Microvascular insufficiency and in turn, tissue ischemia and necrosis, contribute to a variety of chronic diseases, and can be an adverse outcome of common reconstructive and plastic surgeries. The goal of this project is to advance a novel ultrasound-based technology to induce neovascularization directly in vivo, and thereby enhance local tissue perfusion. Acoustic patterning utilizes radiation forces associated with an ultrasound field to rapidly and non-invasively organize cells or microparticles volumetrically into defined geometric assemblies. We have shown that in vitro acoustic patterning of endothelial cells within collagen hydrogels leads to the formation of three-dimensional microvascular networks, and that acoustic field parameters employed for patterning influence microvessel morphology. In our recent studies, in vivo acoustic patterning of endothelial cells within injectable hydrogels resulted in formation of perfused microvascular networks in a murine model, providing the first proof- of-concept demonstration that non-invasive, acoustic cell patterning can be used to fabricate functional microvascular networks directly in vivo. An important advantage of ultrasound is its ability to propagate through tissue as an acoustic beam, thus offering avenues to rapidly translate this technology toward in vivo tissue regeneration. Research and development in three key areas are necessary to advance acoustic patterning towards clinical translation: i) development of systematic protocols to fabricate functional microvessel networks within patterned hydrogels, ii) engineering innovative instrumentation for acoustic patterning in vivo, and iii) demonstrated efficacy of the technology in a preclinical model. We address these key areas as follows. Aim 1 will identify sets of acoustic parameters, along with hydrogel and cell combinations, that give rise to functional microvascular networks, in a manner that allows for predictable control over the morphology of three-dimensional microvascular networks. Aim 2 will advance two acoustic instrumentation systems, a dual-transducer system and a phase holographic lens transducer, to provide versatile systems for efficient, site-specific patterning in vivo. Aim 3 will evaluate the efficacy of our in vivo acoustic patterning strategies using mouse models of tissue vascularization and ischemia. Completion of this project will advance in vivo acoustic patterning technologies for tissue vascularization to address a range of clinically relevant scenarios of tissue ischemia.