Development of an artificial vessel lined with human vascular cells

Abstract

ObjectivesThrombogenity of small-diameter vascular prostheses might be reduced by complete coverage of the luminal surface with vascular cells. We investigated cell seeding on polyurethane vascular prostheses. MethodsThirty polyurethane vascular prostheses were divided into 3 groups of 10 each: group A, diameter of 20 mm and γ-sterilized; group B, diameter of 4 mm and γ-sterilized; and group C, diameter of 4 mm and ethylene oxide sterilized. Human smooth muscle cells, fibroblasts, and endothelial cells were isolated from saphenous vein segments and expanded in culture. Five polyurethane vascular prostheses of each group were seeded with endothelial cells alone (mean, 4.8 ± 1.2 × 106 cells), and the remaining 5 polyurethane vascular prostheses were preseeded with a mixed culture of fibroblasts and smooth muscle cells (mean, 7.7 ± 2.3 × 106 cells), followed by endothelial cell seeding (mean, 4.4 ± 0.9 × 106 cells). Seven days after cell seeding, the polyurethane vascular prostheses were perfused under a pulsatile flow (80 pulses/min, 140/80 mm Hg, and 120 mL/min) for 2 hours. Specimens were taken after each seeding procedure both before and after perfusion and then examined both with a scanning electron microscope and immunohistochemically. ResultsIsolated endothelial cell seeding revealed better initial adhesion in groups A and B than in group C (63% vs 33%). After 7 days, the cells had covered approximately 80% of the luminal surface in groups A and B, whereas group C cells rounded up and lost adhesion. After perfusion testing of group A and B prostheses, only 10% of the surface was still covered with endothelial cells. Preseeding with the mixed culture again revealed a better initial adhesion in groups A and B compared with that in group C (76% vs 41%). In groups A and B endothelial cell seeding (adhesion, 72%) resulted in a confluent endothelial cell layer. The results of immunohistochemical staining were positive for collagen IV, laminin, CD31, and Factor VIII. In group C only isolated cells were found after each seeding procedure, which rounded up and vanished during the next days. Perfusion testing of group A and B prostheses revealed that the confluent cell layer remained stable, with only small defects (<10% of the surface). The cells stained positivively for endothelial nitric oxide synthase. ConclusionSeeding of a mixed culture out of fibroblasts and smooth muscle cells resulted in improved endothelial cell adhesion and resistance to shear stress. This outcome was caused by an increased synthesis of extracellular matrix proteins. Cell attachment was better on γ-sterilized polyurethane vascular prostheses compared with on those undergoing ethylene oxide sterilization.