Prevention of tissue calcification on bioprosthetic heart valve by using epoxy compounds: a study of calcification tests in vitro and in vivo.

Calcification is the principal cause of the clinical failures of the bioprosthetic heart valves fabricated from glutaraldehyde pretreated porcine aortic valves or bovine pericardium. In this paper, we compared the calcification on various types of bovine pericardiums pretreated with two hydrophilic epoxy compounds adding GA post-treatment (EP 1 and EP 2), glutaraldehyde (GA)- and nontreated pericardium (Fresh), respectively, by in vitro and in vivo tests. Significant decrease of calcification was found by pretreatment with both epoxy compounds rather than with glutaraldehyde: 0.250 +/- 0.001 (Fresh), 0.276 +/- 0.058 (EP 1), 0.302 +/- 0.071 (EP 2), and 0.478 +/- 0.172 (GA) micrograms (Ca)/mg (dried tissue), respectively, after 20 days dipping in a simulating serum solution in vitro; 115.13 +/- 60.11 (Fresh), 129.84 +/- 51.08 (EP 1), 167.39 +/- 20.81 (EP 2), and 205.19 +/- 16.86 (GA) micrograms/mg, respectively, after 3 months subcutaneous implantation in rabbits. The in vitro method for evaluating calcification designed by us gave the similar order among four samples with that obtained by in vivo test. Because the bovine pericardium pretreated with the epoxy compounds adding GA post-treatment possesses the greater tenacity than that pretreated only with epoxy compounds or GA, meanwhile the calcification is also significantly decreased with this pretreatment, it may be expected that the bovine pericardium with this pretreatment will have the greater anticalcification and durability in dynamic stress.     

Onset and progression of experimental bioprosthetic heart valve calcification

Calcification, the major cause of bioprosthetic heart valve failures, is a serious clinical problem with uncertain pathogenesis. The objectives of the present study were to define the progressive chemical and morphologic sequence of mineralization in glutaraldehyde-treated porcine aortic valve cusps implanted subcutaneously in rats and to compare the pathology and pathophysiology of calcification in subcutaneous implants with that of orthotopic valve replacements in calves. Cusps were implanted subcutaneously in 3-week-old rats for 24 hours to 18 weeks. Cuspal calcium was 114 +/- 18 micrograms/mg of dry weight (mean +/- SEM) at day 21 and 218 +/- 6 at day 56 of implantation and unchanged thereafter. The earliest mineral deposits, noted at 48 hours, were associated with devitalized porcine connective tissue cells, but by 7 days, mineral deposits also involved collagen bundles. Scanning electron microscopy with energy-dispersive x-ray analysis demonstrated predominant accumulation in the spongiosa with a spongiosa to fibrosa energy-dispersive x-ray analysis count ratio of calcium of 15 at 21 days. In stent-mounted glutaraldehyde-preserved porcine valves implanted in five calves as mitral replacements for 69 to 142 days, cuspal calcium was 86 micrograms/mg (mean) (range 47 to 128). Calf implants also had cell oriented and collagen calcification predominating in the valvar spongiosa. In both rat subcutaneous and calf mitral valve models, early diffuse calcific microcrystals evolved into confluent nodules that disrupted tissue architecture. It is concluded that calcification of glutaraldehyde-preserved porcine aortic valves implanted subcutaneously in rats begins within 48 hours, earliest deposits are localized to residual porcine connective tissue cells, but latter deposits also involve collagen fibrils, mineralization is most prominent in the spongiosa, the pathology of calcification in rat subcutaneous implants and calf mitral replacements is comparable, suggesting a common pathophysiology, and calcific nodule formation most likely initiates clinical features.     

Treatment of bioprosthetic heart valve tissue with long chain alcohol solution to lower calcification potential

The use of glutaraldehyde-treated biological tissue in heart valve substitutes is an important option in the treatment of heart valve disease. These devices have limited durability, in part, because of tissue calcification and subsequent tearing of the valve leaflets. Components thought to induce calcification include lipids, cell remnants, and residual glutaraldehyde. We hypothesized that treatment of glutaraldehyde-treated bioprosthetic heart valve material using a short and long chain alcohol (LCA) combination, composed of 5% 1,2-octanediol in an ethanolic buffered solution, would reduce phospholipid content and subsequently lower the propensity of these tissues to calcify in vivo. Phospholipid content of glutaraldehyde-treated porcine valve leaflets and bovine pericardium was found to be 10.1 +/- 4.3 (n = 7) and 3.9 +/- 0.48 (n = 2) microg/mg dry tissue, respectively, which was reduced to 0.041 +/- 0.06 (n = 7) and 0.21 +/- 0.05 (n = 4) microg/mg dry tissue, respectively, after LCA treatment. Calcification potential of the treated tissues was assessed using a rat subcutaneous implant model. After 60 days of implantation, calcium levels were found to be 171 +/- 32 (n = 11) and 83 +/- 70 (n = 12) mg/g dry weight for glutaraldehyde-treated porcine leaflets and bovine pericardium, respectively, whereas prior LCA treatment resulted in reduced calcium levels of 1.1 +/- 0.6 (n = 12) and 0.82 +/- 0.1 (n = 12) mg/g dry weight, respectively. These data, taken together, support the notion that treatment of glutaraldehyde-treated tissue with a short and long chain alcohol combination will reduce both extractable phospholipids and the propensity for in vivo calcification.     

New treatments using alginate in order to reduce the calcification of bovine bioprosthetic heart valve tissue

Calcification limits the functional lifetime of cardiac valve substitutes fabricated from glutaraldehyde preserved bovine pericardium. Host factors, mainly younger age, and implant factors, mainly glutaraldehyde cross-linking, are implicated in the calcification process. Glutaraldehyde cross-linking is believed to activate the potential sites in the tissues for biocalcification. In the present work, we investigated the possibility of using alginate azide (AA) instead of glutaraldehyde for the preservation of pericardial tissues in order to enhance the durability of bioprosthetic heart valves. Grafting with poly(GMA-BA) copolymer to the alginate azide cross-linked pericardial (AACPC) tissue was carried out to obtain better stability, strength, and anticalcification properties. The strength property and thermal stability of the AA cross-linked tissues were studied. Calcification studies in rat subdermal models reveal that AA cross-linking reduces the calcification to negligible levels. After 30 days implantation, the calcium content was found to be 10.4 ± 1.2 and 6.1 ± 0.3 μg mg^-1 for untreated AACPC and polymer grafted AACPC, respectively, compared to a value of 100 ± 1.2 μg mg^-1 calcium recorded for control glutaraldehyde cross-linked pericardial (GCPC) tissues.     

Mechanical properties and histology of charge modified bioprosthetic tissue resistant to calcification.

Modification of bioprosthetic heart valves tissue by covalently binding protamine sulphate, results in stable covalent links of protamine to the tissue, conferring resistance to calcification. We report here the morphological evaluation and mechanical properties (elastic modulus and ultimate tensile strength) of protamine-bound bioprosthetic tissue that have high anticalcification potential. Protamine-bound bioprosthetic tissue had significantly higher tissue modulus and ultimate tensile strength values than control tissue groups. However, the mechanical properties and tissue architecture were inferior to those of bioprosthetic tissue.     

Covalent binding of aminopropanehydroxydiphosphonate to glutaraldehyde residues in pericardial bioprosthetic tissue: stability and calcification inhibition studies

Calcification has limited the clinical utility of bioprosthetic heart valves fabricated from either glutaraldehyde-pretreated bovine pericardium or porcine aortic valves. Aminopropanehydroxydiphosphonate (APDP), covalently bound to residual aldehyde groups in glutaraldehyde-treated pericardial bioprosthetic tissue, has been shown to inhibit cardiovascular calcification in the rat subdermal model. Using 3H-labeled glutaraldehyde (GLUT) at a concentration of 0.02 M and 0.14 M 14C-labeled APDP, we assessed the effects of GLUT incubation temperature (4 degrees or 25 degrees C) and pH of the GLUT incubation solution (pH 4.0, 7.4, or 10.0) on the GLUT incorporation step and subsequent APDP binding (24 hr 25 degrees C) into bioprosthetic valve (BPV) tissue (bovine pericardium). Increased incorporation of GLUT and APDP occurred at lower GLUT incubation temperature (GLUT, 346.05 +/- 1.9 nM/mg, 4 degrees C vs 259.76 +/- 1.39 nM/mg, 25 degrees C; APDP, 57.56 +/- 4.43 nM/mg, 4 degrees C vs 36.36 +/- 0.46 nM/mg, mean +/- standard error, at 25 degrees C). There also was a greater incorporation of GLUT but not APDP at the higher glutaraldehyde pretreatment pH (GLUT, pH 10.0, 213.73 +/- 73 nM/mg vs pH 4, 132.08 +/- 43 nM/mg; APDP, pH 10.0, 51.41 +/- 12 nM/mg vs pH 4.0, 49.97 +/- 6 nM/mg). In vivo studies revealed that all groups with treated BPV implanted for 21 days in male 3-week-old CD rats demonstrated a loss of both GLUT (12-50%) and APDP (48-64%) compared to preimplant content. BPV implant calcification was significantly inhibited in all groups treated with APDP compared to control Ca2+ (5.54 +/- 2.1-9.64 + 1.2 micrograms/mg, APDP pretreated, vs 93.64 +/- 11.65 micrograms/mg, control; P less than or equal to 0.001) despite the progressive loss of both GLUT and APDP with time. It is concluded that preincubation of BPV tissue in GLUT at lower temperature (4 degrees C) and higher pH (10.0) enhanced BPV GLUT uptake and subsequent APDP covalent binding. In addition, in the rat subdermal model, BPV tissue calcification was markedly inhibited by APDP, despite a significant loss of bound drug.     

Ultrastructural substrates of dystrophic calcification in porcine bioprosthetic valve failure.

Calcific degeneration is the main cause of porcine bioprosthetic valve failure. This dystrophic phenomenon has been studied by transmission electron microscopy in 26 explants; six normally processed unimplanted xenografts and a pig aortic valve from the slaughterhouse served as controls. Loss of endothelial lining and proteoglycans were a regular finding in all the commercially processed valves. In order to detect initial calcifications, we investigated in particular areas apparently devoid of mineralization at x-ray. Three main ultramicroscopic features were found: 1) intracytoplasmic and interstitial calcospherulae in 22 explants, 2) calcified collagen fibrils in 15, and 3) platelike calcium deposits upon amorphous material in 3. X-ray diffraction and energy-dispersive microanalysis identified Ca2+ deposits as crystals of hydroxyapatite. From these findings there is evidence that debris and membrane fragments of the pig cusp cells represent one of the initial nuclei of calcification.     

The susceptibility of bioprosthetic heart valve leaflets to oxidation

The clinical use of bioprosthetic heart valves (BHV) is limited due to device failure caused by structural degeneration of BHV leaflets. In this study we investigated the hypothesis that oxidative stress contributes to this process. Fifteen clinical BHV that had been removed for device failure were analyzed for oxidized amino acids using mass spectrometry. Significantly increased levels of ortho-tyrosine, meta-tyrosine and dityrosine were present in clinical BHV explants as compared to the non-implanted BHV material glutaraldehyde treated bovine pericardium (BP). BP was exposed in vitro to oxidizing conditions (FeSO₄/H₂O₂) to assess the effects of oxidation on structural degeneration. Exposure to oxidizing conditions resulted in significant collagen deterioration, loss of glutaraldehyde cross-links, and increased susceptibility to collagenase degradation. BP modified through covalent attachment of the oxidant scavenger 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine (DBP) was resistant to all of the monitored parameters of structural damage induced by oxidation. These results indicate that oxidative stress, particularly via hydroxyl radical and tyrosyl radical mediated pathways, may be involved in the structural degeneration of BHV, and that this mechanism may be attenuated through local delivery of antioxidants such as DBP.     

一种新型抗钙化处理的人工生物瓣膜流体力学性能

目的 评价一种新型生物瓣膜的体外流体力学性能,并与传统生物瓣膜及机械瓣膜进行比较.方法 将测试瓣膜分成三组:新型生物瓣组(GA SOB处理牛心包瓣),传统生物瓣组(单纯GA处理牛心包瓣),机械瓣组(双叶瓣),每组分别选21号、25号、29号三种型号,采用清华大学TH-1200脉动流测试仪,按照ISO5840瓣膜检测标准进行流体性能检测,包括跨瓣压差、返流量、返流百分比及有效开口面积,并进行组间的分析、比较.结果 新型生物瓣膜的前向流跨瓣压差较传统生物瓣小17%～30%,较机械瓣小23%～50%;新型生物瓣的有效开口面积较传统生物瓣和机械瓣分别大13%～37%和36%～50%;新型生物瓣的返流量较传统生物瓣大1.2～2.0 mL,约3%～6%;较机械瓣小0.9～2.8mL,约1.3%～5%.结论 新型人工生物瓣膜具有良好的血流动力学性能.     

Retraction of bioprosthetic heart valve cusps: a cause of wide-open regurgitation in right-sided heart valves

Most failures of bioprosthetic heart valves in children are due to stenosis secondary to thrombus, calcific deposits, or tissue ingrowth. Valve failures due to regurgitation typically involve cuspal detachment, tears, or perforations. We present four cases of prosthetic valve regurgitation in children caused by cuspal retraction without stenosis and describe the morphologic findings related to the valves at autopsy or explantation. A mononuclear cell and giant cell response to the cusps of the valve was a striking finding in one patient.     

Decreased tissue reaction to bioprosthetic heart valve material after L-glutamic acid treatment. A morphological study.

Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).     

Collagen fiber disruption occurs independent of calcification in clinically explanted bioprosthetic heart valves

The durability of bioprosthetic heart valves (BHV) is severely limited by tissue deterioration, manifested as calcification and mechanical damage to the extracellular matrix. Extensive research on mineralization mechanisms has led to prevention strategies, but little work has been done on understanding the mechanisms of noncalcific matrix damage. The present study tested the hypothesis that calcification-independent damage to the valvular structural matrix mediated by mechanical factors occurs in clinical implants and could contribute to porcine aortic BHV structural failure. We correlated quantitative assessment of collagen fiber orientation and structural integrity by small angle light scattering (SALS) with morphologic analysis in 14 porcine aortic valve bioprostheses removed from patients for structural deterioration following 5-20 years of function. Calcification of the explants varied from 0 (none) to 1+ (minimal) to 4+ (extensive), as assessed radiographically. SALS tests were performed over entire excised cusps using a 0.254-mm spaced grid, and the resultant structural information used to generate maps of the local collagen fiber damage that were compared with sites of calcific deposits. All 42 cusps showed clear evidence of substantial noncalcific structural damage. In 29 cusps that were calcified, structural damage was consistently spatially distinct from the calcification deposits, generally in a distribution similar to that noted in porcine BHV subjected to in vitro durability testing. Our results suggest a mechanism of noncalcific degradation dependent on cuspal mechanics that could contribute to porcine aortic BHV failure.     

Retardation of calcification of bovine pericardium used in bioprosthetic heart valves by phosphocitrate and a synthetic analogue

The purpose of this study was to determine if phosphocitrate (PC), a naturally occurring inhibitor of calcification, and its synthetic analogue, N-sulpho-2-amino tricarballylate (SAT), administered either by daily injection or local delivery via Alzet osmotic minipump, could inhibit calcification of glutaraldehyde-preserved bovine pericardium used in bioprosthetic heart valves, subcutaneously implanted in rats. Local drug delivery, but not systemic administration, was effective. PC, administered by Alzet minipump (12 mg.kg-1.day-1), inhibited calcification significantly (tissue calcium = 5 +/- 2 micrograms/mg dry tissue, mean +/- SEM), compared with untreated or saline-treated controls (89 +/- 9 and 49 +/- 9 micrograms/mg, respectively). SAT, administered by the same route at both the same and a higher molar dosage, was less potent (tissue calcium = 26 +/- 9 micrograms/mg and 17 +/- 5 micrograms/mg, respectively). PC and SAT therapy were not associated with adverse effects. We conclude that locally administered PC and SAT can inhibit intrinsic calcification of bovine pericardium, with PC being more potent.     

Mitigation of bioprosthetic heart valve degeneration through biocompatibility: in vitro versus spontaneous endothelialization

BACKGROUND: Glutaraldehyde-related cytotoxicity and transanastomotic ingrowth inhibition prevent the spontaneous endothelialization of bioprosthetic heart valves. In order to evaluate the ability of improved biocompatibility to reduce tissue degeneration, conventionally fixed aortic root prostheses were both glutaraldehyde-detoxified and in vitro endothelialized. METHODS: Entire aortic roots were fixed in 0.2% glutaraldehyde (GA) (control group) and either detoxified in acetic acid-buffered urazole (0.1 M) or detoxified and in vitro lined with cultured, autologousjugular vein endothelial cells. The valved roots were inserted in the distal aortic arch of 15 juvenile Merino sheep for a period of 12 weeks. Upon explant, leaflets, sinuses and aortic wall of the prostheses were analysed by SEM to assess the surface endothelium, histologically regarding tissue inflammation, and by atomic absorption spectrophotometry to determine the content of tissue calcium. RESULTS: There was no endothelium on control grafts, except for a short anastomotic pannus. The detoxified group showed an incomplete patchy endothelium on the aortic wall but hardly any on the leaflets, whereas, the in vitro lined group had aortic wall, sinuses and most of the leaflets confluently endothelialized. Tissue inflammation was prominent in the control group and least expressed in the endothelialized group (p < 0.05). Detoxification significantly reduced leaflet calcification. In the aortic wall, both detoxification and endothelial lining were required to significantly mitigate calcification. CONCLUSION: In the 12 week circulatory sheep model, the calcium mitigating effect of detoxification was more pronounced than that of in vitro endothelialization. Nevertheless, there was a distinct overall benefit if detoxification was combined with endothelialization.     

Mechanisms of bioprosthetic heart valve failure: fatigue causes collagen denaturation and glycosaminoglycan loss.

Bioprosthetic heart valve (BPHV) degeneration, characterized by extracellular matrix deterioration, remodeling, and calcification, is an important clinical problem accounting for thousands of surgeries annually. Here we report for the first time, in a series of in vitro accelerated fatigue studies (5-500 million cycles) with glutaraldehyde fixed porcine aortic valve bioprostheses, that the mechanical function of cardiac valve cusps caused progressive damage to the molecular structure of type I collagen as assessed by Fourier transform IR spectroscopy (FTIR). The cyclic fatigue caused a progressive loss of helicity of the bioprosthetic cuspal collagen, which was evident from FTIR spectral changes in the amide I carbonyl stretching region. Furthermore, cardiac valve fatigue in these studies also led to loss of glycosaminoglycans (GAGs) from the cuspal extracellular matrix. The GAG levels in glutaraldehyde crosslinked porcine aortic valve cusps were 65.2 +/- 8.66 microg uronic acid/10 mg of dry weight for control and 7.91 +/- 1.1 microg uronic acid/10 mg of dry weight for 10-300 million cycled cusps. Together, these molecular changes contribute to a significant gradual decrease in cuspal bending strength as documented in a biomechanical bending assay measuring three point deformation. We conclude that fatigue-induced damage to type I collagen and loss of GAGs are major contributing factors to material degeneration in bioprosthetic cardiac valve deterioration.     

Immune response in patients receiving a bioprosthetic heart valve: lack of response with decellularized valves

Conventional biological heart valves treated with glutaraldehyde (GA) reveal a limited lifespan due to calcification. This is assumed to be an immune response initiated process, which is not seen with decellularized valves. However, their immunological potential is still a matter of debate. Therefore, serum samples from patients undergoing heart valve surgery were obtained before (Pre), after (Post), and 9-12 months after operation (Follow Up). Immunoglobulin G (IgG) and M (IgM) antibodies against porcine collagen I and α-Gal (Gal-alpha1,3-Gal-beta1,4-GlcNac-R) were determined for decellularized and GA treated valves. Antibody titers for collagen type I revealed no significant alteration for both types of valves. However, a considerable anti-α-Gal antibody response was observed in patients with GA-treated porcine valves. In detail, IgM antibodies were increased during follow up (p<0.05), whereas decellularized valves revealed a minor decrease in the IgM response (p<0.001). IgG antibodies were considerably increased with GA-treated porcine (p<0.05) and bovine (p<0.01) xenografts, whereas there was lack of response with decellularized valves. This indicates that GA treatment is not sufficient to eliminate immune response to the α-Gal epitope completely. Future investigations will have to verify whether immune response to α-Gal can be linked to the limited durability of conventional valves.     

The evolution of bioprosthetic heart valve design and its impact on durability.

Bioprosthetic heart valves have evolved over the years into remarkably useful and predictable devices. During this process, a number of specific designs have come and gone, and a few have remained. Many design changes were successful, and many were not. This article will describe the successes and failures of the various bioprosthetic valve designs and will detail the specific reasons why a particular design change succeeded or failed to improve bioprosthetic valve performance.