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 nanoscale networks inhibits blood vessels outgrowth, required for oxygen and nutrient delivery. Despite advancements in the biofabrication of small channels (0.1-1 mm), achieving vascularization (with capillaries down to 10 µm) throughout cm-scale bioprinted constructs remains a critical bottleneck. Herein, phase separating is integrated, cell-interactive gelatin-norbornene hydrogels with volumetric bioprinting to generate architecturally defined centimeter-scale constructs with 0.1-1mm scale printed channels and interpenetrating micron-scale porosity. This novel approach produced 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. Vascular structures formed in the pore spaces with feature sizes on the scale of capillaries (<10 µm), crucial to provide oxygen and nutrients to all regions of the hydrogel. The networks remained stable for over 14 days, outperforming classical nanoporous biomaterials. Vascular networks are perfusable in this custom-made bioreactor system and exhibited extended vessel outgrowth under perfused culture conditions. These complex hydrogel-based constructs with engineered multi-scale vascular networks have potential for generating actively perfusable advanced tissue models.