journal of the mechanical behavior of biomedical materials 21 (2013) 149166

Available online at www.sciencedirect.com

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Research Paper

Microstructure-based constitutive modeling for the large

intestine validated by histological observations

Dimitrios P. Sokolisn, Sofia G. Sassani

Laboratory of Biomechanics, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

art i cle i nfo ab st rac t

Article history: Other than its transport role, the large bowel performs numerous sophisticated functions, e.g.
Received 18 July 2012 water, electrolyte, and vitamin absorption, optimized by its contractile properties and passive
Received in revised form recoil capacity, but these properties have attracted limited attention than has been the case
12 February 2013 for other parts of the gastrointestinal tract. Accordingly, we investigated in vitro the pseudo-
Accepted 20 February 2013 elastic properties of tubular specimens from the ascending, mid, and descending colon, and
Available online 1 March 2013 the rectum of healthy Wistar rats under passive quasi-static conditions and a physiologic

Keywords: range of pressures/axial stretches. A neo-Hookean and five-fiber family model was chosen as

Large intestinal biomechanics a microstructure-based material model for its efficiency in producing accurate representations

Inflation/extension of the three-dimensional inflation/extension data in relation to the underlying microstructure.

Computerized histology Guided by our optical microscopy observations, this model took account of isotropic elastin

Anisotropy properties and multi-directional collagen organization, but suffered from parameter covar-

Passive pseudo-elasticity iance. Moreover, the contributions to the total model of the neo-Hookean and circumferential-

Microstructure fiber family were negligible, given the tiny amounts of elastin and circumferentially-arranged
collagen fibers that were disclosed histologically, and the contributions of the diagonal and
radial-fiber families to data representation were similar. The multiaxial response of the
intestinal wall was fit equally accurately but without over-parameterization problems by the
neo-Hookean and three-fiber (diagonal and axial) family model. The preferred alignment
of collagen fibers towards the axial direction bestowed increased axial stiffness to the tissue.
The mid colon was the stiffest region by virtue of its greatest material parameters, as
validated by its higher collagen content than that of the distal regions. The present findings
generate a more cohesive understanding of the large bowel in histomechanical terms, with
potential for clinical and biomedical applications.
& 2013 Elsevier Ltd. All rights reserved.

1. Introduction
The large intestine performs numerous sophisticated functions,
such as absorption of water, electrolytes, and vitamins from
the remaining indigestible food matter, except from serving as a
conduit for the transport of waste products out of the body
(Gregersen, 2002). The geometrical and biomechanical properties
of the large intestine have a direct influence on these functions,
but little is known about them, unlike the small intestine for
which there is a plethora of observations; see Li et al. (2008),
Liao et al. (2010), and references listed therein. The literature on
experiments on its passive properties is limited to simple
elongation data of tissue strips (Yamada, 1970; Watters et al.,
1985a, 1985b; Egorov et al., 2002; Ciarletta et al., 2009) that may
not suffice for a multiaxial quantification, due to the highly
intricate and intertwined organization of the muscular and

n
Correspondence to: 35, Lefkados Street, Athens 15354, Greece. Tel.: 30 210 6597370; fax: 30 210 6597365.
E-mail address: DimitrisSokolis@ath.forthnet.gr (D.P. Sokolis).

1751-6161/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jmbbm.2013.02.016
150 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166
fibrous elements in the multilayered composite wall of the
large intestine, eventuating in a strong coupling amongst the
deformations in the different principal directions, as in other
visceral organs. Rather, cylindrical segments have to be studied
under triaxial loading states that incorporate the physiologic
domain, yet in the report of Itasaka et al. (1992), information on
the behavior of the axial force was missing and the segments
were held at a single axial stretch, so that once again this study
did not provide complete datasets for the determination of
multiaxial biomechanical properties.
It appears that Gao and Gregersen (2000) were the first
authors to define those properties via pressureradiuslength
recordings, but did not endeavor to quantify the nonlinear and
anisotropic response by a three-dimensional (3D) material law.
Nonetheless, it is only after a constitutive law is established for
the organ over a wide deformation range that its complex
transport phenomena can be evaluated on a quantitative basis
under physiologic and pathophysiologic conditions. This gap
was filled by our late study (Sokolis et al., 2011a) that
(a) afforded comprehensive experimental data on the deforma-
tional response of large intestine under a 3D stress field,
derivable by common testing procedures for soft biological
tissue such as inflation/extension testing, and (b) satisfactorily
described those data by the Fung-type exponential model. From
the viewpoint of material model formulation, the next logical
step that we report in this study was the rigorous characteriza-
tion of the biomechanical properties of large bowel via a
microstructure-motivated model and histological guidance,
concentrating as much as possible on the hierarchical struc-
tures of the extracellular matrix and muscle components,
comprising the tissue. The improved definition through the
present data of the function of the normal large intestinal wall
with respect to its underlying microstructure may contribute to
a more detailed understanding of its physiology and pathophy-
siology, with potential for clinical and biomedical applications.
2. Experimental methods
2.1. Preparation of large intestinal tissue
Seven normal large intestines were surgically resected from
healthy adult Wistar rats of 340 g weight that were used in
other physiological experiments; biomechanical results from
five of those have been recently presented in our companion
paper (Sokolis et al., 2011a). Briefly, the intestines were carefully
dissected free of the mesenterium from the ileo-colonic junc-
tion down to the anal canal, removed, and divided into four
segments, the ascending, mid, and descending colon, and the
rectum. Their contents were rinsed by flushing normal saline
through their lumen. Care was taken not to stretch the tissue
during removal or the flushing procedure. Adjacent 34 cm long
tube-form specimens from each region were in turn retained
for histology and biomechanical testing.
2.2. Computerized histology
Unloaded segments of the specimens isolated were formalin
fixed and routinely prepared for light microscopy. Circumfer-
ential and axial (5-mm thick) sections were cut and stained
with hematoxylineosin, orcein, and sirius red to differenti-
ate in the intestinal wall the smooth muscle cell nuclei by
black color, elastin by orange color, and collagen by red color,
respectively. Images were acquired by a digital camera
(Altra20; Soft Imaging System, Munster, Germany) connected
to an optical microscope (Olympus CX31; Olympus, Tokyo,
Japan) and processed with the Image-Pro Plus software (v4.5;
Media Cybernetics Inc, Silver Spring, MD, USA). The orienta-
tion of muscle, elastin, and collagen was inspected and their
area densities were assessed in six characteristic sites across
the perimeter or length of each specimen and at three cross-
sections. The percent area of each layer relative to the entire
wall was additionally measured at the same regions and
cross-sections.
2.3. Biomechanical methods
The experimental setup and testing methods employed were
alike to those in (Sokolis et al., 2011a). The basic parts of our
setup were: (a) an organ bath with calcium-free Krebs solu-
tion and 0.25% EGTA, kept at 37 1C and bubbled with carbo-
gen, hosting the specimens; (b) a syringe pump (sp100i2;
World Precision Instruments, Hertfordshire, UK), pressurizing
them between 0 and 15 mmHg at a 0.15 mmHg/s rate, as
measured by a transducer (BLPR; World Precision Instru-
ments), while at a constant axial stretch; (c) a laser micro-
meter (LS-3100; Keyence Corp, Osaka, Japan), recording the
specimens external radius; (d) a load cell (Fort 100; World
Precision Instruments) and a micrometer (Tesa Technology,
Renens, Switzerland), which measured the axial force to
which the specimens were subjected and their extension;
(e) a transducer (Transbridge 4M; World Precision Instruments)
that amplified our data and a data acquisition card (DAQmx
6009; National Instruments, Austin, TX, USA) that stored them
on a nearby computer by use of an acquisition interface
(Labview v7.1; National Instruments). In a typical experiment,
the specimens were preconditioned by five inflation/deflation
cycles at each axial pre-stretch from 100% to 130% of their
undeformed length in 10% steps. The inflating data of the final
steady-state loop were considered for processing.
The undeformed dimensions of our large intestinal speci-
mens were determined on photos of radially-cut rings, taken
by a Stemi 2000C stereoscope (Carl Zeiss Optical, Chester, VA,
USA), as previously (Sokolis et al., 2011a).
2.4. Material models
The intestinal wall was treated as a nonlinear, pseudo-elastic,
homogeneous, and anisotropic material subjected to finite
deformation. The pseudo-elasticity hypothesis was supported
by two facts: that (a) the hysteresis loops were slim, implying
predominance of the elastic components and (b) their form/
shape was almost insensitive to strain-rate variations, at least
within the two orders of magnitude studied. In the following,
only the inflating limbs of the hysteresis are shown and the
wall tissue was regarded as being perfectly hyper-elastic in
loading. The analytical formulation for the problem of a
residually-stressed thick-walled cylinder, subjected to internal
pressurization and axial force, is outlined in our companion
paper (Sokolis et al., 2011a); see also Humphrey (2002).
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166 151

We have already reported therein the results of fitting our m,n

X exp exp
2 mod 2
RSS wPij Pij Pmod F
ij wij Fij Fij , 4
inflation/extension data by the four-parameter Fung-type SEF, i,j 1
this being an empirical exponential function, whose exponent
is a quadratic function of the circumferential and axial Green between the experimentally-recorded (exp) and theoretically-
strains. A neo-Hookean and five-fiber family SEF was chosen modeled (mod) values of transmural pressure and axial force,
to quantify the response of large intestinal tissue via a using the NelderMead nonlinear regression technique in
exp
microstructure-based model. This model decomposes the SEF MicroCal Origin (v8.5; OriginLabs Corp.); wPij 1=Pij 2 and
F exp 2
into an isotropic term for elastin and an anisotropic for collagen wij 1=Fij were weight factors normalizing the squared
p
X differences. The root-mean-square error e w2 was an
j
W Wiso Wanis mI1 3 Wanis , 1 estimate of the quality of fitting, where w2 was the RSS divided
j d, d0 , a, c, r
by the total number of experimental points minus the
where W was the resultant SEF for the entire wall under passive number of material parameters. Parameters were subject to
conditions, supposing absent active tone by smooth muscle thermodynamic constraints, guaranteeing physically realistic
cells, and Wiso , Wanis were the elastic and collagen-related parameter values and the positive-determinateness of the
terms. The neo-Hookean model was a reasonable choice for SEF. Data were taken every 0.025 mmHg, from 1 to 15 mmHg,
mathematically characterizing the isotropic nature of elastin in for three axial stretch ratios (110130%), resulting in a total
terms of material parameter m with stress units and the first number of approximately 1800 points used for fitting. Due to
invariant of the right CauchyGreen tensor, i.e. the sum of the significant bending, which led to near-zero and negative axial
three principal squared stretches, I1 l2y l2z l2r . The aniso- force values, our recordings for the unstretched specimen
tropic term for the collagen network in Eq. (1) was simulated by length (100%) were not included in the fitting procedure. This
the sum of two diagonally (identified by superscripts d and d0 ), was repeated for a variety of initial parameter values, with
axially (a), circumferentially (c), and radially-arranged (r) fiber the intention of ascertaining that the global and not a local
families, where minimum had been reached.
The choice of constitutive model in a material characteriza-
kd1 n h 0 i o
0
kd1 n h i o 0 tion study for a particular tissue is dictated by a variety of
Wdanis exp kd2 l2d 12 1 , Wdanis d0
exp kd2 l2d0 12 1 ,
4kd2 4k2 criteria other than its descriptive capacity, e.g. the number of
2a parameters and their microstructural interpretation. A comple-
mentary assessment was made to substantiate the need for
ka1     inclusion of the parameters of the neo-Hookean and five-fiber
Waanis exp ka2 l2z 12 1 , 2b
4ka2 family SEF (Eqs. (1)(3)), given that the smallest number of
parameters should be employed in general to circumvent
kc1     problems of numerical instability with computational imple-
Wcanis exp kc2 l2y 12 1 , 2c
4kc2 mentations in nonlinear modeling. For this purpose, the stan-
dard fitting procedure was followed, during which the
kr1     parameters of every one of the neo-Hookean and five-fiber
Wranis exp kr2 l2r 12 1 , 2d
4kr2 families were successively zeroed and the ensuing root-mean-
square error compared with that when allowance was made for
were the contributions of the diagonal (Eq. (2a)), axial (Eq. (2b)),
all SEF parameters to vary. Moreover, parameter covariance and
circumferential (Eq. (2c)), and radial-fiber (Eq. (2d)) families.
over-parameterization was established by computing the deter-
These contributions were null under compressive stretches,
0
minant of the correlation matrix for the estimated material
lj o1, j d, d0 , a, c, r. In the preceding, kd1 , kd1 , ka1 , kc1 , and kr1 were
parameters as in Stavropoulou et al. (2009).
material parameters with stress units, specifying the stiffness
0
level independent of deformation, and kd2 , kd2 , ka2 , kc2 , and kr2
were unit-less parameters, specifying the progressive stiffening 2.6. Statistical methods
effect of the multiple-arranged fiber families with increasing
0 0
deformation. Certain equalities, i.e. kd1 kd1 , kd2 kd2 , and ad Parameters are presented as individual values, mean
0
ad a0 , resulted by virtue of the symmetrical and regular values71 standard error, or both. One-way analysis of var-
distribution of the two diagonal-fiber families. The stretch, to iance (ANOVA) and the Tukey multiple-comparison test was
which both diagonal-fiber families were subjected, was calcu- used to compare mean values among the different large
lated by intestinal regions; p values less than 0.05 were considered
as statistically significant. All statistical analyses were performed
ld l2y sin2 a0 l2z cos2 a0 1=2 , 3
with the SPSS v17.0 (SPSS Inc, Chicago, IL, USA).
in terms of angle a0 between the diagonal and axial directions
in the stress-free state, and of the circumferential, ly , and axial,
lz , stretch ratios. 3. Results

3.1. Light microscopic examination and histometric data

2.5. Fitting procedure
The microstructure of the different intestinal segments is
The material parameters within Eqs. (1)(3) were identified by visualized in Figs. 13. In general, there were negligible
minimizing the residual sum of squares (RSS) elastin quantities without a particular organization in the
152 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 1 Four histological sections obtained from the ascending (A), mid (B), and descending colon (C) and the rectum (D) of the
rat through the wall thickness in the circumferential and axial direction, stained with orcein to highlight the elastin fibers
(brown). Higher magnifications (  40) of the boxed regions are placed to the right of the low magnifications (  4), showing
negligible elastin content of all layers throughout the large intestine. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)

large intestinal wall, as noted with orcein staining (Fig. 1).
Histological staining with picro-sirius red (Fig. 2) showed that
the configuration of collagen network differed strikingly among
the large intestinal wall layers. Collagen fibers in the mucosa had
radial orientation, partly because of the buckling of the mucosal
layer, traversing different depths within the layer while small
fibers branched off orthogonally and interconnected the radial
fibers. The submucosa included layers of collagen fibers with a
cross-ply arrangement, but fibers seemed lengthier in the axial
relative to the circumferential section suggesting a preference for
alignment in the axial direction. Collagen fibers run parallel to
the smooth muscle cells in the two muscle layers, appearing
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166 153

Fig. 2 Adjacent sections to those of Fig. 1 from the ascending (A), mid (B), and descending colon (C) and the rectum (D) of the
rat, stained with sirius-red to highlight collagen fibers (red), showing declining collagen content of the mucosa distally in the
large intestine and invariant content of the remaining layers. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

with circumferential orientation in the inner and with axial
orientation in the outer muscle (see hematoxylineosin stained
sections in Fig. 3 for the arrangement of cell nuclei), while
parallel layers of axial ribbons were noted in the serosa. Inter-
connections existed between the collagen networks of the
various layers, thereby providing structural continuity to the
intestinal wall.
Morphometric evaluation showed elastin concentrations
less than 5% in all layers and regions and a much greater
collagen concentration in the submucosa than the mucosa
and muscle layers (Fig. 4). Collagen content of the mucosa
was significantly reduced in the two distal regions although
that of the other layers was invariant, leading to a similar
regional distribution in the content of the entire wall, despite
154 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 3 Adjacent sections to those of Fig. 1 from the ascending (A), mid (B), and descending colon (C) and the rectum (D) of the rat,
stained with hematoxylineosin to highlight the cell nuclei (black). Note the gradual increase in thickness, area, and folding of the
mucosa caudally, while morphological parameters of the submucosa and adventitia remain mostly invariant in all regions. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the progressive thickening of the epithelium with respect to
the other layers distally.
3.2. Fitting results: neo-Hookean and five-fiber family SEF
In Fig. 5, representative examples are shown from our
pressureradiusforce measurements for the four large
intestinal regions, noting that the in situ axial pre-stretch was
110% everywhere. Despite marked regional differences in the
inflation/extension response, a common finding in all regions
was the remarkable nonlinearity of the pressureradius data,
contrasting the almost linear relationship of the forceradius
data. Axial force declined with pressure at small axial pre-
stretches, while staying invariant at the in-vivo and rising at the
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166 155

Fig. 4 Collagen (A) and elastin (B) area densities, and percentage cross-sectional area (C) of mucosa, submucosa, muscle,
adventitia, and entire wall in the ascending, mid, and descending colon and the rectum of the rat. Symbols z, , #, and y
denote po0.05 relative to the ascending, mid, and descending colon and the rectum, respectively (Tukey test after ANOVA).

large axial pre-stretches, and external radius decreased at all
pressures by increasing the level of axial pre-stretch.
Final numerical results of the optimized material parameters
for the neo-Hookean and five-fiber family SEF are listed in
Table 1 for each of the four regions examined. Even though
allowance should be made for measurement error in our
experimental data, these were well reproduced by the fitted
(solid) curves (Fig. 5). The fits closely agreed with the nonlinearly-
varying pressure vs. radius and the linearly-varying force vs.
radius data, spanning the entire data range. As regards the best-
fit material parameters, the angle of orientation a0 of the
diagonal fibers was invariably less than 45 deg, and the following
inequalities in the material parameters of the fiber families were
found in all regions, i.e. ka1 4kd1 4kc1 kr1 and fkr2 ,kd2 g4fka2 ,kc2 g.
Regional differences in the parameters of only the diagonal-
fiber families and the neo-Hookean function were found
between the two more proximal and distal regions. Our attempts
to further improve the fit were unlikely to be productive. In
particular, we found that use of a quadratic instead of a neo-
Hookean term to account for the anisotropy at low radii,
together with the five-fiber families, was insufficient to more
reliably capture the inflation/extension data and did not result in
lower root-mean-square error values (results not shown).
3.3. Parameter justification and covariance
A drawback of the proposed SEF of Eqs. (1)(3) was that the
fitting procedure did not always converge, suggesting that
this function was over-parameterized. In every case, we
noticed that the parameter error for one or more parameters
was large relative to the parameter value that was close to
zero, and that the parameter dependence was almost unity.
Furthermore, the determinant of the correlation matrix was
much less than the 104 limit set for determining over-
parameterization (Table 1), and we decided to remove the
parameters of the radial-fiber family that had a similar
contribution to the fitting of pressureradius data as that of
the diagonal-fiber family. The situation was improved dras-
tically, but even with the reduced model there were still cases
when the value of the determinant of the correlation matrix
was indicative of parameter covariance (Table 2; see Fig. 6 for
examples of fitting with the neo-Hookean and four-fiber
family SEF). These findings recommended fixing or probably
removing the parameters whose dependency was close to
one, since the fit did not seem to depend upon those
particular parameters.
Extensive modeling studies were accordingly performed,
in which the parameters of each one of the SEF terms were
sequentially set equal to zero. The results are summarized in
Table 3 and demonstrated in Fig. 7, showing the comparison
of experimental to modeled data for a representative rectum
specimen. Judging from the root-mean-square error value,
which is a clear indication of the descriptive capacity of a
model, and from visual inspection, we realized that the four-
fiber family model with no contribution from the neo-
Hookean SEF term provided a similarly good fit to the
156 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 5 Two-dimensional scatter plot of representative intraluminal pressure (left) and axial force (right column) vs. external
radius data at three axial stretches for the different large bowel regions, together with the fits (solid lines) obtained from the
neo-Hookean and five-fiber family SEF. (A and B) Ascending colon: l 0.387 kPa, kd1 13:780 kPa, kd2 7:963, ka1 12:503 kPa,
ka2 4:311, kc1 0:000 kPa, kc2 7:866, kr1 0:000 kPa, kr2 7:021, a0 35 deg, e0.223, det(R) 8  107. (C and D) Mid colon:
l 0.044 kPa, kd1 13:635 kPa, kd2 38:433, ka1 85:833 kPa, ka2 2:741, kc1 0:000 kPa, kc2 13:708, kr1 0:000 kPa, kr2 0:000,
a0 35 deg, e0.297, det(R) 2  108. (E and F) Descending colon: l 0.389 kPa, kd1 0:360 kPa, kd2 15:509, ka1 36:248 kPa,
ka2 2:314, kc1 0:000 kPa, kc2 1.745, kr1 1.752 kPa, kr2 10.475, a0 36 deg, e0.158, det(R) 7  1012. (G and H) Rectum:
l 0.000 kPa, kd1 2.110 kPa, kd2 8.736, ka1 9.669 kPa, ka2 4.004, kc1 1.374 kPa, kc2 0.585, kr1 0.771 kPa, kr2 9.999, a0 35
deg, e0.166, det(R) 4  1010.
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166
Table 1 Fitted material parameters in the neo-Hookean and five-fiber family SEF to the experimental data of the different intestinal regions.

m [kPa] kd1 [kPa] kd2 [] ka1 [kPa] ka2 [] kc1 [kPa] kc2 [] kr1 [kPa] kr2 [] a0 [deg] e [] det(R) []

Ascending colon 0.16970.047$ 9.00171.380$ 8.27470.499# 31.404711.429 4.12571.094 0.39770.106 4.71570.729 1.54170.588 11.73771.354 33.670.8 0.21270.011 (271)  107
Mid colon 0.29570.177 5.95672.660 20.18774.433n& 51.63576.585 1.33170.639 0.98470.530 11.70376.721 1.07070.417 13.66473.249 38.671.7 0.18770.036 (171)  107
Descending colon 0.77670.233n& 1.80470.776n 11.72871.747 47.925721.822 2.59370.250 1.66670.795 0.46470.239 1.13270.456 15.36072.599 34.271.2 0.21070.019 (271)  108
Rectum 0.03870.018$ 5.35171.556 5.65970.870# 6.18672.267 3.59270.690 0.73570.189 3.40371.337 0.80770.251 21.90076.757 39.172.7 0.20470.022 (373)  109

, #, $, & po0.05 vs. ascending colon, mid colon, descending colon, and rectum. Refer to Table 1 in the electronic supplementary material for the individual parameter values.

Table 2 Fitted material parameters in the neo-Hookean and four-fiber family SEF to the experimental data of the different intestinal regions.

m [kPa] kd1 [kPa] kd2 [] ka1 [kPa] ka2 [] kc1 [kPa] kc2 [-] a0 [deg] e [] det(R) []

Ascending 0.25670.050 12.85070.486$& 8.27070.498# 37.839711.753 3.51070.930 1.53370.143 5.35070.353 37.370.4 0.24270.005 (2072)  104
colon
Mid colon 0.33170.182 7.35972.669 18.31374.760n& 50.94276.576 1.34870.668 1.01870.620 12.15575.293$& 39.571.8 0.18470.037 (473)  104
Descending 0.67370.171& 3.60270.870n 9.26471.108 48.449721.732 2.24870.253 0.97070.251 0.78370.580# 36.371.5 0.20770.014 (473)  103
colon
Rectum 0.04370.020$ 3.29670.722n 6.40670.533# 8.84072.144 3.67470.609 1.08370.164 0.97270.371# 40.771.9 0.19870.022 (1074)  104

, #, $, & po0.05 vs. ascending colon, mid colon, descending colon, and rectum. Refer to Table 2 in the electronic supplementary material for the individual parameter values.

157
158 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 6 Two-dimensional scatter plot of representative intraluminal pressure (left) and axial force (right column) vs. external
radius data at three axial stretches for the different large bowel regions, together with the fits (solid lines) obtained from the
neo-Hookean and four-fiber family SEF. (A and B) Ascending colon: l 0.411 kPa, kd1 14.217 kPa, kd2 7.741, ka1 12.481 kPa,
ka2 4.310, kc1 0.000 kPa, kc2 10.730, a0 35 deg, e 0.218, det(R) 4  103. (C and D) Mid colon: l 0.044 kPa, kd1 13.635 kPa,
kd2 38.433, ka1 85.833 kPa, ka2 2.741, kc1 0.000 kPa, kc2 13.708, a0 35 deg, e0.296, det(R) 106. (E and F) Descending
colon: l 0.769 kPa, kd1 2.359 kPa, kd2 10.296, ka1 36.700 kPa, ka2 2.230, kc1 0.000 kPa, kc2 0.000, a0 38 deg, e 0.164,
det(R) 2  102. (G and H) Rectum: l 0.152 kPa, kd1 3.117 kPa, kd2 8.300, ka1 9.516 kPa, ka2 4.014, kc1 1.514 kPa, kc2 0.338,
a0 37 deg, e 0.166, det(R) 9  104.
Table 3 Validation of the material parameters of the neo-Hookean (elastin) and four-fiber (collagen) family SEF terms for the different intestinal regions.

journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

m [kPa] kd1 [kPa] kd2 [] ka1 [kPa] ka2 [] kc1 [kPa] kc2 [] a0 [deg] e [] det(R) []

Four-fiber family SEF

Ascending 0 12.60570.773 8.31771.209 39.501731.355 3.52271.902 2.77470.714 1.76670.604 35.871.3$& 0.25970.025 (474)
Colon  103
Mid Colon 0 8.40173.748 17.91975.133 51.66577.536 1.23870.768 2.00670.927 5.38172.252 39.471.8 0.20070.041 (1073)
 102
Descending 0 4.86571.876 7.34171.488 53.739734.323 2.23470.312 2.76270.963 0.62770.549 36.371.7n 0.25270.027 (2076)
Colon  104
Rectum 0 3.37371.007 6.43570.701 8.60073.065 3.85270.861 1.35170.343 0.88670.571 40.372.7n 0.20370.031 (674)
 103

Neo-Hookean and two-fiber (axial and circumferential) family SEF

Ascending 0.03270.032 0 0 58.644732.890 2.21971.103 0.04770.039 9.18973.260 0 0.75170.022 (873)
Colon  104
Mid Colon 0.46270.298 0 0 66.632711.081 0.99970.603 0.20570.142 5.71772.871 0 0.65070.046 (573)
 102
Descending 0.53970.279 0 0 65.534737.716 1.73870.155 0.66970.527 4.44272.238 0 0.62270.056 (272)
Colon  102
Rectum 0.30870.202 0 0 13.31773.675 2.65470.541 0.27270.172 4.20972.032 0 0.67170.051 (773)
 102

Neo-Hookean and three-fiber (diagonal and circumferential) family SEF

Ascending 0.71770.198 17.13575.208 7.20872.176 0 0 1.94570.696 1.81171.011 31.972.9 0.44770.072 (472)
Colon  103
Mid Colon 0.74870.397 12.46077.605 15.71074.839 0 0 1.52370.903 5.43272.233 36.273.2 0.51470.076 (2075)
 102
Descending 0.97170.273 3.91971.296 9.57071.623 0 0 0.62070.380 52.253750.155 35.372.0 0.56070.037 (777)
Colon  103
Rectum 0.46870.105 2.87670.787 7.44370.593 0 0 0.61970.497 5.13073.789 37.872.8 0.48570.074 (272)
 102

Neo-Hookean and three-fiber (diagonal and axial) family SEF

Ascending 0.70570.059 13.11370.464$& 8.16870.458# 37.359711.316 4.02670.829# 0 0 37.071.0 0.24370.015 (1078)
Colon  103
Mid Colon 0.68070.298 8.12572.956 19.09274.418n$& 47.73677.194 1.45770.642n& 0 0 41.271.5 0.23370.032 (2077)
 103
Descending 0.82270.162 3.43170.750n 9.39871.218# 48.631722.033 2.60370.265 0 0 36.171.9& 0.21870.017 (873)
Colon  103
Rectum 0.28070.047 3.18470.701n 6.82070.600# 7.43471.868 4.12770.620# 0 0 41.971.8$ 0.22870.019 (1076)
 103

, #, $, & po0.05 vs. ascending colon, mid colon, descending colon, and rectum.

159
160 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 7 Two-dimensional scatter plot of representative intraluminal pressure (left) and axial force (right column) versus
external radius data at three axial stretches for the rectum, together with the fit (solid lines) obtained from the (A and B) four-
fiber family SEF kd1 3.296 kPa, kd2 8.048, ka1 9.976 kPa, ka2 3.922, kc1 2.096 kPa, kc2 0.169, a0 37 deg, e 0.171,
det(R) 4  103; (C and D) neo-Hookean and two-fiber (axial and circumferential) family SEF: l 0.090 kPa, ka1 16.083 kPa,
ka2 3.224, kc1 0.061 kPa, kc2 4.422, e 0.720, det(R) 2  103; (E and F) neo-Hookean and three-fiber (diagonal and
circumferential) family SEF: l 0.562 kPa, kd1 3.049 kPa, kd2 9.022, kc1 0.529 kPa, kc2 0.000, a0 36 deg, e0.504,
det(R) 6  103; and (G and H) neo-Hookean and three-fiber (diagonal and axial) family SEF: l 0.449 kPa, kd1 2.610 kPa,
kd2 9.126, ka1 8.243 kPa, ka2 4.355, a0 38 deg, e 0.193, det(R) 2  102.

experimental data, when compared to the entire material family model, that is when setting zero values to the material
model (Table 2); corresponding optimized material para- parameters of circumferential fibers. In contrast, the descrip-
meters of that specimen are provided in Fig. 6. Equally was tive capacity of the neo-Hookean and the (axial and circum-
inferred for the neo-Hookean and (diagonal and axial)-fiber ferential) or the (diagonal and circumferential)-fiber family
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166
SEF was very poor. The pressureradius data were defectively
modeled in the absence of diagonal fibers (Fig. 7C and D) and
the forceradius data were defectively modeled in the
absence of axial fibers (Fig. 7E and F).
As a separate case for a microstructure-based model, we
considered the neo-Hookean and two-fiber family model that
comprised only diagonally-oriented collagen fibers (see
Table 3 in the Supplementary material). In Fig. 8, we see a
tendency for that SEF to somewhat manage the pressure data
but seriously underestimate the force data over the entire
radius range. Although this material model did not suffer
from issues of over-parameterization, its root-mean-square
error was considerably higher than that of the neo-Hookean
and four-fiber family model, indicative of non-viable fitting.
3.4. Optimum choice: neo-Hookean and three-fiber family
SEF
While deletion of either one of the neo-Hookean or the
circumferential-fiber family terms led to similar fits, the
quality of fitting was aggravated when both terms were
removed (results not shown). Overall, the most appropriate
fit to the large bowel data was obtained when using the neo-
Hookean and three-fiber family SEF, i.e. fits were equally good
when setting parameters kc1 and kc2 equal to zero (Fig. 9) than
when the circumferential-fiber family obtained individual
sets of values (c.f. Fig. 6). The fit with this SEF converged for
all specimens tested, with the minimization procedure reach-
ing the absolute minimum value and the SEF was never over-
parameterized. Best-fit values of the material parameters
for each intestinal region are listed in Table 4. It is noted that
the SEF parameters were very similar to those of the neo-
Hookean and four-fiber family SEF and the presence of axial
and diagonal fibers (with orientation angles a0 40 deg) only
implied axial reinforcement and pronounced anisotropy.
In addition, the greater parameter values of the diagonal-
fiber families for the ascending and mid colon compared to
the descending colon and rectum were suggestive of prox-
imal tissue stiffening.
4. Discussion
4.1. Interpretation of biomechanical testing findings in
relation to prior studies
The biomechanical properties of the large bowel modulate a
broad spectrum of phenomena, including water and vitamin
absorption from the remaining indigestible food matter and
transport of waste material out of the body, which have a
decisive impact on gastrointestinal function in health and
disease (Gregersen, 2002). Steady progress has been made the
past two decades in understanding the biomechanical prop-
erties of small intestine; refer to the numerous investigations
by Gregersen and colleagues, cited by Li et al. (2008) and Liao
et al. (2010). By contrast, there has been only restricted
interest in the respective properties of the large intestine.
Most previous attempts to determine the biomechanical
properties have employed indices, such as tensile strength,
ultimate extensibility, wall stiffness, etc. Nonetheless, these
161
uniaxial loading approaches (Yamada, 1970; Watters et al.,
1985a, 1985b; Itasaka et al., 1992; Egorov et al., 2002; Ciarletta
et al., 2009) may not be suitable for robust material char-
acterization, because of the lack of a satisfactory methodol-
ogy to yield a 3D description of the multiaxial tissue
response.
A strongly nonlinear pressureradius behavior was
observed in the large intestine as well as anisotropy between
the circumferential and axial directions, as is typically the
case with biological soft tissues (Humphrey, 2002; Sacks and
Sun, 2003). The nonlinear pseudo-elastic response of the
large bowel is important for its function, since wall compli-
ance at low to physiologic pressure levels (05 mmHg) is
related to its storage capacity and the increasing resistance
at higher pressures serves to protect against over-distention
and rupture of the tissue. Consistent with Watters et al.
(1985a), we found that the rat colon was axially stretched by
110% in situ, for which our inflation/extension data demon-
strated an almost flat forceradius relationship. Likewise was
noted for the 120% and 130% pre-stretches, this finding
constituting an energetically advantageous behavior, as the
tissue performed little axial work under pressure inflation.
We have reported comparable data for other tissues from the
gastrointestinal (Stavropoulou et al., 2009) and urinary tract
(Sokolis, 2011), unlike cardiovascular tissues for which force
remains constant under inflation only at the in situ axial pre-
stretch; see our recent vein results (Sokolis, in press) and the
discussion by Humphrey (2002).
The increased stiffness of the mid colon documented
by our present and recent work (Sokolis et al., 2011a) is in
agreement with the qualitative observations by Gao and
Gregersen (2000) in their distension studies, as is the aniso-
tropic response, that is the increased stiffness in the axial
than the circumferential direction; see also the uniaxial
tension results by Yamada (1970) and Egorov et al. (2002).
Our data also support the increased circumferential strength
of the rat mid colon and the decreased strength of the rectum
(Watters et al., 1985a). Strength and stiffness are both
dependent on the percentage content of collagen that accord-
ing to our semi-quantitative optical microscopic results was
greatest in the mid colon and smallest in the rectum, given
the constant thickness of the collagen-rich submucosa
throughout the large intestine and the progressively increas-
ing thickness of the entire wall distally (Fig. 4). In humans,
tensile strength fell in the sigmoid colon due to wall thicken-
ing in that region, while internal radius declined distally
(Watters et al., 1985b). A similar distribution of those mor-
phological measures along the large bowel of the rat was
disclosed for both histologically-processed (Fig. 3) and fresh
tissue (Sokolis et al., 2011a). The latter data corroborate the
more detailed distributions of morphometric parameters,
residual strains, and opening angle in the no-load and zero-
stress conditions, provided by Gao and Gregersen (2000).
Finally, our histological observations imply that the aniso-
tropic properties of the large bowel wall were produced by the
high degree of collagen fiber organization along the axial
direction.
The functional significance of the abovementioned find-
ings is as follows. The increased wall compliance circumfer-
entially aids the storage and transport function of feces,
162 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

Fig. 8 Two-dimensional scatter plot of representative intraluminal pressure (left) and axial force (right column) vs. external
radius data at three axial stretches for the ascending, mid, and descending colon, and the rectum, together with the
simulations (solid lines) calculated from the neo-Hookean and diagonal-fiber family SEF. (A and B) Ascending colon:
l 0.673 kPa, kd1 17.822 kPa, kd2 6.810, a0 32 deg, e 0.428, det(R)3  102. (C and D) Mid colon: l 0.078 kPa, kd1 14.517 kPa,
kd2 39.182, a0 35 deg, e 0.629, det(R)2  102. (E and F) Descending colon: l 0.900 kPa, kd1 2.259 kPa, kd2 11.273, a0 38
deg, e 0.598, det(R)6  102. (G and H) Rectum: l 0.646 kPa, kd1 2.835 kPa, kd2 9.386, a0 36 deg, e0.505, det(R) 6  102.

whereas the high wall stiffness axially aids in keeping the those segments, which is vitamin, electrolyte, and water
bowel intact. The increase in wall stiffness from the ascend- absorption for the former segment, and the need to propel
ing to the mid colon may reflect the dissimilar functions of the more-solid colonic contents for the latter. The stiffness
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166 163

Fig. 9 Two-dimensional scatter plot of representative intraluminal pressure (left) and axial force (right column) vs.
external radius data at three axial stretches for the ascending, mid, and descending colon, and the rectum, together
with the simulations (solid lines) calculated from the neo-Hookean and three-fiber (diagonal and axial) family SEF.
(A and B) Ascending colon: l 0.411 kPa, kd1 14.216 kPa, kd2 7.741, ka1 12.484 kPa, ka2 4.310, a0 35 deg, e 0.218,
det(R) 4  103. (C and D) Mid colon: l 0.060 kPa, kd1 13.845 kPa, kd2 37.948, ka1 85.690 kPa, ka2 2.737, a0 35 deg,
e 0.295, det(R) 6  103. (E and F) Descending colon: l 0.769 kPa, kd1 2.351 kPa, kd2 10.318, ka1 36.702 kPa, ka2 2.229,
a0 38 deg, e0.164, det(R) 2  102. (G and H) Rectum: l 0.449 kPa, kd1 2.610 kPa, kd2 9.126, ka1 8.243 kPa, ka2 4.355,
a0 38 deg, e0.193, det(R) 2  102.
164 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166

decrease in the rectum may be linked to its reservoir function

(1078)  103
(2077)  103
(873)  103
(1076)  103
that occurs until the discharge of fecal matter from the body,

det(R) []
and the descending colon provides reserve storage space by
exhibiting a marginally stiffer wall than the rectum.
Table 4 Fitted material parameters in the neo-Hookean and (diagonal and axial)-fiber family SEF to the experimental data of the different intestinal regions.
4.2. Interpretation of material characterization results

, #, $, & po0.05 vs. ascending colon, mid colon, descending colon, and rectum. Refer to Table 4 in the electronic supplementary material for the individual parameter values.
0.24370.015
0.23370.032
0.21870.017
0.22870.019
The identification of a SEF within the theoretical framework
of pseudo-elasticity is the most appropriate method for soft
tissue biomechanical characterization. This was Fungs pro-
e []

position four decades ago [see discussion in (Fung, 1993)] that

was followed by intense activity in the field with an accu-
mulation of seminal publications on constitutive descriptors
of the different classes of biological soft tissue [refer to the
36.171.9&
41.971.8$
37.071.0
41.271.5
a0 [deg]

reviews by Humphrey (2002), Sacks and Sun (2003), Holzapfel

and Ogden (2010)], but only lately have been in focus the
tissues from the gastrointestinal tract.
In recent years, significant effort has been put into devel-
oping microstructure-based material models, whose material
1.45770.642n&

parameters reflect the individual properties of wall compo-

4.02670.829#

4.12770.620#
2.60370.265

nents and their microstructural organization/arrangement.

Ideally, the development of such a material model should
ka2 []

be guided by histological analysis, which has in fact been our

intent in the present study, namely to validate through
computerized light microscopy the implementation of a
microstructure-based SEF in reasonably simulating our
37.359711.316

48.631722.033
47.73677.194

7.43471.868

recent (Sokolis et al., 2011a) and present multiaxial data.

The neo-Hookean and five-fiber family model was initially
ka1 [kPa]

selected for its effectiveness in offering suitable multiaxial

characterization of the large intestine in relation to the under-
lying wall microstructure (Fig. 5 and Table 1). This SEF considered
linear isotropic properties for elastin and nonlinear anisotropic
properties for collagen, organized in five different orientations.
19.09274.418n$&

The intestinal wall was hypothesized as being in its non-

8.16870.458#

9.39871.218#
6.82070.600#

contracting state, with all main microstructural components

arranged in parallel without serial connections and the passive
kd2 []

contribution of the smooth muscle was considered minimum.

Compared to the phenomenological forms implemented in our
recent report, the present microstructure-based form displayed
superior descriptive capability of both the pressureradius and
forceradius data in specimens from all four regions; c.f. Table 1
13.11370.464$&

3.43170.750n
3.18470.701n
8.12572.956

herein with Table 1 in Sokolis et al. (2011a). Despite, however, its

very good descriptive capacity, it employed many more para-
kd1 [kPa]

meters than would be needed. This does not necessarily mean

that these parameters were without physical meaning. Rather, it
indicated that there were infinite solutions of the fit process and
suggested removing the radial-fiber family parameters whose
contribution to the representation of experimental data was
0.70570.059
0.68070.298
0.82270.162
0.28070.047

comparable to that of the diagonal-fiber families.

The neo-Hookean and four-fiber family SEF was first
m [kPa]

proposed and effectively utilized as a constitutive descriptor

of elastic (Gleason et al., 2008) and muscular arteries (Wicker
et al., 2008). It was thereafter extended by our group to
include anisotropic elastin properties, by replacing the neo-
Descending colon

Hookean with a quadratic term, in modeling studies of the

Ascending colon

arterial (elastic and muscular) (Sokolis et al., 2011b) and

venous tissue (Sokolis, in press). Our modeling results for
Mid colon

Rectum

the large intestine suggested however that the low-stress

response generally attributable to elastin was dissimilar from
that for the abovementioned tissues and it was redundant to
 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166
express it by a quadratic function. The neo-Hookean and
four-fiber family SEF was employed by Ciarletta et al. (2009)
for the large intestine, but different to the present micro-
structural interpretation was given to the four-fiber families
(see discussion in Section 4.3 below) without histological
justification, namely circumferential and axial families were
related to the smooth muscle fibers in turn of the inner and
outer muscle layers, and diagonal families were related to
submucosal collagen. Good fits to the data were reported by
the authors, but examination of a small sample number
under uniaxial loading conditions considerably hinders the
utility of these findings for physiological applications and
differences with region were not defined.
We found, nonetheless, that even the neo-Hookean and
four-fiber family SEF was limited by over-parameterization
issues. Our modeling studies (Fig. 7 and Table 3) revealed that
the contribution of axial and diagonal fibers to data fitting
was important and their maintenance was deemed neces-
sary, whereas removing either the neo-Hookean or the
circumferential-fiber family term led to comparable fits,
anticipated from the fact that their parameters obtained
small values in the entire SEF (Table 2). Notice, however, that
when the parameters of both terms were removed, the
quality of the fit was deteriorated significantly, so that only
parameters kc1 and kc2 in Eqs. (1)(3) were zeroed and the
values of the left-over parameters m, kd1 , kd2 , ka1 , ka2 , and a0 were
optimized. The goodness of fitting was analogous to that of
the entire model and there were no longer problems of over-
parameterization (Fig. 9 and Table 4), supporting the validity
of the neo-Hookean and three-fiber (diagonal and axial)
family SEF as a material descriptor of the large bowel.
4.3. Identification of a structurefunction relationship
The biomechanical response of the large bowel stems from the
organization and distribution of the microstructural wall com-
ponents, such as collagen and elastin fibers, and smooth muscle
cells (Gregersen, 2002). These components display individual
biomechanical properties and sequentially carry loads at differ-
ent stress levels, so that the intestinal wall shows a complex and
hard to analyze cumulative response. From histology, we infer an
evidently distinct organization of collagen in the various layers of
the healthy wall (Fig. 2). Mucosal collagen run radially and
submucosal collagen was distinguished by cross-ply fibers with
mainly axial orientation, while collagen in the muscle layers run
almost in parallel to the circumferential and axial muscle cells,
therefore stiffening those layers and restricting their ability to
stretch. Finally, serosal collagen was arranged in a loose interlace
of axial bundles. These distinguishing architectural patterns
appear advantageous for the individual layer functionalities,
contributing: (a) to the load-bearing capacity and flexibility of
submucosa in carrying biaxial loads, (b) to the structural integrity
of intestinal wall by providing mechanical support to the cells
and resistance to overstretching of the mucosal and muscle
layers, and (c) to the tethering of the serosa to the surrounding
tissues.
Several studies have examined the collagen architecture of
the submucosa but not of the other layers, as the greatest part of
collagen in the large bowel is located there and only small
amounts are present in the muscle layers and serosa (see the
165
histometric determinations in Fig. 4). Our qualitative description
of fiber orientation matches the quantitative analysis of Sacks
and Gloeckner (1999) on the porcine small intestinal submucosa.
These authors used small-angle light scattering to quantify gross
collagen fiber structure and evidenced a single preferred popula-
tion of fibers along the long axis of intestine. Two arrays of fibers
running diagonally (at 730 deg) around the wall in a clockwise
and an anticlockwise manner were formerly advocated by
Hiltner and coworkers, using polarizing light microscopy
(Fackler et al., 1981), as well as scanning electron (Orberg et al.,
1982) and transmission electron microscopy (Klein et al., 1983).
Similarly was found by Sacks and Gloeckner (1999) on few
occasions, but the two populations were not adequately distinct,
thus collectively producing a single population that was centered
near the long axis with wide angular distribution.
To establish a structurefunction relationship for the large
intestinal wall tissue, it is important to know which component
relates with a different part of the inflation/extension data. Our
modeling studies suggested that the pressureradius response at
physiologic to high radii may be satisfactorily accounted for by
the diagonal fibers, as their non-incorporation in the SEF
produced insignificant fits of the pressureradius data (Fig. 7C).
Very similar was the contribution of the radial fibers to the
representation of pressure data, so that their inclusion in the SEF
led to severe over-parameterization. This fact and the radial
orientation of this fiber family caused by the mucosal folding
disqualified its use. Next, the flat dependence of force on radius
is what one would expect from an axial arrangement of fibrous
material, so that its absence resulted in insignificant fits of the
forceradius data (Fig. 7F). Finally, the large bowel included small
amounts of elastin and circumferential collagen fibers, hence the
near zero values for parameter m of the neo-Hookean function
(used as a descriptor of the low-stress response) and parameters
kc1 and kc2 of the circumferential-fiber family (used as a descriptor
of the physiologic and high-stress pressureradius response).
4.4. Limitations
Several limitations of our study need to be acknowledged.
Those relating to the protocols used for histomorphometry
have been detailed previously (Sokolis, 2010), along with
those relating to the non-consideration of wall heterogeneity,
namely the determination of layer-specific properties (Sokolis
et al., 2011a). More elaborate than the present microscopic
observations of the collagen fiber orientation and organiza-
tion in the large bowel, afforded by imaging modalities such
as small-angle light scattering and confocal microscopy, are
necessary before developing a purely microstructural consti-
tutive descriptor of its material response. As regards other
experimental aspects of our study, we have performed 3D
testing over a physiologically-relevant deformation range, but
only passively. Multiaxial testing under active conditions is
urgent, especially for the large bowel which is a muscular
duct, offering insight for establishing a solid understanding
of the functional organization of intestinal wall components.
Such a research line remains acute for the majority of soft
biological tissues, even those that have customarily attracted
the attention of researchers, for which complete 3D informa-
tion is currently unavailable in the literature. For a realistic
simulation of the organ, information on the biomechanical
166 journal of the mechanical behavior of biomedical materials 21 (2013) 149 166
response of large bowel wall and of the effect of surrounding
tissues, i.e. of tethering, are important prerequisites. This
paper provides an answer to the first problem.
4.5. Concluding remarks
Although liable to improvements, the neo-Hookean and
three-fiber (diagonal and axial) family model mimics pro-
perly the salient features of the large bowel biomechanical
response. The distinctive collagen organization of the differ-
ent layers may well explain the nonlinearity and anisotropy
of the response of the healthy intestine and highlight the
different functionalities of the collagen fibers. The diagonal
fibers of submucosa were responsible for the exponentially-
shaped pressureradius response, while its axial fibers were
responsible for the flat (horizontal) forcepressure response.
The present basic knowledge of the multiaxial histomecha-
nical properties of large intestinal wall may be applied as a
baseline for comparison with future studies of anastomosed
and diseased bowel segments (Yang et al., 2003; Zhao et al.,
2009). Better insight into the microstructural basis of the
material response will see us through translating the knowl-
edge gained into clinical and biomedical applications.
Appendix A. Supporting information
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.jmbbm.
2013.02.016.
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