Physiological pulsatile flow culture conditions to generate functional endothelium on a sulfated silk fibroin nanofibrous scaffold

Many studies have demonstrated that in vitro shear stress conditioning of endothelial cell-seeded small-diameter vascular grafts can improve cell retention and function. However, the laminar flow and pulsatile flow conditions which are commonly used in vascular tissue engineering and hemodynamic studies are quite different from the actual physiological pulsatile flow which is pulsatile in nature with typical pressure and flow waveforms. The actual physiological pulsatile flow leading to temporal and spatial variations of the wall shear stress may result in different phenotypes and functions of ECs. Thus, the aim of this study is to find out the best in vitro dynamic culture conditions to generate functional endothelium on sulfated silk fibroin nanofibrous scaffolds for small-diameter vascular tissue engineering. Rat aortic endothelial cells (RAECs) were seeded on sulfated silk fibroin nanofibrous scaffolds and cultured under three different patterns of flow conditioning, e.g., steady laminar flow (SLF), sinusoidal flow (SF), or physiological pulsatile flow (PPF) representative of a typical femoral distal pulse wave in vivo for up to 24 h. Cell morphology, cytoskeleton alignment, fibronectin assembly, apoptosis, and retention on the scaffolds were investigated and were compared between three different patterns of flow conditioning. The results showed that ECs responded differentially to different exposure time and different flow patterns. The actual PPF conditioning demonstrated excellent EC retention on sulfated silk fibroin scaffolds in comparison with SLF and SF, in addition to the alignment of cells in the direction of fluid flow, the formation of denser and regular F-actin microfilament bundles in the same direction, the assembly of thicker and highly crosslinked fibronectin, and the significant inhibition of cell apoptosis. Therefore, the actual PPF conditioning might contribute importantly to the generation of functional endothelium on a sulfated silk fibroin nanofibrous scaffold and thereby yield a thromboresistant luminal surface.