| Grafted veins are widely used in coronary artery bypass surgery,lower extremity vascular reconstruction,and fistulas in patients undergoing haemodialysis;however,restenosis is the "Achilles’ heel" of grafted veins.The extravascular stent is wrapped around the grafted vein,thereby providing mechanical strength;it can buffer high pressure and subsequent vasodilation and reduce endothelial damage,aggregation of macrophages and T cells,various inflammatory cells,inflammation,and factor release,thus preventing restenosis.Gelatin has been widely used in various fields,such as medical treatment,food,and biopharmaceuticals.In tissue engineering and regenerative medicine,gelatin is an attractive primary material used for tissue scaffolding,tissue repair,drug delivery,and 3D cell culture.Nanocellulose is a polysaccharide extracted from raw cellulose after acid hydrolysis and is widely used in biomaterials such as 3D cell culture,tissue engineering,diagnosis,drug delivery,and separation.The most significant disadvantage of gelatin is its poor mechanical properties.However,nanocellulose can adjust the mechanical properties of gelatin through forces such as electrostatics,hydrogen bonds,and chain entanglement.Thus,nanocellulose is a potential external scaffold material.The endothelial-mesenchymal transition(EndMT)of grafted vein endothelial cells(EC)is a process in which EC lose their characteristic phenotype and gradually transform into a mesenchymal phenotype,including spindle-like morphological changes and gain greater motility,invasiveness,and contractility.In recent years,many studies have shown that EndMT plays an important role in graft vein remodelling and stenosis.Notably,as the grafted vein adapts to arterial pressure,more and more EC undergo EndMT to "adapt" to the new arterial pressure environment.Extravascular stents can resist EndMT and prevent restenosis,but the mechanism remains unclear.Autophagy is essential for cells to maintain the stability of their internal environment,and it is also an important way for cells to resist their environmental pressures.Studies have found that autophagy is significantly inhibited during EndMT in endothelial cells.The EndMT phenotype was reversed with the autophagy activator rapamycin in vitro,and these findings were validated in vivo in animal experiments.Astragaloside IV(AS-IV)is a lanolin alcohol tetracyclic triterpene saponin extracted from Astragalus membranaceus.AS-IV inhibits cell proliferation,migration,metastasis,and phenotypic transformation by promoting cell autophagy and apoptosis.Therefore,this study puts forward the following scientific hypothesis:the new nanocellulose-gelatin extravascular stent can resist EndMT and inhibit the restenosis of grafted veins by activating the autophagy pathway.After being loaded with AS-IV,the effect of resisting EndMT through the autophagy pathway is enhanced,helping to inhibit graft vein restenosis better.In this study,gelatin was used as the primary material,genipin was used as a crosslinking agent,and nanocellulose was used to adjust the stent’s mechanical properties to prepare a nanocellulose-gelatin stent.The physical and chemical properties were characterised using infrared,stretching force,expansion degree,thermal stability,and a scanning electron microscope,and the biocompatibility of the outer scaffold was characterised by in vivo and in vitro experiments.The co-culture of the outer scaffold with human umbilical vein endothelial cells was performed.We determined cell proliferation ability,DNA damage,and the live/dead experiment.We analysed the toxic response of the outer scaffold to the cells.We immunohistochemically evaluated macrophage infiltration in local tissues and analysed foreign body reactions of external scaffolds to local tissues using in vivo embedding experiments.Pathological sections of multiple organs(heart,liver,spleen,lung,and kidney)were used to analyse the toxic response of external stents to the whole body.The effect of external stents on systemic inflammation was analysed using an enzyme-linked immunosorbent assay(ELISA),blood flow cytometry detection,and spleen PCR and flow cytometry analysis.By establishing a jugular vein-artery graft model in rats,we simulated the pathophysiological changes of grafted veins through the intervention of extravascular stents,detected the blood flow velocity of grafted veins through pathological sections,pathological immunofluorescence,western blot,and RT-PCR,and clarified the passage of external stents’ resistance to EndMT,which inhibits IH.The human umbilical vein endothelial cell EndMT model was constructed via the detection of EndMT cell immunofluorescence double staining,western blot,RT-PCR,and transmission electron microscopy were used to clarify the mechanism of autophagy in EndMT.In vivo and in vitro experiments clarified that extravascular stents prevent restenosis by activating autophagy to resist EndMT and inhibiting IH.By preparing high and low doses of ASIV extravascular stents,we characterised their physical and chemical properties.We tested the drug release rate,demonstrating the feasibility of AS-IV extravascular stents.In vitro cell experiments verified that AS-IV resisted EndMT through the autophagy pathway.In vivo animal experiments clarified that AS-IV enhances the resistance of EndMT through the autophagy pathway and inhibits the effect of IH to prevent restenosis.The nanocellulose-gelatin extravascular stent was successfully constructed,and 10%nanocellulose was selected as the most suitable ratio for the experiment through tensile force and expansion degree experiments;the 10%external stent group significantly inhibited the expansion of the grafted vein(10%NC:0.103±0.006 versus,vein:0.720± 0.010 mm,p<0.05 at 150 mmHg).Fourier transform infrared shows that the hydroxyl groups of nanocellulose and the amino groups in gelatin can form a large number of hydrogen bonds,which makes the absorption peak of gelatin at 3300 cm-1 shift to a lower wave number.The hydrogen bonds between the two benefit the hydrogels’ mechanical properties.Moreover,10%NC had the lowest in vivo degradation rate(10%NC:7.8±0.01%versus 5%NC:14.6 ± 0.01%,0%NC:28.3 ±0.01%,p<0.05).The typical hydrogel-like morphology could be identified via by scanning electron microscopy;by comparison,more nanocellulose enrichment could be observed in the 10%outer scaffold group.The co-cultured cell counting kit 8(CCK8),comet experiment,and live/dead experiment showed that the outer scaffold did not cause apparent toxic damage to the cells.Immunohistochemical results of hematoxylin and eosin(H&E),Masson,iNOS,CD206,and CD68 in locally embedded tissues showed that macrophage infiltration decreased after 3 days.The external stent did not cause an obvious local or foreign-body reaction.H&E and Masson staining of the heart,liver,spleen,lung,and kidney showed that the external stent did not cause toxic damage to the organs of the whole body;the detection of the external stent embedding ELISA,RT-PCR,and flow cytometry showed that the external stent did not cause significant damage to the whole body’s inflammation.In the rat vein graft model,the external stent inhibited intimal hyperplasia(intimal thickness:external stent group:443.0±150.0 μm,graft vein group:676.0 ± 204.7 μm,p<0.05);graft vein CD31/αSMA immune results of fluorescent double staining,western blot,RT-PCR,and in vitro cell experiments showed that the outer scaffold resisted EndMT of grafted veins through autophagy.The AS-IV-loaded extravascular stent was designed to limit the vein expansion in vitro and has a promising drug release rate(30-day cumulative release is 47±0.01%);by constructing a rat vein graft model and AS-IV intervention cell model,the results of H&E staining,Masson staining,immunofluorescence double staining,western blot,RT-PCR and transmission electron microscopy suggested that AS-IVloaded extravascular stents could better inhibit intimal hyperplasia than non-drugloaded(high-dose group:171±67.0 μm versus low-dose group:253±67.0 μm,stent group:445.3±150.0 μm,no stent group:676.4±204.7 μm,p<0.05);AS-IV-loaded external stents enhanced resistance to EndMT through autophagy pathway to prevent restenosis effect.This study explored the mechanical properties of nanocellulose-gelatin hydrogel to limit the expansion of grafted veins under high pressure and provided a broader idea for the application of nanocellulose-gelatin in bioengineering and clinical fields,from cells to animals,local to the whole,inflammation to toxicity,and multi-dimensionally and systematically demonstrated the biocompatibility of extravascular stents.The positive results laid a solid foundation for the future clinical application of nanocellulose-gelatin extravascular stents.The extravascular stent restricts expansion,inhibits local inflammatory release,activates autophagy,resists EndMT,inhibits IH,and prevents restenosis,an important mechanism that will help further clarify the mechanism of action of the extravascular stent.However,it is helpful to clinically optimise the treatment plan of extravascular stents and provide an experimental and theoretical basis for improving the prognosis of patients with vein grafts.Additionally,it clarified that AS-IV enhanced external stents resist EndMT through autophagy,which helps optimise extravascular stents,particularly the application of the drug-loaded extravascular stent in clinical treatment. |
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