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Background: Regeneration of thicker or larger tissues of clinically relevant size remains a challenge due to poor oxygen diffusion into cells that are contained within non-vascularized tissue-engineered constructs. However, without exposing the vascular lining cells to flow, their functionality and in vivo stability are suboptimal. In physiological conditions, hemodynamic shear stress alters cellular morphology and biological activity, especially luminal endothelial cells within blood vessels. In our previous work, we have developed tissue engineered constructs with fibroblast and smooth muscle cells co-cultured with pericyte and endothelial cells within microchannels resulting in the formation of anatomically correct neointimal and neomedial layers. Here, we “prime” the constructs by dynamically perfusing them and determine how flow induced shear stress optimizes the endoluminal surfaces of our tissue-engineered vessels.
Methods: Pluronic F127 fibers, 1.5 mm in diameter, were sacrificed in type I collagen, creating a central looped microchannel. Twenty-four hours following fiber sacrifice, a 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 72 hours, and then perfused at 10 dynes/cm² for an additional 1, 3, 5 or 7 days. After perfusion, scaffolds were fixed and processed for histology.
Results: After a total of 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. Ki67 staining demonstrated statistically significant increased cell proliferation in constructs that were exposed to dynamic perfusion suggesting stimulation by the exposure to shear stress (p=0.0429).
Conclusion: We have successfully created tissue engineered scaffolds with microchannels that support the attachment of fibroblast, smooth muscle, endothelial and pericytes cells which form neointimal and neomedial layers. 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.