Abstract
Forming capillary networks with predefined geometries is a critical challenge in engineering complex three-dimensional tissues in vitro. While bioprinting and microfluidic technologies have enabled vascular tissue fabrication, precise control over capillary-scale vascularization remains limited. In this study, we investigated vascular formation process along hydrogel microchannels to elucidate mechanisms governing capillary-scale lumen formation. Microchannels were fabricated by femtosecond laser ablation in collagen and fibrin hydrogels. We optimized multiphoton lithography parameters to fabricate microchannels within these hydrogels and analyzed vascular formation along the channels. Luminal vascular structures formed readily in 50-μm channels, while vascular formation failed in 20-μm channels under static conditions, suggesting a significant shift in endothelial organization at the cellular scale. Flow stimulation significantly promoted vessel formation through collective endothelial cell migration and adhesion, whereas static conditions induced endothelial-to-mesenchymal transition. These findings provide key insights into capillary-scale vascularization and contribute to the development of more complex architectures with predefined shapes.