Acta Polytechnica CTU Proceedings https://doi.org/10.14311/APP.2024.48.0061 Acta Polytechnica CTU Proceedings 48:61–65, 2024 © 2024 The Author(s). Licensed under a CC-BY 4.0 licence Published by the Czech Technical University in Prague DESIGN OF THE TESTING PROCEDURE FOR INVESTIGATION OF ERYX CONICUS DERMAL ARMOUR CHARACTERISTICS Blanka Žaloudkováa, Petr Koudelkab, Petra Frýdlovác, Mykhailo Drozdenkod, Jan Šleichrtb, Daniel Kytýřb,∗ a Czech Technical University in Prague, Faculty of Transportation Sciences, Na Florenci 25, 110 00 Prague 1, Czech Republic b Czech Academy of Sciences, Institute of Theoretical and Applied Mechanics, Prosecká 809/76, 190 00 Prague 9, Czech Republic c Charles University, Faculty of Science, Viničná 7, 128 44 Prague 2, Czech Republic d Czech Academy of Sciences, Nuclear Physics Institute, 250 68 Řež, Czech Republic ∗ corresponding author: kytyr@itam.cas.cz Abstract. The aim of this work is to develop an experimental method suitable for the mechanical testing of highly non-standard biological samples such as snake skin with osteoderms. The objective of the method is to determine, whether the osteoderms provide a protective function for the animal in its natural environment. For this purpose, a simulation of rodents biting the skin based on uni-axial compressive loading using a synthetic tooth as a penetrator was developed with an emphasis on integration with X-ray scanners to facilitate in-situ testing. To identify and characterise the structure of snake skin and to prove the protective function of osteoderms, all samples were subjected to high resolution X-ray computed tomography. The results of the experiments are presented in the form of stress-strain curves and a map of the tangent modulus. Keywords: Bioinspiration, snake skin, osteoderms, mechanical testing, computed tomography, dermal armour. 1. Introduction Biological materials and animal anatomical features in general have been an inspiration for human protection since immemorial eras. Even though the research in recent years has been more material-driven, studies show that a link can be seen between modern de- fensive structures and structures found in the realm of animals [1]. Thus, studying the natural animal dermal armour can be helpful in dealing with the ul- timate goal of human ballistic protection, which is to find the optimum between the weight of the armour, its flexibility when worn, and, most importantly, the protection it provides against a multitude of threats. A multi-layer or multi-material body armour is one of the approaches often adopted when dealing with such a problem, but there is insufficient data available on ballistic performance [2]. Here, it is important to note that different body parts require different levels of protection, where heavy armour is needed for ex- posed and large profile body parts such as the head or chest, while soft armour is used in other areas to reduce the overall weight and, importantly, to allow sufficient mobility in the areas around joints (i.e., typ- ically shoulders and hips). This is also a result of natural development that originates from anatomi- cal solutions to survival problems in animals, where, e.g., rhinoceros species have thinner skin around their joints to achieve the best ratio of protection versus mobility [3]. Thus, by the increase of protective per- formance in the body armour without the inevitable weight increase with available armour types, it would be possible to provide high-end protection to more parts of the human body than front and back of the torso, which is the normal extent of protection in the commonly used armour vests or plate carriers. In general, for a deeper understanding of all the fac- tors and processes involved in the field of bio-inspired ballistic protection, knowledge gathering methods based on the study of nature and effective transfer of the knowledge to engineering solutions of prob- lems involve research in numerous fields including kinematics [4], morphology [5], material science [6] and structural analysis [7]. In this work, we present an initial study paving the way for in-situ investiga- tion of morphological and mechanical characteristics in Eryx conicus dermal armour as part of research aimed at verifying its existence and determining its role in protecting these snake species in their natural environment. 2. Wildlife story In our study, we focused on rough-tailed sand boas (Eryx conicus). In nature, it can be found in India, Pakistan, Nepal, and Bangladesh. They are stout- bodied and medium sized reaching around 80 cm in length in adulthood, the life expectancy is 20 years or even longer. Their natural habitat includes dry and rainy areas; they shelter in deep cracks and rodent 61 https://doi.org/10.14311/APP.2024.48.0061 https://creativecommons.org/licenses/by/4.0/ https://www.cvut.cz/en B. Žaloudková, P. Koudelka, P. Frýdlová et al. Acta Polytechnica CTU Proceedings burrows. Although they are mostly nocturnal animals, they are also found to be active during the day. They prey on rodents, lizards, ground-feeding birds, and amphibians. Their anatomically unique tail with der- mal armour [8] probably plays a fundamental role in their antipredatory strategy. It is hypothesised that they use it to protect and avoid serious injuries to vital parts of their body. We assume that two-headed snakes of the genus Eryx use their armoured tails as a deceptive target while plundering rodent nests (e. g., gerbils). Indian Gerbil (Tatera indica) is a rodent of the Gerbillinae subfamily that commonly shares territory with snakes. In adulthood, they can grow up to 38 cm with male species slightly larger than females. Their lifespan in captivity can reach up to 7 years; however, when living in the wild, life expectancy is just a few years as many individuals have problems surviving the first year of their life due to the hazard posed by snakes. However, nests are defended by parents with sharp edged incisors (see Figure 1) [9]. The aim of this study is to design a testing method for evaluation the protective function of osteoderms (irregular small bony elements with dimensions about 500×250×150 µm, see Figure 1 red) in the skin of the snake based on localised mechanical experiments cou- pled with detailed CT inspection to quantify relevant biomechanical parameters. As a first step, the ex- periment was designed to investigate the quasi-static interaction of the loading tip with real snake skin with osteoderms. The loading tip was the dimension of the gerbil incisors to make it as real as possible. Snake skin without osteoderms was used as a control. Figure 1. Size comparative CT based visualisation of the gerbil skull with incisors and Eryx conicus cau- dal segment with osteoderms visible as a small bony elements around the body without a tight connection to the axial skeleton together with detail of single osteoderm (in red). 3. Materials and methods 3.1. Skin samples Four samples of the skin surface layer with thicknesses up to 500 µm of rectangular-like shape and dimensions ranging from 7×18 mm to 20×30 mm were harvested from a single individual. In the central (labelled A) and caudal (C) part of the body where the osteoderms of interest (see Figure 1) are predominantly located on the lateral (L) and ventral (V) side of the caudal segment. The samples were then long-term stored in commonly used 70 % ethanol solution [10]. The moderate effect of storage conditions on mechanical properties has been reported [11], but a more signifi- cant effect arises from the drying of the sample. For that reason, the testing procedures were performed in the shortest possible time and the samples were re-hydrated after the mechanical tests prior to X-ray imaging. 3.2. Mechanical loading The skin samples were subjected to mechanical load- ing using an in-house developed compact table-top loading device presented in detail in [12] and specially instrumented for snake skin testing (see Figure 2). The upper steel loading pin with a tip cross-section of 1.25 × 0.4 mm was designed on the basis of the geometry assessed from an X-ray CT scan of the gerbil incisors (see Figure 1) and instrumented by LCM300 (Futek, USA) load-cell with a nominal ca- pacity of 10 N. The lower gliding platform with the skin sample fixed by double-sided scotch tape allowed manual positioning for setting the quasi-regular the testing grid consisting of 14–20 testing points. The experiments were displacement driven up to 500 µm or interrupted when reaching the force limit of the load cell. The loading rate was set to 2 µm s−1. The experiment was carried out using real-time closed loop control software [13] with 200 Hz sampling rate for both force value and encoder position readout. Figure 2. Compact table-top loading device instru- mented for the snake skin testing procedure. 3.3. X-ray imaging The modular in-house designed laboratory CT scan- ner, equipped with linear positioning axes and a rotary stage depicted in Figure 3, allows the exchange of the X-ray detector and source to meet specific measure- ment requirements. For soft, partially ossified skin 62 vol. 48/2024 Testing procedure for Eryx conicus dermal armour investigation tissue, L10321 (Hamamatsu Photonics, Japan) micro- focus X-ray source without additional filtering of the X-ray beam [14] together with Dexela 1512 (Varex Imaging, Germany) CMOS X-ray detector equipped with a CsI scintillator were employed. The native resolution of the detector is 1944 × 1536 at a pixel pitch of 74.8 µm. The imaging was performed with an acceleration voltage of 75 kV and a target current of 150 µA to achieve the best possible quality in the ac- quired radiograms. Each tomographic scan consisting of 1912 equiangular projections took approximately 30 minutes. The cone beam filtered backprojection re- construction algorithm based on the Feldkamp-Davis- Kress method [15] implemented in VG Studio MAX 3.4.1 (Volume Graphics, Germany) was used to ob- tain the resulting 3D images of investigated samples having dimensions of 1536 × 1536 × 1944 voxels and 20 µm effective voxel size. The histogram-based tissue segmentation procedure [16] was applied to identify the location of the osteoderm. Figure 3. In-house designed CT setup with the X-ray source (left), detector (right) and the detail of the skin sample on rotary stage. 3.4. Data evaluation Fully automated evaluation procedure implemented in MATLAB R2023b (MathWorks, USA) was developed to calculate the stress-strain data from the measured force F and displacement u signals. For the calculation of engineering stress σ, the cross-sectional area of the nominal loading tip Atip was used. The engineering strain ϵ was calculated for each measurement point individually due to the non-uniform thickness t of the sample. Here, the thickness was measured as the difference between the tip-baseplate contact tb and the tip-skin contact position ts, when the contact force reached 0.07 N at the beginning of the experiment. The stress and strain were then calculated using the equations σ = F Atip , ϵ = u tb − ts . (1) To quantify the fully non-linear stress-strain be- haviour of the samples, the tangent modulus was chosen as a representative quantity. The tangent modulus was calculated using the polynomial curve fitting in the last 10 % of the strain. The results are graphically represented by stress-strain diagrams and tangent modulus values projected on the 3D image visualisation of the respective sample. 4. Results As a result of this pilot study, the results of the three samples harvested from: i) the ventral side of the cen- tral segment (AV), ii) the ventral side of the caudal segment (CV), and iii) the lateral side of the cau- dal segment (CL), where the osteoderms were then identified only in the CL samples. The strain stress diagrams of the AV sample are depicted in Figure 4. Invalid measurements probably caused by improper triggering of the tip-skin contact point are plotted in grey. 0 1 2 3 4 5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 S tr e ss [ M P a ] Strain [-] Figure 4. Stress-strain diagram of ventral side of central segment (AV) with 11 valid measurements presented in blue, excluded measurements in grey. Based on 11 valid experiments, the values of the individual tangent modulus in the range of 11.48– 18.66 MPa were mapped on the CT visualisation pre- sented in Figure 5. The values are indicated by the colour and diameter of the circle with the centre corre- sponding to the simulated bite point. The mean value of the tangent modulus was calculated as 15.99 MPa with a standard deviation of 1.61 MPa (10.1 %). The strain stress diagrams of the CV sample are depicted in Figure 6. Based on 13 valid experiments, the values of the individual tangent modulus in the range of 7.48–17.39 MPa were mapped on the CT visualisation presented in Figure 7. The mean value of the tangent modulus was calculated as 12.52 MPa with a standard deviation of 3.20 MPa (25.5 %). The strain stress diagrams of the CL sample are de- picted in Figure 8. Based on 3 experiments carried out on osteoderm area, individual tangent modulus values in the range of 19.15–21.70 MPa with a mean value of 20.45 MPa and a standard deviation of 1.27 MPa (6.2 %) were calculated (see Figure 9). The results of 63 B. Žaloudková, P. Koudelka, P. Frýdlová et al. Acta Polytechnica CTU Proceedings Figure 5. Localised tangent modulus magnitudes on ventral side of central segment (AV). 0 1 2 3 4 5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 S tr e ss [ M P a ] Strain [-] Figure 6. Stress-strain diagram of ventral side of caudal segment (CV) with 13 valid measurements presented in blue, excluded measurements in grey. the other 12 experiments were not influenced by os- teoderms. Here, tangent modulus values in the range of 5.85–16.17 MPa with a mean value of 10.19 MPa and a standard deviation of 3.67 MPa (36.1 %) were assesed (see Figure 9). The bite test was performed on three skin samples from a single Eryx conicus individual. Osteoderms were identified by CT scans only on the lateral side of the caudal segment. The average tangent modulus of the samples without osteoderms (AV, CV) was signifi- cantly higher, measuring 14.26 ± 2.45 MPa, compared to the non-ossified part of the CL, which measured 10.19 ± 3.67 MPa. For all bite tests that excluded osteoderms, the tangent modulus was calculated as 12.90 ± 2.92 MPa, while the tangent modulus in osteo- derms reached 20.45 ± 1.27 MPa, although this latter value is not supported by a statistically significant number of measurements. 5. Conclusions Based on the acquired results, it can be concluded that the proposed methodology and experimental equip- Figure 7. Localised tangent modulus magnitudes on ventral side of caudal segment (CV). 0 1 2 3 4 5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 S tr e ss [ M P a ] Strain [-] Figure 8. Stress-strain diagram of lateral side of cau- dal segment (CL) with 3 measurements of osteoderms and 12 measurement not influenced by osteoderms presented in blue, excluded measurements in grey. Figure 9. Localised tangent modulus magnitudes on lateral side of caudal segment (CL). ment including the X-ray imaging chain is a viable solution to the problem of mechanical experiments and imaging of snake skin tissue equipped with der- mal armour. The reconstructed 3D images from the 64 vol. 48/2024 Testing procedure for Eryx conicus dermal armour investigation tomographical measurements show that it is possi- ble to identify the osteoderms using high-resolution X-ray imaging with high confidence and to support the interpretation of the mechanical experiments. By analysing the mechanical data, the presence of osteo- derms was clearly apparent, as the assessed average tangent modulus was twice as high compared to the skin without osteoderms. However, because of the geometry of the specimen and geometry of the simu- lated incisors, it is relatively difficult to measure the mechanical quantities precisely at the predetermined locations. 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