Multi-scale Engineered Vasculature and Hierarchical Porosity via Volumetric Bioprinting-guided Photopolymerization-induced Phase Separation

Vascularization remains a major challenge in hydrogel-based engineered tissues due to the inherent nano-scale porosity of common synthetic and natural biomaterials. Critically, the confinement imposed by the nanoscale network inhibits the outgrowth of blood vessels required for oxygen and nutrient delivery. Despite advancements in biofabrication that enable formation of small channels (0.1-1 mm), achieving vascularization (with capillaries down to 10 micrometers) throughout cm-scale bioprinted constructs remains a critical bottleneck. Herein, we integrated phase separating cell-interactive gelatin-norbornene hydrogels with volumetric bioprinting to generate architecturally defined centimeter-scale constructs with 0.1-1 mm scale printed channels and interpenetrating micron-scale porosity. Our novel approach allowed us to generate freeform construct designs with light-controllable micron-scale and hierarchical porosity. Importantly, this porosity enabled endothelial cell infiltration and microvessel outgrowth deep into the engineered tissue. Micron-scale vascular structures formed in the pore spaces with feature sizes on the scale of capillaries (<10 micrometers), crucial to provide oxygen and nutrients to all regions of the hydrogel. The networks remained stable for over 14 days, outperforming classical nanoporous biomaterials. These complex hydrogel-based constructs with engineered multi-scale vascular networks have substantial potential for the generation of actively perfusable advanced tissue models.