In this study, we utilized USWF technology to rapidly fabricate vascularized 3D collagen-based, engineered tissue constructs in vitro. USWF-induced cell banding within collagen gels resulted in the formation of ~100 µm-long endothelial cell sprouts and co-aligned collagen fibers within 1 day of ultrasound exposure. Within 6–10 days, the endothelial cell sprouts had grown into anastomosing, vascular networks that were found throughout the entire volume of the 3D collagen gel. USWF-exposed collagen constructs contained endothelial cell networks with large, arteriole-sized lumen areas that branched into smaller, capillary-sized, lumen-containing structures, indicating the formation of a complex tree-like network within these collagen constructs. In contrast, endothelial cell sprout formation in sham-exposed constructs was delayed until day 4; network structures found within the sham-exposed samples were absent from gel centers and lacked the tree-like complexity formed within USWF-exposed constructs.
Cell migration, proliferation, and extracellular matrix remodeling are three essential components of neovessel formation in vivo (Risau 1997
). USWF-exposed constructs contained proliferating endothelial cells after 10 days in culture. In contrast, proliferating cells were not observed in day 4 collagen gel constructs. Thus, the initial increase in sprout length in USWF-exposed collagen gels was likely due to endothelial cell migration out of the banded areas without a significant contribution from cell proliferation. Extensive remodeling of collagen fibers into elongated, aligned fibrils was observed on day 1 in USWF-exposed constructs. Capillary formation in vitro has been shown to occur along tracks of aligned collagen fibers (Kirkpatrick et al. 2007
; Korff and Augustin 1999
). Therefore, the early cell-mediated collagen fibril reorganization observed in our USWF-exposed samples is consistent with the rapid migration of endothelial cell sprouts from banded areas along similarly aligned collagen fibers. Additionally, collagen condensation around capillaries is associated with sprout maturation (Kirkpatrick et al. 2007
). Thus, the cell-mediated collagen condensation observed in USWF-exposed gels at days 4 and 10 is consistent with the progression of the endothelial cell sprouts into more mature structures. The presence of proliferating cells at day 10 suggests that increases in sprout length and lumen diameter from day 4 to day 10 were due, at least in part, to cell proliferation. Taken together, these data provide evidence that USWF-mediated organization of endothelial cells into planar bands is sufficient to initiate a cascade of events similar to that which controls capillary formation in vivo (Folkman and Shing 1992
; Risau 1997
The formation of multicellular, planar aggregates of endothelial cells using USWF resulted in the formation of a vascular-like network throughout the entire collagen gel volume. By day 4, USWF-induced cell bands were lined with elongated endothelial cells, effectively producing vessels with large lumen areas from which smaller, lumen-containing, capillary-like structures emerged. Endothelial cells within the interior of the cell bands likely underwent apoptosis (Korff and Augustin 1998
), and apoptotic bodies cleared from the lumen areas with time. These results, coupled with the finding that cell-cell adhesions are formed in endothelial cell-lined sprouting structures, suggest that USWF technology may be used to fabricate 3D constructs that contain vascular channels throughout the gel to accommodate unrestricted blood perfusion upon implantation. Similarly, immediate perfusion of USWF-fabricated tissue constructs in vivo could be established with surgical anastomosis of the large lumen areas to host vasculature upon implantation (Rouwkema et al. 2008
In summary, our results demonstrate that non-invasive organization of endothelial cells using USWF accelerates the formation of capillary-like sprouts compared with sham-exposed collagen gels, and results in the maturation of sprouts into lumen-containing, branching networks throughout the complete volume of the collagen construct. Ultrasound technologies are non-destructive, non-ionizing, inexpensive, and can be adapted to a wide variety of fabrication processes. Design of USWF, by choice of ultrasound frequency and/or use of multiple transducer geometries, can produce more complex cell patterns within hydrogels. Thus, USWF technologies provide a novel approach to vascularize large 3D engineered tissues in vitro.