METHODS: Pluronic F127 fibers, 1.5 mm in diameter, were sacrificed in type I collagen, creating a central looped microchannel. 100μL polyculture cell suspension mixture of 5 x 106 cells/mL of human foreskin fibroblasts and 5 x 106 cells/mL of human aortic smooth muscle cells was seeded into the microchannel. The following day, a 100μL cell suspension of 5 x 105 cells/mL of human placental pericytes and 5 x 106 cells/mL of human umbilical vein endothelial cells was seeded into the microchannel. All constructs underwent daily media changes in static culture for 7 days, and then half were perfused at 10 dynes/cm2 for an additional 7 days. After 14 days, scaffolds were fixed and processed.
RESULTS: After 7 and 14 days of culture, constructs formed intact endoluminal linings along the microchannel with increasing thickness over time. CD31 expressing endothelial cells were noted along the luminal surface after 7 days and throughout the endoluminal lining after 14 days, establishing a neointima. Constructs undergoing static and dynamic culture had robust, vascular linings that spanned the entire microchannel. However, dynamic constructs had a 59% thicker lining in the channel (p=0.0057). Ki67 staining demonstrated statistically significant increased cell proliferation in constructs that experienced dynamic perfusion suggesting stimulation by the shear stress (p=0.0429).
CONCLUSION: Shear stress through dynamic perfusion was used to optimize the development of a layer of vascular lining cells to provide a non-thrombogenic surface to allow continuous blood flow in these tissue engineered vessels. Exposing pre-vascularized engineered tissues to controlled perfusion produces vessels with architecture that more accurately recapitulates the in vivo phenotype and provides a surface for thrombosis-free blood flow, allowing for surgical implantation via microanastomosis.