Studies in the Use of Blood Outgrowth Endothelial Cells to Populate Polycaprolactone Scaffolds for Therapies in Human Heart Valve Disease.

Abstract

BackgroundEndothelial cells line blood vessels and the heart where they release cardio-protective hormones that prevent thrombosis. Available replacements to treat heart valve diseases are limited by the lack of the endothelial cell layer, making them susceptible to calcification and thrombosis, which limits utility and increases the need for multiple replacements[1]. A suggested solution is to adapt tissue engineering techniques to formulate intelligent instructive scaffolds decorated with specific molecules to enhance the adhesion and population of circulating progenitor endothelial cells from the blood.AimIn this study, we aim to modulate nanofibrous scaffolds fabricated from the biodegradable polyster polycaprolactone (PCL) to provide a viable environment for endothelial cells from blood progenitors (blood outgrowth endothelial cells; BOECs) to adhere, proliferate and function successfully. To date we have (i) compared the behaviour of BOECs to endothelial cells isolated from human heart valves (hVECs) under shear conditions, (ii) tested the compatibility of PCL with BOECs by assessing cell viability and inflammatory responses on modified PCL films, and their ability to populate structured PCL scaffolds.MethodsBOECs were isolated from the blood of healthy volunteers by selective plating of peripheral blood mononuclear cells (PBMCs), and phenotyping was assured by measuring the expression of endothelial cell markers using fluorescent activated cell sorting (FACS). hVECs were isolated from human aortic valves by collagenase digestion. Shear stress was studied using a cone and plate model. PCL films were prepared by solvent evaporation method, and sterilized with ethanol for 30 min, or modified by plasma oxidation at 30 w and 0.1 m bar for 30 min. PCL Films were coated with extracellular matrix proteins before cell seeding. After 72 hr of culture, cell viability was measured using alamar blue and cell secretory function measured by the release of CXCL8, endothelin-1 (ET-1) and prostacyclin by ELISA. Finally, the ability of these cells to populate 3D structured nanofibrous PCL scaffolds fabricated by jet spraying technique2 was determined by confocal microscopy of phalloidin stained cells.ResultsBOECs colonies appeared at between 7 and 21 days of culture. FACS analysis of expanded cells showed positive expression of the typical endothelial cell markers CD31, CD90, VE Cadherin, CD44, and CD105. Also, BOECs stained negative for the (non-endothelial) exclusion markers CD14, CD45, and CD133. In shear stress studies, BOECs and hVECs aligned similarly to ventricular flow (ie. directional shear stress). Both cell types displayed similar viability responses when grown on PCL which was ∼40% less than achieved when cells were grown on control glass slides. Modifying PCL with plasma oxidation or extracellular matrix coating did not improve viability of either cell type. Both BOECs and hVECs released CXCL8 and ET-1 when grown on control glass slides which was not increased in cells cultured on PCL. In addition, both cell types released the cardio-protective hormone prostacyclin when grown on glass or PCL, suggesting that they would provide a viable anti-thrombotic surface. Regarding to 3D structures, BOECs were able to populate both modified and unmodified aligned nanofibrous PCL scaffolds, and appeared to align with the direction of the fibres.ConclusionsBOECs expressed the requisite endothelial cell markers and, as we have shown previously, responded appropriately to shear and released CXCL8, ET-1 and prostacyclin 3; suggesting that BOECs are suitable seeding cells for tissue engineering. For the endothelialization of tissue engineered heart valves, the target cells that BOECs need to mimic are hVECs. Our preliminary studies show that BOECs and hVECs profile similarly in population of PCL, alignment with shear stress and with endothelial cell hormone release. In addition, BOECs are able to align with the direction of PCL fibres, mimicking the native valve endothelial cells that were previously reported to align with the direction of the collagen fibers[4]. These results are promising and indicate the potential of BOECs as a cell source to populate PCL nanofibrous scaffolds designed to replace heart valves. Nevertheless, our data shows that standard PCL platforms are not optimal in terms of cell viability. This may be improved by biofunctionalizing PCL with specific molecules to support BOECs adhesion and proliferation. For that, we are currently studying the potential of linking the BOECs specific peptide (TPS) to PCL.