Exploration And Application Of Novel Technology For Bovine Pericardial Immunogenicity Removal

Background:Bioprosthetic heart valves are clinically preferred beyond mechanical valves due to their favorable hemocompatibility and hemodynamic properties.However,the inherent immunogenicity of xenograft biomaterials often affects the durability of bioprosthetic heart valves,removes the cellular components and extracellular antigens in tissues,and reduces the immunogenicity of materials,which are necessary links in the treatment of modern animal-derived materials.However,current methods are insufficient in eliminating immunogenicity from the biomaterials,necessitating the exploration of novel techniques.At present,the pericardium or aortic valve widely used in clinical practice,and the bovine jugular valve duct,are treated with glutaraldehyde.The long-term results of clinical application have shown that calcification is one of the main reasons for the failure of these products.Research suggests that immune response is closely related to calcification of glutaraldehyde crosslinked biological valves.Improving the anti-calcification performance of glutaraldehyde crosslinked biological valves by removing immunogenicity is currently an important direction of research on biological valves.Objective:To improve the anti-calcification property of glutaraldehyde(GA)crosslinked bovine pericardium by constructing a compound immunogenicity removal scheme of the zwitterion detergent Amidosulfobetaine(ASB16)and sodium hydroxide(NaOH).To explore the mechanism of glutaraldehyde(GA)calcification in bovine pericardium.Methods:Using bovine pericardium as the research material,a novel removel immunogenicity technique was constructed by exploring the optimal concentrations of ASB16 and NaOH;Evaluate the efficiency of cell component removal in different schemes by comparing DAPI staining and DNA quantification with traditional acellular detergent Triton X-100;ELISA and Western Blot methods were used to evaluate the removal efficiency of different methods for bovine pericardial antigens and transplantation related antigens;Histological staining and quantitative detection of extracellular matrix components were used to evaluate the effects of different immunogenic removal methods on the structure of bovine pericardium;Evaluate the effects of different immunogenicity removal schemes on the biomechanical properties and thermal stability of bovine pericardium through uniaxial tensile and thermal shrinkage experiments.Evaluate the biocompatibility(immune inflammatory response,anti-degradation ability,and calcification)of different immunogenicity removal schemes using a rat subcutaneous embedding model to verify the excellent immunogenicity removal performance of the new immunogenicity removal scheme(ASB16/NaOH)on bovine pericardium.Construct ASB16/GA bovine pericardium and compare it with GA cross-linked bovine pericardium and Triton X-100/GA bovine pericardium;The structure and mechanical properties of its extracellular matrix and its ability to resist enzymatic hydrolysis were explored;Evaluate the inflammatory response and calcium content through subcutaneous embedding experiments in rats to evaluate the anti-calcification performance of a novel removal immunogenic regimen on glutaraldehyde crosslinked bovine pericardium;Use q PCR to detect the m RNA expression levels of inflammatory factors and calcification factors,and preliminarily explore the relevant mechanisms of calcification.Results:The efficiency of the novel immunogenic removal technology(ASB16/NaOH)in removing cell components was better than that of the traditional detergent(Triton X-100);ASB16/NaOH can remove most of the antigen components,significantly superior to the removal of Triton X-100 and the blocking effect of glutaraldehyde;Using ASB16/NaOH method will not significantly damage the original extracellular matrix structure and components of bovine pericardium,and can have good biomechanical properties and thermal stability;In vivo experiments have shown that ASB16/NaOH bovine pericardium does not cause severe humoral immune and inflammatory reactions in the host at different time points,and has better anti degradation ability compared to native bovine pericardium and Triton X-100 bovine pericardium;ASB16/NaOH bovine pericardium also has better regeneration and anti calcification ability.In order to improve glutaraldehyde bovine pericardium,we constructed(ASB16/NaOH)/GA bovine pericardium(Abbreviated as ASB16/NaOH);ASB16/GA bovine pericardium has excellent mechanical properties and anti enzymatic ability.There was no significant difference compared with GA bovine pericardium;ASB16/GA bovine pericardium can effectively maintain extracellular matrix components without additional cytotoxicity;The subcutaneous embedding results of rats showed that ASB16/GA bovine pericardium has good anti-inflammatory and anti-calcification properties,which can reduce the calcium content of GA bovine pericardium by more than 50%.The m RNA expression of inflammatory factors such as IL-17,IL-6 and TNF-α in ASB16/GA was lower than that in the Triton X-100/GA and GA group.The expression of NF-κB,a calcification related pathway downstream of IL-17,was also significantly lower in ASB16/GA bovine pericardium than in the other two groups.Finally,the detection of NF-κB mediated calcium related protein expression in ASB16/GA bovine pericardium also proved that the expression of ASB16/GA bovine pericardium was lower than that in GA bovine pericardium and Triton X-100/GA bovine pericardium.Conclusion:The use of ASB16 and NaOH composite immunogenicity method can completely remove the cells and antigen components of bovine pericardium,while maintaining the integrity of extracellular matrix(ECM)and improving the biocompatibility of the material.It is a promising technology with ideal clinical application prospects.Using ASB16/NaOH to treat glutaraldehyde bovine pericardium can reduce the inflammatory reaction of glutaraldehyde bovine pericardium and increase its anti-calcification performance,which is expected to improve the durability of biological valves in clinical use.Preliminary exploration of the mechanism by which IL-17 regulates the downstream NF-κB pathway and mediates the expression of calcium related proteins leading to calcification provides a new idea for the future regulation of biological valve calcification prevention.