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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_3655_Библиотеки_им_академика_М_И_Перельмана
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156
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M. Valente et al.
Anticalcication Strategies
The mitigation of bioprosthetic valve calcication can be
achieved in two main ways:
• Free aldehyde neutralization: It is possible to prevent the
toxic effects of free aldehyde residuals by bonding unsaturated glutaraldehyde groups with amino-terminal amino
acids, such as amino-oleic or other advanced patented
technologies that involve also glycol as preventive
medium. They neutralize the “killing” action of the aldehyde residuals against host endothelial cells, allowing the
graft surface to be cell covered and thereby decreasing
any propensity to mineralization.
• Lipid extraction: this method aims to remove the phosphorus component of the early nuclei of calcication by
means of solvents/surfactants. Examples include sodium
dodecyl sulfate (T6), alcohols, polysorbate 80 (Tween
80), or a combination of polysorbate 80 and ethanol as in
Edwards XenoLogiX treatment (Fig.9.4).
Preclinical Testing
The efcacy of these anticalcication agents may be monitored preclinically in several ways:
• Subdermal rat implantation: This rapid method involves
the subcutaneous implantation of tissue pieces
(glutaraldehyde- xed bovine pericardium or porcine
cusps) into the back of Sprague-Dawley rats. Usually,
four round or square pieces—two treated and two
untreated—are applied to the same animal, with the
implant position being rotated in different rats. The ideal
implantation time should be no less than 10–12weeks.
• Circulatory implantation in large animals: This is the second
compulsory step for the preclinical testing of any novel bioprosthetic device, with an orthotopic intracardiac implant
(usually atrioventricular) replacing the native valve. Mitral
valve replacement in juvenile sheep (aged 20weeks), with an
implant time of at least 140–150days, is considered the best
accelerated calcication model for circulatory implants and is
recommended by the FDA.The orthotopic aortic position in
animals is used to test transarterial or transapical aortic valve
implants with bioprosthetic devices (TAVI).
• In vitro: Accelerated calcication can be accomplished
through a pulsatile testing device. In this case, the stented
valve—either porcine or pericardial—is inserted into a
device within a rapid synthetic calcication solution
[Ca×P=130(mg/dl)2], with full opening and closure of
the valve cusps being conducted at 300cycles per minute
(Fig. 9.6). This system has been shown to reproduce
intrinsic calcium phosphate mineralization after
19×106cycles, with results suggesting the presence of
apatite crystals by diffractometry.
9 Anticalcication Strategies toIncrease Bioprosthetic Valve Durability
157
a
c
b
d
Fig. 9.6 Accelerated intrinsic calcication achieved invitro through
pulsatile testing with a rapid synthetic calcication solution, viewed at
X-ray (a) and histology (b). Imaging with scanning electron micros-
Clinical Trials
Testing the efcacy of anticalcic agents in new-generation
bioprosthetic xenografts in clinical setting requires at least
10–15years of follow-up, with echocardiographic monitoring (possibly with the addition of computed tomography) to
detect early mineralization in vivo (Fig. 9.5). A thorough
study of the explants at the time of reoperation or death is
mandatory, not only to establish the cause and mode of failure but also to distinguish the primary causes of SVD (such
as calcication, brous tissue overgrowth, primary tears,
lipid inltration, thrombus formation, cusp hematoma, commissural dehiscence, stent fracture or bending) from secondary causes (such as endocarditis or paravalvular leak). A
rigorous study protocol for pathological investigations is
compulsory, both in animals and in the clinical experience.
This would include gross examination, X-ray, histology,
copy (c) and diffractometry (d) revealed the calcication to be apatite
crystals. From Pettenazzo etal., J Thorac Cardiovasc Surg, 2001
immunohistochemistry investigations, transmission and
scanning electron microscopy, and atomic absorption spectroscopy for monitoring both calcium and phosphorus levels.
Energy-dispersive analysis and diffractometry are also recommended (Fig.9.6).
Preclinical andClinical Tests ofNew
Anticalcication Agents: ThePadua
Experience
Free Aldehyde Radical Neutralization
(Homocysteic Acid Detoxication)
The pericardium was xed in glutaraldehyde for crosslinking, treated with homocysteic acid to bind unsaturated aldehyde groups, and then preserved with a buffered
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M. Valente et al.
a
c
b
d
Fig. 9.7 Subdermal implant with free aldehyde radical neutralization
achieved by homocysteic acid. After 12 weeks, pinpoint calcication
was observed in treated pericardial discs, vs. massive in untreated peri-
glutaraldehyde-free solution. Four discs of pericardium (two
treated, two untreated) were implanted subcutaneously into
the back of 24 Sprague-Dawley rats for 14, 28, 56, and
84days and then submitted to pathology study.
Dystrophic calcication was shown to commence at
28 days and increased with time in both the treated and
untreated animals, with a signicant difference between
the two at 84days (p=0.01). Mineralization was intrinsic
to the graft tissue and involved both cell debris and collagen. Apatite crystals were identied at diffractometry
(Fig.9.7).
The same test was then conducted in the large animal circulatory model (orthotopic tricuspid position). Although the
results were satisfactory, the choice of the tricuspid position
rendered the results questionable, as the right-sided heart is a
low-pressure system and hence unreliable for promoting calcication. The overall results were so convincing, with
regard to homocysteic acid potential to mitigate dystrophic
calcication, that new-generation pericardial prosthetic
cardial discs, at both X-ray (a) and histology with von Kossa stain
(b=pinpoint, c=massive). The diffractometry results were in keeping
with apatite mineralization (d)
valves had been created: the Pericarbon More and the stentless Freedom Solo. They were manufactured by Sorin and
introduced to the market for clinical use.
α-Amino-oleic Acid Detoxication
α-Amino-oleic acid treatment is currently employed in the
manufacture of the Mosaic bioprosthetic valve, a newgeneration porcine valve (Medtronic Inc.). The experiments
were conducted in the mitral position of the sheep model,
comparing Hancock Standard versus Mosaic valve prostheses. As requested by the FDA protocol, the prostheses were
in place for 20weeks. Overall, the degree of mineralization
was low in both cases, most likely because the sheep at
implant were relatively old; nonetheless, the difference in
calcium content of the two valve prostheses after 20weeks
was statistically signicant between the Mosaic and the
Hancock Standard (p<0.01) (Fig.9.8).
Mean Ca content: 5.90 µg/mg dry weight Mean Ca content: 0.88 µg/mg dry weight
Mosaic (140 days)
Hancock Standard (148days)
ab
cd
9 Anticalcication Strategies toIncrease Bioprosthetic Valve Durability
159
Fig. 9.8 Hancock Standard versus Mosaic porcine valves after
20weeks of implantation in the mitral position of growing sheep. Note
the presence of mineralization in Hancock Standard (a, c) compared
Lipid Extraction
with absence of mineralization in Mosaic (b, d), both at X-ray and histology. From Weber P etal., J Thorac Cardiovasc Surg, 2006
the Hancock II the rate of SVD was much lower with valve
failure postponed by a further 5–7years (Fig.9.5). Hence,
Detergents/Surfactants
T6 treatment had resulted in a sharp increase in the porcine
valve xenograft durability, so the patient age threshold for
Surfactant treatment with T6 (sodium dodecyl sulfate) was
introduced as an innovative anticalcication strategy in the
second-generation Hancock porcine valve (Hancock II). The
clinical use of this new device commenced in 1983, and sufcient time has elapsed to establish whether the treatment
was effective in increasing porcine valve xenograft durability. A report of the cumulative experience from the Treviso
and Padua cardiac surgery teams demonstrated that whereas
in the case of the Hancock Standard signicant prosthetic
failure due to SVD had started at 7–8years after implant, in
clinical use of Hancock II could be anticipated by 10years.
Studies of the rare late Hancock II explants disclosed
another unexpected cause of failure in the long term, namely,
lipid inltration with tearing (Fig.9.9). Lipid-mediated macrophage inammation and the expression of metalloproteinase- 9 may contribute to degradation of the cusp extracellular
matrix (Fig.9.10). The monitoring of serum cholesterol and
low-density lipoproteins may be mandatory in patients bearing a bioprosthetic valve xenograft and, if increased, statin
therapy might be warranted.
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M. Valente et al.
a
c
b
d
Fig. 9.9 Transmission electron microscopy of a porcine cusp Hancock
II valve, which failed after 12years because of cusp tearing. Note the
lipid insudation (a) with lipid-laden macrophages (lipid droplets, cho-
lesterol needles) (a–c), similar to foam cells in atherosclerosis.
Calcication of cholesterol needles was also observed (d). From Bottio
etal., J Thorac Cardiovasc Surg, 2003
9 Anticalcication Strategies toIncrease Bioprosthetic Valve Durability
a b
161
Fig. 9.10 Aortic Hancock II explant, 79months after implant. Yellow appearance of the cusps by lipid Insudation (a) and macrophage inamma-
tory reaction against cholesterol clefts at histology (b). From Bottio etal., J Thorac Cardiovasc Surg, 2003
Alcohols
bovine pericardium—treated and untreated—were
implanted subcutaneously in Sprague-Dawley rats for 30
Ethanol has proven to be a very effective means to extract the
phospholipids of cell membranes and thus to act as a “decellularization” treatment. The lipids of cell membranes are
well known as calcium binders, offering the phosphorus
component for the onset of calcium phosphate production.
Ethanol is currently employed in the Epic™ porcine xenograft (St. Jude Medical Inc.) (Fig.9.4).
More recently, an investigation was made with octanediol, a long-chain aliphatic alcohol that possesses a hydrophobic tail to aid its solubility in lipids and a hydrophilic
head to aid its water solubility. The hydrophilic head also
allows to bind cytotoxic aldehyde residues (Fig.9.11). A
combination of 5% 1,2-octanediol with 40% ethanol provides Duranol™. The efcacy of octanediol treatment on
glutaraldehyde-xed bovine pericardium was rst tested in
a subcutaneous rat model. Squares of glutaraldehyde-xed
and 75days. The ndings were impressive, with a clearcut
prevention of mineralization even at 75days, both at X-ray
(score 0–4) (Fig.9.12) and histology. Moreover, the difference in calcium content (monitored with atomic absorption
spectroscopy) was highly signicant (p < 0.0001), with
mean calcium contents at 75 days of 165.61 ± 23.35 in
untreated vs. 2.36±7.38mg/g in treated samples, respectively. By investigating the pericardium with transmission
electron microscopy, the octanediol procedure resulted in
the disappearance of pericardiocytes (=total decellularization) in both unimplanted and implanted tissues (Fig.9.13).
The efcacy of octanediol was subsequently tested in the
accelerated calcication model of a juvenile sheep circulatory implant in the mitral position, in place 20 weeks. A
sharp decrease (25%) in mineralization was achieved
(p<0.001) (unpublished data).
162
•
•
ON
STANDARD
OCTANEDIOL
bd
head
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Fig. 9.11 Mechanism of
phospholipid reduction by
octanediol treatment
Octanediol
molecule
M. Valente et al.
Octanediol
molecule
Fig. 9.12 Octanediol treatment
(ON vs. standard ST) in the
subdermal rat model at X-ray
examination (grade score: 0–4)
at 30 vs. 75days from implant.
From Pettenazzo etal., Eur J
Cardiothorac Surg, 2008
Phospholipid
An untreated pericardial
layer is going to be treated
using Octanediol molecules
Phospholipid Phospholipid
The Octanediol’s lipid-
soluble tail interacts with
the phospholipid’s head
Lipid-soluble tail Water-
The Octanediol’s water-soluble head makes
the phospholipid soluble, namely removeable
by rinsing pericardium layers
soluble
Mean X-Ray score at
30 days:
ST = 3.16 ± 1.1
vs
ON= 0
Mean X-Ray score at
75 days:
ST= 3.58 ± 0.66
vs
ON= 0.33 ± 1.15
before manufacturing
ST
ST
ON
Fig. 9.13 Electron
microscopy images in
untreated (a, b) vs.
a
c
octanediol-treated (c, d)
pericardium, either
unimplanted or explanted
after 30days. Note the
decellularization in the ON
treated pericardium and
Unimplanted
calcication occurring in the
cell debris of the untreated
pericardium. From Pettenazzo
etal., Eur J Cardiothorac
Surg, 2008
30 days
9 Anticalcication Strategies toIncrease Bioprosthetic Valve Durability
163
Decellularization
Decellularization of aortic and pulmonary homografts is one
of the most innovative tissue-engineered means to provide a
very effective antimineralization treatment. This was tested
in the sheep model (Fig.9.14).
Complete decellularization was achieved by 0.5% sodium
deoxycholate and 0.5% sodium dodecyl sulfate, with disappearance not only of endothelial cells from cusps and aortic
wall but also of interstitial cells from cusps and lamellar
units of the tunica media (Fig.9.15).
Fig. 9.14 Unimplanted
decellularized homograft. (a)
Gross view; (b) X-ray
ab
After 420 days of implant, re-endothelial lining and
repopulation in the recipient interstitial cusp cells occurred
(Fig.9.16) as well as in the external tunica media (Fig.9.17).
Thus, the homografts became autografts, as far as cell population is concerned.
Spectroscopy revealed a negligible amount of mineralization, both in the cusps and aortic wall.
Removal of native cells followed by repopulation with recipient cells (self-repopulation) seems to be an effective method to
prevent calcication, transforming the homograft into a vital
autograft and preventing occurrence of immune rejection.
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M. Valente et al.
a
b c
def
Fig. 9.15 At histology absence of cells removed from the aortic lamellar units (a–c) and from the cusps (d–f), after decellularization treatment.
From Della Barbera etal., Cardiovasc Pathol, 2015
9 Anticalcication Strategies toIncrease Bioprosthetic Valve Durability
165
a
b
cd
Fig. 9.16 After implant in sheep, repopularization of endothelial and interstitial cells in the cusps occurred (a–d)
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