Vascularization of Three-Dimensional Collagen Hydrogels Using Ultrasound Standing Wave Fields

The successful fabrication of large, three-dimensional (3-D) tissues and organs in vitro requires the rapid development of a vascular network to maintain cell viability and tissue function. In this study, we utilized an application of ultrasound standing wave field (USWF) technology to vascularize 3-D, collagen-based hydrogels in vitro. Acoustic radiation forces associated with USWF were used to noninvasively organize human endothelial cells into distinct, multicellular planar bands within 3-D collagen gels. The formation and maturation of capillary-like endothelial cell sprouts were monitored over time and compared with sham-exposed collagen constructs, which were characterized by a homogeneous cell distribution. USWF-induced cell banding accelerated the formation and elongation of capillary-like sprouts, promoted collagen fiber alignment and resulted in the maturation of endothelial cell sprouts into lumen-containing, anastomosing networks found throughout the entire volume of the collagen gel. USWF-induced endothelial cell networks contained large, arteriole-sized lumen areas that branched into smaller, capillary-sized structures indicating the development of vascular tree-like networks. In contrast, sprout formation was delayed in sham-exposed collagen gels and endothelial cell networks were absent from sham gel centers and failed to develop into the vascular tree-like structures found in USWF-exposed constructs. Our results demonstrate that USWF technology leads to rapid and extensive vascularization of 3-D collagen-based engineered tissue and, therefore, provide a new strategy to vascularize engineered tissues in vitro.