In Vitro Effect Of Biologically Active Coating On Nitinol Devices Designed For The Closure Of Intracardiac Shunt

Objective: To assess the effect of bioactive coatings on biocompatibility of Nitinol slices designed for the closure of congenital heart defects in vitro.Methods: Binding procedure was performed with covalently immobilized onto the surface of Nitinol slices. Heparin (10mg/ml), r-hirudin (2v/ml) or fibronectin (10mg/ml), respectively, were bound to the basic coating (Chi). In vitro tests were performed on different coating groups: control, uncoating, basic coating, Chi/hep, Chi/r-hir, and Chi/fn. The cytotoxicity of biomaterials was evaluated using the hemolysis test. Anticoagulant activity was studied in a dynamic model with whole blood by partial thrombin time (APTT), prothrombin time (PT), flbrinogen time(FIB), thrombin time (TT)and D-Dimer(DD) respectively. Scanning electron microscopy was used for the manifestation of the surface characterizations such as protein adsorption and platelet adhesion. Humanumbilical vein endothelial cells (HUVECs) were cultured in the presence of tested slices for72h. Adhesion of HUVECs were evaluated with light microscopy and fluorescence microscope by double-staining of vinculin, Ki67, fibronectin and von Willebrand Factor.Results: (1)The hemocompatibility test: Uncoated or coated devices did not influence hemolysis. White blood count (WBC), red blood count(RBC) and platelet count (PC) were all in normal range and there were no evident difference after the slices were incubated with whole blood for 2h.The hemolysis rates was all less than 5%. The PT, APTT and TT were longer in Chi/r-Hir and Chi/Hep than that in other groups. Heparin coating showed shorter in APTT and TT than r-Hirudin coating but PT had no difference(LSD and SNK, P＜0.005). Furthermore, it can be observed there was dose-effect relationship among differential density r-Hir coating (Dunnetts, P＜0.005) but no significant difference in differential density of Chi/Hep coating. Scanning electron microscopy indicated that the protein adsorption, platelet adhesion and thrombus formation were all reduced by the immobilization of Chi/r-Hir and Chi/Hep. (2) Cyto-compatibility test:After 72h of incubation, there were no showing abnormal morphology, HUVECs were able to grow next to all kinds of slices. The cells grown on the well surfaces exhibited a more assembly morphous than that on the slices surfaces. By fluorescence microscope adhering cells were decreased in the order: Chi/Fn coating＞Basiccoating＞Uncoating＞Chi/r-Hir coating＞Chi/Hep coating. Ki67 expression was mainly HUVEC proliferation, decreased in the order:Chi/Fncoating＞basic coating=uncoating＞Chi/r-hir coating＞Chi/hep coating. The cells grown on the surfaces of Chi/r-Hir were not as good as on the Nitinols slices. Nevertheless HUVECs proliferation was inhibited by Chi/hep coating.Conclusion: Proliferation of HUVECs on the nitinol slices could enhanced by Chi/Fn coating, but the PT, APTT and TT did not change after the coating.Thrombus formation were all reduced by the immobilization of Chi/r-Hir and Chi/Hep, furthermore, Chi/r-hir coating decreased thrombogenicity, increased endothelialisation, we drew a conclusion: biocompatibility of flat Nitinol slices designed for the closure of congenital heart defects can be improved by Chi/r-Hir conjugate onto the surface.