Increased in-vitro incubation time of endothelial cells on fibronectin-treated ePTFE increases cell retention in blood flow

Abstract

Endothelial cell (EC) seeding is postulated as a mechanism of improving patency of small calibre vascular grafts. However, the majority of seeded cells are lost within hours following restoration of blood flow. We postulated that incubating EC in-vitro on a graft will improve adherence and resistance to the sheer stresses of pulsatile blood flow. Fibronectin-treated ePTFE (5 cm x 4 mm ID) seeded with Indium-111-labelled autologous canine EC (1.5 x 10(5) cells/cm2) were incubated for four different time periods; 90 min, 24 h, 72 h and 6 days. Incubated grafts were subjected to blood flow of 75 ml/min for 6 h, in a canine ex-vivo arteriovenous shunt circuit. EC retention during perfusion was studied by measuring gamma activity emitted by the grafts. Cell morphology of non-perfused control groups and perfused groups was compared using scanning electron microscopy (SEM). SEM of control grafts showed progressive EC spreading on the ePTFE surface for up to 72 h incubation. Gamma activity was significantly higher at 6 h perfusion in grafts incubated for 72 h (82 +/- 4%) and 24 h (63 +/- 6%) vs. 90 min (34 +/- 13%, p less than 0.05), and between grafts incubated for 72 h vs. 6 days (55 +/- 7%, p less than 0.05). Perfused grafts incubated for 72 h showed unaltered EC morphology on SEM, few cells remained on 90 min incubated grafts. We conclude that incubating EC on fibronectin-treated ePTFE for 72 h in-vitro after seeding improves cell retention during blood flow.