Clinical Anatomy 00:00–00 (2015)
ORIGINAL COMMUNICATION
Elastic Properties of Thiel-Embalmed Human Ankle Tendon and Ligament XIAOCHUN LIAO,1 SANDY KEMP,1 GEORGE CORNER,2 ROOS EISMA,3 ZHIHONG HUANG1* 1
AND
School of Engineering, Physics and Mathematics, University of Dundee, Dundee, DD1 4HN, United Kingdom Department of Medical Physics, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom 3 Centre for Anatomy and Human Identification, College of Art, Science and Engineering, University of Dundee, Dundee, DD1 4HN, United Kingdom
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Thiel embalming is recommended as an alternative to formalin-based embalming because it preserves tissue elasticity, color, and flexibility in the long term, with low infection and toxicity risk. The degree to which Thiel embalming preserves elasticity has so far been assessed mainly by subjective scoring, with little quantitative verification. The aim of this study is to quantify the effect of Thiel embalming on the elastic properties of human ankle tendons and ligament. Biomechanical tensile tests were carried out on six Thiel-embalmed samples each of the peroneus longus, peroneus brevis, and calcaneal tendons, and the calcaneofibular ligament, with strain rates of 0.25%s21, 2%s21, and 8%s21. The stress2strain relationship was calculated from the force-extension response with cross-sectional area and gauge length. Young’s modulus was determined from the stress2strain curve. The results showed that the tendon and ligament elasticity were lower after Thiel embalming than the literature values for fresh nonembalmed tendons and ligament. The biomechanical tensile test showed that the measured elasticity of Thiel-embalmed tendons and ligaments increased with the strain rate. The Thiel embalming method is useful for preserving human ankle tendons and ligaments for anatomy and surgery teaching and research, but users need to be aware of its softening effects. The method retains the mechanical strain rate effect on tendons and ligament. Clin. Anat. 00:000–000, 2015. VC 2015 Wiley Periodicals, Inc. Key words: Thiel embalming; ankle; tendon; ligament; tensile test; elasticity; strain rate
INTRODUCTION Cadavers are widely accepted as the most suitable models in anatomy and surgery for practical teaching of operations and for cutting-edge research into new surgical techniques. Fresh human cadavers are limited in supply and can only be used for a short time. Cadavers can be preserved by freezing, but this only temporarily suspends the process of decay and once the body is defrosted there is a risk of infection. Formalin-based preservation maintains tissue structure and reduces the infection risk. However, formaldehyde stiffens tissues by increasing protein crosslinking. It is also toxic, and respiratory irritation and discomfort are reported by anatomy staff and stu-
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dents during dissection of formalin-fixed cadavers. Owing to these health hazards and working limitations, there is increasing interest in new soft-fix
Contract grant sponsor: SUPA (Scottish Universities Physics Alliance), Health Science Solutions Ltd. *Correspondence to: Zhihong Huang, School of Engineering, Physics and Mathematics, University of Dundee, Dundee, DD1 4HN, UK. E-mail: [email protected] Received 4 February 2014; Revised 24 December 2014; Accepted 6 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ca.22512
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TABLE 1. Cadaver Details Cadaver
Sex
Age at death
Time between death and embalming
Time between embalming and tensile test
A B C
Female Male Female
92 years 68 years 94 years
2 Days 2 Days 6 Days
34 months 31 months 31 months
embalming techniques with the additional benefits of extremely low health risks and maintaining cadavers in a life-like condition for long periods of time (Anderson, 2006; Holzle et al., 2012). In Scotland, surgical procedures have been permitted on human cadavers since 2006 by the Human Tissue (Scotland) Act. This has also increased interest in soft-fix preservation methods that can produce cadavers more suitable for clinical and surgical procedures. Thiel embalming is a soft-fix embalming technique € r Anatomie, developed by Walter Thiel at the Institut fu Graz, Austria (Thiel, 1992). This method maintains tissue softness, color, and flexibility because the levels of formaldehyde in the Thiel embalming fluid are very low (Benkhadra et al., 2011; Jaung et al., 2011; Wilke et al., 2011; Eisma et al., 2013). Thiel-embalmed cadavers can be kept for the three years permitted under the Human Tissue (Scotland) Act 2006 at ambient temperature without significant deterioration. The embalming process comprises an initial perfusion followed by submersion in fluid for at least three months. After that, the bodies can stay submerged or be stored in plastic bags; no refrigeration is necessary. The embalming fluids are based on water, glycol and various salts. More harmful components such as formaldehyde, chlorocresol, and morpholine are also present, but only in very low concentrations (1.44%, 0.35% and 1.15% respectively for arterial infusion), and this, combined with the good biocidal properties of the fluids, makes the cadavers safe to work with. Such cadavers have great potential for training, research and development of new products and techniques (Feigl et al., 2006). However, it is important to understand the limitations of the model and to assess in which aspects and to what extent it is realistic. The mechanical properties of tissues are particularly important for task fidelity during training, and for the interpretation of experimental results prior to clinical trials. A better understanding of these properties will allow judgments about the procedures for which Thiel-embalmed cadavers are suitable to be better informed. Tendons and ligaments are connective tissues with similar compositions, consisting mainly of type I collagen and elastin. Their mechanical properties are generally described as anisotropic, nonlinear and viscoelastic. However, when exercised longitudinally at medium or large strain, they usually exhibit a linear force response against extension. Within this linear elasticity response, the nominal stress is proportional to the nominal strain; the gradient between stress and strain is known as the Young’s modulus or elastic modulus. There has been little research into the effect of Thiel embalming on the mechanical properties of tis-
sues. There have been qualitative observations (Giger et al., 2008; Eisma et al., 2011; Holzle et al., 2012; Eisma et al., 2013), and sonoelastography has demonstrated realistic tissue behavior during ultrasoundguided anesthetic injections (Munirama et al., 2012). A few quantitative studies have been reported, covering a limited range of tissues, structures and mechanical parameters. A study of Thiel-embalmed human flexor digitorum profundus tendons and rat tail tendons showed lower failure stress in Thiel-embalmed than freshly-frozen specimens, along with altered failure characteristics (Fessel et al., 2011). A study on the mechanical properties of bone after Thiel embalming revealed a lower Young’s modulus but no difference in the force needed to break the sample; the Thiel-embalmed specimens also showed more plastic energy absorption (Unger et al., 2010). The mechanical behavior of a spinal segment (including ligaments, joint capsules and intervertebral discs) after Thiel or formalin embalming was compared with that of freshfrozen specimens (Wilke et al., 2011); the Thielembalmed specimens were 22245% more flexible, while the formalin-embalmed ones were approximately five times stiffer. Unlike formalin embalming, Thiel-embalming maintained the nonlinear load–deformation characteristics. The aim of this study was to quantify the effects of Thiel embalming on the elastic properties of human ankle tendons and ligament. The tissues used were the tendons of the peroneus longus and peroneus brevis, the calcaneal tendon, and the calcaneofibular ligament. The results verify that Thiel embalming preserves human tissue elasticity, and they can be compared with values from studies of other preservation methods.
MATERIALS AND METHODS Thiel Specimen Details and Preparation Specimens were collected from Thiel-embalmed cadavers at the local anatomy school. The work was conducted in accordance with Scottish statutory requirements and with the Standard Operating Procedures of the local anatomy school. Samples of peroneus longus, peroneus brevis, and calcaneal tendons and calcaneofibular ligament were collected from both the left and right ankles of three cadavers (Table 1), providing 24 samples in total. The left femoral artery had been used for embalming all three cadavers, which meant that the left leg received more arterial embalming fluid than the right because delivery to the left leg via a small arterial cannula was more localized. All cadavers had previously been used
Elastic Properties of Thieled Tendon and Ligament
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Fig. 1. Thiel samples: (a) peroneus longus, (b) peroneus brevis, (c) calcaneal tendon, and (d) calcaneofibular ligament. The numbered scale is in cm.
for a range of surgical and other procedures, though the ankles were intact when the specimens were dissected and removed. Examples of each type of specimen are shown in Figure 1.
EXPERIMENTAL PROCEDURES A uniaxial extensometer (H5KS, Tinius Olsen, Surrey, UK) with a 5 KN load cell with extension precision of 1 mm was used for the tensile tests. Each sample was mounted with a custom clamp at each end (Fig. 2). To reduce slippage and damage to the sample, a pad of tissue paper was wrapped around each end and pressure was applied to the clamps to expel Thiel fluids. During the tests, moistening fluid was applied to the central section of the samples to keep them
from drying out; the moistening fluid was a dilute version of Thiel embalming fluid used in the local anatomy school (Eisma et al., 2013). The clamping position was adjusted for each sample to ensure a uniform sample profile between the clamps. The load of 5 N was applied before the original sample length was measured. The original crosssectional area was calculated after the sample’s width and thickness had been measured at the central position using digital calipers with a resolution of 0.01 mm. To ensure that the tissue’s true mechanical response was exhibited, no preconditioning was done (Cheng et al., 2009; Quinn and Winkelstein, 2011). A tensile test was repeated at three strain rates for each sample—0.25%s21, 2%s21 and 8%s21—chosen to represent slow, normal and fast loading conditions for human connective tissues. These tests were conducted in the sequence from 0.25%s21 through 2%s21 to 8%s21, with a strain limit of 0.15 to preclude fractures. The tensile tests for each sample and for each strain rate were repeated three or four times to guarantee reproducibility; between successive tests, the sample was placed horizontally for one minute to allow it to recover its shape and mechanical response. Throughout the test, force was recorded against extension in real time by the extensometer.
Data Processing
Fig. 2. (a) Custom-designed clamps and (b) calcaneofibular ligament mounted between the custom clamps with each end wrapped in tissue paper.
Force (F ) data were normalized as nominal stress (r) with cross-section area (A). Extension data (DL) were normalized as nominal strain (‹) with the original length as gauge length (L). The nominal stress vs nominal strain curve was then fitted to the data and
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Fig. 3. (a) Stress2strain response from tensile tests with calcaneofibular ligament from cadaver C left ankle with strain rate of 2%s21 and (b) stress2strain response from tensile test with calcaneal tendon from cadaver A left ankle with three strain rates of 0.25%s21, 2%s21, and 8%s21.
the tangent to this curve was the specimen’s elasticity, Young’s modulus (E), calculated using the equations below (Fig. 3a): F A
(1)
DL L
(2)
r5 ‹5
Dr (3) De Within the toe region the stress-strain relationship shows features of nonlinearity, mainly because some individual fibers have not been fully stretched. Beyond the nonlinear region, most fibers display elastic behavior, with a linear stress-strain relationship and a constant Young’s modulus. A typical stress vs. strain response with the three different strain rates of 0.25%s21, 2%s21, and 8%s21 is illustrated in Figure 3b. There is a greater stress response vs strain when the strain rate is higher. To investigate whether the measured elasticity differed significantly among (a) cadavers, (b) samples from different positions or (c) testing strain rates, two-factor ANOVA was conducted without replication. The measured elasticity values were segmented into subgroups of cadaver (A, B, and C), subgroups of position (left and right), and subgroups of strain rate (0.25%s21, 2%s21, and 8%s21), according to each statistical hypothesis. E5
RESULTS The mean and standard deviation were calculated for each sample’s Young’s modulus under each strain
rate used (Fig. 4). The means and standard deviations of Young’s modulus averaged across the cadaver group are shown in Table 2. In view of the small standard deviation over the mean elasticity for each sample, the mean value was used for the two-factor ANOVA test; a P-value of 0.05 was taken as the criterion of significance. The mean and standard deviation of each subgroup were tabulated with the corresponding F value and P-value (Table 3). The three cadavers did not differ significantly. Samples from the left ankle were generally stiffer than those from the corresponding right ankle, with P-values as low as 3.26E-04. A generally consistent correlation between the tensile strain rate and the measured Young’s modulus was confirmed with F 5 33.14, far above the critical F value of 3.20 for the 0.05 significance level.
DISCUSSION The purpose of this research was to quantify the effect of Thiel embalming on the elastic properties of human ankle tendons and ligament. Formalin embalming results in cross-linking of proteins, which generally makes the embalmed tissue stiffer, discolored, and inflexible. In contrast, Thiel-embalmed tissues are described as life-like with regard to elasticity, color and flexibility, with further advantages of low infection and toxicity risk (Eisma et al., 2011). There has been no previous research on the biomechanical properties of Thiel-embalmed specimens from the human ankle. In the recent literature we found only Fessel’s biomechanical experiments on
Elastic Properties of Thieled Tendon and Ligament
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Fig. 4. Young’s modulus measurements of (a) peroneus longus, (b) peroneus brevis, (c) calcaneal tendons, and (d) calcaneofibular ligament with strain rates of 0.25%s21, 2%s21, and 8%s21.
Thiel-embalmed human flexor digitorum profundus tendons (Fessel et al., 2011). The highest Young’s modulus found in this study (497.20 6 10.68 MPa, mean 6 standard deviation), of the Thiel-embalmed peroneus longusis, was considerably lower than the 6896159 MPa for fresh nonembalmed peroneus longus (Arthrex, 2007). However, Arthrex did not state the number of samples or the age or sex of the donors. The elasticity of 430 6 151 MPa from Fessel’s biomechanical testing of Thiel-embalmed human forearm tendons is comparable with our measurements (Fessel et al., 2011).
A single value reported for the Young’s modulus of nonembalmed peroneus brevis tendon, 149.7 MPa (Carmont and Maffulli, 2007), is between the lower (119.95 6 6.46 MPa) and upper (407.60 6 19.56 MPa) bounds of our measurements. The large difference in literature values for the peroneus longus and brevis is surprising given the similarities in anatomical position and physiological function, suggesting that sample groups or methodology are not comparable between these studies. The Young’s modulus of the calcaneal tendon varies widely, 559.2 6 257.3 MPa, among fresh human cadavers (Louis-Ugbo et al., 2004). Their average was
8%s21 356.93 6 49.54 267.93 6 70.38 2%s 337.55 6 106.40 239.57 6 41.72 0.25%s21 298.49 6 71.70 169.65 6 81.59 8%s 272.82 6 86.61 237.02 6 84.29
Calcaneofibular ligament (Mean 6 STD)
2%s 215.21 6 108.87 190.52 6 74.25
21
0.25%s 106.73 6 66.15 62.23 6 18.96
21
Calcaneal tendon (Mean 6 STD)
Strain rate Left Right
21
Young’s Modulus (MPa)
8%s21 268.67 6 27.58 261.30 6 23.67 2%s21 343.57 6 62.68 303.58 6 66.61 0.25%s21 239.78 6 66.87 203.65 6 113.80 8%s21 346.91 6 84.50 306.32 6 171.25 2%s21 339.96 6 60.45 298.83 6 163.06 0.25%s21 237.63 6 95.64 174.18 6 52.19
Proneous longus (Mean 6 STD) Young’s Modulus (MPa)
Strain rate Left Right
Proneous brevis (Mean 6 STD)
Liao et al. TABLE 2. Young’s Modulus Measured Through Repeated Tensile Tests with Statistical Mean (Mean) and Standard Deviation (STD) Calculated for All 24 Samples with Strain Rates of 0.25%s21, 2%s21, and 8%s21
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considerably higher than the highest Young’s modulus found in our study (362.63 6 21.74 Mpa). However, some of their measurements (335, 279, 203 MPa) are consistent with ours (331.30, 266.03, 198.95 MPa). The Young’s modulus of the Thiel-embalmed calcaneofibular ligament was 278.35 6 89.14 MPa for all three strain rates (Table 2). This is significantly lower than the Young’s modulus of 512.0 6 333.5 MPa for nonembalmed calcaneofibular ligament (Siegler et al., 1988), although the lower parts of the ranges overlapped. The large standard deviation in the measured Young’s modulus could be attributed to the range of donor ages (from 95 to 33 years) in Siegler’s research. Individuals differ significantly in all aspects of anatomy, and any training or product development needs to take this into consideration. The elasticity of the Thiel-embalmed samples in our study were compared quantitatively with nonembalmed samples from the literature at comparable strain rates (Table 4). The elasticity of the Thiel-embalmed specimens are within the range of those for non-embalmed specimens in the literature, which confirms the qualitative evaluations of Thiel-embalmed cadavers. However, the average elasticity values from the Thiel embalming method in our study are lower than those in the literature from non-embalmed specimens; this difference might be expected to affect the results of quantitative investigations. Therefore, it could be better to use fresh biological tissues in critical situations when mechanical behavior and numerical limits are to be tested, for example in product development. During the embalming process, embalming fluid was perfused into the left femoral artery. This means that the left lower limb received fluid more directly than the right and was generally better perfused. This embalming fluid has higher concentrations of alcohol, morpholine, and formalin than the tank fluid in which the body was subsequently submerged, and these components contribute to the preservation of tissue firmness. All the samples from the left ankles had a higher Young’s modulus than those from the right ankles, as confirmed statistically (Table 3), except the peroneus longus from Cadaver B and the peroneus brevis from Cadaver C. This finding supports the view that the initial perfusion of the cadavers is important in preserving tissue elasticity. There were no apparent differences in tendon and ligament elasticity values between Cadavers A and C, which were of the same sex and similar age at death and the same duration of Thiel embalming, though the times between death and embalming differed (2 days and 6 days, respectively). This suggests that a prolonged time between death and embalming has little effect on tendon and ligament elasticity. This hypothesis needs to be tested in future experiments with more Thiel-embalmed samples. Cadavers are typically taken into use 6212 months after embalming. Then, for the following 12218 months, they are used for surgical procedures and alternatively stored in bags or tanks, and
Elastic Properties of Thieled Tendon and Ligament
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TABLE 3. The Mean and Standard Deviation of the Measured Young’s Modulus and ANOVA (Two-Factor Without Replication) Test Results Within Different Cadavers, Different Anatomical Dissection Positions, and Different Tensile Testing Strain Rates Mean 6 STD (MPa) Cadaver
ANOVA
A
B
C
F
P-value
F crit-0.05
246.82 6 103.20
265.00 6 126.07
248.06 6 75.67
0.33
0.72
3.20
Mean 6 STD (MPa) Position
ANOVA
Left
Right
F
280.35 6 95.92
226.23 6 103.15
15.88
P-value
F crit-0.05
3.26E-04
4.12
Mean 6 STD (MPa) Strain rate
0.25%/s
2%s
186.54 6 96.29
ANOVA
21
8%s
283.60 6 96.65
21
289.74 6 82.92
finally they are made available for dissection studies. All three cadavers used in this study were at the end stages of use and close to the 3-year limit for which cadavers can legally be retained under the Human Tissue (Scotland) Act 2006. Our experience suggests that cadavers gradually lose fluid over time, and those at different stages of use have different properties. Further work is needed to quantify how this affects the mechanical properties of tissues. The tensile tests were implemented with three strain rates, 0.25%s21, 2%s21, and 8%s21, 2%s21 representing the physiological strain rate while 0.25%s21 and 8%s21 represented slow and fast load conditions, respectively. For all the Young’s modulus measurements there was a general increase with increased strain rate, and this was especially clear for the calcaneal tendon, as previously reported (Wren et al., 2001). The other three types of samples showed minor discrepancies in the tendency of Young’s modulus to increase with strain rate. This is probably due to sample degradation caused by the accumulation of damage during the tensile test, especially with the strain rate of 8%s21. The calcaneal tendon usually contains larger collagen fibers within its large size than the other sample types (Jarvinen et al., 2004), which possibly helped it to withstand the accumulation of mechanical damage.
F
P-value
F crit-0.05
33.14
1.22E-09
3.20
This study has a number of limitations concerning the Thiel-embalmed human specimens and damage accumulation. First, there were few Thiel-embalmed specimens with uncontrollable differences in sex, age, and condition at death, and in embalming time period, which could affect tissue elasticity. Secondly, damage within the specimens accumulated during the repeated tensile tests, the larger strain rate affecting the accuracy of measurement. It would be advisable to set a lower maximum strain for each sample, taking into consideration the dimensions and condition of preservation of the sample in future tensile tests with different strain rates. Individual gait problems can also induce bias in the elasticity of ankle tendons and ligaments. It will therefore be important in our future studies to conduct microscopic and mechanical analyses of other structures within the ankle region to enhance the reliability of the conclusions.
CONCLUSIONS In this study we found that Thiel embalming of human cadavers tends to soften the ankle tendons and ligament extent little, although our measured elasticity values for Thiel-embalmed samples overlap
TABLE 4. The Measurements of Young’s Modulus of Thiel-Embalmed Samples in Our Study and Nonembalmed Samples From the Literature at Comparable Strain Rates Thiel-embalmed
Nonembalmed
Young’s modulus (MPa)
Strain rate
Young’s modulus (MPa)
Strain rate
Peroneous Longus Peroneous brevis
319.39 6 112.27 221.72 6 85.79
2%s21 0.25%s21
689 6 159 149.7 6 56.3
N.A. N.A.
Calcaneal tendon
202.86 6 84.44
2%s21
559.2 6 257.3
2.67%s21
Calcaneofibular ligament
234.07 6 98.48
0.25%s21
512.0 6 333.5
0.1320.45%s21
Sample
Reference Arthrex, 2007 Carmont and Maffulli, 2007 Louis-Ugbo et al., 2004 Siegler et al., 1988
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somewhat with the ranges for fresh nonembalmed tissue found in the literature. This suggests that Thiel-embalmed tissues are similar enough to fresh tissues to be suitable for many procedures and applications, but users need to be aware of the softening effects of the Thiel embalming method, especially in quantitative biomechanical studies. Thiel-embalmed tendons and ligament have strain rate effects similar to those of the fresh tissues, with the Young’s modulus increasing with the strain rate. This supports subjective observations of the realistic handling of Thielembalmed cadavers.
ACKNOWLEDGMENT The authors wish to thank Dr Clare Lamb for her assistance in dissecting the Thiel tendon and ligament samples, and Miss Amanda Hunter for her assistance in revision. Finally, they would like to express our gratitude to the people who choose to donate their bodies to science.
REFERENCES Anderson SD. 2006. Practical light embalming technique for use in the surgical fresh tissue dissection laboratory. Clin Anat 19:8– 11. Arthrex. 2007. Peroneus Longus Tendon as an ACL Reconstruction Allograft. Arthrex Res Dev. Benkhadra M, Bouchot A, Gerard J, Genelot D, Trouilloud P, Martin L, Girard C, Danino A, Anderhuber F, Feigl G. 2011. Flexibility of Thiel’s embalmed cadavers: The explanation is probably in the muscles. Surg Radiol Anat 33:365–368. Carmont MR, Maffulli N. 2007. Less invasive Achilles tendon reconstruction. Bmc Musculoskel Dis 8. Cheng S, Clarke EC, Bilston LE. 2009. The effects of preconditioning strain on measured tissue properties. J Biomech 42:1360–1362. Eisma R, Lamb C, Soames RW. 2013. From formalin to thiel embalming: What changes? One anatomy department’s experiences. Clin Anat 26:564–571.
Eisma R, Mahendran S, Majumdar S, Smith D, Soames RW. 2011. A comparison of Thiel and formalin embalmed cadavers for thyroid surgery training. Surg-J R Coll Surg E 9:142–146. Feigl G, Fuchs A, Gries M, Hogan QH, Weninger B, Rosmarin W. 2006. A supraomohyoidal plexus block designed to avoid complications. Surg Radiol Anat 28:403–408. Fessel G, Frey K, Schweizer A, Calcagni M, Ullrich O, Snedeker JG. 2011. Suitability of Thiel embalmed tendons for biomechanical investigation. Ann Anat 193:237–241. Giger U, Fresard I, Hafliger A, Bergmann M, Krahenbuhl L. 2008. Laparoscopic training on Thiel human cadavers: a model to teach advanced laparoscopic procedures. Surg Endosc 22:901– 906. Holzle F, Franz EP, Lehmbrock J, Weihe S, Teistra C, Deppe H, Wolff KD. 2012. Thiel embalming technique: A valuable method for teaching oral surgery and implantology. Clin Implant Dent R 14: 121–126. Jarvinen TA, Jarvinen TL, Kannus P, Jozsa L, Jarvinen M. 2004. Collagen fibres of the spontaneously ruptured human tendons display decreased thickness and crimp angle. J Orthop Res 22:1303– 1309. Jaung R, Cook P, Blyth P. 2011. A comparison of embalming fluids for use in surgical workshops. Clin Anat 24:155–161. Louis-Ugbo J, Leeson B, Hutton WC. 2004. Tensile properties of fresh human calcaneal (Achilles) tendons. Clin Anat 17:30–35. Munirama S, Satapathy AR, Schwab A, Eisma R, Corner GA, Cochran S, Soames R, McLeod GA. 2012. Translation of sonoelastography from Thiel cadaver to patients for peripheral nerve blocks. Anaesthesia 67:721–728. Quinn KP, Winkelstein BA. 2011. Preconditioning is correlated with altered collagen fiber alignment in ligament. J Biomech Eng-T Asme 133. Siegler S, Block J, Schneck CD. 1988. The mechanical characteristics of the collateral ligaments of the human ankle joint. Foot Ankle 8:234–242. Thiel W. 1992. The preservation of the whole corpse with natural color. Ann Anat 174:185–195. Unger S, Blauth M, Schmoelz W. 2010. Effects of three different preservation methods on the mechanical properties of human and bovine cortical bone. Bone 47:1048–1053. € ussler K, Reinehr M, Bo € ckers TM. 2011. Wilke H-J, Werner K, Ha Thiel-fixation preserves the non-linear load-deformation characteristic of spinal motion segments, but increases their flexibility. J Mech Behav Biomed Mater 4:2133–2137. GS, Carter DR. 2001. Mechanical Wren TAL, Yerby SA, Beaupre properties of the human achilles tendon. Clin Biomech 16:245– 251.
ORIGINAL COMMUNICATION
Elastic Properties of Thiel-Embalmed Human Ankle Tendon and Ligament XIAOCHUN LIAO,1 SANDY KEMP,1 GEORGE CORNER,2 ROOS EISMA,3 ZHIHONG HUANG1* 1
AND
School of Engineering, Physics and Mathematics, University of Dundee, Dundee, DD1 4HN, United Kingdom Department of Medical Physics, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom 3 Centre for Anatomy and Human Identification, College of Art, Science and Engineering, University of Dundee, Dundee, DD1 4HN, United Kingdom
2
Thiel embalming is recommended as an alternative to formalin-based embalming because it preserves tissue elasticity, color, and flexibility in the long term, with low infection and toxicity risk. The degree to which Thiel embalming preserves elasticity has so far been assessed mainly by subjective scoring, with little quantitative verification. The aim of this study is to quantify the effect of Thiel embalming on the elastic properties of human ankle tendons and ligament. Biomechanical tensile tests were carried out on six Thiel-embalmed samples each of the peroneus longus, peroneus brevis, and calcaneal tendons, and the calcaneofibular ligament, with strain rates of 0.25%s21, 2%s21, and 8%s21. The stress2strain relationship was calculated from the force-extension response with cross-sectional area and gauge length. Young’s modulus was determined from the stress2strain curve. The results showed that the tendon and ligament elasticity were lower after Thiel embalming than the literature values for fresh nonembalmed tendons and ligament. The biomechanical tensile test showed that the measured elasticity of Thiel-embalmed tendons and ligaments increased with the strain rate. The Thiel embalming method is useful for preserving human ankle tendons and ligaments for anatomy and surgery teaching and research, but users need to be aware of its softening effects. The method retains the mechanical strain rate effect on tendons and ligament. Clin. Anat. 00:000–000, 2015. VC 2015 Wiley Periodicals, Inc. Key words: Thiel embalming; ankle; tendon; ligament; tensile test; elasticity; strain rate
INTRODUCTION Cadavers are widely accepted as the most suitable models in anatomy and surgery for practical teaching of operations and for cutting-edge research into new surgical techniques. Fresh human cadavers are limited in supply and can only be used for a short time. Cadavers can be preserved by freezing, but this only temporarily suspends the process of decay and once the body is defrosted there is a risk of infection. Formalin-based preservation maintains tissue structure and reduces the infection risk. However, formaldehyde stiffens tissues by increasing protein crosslinking. It is also toxic, and respiratory irritation and discomfort are reported by anatomy staff and stu-
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2015 Wiley Periodicals, Inc.
dents during dissection of formalin-fixed cadavers. Owing to these health hazards and working limitations, there is increasing interest in new soft-fix
Contract grant sponsor: SUPA (Scottish Universities Physics Alliance), Health Science Solutions Ltd. *Correspondence to: Zhihong Huang, School of Engineering, Physics and Mathematics, University of Dundee, Dundee, DD1 4HN, UK. E-mail: [email protected] Received 4 February 2014; Revised 24 December 2014; Accepted 6 January 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/ca.22512
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TABLE 1. Cadaver Details Cadaver
Sex
Age at death
Time between death and embalming
Time between embalming and tensile test
A B C
Female Male Female
92 years 68 years 94 years
2 Days 2 Days 6 Days
34 months 31 months 31 months
embalming techniques with the additional benefits of extremely low health risks and maintaining cadavers in a life-like condition for long periods of time (Anderson, 2006; Holzle et al., 2012). In Scotland, surgical procedures have been permitted on human cadavers since 2006 by the Human Tissue (Scotland) Act. This has also increased interest in soft-fix preservation methods that can produce cadavers more suitable for clinical and surgical procedures. Thiel embalming is a soft-fix embalming technique € r Anatomie, developed by Walter Thiel at the Institut fu Graz, Austria (Thiel, 1992). This method maintains tissue softness, color, and flexibility because the levels of formaldehyde in the Thiel embalming fluid are very low (Benkhadra et al., 2011; Jaung et al., 2011; Wilke et al., 2011; Eisma et al., 2013). Thiel-embalmed cadavers can be kept for the three years permitted under the Human Tissue (Scotland) Act 2006 at ambient temperature without significant deterioration. The embalming process comprises an initial perfusion followed by submersion in fluid for at least three months. After that, the bodies can stay submerged or be stored in plastic bags; no refrigeration is necessary. The embalming fluids are based on water, glycol and various salts. More harmful components such as formaldehyde, chlorocresol, and morpholine are also present, but only in very low concentrations (1.44%, 0.35% and 1.15% respectively for arterial infusion), and this, combined with the good biocidal properties of the fluids, makes the cadavers safe to work with. Such cadavers have great potential for training, research and development of new products and techniques (Feigl et al., 2006). However, it is important to understand the limitations of the model and to assess in which aspects and to what extent it is realistic. The mechanical properties of tissues are particularly important for task fidelity during training, and for the interpretation of experimental results prior to clinical trials. A better understanding of these properties will allow judgments about the procedures for which Thiel-embalmed cadavers are suitable to be better informed. Tendons and ligaments are connective tissues with similar compositions, consisting mainly of type I collagen and elastin. Their mechanical properties are generally described as anisotropic, nonlinear and viscoelastic. However, when exercised longitudinally at medium or large strain, they usually exhibit a linear force response against extension. Within this linear elasticity response, the nominal stress is proportional to the nominal strain; the gradient between stress and strain is known as the Young’s modulus or elastic modulus. There has been little research into the effect of Thiel embalming on the mechanical properties of tis-
sues. There have been qualitative observations (Giger et al., 2008; Eisma et al., 2011; Holzle et al., 2012; Eisma et al., 2013), and sonoelastography has demonstrated realistic tissue behavior during ultrasoundguided anesthetic injections (Munirama et al., 2012). A few quantitative studies have been reported, covering a limited range of tissues, structures and mechanical parameters. A study of Thiel-embalmed human flexor digitorum profundus tendons and rat tail tendons showed lower failure stress in Thiel-embalmed than freshly-frozen specimens, along with altered failure characteristics (Fessel et al., 2011). A study on the mechanical properties of bone after Thiel embalming revealed a lower Young’s modulus but no difference in the force needed to break the sample; the Thiel-embalmed specimens also showed more plastic energy absorption (Unger et al., 2010). The mechanical behavior of a spinal segment (including ligaments, joint capsules and intervertebral discs) after Thiel or formalin embalming was compared with that of freshfrozen specimens (Wilke et al., 2011); the Thielembalmed specimens were 22245% more flexible, while the formalin-embalmed ones were approximately five times stiffer. Unlike formalin embalming, Thiel-embalming maintained the nonlinear load–deformation characteristics. The aim of this study was to quantify the effects of Thiel embalming on the elastic properties of human ankle tendons and ligament. The tissues used were the tendons of the peroneus longus and peroneus brevis, the calcaneal tendon, and the calcaneofibular ligament. The results verify that Thiel embalming preserves human tissue elasticity, and they can be compared with values from studies of other preservation methods.
MATERIALS AND METHODS Thiel Specimen Details and Preparation Specimens were collected from Thiel-embalmed cadavers at the local anatomy school. The work was conducted in accordance with Scottish statutory requirements and with the Standard Operating Procedures of the local anatomy school. Samples of peroneus longus, peroneus brevis, and calcaneal tendons and calcaneofibular ligament were collected from both the left and right ankles of three cadavers (Table 1), providing 24 samples in total. The left femoral artery had been used for embalming all three cadavers, which meant that the left leg received more arterial embalming fluid than the right because delivery to the left leg via a small arterial cannula was more localized. All cadavers had previously been used
Elastic Properties of Thieled Tendon and Ligament
3
Fig. 1. Thiel samples: (a) peroneus longus, (b) peroneus brevis, (c) calcaneal tendon, and (d) calcaneofibular ligament. The numbered scale is in cm.
for a range of surgical and other procedures, though the ankles were intact when the specimens were dissected and removed. Examples of each type of specimen are shown in Figure 1.
EXPERIMENTAL PROCEDURES A uniaxial extensometer (H5KS, Tinius Olsen, Surrey, UK) with a 5 KN load cell with extension precision of 1 mm was used for the tensile tests. Each sample was mounted with a custom clamp at each end (Fig. 2). To reduce slippage and damage to the sample, a pad of tissue paper was wrapped around each end and pressure was applied to the clamps to expel Thiel fluids. During the tests, moistening fluid was applied to the central section of the samples to keep them
from drying out; the moistening fluid was a dilute version of Thiel embalming fluid used in the local anatomy school (Eisma et al., 2013). The clamping position was adjusted for each sample to ensure a uniform sample profile between the clamps. The load of 5 N was applied before the original sample length was measured. The original crosssectional area was calculated after the sample’s width and thickness had been measured at the central position using digital calipers with a resolution of 0.01 mm. To ensure that the tissue’s true mechanical response was exhibited, no preconditioning was done (Cheng et al., 2009; Quinn and Winkelstein, 2011). A tensile test was repeated at three strain rates for each sample—0.25%s21, 2%s21 and 8%s21—chosen to represent slow, normal and fast loading conditions for human connective tissues. These tests were conducted in the sequence from 0.25%s21 through 2%s21 to 8%s21, with a strain limit of 0.15 to preclude fractures. The tensile tests for each sample and for each strain rate were repeated three or four times to guarantee reproducibility; between successive tests, the sample was placed horizontally for one minute to allow it to recover its shape and mechanical response. Throughout the test, force was recorded against extension in real time by the extensometer.
Data Processing
Fig. 2. (a) Custom-designed clamps and (b) calcaneofibular ligament mounted between the custom clamps with each end wrapped in tissue paper.
Force (F ) data were normalized as nominal stress (r) with cross-section area (A). Extension data (DL) were normalized as nominal strain (‹) with the original length as gauge length (L). The nominal stress vs nominal strain curve was then fitted to the data and
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Liao et al.
Fig. 3. (a) Stress2strain response from tensile tests with calcaneofibular ligament from cadaver C left ankle with strain rate of 2%s21 and (b) stress2strain response from tensile test with calcaneal tendon from cadaver A left ankle with three strain rates of 0.25%s21, 2%s21, and 8%s21.
the tangent to this curve was the specimen’s elasticity, Young’s modulus (E), calculated using the equations below (Fig. 3a): F A
(1)
DL L
(2)
r5 ‹5
Dr (3) De Within the toe region the stress-strain relationship shows features of nonlinearity, mainly because some individual fibers have not been fully stretched. Beyond the nonlinear region, most fibers display elastic behavior, with a linear stress-strain relationship and a constant Young’s modulus. A typical stress vs. strain response with the three different strain rates of 0.25%s21, 2%s21, and 8%s21 is illustrated in Figure 3b. There is a greater stress response vs strain when the strain rate is higher. To investigate whether the measured elasticity differed significantly among (a) cadavers, (b) samples from different positions or (c) testing strain rates, two-factor ANOVA was conducted without replication. The measured elasticity values were segmented into subgroups of cadaver (A, B, and C), subgroups of position (left and right), and subgroups of strain rate (0.25%s21, 2%s21, and 8%s21), according to each statistical hypothesis. E5
RESULTS The mean and standard deviation were calculated for each sample’s Young’s modulus under each strain
rate used (Fig. 4). The means and standard deviations of Young’s modulus averaged across the cadaver group are shown in Table 2. In view of the small standard deviation over the mean elasticity for each sample, the mean value was used for the two-factor ANOVA test; a P-value of 0.05 was taken as the criterion of significance. The mean and standard deviation of each subgroup were tabulated with the corresponding F value and P-value (Table 3). The three cadavers did not differ significantly. Samples from the left ankle were generally stiffer than those from the corresponding right ankle, with P-values as low as 3.26E-04. A generally consistent correlation between the tensile strain rate and the measured Young’s modulus was confirmed with F 5 33.14, far above the critical F value of 3.20 for the 0.05 significance level.
DISCUSSION The purpose of this research was to quantify the effect of Thiel embalming on the elastic properties of human ankle tendons and ligament. Formalin embalming results in cross-linking of proteins, which generally makes the embalmed tissue stiffer, discolored, and inflexible. In contrast, Thiel-embalmed tissues are described as life-like with regard to elasticity, color and flexibility, with further advantages of low infection and toxicity risk (Eisma et al., 2011). There has been no previous research on the biomechanical properties of Thiel-embalmed specimens from the human ankle. In the recent literature we found only Fessel’s biomechanical experiments on
Elastic Properties of Thieled Tendon and Ligament
5
Fig. 4. Young’s modulus measurements of (a) peroneus longus, (b) peroneus brevis, (c) calcaneal tendons, and (d) calcaneofibular ligament with strain rates of 0.25%s21, 2%s21, and 8%s21.
Thiel-embalmed human flexor digitorum profundus tendons (Fessel et al., 2011). The highest Young’s modulus found in this study (497.20 6 10.68 MPa, mean 6 standard deviation), of the Thiel-embalmed peroneus longusis, was considerably lower than the 6896159 MPa for fresh nonembalmed peroneus longus (Arthrex, 2007). However, Arthrex did not state the number of samples or the age or sex of the donors. The elasticity of 430 6 151 MPa from Fessel’s biomechanical testing of Thiel-embalmed human forearm tendons is comparable with our measurements (Fessel et al., 2011).
A single value reported for the Young’s modulus of nonembalmed peroneus brevis tendon, 149.7 MPa (Carmont and Maffulli, 2007), is between the lower (119.95 6 6.46 MPa) and upper (407.60 6 19.56 MPa) bounds of our measurements. The large difference in literature values for the peroneus longus and brevis is surprising given the similarities in anatomical position and physiological function, suggesting that sample groups or methodology are not comparable between these studies. The Young’s modulus of the calcaneal tendon varies widely, 559.2 6 257.3 MPa, among fresh human cadavers (Louis-Ugbo et al., 2004). Their average was
8%s21 356.93 6 49.54 267.93 6 70.38 2%s 337.55 6 106.40 239.57 6 41.72 0.25%s21 298.49 6 71.70 169.65 6 81.59 8%s 272.82 6 86.61 237.02 6 84.29
Calcaneofibular ligament (Mean 6 STD)
2%s 215.21 6 108.87 190.52 6 74.25
21
0.25%s 106.73 6 66.15 62.23 6 18.96
21
Calcaneal tendon (Mean 6 STD)
Strain rate Left Right
21
Young’s Modulus (MPa)
8%s21 268.67 6 27.58 261.30 6 23.67 2%s21 343.57 6 62.68 303.58 6 66.61 0.25%s21 239.78 6 66.87 203.65 6 113.80 8%s21 346.91 6 84.50 306.32 6 171.25 2%s21 339.96 6 60.45 298.83 6 163.06 0.25%s21 237.63 6 95.64 174.18 6 52.19
Proneous longus (Mean 6 STD) Young’s Modulus (MPa)
Strain rate Left Right
Proneous brevis (Mean 6 STD)
Liao et al. TABLE 2. Young’s Modulus Measured Through Repeated Tensile Tests with Statistical Mean (Mean) and Standard Deviation (STD) Calculated for All 24 Samples with Strain Rates of 0.25%s21, 2%s21, and 8%s21
6
considerably higher than the highest Young’s modulus found in our study (362.63 6 21.74 Mpa). However, some of their measurements (335, 279, 203 MPa) are consistent with ours (331.30, 266.03, 198.95 MPa). The Young’s modulus of the Thiel-embalmed calcaneofibular ligament was 278.35 6 89.14 MPa for all three strain rates (Table 2). This is significantly lower than the Young’s modulus of 512.0 6 333.5 MPa for nonembalmed calcaneofibular ligament (Siegler et al., 1988), although the lower parts of the ranges overlapped. The large standard deviation in the measured Young’s modulus could be attributed to the range of donor ages (from 95 to 33 years) in Siegler’s research. Individuals differ significantly in all aspects of anatomy, and any training or product development needs to take this into consideration. The elasticity of the Thiel-embalmed samples in our study were compared quantitatively with nonembalmed samples from the literature at comparable strain rates (Table 4). The elasticity of the Thiel-embalmed specimens are within the range of those for non-embalmed specimens in the literature, which confirms the qualitative evaluations of Thiel-embalmed cadavers. However, the average elasticity values from the Thiel embalming method in our study are lower than those in the literature from non-embalmed specimens; this difference might be expected to affect the results of quantitative investigations. Therefore, it could be better to use fresh biological tissues in critical situations when mechanical behavior and numerical limits are to be tested, for example in product development. During the embalming process, embalming fluid was perfused into the left femoral artery. This means that the left lower limb received fluid more directly than the right and was generally better perfused. This embalming fluid has higher concentrations of alcohol, morpholine, and formalin than the tank fluid in which the body was subsequently submerged, and these components contribute to the preservation of tissue firmness. All the samples from the left ankles had a higher Young’s modulus than those from the right ankles, as confirmed statistically (Table 3), except the peroneus longus from Cadaver B and the peroneus brevis from Cadaver C. This finding supports the view that the initial perfusion of the cadavers is important in preserving tissue elasticity. There were no apparent differences in tendon and ligament elasticity values between Cadavers A and C, which were of the same sex and similar age at death and the same duration of Thiel embalming, though the times between death and embalming differed (2 days and 6 days, respectively). This suggests that a prolonged time between death and embalming has little effect on tendon and ligament elasticity. This hypothesis needs to be tested in future experiments with more Thiel-embalmed samples. Cadavers are typically taken into use 6212 months after embalming. Then, for the following 12218 months, they are used for surgical procedures and alternatively stored in bags or tanks, and
Elastic Properties of Thieled Tendon and Ligament
7
TABLE 3. The Mean and Standard Deviation of the Measured Young’s Modulus and ANOVA (Two-Factor Without Replication) Test Results Within Different Cadavers, Different Anatomical Dissection Positions, and Different Tensile Testing Strain Rates Mean 6 STD (MPa) Cadaver
ANOVA
A
B
C
F
P-value
F crit-0.05
246.82 6 103.20
265.00 6 126.07
248.06 6 75.67
0.33
0.72
3.20
Mean 6 STD (MPa) Position
ANOVA
Left
Right
F
280.35 6 95.92
226.23 6 103.15
15.88
P-value
F crit-0.05
3.26E-04
4.12
Mean 6 STD (MPa) Strain rate
0.25%/s
2%s
186.54 6 96.29
ANOVA
21
8%s
283.60 6 96.65
21
289.74 6 82.92
finally they are made available for dissection studies. All three cadavers used in this study were at the end stages of use and close to the 3-year limit for which cadavers can legally be retained under the Human Tissue (Scotland) Act 2006. Our experience suggests that cadavers gradually lose fluid over time, and those at different stages of use have different properties. Further work is needed to quantify how this affects the mechanical properties of tissues. The tensile tests were implemented with three strain rates, 0.25%s21, 2%s21, and 8%s21, 2%s21 representing the physiological strain rate while 0.25%s21 and 8%s21 represented slow and fast load conditions, respectively. For all the Young’s modulus measurements there was a general increase with increased strain rate, and this was especially clear for the calcaneal tendon, as previously reported (Wren et al., 2001). The other three types of samples showed minor discrepancies in the tendency of Young’s modulus to increase with strain rate. This is probably due to sample degradation caused by the accumulation of damage during the tensile test, especially with the strain rate of 8%s21. The calcaneal tendon usually contains larger collagen fibers within its large size than the other sample types (Jarvinen et al., 2004), which possibly helped it to withstand the accumulation of mechanical damage.
F
P-value
F crit-0.05
33.14
1.22E-09
3.20
This study has a number of limitations concerning the Thiel-embalmed human specimens and damage accumulation. First, there were few Thiel-embalmed specimens with uncontrollable differences in sex, age, and condition at death, and in embalming time period, which could affect tissue elasticity. Secondly, damage within the specimens accumulated during the repeated tensile tests, the larger strain rate affecting the accuracy of measurement. It would be advisable to set a lower maximum strain for each sample, taking into consideration the dimensions and condition of preservation of the sample in future tensile tests with different strain rates. Individual gait problems can also induce bias in the elasticity of ankle tendons and ligaments. It will therefore be important in our future studies to conduct microscopic and mechanical analyses of other structures within the ankle region to enhance the reliability of the conclusions.
CONCLUSIONS In this study we found that Thiel embalming of human cadavers tends to soften the ankle tendons and ligament extent little, although our measured elasticity values for Thiel-embalmed samples overlap
TABLE 4. The Measurements of Young’s Modulus of Thiel-Embalmed Samples in Our Study and Nonembalmed Samples From the Literature at Comparable Strain Rates Thiel-embalmed
Nonembalmed
Young’s modulus (MPa)
Strain rate
Young’s modulus (MPa)
Strain rate
Peroneous Longus Peroneous brevis
319.39 6 112.27 221.72 6 85.79
2%s21 0.25%s21
689 6 159 149.7 6 56.3
N.A. N.A.
Calcaneal tendon
202.86 6 84.44
2%s21
559.2 6 257.3
2.67%s21
Calcaneofibular ligament
234.07 6 98.48
0.25%s21
512.0 6 333.5
0.1320.45%s21
Sample
Reference Arthrex, 2007 Carmont and Maffulli, 2007 Louis-Ugbo et al., 2004 Siegler et al., 1988
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Liao et al.
somewhat with the ranges for fresh nonembalmed tissue found in the literature. This suggests that Thiel-embalmed tissues are similar enough to fresh tissues to be suitable for many procedures and applications, but users need to be aware of the softening effects of the Thiel embalming method, especially in quantitative biomechanical studies. Thiel-embalmed tendons and ligament have strain rate effects similar to those of the fresh tissues, with the Young’s modulus increasing with the strain rate. This supports subjective observations of the realistic handling of Thielembalmed cadavers.
ACKNOWLEDGMENT The authors wish to thank Dr Clare Lamb for her assistance in dissecting the Thiel tendon and ligament samples, and Miss Amanda Hunter for her assistance in revision. Finally, they would like to express our gratitude to the people who choose to donate their bodies to science.
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Eisma R, Mahendran S, Majumdar S, Smith D, Soames RW. 2011. A comparison of Thiel and formalin embalmed cadavers for thyroid surgery training. Surg-J R Coll Surg E 9:142–146. Feigl G, Fuchs A, Gries M, Hogan QH, Weninger B, Rosmarin W. 2006. A supraomohyoidal plexus block designed to avoid complications. Surg Radiol Anat 28:403–408. Fessel G, Frey K, Schweizer A, Calcagni M, Ullrich O, Snedeker JG. 2011. Suitability of Thiel embalmed tendons for biomechanical investigation. Ann Anat 193:237–241. Giger U, Fresard I, Hafliger A, Bergmann M, Krahenbuhl L. 2008. Laparoscopic training on Thiel human cadavers: a model to teach advanced laparoscopic procedures. Surg Endosc 22:901– 906. Holzle F, Franz EP, Lehmbrock J, Weihe S, Teistra C, Deppe H, Wolff KD. 2012. Thiel embalming technique: A valuable method for teaching oral surgery and implantology. Clin Implant Dent R 14: 121–126. Jarvinen TA, Jarvinen TL, Kannus P, Jozsa L, Jarvinen M. 2004. Collagen fibres of the spontaneously ruptured human tendons display decreased thickness and crimp angle. J Orthop Res 22:1303– 1309. Jaung R, Cook P, Blyth P. 2011. A comparison of embalming fluids for use in surgical workshops. Clin Anat 24:155–161. Louis-Ugbo J, Leeson B, Hutton WC. 2004. Tensile properties of fresh human calcaneal (Achilles) tendons. Clin Anat 17:30–35. Munirama S, Satapathy AR, Schwab A, Eisma R, Corner GA, Cochran S, Soames R, McLeod GA. 2012. Translation of sonoelastography from Thiel cadaver to patients for peripheral nerve blocks. Anaesthesia 67:721–728. Quinn KP, Winkelstein BA. 2011. Preconditioning is correlated with altered collagen fiber alignment in ligament. J Biomech Eng-T Asme 133. Siegler S, Block J, Schneck CD. 1988. The mechanical characteristics of the collateral ligaments of the human ankle joint. Foot Ankle 8:234–242. Thiel W. 1992. The preservation of the whole corpse with natural color. Ann Anat 174:185–195. Unger S, Blauth M, Schmoelz W. 2010. Effects of three different preservation methods on the mechanical properties of human and bovine cortical bone. Bone 47:1048–1053. € ussler K, Reinehr M, Bo € ckers TM. 2011. Wilke H-J, Werner K, Ha Thiel-fixation preserves the non-linear load-deformation characteristic of spinal motion segments, but increases their flexibility. J Mech Behav Biomed Mater 4:2133–2137. GS, Carter DR. 2001. Mechanical Wren TAL, Yerby SA, Beaupre properties of the human achilles tendon. Clin Biomech 16:245– 251.
