The Biofunctionalization And Mechanism Of Bioactive Exosome Coating On ZE21B Magnesium Alloy For Vascular Stents

At present,more than 10 million people worldwide die from cardiovascular disease(CVD)and its complications every year,among which atherosclerosis and artery stenosis are the main causes of cardiovascular disease.The commonly used clinical treatment means are stent implantation,which could reshape blood vessels and stabilize blood flow.Due to its own degradability and good biocompatibility,magnesium alloy scaffolds have become the development direction of vascular scaffolds in the future.The re-endothelialization of stent surface is considered to be an effective strategy to prevent late thrombosis and hyperplasia.However,the delayed surface endothelialization defect was the main problem of existing magnesium alloy stent products.Therefore,how to accelerate the surface endothelialization has become a bottleneck problem and research hotspot in this field.In many studies,preparation of biocompatible coatings on the degradable magnesium alloys surface is a more convenient and effective method,which also close to clinical needs.Therefore,the main topic of the research is using exosome,which derived from plasma of healthy volunteers,to prepare bioactive sustained-release coatings on degradable magnesium alloy.Exosome is a kind of nanoscale vesicle released from the cells,containing the genetic material,proteins and lipids,which can regulate the life activities of targeted cells by releasing the contained components.At present,a large number of studies focus on explore the role of exosome in cell proliferation and anti-cancer area,fewer researchers used exosomes as a modification coating to modify the surface of materials.The research conclusions of this dissertation are as follows:(1)For exosomes obtained,the morphology of exosome was observed by transmission electron microscopy,the particle size distribution range of exosome was analyzed by nanoparticle tracking,specific protein expression of exosome was analyzed by western blotting,which all confirmed the obtained exosome met the international characterization of this substance.Since there were no relevant papers studied the preparation process of the exosome coating on materials,the preparation parameters of exosome on the surface of materials have to be selected first.The optimization procedure of the preparation time and the concentration of exosome were confirmed by the proliferation and migration of endothelial cells behavior.It was found that prepared with 25 μg/m L exosome for 12 hours could obtain excellent exosome coating,which had a certain rapid endothelialization and migration properties;(2)On the preparation parameter,bioactive exosome coating on magnesium alloy surface was prepared.The three-dimensional morphology of the coating surface was reconstructed by atomic force microscopy,and the distribution of exosome on the surface was characterized by fluorescence.The hydrophilic improvement of exosome coating was confirmed by water contact angle measurement.The overall thickness of the exosome coating is about 0.64 μm.The better corrosion resistance of the exosome coating was confirmed by electrochemical test,immersion test and p H analysis.The formation mechanism of exosome coating was summarized based on these results;(3)Bioactive exosome coating positively promoted cell proliferation,migration and factor expression(CD31 and NO)of HUVEC.The expression of contractile smooth muscle cells on the exosome active coating was increased,but the proliferation was not obvious.The endothelial cells and smooth muscle cells were cocultured in the extract solutions of each sample,and HUVEC in this environment proliferated rapidly while the proliferation of the smooth muscle was stable.The expression of reactive oxygen species(ROS)and TNF-α of macrophages in the exosome coating extract solution were significantly decreased,suggested that the inflammatory reduced by reducing the ROS level of macrophages.Through animal experiments,it was found that the magnesium alloy implants containing exosomes had uniform tissue adhesion on the surface.(4)To explore the mechanism of exosome coating on the magnesium alloys,the degradation and uptake process were observed.Exosome can be released slowly within 5 to 7 days,and absorbed by target cells through endocytosis.The exosome distributed around the nucleus,lysosome and mitochondria,and then participated in cell division and proliferation.In addition,the degradation of magnesium alloy during the degradation of exosome coating has an equally important effect on cell behavior.Studies have shown that the increased magnesium ion concentration in the environment accelerates the appearance of adhesive spots on the surface of endothelial cells.(5)Magnesium ions in extract solutions reduced the expression of inflammatory factors in macrophages and promoted the trend of M2-type macrophages.Endothelial cells were cultured in conditioned culture medium of macrophages,and the results showed that the conditioned culture medium could promote the proliferation of endothelial cells and the expression of endothelial cell adhesion molecules.This is due to the increase of magnesium ion concentration in the extract solutions,leading to a sharp increase of intracellular magnesium ion.The increased magnesium ion reduced the expression of reactive oxygen species and inflammatory expression of macrophages,and further promoted the initial adhesion and long-term proliferation of endothelial cells.Moreover,the concentration of magnesium ion in the cell changes was closely with the environment,and the cell regulation is a reversible process.In conclusion,the exosome active coating on the surface of cardiovascular stent material can enter into endothelial cells through endocytosis and promote the rapid endothelialization process on the surface of the material.And the degradation products helped to enhance the expression of M2 macrophages,promote the adhesion and proliferation of endothelial cells by reducing the expression of inflammation.