A hybrid compliant vascular graft seeded with microvascular endothelial cells extracted from human omentum.

Abstract

We report the development of a hybrid vascular graft using an innovative compliant poly(carbonate-urea)urethane unlike any previous polyurethane MyoLink as a permanent scaffold. The engineered graft has a hierarchical arterial structure: a monolayer of oriented microvascular endothelial cells (MVECs) and 3-D matrix (human collagen Type 4/dermatan sulfate) bonded onto MyoLink. The grafts' clinical feasibility was evaluated by determining optimal MVEC seeding density, incubation time, viability, and adhesion of these cells when exposed to arterial shear stress. MVECs from human omentum were isolated by a new centrifugation protocol, radiolabeled and seeded onto hybrid graft 2 to 18 x 105 cells/cm(2) for 24 h at 37 degrees C and for 1 to 5 h at 6 x 105 cells/cm(2), washed 3 times, and gamma counted. Qualitative assessment of seeding density/incubation was also undertaken with scanning electron microscopy (SEM) and viability tested with a modified Alamar Blue assay. A pulsatile flow phantom was used to subject the hybrid graft (200 mm length, 5 mm internal diameter) seeded with radiolabeled MVECs (6 x 105 cells/cm(2) at 2 h) to arterial shear and dynamic scintigraphy images acquired in real time using a nuclear medicine gamma camera system during 14 h of perfusion (n = 6). The optimal seeding density was 6 x 105 cells/cm(2), and qualitative SEM confirmed this. Incubating cells for 2 h produced significantly greater cell attachment than was seen for 1 h incubation (p < 0.05), and there was no significant difference in adhesion between cells incubated over the 2 h. Exposure of grafts to acute shear stress resulted in significantly higher proportions of initial cells attached to hybrid grafts compared to native MyoLink grafts (67 +/- 3% versus 55 +/- 2%, p < 0.001). As shown here, tissue engineering of native Myolink grafts significantly reduces the seeding density and incubation time to produce a monolayer onto which cells adhere to better.