Wild boar's tusk enamel: Structure and mechanical behavior

Accepted Manuscript Wild boar's tusk enamel: Structure and mechanical behavior

Xu Wang, Nan Zhang, Yujie Zhong, Fuxue Yan, Bailing Jiang PII: DOI: Reference:

S0928-4931(19)30299-1 https://doi.org/10.1016/j.msec.2019.03.017 MSC 9526

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

23 January 2019 22 February 2019 4 March 2019

Please cite this article as: X. Wang, N. Zhang, Y. Zhong, et al., Wild boar's tusk enamel: Structure and mechanical behavior, Materials Science & Engineering C, https://doi.org/ 10.1016/j.msec.2019.03.017

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ACCEPTED MANUSCRIPT Wild boar's tusk enamel: Structure and mechanical behavior Xu Wang*,1, Nan Zhang1, Yujie Zhong2, Fuxue Yan1, Bailing Jiang1 1

School of Materials Science and Engineering, Xi’an University of Technology, 5

South Jinhua Road, Xi’an 710048, China School of Materials Science and Engineering, Xi’an Shiyou University, Xi’an

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2

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710065, China

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Abstract: Natural bio-ceramics have attracted extensive interests due to its high

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strength and high toughness, which can hardly be achieved in artificial ceramics simultaneously. In this work, the microstructure and properties of the wild boar's tusk

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enamel were investigated. The enamel was found to exhibit a hierarchical structure ranging from the hydroxyapatite (HAP) fibers (single or poly-crystals, nano-scale),

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enamel rods (micro-scale), enamel types (meso-scale) to enamel patterns (macro-scale). It is worth mentioning that the high-density and high-order hierarchical

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nanotwins were observed in the HAP fibers. The mechanical properties of the wild boar's tusk enamel showed strong anisotropy and were higher along the longitudinal

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direction than along the transverse direction. The mechanical properties varied from the dentin-enamel junction (DEJ) to the outer surface. The elastic modulus increased with the distance from the DEJ and then kept invariant. The nano-hardness increased in inner enamel but decreased in outer enamel. There was a peak of nano-hardness in inner enamel area. The fracture toughness showed an opposite tendency. It exhibited high values in inner enamel, but fell in the outer enamel zone. The irregular, 1

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decussating texture of the enamel, as well as the nanotwins/hierarchical nanotwins was considered as the main reason for its excellent mechanical properties. These unique structures of the wild boar's tusk enamel are expected to cast light on the design of medical materials and provide some guidelines to improve their mechanical

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properties.

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Keywords: Wild boar's tusk enamel; Hierarchical structure; HAP; Hierarchical

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nanotwins; Mechanical properties; *

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Corresponding authors. E-mail address: [email protected] (X. Wang)

1. Introduction

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Artificial ceramics have been widely used at extreme conditions due to their superior properties of strength, stiffness, microstructure stability and excellent

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wear-resistance [1-3]. However, the brittleness of synthetic ceramics restricts their wide applications [1, 2]. In comparison, natural bio-ceramics have involved in

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complex and ingenious structure characteristics to fulfill both high strength and high toughness, which are hard to be achieved in artificial ceramics [4-6]. Therefore, it has

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attracted extensive interests and provided useful inspirations for the fabrication of engineering materials [7-10]. The microstructures and properties of tooth have been deeply studied for different species such as human [11], bovine [12, 13], molar of pedetes cafer [12], shark [14] and pig molar cusps [15]. It was found that the teeth were comprised of an internal dentin layer and an external enamel layer [16, 17]. The external enamel was 2

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self-assembled into hierarchical structure, which was comprised of hydroxyapatite (HAP) fibers (nano-), enamel rods (micro-), enamel types (meso-) and enamel patterns (macro-scales) [18]. The first level, HAP fiber, was measured to have a length of ~250-1000 nm extending from dentin-enamel junction (DEJ) to outer

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enamel surface [19]. The HAP fibers were self-assembled parallelly to form the

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enamel rods (the second level) [20]. The enamel rod was the main force unit of the

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enamel and had a diameter of ~5 μm [18]. The section shape of the enamel rod

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presented the hexagon, circular, tainter and keyhole-like section shape, showing a significant difference from all other kinds of animal's teeth [13]. A layer of interrod,

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which existed in the adjacent enamel rods usually surrounded the enamel rods. The enamel rods built different enamel types (the third level) with different arrangements

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and/or orientations, such as radial enamel, decussating enamel, irregular enamel and tangential enamel [12]. Then, the enamel (the forth level) was formed by diverse

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enamel types in three-dimensional space. Similar structures were also found in the seashells [21, 22]. The enamel layer usually shows excellent hardness, good wear

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resistance and high elastic modulus [7, 23] due to its high mineralization degree and gradient structure [24] with various toughening mechanism. For instance, giant panda fed almost on bamboo while the stems of bamboo had high stiffness and strength. In particularly, the tensile strength of bamboo was about six times higher than that of mild steel [25]. Besides, outer surface of shark teeth was enameloid, in which HAP crystals with different sizes formed bunchy structure and self-assembled together by 3

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specific ways to maintain high stress concentration. The research showed that the bite force of shark teeth was three hundred times higher than that of the human teeth [14]. Even different location of a tooth also showed various microstructures. An excellent case was the blarina brevicauda [26], whose enamel was composed of external

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colored enamel and internal colorless enamel. The enamel rods were vertical to each

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other in the internal colorless enamel whereas the HAP fibers were bundled together

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but had no formation of column structure in the external colored enamel. These teeth

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played an important role in masticating food. However, the wild boar's tusk usually acts as a natural attacking weapon. Different from the eating teeth, it was mainly used

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to capture, hunt, break down foods, fight and self-defense. The enamel needs to endure enormous impact and load during service.

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The most key role of the nature weapons was to attack and defense. For example, the scales of teleost fish and pangolin [27-29] exhibited good properties of

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wear-resistance and anti-adhesion against soil/rocks to protect their body from predatory threats [30]. The squid beak was well known for its remarkable hardness

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and large stiffness gradient [31]. In addition, the crayfish mandible was a typical example of such weapons. It was usually used to grasp, cut or crush foods, fight against enemies and suck their fluids in some insects, such as bugs, mosquitos and lice [32, 33]. The perfect combination of hardness and stiffness can principally increase attack and defense efficiency of nature weapon. The tusks, as the only weapons of the wild boar, serve throughout its life. The performance of the protect 4

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layer, i.e. the enamel, is of critical importance. It is necessary to explore the microstructure and damage mechanism of the wild boar's tusk enamel in a deeper degree. The aim of present work is to clarify the enamel structure of the wild boar's tusk

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hydration

conditions

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loading

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by

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under

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and investigate the changing trend of nano-hardness and elastic modulus of enamel

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nano-indentation tests, as well as analyze the damage mechanisms of enamel by

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three-point bending tests. Meanwhile, the underlying relationship between mechanical properties and microstructure will be discussed. This research is expected to provide

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some illuminating insights and new directions for developing high-performance

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2. Material and methods

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synthetic materials.

Wild boar's tusks were purchased from individual suppliers. The tusk of wild

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boar was sectioned along transverse and longitudinal direction with a STX-202A water-cooled low-speed diamond wire-endless saw (Shenyang Kejing, China). The

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section samples were ground using SiC-paper of 1200#, 1500# and 2000#, and carefully polished using diamond polishing paste with size of 2.5 μm. They were etched in 10 wt% phosphoric acid (H3PO4) solution for 1 min, washed with distilled water and dried immediately. Thin occlusal surface specimens for transmission electron microscopy (TEM) investigation were cut from the top of the tusk, and then carefully ground to about 30 μm, and ion beaming milled at 5 kV. 5

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For mechanical characterization, the transverse and longitudinal samples of the wild boar's tusk were prepared under dry and wet conditions, respectively. Some samples were dried in air at room temperature for at least 18 h. The others were placed in distilled water at least 18 h. Nano-indentation tests were performed along

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parallel and perpendicular to the axis of the enamel rods at the load of 20 gf using an

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Agilent G 200 Nano Indenter (Agilent Technologies, Oak Ridge, TN, USA). A rate of

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10 nm/s was used for the loading/unloading process and the hold time was set as 10s.

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A total of 7 samples were measured, respectively. Specimens in dimensions of 25 × 4 × 2 mm3 were cut from the tusk with a wire-endless diamond saw. Three-point

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bending tests were performed with a gauge length of 20 mm at room temperature at a constant displacement rate of 0.5 mm/min using an Instron E 1000 testing machine

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(Changchun Machine, China). The loading direction was perpendicular to the occlusal surface. A total of 5 samples were tested.

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The phase constituent of whole enamel layer was identified with a step-scanning X-ray diffraction (XRD, 7000, Shimadzu, Japan). The microstructure of enamel

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patterns were observed by scanning electron microscopy (SEM, LEO Supra 35, Cral, Zeiss, Germany). A 200 kV Tecnai G2 F20 TEM (FEI, Eindhoven, the Netherlands) was used for electron diffraction analysis and high-resolution TEM (HRTEM) observation. Fast Fourier transformation (FFT) was carried out using the Digital Micrograph package (Gatan, California, USA). 3. Results and discussion 6

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3.1 Hierarchical structure of the wild boar's tusk enamel Fig. 1a shows the appearance of an adult wild boar's tusks. This pair of tusks is well maintained without any decay, and has a length of ~ 15cm. The transverse, longitudinal and occlusal surface of the tusk is indicated in Fig. 1b. Fig. 1c shows the

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longitudinal section of the tusk. The overall section is primarily divided into three

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parts: enamel, dentin and pulp chamber. As shown by the black line, the thickness of

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the outmost enamel is about 200 ± 50 μm. Dentin is the secondary outer layer (the

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area between the black lines and the white lines), which accounts for more than 80% of the total volume of tusk, and is the main carrying organ of the tusk. The inner layer

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is the pulp chamber (inside the white lines). This part is hollow due to the tissue loss during preservation.

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The phase constituents of the enamel is determined by the XRD patterns and displayed in Fig. 2. The enamel is composed of hydroyapatite (HAP) only. It is

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polycrystalline but shows strong <0001> growth direction. Fig. 3a shows the microstructure of the overall tusk. The enamel area, dentin area

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and the DEJ are clearly presented. In the enamel area, the arrangement of enamel rods markedly differs along transverse section (Fig. 3c). Consequently, for easy description, the overall enamel layer is mechanically divided into two parts: the inner enamel and the outer enamel. In the outer enamel, the enamel rods are parallel to each other and perpendicular to the occlusal surface (Fig. 3b). In the inner enamel, the enamel rods are not parallel to each other, but are curved or crossed at a certain angle (Fig. 3d). 7

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There are two characteristic zones, labelled as para-zone (P-zone) and dia-zone (D-zone), existing in the inner enamel area (Fig. 3e). The central axial directions of enamel rods are perpendicular and parallel to the outer surface in the P-zone and D-zone, respectively. Namely, the HAP fibers in P-zone and D-zone are perpendicular

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to each other. The DEJ, as shown in Fig. 3f, is not a straight line but is connected by

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many small concave arcs on the dentin side. This kind of connection effectively

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increases the interface areas between enamel and dentin area, making the two tissues

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bonded more firmly. It is similar to what other researchers have reported in other animal's teeth [34].

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Fig. 4a shows the microstructure of occlusal surface. It presents fish-scale-like patterns. The section of enamel rod approximates to round shape with a diameter of

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~5.0 μm (as shown by the white dotted line in Fig. 4a). The enamel rods are surrounded by a layer of interrod (as pointed by the yellow arrows). The different

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arrangement of the HAP fibers in enamel rods and adjacent interrods are displayed in Fig. 4b and c. It is observed that the HAP fibers in the enamel rods are parallel to the

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axial direction of the enamel rods in the near central axis. As they move away from the central axis, the HAP fibers obviously deflect toward the adjacent interrods. However, in the interrod area, these HAP fibers have experienced significant rotation and are nearly perpendicular to the axis of the enamel rod, as exhibited in Fig. 4c. In order to further investigate the microstructure of the enamel, thin film samples are prepared for TEM study. Fig. 5a presents the TEM micrograph of HAP fibers of 8

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occlusal surface. With the relatively soft protein etched, the HAP fibers (as circled by the white dotted lines) can be clearly observed. According to the diffraction patterns shown in Fig. 5b, it can be obtained that these HAP fibers show different growth directions. Fig. 5c shows the morphology of a typical single HAP fiber. It has a

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diameter of ~500 nm and the corresponding growth direction is [0001], which is in

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agree with the XRD results that the enamel showing strong <0001> growth direction.

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(as pointed by the yellow arrows in Fig. 5a).

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However, it is worth to note that high-density nanotwins are found in the HAP fibers

Fig. 6a exhibits the TEM image of nanotwins in a HAP fiber. The nanotwins are

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widespread in the HAP fibers and end at the boundaries of HAP fibers. The HRTEM micrograph of the representative nanotwin is shown in Fig. 6b. It turns out that the

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( 1121 ) symmetry plane and the twin are symmetrical tilted by 68.8o with reference to the ( 1121 ) twin plane. The Fig. 6c presents the corresponding selected area electron

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diffraction patterns. The incident index is calibrated along [ 1100 ] twining axis. Fig. 6d is the filtered image of the nanotwin shown in Fig. 6b. The crystal lattices on two

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sides of twin boundaries show a slight mismatch implying that twins may not be perfectly symmetrical at the atomic level. The above structural characteristics indicate that there are huge imperfections in the gradient structure of the tusk even down to the atomic scale. As shown in Fig. 7a, some irregular nanotwins and high-order hierarchical nanotwins (pointed by the yellow arrows) are randomly distributed in the HAP fibers. 9

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These high-order hierarchical nanotwins still grow along the ( 1121 ) twin plane, as illustrated in Fig. 7b and c. Based on the investigation above, the structure of the wild boar's tusk enamel can be illustrated by Fig. 8. It is self-assembled into hierarchical structure from the HAP

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fibers (single or poly-crystals, nano-scale), enamel rods (micro-scale), enamel types

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(meso-scale) to enamel patterns (macro-scale). The nano HAP fiber is the first level.

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They are bonded together to form the hierarchy structure of enamel rods. The

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arrangements of enamel rods especially with varying orientations, result in decussating bands on the large length-scale. However, as elucidated above, what

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worthy of mentioning is the existence of high-density nanotwins and high-order hierarchical nanotwins in the HAP fibers. Actually, various imperfections, such as

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vacancies, dislocations and nanotwins commonly exist in solid materials [35]. Akin to alloying, low-energy imperfections, such as the twins could tune the properties of

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materials [36].

3.2 Mechanical properties of the wild boar's tusk enamel

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3.2.1 Nano-hardness and elastic modulus Because there are many proteins in the enamel patterns and these proteins usually have effect on the strength and toughness [37, 38], a series of nano-indentation tests are carried out under dry and wet conditions, respectively. As shown in Fig. 9, the nano-hardness and elastic modulus show larger values along the longitudinal (parallel to the enamel rod's axial direction, L) directions than along the 10

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transverse (perpendicular to the enamel rod's axial direction, T) directions and present strong anisotropy under both conditions. The values of nano-hardness and elastic modulus along L (T) directions are 4.23 ± 0.2 GPa and 92.2 ± 3.8 GPa (3.44 ± 0.15 GPa and 45.17 ± 1.54 GPa), respectively.

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Note that the nano-indentation tests in the perpendicular directions to the enamel

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rod are done at distance of ~80 μm from DEJ. According to Fig. 2 and Fig. 5, the

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enamel rods are polycrystalline but show a strong growth tendency along <0001>

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direction. Similar to directionally solidified Ni-based superalloy, there are little transverse grain boundaries but many longitudinal grain boundaries along the growth

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directions of the enamel rods. Therefore, the enamel exhibits strong anisotropy in mechanical properties. However, the nano-hardness and elastic modulus of the enamel

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under dry condition are higher than that under the wet condition in both directions. This is because that the organic materials become soft under the wet condition. The

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nano-hardness and elastic modulus decrease naturally. 3.2.2 Evolution of the mechanical properties along growth direction of the

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enamel

According to Fig. 3, the arrangement of the enamel rods is quite different from DEJ to outer. In order to describe the microstructure easily, it is roughly divided into outer enamel and inner enamel. Actually, it is an integral whole. Based on this, the nano-hardness and elastic modulus are investigated from DEJ to the external surface. The variations of nano-hardness and elastic modulus as a function of the distance 11

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away from DEJ are shown in Fig. 10. It can be seen that the elastic modulus increases with increase of distance and then keeps plateau. The nano-hardness increase at first and then decreases slightly from the DEJ to the occlusal surface. The peak is in inner enamel area where the enamel rods interlock each other forming decussating texture,

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such the defined P-zone and D-zone shown in Fig. 3f. However, the fracture

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toughness shows an opposite tendency. It maintains almost constant at first, but falls

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in the outer enamel zone. These phenomena are definitively enamel-types-dependent.

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The enamel types in inner enamel are decussating and irregular, as shown in Fig. 3d and f. However, enamel types in outer enamel shows paralleled arrangement, as

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shown in Fig. 3b. Cracks in the inner enamel would experience a significant deflection and distortion under the interaction of enamel rods. Higher toughness in the

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inner enamel is expected. However, in the outer enamel, higher nano-hardness and elastic modulus are obtained because of the regular enamel arrangements. Thus, the

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enamel shows high strength in the outer part and high toughness in the inner part. To further explain these phenomena, three-point bending tests are conducted with

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non-standard samples and the fracture morphology is observed. The representative cleavage fracture morphology is shown in Fig. 11. River line patterns can be seen in the enamel zone. The direction of the river line patterns, which means the direction of the cracks propagation, is exhibited by the yellow arrows. Small river line patterns in inner enamel zone are radicalized, as presented by yellow arrows in Fig. 11b. These small individual cracks interact with the neighbors and merge into large steps. Fig. 12

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11c shows the fracture morphology of enamel in the outer zone. The fracture surface is relatively flat comparing to that in inner enamel. Cracks in outer enamel seem to prefer to propagate along the sheaths (interrods). Cracks and bridging can be widely seen.

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However, our focus here is on the cracks morphology in inner enamel area. Fig.

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11d shows the morphology marked by the yellow rectangle in Fig. 11a. At first, cracks

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run through DEJ and then propagate to the enamel area. Because the enamel is brittle

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materials, the fracture behavior obeys the Griffith's theory [39]. The cracks would propagate along the weak interrods. With the continuous propagation, the interrod

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cracks will evolve into enamel rods because of the changes in arrangement of enamel rods. Sometimes, these cracks trapped in the enamel rods, also select to cut the HAP

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fibers again to evolve into interrods cracks. Then, these cracks will experience a significant deflection and distortion under the interaction of HAP fibers, and complex

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tearing ridges are formed, as shown in Fig. 11d. The fracture morphology of two characteristic zones (P & D) also presents obvious difference, as shown in Fig. 11e. In

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the P-zone, where the direction of cracks is parallel to the direction of the enamel rods, the failure is mainly determined by interred-cracking. However, in the D-zone, where direction of cracks is perpendicular to the direction of the enamel rods, the failure mode is mainly caused by cutting off the enamel rods. This kind of decussating texture is an effective pinning of the crack front along the step, and enhances the crack-resistance energy, leading to higher toughness in inner enamel that outer 13

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enamel. Generally, nanotwins widely exist in ductile alloys [40]. Previous experiments have revealed that alloys with nanotwins have ultrahigh strength/hardness, good ductility, and great strain hardening [41]. The nanotwins can also increase resistance

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energy of crack initiation and propagation deflection [42]. Researchers have

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demonstrated that compared to nanotwins, high-order hierarchical nanotwins

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structures can contribute to further improvement of strength of alloys without

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sacrifice of fracture toughness. Undoubtedly, these imperfections can also be extended to bioceramics. Besides, the high-order hierarchical nanotwins in the wild boar’s tusk

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enamel are beneficial defects resulting from natural adaptation of special living environment. Therefore, it can infer that existence of low-energy, high-density

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nanotwins, especially the hierarchical nanotwins in the enamel of the wild boar's tusk might be another reason for its high performance.

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4. Conclusion

In summary, the microstructure of the wild boar's tusk enamel was

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self-assembled into hierarchical structure from the HAP fibers (single or poly-crystals, nano-scale), enamel rods (micro-scale), enamel types (meso-scale) to enamel patterns (macro-scale). High-density nanotwins and high-order hierarchical nanotwins exist in the HAP fiber. The mechanical properties of the wild boar's tusk enamel show strong anisotropy. The nano-hardness and elastic modulus parallel to the enamel rod's axial direction are higher than that perpendicular to the enamel rod's axial direction. The 14

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mechanical properties show variation from the DEJ to the outer surface of the enamel. The elastic modulus increases with increase of distance from the DEJ and then keeps plateau. The nano-hardness increase at first and then decreases slightly from the DEJ to the occlusal surface. There is a peak of nano-hardness in inner enamel area. The

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fracture toughness shows an opposite tendency. It maintains almost constant in inner

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enamel, but falls in the outer enamel zone. The decussating texture of the enamel in

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properties from the DEJ to the outer surface.

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the inner enamel is found to be the main reason for the variation of the mechanical

Acknowledgement

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This work was supported by the National Natural Science Foundation of China (grant numbers 51804252, 51701156), Equipment Pre-Research Foundation of China

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(grant numbers 6140759040102, 6140923040203) and Doctoral Starting Fund of Xi’an University of Technology (grant number 101-451116013).

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Figure captions Figure 1. Macroscopic appearance of the wild boar's tusk (a); (b) schematic illustrations of the growth direction, transverse, longitudinal and occlusal surface of the tusks; (c) macroscopic appearance of the longitudinal section of the tusks

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Figure 2. XRD patterns of occlusal surface of the wild boar's tusk

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Figure 3. SEM micrographs of the overall tusk (a); the outer enamel (b); the overall

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enamel (c); the inner enamel (d) ; the DEJ (e) and P-zone & D-zone (f)

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Figure 4. SEM micrographs of the occlusal surface of the tusk (a) and the HAP fibers in enamel rods and interrods (b); (c) magnified area in (b). The axial directions of the

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HAP fibers in the enamel rods and interrods are indicated by the white arrows and the yellow arrows, respectively.

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Figure 5. TEM micrographs of HAP fibers of occlusal surface (a); (b) the corresponding diffraction patterns of (a); (c) TEM morphology of a typical single

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HAP fiber and (d) the corresponding diffraction pattern of (c) Figure 6. TEM micrograph showing nanotwins in a single HAP fiber (a); (b) the

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HRTEM micrograph of the nanotwin marked by the white rectangle; (c) the corresponding diffraction patterns of (c); (d) the filtered image of (b) Figure 7. TEM micrographs of nanotwins and high-order hierarchical nanotwins in a single HAP fiber (a); (b, c) HRTEM micrographs of the representative high-order hierarchical nanotwins. The insets are the corresponding FFT patterns. Figure 8. Schematic illustrations of the hierarchical structure of enamel of wild boar's 21

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tusks Figure 9. Mechanical properties of enamel of the wild boar's tusk measured by indentation under both conditions along longitudinal (L) and transverse direction (T) Figure 10. Variations in nanohardness, elastic modulus and toughness along the

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transverse direction as a function of the distance from the DEJ towards the occlusal

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surface

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Figure 11. SEM images showing the crack propagation patterns in the overall tusk (a);

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(b) inner enamel; (c) outer enamel; (d) magnified area marked in (a) and (e) P-zone &

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D-zone

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Highlights 1. High-density and high-order hierarchical nanotwins were observed in the HAP fibers of wild boar's tusk enamel. 2. The mechanical properties of the wild boar's tusk enamel showed strong anisotropy and were higher along the longitudinal direction than along the transverse

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direction.

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3. The decussating texture of the enamel in the inner enamel is found to be the main reason for the variation of the mechanical properties from the DEJ to the outer

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surface.

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