In vitro testing of endothelial cell monolayers under dynamic conditions inside a beating ventricular prosthesis

Thromboembolic complications remain a major problem associated with the long-term clinical use of cardiac prostheses. A promising approach toward resolving this predicament is lining the blood contacting surfaces with a functional monolayer of endothelial cells (EC). In developing an endothelialized cardiac prosthesis, the authors in the past focused on establishing a confluent EC monolayer on the luminal surface of ventricular blood sacs. In this study, the authors concentrated on exposing the post confluent monolayers to the dynamic conditions inside a beating ventricle. The cells, derived from either bovine aortae or jugular veins, were grown to post confluence inside fully assembled ventricles on fibronectin or plasma cryoprecipitate coated, textured surfaces. After 11 days of culturing under static conditions, the endothelialized ventricles were connected to a mock loop that was run for 6 and 24 hr at 60 bpm and mean flow rate of 3.2 L/min. The status of the monolayer was evaluated by Alamar Blue assay before and after each run, and the extent of surface coverage was determined visually using bright field microscopic study after cell staining with KMnO₄ and toluidine blue. In addition, morphometric information on cells/polyurethane surface was obtained with a scanning electron microscope. After 6 hr of pumping, cell staining revealed signs of moderate cell loss in fibronectin coated blood sacs, whereas in cryoprecipitate coated bladders the signs of denudation were marginal. In seven ventricles operated for 24 hr, Alamar Blue measurements indicated 35 ± 16% of cell loss from monolayers established on fibronectin coating, but only 4.8 ± 6.25% on cryoprecipitate. Thus, the current study demonstrates the feasibility of maintaining an intact endothelial surface in a beating ventricular prosthesis and indicates that the integrity of the endothelial lining is dependent upon a proper choice of surface macrostructure and protein coating.