Tissue Engineered Small Vessel Conduits – The Anti-Thrombotic Effect of Re-Endothelialization of Decellularized Baboon Arteries: A Preliminary Experimental Study

Abstract

BackgroundThe use of decellularized biological scaffolds for the reconstruction of small-diameter vascular grafts remains a challenge in tissue engineering. Thrombogenicity is an important cause of obstruction in these vessels due to decellularization. Seeding of the decellularized vascular constructs with endothelial cells is therefore a prerequisite for the prevention of thrombosis. The aim of this study was to seed decellularized baboon arteries with endothelial cells and to compare the thrombogenicity to that of decellularized arteries after circulation of blood.Material/MethodsCarotid, radial, and femoral arteries (12 arteries in total) were harvested from 2 Papio ursinus baboons. Ten arteries were decellularized. Normal morphology was confirmed in the control vessels. The effect of re-endothelialization was studied in the vessel scaffolds using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Decellularization resulted in vessel scaffolds with well-preserved extracellular matrix and intact basal membranes. Six of the decellularized vessel scaffolds were seeded with viable human umbilical vein endothelial cells (HUVEC). Luminal endothelialization was established after 7 days in a bioreactor and SEM confirmed confluency. Two control, 4 decellularized, and 6 decellularized re-endothelialized vessel scaffolds were studied in an in vitro flow chamber using baboon blood.ResultsThe decellularized arteries showed an absence of endothelial lining, and an intact basement membrane. The seeding process produced a complete endothelial layer on the surfaces of the arteries. After perfusion with whole blood, no thrombi were formed in the control arteries and re-endothelialized vessels. Widespread platelet activation and adhesion occurred in the decellularized vessels despite a relatively intact basal membrane.ConclusionsThis study supports the development of re-endothelialized tissue engineered small-vessel conduits.

Re‐endothelialization of non‐detergent decellularized porcine vessels

“…In addition, HUVECs were successfully seeded onto NaOH decellularized rat lungs. 21 Rotation re-seeding, which was reported to be advantageous compared to static strategies in previous studies, [43][44][45] enabled the coverage of the entire luminal surface of the vessel ( Figure 5).…”

Strategies in cell‐free tissue‐engineered vascular grafts

“…Generally, two or three treatment methods were combined to remove cell components and avoid excessive immune response, and researchers should be very careful to get a compromise between removing antigenic cellular components and maintaining ECM strength. The standard for determining efficient decellularization should be defined as follows: (a) no nuclei visible upon staining using either hematoxylin and eosin (H&E) or DAPI (4 0 , 6-diamidino-2-phenylindole) staining, (b) less than 50 ng of double-stranded DNA (dsDNA) per mg dry weight of ECM, and (c) a integrated basement membrane should be preserved (Crapo, Gilbert, & Badylak, 2011;Garreta et al, 2017;Meiring, Khemisi, Laker, Dohmen, & Smit, 2017). In addition, an efficient cross-linking treatment is necessary to reconstruct the ECM microstructure.…”

Digital 3D Topographic Microscopy: Bridging the Gaps Between Macroscopy, Microscopy and Scanning Electron Microscopy

“…Vascular healing is evaluated in animal studies through detailed macroscopic observation at necropsy and histologically as well as through SEM analysis of the vascular surface. 13,14 Macroscopy is an indispensable step to study vascular integrity and remodeling and assess for the presence of grossly visible changes that could signal poor healing such as hemorrhage, thrombus formation, stenosis, excessive dilation, and local or downstream embolization and ischemia. Histopathology provides detailed views into the state of healing at the tissue and cellular levels from which key insights on the pathophysiology of adverse events can be derived.…”

“…Scanning electron microscopy is the de facto go-to imaging platform to assess endothelial coverage of vascular surfaces and implanted vascular grafts. [6][7][8][9][10][11][12][13][14][15] It offers the advantage of a very visual 3-dimensional like topographical perspective where the typical cobblestone appearance of confluent endothelial cells can be demonstrated. Alternatively, surface abnormalities indicative of incomplete healing such as presence of thrombus, adherent leukocytes, or uncovered biomaterial can also be evidenced.…”

“…Therefore, establishing the course of endothelium healing is an essential aspect of safety assessment for these devices. 13 The time course of complete vascular healing must be well understood in preclinical animal models to predict healing in the patient population and establish the risk period during which preventative antithrombotic medication should be prescribed.…”