcome bulk hydrogels' limitations in nutrient transport and cellular migration. Current fabrication methods, however, often rely on cytotoxic porogenic agents, limiting scalability and biocompatibility. Herein, a microporous hydrogel bioink comprised of extracellular matrix (ECM) mimicking proteins and biopolymers aqueous two‐phase systems (ATPS) is formulated to simultaneously impart interconnected porosity and tunable anisotropy via embedded 3D bioprinting. Leveraging on the immiscibility between the proteins in the pre‐gel phase and a polyethylene oxide‐based porogen, interconnected porosities of up to 70% are achieved. The stability of the bioink also enables the biofabrication of complex living constructs with interconnected porosity across the entire volume, supporting stem cell viability up to 14 days. Furthermore, ATPS bioinks processing through embedded 3D bioprinting enables the generation of simultaneously anisotropic and interconnected porous living constructs, providing the grounds for controlling cell alignment in 3D. Overall, the formulated inks advance the toolbox of hydrogel bioinks that can be used to generate tunable porosity and anisotropy in biofabricated living constructs across different scales.