| BackgroundCoronary stent implantation is the main means in the treatment of coronary heart disease at present, but restenosis and thrombosis in-stent are the most important and serious complications after percutaneous coronary intervention which have hindered the development of percutaneous coronary intervention. Drug eluting stent which is the most extensively used stent presently could reduce restenosis in-stent by inhibiting proliferation of vascular smooth muscle cells. It also inhibits the proliferation of vascular endothelial cells which will lead to endothelial cell healing delay and stent thrombosis. A novel stent that can reduce stent restenosis and thrombosis is needed to develop in the field of biomedical materials. This research has great theoretical and practical significance. A large number of studies have found that accelerated endothelialization after stent implantation can reduce both restenosis and thrombosis formation. Our previous studies confirmed that CD34 antibody stent although could reduce restenosis and thrombosis by accelerated endothelialization. But the specificity of CD34 antigen selection was not enough. This stent could promote intimal endothelialization. It could not inhibit the proliferation of vascular smooth muscle cells, and the restenosis rate in-stent is still high.ObjectiveWe constructed a novel stent coated with CD34 antibody and vascular endothelial growth factor(VEGF) in our project which could promote endothelial cells. The novel stent double coated by everolimus which can inhibit proliferation of vascular smooth muscle cells. This double-sided coating novel coronary stent was investigated the effect of inhibiting proliferation of smooth muscle cells and promoting endothelialization in vitro and vivo. The study of novel stent in the prevention of in stent restenosis and thrombosis can supply important basic data forclinical application.MethodsFirstly, we bulit 316 L stainless steel sheet coated with CD34 antibody and VEGF(referred to as experimental steel): we prepared compound through the condensation reaction of dopamine and heparin, with fourier transform infrared spectrometer(FTIR) as the characterization of the reaction;we immobilized the polymer coating on the stainless steel sheet via polymerization of dopamine, with X-ray photoelectron spectrograph(XPS) as the characterization; then CD34 antibody and VEGF were coated on the polymer by layer-by-layer self assembly(LBL),and the levels of CD34 antibody and VEGF coatd on the steel sheet were tested qualitatively and quantitatively by Confocal laser scanning microscope( CLSM) and enzyme-linked immunosorbent assay( ELISA). Then we evaluated the biocompatibility of the experimental steel in vitro: we evaluated the blood compatibility in vitro by hemolytic test. We measured the type and quantity of protein absorption,activated partial thromboplastin( APTT) and prothrombin time( PT).We isolated and cultured endothelial progenitor cell(EPC) in vitro to evaluate cell compatibility of the material by 2 methyl thiazole diphenyl tetrazolium bromide salt(MTT) assay and estimate its influence on the differentiation of proliferation of EPC. We designed double-sided coating stent as described above by LBL to immobilization of CD34 antibody and VEGF on the inner layer and everolimus on the outer one. Finally, the bare metal stents(referred to as NIR stents), everolimus stent,CD34 and VEGF coated stent(referred to as single stent) and double coated stent(referred to as experimental stent) were evaluated through animal experiments to clarify that double-sided coating coronary stent. Then we prepared swine coronary artery damage model and evaluated impacts on the intimal hyperplasia and reendothelialization of the four groups of stents: we contrasted percentage of Diameter Stenosis(%DS) of different groups by quantitative coronary angiography( QCA) and percentage of area stenosis(%AS), intimal hyperplasia,reendothelialization under light microscope 4weeks and 12 weeks after stents implantation.ResultsWe bulit the steel coated with CD34 antiby and VEGF successfully: the result of FTIR testified the reaction between dopamine and heparin; the poly-dopamine polymer on the surface was conformed by XPS; the results of CLSM and ELISA showed that CD34 antibody and VEGF were coated on the surface successfully qualitatively and quantitatively respectively. The experimental steel had good blood compatibility in vitro: the experimental steel has qulified hemolysis rate; the amount of albumin absorbed by the experimental steel was larger than control group(P<0.05)and the experimental steel had longer APTT(P<0.05). The experimental steel showed good cell compatibility in vivo: the amount of growing EPC on the surface of experimental steel was larger than the control group in the MTT test(P<0.01)The experimental steel had the ability to promote the differentiation and proliferation EPC toward EC in vitro: the result of flow cytometer showed that specific marker of EC(CD31 and KDR) were more than the control group after cultivation(both P <0.05).By measuring percentage of Diameter Stenosis(%DS) of each group on 4weeks and 12 weeks after stents implantation, results showed NIR groups were bigger than other groups, single-sided stent was the second, experimental groups were smaller than other groups( P < 0.05), while had no difference compared with everolimus groups on 4 weeks(P>0.05).When compared within groups,there was no statistical difference between the experimental group at 4 weeks and 12 weeks, and the other 3 groups at two time points were significantly different(P < 0.05).We Observed intimal hyperplasia and compared percentage of area stenosis(%AS) under light microscope after HE staining,the results demonstrated that experimental groups and everolimus groups could reduce intimal hyperplasia significantly compared with NIR groups(P<0.05), experimental groups showed less hyperplasia in comparison with single-sided polymer groups and everolimus groups(P<0.05),while there was no difference between experimental groups with everolimus groups on 4 weeks(P >0.05).And we found that the endothelialization thickness and distribution of experiment stent groups were observed more uniform under the light microscope.Compared within groups, there was no statistical difference between theexperimental group and everolimus group at 4 weeks and 12 weeks, while the other two groups at 4weeks and 12 weeks were significantly different(P < 0.05).ConclusionNovel double-sided stent coated with CD34 antibody, VEGF and everolimus has good biocompatibility in vitro, which can promote EPC differentiation and proliferation of EC and reduce intimal hyperplasia in the scaffold surface, and accelerate endothelialization. Moreover, the double-sided stent gain good treatment effect in 4 weeks. Our research has provided a solid theoretical basis and experimental basis for the research of novel stent in prevention of restenosis and thrombosis in-stent.Significance and application value of the ResearchAt present there is no stent reported coated with CD34 antibody and VEGF jointly promoting endothelial cells to accelerate endothelialization, while taking advantage of everolimus to inhibit the proliferation of smooth muscle cells to maximum limit to prevent restenosis and thrombosis in-stent. We constructed a novel double-sided stent coated with CD34 antibody and VEGF on the inner layer and everolimus on the outer one to solve the bottleneck problem oft restenosis and thrombosis in-sten, to reduce complications after coronary stent implantation and improve the patients prognosis, to improve the coronary heart disease patients’ survival rate. Application of the new coronary stent that could reduce cardiac events rate and revascularization rate may also be a great saving of national health resources.It would bring huge economic and social benefits, and provide theoretical and experimental basis for the development and clinical application of the domestic high performance new coronary stent. In addition, the research will supply great significance for the development of vascular surgery repair materials. This is significant for the advancement of science and technology, the upgrading of the industrial structure and the upgrading of the medical coronary stent in the field of biomedical implants. |
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