Abstract 11764: A Novel Approach for Pre-Implant Endothelialization of Xenogeneic Decellularized Vascular Tissues by Human Endothelial Cells Utilizing Tissue Surface Modification and Dynamic Culture

Abstract

Introduction: Autologous grafts for revascularization might be limited due to the quality of vessels. Decellularized xenogeneic vascular grafts potentially substitute for autologous grafts. We have been developing a decellularization method based on high hydrostatic pressure (HHP) which confers highly preserved extracellular structure. For broader application of the HHP-decellularized vascular graft, pre-implant endothelialization of the grafts by human endothelial cells would be anticipated which may prevent clot formation and graft occlusion. Hypothesis: We hypothesized to achieve sufficient ex vivo endothelialization of HHP-decellularized tissues by virtue of chemical endothelial surface modification and dynamic culture recapitulating blood flow. Methods: Thin slices of bovine aortic endothelium were decellularized by HHP. We divided the tissues in 2 groups: coated with E8 fragment of human laminin 411 (hL411) holding high affinity with endothelial cells and coated with gelatin (Gel). We directly seeded human umbilical vein endothelial cells (HUVECs) with a density of 2 х10 6 cells/cm 2 , then divided the tissues in 2 subgroups: dynamic culture (20 cm/s) and static culture. We kept the culture for up to 2 weeks and evaluated the area coverage by staining of live cells by Calcein-AM, histological analysis, and scanning electron microscopy (SEM). We tested clot formation using human whole blood. Results: hL411/dynamic group exhibited significantly higher area coverage compared to those of other groups which almost reached 100% [coverage at 2 weeks: Gel/static vs hL411/static vs Gel/dynamic vs hL411/dynamic: 57.3±4.1 vs 61.3±2.7 vs 74.7±19.9 vs 99.3±0.7 %, p<0.001(vs NC/static), n=6]. Immunostaining for CD31 and SEM revealed sufficient coverage with endothelial cells in hL411/dynamic. Clot formation assay showed lower clot formation in hL411/dynamic group than in hL411/static (p=0.014) and mock without hL411 coating (p=0.006) (n=3). Conclusions: Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization accompanied with the prevention of clot formation which would promote the clinical use of endothelialized vascular grafts in the future.