Abstract 7: Utilizing Shear Stress to Optimize Endoluminal Linings withing Pre-Vascularized Engineered Tissues

Abstract

PURPOSE: 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. Another major obstacle in the ability to precisely replicate the intricate design of the vascular system is due to a lack of proper endothelialization on the luminal surface of vessels. 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 fabricated tissue engineered constructs with microvasculature comprised of anatomically correct neointimal and neomedial layers. Here, we “prime” these 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, were sacrificed in type-I collagen, creating a central looped microchannel. Twenty-four hours following fiber sacrifice, a cell suspension mixture of normal human dermal fibroblasts and human aortic smooth muscle cells was seeded into the microchannel. The following day, another cell suspension of human placental pericytes and human umbilical vein endothelial cells was seeded into the microchannel. All constructs underwent daily cell media changes in static culture for 72 hours, and then perfused at 10 dynes/cm2 for an additional 1, 3, 5 or 7 days using a peristaltic pump in a bioreactor. Scaffolds were processed for histology and immunohistochemical analysis. Images were quantified using ImageJ (NIH). A two-tailed unpaired t-test was used to compare variables between experimental groups RESULTS: After culture, all 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. Representative slides were taken from each construct, and the area of robust cellular lining was measured and normalized to channel diameter. Perfused constructs had a 59% significantly thicker lining in the channel than compared to constructs cultured under static conditions (p=0.0057). In addition, cellular proliferation (measured by calculating the ratio between Ki67 and DAPI) was significantly increased in perfused constructs (p=0.0429). Taken together, these data suggest that shear stress plays a significant role in the proliferation and maturation of the vascular lining. 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.