Understanding the role of mechanical forces on cell behavior is critical for tissue engineering, regenerative medicine, and disease initiation studies. Current hemodynamic bioreactors are largely limited to 2D substrates or the application of general flow conditions at a tissue level, which eliminates the investigation of some essential physiological and pathological responses. One example is the mesenchymal transformation of endothelial cells in response to shear stress. Endothelial to mesenchymal transformation (EndMT) is a valve morphogenic mechanism associated with aortic valve disease initiation. The aortic valve experiences oscillatory shear on the disease-susceptible fibrosa, and the role of hemodynamics on adult EndMT is unknown. The goal of this work was to develop and characterize a microfluidic bioreactor that applies physiologically relevant laminar or oscillatory shear stresses to endothelial cells and permits the quantitative analysis of 3D cell-extracellular matrix (ECM) interactions. In this study, porcine aortic valve endothelial cells were seeded onto 3D collagen I gels and exposed to different magnitudes of steady or oscillatory shear stress for 48 hours. Cells elongated and aligned perpendicular to laminar, but not oscillatory shear. Low steady shear stress (2 dyne/cm2) and oscillatory shear stress upregulated EndMT- (ACTA2, Snail, TGFB1) and inflammation- (ICAM1, NFKB1) related gene expression, EndMT-related (αSMA) protein expression, and matrix invasion when compared with static controls or cells exposed to high steady shear (10 and 20 dyne/cm2). Our system enables direct testing of the role of shear stress on endothelial cell mesenchymal transformation in a dynamic, 3D environment and shows that hemodynamics regulate EndMT in adult valve endothelial cells.

Heart valve disease is an increasing clinical burden for which there is no effective treatment outside of prosthetic replacement. Over the last 20 years, clinicians have increasingly preferred the use of biological prosthetics to mechanical valves despite their superior durability because of the lifelong anticoagulation therapy that is required. Mechanical valve surface engineering has largely focused on being as non-thrombogenic as possible, but despite decades of iteration has had insufficient impact on the anticoagulation burden. In this study, we systematically evaluate the potential for endothelialization of the pyrolytic carbon surface used in mechanical valves. We compared adsorbed adhesion ligand type (collagen I, fibronectin, laminin, and purified adhesion domain fragments GFOGER and FN7-10) and concentration on endothelial adhesion rates and adhesion strength on Medtronic-Hall prosthetic valve surfaces. Regardless of ligand type or concentration, endothelial adhesion strengthening was insufficient for their intended ultra-high shear stress environment. We then hypothesized that microfabricated trenches would reduce shear stress to tolerable levels while maintaining endothelial access to the flow stream, thereby promoting a confluent and anticoagulant endothelial monolayer. Computational fluid dynamics simulations predicted an empirical relationship of channel width, depth, and spacing that would maintain interior surface shear stress within tolerable levels. Endothelial cells seeded to confluence in these channels retained a confluent monolayer when exposed to 600 dynes/cm2 shear stress for 48 hours regardless of applied adhesive ligand. Furthermore, sheared EC expressed a mature anti-coagulant profile, including endothelial nitric oxide synthase (eNOS), VE-cadherin, and significantly downregulated plasminogen activator inhibitor-1 (PAI-1). As a final test, channeled pyrolytic carbon surfaces with confluent EC reduced human platelet adhesion 1000-fold over pyrolytic carbon alone. These results advance a promising biohybrid approach to enable active moderation of local coagulative response in mechanical heart valves, which could significantly extend the utility of this important treatment for heart valve disease.