Background: The aim of this study was to directly compare the effects of Thiel embalming on the mechanical properties of three human tendons to similar tendons from the fresh-frozen, ipsilateral foot of the same cadavers. Method: Following pre-conditioning, biomechanical tensile tests were conducted on tibialis anterior, peroneus longus and Achilles tendons at a strain rate of 5 mm/minute, using an optical extensometer for the direct measurement of sample strain. Results: Qualitatively, all tested tendons exhibited similar mechanical behaviour characterised by an initial toe region followed by a region of linear behaviour. Quantitatively, however, averaging the results for the three cadaveric samples revealed consistent differences across all tendons in the case of unloading stiffness. Conclusion: Thiel embalming method is suitable for preservation of human tendons, with embalmed tendons appearing to have similar mechanical behaviour compared to their fresh frozen counterparts. Although quantitative differences do exist, these do not disprove the use of Thiel embalmed tendons in comparative studies once care is exercised when utilizing these for biomechanical testing. However, in vitro results used to infer in vivo function should always be treated with caution.peer-reviewe

Chatzistergos, Panagiotis

Chockalingam, Nachiappan

Formosa, Cynthia

Gatt, Alfred

Gatt, Ruben

Grima, Joseph N.

Schembri-Wismayer, Pierre

OAR@UM

Acta Scientific Orthopaedics (ISSN: 2581-8635)     Volume 4 Issue 11 November 2021The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot TendonsAlfred Gatt1,2*, Nachiappan Chockalingam1,2, Panagiotis Chatzister-gos2, Ruben Gatt3, Pierre Schembri-Wismayer4, Joseph N Grima3 and Cynthia Formosa1,21Podiatry Department, Faculty of Health Sciences, University of Malta, Msida, Malta2Centre for Biomechanics and Rehabilitation Technologies, Staffordshire University, Stoke-on-Trent, England, United Kingdom3Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, Msida, Malta4Department of Metamaterials, Faculty of Science, University of Malta, Msida, Malta*Corresponding Author: Alfred Gatt, Podiatry Department, Faculty of Health  Sciences, University of Malta, Msida, Malta.Research ArticleReceived: September 23, 2021Published: October 12, 2021© All rights are reserved by Alfred Gatt., et al. AbstractBackground: The aim of this study was to directly compare the effects of Thiel embalming on the mechanical properties of three human tendons to similar tendons from the fresh-frozen, ipsilateral foot of the same cadavers. Method: Following pre-conditioning, biomechanical tensile tests were conducted on tibialis anterior, peroneus longus and Achilles tendons at a strain rate of 5 mm/minute, using an optical extensometer for the direct measurement of sample strain. Results: Qualitatively, all tested tendons exhibited similar mechanical behaviour characterised by an initial toe region followed by a region of linear behaviour. Quantitatively, however, averaging the results for the three cadaveric samples revealed consistent differ-ences across all tendons in the case of unloading stiffness.Conclusion: Thiel embalming method is suitable for preservation of human tendons, with embalmed tendons appearing to have similar mechanical behaviour compared to their fresh frozen counterparts. Although quantitative differences do exist, these do not disprove the use of Thiel embalmed tendons in comparative studies once care is exercised when utilizing these for biomechanical testing. However, in vitro results used to infer in vivo function should always be treated with caution. Keywords: Biomechanical Properties; Human Fresh-Frozen; Thiel Embalmed Foot TendonsIntroductionIn humans, testing for the mechanical properties of tendons is often performed in cadaveric fresh-frozen samples. The use of these samples, which are stored at -20oC until they can undergo testing, is considered as the gold-standard in this type of research. The use of fresh-frozen samples, however, has two very impor-tant limitations: thawed tissue has a limited time period during which testing can be performed, thus placing considerable time-constraints and pressure on the investigator, consequently raising the possibilities of human error during testing which could ad-versely affect results. Secondly, freezing only delays the decaying Citation: Alfred Gatt., et al. “The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot Tendons". Acta Scientific Orthopaedics 4.11 (2021): 19-23.process and once thawed, the risk of possible infection for the in-vestigators is increased [1]. In order to overcome these limitations, several preservation techniques including formalin fixation, Thiel-fixation, and alcohol-glycerine fixation, are reported [1]. Thiel fixation has been claimed to reduce risks of infection whilst maintaining tissue suppleness for a long period of time without the need for refrigeration, thus leaving the tissue looking ‘normal’. The colour, consistency and transparency of the tissue are thus very well preserved [2].This ‘soft-fix’ method has become increasingly popular in anatomy laboratories; however their use in biomechanical testing is rather limited due to a lack of scientific data arising out of ap-propriate studies. The importance of determining the mechanical characteristics of these tendons becomes evident when one con-siders that these Thiel embalmed specimens have the potential to be utilised in biomechanical testing, for example following the de-velopment of new surgical techniques. Although there appears to be general consensus that Thiel embalmed soft tissue may be 22 - 45% more flexible [3] than fresh-frozen specimens, possibly due to dis-organisation of muscle fibre following considerable fragmenta-tion of the muscle proteins [4], few studies have been conducted to confirm, or otherwise, this hypothesis.To date, there is a paucity of evidence as to whether this method of embalming leaves tendons with appropriate mechanical charac-teristics that enable them to be utilized for biomechanical testing, with very few studies on individual Thiel embalmed tendons hav-ing been conducted. Fessel., et al. [5], who investigated the digito-rum profundus and rat-tail tendons, which demonstrated a lower failure stress when compared with their fresh-frozen counterparts, whilst Liao., et al. [6] mechanically tested human peroneus brevis and peroneus longus tendons, report that Thiel embalming pre-serves human tissue elasticity. Both these studies have some limi-tations; while the former compares only 1 type of human tendon, the latter study’s two tendons’ characteristics from 3 cadavers were compared with data from the literature. Another study on 7 human Achilles tendons reports that Thiel embalming significantly alters the Young’s modulus of human tissue [7]. Aim of the StudyThe aim of this study was to quantify and directly compare the effects of Thiel embalming on the mechanical properties of three human tendons. Throughout this study, the Thiel embalmed ten-dons were directly compared to similar tendons from the ipsilat-eral foot of the same cadavers following a method of preservation specifically developed for this study that has never been previously reported in literature. MethodsThis research was approved by the University Research and Ethics Committee of the University of Malta prior to data collec-tion. Human tissue utilized throughout this study were received and stored at the Anatomy Department at the same University, fol-lowing strict guidelines as outlined within the local legislation that strictly followed the European Union directives pertaining to the handling of human tissues.The feet from three cadavers voluntarily donated for scientific research were utilised throughout the study. A technique for the fresh-frozen preservation of one foot, and Thiel embalming of the other, was developed specifically for this study as follows. Prior to preservation, each cadaver had a single leg amputation 20 cm proximal to the medial malleolus, which was then maintained in a frozen state at -20oC. Each cadaver was then maximally perfused through the femoral artery with Thiel embalming fluid through a plastic tube inserted into the proximal and distal segments of the femoral artery sectioned just below the inguinal ligament. A sec-ond tube was inserted into the femoral vein at the same level and left to drain into the embalming table sump. Saline was pumped into the cadaver until the fluid which ran out of the venous drain was free of blood. Thiel solution was then injected into the cadaver. External embalming was carried out in a tank. Visual inspections for significant differences between the right and left foot of each cadaver was carried out to ensure similarity. Signs of significant deformity and past surgery would have ren-dered these specimens unusable.Before testing, the fresh-frozen samples were allowed to thaw for 24 hours prior to dissection. The embalmed feet, on the other hand, had no need for any specific preparation. The tendons of the tibialis anterior, peroneus longus, together with the Achilles ten-don, were harvested by blunt dissection. The same part of the ten-don was prepared for testing for each specimen. All testing was performed in the same laboratory, at 18oC and 57% humidity. Each end of the tendon was wrapped in a thin piece of cloth and then mounted on the load frame using purpose-built compression grips as per Fessel., et al [5]. Consequently, due dili-gence was maintained when tightening these clamps in order not to damage the tendons.20The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot TendonsCitation: Alfred Gatt., et al. “The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot Tendons". Acta Scientific Orthopaedics 4.11 (2021): 19-23.These clamps, in turn, had previously been mounted on both ends of a Testometric (Lincoln, United Kingdom) M350-20CT mate-rials testing device, which had a 1000N load cell (type DBBMTCL). Each tendon was marked with an indelible marker to facilitate optical referencing [8] for a two-camera Zwick extensometer, cali-brated as per manufacturer instructions, thus providing markers that facilitated the direct measurement of sample strain (Figure 1).Pre-conditioning of the tendon was performed by applying a tensile load up to 10N, held for 30 seconds and released back to 0N, at a load rate of 0.25 N/sec. This was repeated for 10 times to ensure the comparative results are not influenced by the loading history of the samples [8,9].After preconditioning the samples were subjected to one load/unload cycle of tensile loading to a maximum force of 100 N. Both loading and unloading were performed by imposing a constant dis-placement rate (grip-to-grip) of 5 mm/min. After synchronisation force data exported from the load frame and strain data from the video extensometer were used to draw the force/strain graphs for each sample and to measure their maxi-mum strain (i.e. strain for 100 N tension force), hysteresis ratio and stiffness for loading and unloading. Hysteresis ratio was calculated by subtracting the area below the force/ strain graph for unloading from the respective area for loading and divided by the area be-low loading. Stiffness for loading and unloading were calculated as the slope of the force/strain graph for force between 80 N and 100 N for the loading and unloading segments respectively. A paired sample t-test was also performed using Microsoft Excel to assess the statistical significance of the differences found between fresh frozen and Thiel embalmed samples.ResultsNine tendons (3 fresh frozen and 3 Thiel-embalmed) from 3 cadavers (2 females, 1 male, mean age 84 years) were utilized throughout this study.Eight out of the nine tests were completed successfully. An error in data collection during the testing of tibialis anterior sample #2 led to its elimination from the rest of the analysis (Figure 2). Figure 2: The force/strain graphs for all pairs of Thiel em-balmed (T) and fresh frozen (FF) samples of tibialis anterior (first row), Achilles (second row) and peroneus longus (third row) tendons included in this analysis. Results for tendons harvested from cadaveric sample 1, 2 and 3 are shown in first, second and third column respectively. The graph for the fresh frozen tibialis anterior sample #2 is also shown for complete-ness. A qualitative comparison between the force/ strain graphs of Thiel embalmed tendons and their fresh frozen counterparts indi-21The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot TendonsCitation: Alfred Gatt., et al. “The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot Tendons". Acta Scientific Orthopaedics 4.11 (2021): 19-23.Figure 1: Fresh frozen tibialis posterior tendon undergoing testing in the tensile testing machine.cates that all tested tendons exhibit similar mechanical behaviour characterised by an initial toe region followed by a region of linear behaviour (Figure 2). In quantitative terms, averaging the results for the three cadaveric samples revealed consistent differences across all tendons in the case of unloading stiffness (Table 1). More specifically unloading stiffness for Thiel embalmed specimens was 40%, 62% and 14% higher than the stiffness of their fresh frozen counterparts for tibialis anterior, Achilles and peroneus longus ten-don respectively. Paired samples t-test showed that the aforemen-tioned difference was statistically significant in the case of Achilles tendon (Table 1).Sample #1 #2 #3 Average STDEV P-valueAge (y) 84 83 85 84 -Sex (M/F) Female Female Male - - -Max strainTibialis anteriorT 0.019 - 0.013 0.016 0.004-FF 0.030 0.054 0.010 0.020 0.014AchillesT 0.127 0.030 0.013 0.057 0.0620.62FF 0.094 0.032 0.021 0.049 0.039Peroneus longusT 0.026 0.040 0.008 0.024 0.0160.69FF 0.021 0.028 0.017 0.022 0.005Hysteresis ratioTibialis anteriorT 0.19 - 0.38 0.28 0.13-FF 0.49 0.70 0.02 0.25 0.33AchillesT 0.81 0.16 0.21 0.39 0.360.30FF 0.42 0.18 0.09 0.23 0.17Peroneus longusT 0.09 0.18 0.01 0.09 0.080.04FF 0.16 0.31 0.16 0.21 0.08Stiffness Loading (N)Tibialis anteriorT 6901 - 8765 7833 1318-FF 3327 2542 15897 9612 8888AchillesT 1550 3999 15712 7087 75690.43FF 2353 3205 7495 4351 2756Peroneus longusT 5931 6623 11820 8125 32190.64FF 6596 7137 8538 7424 1002Stiffness Unloading (N)Tibialis anteriorT 7932 - 19141 13536 7925-FF 6869 16136 12447 9658 3944AchillesT 7625 7283 13169 9359 33040.03FF 4458 4588 8308 5785 2186Peroneus longusT 7644 11070 13900 10871 31330.58FF 8981 11095 8357 9478 1435Table 1: The experimental results for the Thiel embalmed (T) and fresh frozen (FF) tendons from all three cadaveric samples included in this study. The P-values calculated using paired samples t-test are also presented for all tendons with the exception of tibialis anterior. P-values lower than 0.05 are underlined to highlight statistically significant differences between Thiel embalmed and fresh frozen samples. Paired samples t-test also revealed statistically significant dif-ferences in terms of hysteresis ratio in the case of peroneus longus, with Thiel embalmed specimens having 56% lower average hys-teresis ratio compared to their fresh frozen counterparts (Table 1). In contrast to the results for peroneus longus average hysteresis ratios for Thiel embalmed tibialis anterior and Achilles tendons ap-22The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot TendonsCitation: Alfred Gatt., et al. “The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot Tendons". Acta Scientific Orthopaedics 4.11 (2021): 19-23.peared to be 12% and 73% higher compared to their fresh frozen counterparts respectively. However, the aforementioned differenc-es were not statistically significant (p > 0.05). DiscussionThis study differed significantly from the few previous studies that compared Thiel to fresh frozen samples in the method em-ployed in order to obtain samples from the same cadaver, which could consequently allow for direct comparison. Furthermore, this study has utilized the most number of tendons for direct compari-son.Although it is recognized that the two feet of the same person are not necessarily identical, the use of contralateral feet in a set ensured that these paired feet would have been subjected to the same number of loading cycles in their lifetime. Also, the possibility of tendons being affected by disease would be similar to each foot.One of the most challenging aspects of tendon mechanical test-ing is rigidly gripping the samples without damaging them or al-tering their mechanical properties [10,11]. According to literature excessive compression and the use of friction based grips is likely to cause damage to the tissue itself, whilst the use of freeze-clamps with liquid nitrogen can significantly affect the mechanical behav-iour of the samples if the freezing areas are not properly controlled [10,11]. Considering the challenges associated with freezing-clamps it was decided that their use would complicate the experi-ment beyond that merited by the scope of the research in the first place. Moreover, since the aim of the study was of a comparative nature which did not include the yield point allowed performing the tests at a relatively low tension force (i.e. 100 N). This enabled rigidly gripping the samples without the need for substantial com-pression of the samples within the grips minimising the risk of damage. The effect of possible slippage of the samples out of the grips was eliminated by directly measuring tendon elongation us-ing optical extensometer.ConclusionIn qualitative terms Thiel embalmed tendons appeared to have similar mechanical behaviour compared to their fresh frozen coun-terparts, however quantitative differences do exist. The differences found do not disprove the use of Thiel em-balmed tendons in comparative studies. In any case in vitro results used to infer in vivo function should always be treated with caution.23The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot TendonsCitation: Alfred Gatt., et al. “The Biomechanical Properties of Human Fresh-Frozen vs Thiel Embalmed Foot Tendons". Acta Scientific Orthopaedics 4.11 (2021): 19-23.Bibliography1. Unger S., et al. “Effects of three different preservation methods on the mechanical properties of human and bovine cortical bone”. Bone 47.6 (2010): 1048-1053.2. Thiel W. “The preservation of the whole corpse with natural color”. Annals of Anatomy 174.3 (1992): 185-195.3. Wilke HJ., et al. “Thiel-fixation preserves the non-linear load-deformation characteristic of spinal motion segments, but in-creases their flexibility”. The Journal of the Mechanical Behav-ior of Biomedical Materials 4.8 (2011): 2133-2137.4. Benkhadra M., et al. “Flexibility of Thiel’s embalmed cadavers: the explanation is probably in the muscles”. Surgical and Ra-diologic Anatomy 33.4 (2011): 365-368.5. Fessel G., et al. “Suitability of Thiel embalmed tendons for bio-mechanical investigation”. Annals of Anatomy 193.3 (2011): 237-241.6. Liao X., et al. “Elastic properties of Thiel-embalmed human ankle tendon and ligament”. Clinical Anatomy 28.7 (2015): 917-924.7. Verstraete MA., et al. “Impact of drying and thiel embalming on mechanical properties of achilles tendons”. Clinical Anato-my 28.8 (2015): 994-1001.8. Rigozzi S., et al. “Local strain measurement reveals a varied regional dependence of tensile tendon mechanics on glycos-aminoglycan content”. Journal of Biomechanics 42.10 (2009): 1547-1552.9. Maganaris CN. “Tendon conditioning: artefact or property?” Proceedings of the Royal Society B: Biological Sciences 270.1 (2003): S39-42.10. Chatzistergos PE., et al. “The Fracture Stress of Rat Achilles Tendons”. Scandinavian Journal of Laboratory Animal Science 37 (2010): 149.11. Riemersa DJ and Schamhardt HC. “The cryo-jaw, a clamp de-signed for in vitro rheology studies of horse digital flexor ten-dons”. The Journal of Biomechanics 15.8 (1982): 619-620.Volume 4 Issue 11 November 2021©  All rights are reserved by Alfred Gatt., et al.

The biomechanical properties of human fresh-frozen vs thiel embalmed foot tendons

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The biomechanical properties of human fresh-frozen vs thiel embalmed foot tendons

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