Designing ultrasound fields to control the morphology of engineered microvessel networks

Abstract

Spatial patterning of endothelial cells using ultrasound standing wave fields (USWF) can promote extensive microvessel network formation throughout the volume of three-dimensional collagen hydrogels. The goal of this study was to identify acoustic exposure parameters that generate specific initial spatial patterns of cells, in order to control resultant microvessel morphology. Endothelial cells were suspended in soluble collagen and exposed to a 1-MHz continuous wave USWF for 15 min during collagen gel polymerization. Samples were exposed to peak USWF pressures of 0, 0.1, 0.2, or 0.3 MPa. Samples were either imaged immediately post-exposure using high frequency ultrasound or cultured for ten days. Analysis of B-mode ultrasound images confirmed differences in initial cell band spacing and cell band density between the four exposure pressures tested. After ten days in culture, USWF-induced cell patterning resulted in three distinct microvessel morphologies. Specifically, 0.1 MPa exposure resulted in capillary-like networks, 0.2 MPa exposure resulted in non-branching vessel structures, and 0.3 MPa exposure resulted in hierarchical branching microvessel networks. Spatial characteristics of initial cell bands were then correlated with resulting microvessel network morphology. Results of these investigations allow for the capability to predictively, reproducibly, and rapidly form microvessel networks of known morphology within 3D engineered hydrogels.