Influence of a freeze–thaw cycle on the stress–stretch curves of tissues of porcine abdominal organs

Inﬂuence of a freeze–thaw cycle on the stress–stretch curves of tissuesof porcine abdominal organs N.. Experimental results show strong effects of the realistic freeze–thaw cycle on the

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Inﬂuence of a freeze–thaw cycle on the stress–stretch curves of tissues

of porcine abdominal organs

N Huynh Nguy na,b,f, M Tu n Du’o’nga,b, T Ngo

_ c Tr n

a,c,d, P Tınh Pha

_ m

a,c,

O Grottked, R Tolbae, M Staata,n

aFaculty of Medical Engineering and Technomathematics, Aachen University of Applied Sciences, (FH Aachen), Heinrich-Mußmann-Str 1, 52428 J¨ ulich, Germany

bHanoi University of Science and Technology, Hanoi, Vietnam

cHanoi Architectural University, Hanoi, Vietnam

dDepartment of Anesthesiology, RWTH Aachen University Hospital, Aachen, Germany

eInstitute for Laboratory Animal Science, RWTH Aachen University Hospital, Aachen, Germany

fMax-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany

a r t i c l e i n f o

Article history:

Accepted 5 July 2012

Keywords:

Liver

Spleen

Freeze–thaw process

Decomposition

Autolysis

a b s t r a c t

The paper investigates both fresh porcine spleen and liver and the possible decomposition of these organs under a freeze–thaw cycle The effect of tissue preservation condition is an important factor which should be taken into account for protracted biomechanical tests In this work, tension tests were conducted for a large number of tissue specimens from twenty pigs divided into two groups of 10 Concretely, the first group was tested in fresh state; the other one was tested after a freeze-thaw cycle which simulates the conservation conditions before biomechanical experiments A modified Fung model for isotropic behavior was adopted for the curve fitting of each kind of tissues Experimental results show strong effects of the realistic freeze–thaw cycle on the capsule of elastin-rich spleen but negligible effects on the liver which virtually contains no elastin This different behavior could be explained by the autolysis of elastin by elastolytic enzymes during the warmer period after thawing Realistic biomechanical properties of elastin-rich organs can only be expected if really fresh tissue is tested The observations are supported by tests of intestines

1 Introduction

Besides tests in vivo, biological soft tissues are also tested

ex-corporally for identifying mechanical properties Therefore, tissue

preservation by a freeze–thaw cycle is needed for time consuming

experiments However, the effect of this preservation on the

mechanical behavior of abdominal organs has not been always

comprehended The preservation of tissue by freezing is normally

accompanied by cooling cycles during the preparatory work of

protracted biomechanical tests Freezing is suspect to micro-changes

of the tissue structure Freeze–thaw effects have been tested

mechanically mainly for organs which have a clear mechanical

purpose like tendons, full spine segments and arteries Very few

published such tests have been found for the abdominal organs

which have a non-mechanical purpose such as spleen, liver, and

kidney In compression tests no remarkable difference was observed

between porcine livers that have never been frozen and livers that

have been frozen for 24 h then thawed (Tamura et al., 2002) In

contrast to this it is found that the freeze–thaw process decreases

the strength of the porcine liver capsule but the strength of the human one can be unchanged or increased (Brunon et al., 2010) The ultimate strain seems to be increased by the freeze–thaw process for human organs and possibly for porcine liver capsules (Brunon et al.,

2010) The opposite was found for bovine liver (Santago et al., 2009) and for kidney (Nicolle and Palierne 2010) For other tissue such as menisci, four freeze–thaw cycles signiﬁcantly decreased the intrinsic resistance of the material (Lewis et al., 2008)

Freezing affects the structure and mechanical properties of the porcine femoral artery (Venkatasubramanian et al., 2006) How-ever, freezing caused a signiﬁcant increase in the average elastic modulus in the physiological regime The exact mechanisms for these changes are not known; some evidence suggest that bulk redistribution of water, changes of weight and ﬁber alignment could be important underlying phenomena Damage to the extra cellular matrix (ECM) and loss of smooth muscle cell viability could also play an important role (Venkatasubramanian et al.,

2006) However, there is good evidence that the contribution of smooth muscle cells to the elastic properties of living blood vessels is very small (Burton, 1951)

In order to partially support car crash research, both fresh and preserved (by a freeze–thaw cycle) tissues of abdominal organs are examined because solid organs are the most frequently

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Corresponding author Tel.: þ49 241 6009 53120; fax: þ49 241 6009 53199.

E-mail address: m.staat@fh-aachen.de (M Staat).

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injured abdominal organs in both frontal and side impact

colli-sions (Franklyn et al., 2002) Following accidents the liver has

been identiﬁed to be the most frequently injured abdominal

organ and the next one is the spleen It is known that porcine

organs have approximately the same size as human ones (Kent

et al., 2006) and have similar plumbing The abdominal anatomy

of the swine is reasonably similar to human anatomy (Ibrahim

et al., 2006), such as the organ structures and functions Hence, in

crash tests abdominal characteristics of humans can be

investi-gated by using the porcine organs as surrogates Thus, the

decomposition during thawing after postmortem frozen storage

and its effect on the mechanical properties of organs is

investi-gated in this study The results show that the thawing and the

time elapsed thereafter can signiﬁcantly change the mechanical

properties of speciﬁc tissues This helps explain why spleen is

more frequently lacerated than liver in crash tests in apparent

discrepancy to real accidents

2 Materials and methods

In our experiments, the liver tissues showed nearly isotropic behavior The splenic

tissues may show a stronger anisotropic response However, in our study, all tests of the

spleen were only conducted along the longitudinal direction of the organs Moreover,

the purpose is to investigate the inﬂuence of preservation on mechanical properties of

tissues Therefore, only data from tension tests are required to obtain material

properties Thus, a modiﬁed Fung formulation ( Duong et al., 2012 ) for isotropic models

was adopted for parameter identiﬁcation Statistical analyses were carried out to

investigate the differences between frozen-thawed and fresh specimens.

2.1 Material model

The strain energy function of Fung’s model in a general form is

where A 6 6has the form of a (dimensionless) orthotropic elasticity matrix, C has

the dimensions of a modulus Voigt’s notation of the Green-Lagrange strain vector

and the second Piola-Kirchhoff stress is E ¼ ½E 11 ,E 22 ,E 33 ,2E 12 ,2E 23 ,2E 31 T and

S ¼ ½S 11 ,S22,S33,S12,S23,S31 T , respectively.

In principal coordinates the exponent Q ¼ E T

AE becomes

Q ¼ A 11 E211þ A 22 E222þ A 33 E233þ 2A 12 E 11 E 22 þ 2A 23 E 22 E 33 þ 2A 13 E 11 E 33 : ð2Þ

The second Piola-Kirchhoff stress is calculated from the energy function as

S ¼@W

@E ¼ 2CðAEÞe

Q

The symmetric constitutive matrix is obtained as

C¼ @S

@E¼ 2CAe

Q

with the dyadic product .

An isotropic Fung model is used ( Duong et al., 2012 ) with a symmetric A in the

constitutive matrixC Hence, if A11¼ A22¼ A33and A12¼ A13¼ A23 the exponent

term (2) can now be expressed as

Q ¼ A 11 ðE211þ E222þ E233Þ þ 2A 12 ðE 11 E22þ E 11 E33þ E 22 E33Þ: ð5Þ

The constitutive matrix (4) is positive deﬁnite if C 40; A11 4A12 4 0 In this

case the modiﬁed Fung isotropic model will be stable According to (4), the ratio

between the initial tangent stiffness of decomposed tissues and fresh tissues is

deﬁned as

k0¼ @s decomposed

@

l ¼ 1

@sfresh

@

l ¼ 1

¼ðCA11Þdecomposed ðCA 11 Þ fresh

ð6Þ

2.2 Preparation of specimens

Twenty three-month-old pigs with a weight of 34 72.4 kg (mean7SD) were

euthanized and ﬁnalized after having been used in another experiment ( Grottke

et al., 2010 ) No swine were sacriﬁced for this project Organs were retrieved

post-mortem All organs were harvested with major blood vessels left intact and tested

extracorporeally These swine were divided into two groups of 10 animals: the

fresh and the frozen–thawed ones The fresh organs were brought to the

laboratory within 15 min Each ex-vivo test was performed within 4 h after organ

For the frozen–thawed group, the organs were frozen at 18 1C Here we consider the decomposition processes in a cycle of storage at room temperature

(RT¼ 720 1C to þ25 1C, wetted with normal saline solution), freezing, thawing,

storage in a refrigerator and ﬁnally at RT again The temperature settings and times ( Table 1 ) have been applied This is the basic cycle with freezing the organs

6 h after harvesting Separately, the beginning of thawing is set All organs are subject to the same temperature settings and time intervals.

The lower face of the spleen is not used for experiments because of a dense presentation of arteries and veins In contrary, both lower face and upper face of liver are exploited All specimens are cut into a rectangular shape of 50–65 mm long and 15 mm wide The thickness of the specimens is smaller than 2.5 mm (including capsule and parenchyma).

A single column testing machine Zwick/Roell Z0.5 is adopted for experiments The free test length of specimen is 24 mm The loading rate is of 120 mm/min., i.e the strain rate is around 0.08/s.

To adopt the modified Fung model above all coefficients must be determined from a curve fitting process for nonlinear least squares problems Data analysis was performed by using MATLAB All material parameters were obtained by using

a nonlinear algorithm, such as a subspace trust region method that is based on the interior-reﬂective Newton method.

2.3 Stress measures for large deformation and large displacements

In this section stress and strain are brieﬂy introduced for tension tests of isotropic incompressible materials The tension test generates uniaxial Cauchy stressrand deformation gradient F as

r¼

s 0 0

0 0 0

0 B 1 C , F ¼

0 l1=2 0

0 0 l1=2

0 B

1 C

where the stretchl¼ l/L is the ratio between deformed length l and reference length L.

The paper investigates organ surface including capsule and parenchyma These components contain much connective tissues components but less vascular Hence, spleen and liver capsules can be considered as isotropic materials.

2.4 Statistical analysis

Since there are three material parameters in the formula of the strain energy, the

goodness-of-fit adjusted R2 value will be used in the fitting procedure The effects of decomposition on the biomechanical properties of porcine abdominal organs are examined by analyzing the statistical significance of group differences These

statistical differences between two groups were computed using Student’s t-test at

the level of significance a¼ 0.05 making the confidence level 95% Thus, three material coefficients, the ultimate stress and the ultimate stretch of each group were statistically analyzed in our study For the spleen we assumed that the mean value of the ultimate stretch of the fresh tissue is larger than the one of the decomposed, therefore a one-sided test with a corresponding null-hypothesis was adopted for this case only For the others, a two-sided test assuming for the null-hypothesis that the means of the two groups are equal To get risks of taking the null-hypotheses as correct even if they are not the beta errors (b) were calculated by using power analysis All data is represented in mean7SD (standard deviation).

3 Experimental results The ultimate tensile Cauchy stresses of the tissues in tension test, sult, were computed from the experimentally determined rupture forces according to

Table 1 Temperature cycles.

Assumed (actual) decomposition Simulation of decomposition Time Period Temperature (1C) Period (h) Temperature (1C)

some months 21 4 20 18 to 25

1 h þ 18 to þ 25

11 h þ 4

7–9 h þ 19 to þ 23 1 þ 20 to þ 25

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where F is the tensile fracture force; A is the cross section of

specimen in reference conﬁguration; lult is ultimate tensile stretch (calculated from measurements of clamp to clamp,

Fig 1) The mean values are listed inTable 2

3.1 Spleen

All tissue test curves have been ﬁtted under the convexity constraints of the isotropic Fung model

Table 2 shows a signiﬁcant difference between the ultimate stress of the fresh and the frozen–thawed splenic tissue,

(po0.05 ¼a) The ultimate stretch (1.738670.1400) of the fresh tissue is much larger than the one of the frozen–thawed tissue

(1.303270.0610), (pE140.05,br10 7

).Fig 2shows that there was a signiﬁcant difference between the material curves of fresh and the frozen–thawed tissues especially in the ‘‘toe region’’ This assures a strong effect of the freeze–thaw process on spleen tissue

Table 3shows the mean constitutive parameters of the fresh and the frozen–thawed tissues Signiﬁcant differences in constitutive

parameters (po0.05) were found and the resulting stretch–stress

curves (mean curves) are shown (Fig 3) These curves reﬂect the mean of all mechanical data from the specimens

3.2 Liver

On contrary, there is no statistically signiﬁcant difference between the ultimate stresses of the fresh and the frozen– thawed liver tissue (Table 2) We cannot reject the null Fig 1 Tension test—ruptured fresh spleen specimen.

Table 2

Mean values of ultimate Cauchy stresses and stretches.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

λ

Experimental Fitted

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

λ

2)

Experimental Fitted

Fig 2 Curve-ﬁtting for the fresh (a) and the freeze–thaw (b) splenic tissues—tension tests (not all tests presented in the ﬁgures).

Table 3

Statistics of curve ﬁtting process for spleen tissues.

Fresh, n¼ 51 0.2403 70.1423 0.613670.0729 0.531670.1309 98.6270.94

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hypothesis because p¼ 0.200840.05 Similarly, there was no

statistically signiﬁcant difference in ultimate stretches

(p¼ 0.798640.05,b¼ 0.908) The mean material coefﬁcients from

ﬁtting phase for the fresh and the frozen–thawed tissues are

shown inTable 4

The mean curves (parameters inTable 4) for the representative

fresh and frozen–thawed liver tissue are plotted (Fig 4) No

signiﬁcant difference between the stress–stretch curves is observed

4 Discussion

The key ﬁndings are

Decomposition takes place by autolysis in the thawed organ

There was no decomposition in liver which contains no elastin

Strong decomposition occurs in elastin-rich spleen, which

tends to make the material curve of spleen similar to the

curve of liver Decomposition increases the initial stiffness and

reduces the maximum stretch of the spleen capsule

Elastin-rich tissue must be tested as fresh as possible; storage

of other tissues may be possible

For spleen, there were statistically signiﬁcant differences in

stresses and stretches between the fresh and the frozen–thawed

tissues The freeze–thaw process seems to have a strong effect on

the mechanical properties of spleen by making the organ more

rigid in the physiological range and in particular on ultimate

values On the contrary, our experimental results show that the

mechanical properties of liver tissues are almost not affected It is

also reported that no remarkable difference was observed

between thawed and fresh liver (Tamura et al., 2002) In contrast,

it was found that the freeze–thaw process decreases the strength

of the porcine liver capsule, but increases the ultimate strain

(Brunon et al., 2010) The opposite was found for bovine liver (Santago et al 2009) The ﬁndings in (Brunon et al., 2010) for human and porcine liver are contradicting each other With respect or our ﬁndings (Fig 4) it could be assumed that no effect

of freezing on liver may have been observed (Brunon et al., 2010)

Roach and Burton (1957)digested collagen in blood vessels using formic acid (1 h), and digested elastin using trypsin (22 h) They found that collagen contributed mainly to the stiff quasi-linear region of the nonlinear stress–stretch curve while elastin contrib-uted mainly to its low-stiffness toe part Similar trends under collagenase and elastase have been found also in biaxial tests (Gundiah, 2004) The trend of the stress curve (Fig 5) over the hoop stretch in arteries for digested elastin resembles our observations for spleen after the freeze–thaw cycle Moreover, same as observed for freezing (Venkatasubramanian et al., 2006) it was found in (Roach and Burton, 1957) that digestion of elastin changes the stiffness in the toe region and leads to an increase of the diameter of the arteries This suggests that a decomposition of elastin has taken place in the spleen during the periods around room temperature in the cycle after thawing because collagen is decomposed at a slower rate Contrary to spleen capsules with higher elastin content, liver capsules contain a large number of collagen ﬁbers but virtually no elastin ﬁbers for many species (Neuman and Logan, 1950) This would explain why no effect of the freeze–thaw cycle has been observed for liver Furthermore, for the spleen the ratio of the initial stiffness between the decomposed and the fresh capsules has

changed by a factor of six (k0¼ 6.3) In contrast for the liver, this

value is nearly unchanged (k0¼ 0.68) or has changed very little compared to the spleen tissue

Freezing and thawing often break down cell membranes allowing autolytic membrane-bound enzymes to react with their natural substrates (Huss, 1995) We hypothesize that the freeze– thaw cycle does not directly inﬂuence the tissue mechanics by micro-changes but it accelerates autolysis by the elastolytic enzymes which reduce the fraction of intact elastin ﬁbers in the

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

λ

2]

Fresh Decomposed

Fig 3 Curve-ﬁt representatives for fresh and frozen–thawed spleens.

Table 4

Statistics of curve ﬁtting process for liver tissues.

Fresh, n¼ 49 0.7303 70.4489 0.519470.0462 0.357870.1185 99.00 70.76

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

λ

Fresh Decomposed

Fig 4 Representative stress–stretch curves for fresh and frozen–thawed liver.

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warmer periods after thawing This decomposition shifts the

composition of non-degenerated proteins in spleen in the

direc-tion of composidirec-tion found in liver and both tissues become

similar in their quasi-static mechanical response (Fig 6)

The concentration on freezing in some investigations leads to

lesser control of the possible autolysis particularly in times after

thawing In the otherwise meticulous paper (Brunon et al., 2010),

the adult porcine liver has been bought from the local butchery,

within 4 or 5 days after euthanasia and the human organs have been

kept ‘‘fresh’’ for up to 5 day after death In case of spleen such a

procedure would probably lead to such an amount of decomposition

that there may not remain any margin to show an effect of thawing

Therefore, it is not surprising that there are so many conﬂicting

ﬁndings about freezing of tissue in the literature if the

decomposi-tion by elastolytic enzymes is neglected The present study has been

designed differently because it has tested fresh tissue directly after

euthanasia and has included hold times at þ4 1C to þ8 1C and room

temperature in the freeze–thaw cycle

There is some indication that porcine abdominal organs may have

tissues with higher strength and higher elasticity than human organs

(Stingl et al., 2002) Thus, it is expected that the results can be applied

qualitatively to human organs but quantitatively only with unknown

accuracy It is planned to validate the new ﬁndings about

decom-position under other preservation conditions and with respect to

other organs First tests with different sections of elastin-rich sheep,

porcine and human intestines support our hypothesis although the

behavior of these layered tissues is more complex (Tr n et al.,

submitted for publication) Anisotropy of porcine intestinal tissues changes along the gastrointestinal tract While the porcine jejunum tissue is approximately isotropic, the colonic tissues are strongly orthotropic Only little difference was observed between fresh and thawed porcine jejunum Decomposition had a large impact on circumferential samples of porcine sigmoid and rectum, which becomes stiffer whereas the effect on longitudinal samples seems

to be smaller (Tr n et al., submitted for publication) Our tests with kidney propose that there is no strong decomposition but the statistical basis was not sufﬁcient for publication

Conflict of interest None

Acknowledgments The authors thank TRW Automotive GmbH, Alfdorf, Germany, for support of the project and the permission to use the data presented in this paper

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Duong, M.T., Nguyen, N.H., Staat, M., 2012 Finite element implementation of a 3D Fung-type model In: Holzapfel, G.A., Ogden, R.-W (Eds.), ESMC-2012–8th European Solid Mechanics Conference Verlag d Technischen Universit ¨at Graz, Graz.

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Grottke, O., Braunschweig, T., Philippen, B., Gatzweiler, K.H., Gronloh, N., Staat, M., Rossaint, R., Tolba, R., 2010 A new model for blunt liver injuries in the swine European surgical Research 44, 65–73.

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Lewis, P.B., Williams, J.M., Hallab, N., Virdi, A., Yanke, A., Cole, B.J., 2008 Multiple freeze–thaw cycled meniscal allograft tissue: a biomechanical, biochemical, and histologic analysis Journal of Orthopaedic Research 26 (1), 49–55 Neuman, R.E., Logan, M.A., 1950 The determination of collagen and elastin in tissues Journal of Biological Chemistry 186 (2), 549–556.

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of arteries Canadian Journal of Biochemistry and Physiology 35, 681–690 Santago, A.C., Kemper, A.R., McNally, C., Sparks, J.L., Duma, S.M., 2009 Freezing affects the mechanical properties of bovine liver Biomedical Sciences Instru-mentation 45, 24–29.

Stingl, J., Ba´^ca, V., ^Cech, P., Kovanda, J., Kovandova´, H., Mandys, V., Rejmontova´, J., Sosna, B, 2002 Morphology and some biomechanical properties of human liver and spleen Surgical and Radiologic Anatomy 24, 285–289.

Tamura, A., Omori, K., Miki, K., Lee, J.B., Yang, K.H., King, A.I., 2002 Mechanical characterization of porcine abdominal organs Stapp Car Crash Journal 46, 55–69.

Tr n, T.N., Nova´cˇek, V., Turquier, F., Klinge, U., Tolba, R.H., Bronson, D., Miesse, A., Whiffen, J, Staat, M Characterizing the mechanical properties of intestinal tissues Submitted for publication.

Venkatasubramanian, R.T., Grassl, E.D., Barocas, V.H., Lafontaine, D., Bischof, J.C.,

2006 Effects of freezing and cryopreservation on the mechanical properties of

0

0.1

0.2

0.3

0.4

λ

2)

Decomposed Spleen Decomposed Liver

Fig 6 Representative curves for the decomposed spleen and the liver tissues.

0

0.05

0.1

0.15

0.2

Hoop stretch

Elastin trypsin−digested Fresh

Collagen formic−acid−digested

Fig 5 Role of elastin and collagen for stress–stretch curve of arteries ( Roach and

Burton, 1957 ).

a nonlinear algorithm, such as a subspace trust region method that is based on. .. Biomechanical response of the pediatric abdomen, part 1: development of an experimental model and quantiﬁcation of structural response to dynamic belt loading Stapp Car Crash Journal 50, 1–26.

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