Spatial patterning of endothelial cells and vascular network formation using ultrasound standing wave fields.

The spatial organization of cells is essential for proper tissue assembly and organ function. Thus, successful engineering of complex tissues and organs requires methods to control cell organization in three dimensions. In particular, technologies that facilitate endothelial cell alignment and vascular network formation in three-dimensional tissue constructs would provide a means to supply essential oxygen and nutrients to newly forming tissue. Acoustic radiation forces associated with ultrasound standing wave fields can rapidly and non-invasively organize cells into distinct multicellular planar bands within three-dimensional collagen gels. Results presented herein demonstrate that the spatial pattern of endothelial cells within three-dimensional collagen gels can be controlled by design of acoustic parameters of the sound field. Different ultrasound standing wave field exposure parameters were used to organize endothelial cells into either loosely aggregated or densely packed planar bands. The rate of vessel formation and the morphology of the resulting endothelial cell networks were affected by the initial density of the ultrasound-induced planar bands of cells. Ultrasound standing wave fields provide a rapid, non-invasive approach to pattern cells in three-dimensions and direct vascular network formation and morphology within engineered tissue constructs.

Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound.

Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.

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.

Controlling the spatial organization of cells and extracellular matrix proteins in engineered tissues using ultrasound standing wave fields.

Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to noninvasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional (3-D) collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of 3-D collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization compared with sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro.