Research On The Anti-coagulation And Anti-calcification Technology And Performance Of Valve Material

Background:Mechanical valves are the most widely used type of artificial heart valve due to their long-term durability.However,their hemodynamic and anticoagulant properties are inferior to those of bioprosthetic valves,making them more susceptible to thrombosis and requiring lifelong anticoagulation therapy.Therefore,improving the anticoagulant properties of mechanical valves is of great significance.Methods:In this study,we used laser etching technology to construct optimized grooved patterns on the surface of pyrolytic carbon material of mechanical valves,and evaluated the fluid dynamic characteristics of patterned mechanical valves before and after the patterned modification in an in vitro simulated circulatory system for two valve sizes of 23 mm and 29 mm.In order to further investigate the effect of surface patterning on the anticoagulant properties of mechanical valves,patterned mechanical valves were implanted in the sheep aortic position with domestically produced bileaflet mechanical valves as the control group,and their safety and anticoagulant properties were observed.Hematological and ultrasound examinations were performed before surgery,7 days after surgery,and 1,3,and 6 months after surgery.Results:The fluid dynamic indicators of the mechanical valves before and after the patterned modification were in accordance with the general rules:under the same valve size,as the simulated cardiac output increased,the average transvalvular pressure difference and effective orifice area of the valve increased,and the regurgitation percentage decreased.In addition,compared with the unpatterned mechanical valves,the patterned construction can change the flow field passing through the valve leaflet surface.In vivo implantation experiments in large animals showed that all experimental animals survived until the 6-month sampling node,and hematological and ultrasound reports showed no significant impact of the patterned mechanical valves on the health of the sheep.The results also showed that the grooved patterns constructed on the surface of the mechanical valve can effectively reduce the formation of attachments and thus reduce the risk of mechanical valve thrombosis.Conclusion:Based on the above experimental results,we believe that surface patterning of mechanical valves is an effective method to improve their anticoagulant properties.In clinical applications,surface patterning technology can be used to avoid thrombotic complications after mechanical valve implantation,providing new ideas and methods for the clinical application of mechanical valves.Background and PurposeValvular heart disease is one of the leading causes of morbidity and mortality worldwide,and valve replacement surgery is an effective treatment.Currently,the two commonly used types of artificial heart valves are mechanical and biological,and the choice between them remains a dilemma.The main advantage of mechanical valves is their long-term durability,but they require lifelong anticoagulation therapy to prevent the risk of thrombosis.Biological valves have excellent biocompatibility and hemodynamic performance,with less thrombosis and no need for lifelong anticoagulation,but their durability is reduced due to the degradation of biological materials,mainly caused by calcification.Although many studies have provided different anti-calcification methods,most of them target a single calcification target,and the treated tissue is still preserved in glutaraldehyde,exposing it to an environment that can easily cause calcification again.Our study aims to select the optimal combination treatment method for anti-calcification based on the existing calcification mechanism,by removing calcification factors from multiple dimensions and using aldehyde-free preservation techniques.MethodsThe cell membrane of bovine pericardium biological material was destroyed by treatment with an isotonic solution,and then further decellularized by adding surfactant Triton X-100,sodium deoxycholate(SD),and sodium dodecyl sulfate(SDS)to remove the heterologous antigen of biological material.The tissue was then neutralized with a mixed soIution of saturated sodium bisulfite(SBS)and sodium borohydride with ethanolamine(capping reagent)to remove the unbound aldehyde groups.Finally,the anticalcification treated biological material was stored in glycerol at different concentrations,and a total of 12 treatment combinations were divided.Conventional Glut fixation was used as the control group.Uniaxial tensile tests were performed to evaluate the biomechanical properties of the materials,and a subcutaneous implantation model was established in rats.The implanted samples were collected at 21 and 60 days after surgery,and the calcium ion content was measured using inductively coupled plasma.The morphology of the bovine pericardial cells and matrix fibers was evaluated by histological staining with hematoxylin and eosin.ResultsCompared with the Glut group,the results of the rat subcutaneous implantation experiment at 21 and 60 days showed that all 12 anti-calcification methods significantly reduced the tissue calcification level.Among them,the anti-calcification effect of Triton X-100 decellularization group with capping reagent neutralization and 75%glycerol storage was the lowest,while in the SD decellularization group,the anti-calcification effect of SBS neutralization and 100%glycerol storage was the lowest,and in the SDS decellularization group,the anti-calcification effect of capping reagent neutralization and 75%glycerol storage was the lowest.The results of the uniaxial tensile test showed a slight decrease in the tensile strength and elongation at break of the experimental group,but it did not significantly affect the tissue’s biomechanical properties.The histological staining results showed that the tissue treated with Glut contained a large number of cell nuclei,most of which disappeared after decellularization with Triton X-100,while the tissue treated with SD and SDS still had visible cell nuclei after decellularization.ConclusionThis study proposes a new anti-calcification treatment method,which achieves this through reducing phospholipids,neutralizing residual aldehydes,and storing glycerol in three aspects.Eventually,three combinations of treatment processes with good anticalcification effects were screened out,namely:0.5%Triton X-100+capping agent+75%glycerol,1%SD+SBS+100%glycerol,and 1%SDS+capping agent+75%glycerol.These treatment methods are expected to improve tissue durability and play an important role in clinical applications.In addition,these treatment methods provide new ideas and methods for further research in related fields and have broad significance and application prospects.ObjectiveBased on the above research results,it is expected that the anti-calcification treatment method that reduces phospholipids,neutralizes aldehyde groups,and stores glycerol combinations can improve the clinical durability of bovine pericardium and prolong the lifespan of bioprosthetic heart valves.However,previous studies only examined the anticalcification performance,mechanical properties,and matrix structure of the treated tissue,and did not further explore the physicochemical characterization and biocompatibility of this method.Therefore,this section of the research conducts a more comprehensive study on the three selected anti-calcification methods with good effectiveness to provide biocompatible materials that meet safety requirements for clinical trials.Methods1、Fresh bovine pericardium is treated with Glut fixation,TX-DNG,SD-DNG,and SDSDNG to obtain experimental materials from different groups.The physicochemical characterization is evaluated through water content determination and Fourier transform infrared spectroscopy detection.2、The fiber and cell structures in the tissue are observed using scanning electron microscopy and transmission electron microscopy.3、The biocompatibility of the bovine pericardium treated by different groups is evaluated through in vitro analysis,including cell compatibility and blood compatibility.Cell compatibility is evaluated through cell toxicity experiments,while blood compatibility includes four coagulation tests,in vitro dynamic coagulation analysis of thrombosis formation,in vitro hemolysis evaluation,platelet adhesion analysis of platelets,and immunological testing.4、A rat subcutaneous implantation model is used for 21 and 60 days.The tissue calcification level is analyzed qualitatively and quantitatively through Von Kossa staining and calcium content detection.The inflammation reaction after the implantation of the samples in rats’ subcutaneous tissue is evaluated through HE staining.5、In vitro pulsatile flow test was performed to compare the hemodynamic parameters of different groups of bioprosthetic valves.6、To comprehensively evaluate its biocompatibility,each group of bovine pericardium is sewn into a bioprosthetic heart valve and implanted in sheep through mitral valve replacement surgery for in vivo evaluation.Hematological conditions and echocardiography are tested to assess its clinical safety and practicality.ResearchResults1、The water content of the SD-DNG group decreased significantly compared to the control group after treatment,and the appearance remained flat after drying to the dry weight.There was no statistical difference in water content between the TX-DNG,SDS-DNG groups and the Glut group.Fourier transform infrared spectroscopy showed that all experimental groups maintained the 5 characteristic absorption peaks of collagen fibers.2、Electron microscopy results showed that the TX-DNG,SD-DNG,and SDS-DNG groups removed a large amount of cellular structures and soluble protein components from the tissue compared to the Glut group.The fiber structures of the TX-DNG and SD-DNG groups were compact and aligned,while the fiber structure composition of the SDS-DNG group showed some degree of disorder.The fiber distance of the SD-DNG group was closer.3、Cell toxicity results showed that with prolonged culture time,the Glut group had a large number of cell deaths,while the SD-DNG group maintained a relatively high cell growth rate.The cell growth status of the TX-DNG and SDS-DNG groups worsened.The blood compatibility results showed that the anti-calcification treatment group did not affect the material’s coagulation function,and the hemolysis rate was less than 5%,which meets international standards.It did not activate blood platelets or immune reactions.4、The HE staining results of the bovine pericardium implanted for 21 and 60 days showed no obvious inflammatory reaction in the experimental group.The Von Kossa staining results and calcium content measurement results were consistent,and the TX-DNG,SDDNG,and SDS-DNG groups significantly reduced calcification.5、The results of pulsatile flow test in vitro showed that all tested valves met the ISO 5840 international standard.Compared with Glut group,the effective orifice area was increased,the regurgitation percentage was decreased,and the mean pressure gradient was decreased in TX-DNG and SD-DNG groups.The effective orifice area and mean pressure gradient in SDS-DNG group were similar to those in Glut group,and the percentage of regurgitation was decreased.6、After three months of implantation in sheep,the blood and ultrasound indicators of the TX-DNG,SD-DNG,and SDS-DNG valve groups were within the normal physiological range.ConclusionThis experiment comprehensively evaluated the physicochemical and biological properties of bovine pericardium after anti-calcification treatment.The results showed that the modified treatment did not destroy the three-stranded helical structure characteristic of bovine pericardium collagen and had excellent anti-calcification properties.In vitro experiments demonstrated good blood compatibility of each group.In vivo experiments in large animal mitral valve replacement models showed that the anti-calcification-treated biological valves exhibited good safety and practicality.Compared with the TX-DNG and SDS-DNG groups,the SD-DNG group’s fiber structure was more tightly cross-linked,had better cell compatibility,and had greater clinical application value.