J. Bunvrhan~rr Vol. 19. No I. pp ?7-3i. 1986 @xl-9290 86 13 w + .

cQ
PnntcdI” GrcarBnum C 1986 Pcrgamon Press Ltd.

AN APPROACH TO QUANTIFICATION OF BIAXIAL TISSUE

STRESS-STRAIN DATA

FRANK C. P. YIN, PAUL H. CHEW and SCOTT L. ZEGER

Departments of Medicine, Biostatistics, and Physiology, The Johns Hopkins Medical Institutions,
Baltimore, MD 21205, U.S.A.

Abstract-Delineation of the mechanical properties of biologic tissues is one of the cornerstones of

biomechanics. Abundant data from uniaxial tests exist but these cannot be extrapolated to describe three-
dimensional properties of tissue. Biaxial stress-strain studies have been performed using skin, blood vessels
and pericardium. Quantitative description of tissue properties in these studies has employed either
polynomial or exponential strain-energy functions. Interpretation of these data, however, is difficult because
of wide variability of the estimated coefficients of these functions. This variability has been attributed to
experimental noise, numerical instabilities in the algorithms, or to strain-history dependence. No systematic
method has been proposed to evaluate the variability. This paper describes a statistically based approach to
assessing the sources of and accounting for variability of coefficients in describing biaxial stress-strain data.
Our data are from canine pericardium subjected to various combinations of simultaneous biaxial stretching.
We first determine a suitable strain-energy function with the least number of free parameters that will fit the
data reasonably. We then perform residual analysis to see if standard statistical methods can be used to assess
the variability. If not, we use a nonparametric method called bootstrapping that is suitable for assessing the
uncertainty in the coefficients. Using a five-parameter exponential strain-energy function, pericardial tissue is
found to be strain-history dependent and anisotropic. These findings cannot be attributed to either
experimental noise or instability in the numerical algorithms.

INTRODUCTION nomials or exponentials of the strains (Blatz er al.,

Delineation of the mechanical properties of biologic
tissues is one of the foundations of biomechanics. The
properties of a variety of tissues have been described
under uniaxial or biaxial loading conditions (Chuong
and Fung, 1983; Demer and Yin, 1983; Doyle and
Dobrin, 1971; Fung, 1973, Fung et al., 1978, 1979;
Hildebrandt et al., 1969; Lanir and Fung, 1974;
LoefAer and Sagawa, 1975; Manak, 1980; Parmley et
al., 1973; Pate1 et cl., 1973; Pinto and Fung, 1973;
Sonnenblick, 1974; Spurgeon et al., 1977; Tong and
Fung, 1976; Vaishnav et al., 1972; Vito and Hickey,
1980; Wiegner and Bing, 1981; Wineman et al., 1979;
Yin and Fung, 1971). Uniaxial tests are relatively easy
to perform and interpret but cannot provide a unique
description of the tissue’s three-dimensional constitut-
ive properties. While the general three-dimensional
constitutive properties also cannot be deduced from
biaxial data alone, in some cases they can be inferred
when biaxial data are combined with those from
another type of test such as torsion or shear. Thus,
biaxial tests are a step towards a full description of the
constitutive properties of tissue.
The challenge facing workers in the field is to
describe the material properties as accurately and
concisely as possible. Since most biological tissues are
nonlinear and undergo largedeformations, most of the
attempts at quantitative descriptions have utilized
strain-energy functions that involve either poly-
Received 22 October 1984; in recked form 25 June 1985.
F.C.P. Yin is an Established Investigator of the American
Heart Association.
1969; Dehoff, 1978; Demiray, 1972; Gou, 1970;
Hudetz, 1979; Fung, 1973; Snyder, 1972; Vito, 1973;
Vaishnav, 1980). For both types of functions estim-
ation of the coefficients entails use of nonlinear
optimization techniques. Reliable interpretation of
these estimated coefficients has been hampered, how-
ever, by wide variability (sometimes of several orders
of magnitudes) even under nearly identical test con-
ditions (Tong and Fung, 1976; Fung et al., 1979). This
variability has been ascribed to either experimental
noise, numerical instabilities in the algorithms or true
tissue variability (Fung et ai., 1979). No systematic
method has been proposed either to evaluate the
sources of the variability or to take the variability into
account. Both must be done before reliable interpret-
ations of tissue properties can be made.
We examined the difficulties related to quantifi-
cation of biaxial data, specifically with respect to
assessing the variability of the coefficients. In this
paper we describe an approach to these problems and
illustrate its application to biaxial test results in
pericardial tissue from dogs. The major steps in our
approach are: (1) an objective method to determine
whether a selected strain-energy function is suitably
parameterized, (2) residual analysis to assess whether
the errors between the predicted and observed stresses
follow a Gaussian (normal) distribution and are
independent, and (3) assessment of the variability of
the material coefficients and/or the resulting strain
energy function by either classical statistical methods
or by a nonparametric method called bootstrapping.
This approach allows us both to judge the suitability of
a particular strain-energy function and to estimate the

27
28 F. C. P. YIN,P. H. CHEWand S. L. ZEGER

uncertainties of its coefficients. It thereby improves the

reliability of our interpretations of material properties
from biaxial tests.

METHODS

Data acquisition
A fresh pericardial specimen approximately 4
x 4 cm overlying the anterior wall of the left ventricle
was obtained from a mongrel dog anesthetized with
pentobarbital. Prior to removal from the animal, the
head-tail and transverse directions were marked. The
specimen was then excised and prepared for mounting 0.2 0.4
in our biaxial test apparatus (Demer and Yin, 1983). X-STRAIN
Four arrays of silk thread were sewn directly through
the tissue to form a trampoline-like arrangement. Each Fig. 1. Schematic representation of the three different load-
ing protocols (strain trajectories) to which the tissue was
array was composed of a single strand arranged such
subjected. For both the loading and unloading portions of the
that 4-6 nearly parallel loops were formed. The cycle, the ratio of the strains was kept constant. Because the
specimen was aligned so that it was loaded in the tissue exhibits hysteresis between the loading and unloading
previously marked directions. During this preparation portions of the cycle, that is, different stresses at the same
the tissue was floated in room temperature solution of value ofstrain depending on whether the tissue is stretched or
unstretched, the data analysis in this paper is restricted only to
the following composition (mM): Na, 142; K, 3.9; Cl,
the loading portion of the cycle. Thus, each protocol consists
12l;PO4,0.4;Ca, IS;Mg, 1.0; HCOJ,28.0;glucose, 10. of a trajectory in the x-strain vs y-strain plane.
The solution was freshly prepared at the start of each
study and was bubbled with 95 % O2 and 5 y0 CO2 to
cycles such that for each cycle the ratio of the strains
maintain pH at 7.4. During the study, the temperature
was constant (see Fig. 1). Subsequently we will call
in the test chamber was maintained at 37°C.
each loading procedure a ‘strain trajectory’. Ten
The apparatus consists of two independent servo-
replications of each of the three trajectories were
controlled linear motors connected to mutually per-
performed in random sequence. For each replication,
pendicular wormgears. Two carriages that can move
we first preconditioned the specimen by sequentially
towards or away from one another are attached to each
loading and unloading it along the selected trajectory
wormgear. Each thread array is attached to a carriage
until the force-deformation curves became repro-
thereby allowing independent stretching or unstretch-
ducible (usually 4-6 cycles). Then one cycle was
ing of the tissue in two orthogonal directions. A force
recorded for subsequent analysis. To minimize viscous
transducer is attached to one of each of the pairs of
effects, we imposed the loading-unloading cycles so
carriage to measure force along each axis. The feed-
that 50 s were used for completion of a cycle. At the end
back signal that drives the motors can be either force,
of the study, the thickness of the specimen was
length or velocity. To minimize edge effects, deform-
estimated by obtaining its volume displacement by
ations for calculations of strains were measured in a
comparing its weight in air with that submerged in
central region of about 1 x 1 cm that was demarcated
saline.
by four small carbon paper markers attached to the
tissue with a small dot of cyanoacrylate. The move- Data analysis
ment of these markers was tracked by a pair of video
The recorded analog data of central-region dimen-
cameras and processed with video dimension analyzers
sions, outer-edge dimensions, and outer-edge forces
whose output is an analog voltage proportional to
were digitized with a 12-bit convertor interfaced to a
length as described by Yin et al. (1972). Unstressed
Data General Eclipse S/l 30 computer. The digitizing
dimensions were obtained by unloading the specimen
rate was adjusted so that each loading portion of the
until we could detect a slight degree of slack in the
cycleconsisted ofat least 1OOOdatapoints. Outliers for
thread arrays. We then continued to move thecarriages
each channel of force and length were removed using a
closer to one another and verified that there was no
running median filter with a span equal to nine
further decrease in the measured dimensions in either
consecutive data points. To keep the file sizes man-
direction. In order to calculate the stresses acting in the
ageable for the nonlinear estimation, we then retained
central region it was also necessary to measure the
every tenth point. This reduced file was the one used
deformations of the outer edges of the tissue. This was
for subsequent analysis. From the filtered data we
done with a pair of linear voltage displacement trans-
calculated the Green’s strains in the central region. The
ducers mounted to the force carriages.
strains are related to the stretch ratios i. (deformed
We wished to examine the effects of different loading
length divided by unstressed length) by
procedures. Thus, we subjected the specimen to three
different simultaneous biaxial loading and unloading E, = O.S(iz - 1) and E, = 0.5($ - I). (1)
 Quantification of biaxial tissue stress-strain data 29

that we felt would give reasonable fits to the observed

1 principal orthotropic directions of the specimen prior
FIOihLi:sEAR
I OPTIMIZATION / to mounting in the test apparatus. Therefore, the
I ASSESS I shearing may have been introduced. The effects of such
~A?AMElERIZATION
I OF MODEL
I shearing on our tests are considered in Appendix 2. As
i I
I
I ANALYZE
RESIDUALS / how much the stretching axes were rotated from the
‘NT~~P~~FC~~ION
I line the trajectory lies. The relative strains that we used
Fig. 2. Flow chart of the key components ofour data analysis
approach.
The Kirchhoff stresses acting in the central region were
estimated by assuming that the forces acting at the
outer edges were transmitted proportionally to the
central region. The area on which the force acted was
estimated by assuming that the measured thickness
was uniform across the tissue and that the tissue was
incompressible.
Method Jor quant$ication of biaxial test results
The approach to be outlined is based upon the
concept of a strain-energy function as previously
proposed for other tissues (Tong and Fung, 1976;
Fung et al., 1979). Since the tissue has different
stress-strain relationships during loading and unload-
ing (hysteresis), a complete description should take
into account ,both phases of the cycle. If one wished to
describe the tissue behavior for both loading and
unloading, one could, in theory, derive a directionally
dependent strain-energy function. The same general
approach would be applicable to both the loading and
unloading phases. For illustrative purposes, however,
we will restrict our attention to the loading phase only.
Recognizing that the strain-energy function derived in
this manner is, strictly speaking, incomplete, Fung er
al. (1979) proposed that it be called a ‘pseudo strain-
energy function’. Details of the individual steps of our
approach are presented in Appendix 1 and outlined in
Fig. 2-the following is a brief outline of the major
steps of the approach.
(a) Selection of strain-energy function. The pseudo
strain-energy functions that we used were variations of
the exponential form suggested in previous studies by
Fung et al. (1979) and Tong and Fung (1976). The
requirements that the strain-energy function be posit-
ive definite, symmetric and concise dictated these
choices. We chose the simplest form of exponentials
data.
Our methodology did not allow us to determine the
principal strain directions may not have coincided with
the orthotropic directions. If this were true, some
shown in Table 1 (Appendix 2), if uniform biaxial
strains (such as the path labelled A in Fig. 1) were
imposed, no shear terms would result regardless of
principal axes. For nonuniform biaxial strains, e.g.
trajectories B and C of Fig. 1 with unequal strains,
there will be shear terms introduced-their magnitude
depending upon the relative rotation between the
orthotropic and strain axes. Note that the direction of
the shear depends upon whichside of the equal strain
for trajectories B and C (45 and 33 %) were very close
to the 1.0 and 0.75 values listed in the table. Hence, the
shear strains for a 15, 30 and 45” misalignment would
be about f 3, f 6 and + 9”, respectively. The smallest
shear strain might be too small to be evident by
inspection but would also contribute very little to the
overall strain energy. The two larger shears, if they were
present, should have been easily seen during the test.
Since we observed no gross evidence of shearing
during any of the loading protocols, we presumed that
we had loaded very closely to the principal directions.
We, therefore, chose to fit the data with functions that
did not contain shear terms.
(b) Estimation of model parameters. A nonlinear
estimation technique based upon the Marquardt al-
gorithm, modified to handle biaxial data, was first used
to estimate the parameters of the function in equation
(2) (Patitucci, 1983). Details of this modtied optimiz-
ation technique are presented in Appendix 1.
(c) Assessment oj over-parameterizarion. Once the
parameters of the function that best fit the data were
obtained, we needed to assess whether the function was
over-parameterized. This was done using objective
criteria based upon the correlation matrix for the
estimated parameters obtained during the numerical
estimation. The elements of this matrix and the criteria
used to ascertain overparameterization are also pre-
sented in Appendix 1. If the model was found to be
overparameterized, one or more of the parameters
were deleted and the optimization was performed
again using the reduced function. This was repeated
until (1) deletion of a parameter no longer enabled the
model to fit the data or (2) the model fulfilled the
criteria for not being overparameterized. In some
cases, a completely new function may have to be
chosen.
(d) Residual analysis. Once a suitably paramete-
rized function was selected and its parameters es-
timated, the next step was to perform residual analysis
to see if the errors between the predicted and observed
30 F. C. P. YIN,P. H. CHEW and
stresses were independent and followed a normal
(Gaussian) distribution, If they did, classical statistical
methods could be used to estimate the confidence
intervals for each parameter (Gnanadesikan, 1977;
Draper and Smith, 1981). If the errors were not
independent and normally distributed, nonparametric
methods for obtaining confidence intervals needed to
be used.
(e) Bootstrapping. We found that most of the data
did not fullill the criteria that enabled us to assess
variability by standard statistical means. We, therefore,
adopted a recently described, nonparametric technique
called bootstrapping (Efron, 1979) to assess the vari-
ability of the pertinent parameters of the strain-energy
function that we finally chose. The idea behind boot-
strapping is to generate a set of data having the same
stochastic properties as the actual experimental data.
This was done by creating a pool of all of the errors
between the predicted and observed stresses cor-
responding to each pair of strains for every replication
ofevery trajectory. From this pool, errors were selected
and added to the predicted stresses obtained by fitting
the function to all of the experimental data. One hundred
stress-strain files were generated in this manner al-
though there is no Iimit to the number of such data sets
that could be generated. Details of the bootstrapping
method are also presented in Appendix 1.
Once the desired number of bootstrapped files were
obtained, nonlinear optimization was performed on
each generated file. In our case this resulted in 100 sets
of parameters. Having this large number enabled us to
assess the variability of the individual parameters for a
REPLICATION NO.
REPLICATION NO.
Fig. 3. Examples of the estimated exponential coefficients for each of the ten replications of strain trajectory
A. Left hand panelsare the results using the overparameterired seven-parameter
hand panels are the results using the five-parameter
S. L. ZEGER
selected trajectory. For example, if the 100 values for
two parameters did not overlap, we concluded that
they were statistically distinct. This enabled us to
assess, for example, anisotropy of the tissue.
To assess strain-history dependence we elected to
account for all the parameters by calculating the strain-
energy corresponding to the three different trajectories
at common values of the strains. To avoid extrapolat-
ing beyond the range of the experimental data, we
selected equal x and y strains such that their mean
squared value equalled the largest mean squared value
of the actually imposed strains over the three trajec-
tories tested. The strains used for this comparison,
therefore, may not have been traversed by each
particular loading trajectory. Nevertheless, assessing
results in this fashion accounts for all of the parameters
and tests the hypothesis that the same function can be
derived from data obtained along different trajectories,
i.e. that the tissue does not have this type of strain-
history dependence. Nonoverlap of the strain-energies
for the different trajectories implies that the differences
between strain trajectories are statistically significant,
that is, the tissue properties are strain-history
dependent.
RESULTS
For each strain trajectory the loading portions of the
stress-strain relationships in both the x and y direc-
tions for all ten randomly selected replications were
superimposable. Thus, after the tissue was pre-
conditioned, its mechanical properties were stable and
i 2 3 4 5 9 9 10
REPLICATION NO.
1 20
0
12 3 4 5 6 7 9 9 10
REPLICATION NO.
function (equation 2). Right
function (equation 3).
 Quantification of biaxial tissue stress-strain data
reproducible for the several hour duration of the study.
Since the uniaxial pericardial stress-strain relation-
ship appeared similar to that for skin, we first ex-
amined the seven-parameter combined polynomial-
exponential model that Tong and Fung (1976) used to
model skin
+0X exp[A,Ef + AyE; + ZA,,E,E,]. (2)
We found that, even though this function fitted the
observed data well, it was over-parameterized. Using
the methodsdescribed in Appendix 1 we found that the
A,, term was highly correlated with the A, and A,
terms. Thus, we could reduce the over-
parameterization by dropping this term. In addition,
we also slightly modified the function to the following
form
IV= B,(E_;+E;)+B2ExE,
+0.5C (exp [A, Ez + A, Ei] - I). (3)
Examination of the correlation matrix for this model
indicated that there was still some correlation between
the C and A, and A, coefficients but it was not
sufficient to cause any numerical instabilities. Any
further reduction in numbers of parameters, further-
more, would not enable us to obtain good fits to the
data. Therefore, subsequent analysis was performed
with this function.
An example of the difficulties that could arise when
the overparameterized function (equation 2) was used
to fit the data is presented in Fig. 3 where the values of
the exponential coefficients for all ten replications of
one of the strain trajectories for both the seven and
five-parameter strain-energy functions are shown.
Note the wide variability in the coefficients among the
replications when the overparameterized seven-
parameter function is used. This variability does not
occur in the five-parameter model.
Representative observed and predicted loading por-
tions of the stress-strain curves using the simpler five-
parameter function for one replication in both the x
and y directions for each of the three trajectories are
shown in Fig. 4. Only one of the replications is shown
since the data from successive trials were nearly
superimposable on the first. This figure indicates that
the simpler five-parameter function fits the experimen-
tal data quite well over the strain range examined.
The residuals for the stress in both x and ydirections
for one trajectory are shown in the upper panels of
Fig. 5. It is clear that the residuals are not independent
of one another. Positive and negative residuals tend to
appear in bunches. This correlation is due to the
imperfect fit of the function. The smooth and rough
components of the residuals are shown in the lower
panels. The similar pattern found in the smooth
residuals for each case indicates that the five-parameter
function systematically misfits the data. Because the
residuals were not independent and normally dis-
tributed, we could not use theoretical techniques to
31
24~ li
x
4t
I
0 ,
0.1 0.3 : 4
0.0 0.2
X STRAIN
Fig. 4. Stress-strain relations in both the x and 1 directions
for one replication. The labels for each curve correspond to
the strain trajectories shown In Fig. I. Symbols are the
observed data and solid lines are the predicted results from
the five-parameter function, see squation (3) in text.
estimate the uncertainties in the parameters in terms of
confidence contours. We, therefore, used the boot-
strapping method.
Note two other items illustrated in Fig. 5. First, the
variability in the rough residuals increased at the
higher strains (the increase was almost a factor of 2).
That is, residuals tended to be larger at the larger
strains. This changing variance could be accounted for
by introducing a weighting matrix as in equation (A7)
(Appendix 1).Since the absolute residuals were small in
our case, we found that the effect of ignoring the
unequal variances was small. We have therefore set the
weighting matrix to be the identity matrix in all of our
calculations to facilitate computation.
Second, there are two large residuals in both the x
and y directions at the high strain level. Similar such
large residuals were observed in the other replications.
We believe these may be associated either with tears in
the tissue that are indiscernible by eye or with small
fluctuations in the length feedback signals. Regardless
of the cause, we needed to assess whether the results of
our analysis depended strongly on these few points.
This was done by repeating the analysis with these
outliers removed (points with residuals greater than
three standard deviations from the mean were deleted).
The estimates of both A, and A, were slightly higher
and the estimates of C were slightly lower with the
outliers removed. However, the estimated strain en-
ergies at equal strains were nearly identical to those
with the outliers left in the data. Thus, performing the
analysis with or without the outlying points had no
significant effect on our interpretation of the results.
Therefore, all the subsequent results are shown for the
case where the outliets were not removed from the
data.
Assessment of the strain trajectory dependence is
based upon calculations of the strain energy at ident-
ical strain pairs for the different trajectories. The
panels in Fig. 6 display the strain-energy for 100
bootstrapped replications at equal x and y strains of
32 F. C. P. YIN.P. H. CHEW and S. L. ZEGER

“a
T 6

5 4
-1
3
0 2
;;
2 0

8
L -2

?I
-4
x

0.16 0.24 0.32 0.40 0.20 0.32 0.36 0.40 0.44

X STRAIN Y STRAIN

F
In
-5 E-3.0 1
x I I I I I I I ’
0.16 0.24 0.32 0.40 0.28 0.32 0.36 0.40 0.44
X STRAIN Y STRAIN

Fig. 5. The x-stress residuals (left panels) and y-stress residuals (right panels) for one replication of
trajectory A. The upper panel shows the entire residual and the lower panel shows the smooth and rough
components of the residual.

obtained from each of the original replications. These

o!
EXPERIMENTAL BOOTSTRAPPEO
RESULTS
Fig. 6. Illustration of the strain-energies that would be
obtained at equal x and y-strains of 0.45 using the estimated
coefficients for the five-parameter model. The left-hand set of
data are the values obtained using the coefficients estimated
from each of the ten experimental replicattons. The different
symbols denote the different strain trajectories. The right-
hand set of data are the results from one hundred bootstrap-
ped values of the coefficients. The non-overlap of the energies
for each trajectory indicates statistically significant depen-
dence of the strain-energy (material property) on the loading
protocol.
0.45 for each of the three strain trajectories. The strain
energies corresponding to the loading portions for the
three trajectories do not overlap. This indicates that
the strain-energies corresponding to each strain trajec-
tory are statistically distinct. That is, even when one
restricts attention to the loading portion of the cycle,
there is strain-history dependence of the tissue proper-
ties. Also shown in this figure are the strain-energies
. . .-:. .
values generally lie within the bootstrapped values for
each of the respective trajectories supporting the
statistical validity of the bootstrapping method.
The 100 bootstrapped values of the two exponential
parameters for trajectories B and C of Fig. 1 are shown
in Fig. 7. The nonoverlap of the data for each trajec-
tory enables us to reliably conclude that the differences
between the parameters are statistically significant and
cannot be attributed to noise in the experimental data
RESULTS or in the numerical algorithm. The difference between
the parameters implies that the specimen is anisot-
ropic. Determination of the direction of greater stiff-
ness may sometimes be difficult since the form of the
strain-energy function may result in the relative sizes of
the two coefficients reversing when trajectories on
either side of the uniform biaxial line (path A in Fig. 1)
are used. For the data for trajectory A for this
specimen, however, A, exceeded A,. This finding by
itself implies that the x-direction (head-tail) is stiffer
than the y-direction (transverse). In addition, since the
values A, exceeded A, for both of the other trajec-
tories, this is further evidence that this specimen was
stiffer in the x (head-tail) direction than in the
transverse direction.
DKCUSSION
As mentioned in the introduction, one of the
problems that has beset investigators in biomechanics
 Quantification
0 0
3 13 23 30
AX (gm/mm2)
Fig. 7. Plots of 100 bootswapped values of the exponential coeficients, A, vs A,. for the five-parameter
strain-energy function for the strain trajectories B and C of Fig. 1. None of rhe values touches the line of
identity indicating that the differences between coefficients is staristically significant for each trajectory.
relates to quantification and interpretation of biaxial
stress-strain data. Given any parametric form of a
strain-energy function, it is a fairly straightforward
task to estimate the parameters by nonlinear optimiz-
ation techniques. The difficulty lies with interpretation
of the parameters. Using a larger number of para-
meters leads to better fit of the model to the data. This
better fit is attained at the expense of increasing
difficulty in interpreting unequivocally the meaning of
each parameter. The problem with overparameterized
models has been clearly pointed out by Fung (1973)
and is, in fact, one major reason that the exponential
rather than the polynomial form of the strain-energy
function has been advocated. Even when an exponen-
tial function was used, however, most tissues still
displayed wide variability in the coefficients among
replications of identical tests as shown in Tong and
Fung (1976) and Fung ef nl. (1979). As our study
demonstrates, this variability may be due to an over-
parameterized exponential function. The data of Fig. 3
show wide variability in the coefficients even among
replications ofan identical test. Moreconsistent results
are obtained when a more suitably parameterized
function is used.
The approach that we have presented here and
illustrated by an example ofdata for a highly nonlinear
tissue should be general enough to allow assessment of
any parametric strain-energy function. It allows ob-
jective evaluation of the suitability of the model in
terms of the correlation matrix for the estimated
parameters. If the model is overparameterized, this will
be evident in this matrix and another model with fewer
parameters should be chosen. Once a suitable model is
selected, the remainder of the approach will enable one
to ascertain some of the sources of variability in the
parameters. Once we can assess the variability with
judiciously selected experimental approaches such as
those described here, we should be able to answer such
questions as whether a specimen is anisotropic,
whether the material properties depend upon the
loading pathway, how real the differences in para-
meters are between specimens or groups of specimens,
etc.
of biaxial tissue stress-sIram data 33
I
-,
40 2 :0 23 30 43
AT (gm/mm*)
We have demonstrated the application of an alter-
native method for determining confidence intervals
that allows assessment of the variability in spite of an
imperfect fit to the data. This bootstrapping technique
is rather new in the field of statistics but is gaining
wider recognition as a potentially powerful method of
dealing with difficulties such as those illustrated by our
data. The agreement in the degree of variability
observed between the results of the actual replications
and those obtained after bootstrapping lends confi-
dence to the validity of this method.
Using the greater reliability and insight provided by
bootstrapping we found that the pericardial data fitted
by the function we selected has strain-history depen-
dence in that the properties differ according to the
exact boundary conditions imposed during stretching.
This history dependence, if borne out by further
studies in a wider range of specimens, makes com-
parison among different specimens even more difficult.
First, in order to compare different specimens, we
would need to test each one over the same exact
loading protocols. Otherwise differences could be
attributed to different trajectories. Second, unless we
know the in tko strain history undergone by the
specimen, we have no assurance that the strain path
selected for testing in Ctro bears any resemblance to
that undergone in the natural state. Extrapolation
from the isolated data to the physiologic condition
would be difficult.
It must be emphasized that conclusions based on the
approach mentioned above apply only to the particular
strain-energy function that is selected. The function
that we selected was chosen on the basis of theoretical
considerations of positive definiteness, symmetry etc.
This ud hoc method of selecting a function may not be
the optimal. For example one could conceive specific
tests that could be done to help guide one’s choice of
function. These considerations, however, are beyond
the scope of this paper. The function that we selected
implicitly carries the assumption that the tissue is
isotropic in the low strain ranges where it is highly
distensible and that it manifests anisotropy only at the
higher strain ranges. We feel that this is a reasonable
34 F. C. P. YIN, P. H. CHEW and S. L. ZEGER
assumption since the tissue is so highly distensible at
low strains that it is very difficult to accurately measure
the forces in either direction. It very well may be the
case that a particular function fits the data well in some
least squares sense, is not overparameterized. and yet
shows systematic misfit to the data. In fact, the 5-
coefficient strain-energy function that we selected to fit
the pericardial data had these limitations as evidenced
in the residuals. Choosing another function might have
enabled us to alleviate this difficulty.
The imperfect fit of the data to the function could
also be caused, in part, by our neglecting shear terms.
We feel, however, that the contribution of any possible
shearing was small. First, the resistance of the tissue for
shear is probably less than that for extension so that
the shear coefficient(s) should be small compared to the
extensional strain coefficients. This is due to the
structural arrangement of pericardial tissue in which
the structural components are loosely imbedded in the
ground substance. An analogy would be the behavior
ofa nylon net. In addition, as shown in Appendix 2 and
discussed earlier, there was likely very little shear in our
specimen or we would have easily seen it during the
test. Nevertheless, until we can ascertain the principal
directions prior to performing these kinds of studies,
this point remains unresolved.
We should again point out that we have restricted
ourselves to the loading portion of the stress-strain
curve. Clearly this tissue has hysteresis. A function that
completely describes the tissue properties should take
this behavior into account as well as other behavior
such as strain-rate dependence, etc. Our intention was
not to formulate a complete function but rather to
demonstrate an approach that could be applied to a
general strain-energy function. Extension to analyzing
the unloading portion of the data should be
straightforward if one modifies the function to have
directionality.
Acknowledgement-Supported in part by grant No. P50 HL
17655lO from the National Heart, Lung and Blood Institute.
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 Qu.mtifi~tion of biaxial ussuc stress-stram data 35

structural correlates of canine pericardium. Ctrculution noise is less. Note that in this formulation we can allow the
Rrs. 49. 807-8 14. experimental vartability as measured by the covariance matrix
Wineman, A., Wilson, D. and Melvin, J. W. (1979) Material C(E,) to depend on the strains, E.
rdentification of soft tissue using membrane inflation. Using the above criterion for quality of a fit, a non-hncar
J. Eiomechanics 12, 841-850. optimization procedure was then used to find B. The first step
Yin, F. C. P. and Fung, Y. C. (1971) Mechanical properties of was to select starting values, 8.. This was done by evaluating
isolated mammalian ureteral segments. Am. 1. Ph~stol. 221, the function D(B) on a coarse grid for p and choosing the
14861493. parameter values that minimized D. A Marquardt nonlinear
Yin, F. C. P.,Tompkins, W. R.. Peterson, K. L. and Intaghetta, least-squares algorithm, modified to handle bivariate data
M. (1972) A video dimension analyzer. IEEE Trans. (Patitucci, 1983). was then used. To describe this modifi-
Biowd. Engng 1. 376381. cation, we require the following notatton
Let x = (E,,. E,,. E,,. E,z. E,.. E,.)‘Z
2”I(I (AS)
APPESDIX 1. ,MATHEMATICAL DETAILS OF THE DATA Y = (S,,. s,,,s,2*s,,. s,.. SJ;
Zn1 I
ANALYSIS METHODOLOGY
b =(c,,. c,, . (:,I. E”?* E,..Er.)‘:
2”* I
This appendix describes the details of the various steps used
for analysis of our biaxial data. The individual steps are
(1) nonlinear optimization to estimate the parameters of a
selected strain-energy function; (2) description ofan objective
method to determine if that function is overparameterized;
(3) residual analysis to see if the variability of the parameters
of the suitable function can be assessed by standard statistical Note that A defined in this manner isa block diagonal matrix
methods or whether nonparametric methods for this assess- such that thecovarianceofl’&dis the2n x 2nidentitymatrix.
ment need to be employed; (4) a nonparametric method for The observed data can be transformed using M into the forms
assessing variability called bootstrapping is described.
Following standard matrix notation, boldfaced Latin or X* = ,$IX and Y* = $lY. (A7)
underrored Greek symbols will denote vector or matrix following which Marquardt’s algorithm was used. However,
quantities and the prime symbol will denote the transpose of C(E&and hence M are unknown and must beestimared from
the matrix. As in standard statistical notation, symbols with a the data. We have assumed that C(E,) is constant within each
circumflex will denote estimated quantities. An asterisk of a few ranges of E. The size of the regions was chosen to
denotes a modification needed for handling bivariate encompass a sufficient number of points (roughly n p). Given
regression. an estimate b ofb, we estimated the matrix C(E,, 8). where E,
is in the region m by
(I) Bicariatr nonlinear oprimization

For the selected strain-energy function W= W(E,. E,) the C, = (l/N,) ; [S, -f(E,.B)][S,-f(E,.B)]’ (A8)
stress-strain relations in the x and y directions are the first K=l
partial derivatives of W with respect to its respective strain where the sum is over the N, points in the mth region. The
components. This can be expressed as a system of two estimation of the covariance matrices and the application of
nonlinear equations as follows the Marquardt algorithm are then iterated until convergence.
Having obtained the convergent solutions, we then as-
S = HE, Bl = [JB’(E, @)/a&, aB’(E. B)lZF,] (Al)
certained whether the solution represented a global minimum
where S’ = (S,, S,) is a vector of the Kirchhoff stresses in the in the p-parameter space. This was done by obtaming the
x and y directions and E’ = (E,, E,) is the vector of the convergent solutions for a range of initial values covering at
Green’s strains in the x and ydirections;and /3 is a p x 1vector least two orders of magnitude for each of the p parameters. If
of p unknown parameters which characterizes the strain- multiple different convergent solutions were found, we chose
energy function. The experimental data for a single specimen the solution that had the lowest absolute value of the mean-
are comprised of n sets of stress and strain observations squared error.
(E,,S) i=l,2 ,..., n. W)
(2) Assessment of o~rr-purameterization
As with other least-squares techniques, we assume that the Having obtained the solution with the lowest mean squared
data satisfy equation (Al) except for additive observational
error, the next step was to verify that the function selected was
error, i.e. that not over-parameterized. This was done by first calculating the
S, = f(E,,p)+e, (A3) covariance matrix for the estimated parameter values defined
as
where c; = (e,,; cy,) is a vector of errors assumed to have
A = (Z*‘Z*)-’ where
expectation 0 and covariance matrix C(E,). Note that because
we have two-dimensional stress and strain vectors, we needed
to use bivariate nonlinear regression.
Our approach to estimating the parameters, b, was to z= pLf,(Ef,B,l@,. . ,Zhj,(E:.BYC’B,
minimize a measure ofdeviation of the observed stresses from ~.Li/,(E:,
BVV,. . 2LE’r 8,/L’& (A9)
those predicted by the fitted model. That is, we minimized the ~~j,(CvB)IV,~. c?f,(C.B)lV,
[ I
weighted sum of squares
The elements of thecorrelation matrix R were then calculated
as
D(B) = i; (S, -f(E,. B))‘C(E,)- ’ (Si - f(Ei. B)) (A4)
,=1 R,j = A,j/(AiiAj,)“2. (AlO)

where C(E,) is a consistent estimate of the covariance matrix, A function that is over-parameterized will have a ‘nearly
C(Ei). For a known covariance matrix the /I which minimize singular’ correlation matrix. Since this determination is
D(B) in equation (A4) have the smallest degree of uncertainty arbitrary, we decided to use the criterion proposed by Belsley
as n gets large. More intuitively, D(b) is a criterion which gives et al. (1980). Therefore, we defined functions for which the
more weight to stress-strain data where the experimental determinant of the correlation matrix was less than 10e4 as
36 F. C. P. YIN. P. H. CHEW and S. L. ZEGER

being over-parameterized. It usually was the case that an more nearly satisfy the necessary assumptions and repeat the
overparameterized model also had multiple convergent sol- above steps. Still another approach is to use a nonparametric
utions depending on the set of initial values. To reduce an method to assess the variability in spite of the limitations of
over-parameter&d function we eliminated one of a pair of the model. We have adopted the latter and chose a procedure
highly correlated parameters and retained those parameters known as bootstrapping. This was introduced by Efron
that were essential. This process was repeated until the (1979) and is discussed further in Diaconis and Efron (1983).
correlation matrix was less nearly singular and we could still The idea of bootstrapping is to mimic the process that
obtain a reasonably good fit to the data. Only a solution set generated the original data thereby obtaining a ‘bootstrap’
that was not overparameterized for the selected model was replicate that has similar statistical properties to the original
then carried to the next step of residual analysis. sample. This procedure is repeated many times obtaining
many independent replicates. Each of the generated data sets
is analyzed like the original one and the variability among the
(3) Residual Analysis resulting coefficients is taken to represent the uncertainty in
Our ultimate goal was to arrive at a statistical assessment of the actual estimates.
the uncertainty of each particular parameter as well as the The key step is to determine a procedure for regenerating a
strain-energy function. If certain statistical assumptions are set of stress-strain data that are stochastically similar to the
fulfilled, theoretical calculations of confidence intervals can original set. In our protocol, we repeated each loading a
be made to assess the uncertainty (Draper and Smith, 1981). number of times. We assumed that the observed stresses for a
The major assumption underlying confidence interval calcu- single loading could be represented as the sum of the actual
lations is that the errors between the observed and predicted stress, a smooth error representing systematic deviations of
stresses are approximately independent and normally dis- the model from reality and rough or random errors mainly
tributed Gaussian variates with expectation 0 and covariance representing experimental noise. We created a pool of smooth
closely estimated C(E). error curves comprised of smoothed versions of the residuals
If this assumption is satisfied, from each of the loadings. A pool of rough errors was
comprised of the residuals after the smooth part was re-
a*=&5
moved. A bootstrap stress-strain curve was then generated by
will be comprised of 2n approximately independent Gaussian adding a randomly selected set of smooth residuals and a
variates with mean 0 and variance I. To assess the indepen- random set of rough residuals to the predicted values
dence of the adjusted residuals, d*, we plotted the x and y obtained from fitting the model to all of the original data.
components of the residuals, a:, and 6;. against each other. To generate a bootstrap confidence region we use the
We checked for unequal variances by plotting 6,: and Ss following steps: (I)calculate the parameter fi and the re-
against E,; and E,. We further checked whether the b*s were siduals ~5:. . . . 15:from the observed stresses and strains as
approximately Gaussian using Q-Q plots as described in discussed above, (2) randomly select with replacement from
Gnanadesikan (1977). In these plots the ordered data are
plotted against their expected values under the assumption
that the data are Gaussian. An approximately straight line Table I. Illustration of the influence of different degrees of
results when the data are nearly Gaussian. Details about rotation of the axes ofstretching from the principal directions
methods for residual analysis are given in Belsley et al. (I 980). as a function of the relative strains imposed for various biaxial
loading trajectories
(4) Assessment of rariabiliry
a. Conjdence conzours. If the residuals reasonably satisfy
EX 4 (deg:ees) E,. E,.
the assumptions stated above, we could estimate the un-
certainty in the parameters using previously described
I.0 0.0 IS 0.933 0.067 -0.500
theoretical methods for calculating the confidence contours
30 0.750 0.250 - 0.866
(Draper and Smith, 1981). There are two such approaches to
45 0.500 0.500 -1.000
estimatingconfidencecontours. In the first weassume that the
function 0(/I) is quadratic in the neighborhood of the 1.0 0.2 I5 0.946 0.254 - 0.400
solution, j, and that the joint distribution of b’ is Gaussian 30 0.800 0.400 -0.693
and has elements with mean equal to 0 and variance equal 45 0.600 0.600 -0.800
to 1. Then 1.0 0.5 I5 0.966 0.533 -0.250
Prob[(@-b)A-‘(B-fi)/p < F@.2n-p, I -z)] r I -a 30 0.875 0.623 -0.433
45 0.750 0.750 -0.500
(All)
1.0 0.75 I5 0.983 0.767 -0.125
where F(p, 2n -p, I -a) is the 100 x (I -a) percentile of the
30 0.937 0.813 -0.217
F-distribution with p and Zn-p degrees of freedom
45 0.875 0.875 -0.250
(Morrison, 1976). The confidence contour encloses the set of
Bs which satisfy the inequality in the bracket. Note that the I.0 1.0 I5 1.0 1.0 0
confidence contours using this approach are ellipsoids. 30 I.0 1.0 0
In the second approach we choose the contours to enclose 45 I.0 1.0 0
the set of Bs such that 0.75 1.0 I5 0.767 0.983 0.125
30 0.813 0.937 0.127
0(/I) = D(b){ I + (p/2n -p))F(p, 2n -p. 1 -a);. (AI2)
45 0.875 0.875 0.250
This involves evaluating the surface D(B) over a fine enough
0.5 I.0 I5 0.533 0.966 0.250
grid so as to define the contour. It is computationally more
30 0.625 0.875 0.433
extensive than the first approach, but does not require the
45 0.750 0.750 0.500
assumption that D(b) is quadratic near b. Both of the above
approaches, however, assume that the errors are approxi- 0.2 1.0 15 0.254 0.946 0.400
mately Gaussian and independent and that C(E) is known. 30 0.400 0.800 0.693
See Morrison (1976) for additional details. 45 0.600 0.600 0.800
b. Boorszrapping. If the residuals are not independent and 0.0 I.0 I5 0.067 0.933 0.500
Gaussian in distribution, the above methods are not valid. 30 0.250 0.750 0.866
Several alternatives are then possible. One is to transform the 45 0.500 0.500 1.00
data and!or select an alternate model for which the residuals
 Quantilication of biaxial tissue stress-strain data 37

the original residuals, ~5:. a set of n bootstrap residuals, 6:‘. by the coordinate transiormation relations which are
!=I... n; (3) createa set ofbootstrapstresses.S: = f(E,. b)
E, = E,cos2~+Ersinzx
+e(E,)6:‘, i= 1 n; (4) re-estimate the parameter, /I,
from the bootstrapped stresses, S,’ and the actual strains E, E, = -E,sin’ztEycoszz
and denote the new estimates 8:; (5) repeat steps (2) to (4) K
y,, = (E,-E,)sin2x
times obtaining) j?:, k = 1,2, m; (6) define the 95”,
confidence contour as the smooth contour that encloses 95 of0 where E, and E, denote the principal strain components
of the B*s. The choice of K of course depends on the (there are no shear strains in this system) and E;. E;. and y;,
dimension of 8. However, it is often of the order of 100. denote the normal and shear strain components, respectively
in the stretching coordinate system.
Table I illustrates how different degrees of rotation of the
stretching coordinate system with respect to the princtpal
APPENDIX 2. EFFECT OF STRETCHING ALOSG SON- direction would influence the relative strains in the IWO
PRINCIPAL DIRECTlOSS orthogonal directtons in biaxial tests like those described
herein. A range of stretches varying from the two extremes of
The purpose of this appendix is to illustrate the influence of strip biaxial conditions in which there is only strain in one
stretching the tissue along directions that do not coincide with direction, to uniform biaxial where the strains in the two
its principal orthotropic directions. For simplicity and to directions are equal, are shown. The entries in the table are
match the conditions of our biaxial testing we will consider a normalized to a unit of strain. For example, a strip biaxial test
planar case-the extension to the three-dimensional case is in the x principal direction would consist of 1 unit of x-strain,
straightforward. Let the x and J refer to the principal E,, whereas a uniform biaxial test would consist of I unit of
orthotropic coordinate system and x’ and y’ to the principal strain in both the x and )’ directions. The shear strain is also
directions in which the tissue is stretched. The angle between expressed in normalized units, e.g. if one unit of strain
the two coordinate systems is denoted by a. The relationship represents 20”, of strain, the shear strain for a 15’ rotation
between the strain components in the two systems is governed would be -0.4 x 0.20 = - 0.08 rad.