| Heart valvular disease(HVD)is the main cause of morbidity and mortality worldwide,which is related to population aging and congenital dysfunction.1 Statistics show that there were about 400,000 patients with heart valve replacement worldwide in 2016 and with the aging of the population,the total number of patients is expected to reach 850,000 in 2050.At present,valve replacement is the most effective solution for severe valve dysfunction.Currently,about 55% of valve replacement operations worldwide performed annually use mechanical valves,while the remaining 45% adopt biological valves.Mechanical valves are mostly made of metal materials and require permanent anticoagulation after the operation,which are not suitable for high-risk patients such as the elderly.Biological heart valves(BHVs)include bovine,porcine or equine pericardium,bovine jugular vein or porcine heart valves.Compared with mechanical valves,BHVs boast the advantages of low thrombosis rate and no need for lifelong anticoagulation.However,there are some common defects such as poor fatigue resistance and calcification.There are roughly 1.5 million children with congenital heart disease in China,and nearly 200,000 newborns suffer from this disease every year.Among them,about 30% suffer from complicated congenital heart diseases,and about 5%-10% need to reconstruct the pulmonary artery to achieve a radical cure.There is still a lack of ideal materials and products for pulmonary artery reconstruction.Artificial valved conduits can be used to reconstruct the pulmonary artery to save children's lives and improve surgical results.Common biological tissue materials mainly include autologous pericardium,heterogeneous pericardium,heterogeneous aortic valve.Amid the improvement of material life and the aged tendency of the population,the average age of patients undergoing valve surgery tends to increase gradually.Transcatheter Aortic Valve Implantation(TAVI)has become a new choice for the treatment of heart valve diseases,enjoying the merits of no thoracotomy,no need for extracorporeal circulation,minimal invasion and fast recovery,thus the application of biological heart valves become more and more extensive.Due to the long-term poor durability of interventional valves,they are currently only used in elderly critically ill patients,and their surface properties need further enhancement.Bovine pericardium,as an implant material for cardiovascular surgical repair,has experienced more than 40 years of clinical application and appears to be more convenient,economical,safe and available than other artificial repair materials.In recent years,biological valves made of chemically modified bovine pericardial tissue materials have become the mainstream product regarding artificial biological valves due to their excellent durability.In view of our achievements in the design of a new generation of improved stentless porcine valves and the research on anti-calcification biological heart valves as well as the jointly development of a new type of complete biological pulmonary valved conduit and a complete biological aortic valve,now we need to explore experimental methods to observe the anti-calcification effect,physicochemical properties and histocompatibility of the new bovine pericardial biomaterial.It is necessary to establish a feasible experimental animal model and verify the safety and effectiveness of the application of the new type of complete biological conduit and complete biological valve after implantation in experimental animals,so as to provide a basis for the further clinical application.Objective:The new biological bovine pericardium materials,dealed with the cells,glutaraldehyde(GA)crosslinking curing and 2.3 butanediol(BD)anti-calcification modification,designed with bionic appearance and structure,and was covered with a flexible elastic valve frame.Through the implantation of the pulmonary valve and aortic valve in sheep,its effectiveness and safety can be observed.1.Anticalcification effect,physical and chemical properties and blood compatibility of the new complete biomaterial.2.A large animal experiment of the new complete biological pulmonary valved conduit.3.A large animal experiment of the new complete biological aortic valve.Methods:1.After decellularization,bovine pericardium was cross-linked with glutaraldehyde(GA)and modified with butanediol(BD)to resist calcification respectively,the new biomaterial was subjected to ninhydrin test,collagenase digestion test,plasma protein adsorption test and platelet adhesion test to detect its effects of collagen cross-linking degree and blood compatibility.Acute systemic toxicity test,subacute toxicity test,genetic toxicity test and mechanical property test of bovine pericardial biomaterials were respectively determined to observe the physical and chemical properties of the new bovine pericardial biomaterials.After embedding biomaterials in rats for 8 weeks,they were taken out for HE staining and alizarin red staining to observe the calcification degree of the biomaterial.2.The new type of complete biological pulmonary valved conduit is composed of bovine pericardium treated with decellularization and anti-calcification modification with GA and BD,and proximal and distal conduits based on bionic structural design.The proximal conduit is sewn with polyester suture with a trileaflet heart valve.In this experiment,we planned to perform pulmonary valve replacement on sheep under cardiopulmonary bypass with a beating heart.Fourteen sheep have been implanted with valved conduits.On 1,3,7,30,90 and 180 days after the operation,we conducted the observation respectively.The hemodynamic performance(blood flow velocity,regurgitation area,and pressure gradient across the valve)of the implanted animals were observed,and the structural changes(whether the valve was opened and closed well)were evaluated by ultrasound.Other items include: adverse events after implantation(death,infection,abnormal behavior);inflammatory reaction around the implant: adhesion,infection and necrosis of surrounding tissues;number of leukocytes,lymphocytes,cells,neutrophils,etc;pathological changes of artificial biological conduit and surrounding tissues;thrombosis: thrombosis of valved conduit and organs;hemolytic reaction: the changes of red blood cell morphology and hemoglobin quantity,etc;organ lesions: morphological changes of heart,lung,liver,kidney,spleen,etc;calcification of valve leaflets and conduits and determination of calcium content.Thus,we evaluated the safety and effectiveness of the new type of complete biological pulmonary valved conduit.3.The new complete biological aortic valve adopts bovine pericardial tissue.Through decellularization treatment,GA and BD anti-calcification modification treatment,it is manufactured by adopting flexible elastic valve frame and bionic structure design and sewing process.In this experiment,sheep have undergone aortic valve replacement under extracorporeal circulation.10 cases have been implanted with the complete biological aortic valve and 1 case of control group has adopted the biological heart valve(USA,Edward).On 1,3,7,30,90,180 days after the operation,we have conducted the observation.Observation targets: effectiveness after implantation: hemodynamic performance after implantation(blood flow velocity,regurgitation area,pressure gradient across valve);structural changes after implantation(whether the valve was opened and closed well).Evaluation indicators after implantation: adverse events: death,infection and abnormal behavior of all sheep;inflammatory reaction around the implant: whether there were adhesion,infection and necrosis between the artificial biological valve and the surrounding tissues;number of leukocytes,lymphocytes,cells,neutrophils,etc;pathological changes of artificial biological valve and surrounding tissues;thrombosis: observed the changes of blood coagulation and investigated whether there was thrombosis in valve and main organs;hemolytic reaction: observed the changes of red blood cell morphology and hemoglobin quantity,etc;organ lesions: morphological changes of heart,lung,liver,kidney,spleen,etc.;calcification of valve ring and leaflet and determination of calcium content.Thus,we evaluated the safety and effectiveness of the new type of complete biological aortic valve.Results:1.After the bovine pericardium has undergone decellularization and GA cross-linking and anti-calcification modification by BD,There was no obvious abnormality in acute systemic toxicity test,subacute toxicity test and genetic toxicity test.Mechanical properties showed that the elongation at break and tensile load of bovine pericardium in GA+BD group increased compared with that in GA group,and there was no significant difference in tensile load.Compared with the untreated group,the value of ninhydrin in GA+BD group decreased significantly,and that in GA+BD group decreased further.After GA treatment,the undigestibility of bovine pericardium was significantly increased compared with that of the control group.After GA+ BD treatment,the undigestibility was further increased and its anti-digestibility was further enhanced.The protein adsorption rate of GA group was significantly higher than that of control group,and was lower than that of control group after BD treatment.The platelet adhesion rate of GA+BC group was lower than that of control group,but there was no significant difference between the two groups.The results of embedding experiment showed that no obvious calcification was found in the new biomaterials,while the tissue calcification was obvious in the control group2.A total of 14 sheep pulmonary arteries were implanted with the complete bioprosthetic pulmonary valved conduit in situ,without accidental death and serious complications.Twelve of them were executed according to the plan,and two were still alive and healthy.After implantation,the valve opened and closed well,and there was no serious stenosis or incomplete closure during the experiment.There was no significant change in the percentage of pulmonary valve regurgitation measured by transthoracic echocardiography(TTE)in surviving sheep.The central venous pressure(CVP),mean aortic pressure(MAP)and other indicators were not significantly different from the preoperative measurements.Compared with preoperative data,there was no significant change in the pressure gradient across the valve of the pulmonary artery valve after implantation.The calcium content of all valve leaflets and vascular tissues was less than 10 ?g/mg except 2 cases with elevated calcium content.No obvious thrombosis or thromboembolism was found in all organs during the autopsy.No obvious adverse events such as blood cell damage,liver and kidney injury,inflammatory reaction and the like were found in laboratory examination and autopsy during follow-up.Histopathological examination showed no obvious abnormality.3.Nine complete biological aortic valves and one control biological aortic valve were implanted in situ.Animals were implanted surgically,fed after operation and collected data according to the experimental scheme.One sheep in the experimental group died unexpectedly on the 167 th day.The sheep in the control group died unexpectedly on the 172 nd day.The rest of the animals survived normally to the expected time,and were executed,data collected and autopsied as planned.After implantation,the valve opened and closed well,and there was no serious stenosis or insufficiency in the whole experiment.There was no significant difference regarding hemodynamic indexes such as regurgitation percentage of viable,effective orifice area(EOA),aortic systolic pressure(ASP),diastolic aortic pressure(DAP),mean aortic pressure(MAP),left ventricular systolic pressure(LVSP),and left ventricular end diastolic pressure(LVEDP).The average calcium content of the artificial complete biological heart valve leaflets implanted into experimental animals was 2.01±1.02?g/mg.The highest one was 66.81?g/mg.The calcium content in the control group was 2.20?g/mg.The average calcium content of the artificial complete biological heart valve ring implanted into experimental animals was 2.53±1.75?g/mg.The highest calcium content was 43.57?g/mg in the experimental group and 71.20?g/mg in the control group.No obvious thrombosis or thromboembolism was found in all organs during the autopsy.No obvious adverse events such as blood cell damage,liver and kidney injury,inflammatory reaction and the like were found in laboratory examination and autopsy during follow-up.Conclusion:1.This experiment aimed at a new type of complete biological bovine pericardial material treated by decellularization and GA cross-linking and anti-calcification modification by BD,There was no obvious abnormality in acute systemic toxicity test,subacute toxicity test and genetic toxicity test.Through the ninhydrin experiment and collagenase digestion experiment,the collagen crosslinking degree of decellularized scaffold material can be improved.The plasma protein adsorption experiment and platelet adhesion experiment verified that the treatment can improve the blood compatibility of bovine pericardial biomaterials.By observing HE staining and alizarin red staining after embedding in rats,the calcification degree of bovine pericardial biomaterial can be effectively reduced.GAand BD treatment can further improve the physical and chemical properties of bovine pericardium materials,which is conducive to improving blood compatibility and slowing calcification.Meanwhile,it provides an important theoretical basis for the application of GAand BD treatment methods in the research of tissue engineering valve stent materials.2.The sheep model and experimental method of beating pulmonary artery replacement under extracorporeal circulation have been established in this experiment,which can be used for experimental research on a new type of anti-calcification pulmonary artery valved conduit,and also lays a foundation for the research and development of a new type of pulmonary artery valved interventional system and in vivo research.There were no obvious complications and adverse events after the implantation of the new type of complete biological pulmonary valved conduit,and it delivered an excellent anti-calcification effect.The follow-up data of 6 months after the operation showed that the animal implantation of the new type of complete biological pulmonary valved conduit is safe and meets the requirements of safety and effectiveness of medical device implantation,and the long-term post-implantation effect needs further observation.3.The sheep model and experimental method of aortic valve replacement under extracorporeal circulation have been established in this experiment,which can be used for experimental research of new anti-calcification valve,and also lays a foundation for research and development of new TAVI interventional valve system and in vivo research.There were no obvious complications and adverse events after the implantation of the new type of complete biological aortic valve.One case died unexpectedly in the experimental group(maybe caused by hemorrhage).No obvious thrombosis and thromboembolism was found in all organs in the autopsy of those experimental animals.The results showed that the product delivered excellent blood compatibility with conventional anticoagulation as well as anti-calcification effect except for some focal calcification.The follow-up data of 6 months after the operation showed that the animal implantation of the new type of complete biological aortic valve is safe and meets the requirements of safety and effectiveness of medical device implantation,and the long-term post-implantation effect needs further observation. |
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