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Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
Journal of Bionic Engineering 14 (2017) 356–368
Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales Peng Liu1, Deju Zhu1, Junwen Wang2, Tinh Quoc Bui3 1. College of Civil Engineering, Hunan University, Changsha 410082, China 2. College of Civil Engineering, Tongji University, Shanghai 200092, China 3. Department of Civil and Environmental Engineering, Tokyo Institute of Technology, Tokyo 108-233, Japan
Abstract This work is conducted to investigate the hierarchical structure, mechanical behavior and fracture resistance of grass carp scales with different water contents (hydrated and dehydrated) and load conditions (uniaxial, biaxial and punch tests). The whole cross-section of scales is investigated, and it is found that the bony layer displays discontinuity and partly embeds in collagen layer. Four different locations are considered under both tensile and punch tests. The results of the uniaxial tensile test show a correlation between the failure mode and the distribution of surface morphology on scales. The biaxial test results show that there are minor differences in the tensile strength and the Young’ modulus compared with those of the uniaxial tests, but the ultimate strain is about 20% – 50%. Puncture tests are also conducted with different size of needles and different hardness silicon rubbers as substrate. The results show that the puncture force and deformation are dependent on the size of needle and the hardness of substrate. The failure pattern of scales is related to the water content. Radial cracks occur in the bony layer of hydrated scale, and the collagen fibers twist around the puncture site. However, the shear failure occurs in the bony layer of dehydrated scale. Keywords: grass carp scale, mechanical behavior, biaxial test, penetration resistance Copyright © 2017, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved. doi: 10.1016/S1672-6529(16)60404-3
1 Introduction Natural materials have been drawing people’s attention because of their unique and optimized functions during long term evaluation. Revealing the relationship between structure and material behavior of biological materials can provide valuable information and knowledge to the design of bio-inspired engineering materials in novel ways[1]. These materials like nacre, teeth, and bone, are hard and stiff due to high mineral contents, and display surprising energy absorption ability[2], but they are not flexible and have high density[3]. Other material exoskeletons of crustaceans such as lobster[4], crab[5], contain highly mineralized chitin fibers showing low hardness. Some low mineralized materials like fish scales exhibit both light-weight and local damage in nature. Specially, teleost fish scales (like striped bass) are flexible, and provide a specific resistance to penetration[6]. Corresponding author: Deju Zhu E-mail: [email protected]
The structural and mechanical behaviors of fish scales have been studied, and it is suggested that fish scales can be classified into four types: placoid, ganoid, cosmoid and elasmoid[7]. The observation of morphology of scales can provide some information for classification[8] as well, the non-smooth surface with corrugations is beneficial to enhance flexible strength and redistribute the strain[9]. The hierarchical structure of scales is various, but most scales besides Polypterus senegalusa (four-layer gradient structure: ganoine, dentine, isopedine and bone[10]) have similar hierarchical structures. These structures generally consist of two main layers: a mineralized, hard external layer composed of type I collagen fibrils with a high content of hydroxyapatite[11], and an un-mineralized, soft internal layer composed of type I collagen fibrils[12]. Collagen fibers (bundles of fibrils) are arranged in a twisted plywood pattern with different directions of fibers across lamellae[13,14].
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
The mechanical behavior and failure mode of scale are related to the structure, Ikoma et al.[12] analyzed the mechanical properties of scales from red sea bream, Pagrus major, and showed that the tensile strength is up to 93 MPa, the failure occurs due to the sliding of collagen lamellae and pulling out of collagen fibers. Bruet et al.[10] studied the structure of P. senegalus scales with experiments and finite element simulations, which is indicated that their unique structure and property can inspire new design ideas for modern flexible body amour. As a biomaterial, the mechanical properties of scales are significantly affected by water content[15]. In a hydrated condition, scales from the alligator gar, Atractosteus spatula, and P. major scales have similar ultimate tensile strengths, up to 100 MPa[16] and 93 MPa[12], respectively, which is approximately three times higher than the strength of other scales, such as Arapaima gigas scales (25.2 ± 7.3 MPa)[15], and scales from the common carp, Cyprinuscarpio, (~30 MPa)[3]. When the scales are dried, the tensile strength and modulus of scales dramatically increases, but the failure strain decreases. The tensile strength of dry A. gigas scales is about twice higher than that of hydrated ones, and the elastic modulus is up to 12 times higher[15]. Recently, studies have found one interesting feature as the fish scale behaves anisotropic properties. Zhu et al.[6] experimented the tensile strength of striped bass scales in three directions (0˚, 45˚, 90˚) at the “focus” location, and found that striped bass scales display in-plane anisotropic behavior. Liu et al.[17] further examined and found that both the hydrated and dehydrated grass carp scale reveal an anisotropic behavior at “focus” location. Similar behavior of scales is also observed from the alligator gar, A. spatula[16]. Wang et al.[18] estimated that the structure anisotropic plays an important role in avoiding the indentation attacks with finite element analysis. Gil-Duran et al.[19] found that Megalops Atlanticus scale shows the highest stress strength along with the fish length. Yu et al.[20] examined that the Dabryanus scales are much lower than those of other reported fish scales, and they increase the flexibility. For grass carp, a freshwater species, this is native to lake and rive in Asian. Some researchers have studied the swimming mode and eating inhabits of grass carp. Shi et al.[21] suggested that the low digestive capacity allows the grass carp to maintain its locomotory capacity. In this paper, we describe the whole cross-section of C.
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idella scales and its surface via optical microcopy and Scanning Electron Microscopy (SEM). We investigate the failure types of scale at different location. We also present new results for biaxial tensile tests on scale specimens and show the development of strain distribution with Digital Image Correlation (DIC). A penetration resistance experiment is implemented and failure mechanism is investigated in hydrated and dehydrated condition. Some conclusions drawn from this work are reached, which may facilitate the biomimetic design of a hierarchical protective system with superior performance.
2 Materials and methods 2.1 Materials In this work, the scales are taken from the lateral central area of a grass carp about 5 kg weight, bought from aquaculture market, China. Grass carp scale has about 30 mm in length at longitudinal direction (anteroposterior axis), and about 25 mm in width at transversal direction (ventrodorso axis). The lateral line scales, which characterize a main channel in the center, are not considered in our tests. And five samples are used in each test. 2.2 Sample preparation and test procedures 2.2.1 Uniaxial tensile test The scales were removed from fresh grass carp with tweezers and stored in a freezer at −20 ˚C until tested. Before testing, the scales were taken from the freezer and placed in a water bath at room temperature about 20 ˚C for about 10 minutes for thawing out. The dog-bone-shaped samples are shaped by four areas of the scale involving the posterior, anterior, and lateral (ventro-lateral and dorso-lateral) regions (Fig. 1a) by using a custom-made stainless steel cutter (Fig. 1b). The final specimens have a gauge length of 4 mm, width of 1.5 mm, and the measured thicknesses at posterior, anterior, and lateral are about 0.25 mm, 0.26 mm, and 0.33 mm, respectively (as shown in Fig. 1c). The hydrated samples were kept in hydrated conditions during preparation and testing. The dehydrated samples were prepared by immersing scales in solutions with different contents of alcohol (30%, 50%, 70%, 90%, and 99%) for 2 minutes at each step, and then air-drying the scales for 24 hours. The water contents of hydrated and dehydrated scales are determined as 38.9 ± 2.5% and
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Fig. 1 Sample preparation for tensile test. (a) Fabricating the specimens; (b) customer-made steel cutter; (c) dimensions of test sample.
4.3 ± 0.7%, respectively, with the use of a moisture analyzer (DSH-50-1, Yue Ping Scientific Instrument Co., Ltd, China). The collagen layer samples were prepared by simply peeling the collagen layer off of hydrated scales, which is possible due to the layered structure and weak interfaces between layers of the scale, particularly at the mineralization front or interface between the bony and collagen layers where the scales tend to delaminate. While the collagen material at the separation site might be partially damaged, the peeling force is insufficient to damage the rest of the collagen layer and to alter its overall mechanical properties[6]. The remaining collagenous material (0.11 mm – 0.13 mm thick) was tested in tension along the different locations The samples were then mounted on a miniature loading stage (MTI Instruments, Inc., USA) with a load transducer of 450 N capacity, which was placed under an upright, reflected light optical microscope (BX-51P, Olympus) equipped with a CCD camera in order to monitor deformations and failure modes of the specimens. Images were captured throughout the entire test every 1 second using the CCD camera. All specimens were loaded in tension at a rate of 0.05 mm·s−1 (corresponding to strain rate of 1.25×10−2 s−1) up to complete failure. 2.2.2 Biaxial tensile test Hydrated whole scale specimens were cut from focus of scales with a cruciform-shaped cutter made of hardened stainless steel. As shown in Fig. 2a, the specimens are 9 mm long and 3 mm wide in both the x and y directions, and the thickness is about 0.43 mm – 0.45 mm. The x-direction is along the anteroposterior axis, while the y-direction is along the
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Fig. 2 Biaxial tensile test. (a) The dimension of cruciform specimen; (b) the specimen with speckle pattern clamped in grips.
dorso-ventral axis. The water on the surface of the scale was first removed by patting the samples with filter paper. Hydrophobic black-paint was then spread on the collagen layer side (basal surface) of the specimen by using an airbrush with a 0.3 mm nozzle to produce a speckle pattern for DIC analysis (Fig. 2b). The painted specimens were immersed in water for 12 hours to fully hydrate the samples. The water content of specimens is measured as 38 % ± 3.5%, which is close to that of hydrated specimens used in uniaxial tests. All biaxial tensile tests were conducted on an In-situ Planar Biaxial Fatigue Testing System (IPBF, CARE Co. Ltd, http://www.care-mc.com/) with a 500 N load sensor and at a loading rate of 0.1125 mm·s−1 (corresponding to a strain rate of 1.25×10−2 s−1). An optical microscope equipped with a 3MP color digital camera was used to acquire digital images at 60 fps (frames per second) and record the deformation of specimens throughout the duration of the test. A modified open-source 2D-DIC MATLAB software (ncorr_v1.2, http://www.ncorr.com/) was used to carry out image analysis and obtain the strain distribution of test samples. 2.2.3 Puncture test The puncture tests were conducted on the same miniature loading stage (MTI Instruments, Inc., USA) with a load transducer of 450 N capacity (Fig. 3a), following the similar setup and procedure described in Ref. [6]. The puncture tests were conducted in a quasistatic loading condition with a relatively slow displacement rate of 0.05 mm·s−1. All the tests were conducted in hydrated condition. In order to investigate the effects of substrate stiffness and needle size on puncture resistance of scales, two different silicon rubbers (cut into 20 mm × 20 mm × 10 mm block to fit in the U-shaped support) as the substrate were applied to simulate the dermis and flesh (Fig. 3b). And two dif-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
ferent sizes of sharp needle (the tip diameters of needles 1 and 2 are ~90 μm, ~50 μm, respectively，as shown in
(a) a
Force, displacement Steel needle to simulate a sharp Fish scale Silicon substrate to simulate Steel base dermis and flesh (c) c Needle 1 (b) b
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Fig. 3c) were used to simulate the puncture of a sharp tooth acting on individual scales. On other one hand, to obtain the stiffness of two different silicon rubbers, the mechanical property parameters of substrates were measured by spherical indention[22], using a 4.66 mm steel ball (Fig. 4a). And the indention was simulated with 2D axisymmetric FEM model by ANSYS software (ANSYS Inc., Houston, Pennsylvania, USA) (Fig. 4b). The silicon rubber was modeled as a Neo-Hookean material[22]. According to the results of fitting between the experiment and finite element method, we lastly determined that the shear modulus μ for hard rubber and soft rubber are 0.65 MPa and 0.37 MPa, respectively. Finally, failure patterns at puncture site were observed by optical microscopy and SEM.
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Fig. 3 The designs of puncture test. (a) Actual setup; (b) schematic diagram[22]; (c) optical microscopy image for needles.
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Fig. 4 (a) Axisymmetric finite element model of the ball indentation test; (b) the model was used to fit the experiment curves, yielding the shear modulus of the Neo-Hookean substrates.
Fig. 5 The hierarchical structure of fish scales from grass carp. (a) A whole fish; (b) overlapping scales; (c) individual scale; (d) cross-section (from posterior to anterior through focus); (e) removal of the bony layer reveals the collagen fibrils, obtained by Scanning Electron Microscopy (SEM); (f) collagen lamellae are arranged in a spiral pattern with an angle of about 28˚ – 31˚; (g) schematics of the spiral pattern.
3.1 The hierarchical structure The grass carp, C. idella, scales display a characteristic hierarchical structure (Fig. 5), similar to many other structural biological materials[23]. The fish is covered with a large number of overlapping scales and any given point on the surface of its body is covered with 3 layers of scales (see Figs. 5a and 5b). An individual scale taken from the central area of the fish is a thin plate with an irregular pentagonal shape (Fig. 5c). Like other teleost fish scales, such as those from C. carpio[17] and M. saxatilis[6], grass carp scales are composed of two layers: a highly mineralized external bony layer and a soft internal collagen layer. Fig. 5d shows a cross-section of a scale along the midline and from the anterior to the posterior ends, including the tapered scale edges and elevated central focus. The thickness ratio of the “bony” layer and “collagen” layer varies along the anteroposterior axis, and at the edges of the scale, there is only bony layer. It was also observed that the bony layer is continuous from the anterior region to the focus, but there are some discontinuities in the bony layer from the focus to the posterior region. At the focus, the bony layer partly embedded in collagen layer, and the thickness of collagen layer reduced to zeros at edge. The internal layer contains collagen fibers, which are arranged in a twist-plywood pattern with an angle of [18] ~28˚ – 31˚ between lamellae (Figs. 5e–5g), lower than that of Carassiusauratus (spiral angle about 60˚)[24] and A. gigas (spiral angle 60˚ – 75˚)[7].
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Fig. 6 The surface characteristics of scales at different areas. (a) Focus; (b) lateral; (c) posterior; (d) anterior.
3.2 Surface morphology The surface morphology of grass carp scales was observed using optical microcopy (Olympus BX-51P microscope in standard mode), as shown in Fig. 6. There are mineralized ridges (or “circuli”) and grooves around the focus (Fig. 6a), as well as gaps in the bony layer (or “radii”) with intrusion of the collagen layer, which spread outwards from the focus to the anterior and posterior areas[8]. Surface features of the ventro-lateral region are similar to those of the dorso-lateral region (Fig. 6b), with circuli and grooves aligned with the anteroposterior axis. The posterior region consists of radii and crescent-shaped granules (with possible hydrodynamic function)[6], and without the formation of circuli (Fig. 6c). The continuity of circuli in the anterior area, which may provide mechanical anchoring of the scale, is interrupted by radii, which may increase scale flexibility[6] (Fig. 6d). The surface morphology at different regions of the scale is different, which affects the mechanical properties of the scale, and will be further discussed in the part of results. 3.3
Tensile behavior and fracture mechanism of scales Fig. 7 shows stress-strain curves for hydrated samples (whole scale and collagen layer) at four scale locations: posterior, anterior, ventro-lateral, and dorso-lateral. In general, a quasi-linear region can be observed on the stress-strain curves for hydrated whole scales at the different locations, and then samples soften slightly before reaching the first peak stress of ~15 MPa – 35 MPa, after which the stress drops suddenly due to fracture of the bony layer and progressive delamination between the bony and collagen layers, as observed by optical microscopy. After this stage, the collagen layer continues to bear the tensile force via re-orientation and pulling out of collagen plies, yielding
step-like patterns on the stress-strain curve until the collagen fibers fracture completely at a strain of 0.3 – 0.4. Similar failure behavior was also observed for scales from A. gigas[15] and M. saxatilis[6]. The weak interface between the external and internal layers of the scale is also found among other biomaterials[25], which can prevent catastrophic failure. The stress-strain curves for hydrated collagen layer samples are different than those for whole scale samples (Fig. 7). In the initial region, the tensile stress increases relatively slowly before reaching the elastic region, which exhibits an increase in slope. The Young’s modulus of the collagen layer is defined as the slope of the curve in the elastic region. Before reaching the tensile strength (peak stress), the stress-strain response exhibits nonlinearity due to random and progressive failure of collagen filaments. Beyond this point, the stress decreases rapidly until complete failure of samples. The stress-strain curves are analyzed to measure the Young’s modulus, tensile strength, ultimate strain, and toughness for all specimens. Toughness is evaluated using the area under the stress-strain curve. Tables 1 and 2 give the mechanical properties of hydrated whole scale and collagen layer samples at four scale locations. The Young’s modulus of whole scale is 1.15 – 1.46 times greater than that of the collage layer. The tensile strength of whole scale is only ~46% – 66% of the strength of the collagen layer samples at the same locations. The difference of the ultimate strain between whole scale and collagen layer samples is insignificant, but the toughness of the collagen layer is higher than of that the whole scale. Scales exhibited in-plane anisotropic mechanical properties in terms of tensile strength, ultimate strain and toughness. The Young’s modulus of scale show no significant difference at four regions, as the Young’s modulus at posterior (Table 2), anterior, ventro-lateral and dorso-lateral locations were 0.30 ± 0.03 GPa, 0.28 ± 0.09 GPa, 0.30 ± 0.08 GPa and 0.30 ± 0.06 GPa, respectively. However, the results[18] suggested that the Young’s modulus are 0.30 ± 0.06 GPa, 0.24 ± 0.03 GPa, 0.27 ± 0.03 GPa respectively at three different directions (0˚, 45˚, 90˚) at focus, so scale shows anisotropic mechanical behavior in-plane. The Young’s modulus, tensile strength, ultimate strain and toughness of collagen layer samples are very similar at each of four different locations and three different directions at focus (Tables 1 and 2), indicating that differences in the me-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
at the ventro-lateral and dorso-lateral locations is higher than that at other locations (Table 1), and this may be associated with the distribution of surface features on
Stress (MPa)
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Stress (MPa)
Stress (MPa)
chanical properties of whole scale samples at the various locations are caused by the bony layer. Further, the ultimate strain of whole scale samples
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Fig. 7 Stress-strain curves of hydrated whole scales and collagen layers at the locations of (a) posterior; (b) anterior; (c) ventro-lateral; (d) dorso-lateral. Table 1 The tensile strength and ultimate strain for hydrated scales, hydrated collagen layers and dehydrated scales Tensile strength (MPa)
Location Focus (0˚)
Hydrated scales *
Ultimate strain (mm/mm)
Hydrated collagen
Dehydrated scales
Hydrated scales
Hydrated collagen
Dehydrated scales
37.36 ± 1.78
68.13 ±3.46
120.42±10.16
0.24 ± 0.07
0.29 ± 0.07
0.15 ± 0.03
Focus (45˚)*
39.21 ± 2.85
68.35 ± 6.45
106.71± 0.52
0.27 ± 0.07
0.31 ± 0.03
0.14 ± 0.02
Focus (90˚)*
38.00 ± 1.93
60.12 ± 6.46
125.10 ± 6.27
0.30 ± 0.05
0.31 ± 0.02
0.17 ± 0.03
Posterior
28.61 ± 2.70
61.95 ± 5.46
76.49 ± 6.82
0.24 ± 0.02
0.29 ± 0.03
0.17 ± 0.03 0.18 ± 0.04
Anterior
34.72 ± 4.94
64.84 ± 5.46
103.30 ± 6.63
0.26 ± 0.05
0.32 ± 0.04
Ventro-lateral
41.21 ± 2.83
63.08 ± 7.61
123.10 ± 5.08
0.33 ± 0.03
0.31 ± 0.05
0.18 ± 0.00
Dorso-lateral
41.38 ± 3.28
62.81 ± 3.67
122.91 ± 3.63
0.33 ± 0.04
0.31 ± 0.06
0.18 ± 0.01
Table 2 The Young’s modulus and toughness for hydrated scales, hydrated collagen layers and dehydrated scales Location
Young’s modulus (GPa)
Toughness (MPa)
Hydrated scales
Hydrated collagen
Dehydrated scales
Hydrated scales
Hydrated collagen
Dehydrated scales
Focus (0˚)*
0.30 ± 0.06
0.20 ± 0.04
2.05 ± 0.16
8.03 ± 0.98
11.16 ± 1.40
16.24 ± 1.53
Focus (45˚)*
0.24 ± 0.03
0.21 ± 0.02
2.35 ± 0.55
9.69 ± 2.04
11.50 ± 1.92
15.07 ± 1.08
Focus (90˚)*
0.27 ± 0.03
0.22 ± 0.01
2.17 ± 0.28
8.20 ± 0.67
11.47 ± 1.90
19.13 ± 3.35
Posterior
0.30 ± 0.03
0.23 ± 0.03
1.29 ± 0.12
5.17 ± 0.87
10.20 ± 0.94
10.39 ± 2.20
Anterior
0.28 ± 0.09
0.19 ± 0.02
1.68 ± 0.30
7.26 ± 0.46
13.10 ± 1.91
14.96 ± 1.63
Ventro-lateral
0.30 ± 0.08
0.22 ± 0.03
2.67 ± 0.26
9.81 ± 0.91
12.25 ± 2.89
19.83 ± 2.66
Dorso-lateral
0.30 ± 0.06
0.23 ± 0.02
2.63 ± 0.34
9.92 ± 1.62
11.86 ± 1.86
17.84 ± 2.01
(* The data are adapted from the Ref. [17] .In Tables 1 and 2, standard two-tailed, unpaired two-sample Student’s t-tests assuming unequal variances were used to compare the mean values reported for the tensile tests.)
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scales. Since gaps (or radii) in the bony layer in the posterior and anterior areas are vertical to the direction of tension, the damage occurs more easily along the radii, such as the failure forms at posterior and anterior. These discontinuities cause both lower tensile strength and ultimate strain in the anterior and posterior areas than in other regions. On the other hand, multiple cracks in the bony layer are formed when the radii are parallel to the direction of tension or are absent. These randomly distributed cracks reduce local damage to the bony layer and increase the deformation of whole scales in tension, so that the failure strains at the ventro-lateral and dorso-lateral locations were up to 0.33 ± 0.03 and 0.33 ± 0.04, respectively, which were higher than at other locations. In fact, the damage mechanism in the lateral regions of scales can be divided into three steps. Firstly, as the existing multiple circuli and grooves are parallel to the direction of tension and radii are absent (as shown in Fig. 8a).The multiple cracks form in the bony layer of the scale as there are some differences on the extension between the bony layer and collagen layer when subjected to tension (Fig. 8b). Subsequently, with an increase in tensile deformation, these cracks continue to propagate and widen until the bony layer debonds from (a) 160
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Fig. 8 The fracture mechanism at the ventro-lateral and dorsolateral for hydrated scales. (a) The intact specimen; (b) microcrack forming; (c) bony layer fracture and debonding from the collagen layer as well as collagen fibers rotation; (d) complete fracture of collagen layer. (b) 160
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the collagen layer and completely failed (Fig. 8c). Lastly, only the collagen layer bears the tensile load and the fibers in the collagen layer start to reorient, delaminate and pull out until complete failure (Fig. 8d). The water content has an important effect on the mechanical behavior of biomaterials[3,26]. Here, we conducted tensile tests on dehydrated whole scale samples from different locations. As shown in Fig. 9, the stress values of dehydrated scales significantly increased
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Fig. 9 Stress-strain curves of hydrate and dehydrated scales at (a) posterior; (b) anterior; (c) ventro-lateral; (d) dorso-lateral.
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
3.4 Biaxial test for individual scales To examined the anisotropic mechanical behavior of grass carp scales. We further conducted the biaxial test for individual scales. As shown in Fig. 10, the stress-strain curves for specimens under biaxial loading are similar to those under uniaxial loading, which exhibits linear elastic behavior before reaching the first stress drop due to the initial failure of the bony layer. Before reaching the peak stress, the stress-strain responses display a saw-tooth pattern caused by the progressive failure of the collagen layer. The failure mechanism of scales under biaxial loading is similar to that under uniaxial loading, and the primary difference is that the bony layer does not fracture simultaneously in both the x and y directions. This is due to the non-uniform surface morphology of the bony layer and weak interface between the bony and collagen layers.
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in the quasi-linear region with a larger slope than that of hydrated scales, and then reached a post-yield (strain hardening) region before a dramatic drop to zero stress due to complete failure. In fact, when the stress decreased rapidly, the bony and collagen layers started to fracture at almost the same time, as observed by optical microscopy, which is different from the failure behavior of hydrated scales, as discussed above. This is due to the fact that dehydrated scales become brittle and the inter face strength between the bony and collagen layers might be enhanced as well. The elastic modulus and ultimate strength in the dehydrated condition dramatically increase compared to that of the hydrated state (Tables 1 and 2). With the lack of water content, the scales become more brittle, and the failure strain of dehydrated scales is reduced to about 50% that of hydrated scales. The elastic moduli of dehydrated scales at the ventro-lateral and dorso-lateral locations (up to 2.67 ± 0.26 GPa and 2.63 ± 0.34 GPa, respectively) are about 9 times higher than those of hydrated samples. Also, the tensile strength of dehydrated scales at the ventro-lateral and dorso-lateral locations (123.10 ± 5.08 MPa and 122.91 ± 3.63 MPa, respectively) is more than 3 times higher than that of hydrated samples. As well, the lowest elastic modulus and strength were measured at the posterior location (1.29 ± 0.12 GPa and 76.49 ± 6.82 MPa, respectively) of dehydrated scales, but are still about 2.4 and 4 times greater than those of hydrated scales, respectively.
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0.4 0.6 Strain (mm/mm)
0.8
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Fig. 10 Stress-strain curves of fish scales under biaxial tensile loading in the (a) x direction; (b) y direction.
Fig. 11 The full-field strain distribution of specimens under biaxial loading: (a) εxx; (b) εyy; (c) εM.
Force (N)
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Fig. 12 Puncture force-displacement curves of hydrated and dehydrated scale at locations of (a) focus; (b) posterior; (c) anterior; (d) ventro-lateral; (e) dorso-lateral. The inserted images are puncture sites of hydrated scales.
The tensile strength and failure strain are 42.02 MPa ± 3.36 MPa and 0.20 ± 0.05 in the x-direction, and 39.12 MPa ± 4.20 MPa and 0.18 ± 0.03 in the y-direction. The Young’s modulus in the x-direction is 0.31 GPa ± 0.25 GPa, which is very close to that of 0.30 GPa ± 0.32 GPa in the y-direction. Upon comparison of these results with the mechanical properties at the focus in the 0˚ and 90˚ directions obtained from the uniaxial tests, there are slight differences in tensile strength and Young’s modulus, but the ultimate strain is about 20% – 50% less due to the biaxial state of stress. In order to investigate the development of full-field and local strains in hydrated whole scale samples during biaxial tensile tests, the strains in the x-direction (εxx), y-direction (εyy), and Von-Mises strain (εM) are obtained with DIC (Fig. 11). As shown in Figs. 12a and 12b, the full-field strains εxx and εyy are uniform at the beginning of tests, and by increasing strain, the strains in the four arms of the specimen alter and become different from those in the central area, which is indicated by the dashed lines. However, the strains in the central area become smaller than those in the arm areas when the strains εxx and εyy get larger than 0.09 or 0.08. Additionally, the Von-Mises strain εM is calculated using the
equation, ε M = 2 / 3 ( ε xx2 + ε yy2 ) + 1 / 3γ xy2 [27]. Fig. 11c shows the full-field distribution of εM from 0.05 to 0.30. It is obvious that the εM of the arm areas increases faster than that of the central region. And the εM at the central area of the specimen is uniform until it is increased up to 0.15, when the collagen layer detached from the bony layer. When the εM at the central region increased further from 0.20 to 0.30, the distribution field is divided into two regions, which are separated in Fig. 11c by the black dashed line with an angle of about 45˚ with respect to the x-direction. This phenomenon may be caused by the fact that the bony layer in the y-direction fails at εyy = 0.1, which is less than the failure strain of the bony layer in the x-direction (εxx = 0.14), as shown in Fig. 11 3.5 Puncture resistance of individual scales As shown in Fig. 12a, at focus of fish scale, the penetrating forces drop down slightly when the damage of bony layers occurs, and then rise up until the whole scales are penetrated completely. However, the puncture forces at the other locations steadily increase until scales are fully penetrated (Figs. 12b–12e). According to Fig. 13, the maximum puncture force is about 21.6 N at focus, which is higher than those at
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
posterior (~15.4 N), anterior (~17.2 N), ventro-lateral (~16.1 N), dorso-lateral (16.4 N), and the ventro-lateral area has a resistance to penetration which is similar to dorso-lateral area. As the thickness varies among different locations of the scale, we introduce the normalized puncture stiffness Kp (that is the slope of force-displacement divided by the thickness at the puncture location) to make a direct comparison. The Kp at the focus of hydrated scale is ~24.78 N·mm−2, which is much larger than those of posterior (~20.7 N·mm−2), anterior (~18.45 N·mm−2), ventro-lateral −2 (~18.02 N·mm ), dorso-lateral (~19.3 N·mm−2). So the focus area of hydrated scales exhibits the best penetration resistance. On the other hand, as compared with the hydrated samples and dehydrated samples, the puncture forces at the same location are found to be similar. The same is true to the deformation as shown in Fig 13. The puncture force of dehydrated samples, however, directly drops down when it reaches the peak. In addition, as shown in Fig. 13c, the Kp at the focus of dehydrated scale is up to 40.7 N·mm−2, which is close to the Kp values at anterior, ventro-lateral, and dorso-lateral (40.5 N·mm−2, 39.8 N·mm−2, 38.4 N·mm−2, respectively), but much higher than that at the anterior (28.8 N·mm−2).The Kp values at the anterior, ventro-lateral, dorso-lateral of dehydrated scales are about two times larger than those of hydrated ones, and about 1.5 and 1.2 times as large as those of the focus and posterior of hydrated ones, respectively. The failure type at puncture site is cross-like for bony layer (Fig. 14a), and the local damage was explained in detail in Ref. [6]. Here, we further investigate the failure of collagen fibers through the SEM image. At the puncture site, the collagen fibers are delaminated and fractured, while the collagen fibers around the puncture site are reoriented (Fig. 14b). Although the collagen
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fibers produced small radical cracks after the bony layer was penetrated, the rotation of fibers, observed in tension[28], twist around the puncture site and prevent the radical cracks from continuing to grow. So the local penetration failure of scale is due to the rotation of spiral pattern collagen fibers. The dehydrated scale becomes brittle because of loss of water contents, and this attributes to the shear failure of scales under puncture. As shown in Fig. 14d, three radical cracks formed in the collagen layer, the fibers fracture occurs without twining compared with the hydrate scales. Furthermore, the failure modes of collagen fibers depend on the water contents but do not on the puncture locations. Fig. 15 shows a comparison of the puncture resistance of hydrated and dehydrated scales tested with (a) 28.00
Hydrated scale
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Fig. 13 Mechanical properties of hydrated and dehydrated scales under puncture. (a) Maximum puncture force; (b) maximum deformation; (c) normalized puncture stiffness.
Fig. 14 The SEM images of (a) hydrated bony layer; (b) hydrated collagen layer; (c) dehydrated bony layer; (d) dehydrated collagen layer at the focus of scales punctured by needle 1 with soft rubber.
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(b) 30
Stiff rubber Soft rubber
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Fig. 15 Puncture force-displacement curves at the focus of scale with different substrates and needles. (a) Hydrated scale and (b) dehydrated scale punctured by needle 1; (c) hydrated scale and (b) dehydrated scale punctured by needle 2.
different substrates and needles. As shown in Figs. 15a and 15c, under hydrated condition, the puncture force-deformation curves of scales exhibit load drops, where the bony layer is penetrated. The behavior resulted from the weak interface between bony layer and collagen layer. Because the tip size of needle 1 is larger than the needle 2, needle 1 brings more deformation of substrate, in other words, the larger size of needle introduces more “blunt damage”. The puncture force of dehydrated scales is very similar to the hydrated scales, but lower deformation of scale is discovered when the same size of needle is used to puncture the scales. The puncture force steadily increases until the whole scale is penetrated (Figs.15b and 15d). Moreover, the puncture force-deformation curves of scales tested on the stiff rubber show a steeper slope than those on the soft rubber, and the soft substrate undergoes more deformation to resist puncture. The maximum puncture forces of hydrated scales are similar for both soft rubber and hard rubber, that is, the stiffness of substrate only affects the maximum puncture force slightly, which is in consistence with the conclusion
given by Zhu et al.[22].
4 Conclusion In this study, we characterized the multiscale structure of grass carp scales and investigated the mechanical behavior in uniaxial, biaxial and punch test with various experimental parameters. The following conclusions can be made: (a) Grass carp scales consist of two layers, a hard outer bony layer and a soft inner collagen layer. There are some discontinuities in the bony layer due to the intrusion of collagen layer. (b) Under uniaxial tensile loading, whole scales exhibit in-plane anisotropic behavior, while the collagen layer displays isotropic behavior. Multiple cracks are formed in the ventro-lateral and dorso-lateral regions allowing for a larger failure strain than in the other regions due to different surface morphology. (c) Under biaxial tensile loading, the bony layer does not fracture simultaneously in both x and y directions, and the tensile strength and the Young’s modulus are similar to those under uniaxial loading, but the fail-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
ure strain gets much higher. (d) According to the results of puncture test, it is found that a needle with a small tip is easy to penetrate the scale because of high stress concentration; while a needle with a larger tip tends to cause large deformation of substrate and may cause blunt injury. Moreover, the peak puncture force is independent to the stiffness of substrate. (e) According to the observations of the puncture failure types, we found the local damage mechanism of hydrated scales subjected to sharp needle is that the bony layer produces radial cracks during the process of penetrating the scales and the cracks cause the occurrence of radial damage in collagen layer. Moreover, the radial damage causes spiral cracks and leads to reorientation of collagen fibers around the fracture region. However, dehydrated scales become more brittle, and shear failure occurs in the bony layer.
Acknowledgment The project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry; and the Joint Foundation of Equipment Development and State Education Ministry for Outstanding Researcher.
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Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales Peng Liu1, Deju Zhu1, Junwen Wang2, Tinh Quoc Bui3 1. College of Civil Engineering, Hunan University, Changsha 410082, China 2. College of Civil Engineering, Tongji University, Shanghai 200092, China 3. Department of Civil and Environmental Engineering, Tokyo Institute of Technology, Tokyo 108-233, Japan
Abstract This work is conducted to investigate the hierarchical structure, mechanical behavior and fracture resistance of grass carp scales with different water contents (hydrated and dehydrated) and load conditions (uniaxial, biaxial and punch tests). The whole cross-section of scales is investigated, and it is found that the bony layer displays discontinuity and partly embeds in collagen layer. Four different locations are considered under both tensile and punch tests. The results of the uniaxial tensile test show a correlation between the failure mode and the distribution of surface morphology on scales. The biaxial test results show that there are minor differences in the tensile strength and the Young’ modulus compared with those of the uniaxial tests, but the ultimate strain is about 20% – 50%. Puncture tests are also conducted with different size of needles and different hardness silicon rubbers as substrate. The results show that the puncture force and deformation are dependent on the size of needle and the hardness of substrate. The failure pattern of scales is related to the water content. Radial cracks occur in the bony layer of hydrated scale, and the collagen fibers twist around the puncture site. However, the shear failure occurs in the bony layer of dehydrated scale. Keywords: grass carp scale, mechanical behavior, biaxial test, penetration resistance Copyright © 2017, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved. doi: 10.1016/S1672-6529(16)60404-3
1 Introduction Natural materials have been drawing people’s attention because of their unique and optimized functions during long term evaluation. Revealing the relationship between structure and material behavior of biological materials can provide valuable information and knowledge to the design of bio-inspired engineering materials in novel ways[1]. These materials like nacre, teeth, and bone, are hard and stiff due to high mineral contents, and display surprising energy absorption ability[2], but they are not flexible and have high density[3]. Other material exoskeletons of crustaceans such as lobster[4], crab[5], contain highly mineralized chitin fibers showing low hardness. Some low mineralized materials like fish scales exhibit both light-weight and local damage in nature. Specially, teleost fish scales (like striped bass) are flexible, and provide a specific resistance to penetration[6]. Corresponding author: Deju Zhu E-mail: [email protected]
The structural and mechanical behaviors of fish scales have been studied, and it is suggested that fish scales can be classified into four types: placoid, ganoid, cosmoid and elasmoid[7]. The observation of morphology of scales can provide some information for classification[8] as well, the non-smooth surface with corrugations is beneficial to enhance flexible strength and redistribute the strain[9]. The hierarchical structure of scales is various, but most scales besides Polypterus senegalusa (four-layer gradient structure: ganoine, dentine, isopedine and bone[10]) have similar hierarchical structures. These structures generally consist of two main layers: a mineralized, hard external layer composed of type I collagen fibrils with a high content of hydroxyapatite[11], and an un-mineralized, soft internal layer composed of type I collagen fibrils[12]. Collagen fibers (bundles of fibrils) are arranged in a twisted plywood pattern with different directions of fibers across lamellae[13,14].
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
The mechanical behavior and failure mode of scale are related to the structure, Ikoma et al.[12] analyzed the mechanical properties of scales from red sea bream, Pagrus major, and showed that the tensile strength is up to 93 MPa, the failure occurs due to the sliding of collagen lamellae and pulling out of collagen fibers. Bruet et al.[10] studied the structure of P. senegalus scales with experiments and finite element simulations, which is indicated that their unique structure and property can inspire new design ideas for modern flexible body amour. As a biomaterial, the mechanical properties of scales are significantly affected by water content[15]. In a hydrated condition, scales from the alligator gar, Atractosteus spatula, and P. major scales have similar ultimate tensile strengths, up to 100 MPa[16] and 93 MPa[12], respectively, which is approximately three times higher than the strength of other scales, such as Arapaima gigas scales (25.2 ± 7.3 MPa)[15], and scales from the common carp, Cyprinuscarpio, (~30 MPa)[3]. When the scales are dried, the tensile strength and modulus of scales dramatically increases, but the failure strain decreases. The tensile strength of dry A. gigas scales is about twice higher than that of hydrated ones, and the elastic modulus is up to 12 times higher[15]. Recently, studies have found one interesting feature as the fish scale behaves anisotropic properties. Zhu et al.[6] experimented the tensile strength of striped bass scales in three directions (0˚, 45˚, 90˚) at the “focus” location, and found that striped bass scales display in-plane anisotropic behavior. Liu et al.[17] further examined and found that both the hydrated and dehydrated grass carp scale reveal an anisotropic behavior at “focus” location. Similar behavior of scales is also observed from the alligator gar, A. spatula[16]. Wang et al.[18] estimated that the structure anisotropic plays an important role in avoiding the indentation attacks with finite element analysis. Gil-Duran et al.[19] found that Megalops Atlanticus scale shows the highest stress strength along with the fish length. Yu et al.[20] examined that the Dabryanus scales are much lower than those of other reported fish scales, and they increase the flexibility. For grass carp, a freshwater species, this is native to lake and rive in Asian. Some researchers have studied the swimming mode and eating inhabits of grass carp. Shi et al.[21] suggested that the low digestive capacity allows the grass carp to maintain its locomotory capacity. In this paper, we describe the whole cross-section of C.
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idella scales and its surface via optical microcopy and Scanning Electron Microscopy (SEM). We investigate the failure types of scale at different location. We also present new results for biaxial tensile tests on scale specimens and show the development of strain distribution with Digital Image Correlation (DIC). A penetration resistance experiment is implemented and failure mechanism is investigated in hydrated and dehydrated condition. Some conclusions drawn from this work are reached, which may facilitate the biomimetic design of a hierarchical protective system with superior performance.
2 Materials and methods 2.1 Materials In this work, the scales are taken from the lateral central area of a grass carp about 5 kg weight, bought from aquaculture market, China. Grass carp scale has about 30 mm in length at longitudinal direction (anteroposterior axis), and about 25 mm in width at transversal direction (ventrodorso axis). The lateral line scales, which characterize a main channel in the center, are not considered in our tests. And five samples are used in each test. 2.2 Sample preparation and test procedures 2.2.1 Uniaxial tensile test The scales were removed from fresh grass carp with tweezers and stored in a freezer at −20 ˚C until tested. Before testing, the scales were taken from the freezer and placed in a water bath at room temperature about 20 ˚C for about 10 minutes for thawing out. The dog-bone-shaped samples are shaped by four areas of the scale involving the posterior, anterior, and lateral (ventro-lateral and dorso-lateral) regions (Fig. 1a) by using a custom-made stainless steel cutter (Fig. 1b). The final specimens have a gauge length of 4 mm, width of 1.5 mm, and the measured thicknesses at posterior, anterior, and lateral are about 0.25 mm, 0.26 mm, and 0.33 mm, respectively (as shown in Fig. 1c). The hydrated samples were kept in hydrated conditions during preparation and testing. The dehydrated samples were prepared by immersing scales in solutions with different contents of alcohol (30%, 50%, 70%, 90%, and 99%) for 2 minutes at each step, and then air-drying the scales for 24 hours. The water contents of hydrated and dehydrated scales are determined as 38.9 ± 2.5% and
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a (a)
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Fig. 1 Sample preparation for tensile test. (a) Fabricating the specimens; (b) customer-made steel cutter; (c) dimensions of test sample.
4.3 ± 0.7%, respectively, with the use of a moisture analyzer (DSH-50-1, Yue Ping Scientific Instrument Co., Ltd, China). The collagen layer samples were prepared by simply peeling the collagen layer off of hydrated scales, which is possible due to the layered structure and weak interfaces between layers of the scale, particularly at the mineralization front or interface between the bony and collagen layers where the scales tend to delaminate. While the collagen material at the separation site might be partially damaged, the peeling force is insufficient to damage the rest of the collagen layer and to alter its overall mechanical properties[6]. The remaining collagenous material (0.11 mm – 0.13 mm thick) was tested in tension along the different locations The samples were then mounted on a miniature loading stage (MTI Instruments, Inc., USA) with a load transducer of 450 N capacity, which was placed under an upright, reflected light optical microscope (BX-51P, Olympus) equipped with a CCD camera in order to monitor deformations and failure modes of the specimens. Images were captured throughout the entire test every 1 second using the CCD camera. All specimens were loaded in tension at a rate of 0.05 mm·s−1 (corresponding to strain rate of 1.25×10−2 s−1) up to complete failure. 2.2.2 Biaxial tensile test Hydrated whole scale specimens were cut from focus of scales with a cruciform-shaped cutter made of hardened stainless steel. As shown in Fig. 2a, the specimens are 9 mm long and 3 mm wide in both the x and y directions, and the thickness is about 0.43 mm – 0.45 mm. The x-direction is along the anteroposterior axis, while the y-direction is along the
3 9 15
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Fig. 2 Biaxial tensile test. (a) The dimension of cruciform specimen; (b) the specimen with speckle pattern clamped in grips.
dorso-ventral axis. The water on the surface of the scale was first removed by patting the samples with filter paper. Hydrophobic black-paint was then spread on the collagen layer side (basal surface) of the specimen by using an airbrush with a 0.3 mm nozzle to produce a speckle pattern for DIC analysis (Fig. 2b). The painted specimens were immersed in water for 12 hours to fully hydrate the samples. The water content of specimens is measured as 38 % ± 3.5%, which is close to that of hydrated specimens used in uniaxial tests. All biaxial tensile tests were conducted on an In-situ Planar Biaxial Fatigue Testing System (IPBF, CARE Co. Ltd, http://www.care-mc.com/) with a 500 N load sensor and at a loading rate of 0.1125 mm·s−1 (corresponding to a strain rate of 1.25×10−2 s−1). An optical microscope equipped with a 3MP color digital camera was used to acquire digital images at 60 fps (frames per second) and record the deformation of specimens throughout the duration of the test. A modified open-source 2D-DIC MATLAB software (ncorr_v1.2, http://www.ncorr.com/) was used to carry out image analysis and obtain the strain distribution of test samples. 2.2.3 Puncture test The puncture tests were conducted on the same miniature loading stage (MTI Instruments, Inc., USA) with a load transducer of 450 N capacity (Fig. 3a), following the similar setup and procedure described in Ref. [6]. The puncture tests were conducted in a quasistatic loading condition with a relatively slow displacement rate of 0.05 mm·s−1. All the tests were conducted in hydrated condition. In order to investigate the effects of substrate stiffness and needle size on puncture resistance of scales, two different silicon rubbers (cut into 20 mm × 20 mm × 10 mm block to fit in the U-shaped support) as the substrate were applied to simulate the dermis and flesh (Fig. 3b). And two dif-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
ferent sizes of sharp needle (the tip diameters of needles 1 and 2 are ~90 μm, ~50 μm, respectively，as shown in
(a) a
Force, displacement Steel needle to simulate a sharp Fish scale Silicon substrate to simulate Steel base dermis and flesh (c) c Needle 1 (b) b
Load cell
Needle
Substrate Needle 2 50 µm
Fig. 3c) were used to simulate the puncture of a sharp tooth acting on individual scales. On other one hand, to obtain the stiffness of two different silicon rubbers, the mechanical property parameters of substrates were measured by spherical indention[22], using a 4.66 mm steel ball (Fig. 4a). And the indention was simulated with 2D axisymmetric FEM model by ANSYS software (ANSYS Inc., Houston, Pennsylvania, USA) (Fig. 4b). The silicon rubber was modeled as a Neo-Hookean material[22]. According to the results of fitting between the experiment and finite element method, we lastly determined that the shear modulus μ for hard rubber and soft rubber are 0.65 MPa and 0.37 MPa, respectively. Finally, failure patterns at puncture site were observed by optical microscopy and SEM.
Force (N)
3 Results and discussion 10 mm
Asymmetry boundary condition
Fig. 3 The designs of puncture test. (a) Actual setup; (b) schematic diagram[22]; (c) optical microscopy image for needles.
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Fig. 4 (a) Axisymmetric finite element model of the ball indentation test; (b) the model was used to fit the experiment curves, yielding the shear modulus of the Neo-Hookean substrates.
Fig. 5 The hierarchical structure of fish scales from grass carp. (a) A whole fish; (b) overlapping scales; (c) individual scale; (d) cross-section (from posterior to anterior through focus); (e) removal of the bony layer reveals the collagen fibrils, obtained by Scanning Electron Microscopy (SEM); (f) collagen lamellae are arranged in a spiral pattern with an angle of about 28˚ – 31˚; (g) schematics of the spiral pattern.
3.1 The hierarchical structure The grass carp, C. idella, scales display a characteristic hierarchical structure (Fig. 5), similar to many other structural biological materials[23]. The fish is covered with a large number of overlapping scales and any given point on the surface of its body is covered with 3 layers of scales (see Figs. 5a and 5b). An individual scale taken from the central area of the fish is a thin plate with an irregular pentagonal shape (Fig. 5c). Like other teleost fish scales, such as those from C. carpio[17] and M. saxatilis[6], grass carp scales are composed of two layers: a highly mineralized external bony layer and a soft internal collagen layer. Fig. 5d shows a cross-section of a scale along the midline and from the anterior to the posterior ends, including the tapered scale edges and elevated central focus. The thickness ratio of the “bony” layer and “collagen” layer varies along the anteroposterior axis, and at the edges of the scale, there is only bony layer. It was also observed that the bony layer is continuous from the anterior region to the focus, but there are some discontinuities in the bony layer from the focus to the posterior region. At the focus, the bony layer partly embedded in collagen layer, and the thickness of collagen layer reduced to zeros at edge. The internal layer contains collagen fibers, which are arranged in a twist-plywood pattern with an angle of [18] ~28˚ – 31˚ between lamellae (Figs. 5e–5g), lower than that of Carassiusauratus (spiral angle about 60˚)[24] and A. gigas (spiral angle 60˚ – 75˚)[7].
Journal of Bionic Engineering (2017) Vol.14 No.2
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b (b)
400 µm
400 µm c (c)
(d) d 3 mm 80 µm
400 µm
Fig. 6 The surface characteristics of scales at different areas. (a) Focus; (b) lateral; (c) posterior; (d) anterior.
3.2 Surface morphology The surface morphology of grass carp scales was observed using optical microcopy (Olympus BX-51P microscope in standard mode), as shown in Fig. 6. There are mineralized ridges (or “circuli”) and grooves around the focus (Fig. 6a), as well as gaps in the bony layer (or “radii”) with intrusion of the collagen layer, which spread outwards from the focus to the anterior and posterior areas[8]. Surface features of the ventro-lateral region are similar to those of the dorso-lateral region (Fig. 6b), with circuli and grooves aligned with the anteroposterior axis. The posterior region consists of radii and crescent-shaped granules (with possible hydrodynamic function)[6], and without the formation of circuli (Fig. 6c). The continuity of circuli in the anterior area, which may provide mechanical anchoring of the scale, is interrupted by radii, which may increase scale flexibility[6] (Fig. 6d). The surface morphology at different regions of the scale is different, which affects the mechanical properties of the scale, and will be further discussed in the part of results. 3.3
Tensile behavior and fracture mechanism of scales Fig. 7 shows stress-strain curves for hydrated samples (whole scale and collagen layer) at four scale locations: posterior, anterior, ventro-lateral, and dorso-lateral. In general, a quasi-linear region can be observed on the stress-strain curves for hydrated whole scales at the different locations, and then samples soften slightly before reaching the first peak stress of ~15 MPa – 35 MPa, after which the stress drops suddenly due to fracture of the bony layer and progressive delamination between the bony and collagen layers, as observed by optical microscopy. After this stage, the collagen layer continues to bear the tensile force via re-orientation and pulling out of collagen plies, yielding
step-like patterns on the stress-strain curve until the collagen fibers fracture completely at a strain of 0.3 – 0.4. Similar failure behavior was also observed for scales from A. gigas[15] and M. saxatilis[6]. The weak interface between the external and internal layers of the scale is also found among other biomaterials[25], which can prevent catastrophic failure. The stress-strain curves for hydrated collagen layer samples are different than those for whole scale samples (Fig. 7). In the initial region, the tensile stress increases relatively slowly before reaching the elastic region, which exhibits an increase in slope. The Young’s modulus of the collagen layer is defined as the slope of the curve in the elastic region. Before reaching the tensile strength (peak stress), the stress-strain response exhibits nonlinearity due to random and progressive failure of collagen filaments. Beyond this point, the stress decreases rapidly until complete failure of samples. The stress-strain curves are analyzed to measure the Young’s modulus, tensile strength, ultimate strain, and toughness for all specimens. Toughness is evaluated using the area under the stress-strain curve. Tables 1 and 2 give the mechanical properties of hydrated whole scale and collagen layer samples at four scale locations. The Young’s modulus of whole scale is 1.15 – 1.46 times greater than that of the collage layer. The tensile strength of whole scale is only ~46% – 66% of the strength of the collagen layer samples at the same locations. The difference of the ultimate strain between whole scale and collagen layer samples is insignificant, but the toughness of the collagen layer is higher than of that the whole scale. Scales exhibited in-plane anisotropic mechanical properties in terms of tensile strength, ultimate strain and toughness. The Young’s modulus of scale show no significant difference at four regions, as the Young’s modulus at posterior (Table 2), anterior, ventro-lateral and dorso-lateral locations were 0.30 ± 0.03 GPa, 0.28 ± 0.09 GPa, 0.30 ± 0.08 GPa and 0.30 ± 0.06 GPa, respectively. However, the results[18] suggested that the Young’s modulus are 0.30 ± 0.06 GPa, 0.24 ± 0.03 GPa, 0.27 ± 0.03 GPa respectively at three different directions (0˚, 45˚, 90˚) at focus, so scale shows anisotropic mechanical behavior in-plane. The Young’s modulus, tensile strength, ultimate strain and toughness of collagen layer samples are very similar at each of four different locations and three different directions at focus (Tables 1 and 2), indicating that differences in the me-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
at the ventro-lateral and dorso-lateral locations is higher than that at other locations (Table 1), and this may be associated with the distribution of surface features on
Stress (MPa)
Stress (MPa)
Stress (MPa)
Stress (MPa)
chanical properties of whole scale samples at the various locations are caused by the bony layer. Further, the ultimate strain of whole scale samples
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Fig. 7 Stress-strain curves of hydrated whole scales and collagen layers at the locations of (a) posterior; (b) anterior; (c) ventro-lateral; (d) dorso-lateral. Table 1 The tensile strength and ultimate strain for hydrated scales, hydrated collagen layers and dehydrated scales Tensile strength (MPa)
Location Focus (0˚)
Hydrated scales *
Ultimate strain (mm/mm)
Hydrated collagen
Dehydrated scales
Hydrated scales
Hydrated collagen
Dehydrated scales
37.36 ± 1.78
68.13 ±3.46
120.42±10.16
0.24 ± 0.07
0.29 ± 0.07
0.15 ± 0.03
Focus (45˚)*
39.21 ± 2.85
68.35 ± 6.45
106.71± 0.52
0.27 ± 0.07
0.31 ± 0.03
0.14 ± 0.02
Focus (90˚)*
38.00 ± 1.93
60.12 ± 6.46
125.10 ± 6.27
0.30 ± 0.05
0.31 ± 0.02
0.17 ± 0.03
Posterior
28.61 ± 2.70
61.95 ± 5.46
76.49 ± 6.82
0.24 ± 0.02
0.29 ± 0.03
0.17 ± 0.03 0.18 ± 0.04
Anterior
34.72 ± 4.94
64.84 ± 5.46
103.30 ± 6.63
0.26 ± 0.05
0.32 ± 0.04
Ventro-lateral
41.21 ± 2.83
63.08 ± 7.61
123.10 ± 5.08
0.33 ± 0.03
0.31 ± 0.05
0.18 ± 0.00
Dorso-lateral
41.38 ± 3.28
62.81 ± 3.67
122.91 ± 3.63
0.33 ± 0.04
0.31 ± 0.06
0.18 ± 0.01
Table 2 The Young’s modulus and toughness for hydrated scales, hydrated collagen layers and dehydrated scales Location
Young’s modulus (GPa)
Toughness (MPa)
Hydrated scales
Hydrated collagen
Dehydrated scales
Hydrated scales
Hydrated collagen
Dehydrated scales
Focus (0˚)*
0.30 ± 0.06
0.20 ± 0.04
2.05 ± 0.16
8.03 ± 0.98
11.16 ± 1.40
16.24 ± 1.53
Focus (45˚)*
0.24 ± 0.03
0.21 ± 0.02
2.35 ± 0.55
9.69 ± 2.04
11.50 ± 1.92
15.07 ± 1.08
Focus (90˚)*
0.27 ± 0.03
0.22 ± 0.01
2.17 ± 0.28
8.20 ± 0.67
11.47 ± 1.90
19.13 ± 3.35
Posterior
0.30 ± 0.03
0.23 ± 0.03
1.29 ± 0.12
5.17 ± 0.87
10.20 ± 0.94
10.39 ± 2.20
Anterior
0.28 ± 0.09
0.19 ± 0.02
1.68 ± 0.30
7.26 ± 0.46
13.10 ± 1.91
14.96 ± 1.63
Ventro-lateral
0.30 ± 0.08
0.22 ± 0.03
2.67 ± 0.26
9.81 ± 0.91
12.25 ± 2.89
19.83 ± 2.66
Dorso-lateral
0.30 ± 0.06
0.23 ± 0.02
2.63 ± 0.34
9.92 ± 1.62
11.86 ± 1.86
17.84 ± 2.01
(* The data are adapted from the Ref. [17] .In Tables 1 and 2, standard two-tailed, unpaired two-sample Student’s t-tests assuming unequal variances were used to compare the mean values reported for the tensile tests.)
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scales. Since gaps (or radii) in the bony layer in the posterior and anterior areas are vertical to the direction of tension, the damage occurs more easily along the radii, such as the failure forms at posterior and anterior. These discontinuities cause both lower tensile strength and ultimate strain in the anterior and posterior areas than in other regions. On the other hand, multiple cracks in the bony layer are formed when the radii are parallel to the direction of tension or are absent. These randomly distributed cracks reduce local damage to the bony layer and increase the deformation of whole scales in tension, so that the failure strains at the ventro-lateral and dorso-lateral locations were up to 0.33 ± 0.03 and 0.33 ± 0.04, respectively, which were higher than at other locations. In fact, the damage mechanism in the lateral regions of scales can be divided into three steps. Firstly, as the existing multiple circuli and grooves are parallel to the direction of tension and radii are absent (as shown in Fig. 8a).The multiple cracks form in the bony layer of the scale as there are some differences on the extension between the bony layer and collagen layer when subjected to tension (Fig. 8b). Subsequently, with an increase in tensile deformation, these cracks continue to propagate and widen until the bony layer debonds from (a) 160
(b)
(a)
(d)
(c)
Fig. 8 The fracture mechanism at the ventro-lateral and dorsolateral for hydrated scales. (a) The intact specimen; (b) microcrack forming; (c) bony layer fracture and debonding from the collagen layer as well as collagen fibers rotation; (d) complete fracture of collagen layer. (b) 160
Whole scale (Posterior) Dehydrated Hydrated
120
the collagen layer and completely failed (Fig. 8c). Lastly, only the collagen layer bears the tensile load and the fibers in the collagen layer start to reorient, delaminate and pull out until complete failure (Fig. 8d). The water content has an important effect on the mechanical behavior of biomaterials[3,26]. Here, we conducted tensile tests on dehydrated whole scale samples from different locations. As shown in Fig. 9, the stress values of dehydrated scales significantly increased
120
80
80
40
40
0 0.0
0.1
(c) 160
0.2 0.3 Strain (mm/mm)
0.4
0.5
0
80
40
40
0.1
0.2 0.3 Strain (mm/mm)
0.1
0.4
0.5
0
0.2 0.3 Strain (mm/mm)
0.4
0.5
Whole scale (Dorso-lateral) Dehydrated Hydrated
120
80
0 0.0
0.0
(d) 160
Whole scale (Ventro-lateral) Dehydrated Hydrated
120
Whole scale (Anterior) Dehydrated Hydrated
0.0
0.1
0.2 0.3 Strain (mm/mm)
0.4
0.5
Fig. 9 Stress-strain curves of hydrate and dehydrated scales at (a) posterior; (b) anterior; (c) ventro-lateral; (d) dorso-lateral.
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
3.4 Biaxial test for individual scales To examined the anisotropic mechanical behavior of grass carp scales. We further conducted the biaxial test for individual scales. As shown in Fig. 10, the stress-strain curves for specimens under biaxial loading are similar to those under uniaxial loading, which exhibits linear elastic behavior before reaching the first stress drop due to the initial failure of the bony layer. Before reaching the peak stress, the stress-strain responses display a saw-tooth pattern caused by the progressive failure of the collagen layer. The failure mechanism of scales under biaxial loading is similar to that under uniaxial loading, and the primary difference is that the bony layer does not fracture simultaneously in both the x and y directions. This is due to the non-uniform surface morphology of the bony layer and weak interface between the bony and collagen layers.
(a) 50
Stress (MPa)
40 30 20 10 0
0.0
0.1
(b) 50
0.2 Strain (mm/mm)
0.3
0.4
40 Stress (MPa)
in the quasi-linear region with a larger slope than that of hydrated scales, and then reached a post-yield (strain hardening) region before a dramatic drop to zero stress due to complete failure. In fact, when the stress decreased rapidly, the bony and collagen layers started to fracture at almost the same time, as observed by optical microscopy, which is different from the failure behavior of hydrated scales, as discussed above. This is due to the fact that dehydrated scales become brittle and the inter face strength between the bony and collagen layers might be enhanced as well. The elastic modulus and ultimate strength in the dehydrated condition dramatically increase compared to that of the hydrated state (Tables 1 and 2). With the lack of water content, the scales become more brittle, and the failure strain of dehydrated scales is reduced to about 50% that of hydrated scales. The elastic moduli of dehydrated scales at the ventro-lateral and dorso-lateral locations (up to 2.67 ± 0.26 GPa and 2.63 ± 0.34 GPa, respectively) are about 9 times higher than those of hydrated samples. Also, the tensile strength of dehydrated scales at the ventro-lateral and dorso-lateral locations (123.10 ± 5.08 MPa and 122.91 ± 3.63 MPa, respectively) is more than 3 times higher than that of hydrated samples. As well, the lowest elastic modulus and strength were measured at the posterior location (1.29 ± 0.12 GPa and 76.49 ± 6.82 MPa, respectively) of dehydrated scales, but are still about 2.4 and 4 times greater than those of hydrated scales, respectively.
363
30 20 10 0
0.0
0.2
0.4 0.6 Strain (mm/mm)
0.8
1.0
Fig. 10 Stress-strain curves of fish scales under biaxial tensile loading in the (a) x direction; (b) y direction.
Fig. 11 The full-field strain distribution of specimens under biaxial loading: (a) εxx; (b) εyy; (c) εM.
Force (N)
Force (N)
Force (N)
Force (N)
Force (N)
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Fig. 12 Puncture force-displacement curves of hydrated and dehydrated scale at locations of (a) focus; (b) posterior; (c) anterior; (d) ventro-lateral; (e) dorso-lateral. The inserted images are puncture sites of hydrated scales.
The tensile strength and failure strain are 42.02 MPa ± 3.36 MPa and 0.20 ± 0.05 in the x-direction, and 39.12 MPa ± 4.20 MPa and 0.18 ± 0.03 in the y-direction. The Young’s modulus in the x-direction is 0.31 GPa ± 0.25 GPa, which is very close to that of 0.30 GPa ± 0.32 GPa in the y-direction. Upon comparison of these results with the mechanical properties at the focus in the 0˚ and 90˚ directions obtained from the uniaxial tests, there are slight differences in tensile strength and Young’s modulus, but the ultimate strain is about 20% – 50% less due to the biaxial state of stress. In order to investigate the development of full-field and local strains in hydrated whole scale samples during biaxial tensile tests, the strains in the x-direction (εxx), y-direction (εyy), and Von-Mises strain (εM) are obtained with DIC (Fig. 11). As shown in Figs. 12a and 12b, the full-field strains εxx and εyy are uniform at the beginning of tests, and by increasing strain, the strains in the four arms of the specimen alter and become different from those in the central area, which is indicated by the dashed lines. However, the strains in the central area become smaller than those in the arm areas when the strains εxx and εyy get larger than 0.09 or 0.08. Additionally, the Von-Mises strain εM is calculated using the
equation, ε M = 2 / 3 ( ε xx2 + ε yy2 ) + 1 / 3γ xy2 [27]. Fig. 11c shows the full-field distribution of εM from 0.05 to 0.30. It is obvious that the εM of the arm areas increases faster than that of the central region. And the εM at the central area of the specimen is uniform until it is increased up to 0.15, when the collagen layer detached from the bony layer. When the εM at the central region increased further from 0.20 to 0.30, the distribution field is divided into two regions, which are separated in Fig. 11c by the black dashed line with an angle of about 45˚ with respect to the x-direction. This phenomenon may be caused by the fact that the bony layer in the y-direction fails at εyy = 0.1, which is less than the failure strain of the bony layer in the x-direction (εxx = 0.14), as shown in Fig. 11 3.5 Puncture resistance of individual scales As shown in Fig. 12a, at focus of fish scale, the penetrating forces drop down slightly when the damage of bony layers occurs, and then rise up until the whole scales are penetrated completely. However, the puncture forces at the other locations steadily increase until scales are fully penetrated (Figs. 12b–12e). According to Fig. 13, the maximum puncture force is about 21.6 N at focus, which is higher than those at
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
posterior (~15.4 N), anterior (~17.2 N), ventro-lateral (~16.1 N), dorso-lateral (16.4 N), and the ventro-lateral area has a resistance to penetration which is similar to dorso-lateral area. As the thickness varies among different locations of the scale, we introduce the normalized puncture stiffness Kp (that is the slope of force-displacement divided by the thickness at the puncture location) to make a direct comparison. The Kp at the focus of hydrated scale is ~24.78 N·mm−2, which is much larger than those of posterior (~20.7 N·mm−2), anterior (~18.45 N·mm−2), ventro-lateral −2 (~18.02 N·mm ), dorso-lateral (~19.3 N·mm−2). So the focus area of hydrated scales exhibits the best penetration resistance. On the other hand, as compared with the hydrated samples and dehydrated samples, the puncture forces at the same location are found to be similar. The same is true to the deformation as shown in Fig 13. The puncture force of dehydrated samples, however, directly drops down when it reaches the peak. In addition, as shown in Fig. 13c, the Kp at the focus of dehydrated scale is up to 40.7 N·mm−2, which is close to the Kp values at anterior, ventro-lateral, and dorso-lateral (40.5 N·mm−2, 39.8 N·mm−2, 38.4 N·mm−2, respectively), but much higher than that at the anterior (28.8 N·mm−2).The Kp values at the anterior, ventro-lateral, dorso-lateral of dehydrated scales are about two times larger than those of hydrated ones, and about 1.5 and 1.2 times as large as those of the focus and posterior of hydrated ones, respectively. The failure type at puncture site is cross-like for bony layer (Fig. 14a), and the local damage was explained in detail in Ref. [6]. Here, we further investigate the failure of collagen fibers through the SEM image. At the puncture site, the collagen fibers are delaminated and fractured, while the collagen fibers around the puncture site are reoriented (Fig. 14b). Although the collagen
365
fibers produced small radical cracks after the bony layer was penetrated, the rotation of fibers, observed in tension[28], twist around the puncture site and prevent the radical cracks from continuing to grow. So the local penetration failure of scale is due to the rotation of spiral pattern collagen fibers. The dehydrated scale becomes brittle because of loss of water contents, and this attributes to the shear failure of scales under puncture. As shown in Fig. 14d, three radical cracks formed in the collagen layer, the fibers fracture occurs without twining compared with the hydrate scales. Furthermore, the failure modes of collagen fibers depend on the water contents but do not on the puncture locations. Fig. 15 shows a comparison of the puncture resistance of hydrated and dehydrated scales tested with (a) 28.00
Hydrated scale
Dehydrated scale
21.00 14.00 7.00 0.00
Focus
Posterior Anterior Sample location
Ventrolateral
Dorsolateral
(b) 6.00
Hydrated scale
Dehydrated scale
4.50 3.00 1.50 0.00
Focus
Posterior Anterior Sample location
(c) 50.00
Hydrated scale
Ventrolateral
Dorsolateral
Dehydrated scale
37.50 25.00 12.50 0.00
Focus
Posterior Anterior Sample location
Ventrolateral
Dorsolateral
Fig. 13 Mechanical properties of hydrated and dehydrated scales under puncture. (a) Maximum puncture force; (b) maximum deformation; (c) normalized puncture stiffness.
Fig. 14 The SEM images of (a) hydrated bony layer; (b) hydrated collagen layer; (c) dehydrated bony layer; (d) dehydrated collagen layer at the focus of scales punctured by needle 1 with soft rubber.
Journal of Bionic Engineering (2017) Vol.14 No.2
366 (a) 30
(b) 30
Stiff rubber Soft rubber
20
20
10
10
Stiff rubber Soft rubber
Hydrated scale Needle 1 0
0
1
(c)16
2 3 Displacement (mm)
4
Dehydrated scale Needle 1 5
Stiff rubber Soft rubber
0
0
2 3 Displacement (mm)
12
8
8
4
0
1
2 3 Displacement (mm)
4
5
5
Dehydrated scale Needle 2
Hydrated scale Needle 2 0
4
Stiff rubber Soft rubber
12
4
1
(d)16
0
0
1
2 3 Displacement (mm)
4
5
Fig. 15 Puncture force-displacement curves at the focus of scale with different substrates and needles. (a) Hydrated scale and (b) dehydrated scale punctured by needle 1; (c) hydrated scale and (b) dehydrated scale punctured by needle 2.
different substrates and needles. As shown in Figs. 15a and 15c, under hydrated condition, the puncture force-deformation curves of scales exhibit load drops, where the bony layer is penetrated. The behavior resulted from the weak interface between bony layer and collagen layer. Because the tip size of needle 1 is larger than the needle 2, needle 1 brings more deformation of substrate, in other words, the larger size of needle introduces more “blunt damage”. The puncture force of dehydrated scales is very similar to the hydrated scales, but lower deformation of scale is discovered when the same size of needle is used to puncture the scales. The puncture force steadily increases until the whole scale is penetrated (Figs.15b and 15d). Moreover, the puncture force-deformation curves of scales tested on the stiff rubber show a steeper slope than those on the soft rubber, and the soft substrate undergoes more deformation to resist puncture. The maximum puncture forces of hydrated scales are similar for both soft rubber and hard rubber, that is, the stiffness of substrate only affects the maximum puncture force slightly, which is in consistence with the conclusion
given by Zhu et al.[22].
4 Conclusion In this study, we characterized the multiscale structure of grass carp scales and investigated the mechanical behavior in uniaxial, biaxial and punch test with various experimental parameters. The following conclusions can be made: (a) Grass carp scales consist of two layers, a hard outer bony layer and a soft inner collagen layer. There are some discontinuities in the bony layer due to the intrusion of collagen layer. (b) Under uniaxial tensile loading, whole scales exhibit in-plane anisotropic behavior, while the collagen layer displays isotropic behavior. Multiple cracks are formed in the ventro-lateral and dorso-lateral regions allowing for a larger failure strain than in the other regions due to different surface morphology. (c) Under biaxial tensile loading, the bony layer does not fracture simultaneously in both x and y directions, and the tensile strength and the Young’s modulus are similar to those under uniaxial loading, but the fail-
Liu et al.: Structure, Mechanical Behavior and Puncture Resistance of Grass Carp Scales
ure strain gets much higher. (d) According to the results of puncture test, it is found that a needle with a small tip is easy to penetrate the scale because of high stress concentration; while a needle with a larger tip tends to cause large deformation of substrate and may cause blunt injury. Moreover, the peak puncture force is independent to the stiffness of substrate. (e) According to the observations of the puncture failure types, we found the local damage mechanism of hydrated scales subjected to sharp needle is that the bony layer produces radial cracks during the process of penetrating the scales and the cracks cause the occurrence of radial damage in collagen layer. Moreover, the radial damage causes spiral cracks and leads to reorientation of collagen fibers around the fracture region. However, dehydrated scales become more brittle, and shear failure occurs in the bony layer.
Acknowledgment The project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry; and the Joint Foundation of Equipment Development and State Education Ministry for Outstanding Researcher.
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