Study On Degradation Behaviors And Biological Properties Of A Novel Absorbable Zinc-Based Alloy Vascular Stent

Cardiovascular diseases（CVDs）are the number one killer worldwide,and the mortality of CVDs is higher than that of tumors or other diseases.Coronary atherosclerotic heart disease,commonly abbreviated as“coronary heart disease”is the leading cause of CVDs death,and the incidence of coronary heart disease is increasing year by year in the word,which seriously harms human health.The second generation of drug-eluting stents（DESs）has currently become the first choice for percutaneous coronary intervention in most patients,because of their excellent biosafety and efficacy.However,they are made of inert metal materials（i.e.,316L stainless steels,cobalt-chromium alloys,etc.）and will remain in patients for a lifetime as a foreign body,which may lead to many side effects including chronic inflammation,late-stage thrombosis and impaired vasomotion function,etc.Therefore,to overcome the drawbacks of permanent stents,a new generation of bioresorbable cardiovascular stents made of biodegradable materials is being extensively developed worldwide.Zinc（Zn）,as one of the most important essential trace elements,not only participates many basic biological functions in human beings,but also has anti-oxidant,antithrombotic,anti-inflammatory and antiatherogenic properties.In addition,quite a number of studies have indicated that metal Zn as a promising candidate material for bioresorbable cardiovascular stents due to its excellent biocompatibility and appropriate biodegradability.However,the mechanical strength of pure Zn is too weak,which fails to meet the mechanical criteria for bioabsorbable metal stent;Currently,cytotoxicity tests are mainly used to study the biocompatibility of bioabsorbable stent materials,lacking systematic studies from cells to tissues and individuals,and the long-term studies in vivo of Zn-based alloy stents have not been reported yet;In addition,restenosis is one of the main problems in cardiovascular stent application,and the proliferation and migration of smooth muscle cells（SMCs）and extracellular matrix deposition contribute to restenosis,but there is very limited study on how Zn and Zn-based alloys may affect SMCs behaviors and mechanisms.To solve the above problems,a ternary zinc-based alloy was obtained by alloying and heat treatment technology,and the mechanical properties,degradation behaviors and biological properties of the zinc-based alloy material and vascular stent were studied in vitro and in vivo.The main study contents and conclusions of this work are as follows:（1）Preparation,characterization and in vitro degradation of Zn-based alloy vascular stent materialIn the present work,by adding trace Mg and Cu in Zn matrix and using heat treatment technology,a new kind of hot extruded Zn-Mg-Cu alloy was obtained.The actual chemical composition of the Zn-based alloy was detected by inductively coupled plasma emission spectrometry（ICP-OES）,proving that the Zn-based alloy was Zn-0.02Mg-0.02Cu alloy.Compared with the hot extruded pure Zn,the spherality-like second phase particles were formed in Zn-0.02Mg-0.02Cu alloy,and Zn-0.02Mg-0.02Cu alloy exhibited a more uniform and refine microstructure.In addition,the mechanical properties of Zn-0.02Mg-0.02Cu alloy,including hardness,yield strength and ultimate tensile strength were significantly improved,while the elongation was decreased obviously.In vitro corrosion studies showed that the uniform corrosion morphologies of Zn-0.02Mg-0.02Cu alloy,and the degradation rate of Zn-0.02Mg-0.02Cu alloy was faster than that of pure Zn.The energy dispersive spectrometer revealed that the corrosion products were ZnO and Zn（OH）₂ in the initial stage of corrosion,and late corrosion mainly produced ZnCO₃ and Ca/P.（2）Biocompatibility and antibacterial properties of the Zn-based alloyZn-0.02Mg-0.02Cu alloy extracts could slightly promote human umbilical veins endothelial cells（HUVECs;EA.HY926）viability after 1 day of culture compared with control,suggesting a good cytocompatibility.In the development of zebrafish embryos and blood vessels,no obvious malformations and angiodysplasia were observed in the Zn-0.02Mg-0.02Cu alloy group,which showed that Zn-0.02Mg-0.02Cu alloy had excellent biocompatibility for the development of zebrafish embryos and blood vessels.Subcutaneous implantation in rat showed that Zn-0.02Mg-0.02Cu alloy had good histocompatibility without severe inflammatory response.The hemocompatibility test showed that Zn-0.02Mg-0.02Cu alloy exhibited a very low hemolysis rate,and could significantly inhibit platelet adhesion,aggregation and activation.In addition,Zn-0.02Mg-0.02Cu alloy showed a strong antibacterial effect on Escherichia coli and Staphylococcus aureus.（3）Corrosion behavior and biocompatibility evaluation in vivo of Zn-based alloy vascular stentsZn-0.02Mg-0.02Cu alloy stents were implanted in the carotid arteries of New Zealand white rabbits to investigate the biosafety and degradation behavior of Zn-based alloy vascular stents for the first time.Doppler ultrasonography showed that during the study period,the stents were visible and all stented vessels were complete patency without thrombus formations or obvious intimal hyperplasia at the implantation sites.The stents could rapid endothelialization at 1 week of implantation,and remained stable appearance up to 12 months,showing a low risk of cytotoxicity and thrombosis.The stents corroded slowly and no obvious intimal hyperplasia was observed for 6 months,after which corrosion accelerated at 6 to 12 months,resulting in the slight increase of neointimal area without severe intimal hyperplasia.Immunohistochemistry and Masson staining showed that after 12 months of implantation,the density of SMCs in the neointima near the struts was low with a small amount of muscle fiber secretion,while abundant collagen was observed in the whole neointima.Histopathological and macroscopic evaluation of the organs of stented rabbits revealed no accumulation of degradation products in the organs and systemic toxicity compared to the healthy unstented rabbits,and ICP-OES analysis revealed no obvious Zn²⁺deposits in the organs and serum of stented rabbits at 12 months of implantation.（4）Biological behaviors and mechanisms of the Zn-based alloy on smooth muscle cellsSMCs showed an unhealthy round and dead morphology on Zn-0.02Mg-0.02Cu alloy surface.SMCs were cultured in 100%and 75%Zn-0.02Mg-0.02Cu extracts with cytotoxicity,while 50%and 25%extracts inhibited SMCs activity without cytotoxicity.25%and 12.5%extracts significantly inhibited the migration of SMCs.After 1 day of culture with 50%extract,the expression of actin microfilament in SMCs was very weak,and SMCs showed a round and shrunken shape with much smaller cell area compared with the control.In addition,after 1 day of culture with 50%extract,reactive oxygen species（ROS）levels significantly enhanced in the SMCs compared with the control.Although the gene expression level of antioxidant catalase（CAT）increased,glutathione peroxidase（GPx1）and superoxide dismutase（SOD1）gene expression levels decreased.The balance between the production of ROS and the clear of antioxidant enzymes was broken,which may lead to the oxidative damage and even fracture of cell DNA,because of cell oxidative stress.In the present work,the DNA damage and fracture of SMCs were presented with DNA trailing and smear at 1 day of culture with 50%extracts,resulting in the increase of p53 gene expression,and thus further increased the expression level of Bax gene,and decreased the expression level of Bcl-2 gene,leading to the up-regulation of Bax/Bcl-2 ratio,and inducing SMCs apoptosis.