MMP AND VEGF REGULATION IN ENDOTHELIAL CELL-SEEDED UBM EXTRACELLULAR MATRIX IN A BIOREACTOR

Abstract

Introduction Cardiovascular diseases including aneurysms, atherosclerosis, ischemia, and cerebral haemorrhages are some of the leading causes of death in the western world. Most cardiovascular diseases are treated with arterial substitutes such as the internal thoracic artery, autologous vein and synthetic grafts. However the Internal thoracic artery, due to it location leaves its application only in coronary artery bypassing, resulting in the saphenous vein graft being utilised in most other bypass surgery if available. The current synthetic substitutes such as ePTFE and Dacron can result in a compliance mismatch and lead to increased wavereflection loading and irregular pressure loading on the heart. These problems underline a need for the advancements in alternative arterial substitutes such as improved synthetic materials and tissue engineered grafts. The developments of tissue engineering grafts have remodeling capabilities, which may reduce the influence of compliance mismatch and pressure irregularities on the heart. This study looks at the development of Urinary Bladder Matrix (UBM) which is an Extracellular matrix material derived from a porcine urinary bladder. Similar materials such as small intestine submucosa (SIS) have previously been examined by Badylak et al 1989 and Yamada 2001 (Roeder et al 2001) in animal models in which segments of the aorta were replaced. These studies showed promising results. The main aim of this research is the examination of matrix metalloproteinase (MMP) and vascular endothelial growth factor (VEGF) gene expression in Human Aortic endothelial cells (HAEC’s) when attached to Urinary Bladder Matrix (UBM) under aortic steady flow conditions. Methods The input boundary aortic shear flow conditions were developed form a realistic aortic arch model. The model was reconstructed from CT data, using MIMICS software, and computational fluid dynamics (CFD) was carried out using Gambit and Fluent software packages. Results for aortic shear flow were calculated below the renal arteries. These results were applied to a bioreactor as inlet flow conditions. The ECM material was constructed into a 4-ply 20mm diameter construct/graft using a vacuum technique. The ECM tubes were seeded with human aortic endothelial cells (HAEC) at a density of 0.4x10/cm. The seeded tubes were placed into the Bioreactor flow chamber and steady shear flow was applied for 24 hours. MMP and VEGF gene expression, known indicators of angiogenesis and arterial remodeling were measured using real time RT-PCR. Results The CFD studies concluded an averaged aortic shear stress of 1Pa below the renal arteries (figure1a). The ECM tubes showed high levels of cell attachment and proliferation on the surface determined with a live/dead assay after the flow tests. Gene expression of MMP was downregulated after the application of the steady flow indicating the possible inhibitory response to endothelial cell degradation of the UBM collagen based material following the application of steady shear flow.