Prevention of calcification in bioprosthetic heart valves: challenges and perspectives

Transcription

1 Review 1. Introduction 2. Current status and presentation of bioprosthetic heart valves 3. Macroscopic alterations in explanted bioprosthetic heart valves 4. Factors that influence bioprosthetic heart valve calcification and experimental models 5. Glutaraldehyde as a villain and treatments targeting Glut 6. Tissue components influence calcification of bioprosthetic heart valves 7. Expert opinion and conclusion Ashley Publications General Prevention of calcification in bioprosthetic heart valves: challenges and perspectives Dan T Simionescu Cardiovascular Implant Research Laboratory, Department of Bioengineering, Clemson University, 501 Rhodes Research Center, Clemson, SC , USA Surgical replacement with artificial devices has revolutionised the care of patients with severe valvular diseases. Mechanical valves are very durable, but require long-term anticoagulation. Bioprosthetic heart valves (BHVs), devices manufactured from glutaraldehyde-fixed animal tissues, do not need longterm anticoagulation, but their long-term durability is limited to years, mainly because of mechanical failure and tissue calcification. Although mechanisms of BHV calcification are not fully understood, major determinants are glutaraldehyde fixation, presence of devitalised cells and alteration of specific extracellular matrix components. Treatments targeted at the prevention of calcification include those that target neutralisation of the effects of glutaraldehyde, removal of cells, and modifications of matrix components. Several existing calcification-prevention treatments are in clinical use at present, and there are excellent mid-term clinical follow-up reports available. The purpose of this review is to appraise basic knowledge acquired in the field of prevention of BHV calcification, and to provide directions for future research and development. Keywords: bioprosthetic heart valve, calcification, collagen, elastin, glutaraldehyde, mineralisation Expert Opin. Biol. Ther. (2004) 4(12): Introduction As we settle into the twenty-first century, cardiovascular diseases continue to impact morbidity and mortality, and maintain their position as the number one killer of the civilised world [201]. Valvular pathology is a significant chapter of cardiovascular diseases, but very little is known about the mechanisms involved in the onset of this pathology, and there is no medication available to limit its progression. The most common therapeutic procedure for the treatment of valvular pathology is the surgical replacement of defective heart valves. Diseased human heart valves are replaced by engineered devices that include mechanical valves or valves made from biological tissues. This practice started in the early 1970s and it is estimated at present that 275,000 valve replacements are performed annually worldwide [1]. Mechanical valves represent slightly more than half of these, with the remainder being tissue valves. The most physiological tissue prostheses are the pulmonary autograft valves (the result of a surgical procedure whereby the patient s own pulmonary valve is transplanted into the aortic position) and the human allograft valves (sterilised, cryopreserved cadaveric valves obtained from humans). Allografts exhibit excellent durability after implantation, but are not readily available and represent only a small percentage of total valve replacements. Heterograft valves (xenografts), fabricated from glutaraldehyde-treated porcine aortic valves or from bovine pericardium, represent the largest proportion of biological replacement valves / Ashley Publications Ltd ISSN

2 Prevention of calcification in bioprosthetic heart valves: challenges and perspectives Heart valve replacement offers an excellent improvement in the quality of life for thousands of patients. The main issue that emerged during clinical investigations of replacement heart valves was limited durability of these devices. Reoperation following valve replacement surgery, for the purpose of retrieving and replacing the defective device, is a relatively common event and occurs within years after initial valve surgery [2]. Clearly, a second open-heart surgery is undesirable to the patient and is prone to high clinical risks. Although mechanical valves may last longer, they have a high rate of thromboembolism and require an almost indefinite anticoagulant therapy. Without disregarding the excellent results obtained with mechanical valves, the focus of this paper is on the biological tissue valves, most commonly referred to as bioprosthetic heart valves (BHVs). Careful analysis of explanted tissue valves has shown that the predominant aspect contributing to dysfunction of biological heart valve replacements is structural deterioration and calcification of the tissue component. Histological, ultrastructural and biochemical aspects of degenerated explanted BHVs are similar to those of native human diseased heart valves [3]. The major processes that contribute to this new pathology of replacement heart valves are tissue calcification and mechanical damage. Calcification may occur independent of mechanical damage [4], but may also be accompanied by tissue abrasion, tearing and perforations. Numerous design and manufacturing improvements have been made to reduce the incidence of mechanical damage [5]; however, it is too early for a full clinical evaluation and comparison of these new designs with earlier models. Moreover, in spite of major design developments, it is apparent that calcification remains an important cause of dysfunction in biological valves. The unique pathology of BHVs presents serious challenges to academic scientists, industry representatives and clinicians alike. These challenges incorporate a convoluted interplay of interests between basic research, commercial benefits and the need to provide adequate healthcare. When compared with other biological therapies, there is a surprising paucity of basic knowledge on calcification mechanisms in BHVs, as well as mechanisms of action of treatments that influence calcification. This interplay of interests is also reflected by the confusing terminology assigned to the efficacy of treatments, as these range from the more modest mitigation, delay, retardation or reduction of calcification to prevention, calcificationresistance, and culminate with complete inhibition and anticalcification. This review will use the term prevention. Numerous approaches for the prevention of calcification in BHVs have been bench -tested during the last 30 years, but few of them have been licensed and approved by regulatory agencies and have reached bedside or clinical applications. It is not the purpose of this review to rank or endorse any treatments aimed at prevention of calcification in BHVs. Comparison between different treatments at this point is not realistic because most of them have ill-defined mechanisms of action and as yet unproven long-term clinical efficacy. Moreover, each treatment targets different tissue structures and not all experimental data are available in peer-reviewed publications. This paper will review basic knowledge acquired in the field of prevention of BHV calcification and provide some directions for future research and development. 2. Current status and presentation of bioprosthetic heart valves BHVs come in different designs, shapes and colours, and could be largely categorised into those that rely on a support structure (stent) for functioning and those that are used in surgery without stents (stentless). Stented prostheses are constructed from porcine aortic valves or bovine pericardium treated with % neutral buffered glutaraldehyde, which are then mounted onto supporting structures that mimic the valve anatomy. Glutaraldehyde, a highly reactive water-soluble dialdehyde, crosslinks tissue proteins by reacting with available amine groups, and in doing so greatly reduces the rate of in vivo enzymatic degradation by host cells. Moreover, glutaraldehyde reduces the antigenicity of the tissue and sterilises the prostheses. However, glutaraldehyde fixation is also a main cause for the lack of long-term durability of BHVs (see below). The development of BHVs has been a continuing process in the last three decades. The first generation of BHVs were fixed with glutaraldehyde at high pressure and were not treated with any calcification-prevention agent. These BHVs are highly prone to structural and calcific degeneration, and are not expected to last longer than years. Existing BHVs utilise very low (or zero)-pressure fixation and incorporate chemical treatments that have been shown in animal studies to delay or prevent calcification. For stented valves, Medtronic (Minneapolis, MN, USA) employs sodium dodecyl sulfate (T6), α-amino oleic acid (AOA ) and toluidine blue (Intact ) as calcification-prevention agents targeted at valvular cusps. Other treatments include ethanol for the Epic valves (St. Jude Medical, Minneapolis, MN, USA), as well as ethanol and Tween-80 (XenoLogiX ) for the Carpentier-Edwards valves (Edwards Lifesciences Corporation, Santa Ana, CA, USA). More recently, stentless porcine valves have gained popularity in cardiac surgery and are considered an attractive alternative to stented valves, mainly because of the absence of obstructive stents and strut posts. In most models available to date, the device is derived from the whole porcine aortic root (valve cusps attached to the native sinus and a portion of the ascending aorta), fixed in glutaraldehyde at low pressure and treated with calcification-prevention reagents. The current generation of stentless BHVs, most of which incorporate calcification-prevention treatments, includes the St. Jude Medical Toronto SPV (treated with BiLinx, ethanol for the cusps and AlCl 3 for the wall); Medtronic Freestyle Aortic Root Bioprosthesis (AOA); Edwards Lifesciences Prima Plus (XenoLogiX); CryoLife-O Brien stentless porcine aortic valve (without calcification-prevention treatment); Shelhigh 1972 Expert Opin. Biol. Ther. (2004) 4(12)

3 Simionescu Figure 1. BHV pathology. A) First-generation stented pericardial BHV, not implanted. Top view of the open valve outflow surface showing the three pericardial leaflets sutured to the support. B) Pericardial BHV, explanted after 7 years of intracardiac functioning, showing abrasions at the cusps base (1), commissural tears with leaflet prolapse (2) and nodular calcification (3). BHV: Bioprosthetic heart valve. No-React Stentless Bioprosthesis Biocor PSB/St. Jude Medical (No-React, detoxification, surfactant/heparin). Although promising, follow-up durability data for these new models are limited to only 7 8 years in the clinical setting, and there is conflicting evidence that the use of stentless valves results in improved clinical performance over stented bioprostheses [1]. The hopes and expectations are that the low-pressure tissue fixation and the addition of calcification-prevention agents will affect durability favourably, but many more years of detailed clinical follow-up are required to ascertain these expectations fully. 3. Macroscopic alterations in explanted bioprosthetic heart valves A wide variety of changes are noticeable in explanted BHVs, as outlined in Figure 1: abrasions at the cusps base, commissural tears with leaflet prolapse, and calcification. Throughout the years, attempts have been made at improving BHV performance at each of these levels. Implementing less traumatic techniques for tissue mounting, the use of flexible stents, the covering of stents with a thin layer of pericardium and the introduction of stentless valves [6,7] have greatly reduced the incidence of tears and abrasions in newer generations of BHVs [8]. Despite excellent design development, structural valve deterioration and calcification are still considered the main cause of BHVs replacement [9], and may possibly represent a threat to the new generation of BHVs. Due to its great clinical impact, ample research efforts have been directed towards understanding and reducing degenerative calcification of BHVs. 4. Factors that influence bioprosthetic heart valve calcification and experimental models The major factors involved in BHV calcification are hostrelated factors, mechanical stress, chemical treatment and implant composition. It is well known that host factors, such as age of recipient and altered calcium metabolism, are major determinants, as BHV calcification occurs at a highly accelerated rate in children and growing adults, as well as in patients with renal failure [10]. This was explained by the pro-calcific metabolism of younger patients, as compared with adults, which includes higher levels of blood calcium, phosphate, bone proteins, and enhanced parathyroid hormone and vitamin D metabolism [11]. In addition, a recent retrospective study showed that hypercholesterolaemia could also be a risk factor for BHV calcification [12]. As BHVs calcify less after being implanted in the right heart as compared with the left heart, it is believed that mechanical stress can also influence BHV calcification. In addition, within each BHV cusp, calcification appears to be concentrated in areas of high stress [4]. Improvements in valve designs that reduce valvular stress, such as flexible stents, are believed to reduce calcification, but definitive proof is pending future clinical studies. There are numerous in vitro studies showing that calcification in BHVs can occur through passive mechanisms [13-20]. However, due to inherent drawbacks of these models, more relevant animal models have been developed for the study of BHV calcification, the most popular being subcutaneous implantation in rats or mice, and valve replacement in sheep or calves [21]. Both models attain calcification of BHV tissues in a relatively short interval (several weeks in subdermal implants and several months in intracardiac implants), and the morphology of calcified deposits closely resembles those found in calcified BHVs explanted from patients. The subdermal model has the advantage of relative ease of use and low cost, but does not involve direct exposure of BHV tissue to flowing blood and haemodynamic stress. Conversely, intracardiac implants in large animals more closely mimic human implants, but are more technically demanding and very expensive. Rat subdermal implants have been used as screening tests for calcification-prevention treatments of BHVs, and in most studies, treatments efficient in the subdermal model also reduced calcification in circulatory implants. Although animal models provide a plethora of information about kinetics and factors involved in calcification, we still do not fully understand the mechanisms underlying calcification of BHVs in patients. The role of host cells in BHV calcification is still a matter of debate [22]. Prevention of cell infiltration in subdermally implanted BHV cusps (by enclosing tissues in Millipore chambers before implantation) did not affect calcification in animal models, suggesting a passive, rather than cell-mediated, process [23]. Moreover, graft rejection is not thought to play a major role in bioprosthetic valve mineralisation because BHV tissues implanted in nude mice (T cell-deficient) calcify to the same degree as implants in normal mice [24]. However, evidence also exists for the role of host reactions in BHV calcification. These include recent reports showing the involvement of low-level immune reactions [25-27] and the fact that inhibition of local proteases Expert Opin. Biol. Ther. (2004) 4(12) 1973

4 Prevention of calcification in bioprosthetic heart valves: challenges and perspectives reduces calcification [28]. Without disregarding the importance of the above-mentioned factors, two determinants of BHV calcification have received the utmost attention: glutaraldehyde fixation and tissue composition. 5. Glutaraldehyde as a villain and treatments targeting Glut Glutaraldehyde crosslinking of connective tissues, a process used to render animal tissues inert, non-biodegradable and non-antigenic, has revolutionised the surgical treatment of valvular disease by providing an excellent alternative to mechanical valves and promising long-term use without the need for lifelong anticoagulation. Paradoxically, glutaraldehyde fixation also encourages calcification. The ability of glutaraldehyde to induce BHV calcification has been clearly demonstrated by numerous experiments and has achieved a villain status [29]. Fresh, non-crosslinked tissues are degraded and resorbed rapidly after implantation, without calcification. This suggests that tissues incapable of remodelling will ultimately calcify, and that changes induced by glutaraldehyde are responsible for tissue calcification. In normal cusps, valvular interstitial cells and resident macrophages actively participate in maintaining tissue architecture via constant remodelling and scavenging [30]. It is known that scavenging of cell debris, a normal physiological tissue function, determines the fate of connective tissues. In tissues where scavenging mechanisms are limited (such as chemically fixed tissues), cell debris accumulate calcium rapidly and mediate calcification of adjacent collagen fibres. The presence of macrophages in calcified atherosclerotic plaques [31], calcified aortic aneurysms [32] and implanted BHVs [33] is good evidence for the body s attempt to rescue and scavenge calcifying tissues; however, the amplitude of calcification apparently overpowers these scavenging efforts. Biomaterials derived from chemically stabilised connective tissues probably calcify because of the lack of remodelling and scavenging. Besides preventing tissue remodelling, glutaraldehyde fixation directly induces cell death and formation of cellular debris, which in turn can serve as foci for calcification (see below). Incomplete glutaraldehyde binding to tissue proteins also yields cytotoxic aldehyde group residuals, which could also induce calcification. Therefore, prevention of calcification was approached by using non-aldehyde crosslinkers, neutralisation of free aldehyde groups and detoxification of glutaraldehyde residuals. 5.1 Calcification-prevention by using non-glutaraldehyde fixation chemistry In support of the glutaraldehyde hypothesis, it is remarkable that tissue crosslinking and stabilisation without the use of glutaraldehyde (non-glutaraldehyde fixation) does not induce tissue calcification in animal models [34]. Alternative crosslinking chemistries include: dye-mediated photo fixation [35-37] (PhotoFix, Carbomedics, Austin, TX), which crosslinks proteins at the aromatic amino acids level using a mild oxidation reaction catalysed by a light-sensitive dye carbodiimide-based fixation [38] (Ultifix, Medtronic, Minneapolis, MN), which involves carboxyl group activation with carbodiimides and crosslinking to free amine groups Other non-glutaraldehyde fixation procedures include the use of epoxy [39], diphenylphosphorylazide [40], acyl azides, cyanamide [41] or diisocyanates [42]. In addition, physical methods, such as ultraviolet light [43] and dehydration [11], were proposed for crosslinking of BHV tissues. Tissues treated with most of the above-mentioned processes exhibit good calcification-prevention properties, when compared with glutaraldehyde. It is assumed that these approaches are not inducing calcification simply because they do not employ the use of glutaraldehyde. Moreover, it is not apparent whether cells and matrix changes, which have been described as being responsible for calcification of BHV components, are effectively being prevented using these non-glutaraldehyde crosslinking procedures. For example, it is plausible to hypothesise that all of the non-glutaraldehyde crosslinking approaches will eventually devitalise cells, but it is difficult to imagine that such cells will not undergo calcification. None of these alternative crosslinking methods are in clinical use at present. These studies suggest that the chemical nature of glutaraldehyde may induce calcification, and that alternatives to glutaraldehyde fixation warrant further investigation. 5.2 Calcification-prevention treatments targeted at glutaraldehyde Besides the actual glutaraldehyde crosslinks, it is apparent that residual, loosely bound glutaraldehyde or unreacted aldehyde groups are also involved in BHV calcification. Neutralisation of free aldehyde groups with compounds that possess reactive primary amines and rinsing residual glutaraldehyde has been shown to reduce calcification in animal models. Several amino acids are included in this category, such as glutamine, glycine, homocysteic acid [44,45] and lysine [46-49]. AOA, a potent calcification-prevention agent for cusp and pericardial tissues [50], may be active as a glutaraldehyde-neutralising agent, but it may also influence calcification by mechanisms related to the reduction of calcium diffusion through tissues [51]. Similarly, other compounds described as calcification-prevention treatments have primary amine groups, such as amino-biphosphonates [52] and toluidine blue [53], but it is not clear whether their effect is related to aldehyde neutralisation alone. Biphosphonates, for example, are also believed to act as crystal poisons [54] by binding to preformed hydroxyapatite and preventing further increase in crystal size [55]. Urazole, a secondary amine compound [47,56], and possibly the No-React treatment (St. Jude proprietary detoxification process), may reduce calcification [57] by efficiently rinsing residual glutaraldehyde molecules from tissues Expert Opin. Biol. Ther. (2004) 4(12)

5 Simionescu 6. Tissue components influence calcification of bioprosthetic heart valves The main tissues used for the construction of BHVs are valve cusps, aortic wall and pericardium. All of these tissues are composed of specific cells embedded in an extracellular matrix composed of varying proportions of collagen, elastin, noncollagenous proteins and glycosaminoglycans. For example, cusp tissue is composed of 40% collagen, 4% glycosaminoglycans and a small amount of elastin. The adjacent aortic wall, however, is made of only 15% collagen, 50% elastin and small levels of glycosaminoglycans. Pericardial tissue is almost 90% collagen. It is of interest that after glutaraldehyde fixation, these tissues calcify to different extents in animal models of calcification [58]. Moreover, calcium deposition in fixed tissues can occur on different components. Devitalised cells are involved in calcification of all glutaraldehyde fixed tissues used in manufacturing of BHVs [59]; collagen also calcifies readily in cusp and pericardium [54], but elastin calcifies more severely in implants that contain portions of aortic wall [60]. Some of these components are natural substrates of calcification in normal bone physiology, whereas others are only involved in pathological calcification. During bone formation and mineralisation, bone cells undergo apoptosis, release matrix vesicles that accumulate calcium and promote collagen calcification [61]. In vascular pathology, it is well known that medial aortic calcinosis (calcification of elastic fibres in the media) is mainly associated with elastin, and may also involve newly formed bone cells [62]. Glycosaminoglycans and non-collagenous proteins seem to be involved mainly in the regulation of calcification processes [63], but these roles are still disputed. In normal bone formation, calcium accumulation is preceded by enzymatic degradation of proteoglycans (PGs) [64], suggesting that they serve as a role in inhibiting and regulating the progression of calcification. Other regulatory proteins, such as matrix γ-carboxyglutamic acid (Gla) protein [65] and osteopontin [66], may play a role in BHV calcification, but their role is still under investigation. Overall, these data suggest that most matrix components present in tissues used for fabrication of BHVs are capable of calcifying, possibly each component having different affinity for calcium and kinetics of calcium accumulation. Due to the vital role of cells and matrix components, prevention of BHV calcification was approached by attempts to remove or extract cells, by structural modification of collagen and elastin, and by stabilisation or addition of natural calcification inhibitors. 6.1 Calcification of glutaraldehyde fixed cells mechanisms and prevention Glutaraldehyde was originally introduced as a fixative for transmission electron microscopy (TEM) > 35 years ago [67] and is still in use for ultrastructural studies. Whereas high glutaraldehyde concentrations (2 3%) are used for TEM, for BHV preparation, tissues are treated with % neutral buffered glutaraldehyde. Long exposure to such low dilutions of glutaraldehyde induces adequate (but not optimal) preservation of collagenous structures, but also produces severe cell alterations, which result in formation of cell debris that resemble matrix vesicles formed by bone cells [68,69]. The degree of ultrastructural integrity of cells in BHVs correlates well with their ability to nucleate calcification [70,71]. Figure 2 depicts a segment of a cell membrane, putative component of a matrix vesicle. Transmembrane proteins are embedded in a double-layered lipid membrane, which exposes towards the internal aspect, a high number of acidic phospholipids such as phosphatidyl serine. Passive transporters and ATP-mediated active pumps maintain normal calcium homeostasis, which sustains a 10,000-fold calcium ion concentration gradient in the external compartment (EXT), as compared to the cytoplasm. Calcium accumulation in matrix vesicles occurs by illdefined mechanisms, which probably involve alterations in calcium homeostasis and massive calcium influx [54]. Calcium ions may concentrate on the interior aspect of the membrane by binding to acidic phospholipids and calcium-binding proteins. Inorganic phosphate ions, produced by phosphatases, combine with calcium ions immobilised onto the cell membrane. A continuous influx of calcium ions leads to the formation of hydroxyapatite. Calcification of cell debris in BHVs may possibly take place via these same mechanisms. Some of the main calcification-prevention treatments aim to extract cell lipids, thus removing potential sites of calcification. These treatments include the use of organic solvents, such as ethanol [72] and chloroform/methanol [73], and detergents, such as sodium dodecyl sulfate [74], Tween 80 [75] and Triton X-100 [76]. It is therefore possible that tissues from which cell debris has been removed would benefit from a delay in initiation of calcification. However, it is not evident to what extent these treatments effectively reduce calcification of collagen or elastin, or whether they can prevent readsorption of lipoproteins from blood. Moreover, there is a good deal of evidence for host cell infiltration in implanted BHVs [33,61,77,78]. These cells may be entrapped in the glutaraldehyde-fixed matrix and eventually die, to possibly induce late calcification of BHVs. 6.2 Calcification of collagen and elastin mechanisms and prevention Collagen fibres are present in all tissues used for BHV and provide the architectural framework and mechanical strength required for function in a BHV. Collagen molecules (Figure 3) are staggered within the collagen fibre, creating an apparently empty area, also known as a hole zone, in which PGs reside [79], presumably protecting collagen from calcification. This same area has been shown to be the initial site of calcium deposition onto bone collagen fibres [80,81]. Matrix metalloproteases (MMPs) [82] and glycosaminoglycans-degrading enzymes [83] can degrade PGs and expose sites prone to calcification. Progression of collagen calcification can lead to the formation of hydroxyapatite, which, in turn, can grow into large deposits. Expert Opin. Biol. Ther. (2004) 4(12) 1975

6 Prevention of calcification in bioprosthetic heart valves: challenges and perspectives EXT INT A 1 ATP Ethanol pretreatment of glutaraldehyde-fixed porcine aortic valve cusps [73] and bovine pericardium [75] significantly reduces calcification in experimental models. This anticalcification mechanism is hypothesised to be related to the extraction of cholesterol and phospholipids, to permanent alterations in collagen structure [73], and to a putative interaction with glutaraldehyde [72]. However, this effect is not limited to ethanol alone. Similar results were obtained with ether, methanol, chloroform/methanol and isopropanol [75]. Despite complete removal of lipids and alterations in collagen structure, ethanol is not 2 3 Ca 2+ PO 2 3- Acidic phospholipids Hydroxyapatite B C D Figure 2. Cell calcification. A) Schematic diagram showing hypothetical events associated with cell calcification. See details in section 6.1. B) TEM picture showing a calcified cell in a 21-day subdermal implant of glutaraldehyde-fixed pericardium. Calcium deposits appear as dark needle-shaped crystals. C) and D) show higher magnifications of cell debris and matrix vesicle calcification. Original magnification, B) 30,000, C) and D) 70,000. ATP: Phosphatase substrate; Ca: Calcium; EXT: External compartment; INT: Internal aspect; PO: Inorganic phosphate; TEM: Transmission electron microscopy. equally effective in preventing calcification of the BHV aortic wall. This may reflect the relatively small contribution of collagen to wall calcification, as compared with elastic fibres, which are not affected by ethanol treatments [84]. Elastic fibres are abundant structural proteins in the vascular wall and also functional components of aortic cusps [85]. There is ample evidence showing that degeneration and calcification of elastic fibres occurs in BHVs, as well as in human allografts and pathological vascular calcification [31,86-90]. Ultrastructurally, elastic fibres consist of a core composed of Expert Opin. Biol. Ther. (2004) 4(12)

7 Simionescu B A PG 'Hole zone' crosslinked elastin molecules and a microfibrillar component located mainly around the periphery of the amorphous component [91]. The external coating, rich in acidic components such as fibrillin, is believed to protect elastin from calcification [92,93]. Disruption of elastin fibre integrity has been implicated in the initiation and progression of aortic calcification, and ultrastructural studies have shown that in BHVs, this protective coating is not fully preserved [94]. Thus, the hypothesised pathway by which elastin calcifies in BHVs 1 Figure 3. Collagen calcification. A) Schematic diagram showing hypothetical events associated with collagen calcification. See details in section 6.2. B) TEM picture showing a cross-section through a calcified collagen fibre in a 21-day subdermal implant of glutaraldehyde-fixed pericardium. Collagen fibres appear as round white structures, and calcium deposits as dark needle-shaped crystals. C) Shows a higher magnification of a single collagen fibre sectioned longitudinally. Original magnification, B) 30,000 and C)70,000. PG: Proteoglycan; TEM: Transmission electron microscopy. C 3 (Figure 4) involves degradation of the protective coating containing elastin-associated microfibrils, PGs, and exposure of the elastin core to the calcium-rich extracellular matrix milieu. Consequently, elastin binds calcium ions (circles) followed by inorganic phosphate ions, leading to the formation of hydroxyapatite on the surface of the elastic fibre. Elastin has particular properties that set it apart from other matrix components. Glutaraldehyde does not chemically react with elastin because the mature protein does not 2 Expert Opin. Biol. Ther. (2004) 4(12) 1977

8 Prevention of calcification in bioprosthetic heart valves: challenges and perspectives A B EAMF and PG possess sufficient reactive amine groups [95]. Paradoxically, glutaraldehyde fixation is not required for elastin to calcify [96]. Elastin has been shown to possess a unique structure that facilitates calcium binding [97]. Moreover, the lack of reactivity of glutaraldehyde with elastin also implies that elastin is not protected against the activity of degradative enzymes, such as MMPs [98]. The consequences of this lack of fixation are vital, as progressive degradation of elastin may further aggravate calcification and sustain alterations of mechanical properties of cusp [85] and aortic wall segments of BHVs. Elastin degradation products could possibly elicit immune responses and trigger unwanted reactions in host cells such as protease release and apoptosis [99-101]. In addition, there seems to be a direct correlation between elastin degradation and calcification [102], and local delivery of MMP inhibitors significantly reduced calcification of subdermally implanted elastin [28]. EL Figure 4. Elastin calcification. A) Schematic showing hypothetical events associated with calcification of elastic fibres. See details in section 6.2. B) TEM picture showing elastin-associated calcification in a 21-day subdermal implant of glutaraldehyde-fixed porcine aorta. Calcium deposits appear as dark needle-shaped crystals. C) Shows a higher magnification of the area outlined by dashed lines in B). Original magnification, B) 30,000 and C) 65,000. EAMF: Elastin-associated microfibrils; EL: Elastin; PG: Proteoglycans; TEM: Transmission electron microscopy. C With the advent of stentless BHVs that expose large portions of aortic wall to flowing blood, more effort has been directed towards calcification-prevention treatments targeted at elastin calcification. One of the most studied treatments in this category is the use of aluminium ions. This treatment is part of the BiLinx calcification-prevention process (St. Jude Medical), under clinical evaluation at present. Aluminium ions bind strongly to elastin and induce conformational changes in the elastin structure, which reduces the affinity of elastin towards calcium ions [96]. Aluminium ions also partially stabilise pure elastin against the action of degrading enzymes, and protect it from degeneration and calcification when tested in animal models [102]. However, binding of aluminium to elastin does not seem to be completely irreversible [103], and it is also not yet clear what the effects of aluminium on cell- and collagen-mediated calcification are. In an attempt to improve elastin stabilisation further, tannic acid, a polyphenol belonging 1978 Expert Opin. Biol. Ther. (2004) 4(12)

9 Simionescu to the galloylglucose family, was recently shown to bind to aortic elastin, resulting in an improved resistance to elastase and a significant reduction in calcification of glutaraldehydetreated aorta in the rat subdermal model [98]. Taken together, these results support the hypothesis that protection of elastin from degradation has the potential to extend BHV durability in the clinical setting. 6.3 Role of non-collagenous components Non-collagenous components play important roles in calcium homeostasis within the extracellular matrix. Bone sialoprotein, a bone protein, and bone morphogenetic protein-2, a member of the transforming growth factor cytokine superfamily, are known to participate in the regulation of bone development and maturation [104]. Both of these proteins have also been found in calcific aortic stenosis, indicating that valvular calcification occurs via mechanisms similar to bone formation [105]. Other bone-associated proteins, such as osteopontin, osteocalcin and osteonectin, have also been demonstrated in calcifying atheromatous plaques and in experimentally induced arterial calcification; however, it is believed that these proteins may play a significant role in the regulation (and not initiation) of calcification [106]. In support of this hypothesis, osteocalcin-deficient mice showed increased bone formation without impairing bone resorption [107]. Non-collagenous components have also been shown to associate with BHV calcification [108]. Interestingly, osteopontin (but not osteocalcin, bone sialoprotein or osteonectin) was found to be present in calcified, explanted porcine BHVs [109]. Moreover, glutaraldehyde-fixed cusps showed enhanced calcification when implanted subdermally in osteopontin-null mice, strongly suggesting a regulating role for osteopontin [110]. Overall, these studies point to the involvement of non-collagenous proteins in BHV calcification, and their application as a calcification-prevention strategy merits further investigation. 6.4 Glycosaminoglycans and proteoglycans Glycosaminoglycans (GAGs) are acidic, unbranched polysaccharides present in all vascular connective tissues, including valves, aorta and pericardium [79]. These polysaccharides are bound to core proteins (thus forming PGs) as large polymers that lack protein cores, such as hyaluronic acid. GAGs are distributed in the ground matrix of cusp and wall tissues, and are also associated with the surfaces of collagen and elastic fibres [94]. Loss of GAGs from within the cusp structure has been noted in valves obtained from patients suffering from congenital defects, rheumatic fever and old age, all of which are associated with valve failure [111]. Furthermore, GAGs are also completely lost from implanted BHVs [112], suggesting an insufficient stabilisation by glutaraldehyde. This is not surprising, because GAGs lack the amine functionalities necessary for crosslinking by aldehydes. Experimental studies have shown that GAGs are lost during glutaraldehyde fixation of porcine cusps and walls [94]. In addition, > 80% of tissue GAGs were lost from BHVs after accelerated fatigue testing, as well as after implantation in the sheep circulatory model [113]. Overall, these data showed that glutaraldehyde does not stabilise GAGs. As GAGs within BHVs play an important role in maintaining proper mechanical functions, loss of GAGs may contribute significantly to degeneration of BHVs. Moreover, GAGs are hypothesised to prevent calcium deposition by chelating calcium and preventing hydroxyapatite nucleation [114]. The mechanisms responsible for GAG loss are not fully known, but it is possible that GAGs, which are not crosslinked by glutaraldehyde, may be susceptible to GAG-degrading enzymes present in cusp tissues [83]. Covalent binding of GAGs, such as heparin, was shown to reduce calcification of BHVs in experimental models [115]. Very few attempts have been made to stabilise GAGs in BHVs. As glutaraldehyde is not effective, periodate oxidation was used for crosslinking of valvular GAGs. This is based on the relative specificity of periodate to oxidise geminal diols contained within the structure of GAGs, allowing the formation of aldehyde groups within the GAG chain [116]. These aldehyde groups would then react with amine groups found in the lysine residues of collagen molecules, achieving GAG fixation. Model studies using mixtures of hyaluronic acid and pure type I collagen showed that periodate oxidation induces GAG immobilisation onto collagen fibres. Moreover, when implanted subdermally in juvenile rats, periodate-pretreated, glutaraldehyde-fixed cusps showed 40% less calcification as compared with glutaraldehyde alone [116]. It is not known how various calcification-prevention treatments affect the stability of GAGs. Taken together, these studies showed that GAGs are important functional components of BHV tissues and that new methods of GAG stabilisation deserve further investigation. 7. Expert opinion and conclusion The ideal BHV has to fulfil a number of prerequisites that relate mostly to clinical durability and safety. It must be sturdy enough to last the lifetime of a young patient ( 50 years), simple to implant, widely available, and the design must not allow regurgitant backflow. In addition, it should have a low incidence of thromboembolism, be fully resistant to proteolysis, be non-antigenic and non-toxic. Such a device does not exist at present. Seemingly, BHV calcification after implantation is a highly unregulated process. Calcium deposition, like immunity and coagulation, could be considered an acceptable protective mechanism, with devastating effects when released from homeostatic control. In support of this hypothesis, it is noteworthy that normal bones, in which calcification is highly regulated, contain a maximum of 50% mineral content, whereas highly calcified BHVs can reach > 90% mineral content [64,117]. As described in this paper, numerous approaches for prevention of calcification in BHVs have been described in the literature during the last 30 years, but only a few of them Expert Opin. Biol. Ther. (2004) 4(12) 1979

10 Prevention of calcification in bioprosthetic heart valves: challenges and perspectives Table 1. Calcification-prevention treatments: hypothetical targets and mechanisms of action. Target Treatment Mechanisms Glutaraldehyde PhotoFix, carbodiimides Non-glutaraldehyde fixation UV light, dehydration, cyanamide, acylazide, epoxy Amino-oleic acid Neutralisation and detoxification Amino-biphosphonates Toluidine blue Lysine, homocysteic acid Propylene glycol Urazole Cell debris Ethanol Removal of cellular lipids Sodium dodecyl sulfate Tween 80. Triton X-100 Collagen Ethanol Structural modification Elastin Aluminium chloride Tannic acid Structural modification Stabilisation Other matrix components Osteopontin Addition or preservation of calcification inhibitors Heparin, glycosaminoglycans Periodate Matrix enzymes MMP inhibitors Block degeneration of matrix components Hydroxyapatite growth Biphosphonates Amino-biphosphonates Inhibit crystal growth Crystal poison Calcium transport Amino-oleic acid Block tissue transport of calcium MMP: Matrix metalloprotease. have been studied in detail from a mechanistic point of view. Table 1 shows a summary of hypothetical targets and mechanisms of action of these treatments. Some of these statements are backed up by adequate mechanistic data (ethanol, aluminium, AOA, biphosphonates), while the mechanisms of others are rather speculative. There are numerous unresolved fundamental issues that deserve further exploration. First, we do not understand precisely what mechanisms prevent cardiovascular tissues (valves, aorta, pericardium) from calcifying under physiological conditions. Furthermore, the mechanisms by which these same tissues calcify in pathology elude us. This knowledge is imperative because the better we understand mechanisms of pathological calcification, the easier it will be to devise calcificationprevention treatments for BHVs. Chemical stabilisation of animal tissues is a requirement for the clinical use of BHVs. Despite the fact that glutaraldehyde pretreatment induces calcification, there is no convincing evidence yet for a better alternative. While waiting for substitute chemical crosslinkers to be made available, we should maintain glutaraldehyde as the fixative of choice and try to develop better treatments to prevent calcification. In this respect, we need to invest more effort in understanding how glutaraldehyde changes the properties of cells and matrix components. Because resident cells cannot renew matrix composition in BHVs, the fate and durability of BHVs mainly depend on the mechanical and biochemical longevity of each component. We should not overestimate the ability of glutaraldehyde to equally stabilise all matrix molecules. Although glutaraldehyde efficiently stabilises collagen, there is ample evidence that glutaraldehyde does not crosslink or stabilise elastin and glycosaminoglycans. The consequences of this lack of stabilisation may be elastin degeneration and calcification, and progressive loss of glycosaminoglycans. We should also not underestimate the power of matrix-degrading enzymes (MMPs and glycosaminoglycan-degrading enzymes), whose activity in tissues is reduced by glutaraldehyde, but not completely inhibited. Such enzymes can contribute to elastin degradation and calcification, collagen weakening and removal of calcification inhibitors. With few exceptions, the role of natural inhibitors of calcification has not been fully investigated and warrants further studies. Another matter of controversy is the relevance of animal models for the study of BHV calcification. Subdermal implants in juvenile rats and intracirculatory implants in larger animals may serve as screening tests for calcificationprevention treatments, but we cannot fully anticipate a clinical outcome using these models alone. Furthermore, despite numerous studies supporting the role of glutaraldehyde, cells and matrix components in the calcification of BHVs, we do not yet have all the information to evaluate how these factors interact with host-related factors and mechanical stress. As described above, most matrix components may calcify independently in various physiological and pathological situations, but these components exhibit different calcium-binding 1980 Expert Opin. Biol. Ther. (2004) 4(12)

11 Simionescu affinities and varying kinetics of calcium deposition. We need to critically evaluate whether treatments (that claim to act on a specific substrate) have effects on other components. Therefore, blocking one component may not be sufficient to prevent calcification completely, as another will eventually calcify. As there is sufficient data showing that more than one mechanism may be involved in BHV calcification, exaggerated enthusiasm over a single universal treatment needs to be tempered and focused more on combinations of treatments. Bibliography Papers of special note have been highlighted as either of interest ( ) or of considerable interest ( ) to readers. 1. GUDBJARTSSON T AS, COHN LH: Mechanical/bioprosthetic mitral valve replacement. In: Cardiac Surgery in the Adult. Cohn LH, Edmunds LH Jr (Eds), McGraw-Hill, New York, USA (2003): Updates on BHVs available at present. 2. JAMIESON WR, BURR LH, MUNRO AI, MIYAGISHIMA RT: Carpentier-Edwards standard porcine bioprosthesis: a 21-year experience. Ann. Thorac. Surg. (1998) 66(6 Suppl.):S40-S SCHOEN FJ, LEVY RJ: Founder s Award, 25th Annual Meeting of the Society for Biomaterials, perspectives. Providence, RI, April 28-May 2, Tissue heart valves: current challenges and future research perspectives. J. Biomed. Mater. Res. (1999) 47(4): One of a series of reviews providing updates on the biology and pathology of BHVs. 4. SACKS MS, SCHOEN FJ: Collagen fiber disruption occurs independent of calcification in clinically explanted bioprosthetic heart valves. J. Biomed. Mater. Res. (2002) 62(3): One of a series of papers demonstrating the role of mechanical failure. 5. VESELY I: The evolution of bioprosthetic heart valve design and its impact on durability. Cardiovasc. Pathol. (2003) 12(5): Historical update on the evolution of BHV design issues. 6. DEAC RF, SIMIONESCU D, DEAC D: New evolution in mitral physiology and surgery: mitral stentless pericardial valve. Ann. Thorac. Surg. (1995) 60(2 Suppl.):S433-S438. Acknowledgements 7. DAVID TE: Aortic valve replacement with stentless porcine bioprostheses. J. Card. Surg. (1998) 13(5): GARCIA PAEZ JM, JORGE-HERRERO E: Assessment of pericardium in cardiac bioprostheses. A review. J. Biomater. Appl. (1999) 13(4): One of a series of reviews providing updates on pericardial BHVs. 9. SCHOEN FJ: Future directions in tissue heart valves: impact of recent insights from biology and pathology. J. Heart Valve Dis. (1999) 8(4): CHAMPSAUR G, ROBIN J, TRONC F et al.: Mechanical valve in aortic position is a valid option in children and adolescents. Eur. J. Cardiothorac. Surg. (1997) 11(1): BERREBI AJ, CARPENTIER SM, PHAN KP et al.: Results of up to 9 years of high-temperature-fixed valvular bioprostheses in a young population. Ann. Thorac. Surg. (2001) 71(5 Suppl.):S353-S FARIVAR RS, COHN LH: Hypercholesterolemia is a risk factor for bioprosthetic valve calcification and explantation. J. Thorac. Cardiovasc. Surg. (2003) 126(4): PETTENAZZO E, DEIWICK M, THIENE G et al.: Dynamic in vitro calcification of bioprosthetic porcine valves: evidence of apatite crystallization. J. Thorac. Cardiovasc. Surg. (2001) 121(3): One of a series of papers on in vitro calcification of BHVs. 14. TOMAZIC BB, CHOW LC, CAREY CM, SHAPIRO AJ: An in vitro diffusion model for the study of calcification of bovine pericardium tissue. J. Pharm. Sci. (1997) 86(12): ZIMMERMANN B, WACHTEL HC, NOPPE C: Patterns of mineralisation The author is greatly indebted to Professor R Deac MD, PhD, eminent cardiovascular surgeon and scientist, for providing access to hundreds of explanted bioprostheses and for the introduction to the fascinating field of bioprosthetic heart valves. The author is also profoundly thankful to Professor N Simionescu MD, PhD, illustrious cellular and molecular biologist, for leading his first steps into electron microscopy, and for relentless insights into cardiovascular biology and pathology. in vitro. Cell Tissue Res. (1991) 263(3): RATTNER A, SABIDO O, LE J et al.: Mineralisation and alkaline phosphatase activity in collagen lattices populated by human osteoblasts. Calcif. Tissue Int. (2000) 66(1): AKHOUAYRI O, LAFAGE-PROUST MH, RATTNER A et al.: Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels. J. Cell. Biochem. (1999) 76(2): ANDRE-FREI V, CHEVALLAY B, ORLY I et al.: Acellular mineral deposition in collagen-based biomaterials incubated in cell culture media. Calcif. Tissue Int. (2000) 66(3): DAHM M, PRUFER D, KRUMMENAUER F et al.: In vitro effects of anticalcification treatment on the calcium uptake of bioprosthetic materials. J. Heart Valve Dis. 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