REVIEWS
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 biopros-
thetic and other tissue heart valve substitutes. Tissue
valve calcification is initiated primarily within residual
cells that have been devitalized, usually by glutaralde-
hyde pretreatment. The mechanism involves reaction of
calcium-containing extracellular fluid with membrane-
associated phosphorus to yield calcium phosphate min-
eral deposits. Calcification is accelerated by young recip-
ient 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 physio-
logic mineralization of bone. The most promising pre-
H eart valve substitutes are of two principal types:
mechanical prosthetic valves with components
manufactured of nonbiologic material (eg, polymer,
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metal, carbon) or tissue valves which are constructed, at
least in part, of either human or animal tissue [1, 2].
See page 905
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 bio-
prosthetic valves (generally termed bioprostheses) were
commercially produced and implanted. Today, stent-
mounted glutaraldehyde preserved porcine aortic valves
and bovine pericardial bioprosthetic valves are used
widely, and stentless valves have been introduced. Ap-
proximately 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 me-
chanical 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 pros-
Address reprint requests to Dr Schoen, Department of Pathology,
Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115;
e-mail: fschoen@partners.org.
© 2005 by The Society of Thoracic Surgeons
Published by Elsevier Inc
ventive 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 summa-
rizes current concepts in the pathophysiology of tissue
valve calcification, including emerging concepts of en-
dogenous regulation, progress toward prevention of cal-
cification, and issues related to calcification of the aortic
wall of stentless bioprosthetic valves.
(Ann Thorac Surg 2005;79:1072– 80)
© 2005 by The Society of Thoracic Surgeons
thesis 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 mechan-
ical valve recipients potentiates hemorrhagic complica-
tions. Nevertheless, contemporary mechanical prosthe-
ses are durable.
In contrast, tissue valves have a low rate of thrombo-
embolism 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 dete-
rioration (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, glut-
araldehyde-preserved porcine aortic valves and bovine
pericardial valves). Within 15 years following implanta-
tion, approximately 50% of porcine aortic valves suffer
the major prosthesis-related complication with this type
of valve—tissue failure. The principal underlying patho-
logic process is cuspal calcification; secondary tears fre-
quently 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.
0003-4975/05/$30.00
doi:10.1016/j.athoracsur.2004.06.033
Ann Thorac Surg
2005;79:1072– 80
Fig 1. Extended hypothetical model for the calcification of biopros-
thetic 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 fail-
ure, with tearing or stenosis. The key contributory role of existing
phosphorus in membrane phospholipids and nucleic acids in deter-
mining the initial sites of crystal nucleation is emphasized, and a
possible role for the independent mineralization of collagen is ac-
knowledged. Mechanical deformation probably accelerates to both
nucleation and growth of calcific crystals. Modified by permission
from Schoen FJ: Interventional and surgical cardiovascular pathol-
ogy: clinical correlations and basic principles. Philadelphia: WB
Saunders, 1989.
calcification), but calcific deposits extrinsic to the cusps
may develop in thrombi or endocarditic vegetations
(extrinsic calcification). Calcification is markedly acceler-
ated in younger patients; children and adolescents have
an especially accelerated course, and older patients have
a lower rate of bioprosthetic valve degeneration. Progres-
sive collagen deterioration, independent of calcification,
is also a likely important contributor to the limited
durability of bioprosthetic valves [10, 11]. Bovine pericar-
dial valves also calcify but design-related tearing has
been prominent [12, 13].
Determinants, Mechanisms, and Regulation
Pathological analysis of tissue valve explants from pa-
tients and experiments in animal models using biopros-
thetic heart valve tissue have elucidated many aspects of
the pathophysiology of this important clinical problem.
The current understanding of the determinants, mecha-
nisms, and regulation of tissue calcification is summa-
rized below and in Figure 1.
The most useful experimental models have been or-
thotopic tricuspid or mitral replacements or conduit-
mounted valves in sheep or calves [14, 15], and isolated
REVIEW SCHOEN AND LEVY 1073
CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES
tissue samples implanted subcutaneously in very young,
rapidly growing mice, rabbits, or rats [16 –18]. In both
circulatory and noncirculatory models, bioprosthetic tis-
sue 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 inter-
actions, 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 bio-
material 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 fix-
ation, and high mechanical stress. Calcification is more
rapid and aggressive in the young; the rate of failure of
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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 ac-
counting for the effect of age are uncertain. The acceler-
ated onset of calcific failure in young patients is simu-
lated 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 mech-
anistically similar. It has been hypothesized that the
cross-linking agent glutaraldehyde stabilizes and per-
haps modifies phosphorous-rich calcifiable structures in
the bioprosthetic tissue. These sites are capable of min-
eralization upon implantation when exposed to the com-
paratively high calcium levels of extracellular fluid. Par-
adoxically, high glutaraldehyde pretreatment conditions
(3% glutaraldehyde compared with 0.6% or less presently
used commercially) seem to be protective against calcifi-
cation of bioprosthetic tissue [25].
Calcification of the extracellular matrix structural pro-
teins collagen and elastin has been observed in clinical
and experimental implants of bioprosthetic and ho-
mograft valvular and vascular tissue, and has been stud-
 1074 REVIEW SCHOEN AND LEVY
CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES
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 formalde-
hyde promotes the calcification of collagen sponge im-
plants made of purified collagen but the extent of calci-
fication does not correlate with the degree of cross
linking [27]. In contrast, the calcification of elastin ap-
pears 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, poly-
urethane) [30, 31]. Furthermore, both intrinsic and extrin-
sic 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 fixa-
tion. Normally, the plasma-extracellular calcium concen-
tration is 1 mg/mL (approximately 10⫺3 M); since the
REVIEWS
membranes of healthy cells pump calcium out, the con-
centration 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 glutaraldehyde-
pretreated tissue. The cell membranes and other inter-
cellular structures are high in phosphorus (as phospho-
lipids, especially phosphatidyl serine, and the phosphate
backbone of the nucleic acids); they can bind calcium and
serve as nucleators. Initial calcification deposits eventu-
ally enlarge and coalesce, resulting in grossly mineral-
ized nodules that stiffen and weaken the tissue and
thereby cause a prosthesis to malfunction.
Regulation
Although pathologic calcification has typically been con-
sidered a passive, unregulated, and degenerative pro-
cess, recent studies suggest that the mechanisms respon-
sible 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 in-
ductive 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
Ann Thorac Surg
2005;79:1072– 80
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 arterioscle-
rosis [39, 40]. Specific inhibitors in this context include
osteopontin [41] and high-density liproprotein (the
“good” cholesterol) [42]. The role in pathological miner-
alization of naturally occurring promineralization cofac-
tors, such as inorganic phosphate [43], bone morphoge-
netic 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 noncollag-
enous proteins osteopontin, TGF-␣, and tenascin-C in-
volved in bone matrix formation and tissue remodeling
have been demonstrated in clinical calcified biopros-
thetic 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 calcifi-
cation in vivo and genetic studies in mice. For example,
osteopontin inhibits and proinflammatory lipids and cy-
tokines 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 calcifica-
tion of blood vessels resulted. Moreover, inhibition of
matrix remodeling metalloproteinases inhibits calcifica-
tion of elastin implanted subcutaneously in rats [56].
A potential role for inflammatory and immune pro-
cesses 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 de-
tected 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 re-
sponses 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 extra-
cellular fluid and implantation of valve tissue in congen-
Ann Thorac Surg REVIEW SCHOEN AND LEVY 1075
2005;79:1072– 80 CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES

itally athymic (“nude”) mice, who have essentially no Table 1. Preclinical Studies of Efficacy and Safety
T-cell function, calcification morphology and extent are Study Purpose
unchanged [63]. Clinical and experimental data detecting
antibodies to valve tissue after failure may reflect a Tissue biomechanics Compliance
secondary response to valve damage rather than a cause Strength
of failure. Finite element analysis/ Identification of stress
computer simulation concentrations
Subcutaneous animal Initial screen for
Prevention of Calcification studies (usually rat) anticalcification efficacy
Dose response
Three generic strategies have been investigated for pre-
Mechanism
venting calcification of biomaterial implants: (1) systemic
Toxicity
therapy with anticalcification agents; (2) local therapy
Biomechanical studies of Flow simulation
with implantable drug delivery devices; and (3) bioma- prototype valves Accelerated durability
terial modifications, such as removal of a calcifiable
Morphologic studies of Structural degradation
component, addition of an exogenous agent, or chemical
unimplanted tissue (ie, Matrix integrity
alteration. The subcutaneous model has been widely light, scanning, and
used to screen potential strategies for calcification inhi- transmission electron Surface uniformity and
bition (anticalcification). Promising approaches have microscopy) defects
been investigated further in a large animal valve implant Circulatory implants in In vivo hemodynamics
model. Strategies that appeared efficacious in subcutane- large animals (usually Explant valve pathology
sheep) for calcification,
ous implants have not always proven favorable when
durability, and blood-
used on valves implanted into the circulation (see below). materials interaction
Analogous to any new or modified drug or device, a Systemic pathology
potential antimineralization treatment must meet rigor-
ous efficacy and safety requirements [64]. Investigations
of an anticalcification strategy must demonstrate both the
effectiveness of the therapy and the absence of adverse adjacent to the prosthesis have been investigated. With
effects. The treatment should not impede valve perfor- localized drug delivery the effective drug concentration
mance such as hemodynamics and durability. Adverse would be confined to the site where it is needed (ie, the
effects in this setting could include systemic or local implant) and systemic side effects would thereby be

toxicity, tendency toward thrombosis on infection, induc-
tion 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 con-
tributed 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 con-
tinue 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 im-
planted 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 ani-
mals receiving doses sufficient to prevent bioprosthetic
tissue calcification suffer growth retardation. Thus, the
principal disadvantage of the systemic use of anticalcifi-
cation agents for preventing pathologic calcification is
the consequent inhibition of bone formation. To avoid
this difficulty, coimplants of a drug delivery system
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prevented [69]. Studies incorporating EHBP in nonde-
gradable polymers, such as ethylene-vinyl acetate, poly-
dimethylsiloxane (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 be-
low. Combination therapies using multiple agents may
provide a synergy of beneficial effects to permit simulta-
neous 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
Bisphosphonates
Ethane-1-hydroxy-1, 1 bisphosphonate has been ap-
proved 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-
 1076 REVIEW SCHOEN AND LEVY
CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES
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 osteo-
porosis. Either cuspal pretreatment or systemic or local
therapy of the host with diphosphonate compounds
inhibits experimental bioprosthetic valve calcification
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[71–73].
Trivalent Metal Ions
Pretreatment of bioprosthetic tissue with iron and alumi-
num (eg, FeCl3 and AlCl3) inhibits calcification of sub-
dermal 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 demon-
strated that aluminum chloride prevents elastin calcifi-
cation 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 non-
stented and stented porcine aortic valves [79, 80].
Ann Thorac Surg
2005;79:1072– 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 calci-
fication. 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 phospholip-
ids 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 collage-
nase. 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 con-
nective 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 im-
planted, 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 cal-
cification of bioprosthetic tissues. Moreover, the bio-
chemistry of glutaraldehyde cross-linking is poorly un-
derstood [91]. Therefore, studies have investigated
modifications of and alternatives to conventional glutar-
aldehyde pretreatment [92]. Some investigators have
aimed to improve glutaraldehyde fixation or modify
tissues following conventional treatment with this com-
pound. For example, fixation of bioprosthetic tissue
(cusps and aortic wall) using extraordinarily high con-
centrations of glutaraldehyde (5⫻ to 10⫻ those normally
used) appear to inhibit calcification in subdermal im-
plants [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, borohy-
dride, cyanoborohydride), block residual aldehydes (eg,
L-glutamic acid, glycine, L-lysine, diamine) [95, 96], mod-
ify tissue charge (eg, protamine), and incorporate poly-
Ann Thorac Surg
2005;79:1072– 80
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 glutaral-
dehyde altogether and use other methods of tissue cross-
linking. Nonglutaraldehyde cross-linking of biopros-
thetic tissue with epoxy compounds, carbodiimides, acyl
azide, and other compounds reduces their calcification in
rat subdermal implant studies [97–99]. Epoxy cross-
linking 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 environ-
ment and, with few exceptions, the mechanistic basis for
nonglutaraldehyde approaches has not been established.
One approach investigated clinically illustrates how dif-
ficult 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 ex-
perimental bioprosthetic heart valve calcification, but the
mechanisms responsible for this are not well understood
at this time [102]. Clinical investigation of this technol-
ogy, 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 glutaraldehyde-
pretreated porcine aortic valves (stented and nonstented)
and valvular allografts and vascular segments is ob-
served clinically and experimentally. Mineral deposition
occurs throughout the vascular cross section but is ac-
centuated in the dense bands at the inner and outer
media. As with porcine valve cusps and bovine pericar-
dium, 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, prob-
ably in part owing to its protective effect against calcifi-
cation of elastin, inhibits calcification of the aortic wall.
Combination therapies using multiple agents may pro-
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CALCIFICATION OF TISSUE HEART VALVE SUBSTITUTES
vide 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 calcifi-
cation [107]. Cross-linking by a carbodiimide-based
method [108] and dye-mediated photooxidation also mit-
igated 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 ethylcarbodiimide-
dependent carboxyl-group cross-linking (carbodiimide)
treatment. The possibility that this will be shown to be
mechanistically sound and proven effective with no del-
eterious effects in vivo is indeed exciting.
Conclusions
Calcification of bioprosthetic implants is a clinically im-
portant pathologic process limiting the anticipated dura-
bility and, hence, use of tissue-derived valves. The patho-
physiology of calcification has been characterized and
largely understood through investigation using animal
models; a key common feature is the involvement of
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devitalized cells and cellular debris. Although no clini-
cally useful preventive approach is yet available, several
strategies based on either modifying biomaterials or local
drug administrations appear to be promising in some
contexts. Calcification of an exposed aortic wall may be a
significant problem with some implant types. Interesting
approaches to preventing this problem through synergis-
tic and simultaneous employment of multiple anticalci-
fication therapies or novel tissue treatments are under
investigation. Since some anticalcification approaches
have been used in clinical valves for nearly a decade,
documentation of favorable 15 to 20 year outcomes will
require yet approximately another decade.
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