In vivo acoustic patterning of endothelial cells for tissue vascularization.

Strategies to fabricate microvascular networks that structurally and functionally mimic native microvessels are needed to address a host of clinical conditions associated with tissue ischemia. The objective of this work was to advance a novel ultrasound technology to fabricate complex, functional microvascular networks directly in vivo. Acoustic patterning utilizes forces within an ultrasound standing wave field (USWF) to organize cells or microparticles volumetrically into defined geometric assemblies. A dual-transducer system was developed to generate USWFs site-specifically in vivo through interference of two ultrasound fields. The system rapidly patterned injected cells or microparticles into parallel sheets within collagen hydrogels in vivo. Acoustic patterning of injected endothelial cells within flanks of immunodeficient mice gave rise to perfused microvessels within 7 days of patterning, whereas non-patterned cells did not survive. Thus, externally-applied ultrasound fields guided injected endothelial cells to self-assemble into perfused microvascular networks in vivo. These studies advance acoustic patterning towards in vivo tissue engineering by providing the first proof-of-concept demonstration that non-invasive, ultrasound-mediated cell patterning can be used to fabricate functional microvascular networks directly in vivo.

Ultrasound patterning technologies for studying vascular morphogenesis in 3D.

Investigations in this report demonstrate the versatility of ultrasound-based patterning and imaging technologies for studying determinants of vascular morphogenesis in 3D environments. Forces associated with ultrasound standing wave fields (USWFs) were employed to non-invasively and volumetrically pattern endothelial cells within 3D collagen hydrogels. Patterned hydrogels were composed of parallel bands of endothelial cells located at nodal regions of the USWF and spaced at intervals equal to one half wavelength of the incident sound field. Acoustic parameters were adjusted to vary the spatial dimensions of the endothelial bands, and effects on microvessel morphogenesis were analyzed. High-frequency ultrasound imaging techniques were used to image and quantify the spacing, width and density of initial planar cell bands. Analysis of resultant microvessel networks showed that vessel width, orientation, density and branching activity were strongly influenced by the initial 3D organization of planar bands and, hence, could be controlled by acoustic parameters used for patterning. In summary, integration of USWF-patterning and high-frequency ultrasound imaging tools enabled fabrication of vascular constructs with defined microvessel size and orientation, providing insight into how spatial cues in 3D influence vascular morphogenesis.