Heart valve substitutes are of two principal types:

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1 Calcification of Tissue Heart Valve Substitutes: Progress Toward Understanding and Prevention Frederick J. Schoen, MD, PhD, and Robert J. Levy, MD Department of Pathology, Brigham and Women s Hospital and Harvard Medical School, the Harvard-MIT Division of Health Sciences and Technology, Boston, Massachusetts, and the Abramson Pediatric Research Center, The Children s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania Calcification plays a major role in the failure of bioprosthetic and other tissue heart valve substitutes. Tissue valve calcification is initiated primarily within residual cells that have been devitalized, usually by glutaraldehyde pretreatment. The mechanism involves reaction of calcium-containing extracellular fluid with membraneassociated phosphorus to yield calcium phosphate mineral deposits. Calcification is accelerated by young recipient age, valve factors such as glutaraldehyde fixation, and increased mechanical stress. Recent studies have suggested that pathologic calcification is regulated by inductive and inhibitory factors, similar to the physiologic mineralization of bone. The most promising preventive strategies have included binding of calcification inhibitors to glutaraldehyde fixed tissue, removal or modification of calcifiable components, modification of glutaraldehyde fixation, and use of tissue cross linking agents other than glutaraldehyde. This review summarizes current concepts in the pathophysiology of tissue valve calcification, including emerging concepts of endogenous regulation, progress toward prevention of calcification, and issues related to calcification of the aortic wall of stentless bioprosthetic valves. (Ann Thorac Surg 2005;79: ) 2005 by The Society of Thoracic Surgeons Heart valve substitutes are of two principal types: mechanical prosthetic valves with components manufactured of nonbiologic material (eg, polymer, metal, carbon) or tissue valves which are constructed, at least in part, of either human or animal tissue [1, 2]. See page 905 Address reprint requests to Dr Schoen, Department of Pathology, Brigham and Women s Hospital, 75 Francis St, Boston, MA 02115; fschoen@partners.org. Tissue valves have been used since the early 1960s when aortic valves obtained fresh from human cadavers were transplanted to other individuals (homografts). A decade later, chemically preserved stent-mounted tissue bioprosthetic valves (generally termed bioprostheses) were commercially produced and implanted. Today, stentmounted glutaraldehyde preserved porcine aortic valves and bovine pericardial bioprosthetic valves are used widely, and stentless valves have been introduced. Approximately 85,000 substitute valves are implanted in the United States and 275,000 worldwide each year, of which we presently estimate that approximately half are mechanical and half are tissue, suggesting a shift toward increasingly greater usage of tissue valves over the last decade. Within 10 years postoperatively, prosthesis-associated problems overall necessitate reoperation or cause death in at least 50% to 60% of patients with substitute valves [3, 4]. The rate is similar for mechanical prostheses and bioprostheses; however, the frequency and nature of specific valve-related complications vary with the prosthesis type, model, site of implantation, and certain characteristics of the patient. Specifically, mechanical prosthetic valves have a substantial risk of systemic thromboemboli and thrombotic occlusion, and the chronic anticoagulation therapy required in all mechanical valve recipients potentiates hemorrhagic complications. Nevertheless, contemporary mechanical prostheses are durable. In contrast, tissue valves have a low rate of thromboembolism without anticoagulation, owing to a central pattern of flow similar to that of the natural heart valves and cusps composed of valvular or nonvalvular animal or human tissue. However, a high rate of valve failure with structural dysfunction owing to progressive tissue deterioration (including calcification and noncalcific damage) undermines their attractiveness [2, 5 9]. Structural dysfunction is the major cause of failure of bioprosthetic heart valves (flexible-stent-mounted, glutaraldehyde-preserved porcine aortic valves and bovine pericardial valves). Within 15 years following implantation, approximately 50% of porcine aortic valves suffer the major prosthesis-related complication with this type of valve tissue failure. The principal underlying pathologic process is cuspal calcification; secondary tears frequently precipitate regurgitation. Calcification can also cause pure stenosis owing to cuspal stiffening. Calcific deposits are usually localized to cuspal tissue (intrinsic Dr Schoen discloses that he has financial relationships with CarboMedics, Edwards Lifesciences, Medtronic, and St. Jude Medical by The Society of Thoracic Surgeons /05/$30.00 Published by Elsevier Inc doi: /j.athoracsur

2 Ann Thorac Surg REVIEW SCHOEN AND LEVY 2005;79: CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 1073 Fig 1. Extended hypothetical model for the calcification of bioprosthetic tissue. This model considers host factors, implant factors, and mechanical damage and relates initial sites of mineral nucleation to increased intracellular calcium in residual cells and cell fragments in bioprosthetic tissue. The ultimate result of calcification is valve failure, with tearing or stenosis. The key contributory role of existing phosphorus in membrane phospholipids and nucleic acids in determining the initial sites of crystal nucleation is emphasized, and a possible role for the independent mineralization of collagen is acknowledged. Mechanical deformation probably accelerates to both nucleation and growth of calcific crystals. Modified by permission from Schoen FJ: Interventional and surgical cardiovascular pathology: clinical correlations and basic principles. Philadelphia: WB Saunders, calcification), but calcific deposits extrinsic to the cusps may develop in thrombi or endocarditic vegetations (extrinsic calcification). Calcification is markedly accelerated in younger patients; children and adolescents have an especially accelerated course, and older patients have a lower rate of bioprosthetic valve degeneration. Progressive collagen deterioration, independent of calcification, is also a likely important contributor to the limited durability of bioprosthetic valves [10, 11]. Bovine pericardial valves also calcify but design-related tearing has been prominent [12, 13]. Determinants, Mechanisms, and Regulation Pathological analysis of tissue valve explants from patients and experiments in animal models using bioprosthetic heart valve tissue have elucidated many aspects of the pathophysiology of this important clinical problem. The current understanding of the determinants, mechanisms, and regulation of tissue calcification is summarized below and in Figure 1. The most useful experimental models have been orthotopic tricuspid or mitral replacements or conduitmounted valves in sheep or calves [14, 15], and isolated tissue samples implanted subcutaneously in very young, rapidly growing mice, rabbits, or rats [16 18]. In both circulatory and noncirculatory models, bioprosthetic tissue calcifies progressively with a morphology similar to that observed in clinical specimens, but with markedly accelerated kinetics. The subcutaneous model has emerged as a technically convenient and economically advantageous vehicle for investigating host and implant determinants and mechanisms of mineralization, as well as for screening potential strategies for inhibition of calcification. In general, large animal valve replacements can (1) elucidate further the processes accounting for clinical failures, (2) evaluate the performance of design and biomaterials modifications in valve development studies, (3) assess the importance of blood/surface interactions, and (4) provide data required for approval by regulatory agencies. Despite the potential of in vitro models to elucidate the pathophysiology of biomaterials calcification, such systems have not been generally useful in this regard [19 22]. Determinants The determinants of bioprosthetic valve and other biomaterial mineralization include factors related to (1) host metabolism, (2) implant structure and chemistry, and (3) mechanical factors. Natural cofactors and inhibitors may also play a role (see below). Accelerated calcification is associated with young recipient age, glutaraldehyde fixation, and high mechanical stress. Calcification is more rapid and aggressive in the young; the rate of failure of bioprostheses is approximately 10% in 10 years in elderly recipients, but is nearly uniform in less than 4 years in most adolescent and preadolescent children. Although the relationship is well-established, the mechanisms accounting for the effect of age are uncertain. The accelerated onset of calcific failure in young patients is simulated by the rapid calcification that occurs in young experimental animals. The structural elements of the biomaterial and their modification by processing clearly play an important role. Cells are the predominant location of mineralization (see later) and the usual pretreatment of commercially available bioprostheses with glutaraldehyde, done to improve tissue durability, also potentiate calcification [23, 24]. Calcification of porcine aortic valve and bovine pericardium are qualitatively, quantitatively, and mechanistically similar. It has been hypothesized that the cross-linking agent glutaraldehyde stabilizes and perhaps modifies phosphorous-rich calcifiable structures in the bioprosthetic tissue. These sites are capable of mineralization upon implantation when exposed to the comparatively high calcium levels of extracellular fluid. Paradoxically, high glutaraldehyde pretreatment conditions (3% glutaraldehyde compared with 0.6% or less presently used commercially) seem to be protective against calcification of bioprosthetic tissue [25]. Calcification of the extracellular matrix structural proteins collagen and elastin has been observed in clinical and experimental implants of bioprosthetic and homograft valvular and vascular tissue, and has been stud-

3 1074 REVIEW SCHOEN AND LEVY Ann Thorac Surg CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 2005;79: ied using a rat subdermal model. Collagen and elastic fibers can serve as nucleation sites for calcium phosphate minerals, independent of cellular components [2, 16 18, 26]. Cross-linking by either glutaraldehyde or formaldehyde promotes the calcification of collagen sponge implants made of purified collagen but the extent of calcification does not correlate with the degree of cross linking [27]. In contrast, the calcification of elastin appears independent of whether pretreatment has occurred [28]. Calcification has also complicated the clinical use and experimental investigation of heart valves composed of bovine pericardium [12, 29] and polymers (eg, polyurethane) [30, 31]. Furthermore, both intrinsic and extrinsic mineralization of a biomaterial is generally enhanced at the sites of intense mechanical deformations generated by motion, such as the points of flexion in heart valves [2, 16, 17, 32]. Mechanisms The mineralization process in the cusps of bioprosthetic heart valves is initiated predominantly within nonviable connective tissue cells that have been devitalized but not removed by glutaraldehyde pretreatment procedures [2, 16 18, 33, 34]. This dystrophic calcification mechanism involves reaction of calcium-containing extracellular fluid with membrane-associated phosphorus, causing calcification of the cells. This likely occurs because the normal extrusion of calcium ions is disrupted in cells that have been rendered nonviable by glutaraldehyde fixation. Normally, the plasma-extracellular calcium concentration is 1 mg/ml (approximately 10 3 M); since the membranes of healthy cells pump calcium out, the concentration of calcium in the cytoplasm is normally 1,000 to 10,000 times lower (approximately 10 7 M). However, the physiologic mechanisms for elimination of calcium from the cells are not available in glutaraldehydepretreated tissue. The cell membranes and other intercellular structures are high in phosphorus (as phospholipids, especially phosphatidyl serine, and the phosphate backbone of the nucleic acids); they can bind calcium and serve as nucleators. Initial calcification deposits eventually enlarge and coalesce, resulting in grossly mineralized nodules that stiffen and weaken the tissue and thereby cause a prosthesis to malfunction. Regulation Although pathologic calcification has typically been considered a passive, unregulated, and degenerative process, recent studies suggest that the mechanisms responsible for pathologic calcification may be regulated, similarly to the physiologic mineralization of bone and other hard tissues [35 37]. In normal blood vessels and valves, inhibitory mechanisms outweigh procalcific inductive mechanisms and calcification does not occur. In contrast, in bone and pathologic tissues, the inductive mechanisms dominate. In the process of normal bone calcification, the growth of apatite crystals is regulated by several noncollagenous matrix proteins including: (1) osteopontin, an acidic calcium-binding phosphoprotein with high affinity to hydroxyapatite that is abundant in foci of dystrophic calcification; (2) osteonectin; and (3) osteocalcin [38], and other -carboxyglutamic acid (GLA)- containing proteins, such as matrix GLA protein (MGP). Naturally occurring inhibitors crystal nucleation and growth may also play a role in the regulation of calcific degeneration of natural and bioprosthetic valves and other cardiovascular calcification such as arteriosclerosis [39, 40]. Specific inhibitors in this context include osteopontin [41] and high-density liproprotein (the good cholesterol) [42]. The role in pathological mineralization of naturally occurring promineralization cofactors, such as inorganic phosphate [43], bone morphogenetic protein [44], proinflammatory lipids [35], and other substances (eg, cytokines), as well as inhibitors, is an active area of research [45]. Recent evidence suggests that hypercholesterolemia may be a risk factor in clinical bioprosthetic valve calcification [46, 47]. The noncollagenous proteins osteopontin, TGF-, and tenascin-c involved in bone matrix formation and tissue remodeling have been demonstrated in clinical calcified bioprosthetic heart valves, natural valves, and arteriosclerosis, suggesting that they play a regulatory role in these forms of pathologic calcification in humans [48 50]. Evidence for the active regulation of cardiovascular calcification also derives from tissue culture models of vascular cell calcification, which mimic vascular calcification in vivo and genetic studies in mice. For example, osteopontin inhibits and proinflammatory lipids and cytokines enhance the mineralization of smooth muscle cell cultures [51 53]. In transgenic mouse models, in which the gene for the MGP was knocked out [54] or the osteopontin gene was inactivated [55], severe calcification of blood vessels resulted. Moreover, inhibition of matrix remodeling metalloproteinases inhibits calcification of elastin implanted subcutaneously in rats [56]. A potential role for inflammatory and immune processes has been postulated by some investigators [57 59]. Although (1) experimental animals can be sensitized to both fresh and cross-linked bioprosthetic valve tissues [60, 61], (2) antibodies to valve components can be detected in some patients following valve dysfunction [62], and (3) failed tissue valves often have mononuclear inflammation, no definite role has been demonstrated for circulating macromolecules or cells. In particular, the presence of mononuclear cells in a failed tissue valve does not equate to immunologic rejection. In all papers thus far purporting to demonstrate immune-mediated injury, no evidence is presented to suggest that the mononuclear inflammatory cells are causing functional valve degeneration. Indeed, many lines of evidence suggest that neither nonspecific inflammation nor specific immunologic responses appear to favor bioprosthetic tissue calcification, and no causal or contributory immunologic basis has been demonstrated for bioprosthetic valve calcification or failure. For example, in experiments in which valve cusps were enclosed in filter chambers that prevent host cell contact with tissue but allow free diffusion of extracellular fluid and implantation of valve tissue in congen-

4 Ann Thorac Surg REVIEW SCHOEN AND LEVY 2005;79: CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 1075 itally athymic ( nude ) mice, who have essentially no T-cell function, calcification morphology and extent are unchanged [63]. Clinical and experimental data detecting antibodies to valve tissue after failure may reflect a secondary response to valve damage rather than a cause of failure. Prevention of Calcification Three generic strategies have been investigated for preventing calcification of biomaterial implants: (1) systemic therapy with anticalcification agents; (2) local therapy with implantable drug delivery devices; and (3) biomaterial modifications, such as removal of a calcifiable component, addition of an exogenous agent, or chemical alteration. The subcutaneous model has been widely used to screen potential strategies for calcification inhibition (anticalcification). Promising approaches have been investigated further in a large animal valve implant model. Strategies that appeared efficacious in subcutaneous implants have not always proven favorable when used on valves implanted into the circulation (see below). Analogous to any new or modified drug or device, a potential antimineralization treatment must meet rigorous efficacy and safety requirements [64]. Investigations of an anticalcification strategy must demonstrate both the effectiveness of the therapy and the absence of adverse effects. The treatment should not impede valve performance such as hemodynamics and durability. Adverse effects in this setting could include systemic or local toxicity, tendency toward thrombosis on infection, induction of immunologic effects, or structural degradation, with either immediate loss of mechanical properties or premature deterioration and failure. There are several instances in which an antimineralization treatment contributed to unacceptable degradation of the tissue [65 67]. The essential steps in preclinical validation of the safety and efficacy of an anticalcification strategy are summarized in Table 1. Explant pathology analyses continue to be highly useful, not only in preclinical studies, but also in clinical trials and postmarket surveillance of approved products in general clinical use. Systemic therapy with anticalcification agents may be efficacious but is unlikely to be safe. Sufficient doses of systemic agents used to treat clinical metabolic bone disease, including calcium chelators (eg, diphosphonates such as ethane-1-hydroxy-1, 1 bisphosphonate [EHBP]), can prevent the calcification of bioprosthetic tissue implanted subcutaneously in rats [68]. However, because of their ability to interfere with physiologic calcification (ie, bone growth), systemic drug administration is associated with many side effects in calcium metabolism, and animals receiving doses sufficient to prevent bioprosthetic tissue calcification suffer growth retardation. Thus, the principal disadvantage of the systemic use of anticalcification agents for preventing pathologic calcification is the consequent inhibition of bone formation. To avoid this difficulty, coimplants of a drug delivery system Table 1. Preclinical Studies of Efficacy and Safety Study Tissue biomechanics Finite element analysis/ computer simulation Subcutaneous animal studies (usually rat) Biomechanical studies of prototype valves Morphologic studies of unimplanted tissue (ie, light, scanning, and transmission electron microscopy) Circulatory implants in large animals (usually sheep) adjacent to the prosthesis have been investigated. With localized drug delivery the effective drug concentration would be confined to the site where it is needed (ie, the implant) and systemic side effects would thereby be prevented [69]. Studies incorporating EHBP in nondegradable polymers, such as ethylene-vinyl acetate, polydimethylsiloxane (silicone), and polyurethanes have shown the effectiveness of this strategy in animal models. This approach, however, has not been implemented clinically. Previously investigated strategies for the prevention of tissue valve calcification are summarized in Table 2. The approach that is most likely to yield improved clinical results in the short term involves modification of the substrate, either by removing or altering a calcifiable component or binding an inhibitor. The agents most widely studied for efficacy, mechanisms, lack of adverse effects, and potential clinical utility are summarized below. Combination therapies using multiple agents may provide a synergy of beneficial effects to permit simultaneous prevention of calcification in both cusps and aortic wall, potentially and particularly beneficial in stentless aortic valves, have also been investigated [70]. Inhibitors of Hydroxyapatite Formation Purpose Compliance Strength Identification of stress concentrations Initial screen for anticalcification efficacy Dose response Mechanism Toxicity Flow simulation Accelerated durability Structural degradation Matrix integrity Surface uniformity and defects In vivo hemodynamics Explant valve pathology for calcification, durability, and bloodmaterials interaction Systemic pathology Bisphosphonates Ethane-1-hydroxy-1, 1 bisphosphonate has been approved by the Food and Drug Administration (FDA) for human use to inhibit pathologic calcification and to treat hypercalcemia of malignancy. Compounds of this type probably inhibit calcification by poisoning the growth of calcific crystals and stabilizing bone mineral. Orally ad-

5 1076 REVIEW SCHOEN AND LEVY Ann Thorac Surg CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 2005;79: Table 2. Antimineralization Strategies in Tissue Heart Valve Substitutes Systemic drug administration Localized drug delivery Substrate modification Inhibitors of calcium phosphate mineral formation Biphosphonates Trivalent metal ions Amino-oleic acid Removal/modification of calcifiable material Surfactants Ethanol Decellularization Improvement/modification of glutaraldehyde fixation Fixation in high concentrations of glutaraldehyde Reduction reactivity of residual chemical groups Modification of tissue charge Incorporation of polymers Use of tissue fixatives other than glutaraldehyde Epoxy compounds Carbodiimides Acyl azide ministered bisphosphonates are used to stabilize osteoporosis. Either cuspal pretreatment or systemic or local therapy of the host with diphosphonate compounds inhibits experimental bioprosthetic valve calcification [71 73]. Trivalent Metal Ions Pretreatment of bioprosthetic tissue with iron and aluminum (eg, FeCl 3 and AlCl 3 ) inhibits calcification of subdermal implants with glutaraldehyde-pretreated porcine cusps or pericardium [74, 75]. Such compounds are hypothesized to act through complexation of the cation (Fe or Al) with phosphate, thereby preventing calcium phosphate formation. Both ferric ion and the trivalent aluminum ion inhibit alkaline phosphatase, an important enzyme involved in bone formation, and this may be related to their mechanism for preventing initiation of calcification. Furthermore, recent research has demonstrated that aluminum chloride prevents elastin calcification through a permanent structural alteration of the elastin molecule [76]. Calcium Diffusion Inhibitor Amino-oleic Acid Two- -amino-oleic acid (AOA, Biomedical Design, Inc, Atlanta, GA) bonds covalently to bioprosthetic tissue through an amino linkage to residual aldehyde functions and inhibits calcium flux through bioprosthetic cusps [77, 78]. The AOA is effective in mitigating cusp but not aortic wall calcification in rat subdermal and cardiovascular implants. This compound is used in FDA-approved nonstented and stented porcine aortic valves [79, 80]. Removal or Modification of Calcifiable Material Surfactants Incubation of bioprosthetic tissue with sodium dodecyl sulfate and other detergents extracts the majority of acidic phospholipids [81]; this is associated with reduced mineralization experimentally, probably resulting from suppression of the initial cell-membrane oriented calcification. This compound is used in a commercial porcine valve [82, 83]. Ethanol Ethanol preincubation of glutaraldehyde-crosslinked porcine aortic valve bioprostheses prevents calcification of the valve cusps in both rat subdermal implants and sheep mitral valve replacements [84 86]. Eighty percent ethanol pretreatment (1) extracts almost all phospholipids and cholesterol from glutaraldehyde-crosslinked cusps, (2) causes a permanent alteration in collagen conformation, (3) affects cuspal interactions with water and lipids, and (4) enhances cuspal resistance to collagenase. Ethanol is in clinical use as a porcine valve cuspal pretreatment in Europe, and use in combination with aluminum treatment of the aortic wall of a stentless valve is currently in clinical trials. Decellularization Since the initial mineralization sites are devitalized connective cells of bioprosthetic tissue, decellularization approaches attempt to remove these cells from the tissue, with the intent of making the bioprosthetic matrix less prone to calcification [87, 88]. Such an approach has been used on nonfixed homografts which were clinically implanted, yielding unfavorable results [89, 90]. Improvement or Modification of Glutaraldehyde Fixation Although pretreatment with glutaraldehyde is the most widely used tissue preparation method for bioprosthetic heart valves, previous studies have demonstrated that conventional glutaraldehyde fixation is conducive to calcification of bioprosthetic tissues. Moreover, the biochemistry of glutaraldehyde cross-linking is poorly understood [91]. Therefore, studies have investigated modifications of and alternatives to conventional glutaraldehyde pretreatment [92]. Some investigators have aimed to improve glutaraldehyde fixation or modify tissues following conventional treatment with this compound. For example, fixation of bioprosthetic tissue (cusps and aortic wall) using extraordinarily high concentrations of glutaraldehyde (5 to 10 those normally used) appear to inhibit calcification in subdermal implants [93, 94]. However, tissue fixed by concentrated glutaraldehyde may be stiffer than could be used in a clinically useful bioprosthesis. Agents that reduce the reactivity of chemical groups by reduction (eg, borohydride, cyanoborohydride), block residual aldehydes (eg, L-glutamic acid, glycine, L-lysine, diamine) [95, 96], modify tissue charge (eg, protamine), and incorporate poly-

6 Ann Thorac Surg REVIEW SCHOEN AND LEVY 2005;79: CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 1077 mers (eg, polyethylene oxide, polyethylene glycol) have been used to render the glutaraldehyde-fixed tissue less reactive. Use of Tissue Fixatives Other Than Glutaraldehyde A distinct set of strategies has sought to avoid glutaraldehyde altogether and use other methods of tissue crosslinking. Nonglutaraldehyde cross-linking of bioprosthetic tissue with epoxy compounds, carbodiimides, acyl azide, and other compounds reduces their calcification in rat subdermal implant studies [97 99]. Epoxy crosslinking has generated considerable interest owing to the retained pliability and natural appearance of tissues so treated [100, 101]. Although some of the alternative tissue treatment strategies have met with experimental success, there has been little translation to the clinical environment and, with few exceptions, the mechanistic basis for nonglutaraldehyde approaches has not been established. One approach investigated clinically illustrates how difficult this might be. In a process called dye-mediated photooxidation, tissue is incubated in a photooxidative dye followed by exposure to light of specific wavelengths that are selectively absorbed by the dye, thereby causing cross-linking. Photooxidative preservation inhibits experimental bioprosthetic heart valve calcification, but the mechanisms responsible for this are not well understood at this time [102]. Clinical investigation of this technology, however, met with failure, owing to design-related cuspal tearing [103]. Special Problems Created by an Exposed Aortic Wall Calcification of the aortic wall portion of glutaraldehydepretreated porcine aortic valves (stented and nonstented) and valvular allografts and vascular segments is observed clinically and experimentally. Mineral deposition occurs throughout the vascular cross section but is accentuated in the dense bands at the inner and outer media. As with porcine valve cusps and bovine pericardium, cells are the major sites of initiation of calcific deposits [104]. Extracellular matrix, especially elastin, plays a less prominent role. In the increasingly used nonstented porcine aortic valves which have greater portions of aortic wall exposed to blood than in currently used stented valves, calcification of the aortic wall is potentially deleterious. In the nonstented configuration, calcification could stiffen the root, altering hemodynamic efficiency, cause nodular calcific obstruction, potentiate wall rupture, or provide a nidus for emboli. Some anticalcification agents have differential effects on cuspal and aortic wall mineralization. For example, AOA and ethanol each prevent experimental cuspal but not aortic wall calcification. Conversely, treatment with aluminum compounds is ineffective on cusps but, probably in part owing to its protective effect against calcification of elastin, inhibits calcification of the aortic wall. Combination therapies using multiple agents may provide synergy of beneficial effects to permit simultaneous prevention of calcification in both cusps and aortic wall, potentially and particularly beneficial in stentless aortic valves. For example, reduced valve and wall calcification was demonstrated in vivo in sheep by a differential treatment of ethanol on cusps and aluminum on the aortic walls [105, 106], and diamine treatment followed by extraction of excess glutaraldehyde from porcine aortic valve roots mitigated both cuspal and aortic wall calcification [107]. Cross-linking by a carbodiimide-based method [108] and dye-mediated photooxidation also mitigated both cuspal and aortic wall calcification of porcine aortic valve roots [109, 110]. More recently, and in a study reported in this issue of The Annals, Zilla and colleagues [111] reduced aortic wall calcification in rat subcutaneous implants by treatment of glutaraldehyde-fixed aortic wall segments with AOA followed by an ethylcarbodiimidedependent carboxyl-group cross-linking (carbodiimide) treatment. The possibility that this will be shown to be mechanistically sound and proven effective with no deleterious effects in vivo is indeed exciting. Conclusions Calcification of bioprosthetic implants is a clinically important pathologic process limiting the anticipated durability and, hence, use of tissue-derived valves. 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