Journal of Biomechanics 45 (2012) 2382–2386 Contents lists available at SciVerse ScienceDirect Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com Influence of a freeze–thaw cycle on the stress–stretch curves of tissues of porcine abdominal organs N Huynh Nguy n a,b,f, M Tu n Du’o’ng a,b, T Ngoc Tr n a,c,d, P Tınh Pham a,c, _ _ O Grottke d, R Tolba e, M Staat a,n a Faculty of Medical Engineering and Technomathematics, Aachen University of Applied Sciences, (FH Aachen), Heinrich-Mußmann-Str 1, 52428 Jă ulich, Germany Hanoi University of Science and Technology, Hanoi, Vietnam c Hanoi Architectural University, Hanoi, Vietnam d Department of Anesthesiology, RWTH Aachen University Hospital, Aachen, Germany e Institute for Laboratory Animal Science, RWTH Aachen University Hospital, Aachen, Germany f Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany b a r t i c l e i n f o a b s t r a c t Article history: Accepted July 2012 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 & 2012 Elsevier Ltd All rights reserved Keywords: Liver Spleen Freeze–thaw process Decomposition Autolysis 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 n Corresponding author Tel.: ỵ49 241 6009 53120; fax: ỵ49 241 6009 53199 E-mail address: m.staat@fh-aachen.de (M Staat) 0021-9290/$ - see front matter & 2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.jbiomech.2012.07.008 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) However, 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 N Huynh Nguyˆe~n et al / Journal of Biomechanics 45 (2012) 2382–2386 injured abdominal organs in both frontal and side impact collisions (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 investigated 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 investigated 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 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 WEị ẳ CẵeQ ẳ CẵeE AE 1ị where A6 Â has 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 ẳ ẵE11 ,E22 ,E33 ,2E12 ,2E23 ,2E31 T and S ẳ ẵS11 ,S22 ,S33 ,S12 ,S23 ,S31 T , respectively In principal coordinates the exponent Q ẳ E T AE becomes Q ẳ A11 E211 ỵ A22 E222 ỵ A33 E233 ỵ 2A12 E11 E22 ỵ 2A23 E22 E33 ỵ 2A13 E11 E33 : 2ị The second Piola-Kirchhoff stress is calculated from the energy function as Sẳ @W ẳ 2CAEịeQ : @E Table Temperature cycles Time k Assumed (actual) decomposition Simulation of decomposition Period 6h some months 3–4 days 5–7 h $1 h $ 11 h $ 7–9 h Period (h) 420 12 Temperature (1C) ỵ20 to ỵ 25 18 to 25 ỵ20 to ỵ 25 ỵ4 to ỵ þ20 to þ 25 Temperature (1C) þ21 À 21 þ21 þ4 þ18 to þ 25 þ4 þ19 to þ 23 For the frozen–thawed group, the organs were frozen at À 18 1C Here we consider the decomposition processes in a cycle of storage at room temperature (RT¼ 720 1C to þ 25 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 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 The strain energy function of Fung’s model in a general form is T 2383 ð3Þ In this section stress and strain are briefly introduced for tension tests of isotropic incompressible materials The tension test generates uniaxial Cauchy stress r and deformation gradient F as 0 s 0 l 0 C B B C À1=2 A, r ¼ @ 0 A, F ¼ @ l ð7Þ 0 0 lÀ1=2 where the stretch l ¼ 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 The symmetric constitutive matrix is obtained as Cẳ @S ẳ 2CAeQ ỵ 4CAEị ðAEÞeQ @E ð4Þ with the dyadic product An isotropic Fung model is used (Duong et al., 2012) with a symmetric A in the constitutive matrix C Hence, if A11 ¼A22 ¼ A33 and A12 ¼A13 ¼ A23 the exponent term (2) can now be expressed as Q ¼ A11 E211 ỵ E222 ỵ E233 ị ỵ 2A12 E11 E22 ỵ E11 E33 ỵ E22 E33 ị: 5ị The constitutive matrix (4) is positive definite if C 40; A11 A12 40 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 k0 ¼ @sdecomposed @l l¼1 @sfresh @l lẳ1 1 ẳ CA11 ịdecomposed CA11 ịfresh 6ị 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 a ¼ 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) 2.2 Preparation of specimens Twenty three-month-old pigs with a weight of 34 72.4 kg (mean SD) 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 postmortem 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 Each ex-vivo test was performed within h after organ retrieval: the specimens were kept wet with normal saline during the experiment Experimental results The ultimate tensile Cauchy stresses of the tissues in tension test, sult, were computed from the experimentally determined rupture forces according to sult ¼ F l A ult ð8Þ N Huynh Nguyˆe~n et al / Journal of Biomechanics 45 (2012) 2382–2386 2384 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 in Table 3.1 Spleen All tissue test curves have been fitted under the convexity constraints of the isotropic Fung model Table shows a significant difference between the ultimate stress of the fresh and the frozen–thawed splenic tissue, (p o0.05 ¼ a) The ultimate stretch (1.7386 70.1400) of the fresh tissue is much larger than the one of the frozen–thawed tissue (1.303270.0610), (p E140.05, b r10 À 7) Fig shows 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 shows 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 Tension test—ruptured fresh spleen specimen Table Mean values of ultimate Cauchy stresses and stretches Fresh Frozen–thawed sult (MPa) 0.5246 70.1734 0.2299 70.1113 Spleen Liver 0.8 lult 1.3032 0.0610 1.3330 0.0602 0.8 Experimental Fitted 0.7 0.6 0.7 Experimental Fitted 0.6 0.5 σ (N/mm2) σ (N/mm2) sult (MPa) 0.3231 70.0995 0.1982 70.1198 lult 1.73867 0.1400 1.33777 0.0685 0.4 0.3 0.5 0.4 0.3 0.2 0.2 0.1 0.1 0 1.2 1.4 1.6 1.8 1.2 1.4 λ 1.6 1.8 λ Fig Curve-fitting for the fresh (a) and the freeze–thaw (b) splenic tissues—tension tests (not all tests presented in the figures) Table Statistics of curve fitting process for spleen tissues Organ Fresh, n¼ 51 Frozen–thawed, n ¼56 Adjusted R2 (%) Material constants C (MPa) A11 A12 0.2403 0.1423 1.81667 0.8494 0.6136 0.0729 0.5125 0.001 0.5316 0.1309 0.4272 0.0797 98.62 0.94 98.91 0.91 N Huynh Nguyˆe~n et al / Journal of Biomechanics 45 (2012) 2382–2386 hypothesis because p¼ 0.200840.05 Similarly, there was no statistically significant difference in ultimate stretches (p¼ 0.798640.05, b ¼0.908) The mean material coefficients from fitting phase for the fresh and the frozen–thawed tissues are shown in Table The mean curves (parameters in Table 4) for the representative fresh and frozen–thawed liver tissue are plotted (Fig 4) No significant difference between the stress–stretch curves is observed Discussion The key findings are À Decomposition takes place by autolysis in the thawed organ À There was no decomposition in liver which contains no elastin À 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 À 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 2385 (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 contributed 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 (k0 ¼6.3) In contrast for the liver, this value is nearly unchanged (k0 ¼0.68) 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 0.7 0.35 Fresh Decomposed 0.6 Fresh Decomposed 0.3 0.25 0.4 σ [N/mm2] σ [N/mm2] 0.5 0.3 0.2 0.15 0.2 0.1 0.1 0.05 0 1.2 1.4 1.05 1.1 1.15 1.2 1.25 Fig Curve-fit representatives for fresh and frozen–thawed spleens Fresh, n¼ 49 Frozen–thawed, n¼39 1.35 Fig Representative stress–stretch curves for fresh and frozen–thawed liver Table Statistics of curve fitting process for liver tissues Organ 1.3 λ λ Adjusted R2 (%) Material constants C (MPa) A11 A12 0.7303 0.4489 0.5021 0.4135 0.5194 0.0462 0.5140 0.0074 0.3578 0.1185 0.2333 0.1656 99.00 0.76 98.28 2.20 N Huynh Nguyˆe~n et al / Journal of Biomechanics 45 (2012) 2382–2386 2386 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 0.2 Elastin trypsin−digested Fresh Collagen formic−acid−digested Tension (N/mm) 0.15 0.1 0.05 Conflict of interest None 1.2 1.4 1.6 Hoop stretch 1.8 Fig Role of elastin and collagen for stress–stretch curve of arteries (Roach and Burton, 1957) σ (N/mm2) 0.4 Decomposed Spleen Decomposed Liver 0.2 0.1 1.1 1.2 λ The authors thank TRW Automotive GmbH, Alfdorf, Germany, for support of the project and the permission to use the data presented in this paper References 0.3 Acknowledgments 1.3 1.4 Fig Representative curves for the decomposed spleen and the liver tissues warmer periods after thawing This decomposition shifts the composition of non-degenerated proteins in spleen in the direction of composition 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 or days after euthanasia and the human organs have been kept ‘‘fresh’’ for up to 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 decomposition 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 ỵ4 1C to ỵ8 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 decomposition 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., Brunon, A., Bruye re-Garnier, K., Coret, M., 2010 Mechanical characterization of liver capsule through uniaxial quasi-static tensile tests until failure Journal of Biomechanics 43 (11), 2221–2227 Burton, A.C., 1951 On the physical equilibrium of small blood vessels American Journal of Physiology 164 (2), 319–329 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-20128th ă Graz, European Solid Mechanics Conference Verlag d Technischen Universitat Graz Franklyn, M., Fitzharris, M., Fildes, B., Frampton R., Morris, A., Yang, K.H., 2002 Liver 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affects the mechanical properties of bovine liver Biomedical Sciences Instrumentation 45, 24–29 ^ Stingl, J., Ba´c^ a, 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 arteries Annals of Biomedical Engineering 34 (5), 823–832 ... 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. .. mechanical behaviour of biological soft tissues: observations on kidney tissues Journal of the Mechanical Behavior of Medical Materials 3, 630–635 Roach, M.R., Burton, A. C., 1957 The reason for the. .. 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