Experimental Studies Of Swimbladder-a Novel Biomaterial For Cardiovascular Surgery

Biomaterials are widely used in cardiovascular surgery, among which the xenogaftheart valve and vascular graft are the most popular. Due to the excellent blood flow, lowincidence of thrombosis and embolism and high quality of life after surgery, xenograftvalves are of growing interest as the popular therapeutic procedure especially in thedeveloped countries. But the relatively high occurence of early mechanical failure andcalcification because of the property of the pericardium material have limited theapplication. Big-caliber vascular grafts has been widely applied in the clinical practice andbeen proved to have good performance, However, small-caliber vascular grafts (diameter <6mm) has not been widely accepted because of the low long-term patency caused by itspoor hemocompatability. So people are working on improving the property of biomaterialsor exploring novel biomaterials.The aim of this dissertation is to test the property ofnovel cardiovascular biomaterial-carT’W W[MQbPaddeV baWed SR XKVee dMffeVeRX aWTecXWwhich are physical performance、in-vitro biocompatability and in-vivo trials of rat..Part1Study on physical properties of swimbladderObjective:1、Investigate the biomechanical properties and thermal stability of the swimbladder and analyse the influence of crosslinking with0.625%glutaraldeyde.Methods:1.FaMPYVe$PQaRWM WXVaMR (f), faMPYVe&aYcK] WXVeWW (f) and peak elasticmodulus (Mp) were measured using the uniaxial tensile test.then compare the difference incircumferential and longitudinal direction of fresh and glut-fixed swimbladder, differencebetween glut-fixed swimbladder and bovine pericardium. Describe the fiber alignmentaccording to the mechanical index and the results of VG(Victoria blue-van Gieson)stainand transmission electron microscope in preliminary experiments.2. Thermal denaturationtemperatures of the fresh and glutaraldehyde-treated swim bladder were measured using adifferential scanning calorimeter. The effect of crosslinking with glutaraldehyde-treatmenton the thermal stability of swim bladder was characterized.Result:1. uniaxial tensile test:(1)No matter fresh or glut-fixed，the index off、fandMpwere higher in circumferential direction(circ) than in longitudinal(long) direction.(2)fand Mpin circ direction of glut-fixed swimbladder were higher than the index of samedirection in fresh one, there was no statistic difference inf.(3)fof glut-fixed swimbladderin long direction is apparently higher than the one of the same direction in freshswimbladder, but there was no difference infand Mp.(4)fand Mpof glut-fixed bovine pericardium are apparently higher than the index of fresh pericardium,but no difference inf.(5)fand Mpof swimbladder in circ and long direction are apparently lower thanTeVMcaVdMYQ, bYX XKe VeWYPX Sffare opposite.2. Scan differential calorimeter: The thermaldenaturation temperature of fresh swim bladder is higher than human body temperature.Meanwhile, the thermal denaturation temperature of glutaraldehyde-treated swim bladderis significantly higher than that of fresh swim bladder, which indicating a better thermalstability.Conclusion:1、Fiber alignment of swimbladder are more uniform than pericardium, thecollagen fiber are mainly aligned in circ direction, and elastic fiber are aligned both in twodirection.2、Stiffness of glut-fixed swimbladder is lower than pericardium, and better inelongation performance which is more similar with human heart valve.3.Thermal stabilityof swimbladder， especially after crosslinking, can achieve the human temperaturerequirement.Part2In vitro biocompatibility evaluation of swimbladderObjective:According to international standard for medical devices biocompatibilityevaluation-ISO10993, we study on the in vitro cytotoxicity and hemocompatability ofswimbladder.Methods:1、In vitro cytotoxicity test: Extract swimbladder with MEMmedium(10%FBS). Test the cytotoxicity by MTT method.2、In vitro platelet activationtest：extract swimbladder with human blood, calculate the activated platelet by CD62pcontent.3、In vitro hemolysis test：Extract the swimbladder with sterile0.9%NaCl, mix theextract and blood, then test the hemolytic rate. Bovine pericardium are applied as control inall the test above.Result:1、Cell vitality of L929after contact with100%fresh swimbladder and100%fresh pericardium extract were85.7%and94.2%respectively. Both of the two materialsshowed no cytotoxicity to L929cells.2、CD62p in the group of glut-fixed swimbladderand bovine pericardium are0.94±0.27%and1.73±0.35%respectively. Both of themshowed no influence to the thrombus formation in vitro.3、Hemolytic rate of glut-fixedswimbladder and pericardium were0.2%and0.6%respectively. Both showed no influencein hemolysis.Conclusion: According to in vitro cytotoxicity test and hemocompatability test(plateletactivation test and hemolysis test), swimbladder performed good biocompatibility whichprovided foundation for in vivo test. Part3Calcification test of subcutaneous implantedswimbladder in ratsObjective: Qualitatively and quantitatively analyse the calcification degree ofswimbladder and bovine pericardium in rat subcutaneous implantation model,then comparewith bovine pericardium.Methods:1、According to glut-DSC anticalcification treatment which has been proved tobe effectivt in pericardium material(Edwards Lifesciences Corporation, Patent No.:US6214054B1),we treated the swimbladder and pericardium, then implanted the twomaterials in young rat subcutaneously. Explanted them in7、21、56days, then qualitativelyand quantitatively analysed the calcification degree and described the relationship withimmune response.2、Qualitative analysis of calcification: Study on the calcium depositfrom morphology and biology aspects.3、Quantitative analysis of calcification: Calculatethe calcium content with ICP test.4、 Immunohistochemistry stain： We did theImmunohistochemistry stain for CD68＋macrophage and CD8＋T cell then described therelationship between the immune response and calcification degree.Rsults:1、Caicification degree of swimbladder in every stage were lighter thanpericardium, fibers of swimbladder were intact and aligned uniformly except for thedamage in late stage because of calcification. However, calcium deposition and fiberdamage were found even in the7th day of pericardium. Calcium content was higher inpericardium than swimbladder eapecially in21day stage.2、Immunohistochemistry stainshowed no inflammatory reaction among swimbladder tissue but CD68＋macrophage andCD8＋T cell could been seen in pericardium tissue in every stage.Conclusion：After anti-calcification treatment of DSC method, bovine pericardiumcalcified severely than swimbladder in rat subcutaneous model. Swimbladder has betteranti-calcification property.Part4Novel biomaterial small-caliber vasculargraft-experiment in rat modelObjective: Replace abdominal aorta of rat with small-caliber vascular graft made byswimbladder and bovine pericardium then compare the outcome and hemocompatabilitybetween them.Methods: Replace abdominal aorta of rat with small-caliber vascular graft made byswimbladder and bovine pericardium according to ISO10993standard. Explant them at30、60day stage and compare the following aspects.1: compare the patency and aneurysm formation by320row CT.2、Sduty on the endothelialization with scanning electionmicroscope then compare the endothelialization and the degree of intimal intimalhyperplasia by HE、VB stain and Immunohistochemistry stain with factorⅧ antigen andα-SMA antigen.3、Study on the calcification by X-ray、Micro CT and Von Kossa stain..Results:1、The patency of swimbladder graft in two stage was both100%,with thepatency of pericardium was16.67%in both time.2、Fibers in swimbladder was intact inevery stage，new elasin fiber content were rich in swimbladder. Endothelialization wasfaster and better in swimbladder. Intimal heperplasia could be seen in every anastomoticarea. Intimal growth was not apparently different in30and60stage swimbladder, but wasapparent between two different mateials. Cavity was almost occluded in60day openpericardial vascular graft.3、No calcification was observed in swimbladder and early stageof pericardium. However, in60day explant pericardium sample, calcium deposition andfiber destrction could been observed.Conclusion:Swimbladder has better hemocompatability than pericardium.Small-caliber vascular graft made of swimbladder has excellent two month patency.ConclusionIt was showed in this study that ultimate strength of swimbladder was lower than bovinepericardium,its flexibility and rigidity was better than bovine pericardium.Biocompatability test of swimbladder showed that its cytotoxicity,thrombogenicity andhemolytic capability were satisfactory. Calcification test in rat subcutaneously implantmodel showed calcification of swimbladder underwent anti-calcification procedure wasobviously lower than bovine pericardium with the same procedure. Small-caliber vasculargraft made of swimbladder was studied in rat abdominal aorta transplantation model, theresult showed that in30and60period, the patency, endothelialization capability wassatisfactory and no aneurysm appeared.