Thrombogenecity of decellularized and re-endothelialized baboon arteries

[摘要] English: The use of decellularized biological scaffolds for the reconstruction of small-diameter (< 6 mm) vascular grafts has been the focus of tissue engineering studies. These biological materials constructed through decellularization processes are thought to be ideal graft materials for replacement of diseased vessels. However thrombogenecity is a major cause of obstruction in these vascular constructs and result in early graft occlusion. Seeding of the decellularized vascular constructs with endothelial cells (ECs) is an attractive proposition as the endothelial layer incorporates many of the anti-thrombogenic properties of blood vessels. The aim of this study was to determine if we could successfully re-endothelialize decellularized baboon arteries, thereafter perfuse the newly engineered arteries with whole blood to investigate the possibility of thrombus formation. First, the histology of the decellularized baboon arteries were compared to normal arteries in order to assess the efficiency of the decellularization process. Collagen and elastin fibres were also stained to determine whether the remaining extracellular matrix scaffold was preserved after decellularization. Human umbilical vein endothelial cells (HUVECs) were then cultured under optimal conditions. The viability and proliferation rate of the cultured ECs were assessed using the MTT cell viability and proliferation assay. The cultured endothelial cells were then used to seed the luminal surfaces of decellularized baboon arteries. The confluent endothelial monolayer of the seeded decellularized arteries were then assessed using scanning electron microscopy (SEM) after 7 days. The seeded cells were then detached from the graft surfaces of small sections and tested for viability (metabolic activity and proliferation) using the MTT assay again. Afterwards, normal, decellularized and seeded decellularized arteries were perfused for 2 hours with baboon whole blood collected in 3.2% sodium citrate tubes. Thereafter, small artery sections were examined with SEM to determine whether thrombi was formed on the luminal surfaces of all arteries. Histology examinations showed that the decellularized arteries were not completely cell free. Nuclear and cellular remnants were still retained within the scaffold materials. Histology also revealed that the femoral arteries had retained more nuclear and cellular materials than the carotid and radial arteries which showed much less nuclear material remaining. However, the ECM of the decellularized arteries was preserved after the decllularization processes. Enough viable ECs were obtained in culture to seed three decellularized baboon arteries. After 7 days post seeding, a confluent endothelial monolayer was observed on the luminal surfaces of the decellularized scaffolds using SEM. The perfused normal artery and the seeded decellularized arteries showed no possible thrombus formation on their luminal surfaces. The decellularized arteries however showed wide-spread platelet adhesion and activation on the surface of the ECM. In conclusion, decellularization of arteries was not successfully and proved to be dependent on the thickness of the tissues used. However, the decellularization process produced morphologically preserved extracellular matrix. The endothelialization process was successful since the endothelialization of decellularized vascular grafts does prevent thrombus formation on artery surfaces after perfusion with whole blood, while a decellularized scaffold does promote thrombus formation.

[发布机构] University of the Free State