calcification of tissue heart valves

2005;79:1072-1080 Ann Thorac SurgFrederick J. Schoen and Robert J. Levy

and PreventionCalcification of Tissue Heart Valve Substitutes: Progress Toward Understanding

http://ats.ctsnetjournals.org/cgi/content/full/79/3/1072located on the World Wide Web at:

The online version of this article, along with updated information and services, is

Print ISSN: 0003-4975; eISSN: 1552-6259. Southern Thoracic Surgical Association. Copyright © 2005 by The Society of Thoracic Surgeons.

is the official journal of The Society of Thoracic Surgeons and theThe Annals of Thoracic Surgery

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alcification of Tissue Heart Valve Substitutes:rogress Toward Understanding and Prevention

rederick J. Schoen, MD, PhD, and Robert J. Levy, MDepartment of Pathology, Brigham and Women’s Hospital and Harvard Medical School, the Harvard-MIT Division of Healthciences and Technology, Boston, Massachusetts, and the Abramson Pediatric Research Center, The Children’s Hospital of
hiladelphia, University of Pennsylvania, Philadelphia, Pennsylvania
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alcification plays a major role in the failure of biopros-hetic and other tissue heart valve substitutes. Tissuealve calcification is initiated primarily within residualells that have been devitalized, usually by glutaralde-yde pretreatment. The mechanism involves reaction ofalcium-containing extracellular fluid with membrane-ssociated phosphorus to yield calcium phosphate min-ral deposits. Calcification is accelerated by young recip-ent age, valve factors such as glutaraldehyde fixation,nd increased mechanical stress. Recent studies haveuggested that pathologic calcification is regulated bynductive and inhibitory factors, similar to the physio-
ogic mineralization of bone. The most promising pre-
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righam and Women’s Hospital, 75 Francis St, Boston, MA 02115;-mail: [email protected].

2005 by The Society of Thoracic Surgeonsublished by Elsevier Inc

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entive strategies have included binding of calcificationnhibitors to glutaraldehyde fixed tissue, removal or

odification of calcifiable components, modification oflutaraldehyde fixation, and use of tissue cross linkinggents other than glutaraldehyde. This review summa-izes current concepts in the pathophysiology of tissuealve calcification, including emerging concepts of en-ogenous regulation, progress toward prevention of cal-ification, and issues related to calcification of the aorticall of stentless bioprosthetic valves.

(Ann Thorac Surg 2005;79:1072–80)
© 2005 by The Society of Thoracic Surgeons
eart valve substitutes are of two principal types:mechanical prosthetic valves with components

anufactured of nonbiologic material (eg, polymer,etal, carbon) or tissue valves which are constructed, at

east in part, of either human or animal tissue [1, 2].

See page 905

issue valves have been used since the early 1960s whenortic valves obtained fresh from human cadavers wereransplanted to other individuals (homografts). A decadeater, chemically preserved stent-mounted tissue bio-rosthetic valves (generally termed bioprostheses) wereommercially produced and implanted. Today, stent-ounted glutaraldehyde preserved porcine aortic valves

nd bovine pericardial bioprosthetic valves are usedidely, and stentless valves have been introduced. Ap-roximately 85,000 substitute valves are implanted in thenited States and 275,000 worldwide each year, of whiche presently estimate that approximately half are me-

hanical and half are tissue, suggesting a shift towardncreasingly greater usage of tissue valves over the lastecade.Within 10 years postoperatively, prosthesis-associated

roblems overall necessitate reoperation or cause deathn at least 50% to 60% of patients with substitute valves [3,]. The rate is similar for mechanical prostheses andioprostheses; however, the frequency and nature ofpecific valve-related complications vary with the pros-

ddress reprint requests to Dr Schoen, Department of Pathology,

hesis type, model, site of implantation, and certainharacteristics of the patient. Specifically, mechanicalrosthetic valves have a substantial risk of systemic

hromboemboli and thrombotic occlusion, and thehronic anticoagulation therapy required in all mechan-cal valve recipients potentiates hemorrhagic complica-ions. Nevertheless, contemporary mechanical prosthe-es are durable.

In contrast, tissue valves have a low rate of thrombo-mbolism without anticoagulation, owing to a centralattern of flow similar to that of the natural heart valvesnd cusps composed of valvular or nonvalvular animal oruman tissue. However, a high rate of valve failure withtructural dysfunction owing to progressive tissue dete-ioration (including calcification and noncalcific damage)ndermines their attractiveness [2, 5–9].Structural dysfunction is the major cause of failure of

ioprosthetic heart valves (flexible-stent-mounted, glut-raldehyde-preserved porcine aortic valves and bovineericardial valves). Within 15 years following implanta-

ion, approximately 50% of porcine aortic valves sufferhe major prosthesis-related complication with this typef valve—tissue failure. The principal underlying patho-

ogic process is cuspal calcification; secondary tears fre-uently precipitate regurgitation. Calcification can alsoause pure stenosis owing to cuspal stiffening. Calcificeposits are usually localized to cuspal tissue (intrinsic

Dr Schoen discloses that he has financial relationshipswith CarboMedics, Edwards Lifesciences, Medtronic,
and St. Jude Medical.
0003-4975/05/$30.00doi:10.1016/j.athoracsur.2004.06.033

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1073Ann Thorac Surg REVIEW SCHOEN AND LEVY2005;79:1072–80 CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES

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alcification), but calcific deposits extrinsic to the cuspsay develop in thrombi or endocarditic vegetations

extrinsic calcification). Calcification is markedly acceler-ted in younger patients; children and adolescents haven especially accelerated course, and older patients havelower rate of bioprosthetic valve degeneration. Progres-

ive collagen deterioration, independent of calcification,s also a likely important contributor to the limitedurability of bioprosthetic valves [10, 11]. Bovine pericar-ial valves also calcify but design-related tearing haseen prominent [12, 13].

eterminants, Mechanisms, and Regulation

athological analysis of tissue valve explants from pa-ients and experiments in animal models using biopros-hetic heart valve tissue have elucidated many aspects ofhe pathophysiology of this important clinical problem.he current understanding of the determinants, mecha-isms, and regulation of tissue calcification is summa-ized below and in Figure 1.

The most useful experimental models have been or-hotopic tricuspid or mitral replacements or conduit-

ig 1. Extended hypothetical model for the calcification of biopros-hetic tissue. This model considers host factors, implant factors, andechanical damage and relates initial sites of mineral nucleation to

ncreased intracellular calcium in residual cells and cell fragments inioprosthetic tissue. The ultimate result of calcification is valve fail-re, with tearing or stenosis. The key contributory role of existinghosphorus in membrane phospholipids and nucleic acids in deter-ining the initial sites of crystal nucleation is emphasized, and a

ossible role for the independent mineralization of collagen is ac-nowledged. Mechanical deformation probably accelerates to bothucleation and growth of calcific crystals. Modified by permissionrom Schoen FJ: Interventional and surgical cardiovascular pathol-gy: clinical correlations and basic principles. Philadelphia: WBaunders, 1989.

ounted valves in sheep or calves [14, 15], and isolated m


issue samples implanted subcutaneously in very young,apidly growing mice, rabbits, or rats [16–18]. In bothirculatory and noncirculatory models, bioprosthetic tis-ue calcifies progressively with a morphology similar tohat observed in clinical specimens, but with markedlyccelerated kinetics. The subcutaneous model hasmerged as a technically convenient and economicallydvantageous vehicle for investigating host and implanteterminants and mechanisms of mineralization, as wells for screening potential strategies for inhibition ofalcification. In general, large animal valve replacementsan (1) elucidate further the processes accounting forlinical failures, (2) evaluate the performance of designnd biomaterials modifications in valve developmenttudies, (3) assess the importance of blood/surface inter-ctions, and (4) provide data required for approval byegulatory agencies. Despite the potential of in vitroodels to elucidate the pathophysiology of biomaterials

alcification, such systems have not been generally usefuln this regard [19–22].

eterminantshe determinants of bioprosthetic valve and other bio-aterial mineralization include factors related to (1) hostetabolism, (2) implant structure and chemistry, and (3)echanical factors. Natural cofactors and inhibitors may

lso play a role (see below). Accelerated calcification isssociated with young recipient age, glutaraldehyde fix-tion, and high mechanical stress. Calcification is moreapid and aggressive in the young; the rate of failure ofioprostheses is approximately 10% in 10 years in elderlyecipients, but is nearly uniform in less than 4 years inost adolescent and preadolescent children. Although

he relationship is well-established, the mechanisms ac-ounting for the effect of age are uncertain. The acceler-ted onset of calcific failure in young patients is simu-ated by the rapid calcification that occurs in youngxperimental animals.The structural elements of the biomaterial and theirodification by processing clearly play an important

ole. Cells are the predominant location of mineralizationsee later) and the usual pretreatment of commerciallyvailable bioprostheses with glutaraldehyde, done tomprove tissue durability, also potentiate calcification [23,4]. Calcification of porcine aortic valve and bovineericardium are qualitatively, quantitatively, and mech-nistically similar. It has been hypothesized that theross-linking agent glutaraldehyde stabilizes and per-aps modifies phosphorous-rich calcifiable structures in

he bioprosthetic tissue. These sites are capable of min-ralization upon implantation when exposed to the com-aratively high calcium levels of extracellular fluid. Par-doxically, high glutaraldehyde pretreatment conditions3% glutaraldehyde compared with 0.6% or less presentlysed commercially) seem to be protective against calcifi-ation of bioprosthetic tissue [25].

Calcification of the extracellular matrix structural pro-eins collagen and elastin has been observed in clinicalnd experimental implants of bioprosthetic and ho-
ograft valvular and vascular tissue, and has been stud-


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1074 REVIEW SCHOEN AND LEVY Ann Thorac SurgCALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES 2005;79:1072–80

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ed using a rat subdermal model. Collagen and elasticbers can serve as nucleation sites for calcium phosphateinerals, independent of cellular components [2, 16–18,

6]. Cross-linking by either glutaraldehyde or formalde-yde promotes the calcification of collagen sponge im-lants made of purified collagen but the extent of calci-cation does not correlate with the degree of cross

inking [27]. In contrast, the calcification of elastin ap-ears independent of whether pretreatment has occurred

28]. Calcification has also complicated the clinical usend experimental investigation of heart valves composedf bovine pericardium [12, 29] and polymers (eg, poly-rethane) [30, 31]. Furthermore, both intrinsic and extrin-ic mineralization of a biomaterial is generally enhancedt the sites of intense mechanical deformations generatedy motion, such as the points of flexion in heart valves [2,6, 17, 32].

echanismshe mineralization process in the cusps of bioprostheticeart valves is initiated predominantly within nonviableonnective tissue cells that have been devitalized but notemoved by glutaraldehyde pretreatment procedures [2,6–18, 33, 34]. This dystrophic calcification mechanismnvolves reaction of calcium-containing extracellularuid with membrane-associated phosphorus, causingalcification of the cells. This likely occurs because theormal extrusion of calcium ions is disrupted in cells thatave been rendered nonviable by glutaraldehyde fixa-

ion. Normally, the plasma-extracellular calcium concen-ration is 1 mg/mL (approximately 10�3 M); since the

embranes of healthy cells pump calcium out, the con-entration of calcium in the cytoplasm is normally 1,000o 10,000 times lower (approximately 10�7 M). However,he physiologic mechanisms for elimination of calciumrom the cells are not available in glutaraldehyde-retreated tissue. The cell membranes and other inter-ellular structures are high in phosphorus (as phospho-ipids, especially phosphatidyl serine, and the phosphateackbone of the nucleic acids); they can bind calcium anderve as nucleators. Initial calcification deposits eventu-lly enlarge and coalesce, resulting in grossly mineral-zed nodules that stiffen and weaken the tissue andhereby cause a prosthesis to malfunction.

egulationlthough pathologic calcification has typically been con-

idered a passive, unregulated, and degenerative pro-ess, recent studies suggest that the mechanisms respon-ible for pathologic calcification may be regulated,imilarly to the physiologic mineralization of bone andther hard tissues [35–37]. In normal blood vessels andalves, inhibitory mechanisms outweigh procalcific in-uctive mechanisms and calcification does not occur. Inontrast, in bone and pathologic tissues, the inductiveechanisms dominate. In the process of normal bone

alcification, the growth of apatite crystals is regulated byeveral noncollagenous matrix proteins including: (1)steopontin, an acidic calcium-binding phosphoprotein
ith high affinity to hydroxyapatite that is abundant in c

oci of dystrophic calcification; (2) osteonectin; and (3)steocalcin [38], and other �-carboxyglutamic acidGLA)- containing proteins, such as matrix GLA proteinMGP). Naturally occurring inhibitors crystal nucleationnd growth may also play a role in the regulation ofalcific degeneration of natural and bioprosthetic valvesnd other cardiovascular calcification such as arterioscle-osis [39, 40]. Specific inhibitors in this context includesteopontin [41] and high-density liproprotein (thegood” cholesterol) [42]. The role in pathological miner-lization of naturally occurring promineralization cofac-ors, such as inorganic phosphate [43], bone morphoge-etic protein [44], proinflammatory lipids [35], and otherubstances (eg, cytokines), as well as inhibitors, is anctive area of research [45]. Recent evidence suggests thatypercholesterolemia may be a risk factor in clinicalioprosthetic valve calcification [46, 47]. The noncollag-nous proteins osteopontin, TGF-�, and tenascin-C in-olved in bone matrix formation and tissue remodelingave been demonstrated in clinical calcified biopros-

hetic heart valves, natural valves, and arteriosclerosis,uggesting that they play a regulatory role in these formsf pathologic calcification in humans [48–50].Evidence for the active regulation of cardiovascular

alcification also derives from tissue culture models ofascular cell calcification, which mimic vascular calcifi-ation in vivo and genetic studies in mice. For example,steopontin inhibits and proinflammatory lipids and cy-okines enhance the mineralization of smooth muscle cellultures [51–53]. In transgenic mouse models, in whichhe gene for the MGP was knocked out [54] or thesteopontin gene was inactivated [55], severe calcifica-ion of blood vessels resulted. Moreover, inhibition of

atrix remodeling metalloproteinases inhibits calcifica-ion of elastin implanted subcutaneously in rats [56].

A potential role for inflammatory and immune pro-esses has been postulated by some investigators [57–59].lthough (1) experimental animals can be sensitized tooth fresh and cross-linked bioprosthetic valve tissues

60, 61], (2) antibodies to valve components can be de-ected in some patients following valve dysfunction [62],nd (3) failed tissue valves often have mononuclearnflammation, no definite role has been demonstrated forirculating macromolecules or cells. In particular, theresence of mononuclear cells in a failed tissue valveoes not equate to immunologic rejection. In all papers

hus far purporting to demonstrate immune-mediatednjury, no evidence is presented to suggest that the

ononuclear inflammatory cells are causing functionalalve degeneration.Indeed, many lines of evidence suggest that neither

onspecific inflammation nor specific immunologic re-ponses appear to favor bioprosthetic tissue calcification,nd no causal or contributory immunologic basis haseen demonstrated for bioprosthetic valve calcificationr failure. For example, in experiments in which valveusps were enclosed in filter chambers that prevent hostell contact with tissue but allow free diffusion of extra-
ellular fluid and implantation of valve tissue in congen-


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tally athymic (“nude”) mice, who have essentially no-cell function, calcification morphology and extent arenchanged [63]. Clinical and experimental data detectingntibodies to valve tissue after failure may reflect aecondary response to valve damage rather than a causef failure.

revention of Calcification

hree generic strategies have been investigated for pre-enting calcification of biomaterial implants: (1) systemicherapy with anticalcification agents; (2) local therapyith implantable drug delivery devices; and (3) bioma-

erial modifications, such as removal of a calcifiableomponent, addition of an exogenous agent, or chemicallteration. The subcutaneous model has been widelysed to screen potential strategies for calcification inhi-ition (anticalcification). Promising approaches haveeen investigated further in a large animal valve implantodel. Strategies that appeared efficacious in subcutane-

us implants have not always proven favorable whensed on valves implanted into the circulation (see below).Analogous to any new or modified drug or device, a

otential antimineralization treatment must meet rigor-us efficacy and safety requirements [64]. Investigationsf an anticalcification strategy must demonstrate both theffectiveness of the therapy and the absence of adverseffects. The treatment should not impede valve perfor-ance such as hemodynamics and durability. Adverse

ffects in this setting could include systemic or localoxicity, tendency toward thrombosis on infection, induc-ion of immunologic effects, or structural degradation,ith either immediate loss of mechanical properties orremature deterioration and failure. There are several

nstances in which an antimineralization treatment con-ributed to unacceptable degradation of the tissue65–67].

The essential steps in preclinical validation of theafety and efficacy of an anticalcification strategy areummarized in Table 1. Explant pathology analyses con-inue to be highly useful, not only in preclinical studies,ut also in clinical trials and postmarket surveillance ofpproved products in general clinical use.Systemic therapy with anticalcification agents may be

fficacious but is unlikely to be safe. Sufficient doses ofystemic agents used to treat clinical metabolic boneisease, including calcium chelators (eg, diphosphonatesuch as ethane-1-hydroxy-1, 1 bisphosphonate [EHBP]),an prevent the calcification of bioprosthetic tissue im-lanted subcutaneously in rats [68]. However, because of

heir ability to interfere with physiologic calcification (ie,one growth), systemic drug administration is associatedith many side effects in calcium metabolism, and ani-als receiving doses sufficient to prevent bioprosthetic

issue calcification suffer growth retardation. Thus, therincipal disadvantage of the systemic use of anticalcifi-ation agents for preventing pathologic calcification ishe consequent inhibition of bone formation. To avoid
his difficulty, coimplants of a drug delivery system c

djacent to the prosthesis have been investigated. Withocalized drug delivery the effective drug concentrationould be confined to the site where it is needed (ie, the

mplant) and systemic side effects would thereby berevented [69]. Studies incorporating EHBP in nonde-radable polymers, such as ethylene-vinyl acetate, poly-imethylsiloxane (silicone), and polyurethanes havehown the effectiveness of this strategy in animal models.his approach, however, has not been implementedlinically.

Previously investigated strategies for the prevention ofissue valve calcification are summarized in Table 2. Thepproach that is most likely to yield improved clinicalesults in the short term involves modification of theubstrate, either by removing or altering a calcifiableomponent or binding an inhibitor. The agents mostidely studied for efficacy, mechanisms, lack of adverse

ffects, and potential clinical utility are summarized be-ow. Combination therapies using multiple agents mayrovide a synergy of beneficial effects to permit simulta-eous prevention of calcification in both cusps and aorticall, potentially and particularly beneficial in stentless

ortic valves, have also been investigated [70].

nhibitors of Hydroxyapatite Formation

isphosphonatesthane-1-hydroxy-1, 1 bisphosphonate has been ap-roved by the Food and Drug Administration (FDA) foruman use to inhibit pathologic calcification and to treatypercalcemia of malignancy. Compounds of this typerobably inhibit calcification by poisoning the growth of

able 1. Preclinical Studies of Efficacy and Safety

tudy Purpose

issue biomechanics ComplianceStrength

inite element analysis/computer simulation

Identification of stressconcentrations

ubcutaneous animalstudies (usually rat)

Initial screen foranticalcification efficacy

Dose responseMechanismToxicity

iomechanical studies ofprototype valves

Flow simulationAccelerated durability

orphologic studies ofunimplanted tissue (ie,light, scanning, andtransmission electronmicroscopy)

Structural degradation

Matrix integrity

Surface uniformity anddefects

irculatory implants inlarge animals (usuallysheep)

In vivo hemodynamicsExplant valve pathology

for calcification,durability, and blood-materials interaction

Systemic pathology

alcific crystals and stabilizing bone mineral. Orally ad-



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inistered bisphosphonates are used to stabilize osteo-orosis. Either cuspal pretreatment or systemic or local

herapy of the host with diphosphonate compoundsnhibits experimental bioprosthetic valve calcification71–73].

rivalent Metal Ionsretreatment of bioprosthetic tissue with iron and alumi-um (eg, FeCl3 and AlCl3) inhibits calcification of sub-ermal implants with glutaraldehyde-pretreated porcineusps or pericardium [74, 75]. Such compounds areypothesized to act through complexation of the cation

Fe or Al) with phosphate, thereby preventing calciumhosphate formation. Both ferric ion and the trivalentluminum ion inhibit alkaline phosphatase, an importantnzyme involved in bone formation, and this may beelated to their mechanism for preventing initiation ofalcification. Furthermore, recent research has demon-trated that aluminum chloride prevents elastin calcifi-ation through a permanent structural alteration of thelastin molecule [76].

alcium Diffusion Inhibitor

mino-oleic Acidwo-�-amino-oleic acid (AOA, Biomedical Design, Inc,tlanta, GA) bonds covalently to bioprosthetic tissue

hrough an amino linkage to residual aldehyde functionsnd inhibits calcium flux through bioprosthetic cusps [77,8]. The AOA is effective in mitigating cusp but not aorticall calcification in rat subdermal and cardiovascular

mplants. This compound is used in FDA-approved non-

able 2. Antimineralization Strategies in Tissue Heart Valveubstitutes

ystemic drug administrationocalized drug deliveryubstrate modificationInhibitors of calcium phosphate mineral formation

BiphosphonatesTrivalent metal ionsAmino-oleic acid

Removal/modification of calcifiable materialSurfactantsEthanolDecellularization

Improvement/modification of glutaraldehyde fixationFixation in high concentrations of glutaraldehydeReduction reactivity of residual chemical groupsModification of tissue chargeIncorporation of polymers

Use of tissue fixatives other than glutaraldehydeEpoxy compoundsCarbodiimidesAcyl azide

tented and stented porcine aortic valves [79, 80]. i


emoval or Modification of Calcifiable Material

urfactantsncubation of bioprosthetic tissue with sodium dodecylulfate and other detergents extracts the majority ofcidic phospholipids [81]; this is associated with reducedineralization experimentally, probably resulting from

uppression of the initial cell-membrane oriented calci-cation. This compound is used in a commercial porcinealve [82, 83].

thanolthanol preincubation of glutaraldehyde-crosslinkedorcine aortic valve bioprostheses prevents calcificationf the valve cusps in both rat subdermal implants andheep mitral valve replacements [84–86]. Eighty percentthanol pretreatment (1) extracts almost all phospholip-ds and cholesterol from glutaraldehyde-crosslinkedusps, (2) causes a permanent alteration in collagenonformation, (3) affects cuspal interactions with waternd lipids, and (4) enhances cuspal resistance to collage-ase. Ethanol is in clinical use as a porcine valve cuspalretreatment in Europe, and use in combination withluminum treatment of the aortic wall of a stentless valves currently in clinical trials.

ecellularizationince the initial mineralization sites are devitalized con-ective cells of bioprosthetic tissue, decellularizationpproaches attempt to remove these cells from the tissue,ith the intent of making the bioprosthetic matrix lessrone to calcification [87, 88]. Such an approach has beensed on nonfixed homografts which were clinically im-lanted, yielding unfavorable results [89, 90].

mprovement or Modification of Glutaraldehydeixation

lthough pretreatment with glutaraldehyde is the mostidely used tissue preparation method for bioprostheticeart valves, previous studies have demonstrated thatonventional glutaraldehyde fixation is conducive to cal-ification of bioprosthetic tissues. Moreover, the bio-hemistry of glutaraldehyde cross-linking is poorly un-erstood [91]. Therefore, studies have investigatedodifications of and alternatives to conventional glutar-

ldehyde pretreatment [92]. Some investigators haveimed to improve glutaraldehyde fixation or modifyissues following conventional treatment with this com-ound. For example, fixation of bioprosthetic tissue

cusps and aortic wall) using extraordinarily high con-entrations of glutaraldehyde (5� to 10� those normallysed) appear to inhibit calcification in subdermal im-lants [93, 94]. However, tissue fixed by concentratedlutaraldehyde may be stiffer than could be used in alinically useful bioprosthesis. Agents that reduce theeactivity of chemical groups by reduction (eg, borohy-ride, cyanoborohydride), block residual aldehydes (eg,-glutamic acid, glycine, L-lysine, diamine) [95, 96], mod-

fy tissue charge (eg, protamine), and incorporate poly-



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ers (eg, polyethylene oxide, polyethylene glycol) haveeen used to render the glutaraldehyde-fixed tissue lesseactive.

se of Tissue Fixatives Other Thanlutaraldehyde

distinct set of strategies has sought to avoid glutaral-ehyde altogether and use other methods of tissue cross-

inking. Nonglutaraldehyde cross-linking of biopros-hetic tissue with epoxy compounds, carbodiimides, acylzide, and other compounds reduces their calcification inat subdermal implant studies [97–99]. Epoxy cross-inking has generated considerable interest owing to theetained pliability and natural appearance of tissues soreated [100, 101]. Although some of the alternative tissuereatment strategies have met with experimental success,here has been little translation to the clinical environ-

ent and, with few exceptions, the mechanistic basis foronglutaraldehyde approaches has not been established.ne approach investigated clinically illustrates how dif-cult this might be. In a process called dye-mediatedhotooxidation, tissue is incubated in a photooxidativeye followed by exposure to light of specific wavelengths

hat are selectively absorbed by the dye, thereby causingross-linking. Photooxidative preservation inhibits ex-erimental bioprosthetic heart valve calcification, but theechanisms responsible for this are not well understood

t this time [102]. Clinical investigation of this technol-gy, however, met with failure, owing to design-relateduspal tearing [103].

pecial Problems Created by an Exposed Aorticall

alcification of the aortic wall portion of glutaraldehyde-retreated porcine aortic valves (stented and nonstented)nd valvular allografts and vascular segments is ob-erved clinically and experimentally. Mineral depositionccurs throughout the vascular cross section but is ac-entuated in the dense bands at the inner and outeredia. As with porcine valve cusps and bovine pericar-

ium, cells are the major sites of initiation of calcificeposits [104]. Extracellular matrix, especially elastin,lays a less prominent role. In the increasingly usedonstented porcine aortic valves which have greaterortions of aortic wall exposed to blood than in currentlysed stented valves, calcification of the aortic wall isotentially deleterious. In the nonstented configuration,alcification could stiffen the root, altering hemodynamicfficiency, cause nodular calcific obstruction, potentiateall rupture, or provide a nidus for emboli.Some anticalcification agents have differential effects

n cuspal and aortic wall mineralization. For example,OA and ethanol each prevent experimental cuspal butot aortic wall calcification. Conversely, treatment withluminum compounds is ineffective on cusps but, prob-bly in part owing to its protective effect against calcifi-ation of elastin, inhibits calcification of the aortic wall.
ombination therapies using multiple agents may pro-

ide synergy of beneficial effects to permit simultaneousrevention of calcification in both cusps and aortic wall,otentially and particularly beneficial in stentless aorticalves. For example, reduced valve and wall calcificationas demonstrated in vivo in sheep by a differential

reatment of ethanol on cusps and aluminum on theortic walls [105, 106], and diamine treatment followed byxtraction of excess glutaraldehyde from porcine aorticalve roots mitigated both cuspal and aortic wall calcifi-ation [107]. Cross-linking by a carbodiimide-basedethod [108] and dye-mediated photooxidation also mit-

gated both cuspal and aortic wall calcification of porcineortic valve roots [109, 110]. More recently, and in a studyeported in this issue of The Annals, Zilla and colleagues111] reduced aortic wall calcification in rat subcutaneousmplants by treatment of glutaraldehyde-fixed aortic wallegments with AOA followed by an ethylcarbodiimide-ependent carboxyl-group cross-linking (carbodiimide)

reatment. The possibility that this will be shown to beechanistically sound and proven effective with no del-

terious effects in vivo is indeed exciting.

onclusions

alcification of bioprosthetic implants is a clinically im-ortant pathologic process limiting the anticipated dura-ility and, hence, use of tissue-derived valves. The patho-hysiology of calcification has been characterized and

argely understood through investigation using animalodels; a key common feature is the involvement of

evitalized cells and cellular debris. Although no clini-ally useful preventive approach is yet available, severaltrategies based on either modifying biomaterials or localrug administrations appear to be promising in someontexts. Calcification of an exposed aortic wall may be aignificant problem with some implant types. Interestingpproaches to preventing this problem through synergis-ic and simultaneous employment of multiple anticalci-cation therapies or novel tissue treatments are under

nvestigation. Since some anticalcification approachesave been used in clinical valves for nearly a decade,ocumentation of favorable 15 to 20 year outcomes willequire yet approximately another decade.

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3. Bloomfield P, Wheatley DJ, Prescott RJ, Miller HC. Twelve-year comparison of a Bjork-Shiley mechanical heart valvewith porcine bioprostheses. N Engl J Med 1991;324:573–9.

4. Hammermeister KE, Sethi GK, Henderson WG, Grover FL,Oprian C, Rahimtoola S. Outcomes 15 years after valvereplacement with a mechanical versus a bioprostheticvalve: Final Report of the Veterans Affairs RandomizedTrial. J Am Coll Cardiol 2000;36:1152–8.

5. Schoen FJ, Hobson CE. Anatomic analysis of removed
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2005;79:1072-1080 Ann Thorac SurgFrederick J. Schoen and Robert J. Levy

and PreventionCalcification of Tissue Heart Valve Substitutes: Progress Toward Understanding

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