Measurements of the effects of decellularization on viscoelastic properties of tissues in ovine, baboon, and human heart valves.
ABSTRACT In the development of tissue-engineered heart valves based on allograft decellularized extracellular matrix scaffolds, the material properties of the implant should be ideally comparable to the native semilunar valves. This investigation of the viscoelastic properties of the three functional aortic/pulmonary valve tissues (leaflets, sinus wall, and great vessel wall) was undertaken to establish normative values for fresh samples of human valves and to compare these properties after various steps in creating scaffolds for subsequent bioreactor-based seeding protocols. Torsional wave methods were used to measure the viscoelastic properties. Since preclinical surgical implant validation studies require relevant animal models, the tests reported here also include results for three pairs of both ovine and baboon aortic and pulmonary valves. For human aortic valves, four cryopreserved valves were compared with four decellularized scaffolds. Because of organ and heart valve transplant scarcity for pulmonary valves, only three cryopreserved and two decellularized pulmonary valves were tested. Leaflets are relatively soft. Loss angles are similar for all tissue samples. Regardless of species, the decellularization process used in this study has little effect on viscoelastic properties.
- SourceAvailable from: Pinar Zorlutuna[Show abstract] [Hide abstract]
ABSTRACT: The field of tissue engineering has been growing in the recent years as more products have made it to the market and as new uses for the engineered tissues have emerged, motivating many researchers to engage in this multidisciplinary field of research. Engineered tissues are now not only considered as end products for regenerative medicine, but also have emerged as enabling technologies for other fields of research ranging from drug discovery to biorobotics. This widespread use necessitates a variety of methodologies for production of tissue engineered constructs. In this review, these methods together with their non-clinical applications will be described. First, we will focus on novel materials used in tissue engineering scaffolds; such as recombinant proteins and synthetic, self assembling polypeptides. The recent advances in the modular tissue engineering area will be discussed. Then scaffold-free production methods, based on either cell sheets or cell aggregates will be described. Cell sources used in tissue engineering and new methods that provide improved control over cell behavior such as pathway engineering and biomimetic microenvironments for directing cell differentiation will be discussed. Finally, we will summarize the emerging uses of engineered constructs such as model tissues for drug discovery, cancer research and biorobotics applications.IEEE reviews in biomedical engineering. 12/2012;
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ABSTRACT: Aortic valve (AV) performance is closely linked to its structural components. Glycosaminoglycans (GAGs) are thought to influence the time-dependent properties of living tissues. This study investigates the effect of GAGs on the viscoelastic behaviour of the AV. Fresh porcine AV cusps were either treated enzymatically to remove GAGs or left untreated (control). The specimens were tested for stress relaxation and tensile properties under equibiaxial load conditions. The stress relaxation curves were fitted using a double exponential decay equation and the early relaxation constant (τ(1)) and late relaxation constant (τ(2)) calculated for each specimen. Immunohistochemistry confirmed the successful depletion of both sulphated and non-sulphated GAGs from the AV cusps. A statistical increase in τ(1) was found for both the radial and circumferential directions between the control and -GAGs group (radial, control 17.37s vs. -GAGs 25.65s; circumferential, control 21.47s vs. -GAGs 27.37s). It was also found that τ(1) differed between the two directions for the control group but not after GAG depletion (control, radial 17.37s vs. circumferential 21.47s; -GAGs, radial 25.65s vs. circumferential 27.37s). No effect on stiffness was found. The results showed that the presence of GAGs influences the mechanical viscoelastic properties of the AV but has no effect on the stiffness. The natural anisotropy, which reflects the relaxation kinematics, is lost after GAG depletion.Acta biomaterialia 09/2012; · 5.68 Impact Factor
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ABSTRACT: There is a major need for scaffold-based tissue engineered vascular grafts and heart valves with long-term patency and durability to be used in diabetic cardiovascular patients. We hypothesized that diabetes, by virtue of glycoxidation reactions, can directly crosslink implanted scaffolds, drastically altering their properties. In order to investigate the fate of tissue engineered scaffolds in diabetic conditions, we prepared valvular collagen scaffolds and arterial elastin scaffolds by decellularization and implanted them subdermally in diabetic rats. Both types of scaffolds exhibited significant levels of advanced glycation end products (AGEs), chemical crosslinking and stiffening -alterations which are not favorable for cardiovascular tissue engineering. Pre-implantation treatment of collagen and elastin scaffolds with penta-galloyl glucose (PGG), an antioxidant and matrix-binding polyphenol, chemically stabilized the scaffolds, reduced their enzymatic degradation, and protected them from diabetes-related complications by reduction of scaffold-bound AGE levels. PGG-treated scaffolds resisted diabetes-induced crosslinking and stiffening, were protected from calcification, and exhibited controlled remodeling in vivo, thereby supporting future use of diabetes-resistant scaffolds for cardiovascular tissue engineering in patients with diabetes.Biomaterials 10/2012; 34(3). · 8.31 Impact Factor
Measurements of the Effects of Decellularization
on Viscoelastic Properties of Tissues
in Ovine, Baboon, and Human Heart Valves
Tong Jiao, Ph.D.,1Rodney J. Clifton, Ph.D.,1Gabriel L. Converse, Ph.D.,2and Richard A. Hopkins, M.D.2
In the development of tissue-engineered heart valves based on allograft decellularized extracellular matrix
scaffolds, the material properties of the implant should be ideally comparable to the native semilunar valves.
This investigation of the viscoelastic properties of the three functional aortic/pulmonary valve tissues (leaflets,
sinus wall, and great vessel wall) was undertaken to establish normative values for fresh samples of human
valves and to compare these properties after various steps in creating scaffolds for subsequent bioreactor-based
seeding protocols. Torsional wave methods were used to measure the viscoelastic properties. Since preclinical
surgical implant validation studies require relevant animal models, the tests reported here also include results
for three pairs of both ovine and baboon aortic and pulmonary valves. For human aortic valves, four cryo-
preserved valves were compared with four decellularized scaffolds. Because of organ and heart valve transplant
scarcity for pulmonary valves, only three cryopreserved and two decellularized pulmonary valves were tested.
Leaflets are relatively soft. Loss angles are similar for all tissue samples. Regardless of species, the decellular-
ization process used in this study has little effect on viscoelastic properties.
under tension and flexure.1–15Among them, only a few in-
vestigations report the time-dependent or viscoelastic me-
chanical properties of heart valves tissues.5,6,12,15There are
even fewer reports on the viscoelastic shear properties of
heart valve tissues. Especially for leaflets, because of their
multilayer structure, understanding their viscoelastic shear
properties is very important. Efforts to tissue-engineer heart
valve tissues have led to investigations of the biomechanical
properties of decellularized heart valve tissues. However,
there are no systematic measurements of the viscoelastic
properties of heart valve tissues and the comparison of their
shear properties before and after decellularization.
To obtain the viscoelastic shear properties of heart valve
tissues, both before and after decellularization, we have used
the recently developed torsional wave facility at Brown Uni-
versity to measure the viscoelastic properties of native and
acellular, aortic and pulmonary valves of sheep, baboons, and
humans. By employing high frequencies, the highly frequency-
dependent characteristics that ‘‘tune’’ the cardiovascular
system in the range of expected heart rates (e.g., 1–3Hz) can
be avoided, thereby simplifying tissue comparisons.16–18
ost existing reports on the mechanical response of
cardiovascular materials are focused on their response
To determine whether any animal model might be rele-
vant to human models for preclinical research, this study
includes tests of the viscoelastic properties of aortic/pul-
monary valve tissues from ovine and baboon.
The objectives are (1) to obtain measurements of the vis-
coelastic properties of species-specific native tissues, (2) to
investigate whether decellularization causes degradation of
the material properties that might preclude this approach for
creating scaffolds for tissue-engineered valves, and (3) to
compare human valves to valves from a subhuman primate
and a food stock ungulate.
Materials and Methods
Experimental design and sample preparation
To establish normative values of the viscoelastic properties
of all components of the semilunar heart valves, and to
evaluate the consequences of the process of decellularization,
measurement is made before and after decellularizing allo-
graft semilunar heart valves to create extracellular matrix
(ECM) scaffolds. Table 1 summarizes the experiments that
have been done. The initial tests reported here include results
for three pairs of aortic and pulmonary valves for both ovine
and baboon and four cryopreserved and three decellularized
aortic valves for human. As the human pulmonary valves
1School of Engineering, Brown University, Providence, Rhode Island.
2Cardiovascular Research Institute, Children’s Mercy Hospital, Kansas City, Missouri.
TISSUE ENGINEERING: Part A
Volume 18, Numbers 3 and 4, 2012
ª Mary Ann Liebert, Inc.
are hard to acquire because of their clinical need, only four
cryopreserved and two decellularized human valves were
tested. Including the results for another one or two decel-
lularized pulmonary valves from humans would strengthen
the conclusions. However, from the statistical analysis, it was
found that—provided the power is 80% with the given
number of samples—the difference between the real values
and the measured mean values of the viscoelastic properties
of tissues from the decellularized human pulmonary valve is
much smaller than one standard deviation.
It is well known that vascular structures, including valves,
typically lose compliance with age because of adaptive re-
modeling in response to increased stresses such as hyper-
tension or pathological processes such as degenerative valve
disease. Because this pilot study is focused on the general
viscoelastic properties of tissues from aortic and pulmonary
valves, comparison between age and gender of the samples
are not main factors investigated in this report. Therefore, the
cryopreserved human samples are selected from adults of
either gender and are limited by availability of these gifted
tissues to valves not useable clinically because of minor im-
perfections or inability to clear strict donor criteria. Table 2
gives the age and gender for the human samples.
Fresh hearts from sheep and baboons were cleanly har-
vested and sent in antibiotic saline on ice to our laboratory
(24h). The valves were then dissected in a manner similar to
homograft valve recovery. Valves were kept in lactated ring-
er’s solution, with antibiotics, at 4?C for up to 72h before
testing. Human samples were provided by LifeNet Health
(LNH). They were cryopreserved in a freezing chamber at the
controlled cooling rate of -1?C per minute, utilizing a pro-
grammable controller to a specific endpoint (-40?C or cooler).
The allograft was then transferred to permanent storage in
vapor-phase liquid nitrogen.18The cryopreserved human
samples were shipped to our lab from LNH in a cryoshipper,
which can keep the sample frozen in the vapor of liquid ni-
trogen for 3 or 4 days. To remove the dimethylsulfoxide from
the cryopreserved sample without damaging the tissues, the
clinically validated thawing process was used.17
Intact valves were decellularized using methods previ-
ously described.19Briefly, valves were treated with two cy-
cles of reciprocating hypo/hyper osmolality to fracture cell
membranes and then extracted with a nonionic detergent
(Triton X-100, 0.05%, v/v; Sigma-Aldrich), followed by
treatment with recombinant endonuclease (Benzonase; EMD
Chemicals). Valves were further subjected to separate treat-
ments in an anionic detergent (N-lauroylsarcosine, 1.0%,
v/v; Sigma-Aldrich) and ethanol (40%, v/v; Sigma-Aldrich).
Extraction of organic solvents was then performed using ion
exchange resins (Amberlite XAD 16, Dowex Monosphere
550A Biobeads, IWT-TMD-8; Sigma-Aldrich).
Aortic valves and pulmonary valves are morphologically
similar. They both function bythe opening and closing of three
to the deformation of a thinned region of the vessel wall, called
the sinus. The vessel wall above the sinus is called the great
vessel wall. For each aortic/pulmonary valve, cylindrical
samples were obtained by dermal punch from each of three
leaflets, three sinuses, and three positions on the great vessel
wall. For each leaflet/sinus/great vessel wall, three samples
are cored out (for some valves, the leaflets and sinuses are too
small to obtain three samples, so two samples are cored). Fig-
and the positions where the cylindrical specimens were sam-
pled. Figure 1a and d shows, respectively, the aortic and pul-
monary valve immediately after being thawed. The valves
were opened along the axis as shown in Figure 1b and e. In
these two figures, the leaflets are attached to the sinuses;
however, they were removed from the valve before leaflet
samples were cored out. Each circular hole, shown in Figure 1c
and f, was made when a sample was cored out.
Diameters of the thin cylindrical samples were in the
range 3–5mm. Thicknesses of the samples were quite dif-
ferent, depending on the species, the subject, and which re-
gion the samples were taken from. For the same species,
samples from aortic valves are thicker than those from pul-
monary valves. In general, the leaflet samples were *0.2–
0.4mm thick and samples from the sinus and great vessel
wall were *1–2mm thick. Samples from the great vessel
wall were slightly thicker than those from the sinus. During
testing, the temperature of the environmental chamber is
maintained at 34?C–37?C, with relative humidity above 90%.
For torsional wave experiments,20,21a thin, cylindrical
sample of soft material is placed between two hexagonal
plates: the ‘‘top plate’’ and ‘‘bottom plate’’ shown in the inset
of Figure 2. These two plates are aligned vertically, sharing a
common axis. The bottom plate is mounted on the drive shaft
of a galvanometer that can oscillate about its axis at angles
up to–6? and frequencies up to 2500Hz. To ensure that the
shear strains in the sample are sufficiently small for linear
Table 1. Summary of the Number
of Valves Investigated
Only human samples were cryopreserved.
AV, aortic valve; PV, pulmonary valve.
Table 2. Age and Gender of the Human Samples
Sample No.TypeDonor ID Age (years) Gender
424JIAO ET AL.
viscoelasticity to be a satisfactory approximation, the imposed
rotations are maintained to be less than–0.2?. This assembly is
enclosed in an environmental chamber in order to provide
in vivo conditions. Compared with the soft viscoelastic sample,
the acrylic bottom and top plates can be considered to be
rigid. Therefore, when the bottom plate is oscillated, a torsional
wave propagates up and down through the thickness of the
sample. Based on the wave analysis described previously,20,21
the rotation of the top plate h(h, t) can be expressed as
in which h is the thickness of the sample; x0and h0are the
frequency and amplitude of the rotation of the bottom plate,
respectively; and M (x0, h) and / (x0, h) are, respectively, the
amplification factor and the phase shift that relate the am-
plitude and phase of the motion of the top plate to that of the
bottom plate. The amplification factor and phase shift are
d¼d(x0) ? q0I0x0
c¼c(x0) ? J
) and b¼ sin(d(x0)
in which the complex shear modulus G*(x0) of the sample is
expressed in terms of its magnitude jG*(x0)j and its loss
If the responses M(x0, h) and /(x0, h) are measured over a
range of frequencies, then the moduli jG*(x0)j and d(x0) can
be estimated by means of regression analysis. Values for
these two parameters are readily obtained using an Excel
spreadsheet and minimizing the differences between calcu-
lated and measured values of the amplification factor. Once
jG*j and d are determined, the storage modulus G¢ and the
loss modulus G† can be calculated from G¢=jG*jcos(d) and
The oscillation of the galvanometer is driven by a fre-
quency generator that steps through a sequence of frequen-
cies from a minimum frequency, fmin, to a maximum
frequency, fmax, with steps of Df. At each frequency, the
amplitudes of the rotation of the top and bottom plates are
measured by an optical lever technique as shown in Figures
2 and 3.20,21To measure the sample geometry, a picture of
the sample with both top and bottom plates was taken before
(04-2155) and pulmonary valve (06-4709).
(a) The aortic valve immediately after being
thawed. (b) The aortic valve cut to open
along the axis. The leaflets attached to the
sinuses. They were cut from the valve before
samples are cored out. (c) Thin cylindrical
samples were cored from the leaflet, sinus,
and great vessel wall of this aortic valve, as
shown by the circular holes. (d) The pulmo-
nary valve immediately after being thawed.
(e) The valve cut to open along the axis. The
leaflets are attached to the sinuses. They
were cut from the valve before samples are
cored out. (f) Thin cylindrical samples were
cored from the leaflet, sinus, and great vessel
wall of this pulmonary valve, as shown by
the circular holes.
Cryopreserved aortic valve
Torsional wave experimental setup.
VISCOELASTIC PROPERTIES OF HEART VALVE TISSUES425
and after each test. Using Adobe Photoshop, the ratios of the
thickness and the width of a facet of the top plate to the
thickness and diameter of a sample can be measured, re-
spectively. With the measurement of the thickness and width
of the facet of the top plate, the thickness and diameter of the
sample can be calculated.
The measured amplification factors are determined at
several frequencies. In Figure 4, the top trace is the measured
rotation of the top plate and the bottom trace is the measured
rotation of the bottom plate. Every two periods of the sinu-
soidal wave form corresponds to one period of the oscillation
of the corresponding plate. As the frequency is stepped up,
the angle of rotation of the top plate increases and that of the
bottom plate stays almost the same or even decreases a little.
Eventually, the amplification factor reaches a peak value at
the resonance frequency, fpeak, and then decreases.
Statistical analysis method
As shown in the next part, the test results indicate little
frequency dependence of the viscoelastic properties over the
tested frequency ranges. For each species, the characteristic
viscoelastic moduli for each type of tissue (leaflet, sinus, and
great vessel wall) are obtained by averaging results of tests
on all tissues of the same type, from all the valves. For ex-
ample, four cryopreserved human aortic valves were tested.
For each aortic valve, there are three leaflets. From each
leaflet, two to three samples are tested. Therefore, the vis-
coelastic moduli of the leaflets for cryopreserved human
aortic valves are values averaged over all 35 samples. The
error bar represents the confident interval at 95% confidence
level. To compare the stiffnesses of tissues of different types,
Mann–Whitney U tests, were used to analyze the data.
Stiffnesses of the two types of tissues being compared are
said to be statistically different for p<0.01; otherwise, their
stiffnesses are interpreted as being statistically the same.
Figure 5 shows representative curves of frequency de-
pendence of amplification factors for samples from a human
cryopreserved aortic valve. From these plots, it is evident
that the linear viscoelastic wave analysis provides a re-
markably good fit to the observed frequency dependence of
the amplification factor over a range of frequencies spanning
the peak. The inserts in Figure 5 show the samples sand-
wiched between the top and bottom plates.
For the same top and bottom plates, the resonant fre-
quency depends on the sample size (thickness and diameter)
and the properties of the sample. The frequency range is
usually from 50 to 100Hz for leaflets and 100 to 150Hz for
samples from the sinus and great vessel wall. Test results
indicate little frequency dependence of the viscoelastic
properties over these frequency ranges. Moreover, as indi-
cated earlier, age and gender of the samples are not main
factors investigated in this pilot report. To compare the
mechanical response of the tissues before and after decel-
lularization for different species, we averaged the test results
of each type tissue from the samples at three positions of
Optical layout of torsional wave experiments.
fication factors at successive frequen-
cies, with records of the wave traces at
Typical output of the ampli-
426 JIAO ET AL.
each valve and for all tested fresh or decellularized valves to
obtain characteristic results for the various species. There-
fore, the experimental results of human samples presented
here represent only average behavior. They are not repre-
sentative of specific cohorts of patients in specific age ranges.
Storage moduli determined for all tissues tested are tabu-
lated in Table 3. The p-values from the statistical analysis of
whether moduli for natural tissues and decellularized tissues
are statistically comparable are also shown (Table 3).
Figure 6 shows the experimental results for the three types
of tissues for valves from sheep. Leaflet tissues are shown to
be very pliable for both aortic and pulmonary valves. Stiff-
nesses of sinus and great vessel wall tissues are comparable
(p>0.122); however, the stiffness of pulmonary valve tissues
tends to be lower than that of aortic valve tissues (p<0.009).
As shown in Table 3, the principal effect of decellularization
is that the stiffnesses of sinus and great vessel wall tissues of
pulmonary valves are significantly reduced (p<0.01). Leaflet
stiffness is essentially the same for both aortic and pulmo-
nary valves, either fresh or decellularized. Similarly, tan(d)
values are essentially the same for all tissues, either fresh or
Experimental results for baboon tissues are shown in
Figure 7. In the baboon, both aortic and pulmonary valve
leaflet tissues exhibit much lower stiffness than tissue from
the sinuses and great vessel walls (p<0.002). Additionally,
the pulmonary valve sinus and great vessel wall tissues ex-
hibit lower stiffness than for the correspondent aortic valve
tissues (p<0.007). Figure 7 and Table 3 show that except for
leaflets of pulmonary valves, which tend to be stiffer after
being decellularized (p<0.001), decellularization does not
appear to have a significant effect on the stiffness of any of
the baboon tissues (p>0.034). Tan(d) values are essentially
the same for all tissues and comparable to those for sheep.
Storage modulus and loss tangent values for human heart
valve tissues are shown in Figure 8. For human valves, the
wave theory for leaflet, sinus, and great vessel wall of a
cryopreserved aortic valve from a human. The symbols show
the experimental results and the curves show the fitted re-
sults from the wave analysis.
Typical fit of the experimental results with torsional
Table 3. Storage Modulus of All Types of Tissues
Storage modulus (kPa)
Ovine AV Leaflet 0.350–0.045 0.296–0.0278
PVLeaflet 0.337–0.095 0.373–0.110
Wall 3.003–0.474 1.267–0.208
AV Leaflet 1.297–0.495
Sinus 9.01–1.457 12.94–0.3045
PV Leaflet 0.822–0.216 2.348–0.462
Human AV Leaflet 0.545–0.079
PVLeaflet 0.228–0.022 0.351–0.046
the tissues of aortic and pulmonary valves from sheep. The
bars at the bottom and the top of the plot represent, re-
spectively, the averaged storage modulus and Tan(d), where
d is the loss angle. The error bar shows the range of the
distribution of the results that fall within confident interval at
95% confidence level. AV, aortic value; PV, pulmonary valve.
Experimental results of the viscoelastic properties of
the tissues of aortic and pulmonary valves from baboon
(labels are the same as in Fig. 6).
Experimental results of the viscoelastic properties of
VISCOELASTIC PROPERTIES OF HEART VALVE TISSUES 427
‘‘fresh’’ tissues were cryopreserved as described earlier. Si-
milar to the baboon, human leaflet tissue from both aortic
and pulmonary valves exhibit much lower stiffness than
tissues from the sinus and great vessel wall (p<0.001). Also,
sinus and great vessel wall tissues from aortic valves are
stiffer than those from pulmonary valves (p<0.001). As
shown in Figure 8 and Table 3, after being decellularized, the
stiffness of pulmonary valve leaflets increased (p<0.001),
whereas the stiffness of decellularized sinus of aortic valve
decreased (p<0.007). For the other tissues from human heart
valves, decellularization does not appear to have a signifi-
cant effect on their stiffnesses (p>0.01). Tan(d) values do not
vary significantly over the three types of tissues and appear
to be comparable to those for sheep and baboon.
For comparisons between different species, the measured
viscoelastic properties for fresh/cryopreserved sheep, ba-
boon, and human aortic and pulmonary valve tissues are
shown in Figure 9a and b, respectively. Figure 9a clearly
shows that the viscoelastic properties of the tissues from
human aortic valves are closest to those of baboon. Stiff-
nesses for aortic valve tissues from sheep are significantly
lower than those of the other two species (p<0.001), except
the leaflet, which is comparable to that from human
(p>0.019), but softer than that from baboon (p<0.001). No
significant differences were observed in the viscoelastic
properties of pulmonary valve tissues between these three
species, except that the storage modulus of the leaflet and
great vessel wall are slightly higher in the baboon than in the
other two species.
The first human clinical surgical use of fresh aortic valve
homografts transplanted into the descending thoracic aorta
for clinical amelioration of the consequences of native aortic
valve insufficiency was reported by Gordon Murray in
1956.22As manufactured prosthetic valves gradually evolved
in design and range of choices, the use of homograft valves
began to decline, not due to performance, but rather as a
consequence of logistics, banking, and tissue transport is-
sues.23In the 1980s and 1990s, cardiovascular allograft
tissues became increasingly available, primarily as cryopre-
served valves with variably retained native cell viability.
Especially for pediatric use, the surgical and specific hemo-
dynamic advantages of homograft semilunar cardiac valves
have been recognized.
Although these transplanted valves performed well in the
short to mid term, they have been associated with ultimate
fibrosis, calcification, and failure in a significant proportion
of cases, especially for infants and young children, for whom
retained growth and repair functions would be ideal.23
Ganguly et al. have evaluated the pattern of homograft fail-
ure and the quality of life in patients after homograft im-
plantation.24They found the performance status of 60% of
respondents to be good, 20% to be moderate, and 20% to be
poor. Eleven patients (18.9%) required subsequent redo
valve replacement after initial homograft insertion (pulmo-
nary=6, aortic=5). Therefore, there are continuing efforts to
develop ‘‘tissue-engineered’’ heart valves to achieve viability
combined with relative or actual immune tolerance with the
thought that this would retain the outstanding engineering
design of the native semilunar valve while avoiding the in-
evitable destructive foreign body reaction to transplanted
proinflammatory materials. Simon et al. have reported early
failure of the tissue-engineered porcine heart valve in pedi-
atric patients.25In fact, this case and some other experiments
demonstrate that the lowest level of stimulation was with
thoroughly ‘‘decellularized’’ human tissues.26Therefore, one
conceptual path to an autologous recellularized tissue-en-
gineered valve is to start with an acellular allogeneic ECM
scaffold of proven design derived from decellularized hu-
man allograft. Subsequent ex vivo or in vivo recellularization
could establish a native multicellular host population that
the tissues of aortic and pulmonary valves from human (la-
bels are the same as in Fig. 7).
Experimental results of the viscoelastic properties of
aortic valves from sheep and baboon and from cryopre-
served human samples. (b) Comparison of viscoelastic
properties of fresh pulmonary valves from sheep and baboon
and from cryopreserved human samples.
(a) Comparison of viscoelastic properties of fresh
428JIAO ET AL.
organizes as a living, functioning heart valve tissue that can
grow and remodel, potentially leading to improved wear
However, appealing theoretically, there is risk that any
decellularization process may result in hazardous alterations
of physical properties of the valve itself. Such material and
performance degradations need to be carefully defined,
measured, and related to appropriate standards derived from
similar measurements of functioning human valves. Critical
material properties of semilunar heart valves involve the in-
herent viscoelasticity, evidenced by the known frequency
dependence of the functional components of the valves
(leaflets, sinus walls, and great vessel wall above the valve).27
There are few investigations focused on the shear prop-
erties of heart valve tissues; even fewer reports are on the
viscoelastic shear properties of cardiovascular materials.
Most available reports on the biomechanical properties of
aortic/pulmonary heart valve tissue address their tensile and
flexure behavior. Thubrikar et al.2have investigated the
mechanical properties of aortic valve leaflets from dogs.
They measured the tensile stress–strain curve both in vivo
and in vitro. Vesely and Boughner3have measured the
bending stiffness of porcine xenograft leaflets. Sacks and his
colleagues have investigated the biaxial and the flexural
properties of leaflets from fresh porcine heart valves.4–9With
data from all of these experiments, Sacks10constructed a
structural constitutive model to describe the behavior of
leaflets. As the biomechanical properties of the heart valve
leaflet have been well characterized using conventional
techniques, less mechanical test data are available for tissues
from different parts of the heart valve. Most available data
on the sinus and great vessel wall of both aortic valves and
pulmonary valves are inferred from measurements of tissues
from arterial walls. Hayashi et al.11have used uniaxial and
biaxial tension to get the basic mechanical properties of calf
arterial walls. Silver et al.12–15have obtained the ‘‘pressure–
strain’’ elastic modulus and circumferential elastic modulus
of arterial walls by noninvasively measuring the blood
pressure vs. the diameter change of human aorta. Silver et al.
also measured the uniaxial tension stress–strain curve and
uniaxial tension incremental stress–strain curve of human
Storage moduli reported herein for sinus and great vessel
walls under shear are approximately an order of magnitude
smaller than those obtained for arterial walls by pressure–
diameter measurement and uniaxial tension.13Differences
are likely due to the strains at which the moduli are mea-
sured. The stress–strain curve of a natural tissue subjected to
simple tension is nonlinear, having a very low elastic mod-
ulus in the low strain regime, increasing with progressively
higher strains to a much larger value at larger strains. For
arteries, the walls are in tension initially. The starting strain
of the pressure–diameter measurement corresponds to a
strain of *30%.13Because the tissues are very soft, elastic
moduli in the low strain regime are difficult to measure ac-
curately by uniaxial tension. Consequently, the measure-
ments below strains of 30% are neglected. In torsional wave
experiments, the maximum strain applied is *5%, which is
much smaller than for the other methods. Therefore, the
storage moduli obtained by torsional wave experiments can
be expected to be significantly smaller than the values ob-
tained in other investigations.
When blood flow passes the valves, the valve tissues are
working in the regime of low stresses and strains. Experi-
ments28–31have shown that wall shear stress above 400
dynes/cm2(40 Pa) can damage the endothelial lining of the
aorta, and shear stresses above 950 dynes/cm2can erode the
endothelium from the vessel wall. Aortic wall shear stresses
of *29 dynes/cm2have been measured downstream of xe-
nograft aortic valves in vitro.30,31Aortic valve leaflets are also
covered with an endothelial lining. Weston et al.32have
found that the maximum shear stress on the leaflets is about
79 dynes/cm2. The shear moduli of the heart valve tissues
reported herein are measured under conditions similar to
those at which they function. For leaflets of heart valves in
systole phase, they are stretched up to 30% deformation.
Under this condition, their viscoelastic shear responses can
be expected to be different from those reported here, which
are measured without prestretching. However, the reported
measurements provide simple and systematic comparisons
to elucidate the change of mechanical properties before and
after decellularization. They also provide direct comparison
of differences in the mechanical properties of tissues from
The procedure of cryopreservation may have some impact
on the ECM structure.33,34However, as the fresh human heart
valves are difficult to obtain and cryopreserved heart valves
are still the widely used homografts for implantation, we
compared the viscoelastic properties of cryopreserved human
heart valve tissues before and after decellularization. Several
reports of the effects of decellularization on the quasistatic
mechanical properties of heart valve tissues are available in
the literature.35–41Seebacher et al. reported no change in the
strength of porcine pulmonary valve conduits in uniaxial
tension following decellularization, though stiffness values
were not reported.35Korossis et al. found no difference in the
tensile strength of porcine aortic valve leaflets but did report a
significant decrease in stiffness within the elastin-dominated
region of the stress–strain curve.36Liao et al. investigated the
effects of multiple decellularization protocols on the biome-
chanics of porcine aortic valve leaflets under flexural and
planar biaxial loading conditions. The authors reported de-
creased flexural stiffness regardless of the decellularization
protocol used but also reported increases in areal strain and
tangent modulus under biaxial loading conditions. The rela-
tive magnitude of these changes varied with decellularization
protocol.37Hydrodynamic performance of heart valves trea-
ted by different decellularization protocols has also been in-
vestigated. Dohmen et al. have made in vitro measurements of
the hydrodynamics of decellularized pulmonary porcine
valves and compared with glutaraldehyde and polyurethane
heart valves.38They found that decellularized pulmonary
porcine valves showed the same excellent performance as
polyurethane valve prosthesis. Recently, Bottio et al. have in-
vestigated the hydrodynamic behavior of intact porcine aortic
roots, both before and after decellulariztion.39They have used
four decellularization protocols, TRI-COL, TRI-DOC, sodium
dodecyl sulfate (SDS) 0.03%, and SDS 0.1%. They found that,
except for SDS 0.03%, the other three protocols modified the
systolic and diastolic functions of intact porcine aortic root.
In this report, because the fresh tissues and the decel-
lularized tissues are not from the same valve, comparisons
between fresh and decellularized tissues are not direct.
However, the comparison between the results averaged over
VISCOELASTIC PROPERTIES OF HEART VALVE TISSUES429
three or four fresh/cryopreserved and decellularized valves
appears to provide a statistically significant indication of the
effect of decellularization. Statistical analysis (see Table 3)
shows that for most (i.e., 13 of 18) statistical comparisons of
our experimental results on fresh/cryopreserved and decel-
lularized samples, the decellularization used in this series of
experiments did not cause significant changes in stiffness.
The exceptions are ovine—sinus and great vessel wall of
pulmonary valves; baboon—leaflet of pulmonary valves;
and human—sinus of aortic valves and leaflet of pulmonary
valves. Even for those tissues whose stiffnesses have statis-
tically changed, the magnitude of these changes may not be
large enough to cause any functional consequences. Quinn
et al.19reported comparable hemodynamic performance be-
tween cryopreserved and decellularized pulmonary valves
in a 20-week ovine surgical model. These results suggest that
ECM structural proteins, not cells, are the dominant con-
tributors to the passive viscoelastic properties of semilunar
valves and those ECM properties are typically not altered by
decellularization. By experimental design, this method can-
not test for active cellular contractility but may assess the
passive contribution of cell-based proteins in the ‘‘native
cryopreserved’’ valves.42Clearly, it is important to consider
the effects of a specific decellularization protocol on the
mechanical behavior of heart valves intended for use as tis-
sue-engineering constructs. However, because of differences
in decellularization protocols and biomechanical properties
measured, direct comparison with previous reports cannot
1. For sheep, baboon, and human—and for both aortic
and pulmonary valves—the leaflet is softer than the
sinus and the great vessel wall. The latter two tissues
have similar stiffness.
2. For the same species, tissues from aortic valves are
stiffer than the corresponding tissues from pulmonary
3. Loss angles are similar for all tissue samples.
4. Regardless of species, the decellularization process used
This research was supported by Brown University and by
Children’s Mercy Hospital and NIH–SNPRC Base Grant No.
P51RR013986-11, Southwest National Primate Research
Center (SNPRC) Grants-in-Aid ‘‘Bioengineered Heart Valves:
Baboon Implant Feasibility Study.’’
No competing financial interests exist.
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Address correspondence to:
Tong Jiao, Ph.D.
School of Engineering
Box. D, Engineering
182 Hope St.
Providence, RI 02912
Received: November 22, 2010
Accepted: September 15, 2011
Online Publication Date: October 25, 2011
VISCOELASTIC PROPERTIES OF HEART VALVE TISSUES431