Vascular tissue generation in response to signaling molecules integrated with a novel poly(ɛ-caprolactone)–fibrin hybrid scaffold

Abstract

A major constraint in the use of biodegradable polymer scaffolds for vascular tissue engineering is poor cell adhesion and lack of signals for new tissue generation. The presence of extracellular matrix (ECM) within the scaffold is desirable for growth of endothelial cells and in vitro formation of remodelled vascular conduit. In this study, we have produced a hybrid scaffold by coating porous poly-caprolactone (PCL) film with biomimetic ECM components consisting of fibrin, gelatin, fibronectin, angiogenic growth factors and proteoglycans. Human umbilical vein endothelial cells (HUVECs) adhered, spread, proliferated and survived for long periods in culture on the hybrid scaffold. As compared to bare PCL, enhanced cell adhesion, spreading and cytoskeletal organization were demonstrated on the hybrid scaffold, using confocal microscopy of EC-actin stained with Texas red-conjugated phalloidin. Population doubling of endothelial cells (ECs) on the hybrid scaffold and bare scaffold was estimated as 42 h and 136 h, respectively, as assessed by a 3H-thymidine uptake method. Analysis of proliferating cell nuclear antigen (PCNA) also indicated low proliferation on bare scaffold. Flow cytometric analysis of annexin V-stained cells showed poor survival of ECs on bare PCL as compared to the hybrid scaffold. Deposition of insoluble collagen and elastin was identified on the hybrid scaffold by cells recovered after 15 days and 30 days of EC culture, using fluorochrome-tagged specific antibodies and confocal microscopy, and the fluorescence intensity corresponding to elastin and collagen after 30 days was similar to that of 15 days. The results indicate that ECM deposition by endothelial cells is a regulated process without excessive accumulation after 30 days.