An in Vitro Model to Study Endothelialization of Cardiac Graft Tissues under Flow

Pulmonary valve replacement is performed with excellent resultant hemodynamics in patients that have underlying congenital or acquired heart valve defects. Despite recent advancements in right ventricular outflow tract reconstruction, an increased risk of developing infective endocarditis remains, which has a more common occurrence for conduits of bovine jugular vein (BJV) origin compared with cryopreserved homografts. The reason for this is unclear although it is hypothesized to be associated with an aberrant phenotypic state of cells that reendothelialize the graft tissue postimplantation. The aim of this study was to develop an in vitro model that enables the analysis of endothelial cell (EC) attachment to cardiac graft tissues under flow. In the experiments, EC attachment was optimized on bovine pericardium (BP) patch using human umbilical vein ECs. Different biological coatings, namely gelatin, fibronectin, plasma, or a combination of fibronectin and plasma were tested. After cell adaptation, graft tissues were exposed to laminar flow in a parallel-plate flow chamber. Cell retention to the tissue was analyzed after nuclear staining with YO-PRO-1 and a membranous localization of VE-cadherin. Experiments showed that combined coating with fibronectin and blood plasma together with a two-phased shear pattern resulted in a relevant cell monolayer on BP patch and cryopreserved homograft. For BJV tissue, no adherent cells under both static and shear conditions were initially observed. In conclusion, having established the new flow chamber system we could obtain EC layers on the surface of BP patch and cryopreserved pulmonary homograft tissues. The presented in vitro system can serve as a competent model to study cell phenotypes on cardiac grafts in the close-to-physiologic environment. Moreover, this approach allows broad applications and enables further development by testing more complex conditions.