Bioprosthetic Heart Valve Calcification Research Articles

Biomimetic-modified bioprosthetic heart valves with Cysteine-Alanine-Glycine peptide for anti-thrombotic, endothelialization and anti-calcification

In recent years, bioprosthetic heart valves (BHVs) prepared by cross-linking porcine or bovine pericardium with glutaraldehyde (Glut) have received widespread attention due to their excellent hemocompatibility and hydrodynamic properties. However, the failure of BHVs induced by thrombosis and difficulty in endothelialization still exists in clinical practice. Improving the biocompatibility and endothelialization potential of BHVs is conducive to promoting their anti-thrombosis properties and prolonging their service life. Herein, Cysteine-Alanine-Glycine (CAG) peptide was introduced into the biomimetic BHV materials modified by 2-methacryloyloxyethyl phosphorylcholine (MPC) to improve their anti-thrombosis and promoting-endothelialization performances. MPC can improve the anti-adsorption performance of BHV materials, as well as, CAG contributes to the adhesion and proliferation of endothelial cells on the surface of BHV materials. The results of experiments showed that the biomimetic modification strategy with MPC and CAG reduce the thrombosis of BHV materials and improve their endothelialization in vitro. More importantly, the calcification of BHV significantly reduced by inhibiting the expression of M1 macrophage-related factors (IL-6, iNOS) and promoting the expression of M2 macrophage-related factors (IL-10, CD206). We believe that the valve-modified strategy is expected to provide effective solutions to clinical valve problems.

Biomimetic-modified bioprosthetic heart valves with controlled release of glycyrrhizin acid mediated by the inflammatory microenvironment for anti-thrombotic, anti-inflammatory, and anti-calcification

Artificial heart valve replacement is optimum for treating severe valvular heart disease (VHD). Bioprosthetic heart valves (BHVs) have received widespread attention from patients and doctors due to their good hydrodynamics, hemocompatibility, and minimally invasive implantation. However, the inflammatory microenvironment caused by cytotoxicity of residual aldehyde groups, the immunogenicity of heterogeneous tissue, and implantation damage, have led to problems such as endothelialization difficulties, leaflet thrombosis, and calcification of BHVs, limiting service life and applicability to the population of BHVs. In this study, we introduced chondroitin sulfate (CS) and p-aminophenyl boronic acid-glycyrrhizin acid (PBA-GA) into glutaraldehyde (Glut) cross-linked BHV material, thereby preparing a bionic-modified BHV material (CS-GA-PP), which can remould the inflammatory microenvironment at the injured site. CS can form an extracellular matrix (ECM)-like structure, reducing the immunogenicity and thrombosis of BHVs, at the same time, GA can be released under the inflammatory microenvironment, exerting an anti-inflammatory effect, reducing inflammation and calcification. CS-GA-PP maintained the great structural stability and mechanical properties brought about by cross-linking with Glut. In addition, the strategy significantly improved the anti-thrombotic, anti-inflammatory, endothelialization, and anti-calcification properties of the BHV material. It is expected to provide a new idea and a solution for the intelligent biomedical design of Glut cross-linked BHV material.

Abstract 13187: Inhibition of Advanced Glycation End Products Formation and Serum Protein Infiltration in Bioprosthetic Heart Valves: Investigations of Anti-Glycation Agents

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated bovine pericardium (BP), are the most commonly used heart valve replacements. However, BHV durability is limited by structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition together with serum protein infiltration. This study investigated, for the first time, the application of anti-glycation agents as a way to mitigate AGE-related structural degeneration of BHV. Hypothesis: We investigated the hypothesis that anti-AGE agents (Aminoguanidine (AG), Pyridoxamine (PYR), and N-Acetylcysteine (NAC)) could mitigate BP glycation in model studies, both in vitro and in vivo. Methods: To model AGE accumulation in BHV, we used well-established accelerated in vitro glycation models using BP incubation with or without inhibitors. Incorporation of AGE in BP was monitored using radiolabeled glycation precursors. Changes in collagen structure were examined by two photon confocal microscopy and differential scanning calorimetry. In vivo studies used a juvenile rat subdermal BP implantation model with drug administration via drinking water. Endpoints were AGE formation, collagen structure, thermal denaturation temperature, serum albumin uptake, calcification, and serum glycation marker levels. Results: In vitro studies demonstrated that each of these agents significantly inhibited AGE formation in BP. However, after 28-day rat subdermal BP implantation in vivo, only PYR demonstrated significant inhibition of both AGE and serum albumin accumulation per immunostaining. BHV calcification was not mitigated by PYR, AG or NAC. Conclusions: This study is the first to demonstrate the use of AGE inhibitors in mitigating BHV glycation. PYR demonstrated significant efficacy both in vitro and in vivo, mitigating AGE formation and albumin infiltration in BP. Thus, this study provides a rationale for investigating PYR as a possible candidate for improving the durability and biocompatibility of clinically used BHV, providing the solution for this unmet clinical need.

Mechanisms and Drug Therapies of Bioprosthetic Heart Valve Calcification.

Valve replacement is the main therapy for valvular heart disease, in which a diseased valve is replaced by mechanical heart valve (MHV) or bioprosthetic heart valve (BHV). Since the 2000s, BHV surpassed MHV as the leading option of prosthetic valve substitute because of its excellent hemocompatible and hemodynamic properties. However, BHV is apt to structural valve degeneration (SVD), resulting in limited durability. Calcification is the most frequent presentation and the core pathophysiological process of SVD. Understanding the basic mechanisms of BHV calcification is an essential prerequisite to address the limited-durability issues. In this narrative review, we provide a comprehensive summary about the mechanisms of BHV calcification on 1) composition and site of calcifications; 2) material-associated mechanisms; 3) host-associated mechanisms, including immune response and foreign body reaction, oxidative stress, metabolic disorder, and thrombosis. Strategies that target these mechanisms may be explored for novel drug therapy to prevent or delay BHV calcification.

Bisphosphonates as Potential Inhibitors of Calcification in Bioprosthetic Heart Valves (Review).

As early as 50 years ago, bisphosphonates turned from a water treatment agent into one of the most widely used groups of drugs for the treatment of various diseases of calcium metabolism (bone tissue resorption, oncological complications of neurodegenerative diseases and others). Years of research on bisphosphonates have contributed to the understanding of their molecular and cellular pathways of their action. All bisphosphonates have a similar structure and common properties, however, there are obvious chemical, biochemical, and pharmacological differences between them. Each bisphosphonate has its own unique profile. This review summarizes data on the mechanisms of action of bisphosphonates, demonstrates the experience and prospects for their use for the modification of cardiovascular bioprostheses, since the issue of preventing bisphosphonate calcification has not been settled yet.

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1–182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

Screening analysis of proteolytic enzymes and their inhibitors in the leaflets of epoxy-treated bioprosthetic heart valves explanted due to dysfunction

Bioprosthetic heart valves (BHVs) are known for their lower thrombogenicity rates and excellent hemodynamic parameters similar to native valves. However, the lifespan of these medical devices is limited to 15 years due to the structural valve degeneration. One of the mechanisms underlying functional impairment and calcification of BHVs includes proteolytic degradation of biomaterials. However, proteases found in xenogeneic BHVs tissue remain poorly studied. In this study using the dot blot assay, we have performed a screening analysis of proteolytic enzymes and their inhibitors in the leaflets of five BHVs explanted due to their dysfunction. Five aortic valves (AVs) explanted due to calcific aortic valve disease were studied as a comparison group. The results of the study have demonstrated that at least 17 proteases and 19 of their inhibitors can be found in BHVs. In the AVs 20 proteases and 21 their inhibitors were identified. Small quantitative differences were noted between proteomic profiles of the BHVs and AVs. Matrix metalloproteinases (MMPs) were expressed in BHVs and AVs at comparable levels, but the level of tissue inhibitors of metalloproteinases-1/-2 and RECK protein in implant tissues was lower than in natural valves. Probably, excessive activity of MMPs cannot be counterbalanced by their inhibitors in BHVs and therefore MMPs can degrade prosthetic biomaterial. Moreover, the detection of a wide range of proteolytic enzymes and their inhibitors in the degenerated BHVs suggests the existence of several pathophysiological pathways that can lead to structural valve degeneration.

Prevention of the calcification of porcine heart valve leaflets implanted subcutaneously in rats by an interleukin-8 receptors antagonist (SCH527123)

Bioprosthetic heart valve calcification may result from interactions between the host's immune system and the glutaraldehyde (GA)-fixed tissue that makes up the leaflets. We hypothesized that antagonizing interleukin-8 (IL-8) receptors, CXCR-1 and CXCR-2, may prevent the calcification of GA-fixed porcine aortic valves implanted subcutaneously in rats. Three-week-old Sprague Dawlay males were transplanted with 2 aortic valve leaflets isolated from pigs and implanted into the dorsal wall. Four groups of 3–4 rats (6–8 independent leaflets each time) were compared: glutaraldehyde (GA)-free leaflets, GA-fixed leaflets (0.6% GA for 24 h before implantation), GA-fixed leaflets and treatment of rats with 1 mg/kg SCH5217123 (a CXCR1/2 antagonist) injected once a day locally subcutaneously (SC) or intraperitoneally (IP). Fourteen days later, rats underwent blood leukocyte count and CT-scan, before porcine leaflets explantation and histology and biochemistry analyses. As expected, strong calcification of ectopic valves was induced by the GA. However, we observed a significant about 40% decrease in this effect when rats were treated with the SCH527123. These effects were confirmed by CT-scan acquisitions and Alizarin red staining of valves. Besides, GA pre-incubation of valves significantly increased leukocytes count in rat blood, an effect that is significantly prevented by IP SCH527123 (6.9, 16.6 and 6.8 × 10 9 /L in the GA-free, GA and GA + IP SCH527123 groups respectively, P = 0.0129). Immunohistochemistry of ectopic valves highlighted higher infiltration of CD3 (lymphocytes) and CD68 (macrophages) cells in the GA group compared to the GA-free and GA + SCH527123 groups. Therefore, the calcification of GA-fixed porcine aortic valve leaflets, implanted subcutaneously in rats, is significantly prevented by antagonizing IL-8 receptors with SCH527123. This effect might partly result from an inhibition of the GA-induced leukocytes activation and recruitment into the graft tissue.

Sevelamer Attenuates Bioprosthetic Heart Valve Calcification.

Objective: Sevelamer hydrochloride is a phosphate binder used to treat hyperphosphatemia in chronic kidney disease (CKD) patients that can reduce valvular and vascular calcification. The aim of this study was to examine the effects of sevelamer treatment on calcification in bioprosthetic heart valves (BHVs).Methods: Wister rats were randomly divided into three groups according to sevelamer intake and implantation (sham–sham operation; implant–implantation and normal diet, implant+S implantation, and sevelamer diet). Two kinds of BHVs—bovine pericardium treated with glutaraldehyde (GLUT) or non-GLUT techniques—were implanted in rat dorsal subcutis at 4 weeks. After implantation, sevelamer was administered to the implant+S group. The animals were executed at days 0 (immediately after implantation), 7, 14, 28, and 56. Calcium levels were determined by atomic absorption spectroscopy and von Kossa staining. Serum biochemistry analysis, Western blotting, real-time quantitative polymerase chain reaction, alkaline phosphatase activity measurement, histopathologic analysis, immunohistochemistry, and enzyme-linked immunosorbent assay were conducted to identify the anti-calcification mechanism of sevelamer.Results: Non-GLUT crosslinking attenuates BHV calcification. Serum phosphate and calcium remained unreactive to sevelamer after a 14-day treatment. However, the mean calcium level in the implant+S group was significantly decreased after 56 days. In addition, the PTH level, inflammatory cell infiltration, system and local inflammation, and expression of Bmp2, Runx2, Alp, IL-1β, IL-6, and TNF-α were significantly reduced in the implant+S group.Conclusion: Sevelamer treatment significantly attenuated the calcification of BHVs and had anti-inflammation effects that were independent from serum calcium and phosphate regulation. Thus, sevelamer treatment might be helpful to improve the longevity of BHVs.

Calcification of bioprosthetic heart valves treated with ethylene glycol diglycidyl ether

Highlights. The morphology and elemental composition of calcium deposits formed in the tissues of epoxytreated aortic and mitral bioprostheses do not differ from those in the mineralized matrix of stenotic human aortic valve leaflets. Despite similar elemental composition of mineral deposits in the KemCor and UniLine bioprostheses, the morphology of these calcifications differs between bioprosthetic heart valve substitutes and, apparently, is associated with the specific structure of the fibrous matrix of the biological tissues that are used for their manufacturing.Aim. To analyze the morphology and elemental composition of mineral deposits formed in epoxy-treated aortic and mitral bioprosthetic heart valves made from xenoaortic or xenopericardial material and to compare the obtained findings with the data on calcified human aortic valve.Methods. Leaflets of the mitral and aortic bioprosthetic heart valves KemCor and UniLine (NeoKor, L Russia, Kemerovo) that were explanted due to their failure, as well as leaflets of the calcified native aortic valve were evaluated. The morphology of calcifications was studied by scanning electron microscopy using an S-3400N microscope (Hitachi, Japan). The elemental composition of calcium deposits was studied by electron probe microanalysis using Hitachi S-3400N microscope with energy dispersive spectrometer Bruker XFlash 4010 (Bruker, Germany).Results. Large calcifications located at the internal layers of samples were surrounded by collagen fibers commonly with evident signs of the onset of mineralization. Calcium deposits in the native aortic valve and xenoartic bioprostheses KemCor were located mainly at the spongy layer and had a loose structure, while dense lamellar deposits were found at the leaflets of pericardial bioprostheses UniLine. The elemental composition of calcium deposits showed the presence of Ca, P, O, Mg, and Na in the mineralized regions and the presence of S in the regions of low electron density. The calcium to phosphorus ratio (Ca:P) in the calcifications of the aortic valve leaflets was 1.81 (1.79-1.84; min - 1.48; max - 2.05), whereas the Ca:P ratios in the UniLine and KemCor bioprostheses were 1.78 (1.75-1.86; min - 1.52; max - 2.03) and 1.82 (1.81-1.88; min - 1.71; max - 2.06), respectively. There were no significant differences in the Ca:P ratios between calcifications in the study groups (p&gt;0.05).Conclusion. Calcium deposits detected in epoxy-treated bioprostheses and human aortic valve appeared to be formed under dystrophic calcification. The morphology of calcifications in bioprostheses depended on the type of biological tissue. None correlations between the morphological structure of calcifications and the implantation position were found in bioprosthetic leaflets. The elemental composition of mineral deposits was similar in all study samples.

Bioprosthetic heart valves with reduced immunogenic residuals using vacuum-assisted decellularization treatment

Despite the good hemodynamic characteristics of bioprosthetic heart valves, it is inevitable that they will suffer from calcification and tissue deterioration. Decellularization has been utilized to reduce the immunogenicity and calcification of bioprosthetic heart valves. However, it can take several days or even weeks to obtain the decellularized tissues or organs. Therefore, time-frame should be taken into consideration during the decellularization process. A detergent-enzymatic-method, combined with vacuum, has been proposed as a method of obtaining desirable decellularized heart valves. In this study, heart valves treated under vacuum and normal atmosphere are investigated via histological, biochemical and mechanical analysis. The results show that the decellularization efficiency of heart valves treated under vacuum is enhanced, based on histological staining, DNA contents and α-Gal quantification. The decellularization procedures decrease the contents of the extracellular matrix. However, the mechanical properties, including elastic modulus, fracture tensile strength and fracture strain, show no significant difference between the samples. In vitro cell cytotoxicity experiments indicate the feasibility of further in vivo experiments. Therefore, we conclude that vacuum-assisted decellularization procedures can significantly enhance decellularization efficiency by reducing the decellularization time, without compromising the properties of the heart valves, which is also beneficial in terms of reducing clinical costs. To the best of our knowledge, vacuum is a novel parameter which can be designed into decellularization procedures for heart valves.

Ectopic mineralization in heart valves: new insights from in vivo and in vitro procalcific models and promising perspectives on noncalcifiable bioengineered valves.

Ectopic calcification of native and bioprosthetic heart valves represents a major public health problem causing severe morbidity and mortality worldwide. Valve procalcific degeneration is known to be caused mainly by calcium salt precipitation onto membranes of suffering non-scavenged cells and dead-cell-derived products acting as major hydroxyapatite nucleators. Although etiopathogenesis of calcification in native valves is still far from being exhaustively elucidated, it is well known that bioprosthesis mineralization may be primed by glutaraldehyde-mediated toxicity for xenografts, cryopreservation-related damage for allografts and graft immune rejection for both. Instead, mechanical valves, which are free from calcification, are extremely thrombogenic, requiring chronic anticoagulation therapies for transplanted patients. Since surgical substitution of failed valves is still the leading therapeutic option, progressive improvements in tissue engineering techniques are crucial to attain readily available valve implants with good biocompatibility, proper functionality and long-term durability in order to meet the considerable clinical demand for valve substitutes. Bioengineered valves obtained from acellular non-valvular scaffolds or decellularized native valves are proving to be a compelling alternative to mechanical and bioprosthetic valve implants, as they appear to permit repopulation by the host's own cells with associated tissue remodelling, growth and repair, besides showing less propensity to calcification and adequate hemodynamic performances. In this review, insights into valve calcification onset as revealed by in vivo and in vitro procalcific models are updated as well as advances in the field of valve bioengineering.

Enzyme-oxidative-polymerization method for improving glycosaminoglycans stability and reducing calcification in bioprosthetic heart valves

Glutaraldehyde (GLUT) crosslinked bioprosthetic heart valves (BHVs) might fail within ten years due to progressive degradation and calcification. GLUT cannot stabilize glycosaminoglycans (GAGs), one important component of BHVs. In this current study we developed a new BHVs preparation strategy named as ‘HPA/NT/HRP’ treatment that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA)/neomycin trisulfate (NT) conjugated pericardiums and horseradish peroxidase (HRP)/H2O2 enzyme-oxidative-polymerization method. HPA/TRA-pericardium and HPA-NT conjugation would provide extra phenol groups for enzymatic crosslinking. HPA/TRA conjugated pericardium could be crosslinked by HRP/H2O2 enzyme-oxidative-polymerization. The feeding ratio of HPA-NT was optimized. The GAGs content, collagenase and elastase degradation in vitro, the in vivo GAGs stability and anti-calcification potential of HPA/NT/HRP treated pericardiums were characterized. We demonstrated that HPA/NT/HRP treated pericardiums had sufficiently increased GAGs stabilization and decreased calcification. This new HPA/NT/HRP enzyme-oxidative-polymerization strategy would be a promising method to make BHVs with better GAGs stability and anti-calcification properties.

Rosuvastatin attenuates bioprosthetic heart valve calcification

ObjectiveThere are pathophysiologic similarities between calcification and atherosclerosis because both are the product of an active inflammatory process. The aim of the study was to examine the effects of statin treatment on calcification in commercially available bioprosthetic heart valves. MethodsTwenty Sprague–Dawley rats were fed a high-fat diet to induce hypercholesterolemia during 4 weeks. They were randomly divided into 2 groups according to statin intake (control, n = 10: high-fat diet/statin; n = 10: high-fat diet with statin). Four commercially available tissue valve (Magna Perimount, Carpentier-Edwards, Irvine, Calif; Hancock, Medtronic, Minneapolis, Minn; Mitroflow, LivaNova, London, England; and Trifecta, St Jude Medical, St Paul, Minn) cusp samples (total 320) were implanted in rat dorsal subcutis at 4 weeks. After implantation, rosuvastatin was administered daily to the statin group. The cusps were explanted at 12 weeks, and calcium levels were determined by atomic absorption spectroscopy. Western blotting, histologic, and immunohistochemical analyses were conducted to identify the anticalcification mechanism of the statin. ResultsThe mean calcium level in the control group was significantly higher than in the statin group (P < .01) for all tissue valves (Magna Perimount: 2.67 ± 0.26 mg/g vs 1.31 ± 0.40 mg/g; Hancock: 2.70 ± 0.57 mg/g vs 1.53 ± 0.34 mg/g; Mitroflow: 2.39 ± 0.71 mg/g vs 1.26 ± 0.38 mg/g; Trifecta: 2.54 ± 0.42 mg/g vs 1.63 ± 0.72 mg/g). Inflammatory cell infiltration and interleukin-6 and bone morphogenetic protein 2 expressions were significantly reduced in the statin group. ConclusionsStatin treatment significantly attenuated bioprosthetic heart valve calcification associated with decreasing the levels of interleukin-6 and bone morphogenetic protein 2. Thus, statin treatment might be helpful for the longevity of bioprosthetic heart valves.

Riboflavin photo-cross-linking method for improving elastin stability and reducing calcification in bioprosthetic heart valves.

Glutaraldehyde cross-linked bioprosthetic heart valves might fail due to progressive degradation and calcification. In this study, we developed a new BHVs preparation strategy named as "HPA/TRA/FMN" that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA) conjugated pericardium and riboflavin 5'-monophosphate (FMN) initiated photo-cross-linking method. HPA/TRA-pericardium conjugation would provide extra phenol groups for FMN initiated photo-cross-linking. The feeding ratio of riboflavin 5'-monophosphate was optimized. The collagenase and elastase enzymatic degradation in vitro, biomechanics, calcification, elastin stability in vivo, and macrophage marker CD68 were characterized. We demonstrated that riboflavin photo-cross-linked pericardiums had great collagen and elastin stability, improved mechanical properties, better resistance for calcification, and less CD68 positive macrophages in rat subdermal implantation study. This new riboflavin photo-cross-linking strategy would be a promising method to make BHVs which have better elastin stability, less calcification, and reduced inflammatory response.

Abstract 16744: Advanced Glycation End Products and the Pathophysiology of Bioprosthetic Heart Valve Structural Degeneration

Introduction: Bioprosthetic heart valves (BHV) are widely used to treat severe heart valve disease. BHV function deteriorates after 10-15 years due to structural degeneration (SVD) often involving calcification. SVD occurs regardless of biomaterial, requiring prosthesis replacement. Advanced glycation end products (AGEs) are post-translational, non-enzymatic protein modifications associated with diabetic complications and cardiovascular disease. Serum proteins, such as albumin, are also AGE modified. Hypothesis: AGEs, exacerbated by serum protein infiltration, play a role in SVD. Methods: Clinical BHV explants (n = 45) of glutaraldehyde-crosslinked bovine pericardium (BP, n = 35) or porcine aortic valve (PAV, n = 10) and equivalent unimplanted BHV were probed by immunostaining per semi-quantitative scoring (range 0-4) for generalized AGE, N-carboxymethyllysine (CML, an AGE-receptor ligand), and human serum albumin (HSA), the most abundant serum protein. Demographics comprise ages 34 to 86 (mean: 64.95), 28 males and 17 females, 8 diabetics, and 15 previous and concomitant coronary artery bypass patients (both conditions associated with SVD). BHV calcification was quantified by atomic absorption spectroscopy. BHV glycation was modeled by exposing BP to 50mM glyoxal +/- 5% albumin for 24hr, followed by either immunostaining for CML and albumin or 14 C glyoxal scintillation. Albumin incorporation was verified by mass uptake. Results: All explants showed positive immunostaining for AGE (mean ± SE = 3.52 ± 0.10); most showed HSA (2.30 ± 0.18) and less intense CML (1.55 ± 0.16) staining; unimplanted BHV staining was negative. Score comparisons showed no significant differences for BP vs PAV, coronary bypass or diabetic patients vs all patients. BHV Ca levels (122.01 ± 18.01ug/mg, binned as &lt;30 vs &gt;100ug/mL) did not correlate with AGE immunostaining. In vitro, glyoxal-generated CML and infiltrated albumin were evident throughout BP per immunostaining. 14 C glyoxal uptake by BP was 12.01 ± 0.50nmol/mg after 28 days and was increased 57.2 ± 7.5% by albumin co-incubation. Albumin co-incubation increased BP mass by 4.10 ± 0.19%. Conclusions: AGEs are associated with SVD regardless of BHV type and SVD risk factors. Albumin exacerbates AGE accumulation.

EGCG and enzymatic cross-linking combined treatments for improving elastin stability and reducing calcification in bioprosthetic heart valves.

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H2 O2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H2 O2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019.

Elastin Stabilization Through Polyphenol and Ferric Chloride Combined Treatment for the Enhancement of Bioprosthetic Heart Valve Anticalcification.

The lifetime of bioprosthetic heart valves (BHVs) is limited by the mechanical damage and calcification. The major components of BHVs are collagen and elastin. Collagen could be well protected by glutaraldehyde (GLUT) crosslinking, while elastin is not stabilized and has a high risk of degradation, which could lead to the calcification of BHVs. We aimed to develop methods for stabilizing elastin and decreasing calcification. We investigated the combined tannic acid (TA) or epigallocatechin gallate (EGCG) with ferric chloride to stabilize elastin and prevent calcification. We found that the amount of TA/EGCG bound to elastin was in a time-dependent pattern and this reaction showed better efficiency in acidic condition and ethanol-water mixed solvents. Moreover, Fe3+ could compete with Ca2+ to bind to polyphenol, which could reduce the calcium deposition on BHVs. Cytotoxicity test showed that all extracts from different treatments had similar cell viabilities (85-100%). Through the combined treatments of polyphenol and ferric chloride, the pericardium had a better resistance to elastase degradation and more excellent anticalcification performance.

A correlation between long-term in vitro dynamic calcification and abnormal flow patterns past bioprosthetic heart valves.

In this paper, a long-term in vitro dynamic calcification of three porcine aortic heart valves was investigated using a combined approach that involved accelerated wear testing of the valves in the rapid calcification solution, hydrodynamic assessment of the progressive change of effective orifice area (EOA) along with the transaortic pressure gradient, and quantitative visualization of the flow. The motivation for this study was developing a standardized, economical, and feasible in vitro testing methodology for bioprosthetic heart valve calcification, which would address both the hydrodynamic performance of the valves as well as the subsequent changes in the flow field. The results revealed the failure of the test valves at 40 million cycles mark, associated with the critical decrease in the EOA, followed by the increase in the maximum value of viscous shear stress of up to 52%, compared to the values measured at the beginning of the study. The decrease in the EOA was subsequently followed by the rapid increase in the maximum streamwise velocity of the central orifice jet up to the value of about 2.8m/s, compared to the initial value of 2m/s, and to the value of 2.2m/s in the case of a control valve along with progressive narrowing of the velocity profile for two test valves. The significance of the current work is in demonstrating a correlation between calcification of the aortic valve and spatial as well as the temporal development of abnormal flow features.

The Neglected Villain of Bioprosthetic Degeneration: Inflammatory and Immune Processes.

In an attempt to avoid the destructive process of bioprosthetic heart-valve calcification associated with the use of glutaraldehyde, valves are today prepared using low concentrations of the crosslinking reagent. In this review, we summarize our findings and those of others that confirm that the immunogenicity of such tissue is not sufficiently masked and that a defined humoral response is indeed mounted against a repertoire of antigens unrelated to those associated with vascularized and non—cross-linked xenograft organs. We demonstrate the need for increased cross-linking of tissue to satisfactorily mitigate that response; furthermore, we examine the impact of increased cross-link density on the macrophage as antigen presenting cell with respect to its involvement in both tissue erosion and pannus overgrowth. Finally we present evidence for a role of circulating antibodies in bioprosthesis calcification.