R. S. Hennessy1, S. Jana1, M. Helder1, A. Lerman1 1Mayo Clinic,Rochester, MN, USA
Introduction: Endothelization of decellularized heart valves is required to lower thrombogenicity risk and decrease inflammation of tissue-engineered valves. However, conventional methods of endothelization do not show positive results and can take upwards of 2 weeks to result in a monolayer. These cells are often statically adaptive and do not adhere firmly to the 3-D structure of the heart valve cusp. We hypothesized that the addition of a cell receptive biodegradable material in nanofiber form could increase endothelial cell viability, proliferation, and adherence.
Methods: Porcine aortic valve cusps were decellularized with 1% sodium dodecyl sulfate. After washing and sterilization, the cusps were covered with polycaprolactone (PCL, 9 wt% solution) nanofibers through electrospinning system at a voltage of 17.5 kV and with a distance between the spinneret and collector of 15 cm. The encased cusps and cusps without a nanofiber layer (bare cusp) were then seeded (106 cells/ml) with blood outgrowth endothelial cells (BOECs) and cultured statically with periodic observation. The morphology of the nanofibers and cells on cusps were analyzed with scanning electron microscopy (SEM). The cells on cusps were also stained with Hoechst and imaged with a confocal microscope. Cell counting was performed on confocal images manually and areas covered by cells were calculated with ImageJ software.
Results:The diameter of the electrospun nanofibers was ~200 nm (Fig. a). The bare cusps were covered with an endothelial cell-layer at 14 days of culture (Fig. b) whereas nanofiber-layered cusps formed a monolayer at 4 days (Fig. c). Endothelial cells on bare cusps under SEM had a globular profile and cell-cell junctions were sparse. On nanofiber cusps, SEM showed elongated endothelial cells and the presence of cell-cell junctions. The densities of nuclei shown in Figures d and e obtained through confocal imaging confirmed higher cell proliferation and growth on nanofibrous covered cusps compared to naked cusps. These results are further confirmed by cell number and cell-covered area on these cusps (Fig. f).
Conclusion:A nanofibrous layer made of PCL showed efficacy in improving endothelial cell propagation and growth. The nanofibrous layer also influenced endothelial cells to develop an almost continuous monolayer in 4 days as compared to 14 days in the absence of the nanofibrous layer. Thus, it can be concluded that a biodegradable PCL polymeric nanofibrous layer is helpful to achieving a more robust endothelial layer.
