Bioreactor preconditioning of endothelium for bioengineered vascular constructs

BACKGROUND
Thrombosis is a primary mode of failure for prosthetic vascular grafts used clinically and the use of endothelialized engineered vascular constructs has been proposed to avoid this complication. Despite the key role of endothelial cells in preventing thrombosis, the conditions under which a durable and functional endothelial monolayer can be created has not been defined for engineered vessels. Shear stress is known modulator of endothelial phenotype and function. This study examines the role of shear stress in preconditioning endothelialized vascular constructs.
METHODS:
Autologous sheep endothelial cells were isolated using a novel endothelial progenitor affinity purification column. Progenitors were then expanded into mature endothelial cells (EC) using EGM2 endothelial growth medium and characterized for vWf, Lectin, eNOS, and VEGFR expression. Acellular scaffolds were then seeded with autologous ECs and preconditioned for 9 days to flow and shear stress using a bioreactor system. Three specific protocols were tested: low steady shear stress (SS), high steady SS, and high cyclic SS were investigated. An in vivo arteriovenous bypass model was then used to examine endothelial adherence as well as adherence of blood elements under these preconditioning regimens when exposed to circulating blood for 15 minutes. The luminal surfaces of conditioned grafts were compared using histology and scanning electron microscopy (SEM). Gene expression of endothelial nitric oxide synthase (eNOS) and prostaglandin I synthase was examined by Western blot of lysates from endothelial cells exposed to several preconditioning conditions.
RESULTS:
Histology and SEM of conditioned bioengineered vessels suggest improved EC monolayer confluence when preconditioned under higher shear stress conditions, particularly high cyclic SS when compared vessel constructs that had been subjected to only a static adherence protocol. In addition to improved endothelial adherence, vessel constructs preconditioned to high cyclic shear stress showed over 5 fold less adherence of potentially thrombogenic platelets and leukocytes compared to static EC adhered and non EC adhered grafts. As demonstration of the functional effect of preconditioning, Western blot analysis demonstrated an increase in the protein expression of the endothelial effectors eNOS and prostaglandin I synthase for the high cyclic shear stress conditioned cells compared to cells exposed only to high steady shear stress.
CONCLUSIONS:
Preconditioning of bioengineered vessels with high cyclic shear stress promotes increased endothelial adherence, enhanced resistance to platelets and leukocytes, as well as increased markers of endothelial function when compared to constructs subjected to non cyclic, lower shear stress or absent shear stress conditions. These studies may ultimately establish protocols for the formation of a more durable endothelial monolayer on vascular constructs for clinical use.