structural design and mechanical behavior of alligator (alligator mississippiensis) osteoderms

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  • 5/24/2018 Structural Design and Mechanical Behavior of Alligator (Alligator Mississippiensis) Osteoderms

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    Structural design and mechanical behavior of alligator (Alligator

    mississippiensis) osteoderms

    Chang-Yu Sun, Po-Yu Chen

    Department of Materials Science and Engineering, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd, Hsinchu 30013, Taiwan

    a r t i c l e i n f o

    Article history:Received 9 April 2013

    Received in revised form 12 July 2013

    Accepted 16 July 2013

    Available online 24 July 2013

    Keywords:

    Osteoderm

    Armor

    Composites

    Mechanical property

    Toughening mechanisms

    a b s t r a c t

    Alligator is a well-adapted living fossil covered with dorsal armor. This dermal shield consists of bonyplates, called osteoderms, interconnected by sutures and non-mineralized collagen fibers, providing a

    dual function of protection and flexibility. Osteoderm features a sandwich structure, combining an inner

    porous core and an outer dense cortex, to offer enhancements for stiffness and energy absorbance. In this

    study, we investigated the multi-scale structure and mechanical behaviors of the American alligator (Alli

    gator mississippiensis) osteoderm. Microcomputed tomography was applied to reveal the complex neuro-

    vascular network. Through the observation under optical and scanning electron microscopes, the

    osteoderm was found to consist of woven bone in the dorsal region and lamellar-zonal bone in the ven-

    tral region. Nanoindentation and compressive tests were performed to evaluate the mechanical proper-

    ties of osteoderms. The varying mineral contents and porosity result in a graded mechanical property: a

    hard and stiff dorsal cortex gradually transform to a more compliant ventral base. Three protective mech-

    anisms optimized for alligator osteoderms were proposed and elucidated.

    2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

    1. Introduction

    Many structural biological materials have been extensively

    investigated in recent years due to their superior mechanical prop-

    erties, considering the weak building blocks of which they are

    composed[13]. Currently, flexible and lightweight dermal armors

    have aroused increasing interest due to their intriguing designs for

    protection[4,5], including fish scales[612],turtle shells[1316]

    and armadillo carapaces [17,18]. Fish scales, such as P. senegalus

    [7],A. gigas[8]and A. spatula[10,12], have been widely studied la-

    tely. Despite the differences in material compositions, mineral con-

    tent and thicknesses, they all applied a similar strategy of

    combining a stiffer and harder external region with a softer inter-

    nal base. The scales of these marine species exhibit flexibility

    through interlocking and overlapping[4,5].On the other hand, armadillo carapace [17,18] and turtle shell

    [1316]utilize rather distinct strategies from fish scales. These ar-

    mors share many similar structural features: (1) the main constit-

    uents of these mineralized tissues are bone, consisting of collagen

    fibers and hydroxyapatite minerals; (2) the bony plates are con-

    nected by soft tissues or joints; (3) they are covered by keratinous

    layers on the outer surface; (4) they are both sandwich composites

    with a dense cortex and a porous core. Chen et al.[17]found that

    the non-mineralized collagen fibers are responsible for the

    macroscopic mechanical responses of the armadillo carapace. The

    stretching of these connective fibers between hexagonal plates is

    the major contribution to tensile and shear strengths[17]. In turtle

    shells, the bony segments are juxtaposed with zigzag joints inter-

    locking in between, called sutures. The sutures are three-dimen-

    sional (3-D) and complicated structures with organic tissues

    giving rise to effortless deformation under small loads and trans-

    ferring to stiffer responses after locking under higher degrees o

    movement [15]. Rhee et al. [13]reported that the porous core o

    the turtle shell is made of closed-cell foam, causing the sandwich

    structure to undergo a nonlinear deformation, which leads to a

    higher specific energy absorption compared with the dense cortex

    alone. Recent investigations conducted by Achrai and Wagner[14

    revealed that the dorsal and ventral cortices of the sandwich struc

    ture own various mechanical properties as a result of different fi-ber arrangements. The randomly oriented fibrillar network in the

    dorsal cortex can sustain sharp impact isotropically, while the ply-

    wood arrangement of fibers in the ventral cortex possesses aniso-

    tropic mechanical properties and is beneficial for structura

    support[14]. The turtle carapace also appears to be a functionally

    graded material (FGM) in terms of composition, porosity and

    mechanical properties.

    Crocodilian osteoderm is another interesting topic in natura

    flexible dermal armors. These ancient reptiles have long been con-

    sidered as fierce carnivorous tetrapods with heavily armored skins

    Although they seldom encounter predators, territorial fight

    among the same species can often be deadly because of thei

    1742-7061/$ - see front matter 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.07.016

    Corresponding author. Tel.: +886 3 571 5131x33889.

    E-mail address: [email protected](P.-Y. Chen).

    Acta Biomaterialia 9 (2013) 90499064

    Contents lists available at ScienceDirect

    Acta Biomaterialia

    j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a c t a b i o m a t

    http://dx.doi.org/10.1016/j.actbio.2013.07.016mailto:[email protected]://dx.doi.org/10.1016/j.actbio.2013.07.016http://www.sciencedirect.com/science/journal/17427061http://www.elsevier.com/locate/actabiomathttp://www.elsevier.com/locate/actabiomathttp://www.sciencedirect.com/science/journal/17427061http://dx.doi.org/10.1016/j.actbio.2013.07.016mailto:[email protected]://dx.doi.org/10.1016/j.actbio.2013.07.016http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.actbio.2013.07.016&domain=pdfhttp://-/?-
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    extremely high bite force, reaching 10 kN, the highest value that

    has been reported for living animals to date[19]. Thus, well-devel-

    oped armor designs for excellent mechanical performances are de-

    manded, along with some flexibility for speedy and agile

    movements in order to capture preys. As a matter of fact, the dorsal

    sheaths of crocodilians have been used as armor suits for ancient

    warriors since they are found to repel knives and arrows, and are

    even bulletproof under certain conditions, as discovered recently[20]. However, the microstructure, mechanical properties and

    deformation mechanisms have not been thoroughly investigated.

    In this study, we investigated the osteoderm of American alliga-

    tor (Alligator mississippiensis) by multi-scale structural character-

    izations using materials science approaches. Mechanical

    behaviors were evaluated and related to the structure features at

    varying length scales, and the deformation as well as toughening

    mechanisms of this biological armor when subjected to external

    forces were proposed. We hope this study can provide further

    understanding of biological defensive designs, and offer inspiration

    for novel synthetic armors and advanced composites.

    2. Background

    Reptiles are cold-blooded animals featuring scales that cover

    their whole body. Among them, crocodilians, including crocodiles,

    alligators and caimans, are amazing living fossils which appeared

    150 million years ago and have evolved into one of the most adap-

    tive modern animals on the planet. These large tetrapods possess

    not only keratinous scales on their external surfaces, but also un-

    ique bony plates underneath the keratinous scales for reinforced

    protection, called osteoderms. Crocodilian osteoderms are found

    mainly on the dorsal dermis (also on the abdomen for some spe-

    cies, such as most caimans), sheltering areas from the nuchal to

    the caudal region. These natural armors are composed of mineral-

    ized bony plates which are connected by fibrous tissues, similar to

    armadillo and turtle carapaces. The hierarchical structure of the

    dermal armor of Alligator mississippiensis is schematically pre-sented inFig. 1. The whole armor includes about 70 pieces of bony

    plates. Each plate has a longitudinal keel in the middle. Through a

    transverse cross-section, various structural features are demon-

    strated. The external surface of the bony plates is covered by a thin

    layer of keratinous scutes. These scutes, or scales, cover the dorsal

    armor of alligator as well as all other parts of its body, and may

    vary in shape, composition and formation mechanism. They result

    from morphological transitions through differentiation and

    keratinization of the crocodilians epidermis [21]. Harder and

    tougherb-keratin outer layer coats the osteoderms to provide wear

    resistance, while thea-keratin forms mainly the matrix and hingeregions, acting as a barrier to water and electrolyte exchange

    [21,22]. In addition, connective fibers are found at the junction of

    laterally neighboring bony plates.

    Osteoderm is not an original element in evolutionary for croc-odilians. This type of integumentary skeleton is a plesiomorphic

    trait for tetrapods [23,24], and has been well demonstrated in

    many dinosaurs, such as the renowned stegosaurs [25], ankylo-

    saurs [20], and other extinct relatives such as squamates [26]

    and archosaurs [27]. The osteoderms in various species differ in

    size, shape, ornamentation and functions. In addition to protection

    from claws and teeth of predators, other functions of osteoderms,

    including heat transfer [25,28], mineral storage[29]and locomo-

    tion aid[30], have been suggested. Since these integumentary skel-

    etons of reptiles are not subjected to external forces and do not

    likely undergo bone remodeling, the ages of these animals can be

    estimated by counting the growth marks in the osteoderms corre-

    sponding to the seasonal changes in growth rate[31], which is an

    equally valid yet much more convenient method to obtain ages

    than counting or observing the growth marks in the interior bones

    (e.g. long bones) from living or preserved species [32].

    3. Materials and methods

    3.1. Sample preparation

    A complete dorsal armor of an American alligator was obtained

    from Jernigans Taxidermy (Waco, Texas, USA). The alligator armor

    was prepared using a relatively harmless and natural method

    without using strong chemicals, which may alter the natural state

    of the samples. The longitudinal length of the armor is 0.85 m,

    indicating the animal may have had a body length of1.61.8 m

    since the armored part, excluding the head and about half the tail,accounts for 50% of the length of the entire animal. It it is likely to

    be a mature alligator, 810 years old, depending on the gender. It

    has been reported that male alligators in Texas can be 8 ft long

    at the age of 10 and female alligators 67 ft long at the same age

    [33].

    Keratin coverings on the dorsal surface and dried dermis on the

    ventral surface were removed in order to observe the structural

    Fig. 1. Hierarchical structure of alligator osteoderm from macro-, meso- and micro-, to nanometer-length scales.

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    features of individual osteoderms. All samples prepared for micro-

    structural characterization and mechanical testing were taken

    from the central and caudal parts of the whole armor to maintain

    consistency, since the cervical and transverse terminal osteoderms

    possess rather irregular shapes and non-uniform keel heights. It

    should be noted that the experimental samples are taken from a

    single alligator, and may not be representative of the entire

    species.

    3.2. Elemental analysis

    3.2.1. Ash content measurement

    17 samples sectioned from five mid-dorsal osteoderms (34

    samples from each) with regions varied from the keel to the edge

    were used to determine the average mineral content of the osteo-

    derm by the ash-weight method [17]. Since the samples for the

    measurement were all from the mid-dorsal osteoderms, the results

    may stand for the major portion of the alligator armor. However, it

    should be noted that the mineral content may change for osteo-

    derms at different locations on the body. Samples were dried on

    a hot plate at 105C for 12 h and the dry weights were measured

    using an electronic balance. Samples were then ashed at 600C

    for 24 h and ash weights were measured. The water content and

    ash content (in wt.%) were calculated.

    3.2.2. X-ray diffraction

    X-ray diffraction (XRD) was carried out on powders of ground

    alligator osteoderms utilizing a powder X-ray diffractometer

    (XRD-6000, Shimadzu Co., Kyoto, Japan). A continuous scan using

    Cu Ka1 (k= 0.154 nm) as the radiation source was performed in a

    h2h mode scanning from 2h= 20 to 60, with a step size of

    0.02at a rate of 2min1.

    3.2.3. Electron probe microanalysis

    The localized elemental compositions were analyzed by field-

    emission electron probe microanalysis (FE-EPMA) with a JEOL

    JXA-8500F EPMA (JEOL Ltd., Tokyo, Japan). Three cross-sectional

    samples (10 5 5 mm3) with both dorsal and ventral regions

    of the osteoderm were sectioned and embedded in epoxy followed

    by grinding and polishing. The samples were coated with a thin

    layer of carbon instead of other common conductive coatings such

    as gold or platinum because these heavy metal coatings can se-

    verely suppress the emission of X-rays induced by the incident

    electron, serving as a barrier layer for the signals to come out

    and be detected. Five quantitative measurements were taken from

    the dorsal and ventral region, respectively, for each specimen, and

    the results were then averaged to compare the compositional dif-

    ferences between the two regions.

    3.2.4. Energy-dispersive spectroscopy

    Elemental mapping at interfacial regions was achieved by an

    energy-dispersive spectroscope (EDS) within a field-emission scan-

    ning electron microscope (FE-SEM) (JSM-7600F, JEOL Ltd.). Sam-

    ples were sectioned and ground from the edge of an osteoderm

    and coated with a thin layer of carbon (10 nm) to prevent elec-

    tron charging. X-rays were produced from electron bombardments

    under an accelerating voltage of 10 kV and a working distance of

    15 mm. A silicon-drift detector (SDD) (X-Max SDD, Oxford Instru-

    ments, Abingdon, Oxfordshire, UK) was used to collect the charac-

    teristic X-rays from the sample, while the software AZtec (Oxford

    Instruments) was applied to analyze and map the elementaldistributions.

    3.3. Structural characterization

    3.3.1. Macroscopic observation

    External shape and morphology of osteoderms were taken from

    central and edge regions of the whole armor. For cross-sectiona

    observation, a sample was cut by a hand saw and through the kee

    region followed by grinding and polishing. Photographs of top, bot-

    tom and cross-sectional views were taken by a digital camera.

    3.3.2. Microcomputed tomography (l-CT)l-CT was accomplished by unmonochromatized synchrotron

    hard X-rays with energy ranging from 5 keV to 35 keV at the Na

    tional Synchrotron Radiation Research Center (NSRRC) in Hsinchu

    Taiwan[34]. l-CT images were obtained from a CCD camera (model 211, Diagnostic instruments, 1600 1200 pixels) after convert

    ing the X-rays into visible lights by a scintillator. The resolution o

    the corresponding images (in pixel size) of a 2 lens was

    2.8 lm 2.8 lm. In order to reconstruct 3-D tomographic mod-els, images were collected with a regular step size of 0.3over a to-

    tal 180 rotation of the specimen stage. Xradia software (Xradia

    Inc., Pleasanton, CA, USA) was applied to reconstruct the raw data

    which was then visualized by Amira software (Visualization Sci-

    ence Group, a FEI company, Burlington, MA, USA).

    3.3.3. Optical microscopy and stereoscopy

    Cross-sectional samples through the keel region were ground

    and polished before observing under an optical microscope

    (BX51M, Olympus Co., Tokyo, Japan) equipped with a 0.8 mega-

    pixel digital camera (DP12, Olympus Co., Tokyo, Japan). An inte-

    grated view of the keel cross-section was achieved by combining

    17 micrographs of consecutive regions.

    Stereoscopic images were taken from an Olympus SZX7 Zoom

    Stereomicroscope (Olympus Co., Tokyo, Japan) with a 2.0 mega-

    pixel CCD camera (Infinity 1, Lumenera Co., Ontario, Canada). The

    magnification of the stereoscope ranged from 8to 56.

    3.3.4. Scanning electron microscopy

    Microstructural characterization of the fracture surfaces were

    observed by a FE-SEM (JSM-7600F, JEOL Ltd.). Fracture surfaces

    were created by exerting a bending force through a clamp and a

    wrench. The specimens were coated with a thin layer of platinum

    to enhance electron conductivity on the surface. Secondary elec

    tron images (SEIs) were taken with an accelerating voltage o

    10 kV and a working distance of 10 mm.

    3.4. Mechanical testing

    Schematic representations of specimens prepared for mechani

    cal testing are shown in Fig. 2. The system of coordinates we

    adopted throughout the text is illustrated inFig. 2a. Longitudina

    is defined as the direction along the keel long axis, transverse is re-ferred to the direction perpendicular to the keel and vertical is the

    direction through the thickness of the osteoderm. Longitudinal and

    transverse are both included when the term horizontal i

    referred.

    3.4.1. Nanoindentation

    A sample for nanoindentation was taken from the keel region o

    a caudal osteoderm. The sample was mounted with the longitudi-

    nal cross-sectional area (Fig. 2a) revealed, followed by grinding and

    fine polishing with Al2O3 suspensions from particle size of 1 lm0.3 lm and finally 0.05 lm. The average surface roughness of thefinal sample was 20 nm, measured with atomic force microscopy

    (Dimension Icon, Bruker Corp., Billerica, MA, USA). Nanoindenta-

    tion tests were conducted by using a Hysitron TI900 TriboIndenter(Hysitron Inc., Eden Prairie, MN, USA) with a Hysitron TI-0039

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    Berkovich diamond tip under a load-controlled mode. The radius of

    the tip was 100 nm. The load function can be separated into three

    stages: linear loading, holding and linear unloading, and the dura-tion for each stage was 5 s, with the peak load set to be 1000 lN.The area function of the tip was calibrated before conducting the

    tests with a fused quartz bulk specimen as a standard material

    due to its low elastic-modulus-to-hardness ratio [35]. A series of

    indentations was performed vertically across the cross-section

    (Fig. 2b) with an interval of 300 lm and a total number of 53groups. Each group contained eight indent points forming a 2 4

    rectangular region with a lateral space of 15 lm between neigh-boring indents, which is a significantly large distance compared

    with the indent size to avoid effects of adjacent indents. The hard-

    ness and reduced modulus values of each group were then aver-

    aged. The area function was calculated again after the test and

    showed no significant variations (0.3%) on the tip geometry, sug-

    gesting that the results were reliable even after a large amount ofindentations were performed.

    3.4.2. Compressive testing

    Samples for compressive testing were cut into 2.3

    2.3 4.5 mm3 rectangular pieces by a rotating diamond blade.

    The dimensions were chosen to prevent buckling by the Eulers cri-

    teria. Each facet of the samples was then ground carefully using a

    clamp to ensure that the two surfaces in contact with the upper

    and lower load cells are parallel to each other, while keeping the

    side surfaces perpendicular to the ends as precise as possible to

    eliminate eccentric loading. 80 vertical and 80 horizontal samples

    were prepared, as shown in Fig. 2c. The horizontal samples were

    taken from flat (non-keel) regions, since the large deviation on

    the vertical direction of the keel cross-section may have significantinfluences on experimental results. However, for vertical samples,

    the height 4.5 mm cannot be satisfied at locations far away from

    the keel due to the decrease in thickness from the keel toward

    the edge. Therefore, vertical samples were taken from non-keel re-gions near the keels. The difference in regional distribution be-

    tween the horizontal and vertical samples led to different

    portions of dorsal and ventral regions, which is clearly demon-

    strated inFig. 2c: vertical samples contain more dorsal region than

    the horizontal samples. 40 samples from each group (longitudinal

    and vertical directions) were immersed in Hanks balanced saline

    solution (HBSS) (H2387, SigmaAldrich Co., St Louis, MO) for

    24 h before mechanical testing. The rehydrated samples were

    tested immediately after taking out of HBSS in order to prevent fur-

    ther drying. The other set of samples (40 each direction) were

    tested in ambient dry condition. Compressive tests were conducted

    by using a universal testing machine (Instron 3343 Single Column

    Testing System, Norwood, MA, USA) with a 1 kN load cell at a strain

    rate of

    1

    10

    3

    s

    1

    .

    3.4.3. Flexibility demonstration

    Two adjacent osteoderms were taken from the mid-dorsal re-

    gion of the alligator armor to demonstrate the flexibility of the

    joints. The bony plates were sectioned transversely to better dis-

    play the angles bent by bare hands.

    3.4.4. Whole osteoderm compression

    A transversely cross-sectioned large osteoderm sample with

    keel height of15 mm was used to demonstrate the deformation

    of the sandwich structure under compression. The sample was

    ground at the bottom to create a flat contact surface, followed by

    immersion in HBSS for 24 h before the test. The large-scalecompressive tests were carried out by a universal testing machine

    Fig. 2. Schematic illustrations of sample preparations for mechanical tests. The shades of the color in (b) and (c) denote different regions, i.e. dorsal and ventral region. (a) The

    headtail direction along which the keel is oriented is defined as the longitudinal (or parasagittal) direction. The direction along the lateral row of osteoderm is defined as the

    transverse direction. Both directions are included when horizontal direction is referred. The horizontal direction is perpendicular to the vertical direction. (b) An illustration

    of the direction where nanoindentation tests were performed. (c) An illustration of the locations where compression samples of different orientations were taken. Due to the

    geometrical limitations, vertical samples are taken near the keel and possess more dorsal region, while the horizontal samples are taken mainly within ventral regions.

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    (Instron 4468, Double Column Testing System, Norwood, MA, USA)

    with a 50 kN load cell at a strain rate of1 103 s1.

    4. Results and discussion

    4.1. Macroscopic observation

    An osteoderm from the mid-dorsal region appears in a quadrate

    shape 5 cm in length and width with a parasagittally aligned keelof 1.5 cm in height (Fig. 3a). Osteoderms taken from different

    locations show distinct appearances, in both shape and keel height

    An osteoderm from the transverse terminal is shown inFig. 3b for

    comparison. The large concave regions on the external surface con-

    tain small cavities that connect to the vascular channels, which

    were proposed to be the evidence for the thermoregulation func

    tion of alligator osteoderms[30].The neurovascular foramina ente

    the bony plates from the ventral surface, as shown inFig. 3c, where

    the grooves are traces of bifurcated dorsal median arteries circulat-

    ing across the surface [30]. The transverse edge of an alligato

    osteoderm contains connective collagenous fibers (Fig. 3d) be-tween two adjacent plates, similar to the armadillo carapac

    Fig. 3. Photographs showing the top views of mid-dorsal (a) and transverse terminal (b) osteoderms show different shapes and features. The ventral surface (c) of a mid-

    dorsal osteoderm shows that arterial grooves were used to hold vessel branches. The arrows indicate pits where the artery bifurcates into nutrient foramina entering the

    osteoderm. The edge of the osteoderm between laterally neighboring plates is shown in (d), which was covered by non-mineralized connective fibers. By removing the non-

    mineralized connective fibers, the sutures can be observed in (e). A SEM image in (f) shows the 3-D feature of the sutures, which contain pits that are connected to the

    neurovascular system within the plates.

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    [17]. These fibers account for connectivity and flexibility enhance-

    ments. By removing the connective soft fibers, serrated sutures

    (Fig. 3e) are revealed, which share the same functional design for

    interlocking as the turtle shell[15]. From SEM, we can clearly ob-

    serve the 3-D characteristic of the sutures (Fig. 3f). Numerous tiny

    pits are also shown inFig. 3f, which are connected to the neurovas-

    cular foramina, presumably for vascularization, sensing and nutri-

    ent transportation.

    4.2. Mineral content measurement and elemental analysis

    From the ash-weight measurement, the water content is

    10.70 0.58 wt.% and the mineral content of the dried specimen

    is 65.77 2.26 wt.%, giving 34.23 2.26 wt.% of the dried alligator

    osteoderm to be organic components. The osteoderm of American

    alligator possesses a similar mineral content to that of armadillo

    carapace (65 wt.%)[17]and bovine femur (67 wt.%)[36], which

    is higher than other natural armors such as tortoise shell

    (53 wt.%) [37] and fish scale (46 wt.%) [6], as well as that of

    some mammalian compact bones, for instance, elk antler

    (57 wt.%)[38]. It is also found that the mineral content decreased

    gradually from the keel (67.13 0.58 wt.%) through the transitionregion (65.54 0.56 wt.%) to the edge (62.89 1.60 wt.%) as shown

    inFig. 4a, which is functionally graded since the edge with sutures

    and connective fibers serve for flexibility enhancements, and may

    be a result of evolutionary convergence with the turtle shell[14].

    To confirm the mineral constituents, the crystalline phase was

    detected by XRD. The resulting pattern inFig. 4b can be indexed

    to JCPDS 09-0432, revealing hydroxyapatite as the main compo-

    nent, which is the same as the minerals in bone and other bony tis-

    sues[6,17,38]. Furthermore, localized elemental analysis measuredby FE-EPMA shows that the dorsal region contains more calcium

    (29.58 1.15 wt.%) and phosphorus (13.13 0.68 wt.%) than the

    ventral region (Ca: 24.53 1.62 wt.%, P: 10.38 0.75 wt.%). The re-

    sults indicate that the dorsal region contained more minerals,

    which are primarily non-stoichiometric hydroxyapatite, while the

    ventral region appeared to be less-mineralized and had more or-

    ganic constituents.

    Elemental mapping by EDS under FE-SEM was conducted to

    analyze the compositional difference between the bony plate

    and the connective fibers (Fig. 5). As the secondary electron im-

    age (SEI) in Fig. 5a shows, the area of interest was the interface

    between sutures, which is still a part of the bony plate, and the

    connective fibers at the edge of an osteoderm. Carbon mapping

    in Fig. 5b indicated that both regions contain organic contents,

    which is collagen [24], and that the amount of organic contents

    is obviously much richer in the connective fibers. On the other

    hand, distribution of Ca (Fig. 5c) and P (Fig. 5d) clearly illus-

    trated that hydroxyapatite (Ca10(PO4)6(OH)2), as the major min-

    eral content, is confined in the suture. Therefore, it was

    confirmed that the fibers connecting bony plates together are

    not mineralized.

    4.3.l-CT imaging

    A cross-sectional view in the longitudinal direction of an osteo-

    derm keel reveals a sandwich structure (Fig. 6a), where the porous

    interior is surrounded by compact cortex. A sandwich structure

    also appears in various light-weight designs, such as leaves [39],bird beaks [40] and feathers [41], as well as many defensive de-

    vices against impact and bending, including human skull[39], tur-

    tle shell [14], armadillo carapace [17], fish armor [11] and

    horseshoe crab exoskeleton[1]. The main advantage of sandwich

    structures is to provide high bending stiffness with minimum

    weight. Defensive designs applying this principle also serve the

    function of energy absorbance under impact loads by deformations

    of the cellular core through elastic bending, brittle fracture or plas-

    tic buckling of the walls or trabeculae before undergoing densifica-

    tion[39]. The reduced burden of these lightweight armors can thus

    enhance locomotion along with improvements in mechanical

    properties.

    Since cross-sections can only show porosities and incomplete

    channels, a computed tomographic technique was applied to gain3-D information of the complex neurovascular network in the

    bony plates. By collecting the transmitted X-ray signals and the

    corresponding intensities, we can identify the porosity since the

    absorbance along the X-ray path is different for materials with

    porosity and without porosity. Through rotation during scanning,

    the information of the whole specimen can be obtained and 3-D

    models can be further reconstructed. A sectioned image of the

    reconstructed 3-D model from l-CT scans is shown in Fig. 6b.The neurovascular channels form an intricate 3-D network, where

    the major cavity in the center branches out toward the dorsal and

    ventral regions with much smaller pipes. The bifurcation within

    the dorsal region tends to be more complex compared with the

    ventral region, whereas both regions contain evidences of sea-

    sonal and/or annual growth, indicated by the white arrows inFig. 6b.

    Fig. 4. Minerals in alligator osteoderm. (a) The amount of mineral content withinthe osteoderm was measured by ash-weight method, where the results showed

    decrease from the keel to the edge (keel: 67.13 0.58 wt.%, transition:

    65.54 0.56 wt.%, edge: 62.89 1.60 wt.%). (b) X-ray diffraction (XRD) pattern of

    the alligator osteoderm confirmed that hydroxyapatite is the main mineral

    component.

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    4.4. Microstructural characterization

    4.4.1. Optical microscopy

    Four different regions with different microstructural morpholo-

    gies of bone can be distinguished from the optical micrograph, as

    shown inFig. 7a. At the outer sheath, randomly oriented wovenbone can be observed along with extensive vascularization. The

    vascular channels connect to small pits on the external surface,

    mainly located within the large concave regions, as previously

    shown inFig. 3a and b. These pits and vascular systems are sug-

    gested to be the major evidence of the role of osteoderm in ther-

    moregulation of the body [30]. The second region beneath the

    outer sheath is composed of dense woven bone and scattered

    lamellar rings deposited around the neurovascular channels. An

    SEM image taken from this region is shown inFig. 7b. The patterns

    on the surface indicate collagen fiber bundles being ruptured and

    pulled out, where no preferred orientation can be observed, illus-

    trating bundles entangled in a randomly woven manner. Scattered

    lamellar rings can be recognized in this region. These concentric

    lamellar structures are not likely to be secondary osteons sinceno canaliculi and well-developed vascular systems (Haversian

    and Volkmanns channels) are observed. Moreover, osteoderms

    are not constantly subjected to external loading and bone remod-

    eling may be restricted and limited. The third region contains por-

    ous woven bone with large neurovascular channels. The woven

    bone in this region is similar to that of the outer sheath. The major

    neurovascular foramen and branches mainly locate in this region

    and the large cavities can reach up to hundreds of micrometers

    or several millimeters in diameter. Lamellar bone is also found

    around the channels, forming circular rings. At the bottom, the ba-

    sal region consists of lamellar-zonal bone[42]. This type of bone is

    commonly seen in reptiles, and is related to the poor vasculariza-

    tion, which derives from low metabolic rate[42]. The lamellar-zo-

    nal bones correspond to seasonal or annual growth, where bone

    growth stops or slows down in winters, leaving lines of arrested

    growth (LAGs). The lamellae in this region are not the same a

    the lamellar bone, but rather a parallel-fibered bone, which is con-

    structed by woven collagen fibers with a preferred orientation

    (Fig. 7c). Furthermore, Sharpey-fibered bone is also found mostly

    in the ventral region and sometimes on the edge of dorsal regionextending in oblique directions from the margin to the core with

    wavy or zigzag structures[20,23,24]. This type of bone is derived

    from non-mineralized Sharpeys fibers functioning for connection

    which fuse into the osteoderm and anchor the bony plates to the

    epidermis. The Sharpey-fibered bone in the ventral region indi-

    cated locations where the osteoderm contact the epidermis, while

    those in the dorsal region implied that the genesis of osteoderm

    took place within the skin. It is thus discovered that the alligato

    osteoderm consists of various types of bone, resulting in a hetero

    geneous composition. The differences in fiber orientation at differ-

    ent locations are schematically presented in Fig. 1. It should be

    noted that the structural variations between adjacent regions un

    dergo a gradual change.

    Thin sections of the osteoderm were observed under the stereoscope (Fig. 8). LAGs are clearly observed in the ventral region, while

    only vague annuli can be recognized at the dorsal region because

    no complete halt occurred during growth. The growth rate is also

    different in the two regions. Although this is not a precise quanti-

    tative evaluation due to the limited resolution, we can still distin

    guish that the interval between annual growth marks in the ventra

    region (0.5 mm) is much narrower compared to those in the dor

    sal region (1.1 mm). Hence, we proposed a growth model, as

    shown in the schematic illustration (Fig. 8). The growth rate in

    the keel region is higher than the basal region, leading to a unique

    shape of a ridged keel. In addition, according to Vickaryous and

    Hall [24], the mineralization process of the osteoderm initiate

    from the keel, and then extends radially across the whole plate

    Therefore, combining the non-simultaneity in calcification and

    Fig. 5. Elemental mapping by energy-dispersive spectroscopy (EDS) at the edge of the osteoderm. (a) An SEI image of the area being analyzed, which shows no significant

    distinguishing structural features of the two regions. (b) Carbon mapping indicates that the carbon content is much richer in the connective fibers. (c, d) Calcium and

    phosphate mapping clearly demonstrates the lack of minerals, which is mainly hydroxyapatite, within the connective fibers.

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    the anisotropy in growth rate, it is implied that the degree of min-

    eralization may not be uniform within the entire osteoderm, espe-

    cially along the vertical direction.

    4.4.2. Scanning electron microscopy

    Microstructural characterization of a fractured osteoderm keel

    is shown inFig. 9,where comparisons between the dorsal, ventral

    regions and the non-mineralized collagen fibers are made. At a

    lower magnification, the dorsal region (Fig. 9a) shows a densely

    packed and relatively flat fracture surface, indicating a direct fail-

    ure and rather brittle behavior upon fracture. The mineralized col-

    lagen fiber bundles are fused together and the patterns are

    obscured. Under a more detailed view (Fig. 9b), granular morphol-ogies of mineral aggregates on the surface are observed. Individual

    fibers can hardly be recognized in this region, implying that the fi-

    bers are highly mineralized and form bundles which cannot be eas-

    ily separated. In comparison, the fracture surface taken from the

    ventral region (Fig. 9c) exhibits a fibrous feature. These collagen fi-

    bers were stretched and twisted upon breakage, revealing a more

    ductile behavior. Individual fibrils can be easily distinguished at

    higher magnification (Fig. 9d), where the surfaces of these fibrils

    appear to be smoother compared with those inFig. 9b. It is there-fore suggested that these collagenous fibers should possess a lower

    degree of mineralization, in contrast to those in the dorsal region,

    which corresponds to the growth rate and mineralization process

    as previously proposed in Fig. 8. The SEM observation is also in

    good agreement with the previous EPMA results, which suggested

    mineral content difference in dorsal and ventral regions.

    Fig. 9e shows the microstructural features of the non-mineral-

    ized connective fibers between neighboring bony plates to provide

    flexibility for the dorsal shield. These organic bundles are ran-

    domly oriented, with smooth surface morphologies as shown in

    the higher magnification (Fig. 9f). Characteristic patterns of

    67 nm periodicity in collagen fibrils, which derives from the stag-

    gered molecular arrangement, can also be observed inFig. 9f.

    In summary, the alligator osteoderm is a complex, heteroge-

    neous, hierarchically structured bio-composite with varying de-

    grees of mineralization and porosity at different locations.

    Through a thorough compositional and structural characterization

    at multiple levels with XRD, EPMA, EDS,l-CT, OM (optical micros-copy) and SEM, the whole dermal armor is revealed to be a hybrid

    system of mineralized bony plates and non-mineralized connective

    collagen fibers, whereas each bony plate is also a combination of a

    heavily-mineralized interwoven dorsal cortex and a less-mineral-

    ized parallel-fibered ventral base, with complex 3-D neurovascular

    channels branching from the core of the osteoderm. These features

    are integrated and organized in the schematic illustrations ofFig. 1.

    4.5. Mechanical behavior

    4.5.1. Nano-mechanical evaluation

    Nanoindentation tests along the cross-section of an alligator

    osteoderm reveal the difference in mechanical properties for dorsal

    and ventral regions. Hardness and reduced modulus of each indent

    are calculated from the loaddepth curves according to the Oliver

    Pharr method [43]. Fig. 10a shows the hardness variation across

    the keel while the photo above the plot corresponds to the inden-

    tation position. The average hardness values are 367 94 MPa for

    the ventral region and 690 170 MPa for the dorsal region.

    Fig. 10b shows the change in reduced modulus through the keel

    cross-section, where the trend is the same as the hardness values.

    The ventral region was found to possess an average reduced mod-

    ulus value of 13.9 2.1 GPa, gradually increasing to 20.3 3.4 GPa

    in the dorsal region. Drops at the exterior region for both hardness

    and reduced modulus are observed. The relatively high standarddeviation for both hardness and reduced modulus across the whole

    sample can be related to the non-uniform porosity within the

    osteoderm. The projected area of contact of nanoindentation on

    the specimen is 15 lm2, and if we consider the deformed sur-face profiles around the indents, the area influenced by the inden-

    tation can reach up to 10 lm2. On the other hand, the size of theporosity in the osteoderm spreads over a wide range from sub-

    micrometer to millimeter, where those smaller than tens of

    micrometers may cause serious effects on the indentation results.

    We have avoided the larger pores at the scales from millimeters

    down to several tens of micrometers under the optical microscope

    while choosing the indent positions. However, smaller voids be-

    yond the limit of light microscopy can still exist, which we were

    unable to identify and avoid. Therefore, it is possible that we haveset our indents on or nearby the porosity and affected the results,

    Fig. 6. (a) The sandwich structure of an osteoderm keel cross-section. The porosity

    is caused by the foramina branching system. (b) 3-D image reconstructed froml-CTscans by synchrotron X-rays. The white arrows indicate annual growth of the

    branches.

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    giving rise to the high standard deviation. Furthermore, since the

    porosity is three-dimensional, it is very likely that there are voidsunderneath the indentation surface at any depth, which we cannot

    observe but may have significant effects on the measured values. In

    addition, the osteoderm is composed mainly of woven bone, whichis a loosely structured mineralized tissue. Thus, it is also possible

    (a)

    Woven & lamellar bone

    (b)

    Lamellar-zonal bone

    (c)

    Fig. 7. (a) A combined optical micrograph of an osteoderm keel cross-section. Four regions can be distinguished as marked in the Fig.: outer sheath, woven bone and lamella

    bone, woven bone with neurovascular channels and lamellar-zonal bone. (b) An SEM image of the dorsal region showing tangled woven collagen fiber bundles. (c) An SEM

    image from the ventral region showing parallel-fibered bone (woven bone with a preferred orientation).

    Fig. 8. A schematic drawing demonstrating the proposed growth mechanism of an alligator osteoderm. The insets are stereoscopic images of thin-sectioned samples from the

    dorsal (left) and ventral regions (right). The black arrows indicate growth marks (LAGs or annuli).

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    that we have indented onto the spacing between the collagen fiber

    bundles, resulting in large deviations.

    The distribution of localized mechanical properties is in good

    agreement with the microstructural features in different regions

    previously described in Fig. 7a. The second region (II) from the

    external surface contains highly mineralized, relatively dense wo-

    ven bone and has the highest hardness and reduced modulus;

    whereas less-mineralized lamellar-zonal bone in the basal region

    (region IV) possesses the lowest values in both properties. The

    exterior region (I) and the porous core (III) both seem to have an

    intermediate mechanical property between the dense dorsal re-

    gion and the ventral base, corresponding to their similar microcon-

    stituents (woven bone with high porosity). Fig. 10c shows the

    typical loaddepth curves of the dorsal and ventral regions, illus-

    trating the distinct difference in mechanical properties. Under a

    constant maximum load, it is apparent that the dorsal region

    showed a higher stiffness and hardness with smaller indentation

    depth and larger slope of the unloading path, whereas the ventral

    region showed an advantage of higher energy dissipation esti-

    mated by the area under the curves. The different mechanical

    properties between the dorsal and ventral region may relate to

    the compositional difference in mineral content and variation in

    porosity, as demonstrated in elemental analysis and microstruc-

    tural characterization.

    The combination of hard external region and soft internal re-

    gion is analogous to many biological composites, such as arthropod

    Dorsal Region

    (a) (b)

    Ventral Region

    (c) (d)

    Non-mineralized

    Collagen Fibers

    (e) (f)

    Fig. 9. SEM fractographs at low and high magnifications of the dorsal (a, b) and ventral (c, d) region of an alligator osteoderm keel and the connective fibers (e, f). Different

    morphologies indicate the varied amount of minerals on the collagen fibers: the dorsal region is highly mineralized, the ventral region is less mineralized and the connective

    fibers are non-mineralized.

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    exoskeleton[44]and fish scales[7,8]. This is beneficial for defen-

    sive armors since the stiff and hard exterior can resist penetrations

    by sharp claws or teeth, whereas the compliant interior can arrest

    crack propagation and provide toughness, acting as a cushion by

    absorbing impact energy. However, most of them reported a dis-

    continuity (or extremely steep gradations) in mechanical re-

    sponses between the stiff and compliant layers. In this study,

    hardness and elastic modulus change gradually rather than there

    being discrete interfaces, as found in the scales of alligator gar

    (Atractosteus spatula), which are composed of two distinct materi-

    als (ganoin and bone) [10,12,45]. The alligator osteoderm contains

    only bone but undergoes gradual transitions in microstructuresat different locations due to their growth, leading to mechanical

    functions. The advantages of comprising a homogeneous material

    with mechanical property gradient (i.e. FGM) instead of abrupt

    changes include better stress redistribution and enhanced resis-

    tance to interfacial failure[7].

    4.5.2. Compressive mechanical behavior

    Fig. 11a and b shows the representative stressstrain curves of

    the compressive tests for vertical and horizontal specimens, respec-

    tively, under dry and rehydrated conditions. In the dry condition,

    vertical samples can sustain higher stresses (142.1 21.4 MPa)

    but eventually underwent a direct fracture in a relatively brittle

    manner. On the contrary, the horizontal samples showed lower

    strengths (117.5 15.9 MPa) with several stages of deformation be-fore ultimate failure, fractured in a more ductile mode. It should be

    noticed that for both orientations, the ultimate strengths occurred

    at about the same amount of strain (30%), meaning that both

    directions are able to suffer certain amounts of plastic deforma

    tions. However, the horizontal samples yielded a much higher com

    pressive strain (>40%) before totally breaking down due to th

    impedance to direct failure, providing a greater toughness. Further

    more, the osteoderm samples of both orientations were also tested

    under rehydrated conditions in HBSS to simulate the actual envi

    ronments of the biological system, where both vertica

    (124.2 21.8 MPa) and horizontal samples (88.6 12.5 MPa

    showed a decrease in compressive strength. It was discovered that

    in both dry and rehydrated conditions, the vertical samples exhibited higher strengths than the horizontal samples (dry vertical

    142.1 MPa vs. dry horizontal: 117.5 MPa, rehydrated vertical

    124.2 MPa vs. rehydrated horizontal: 88.6 MPa); moreover, the ver

    tical samples in both conditions failed similarly in a direct and

    rather brittle way, whereas the horizontal samples in both condi

    tions underwent multiple toughening mechanisms upon breakage

    (Fig. 11a and b). This implies that the anisotropic mechanical re-

    sponse is actually employed by the armor within biological sys-

    tems. The compressive mechanical properties are summarized in

    Table 1.

    Typical fracture samples after compressive tests in both direc

    tions are examined under the stereoscope, as demonstrated in

    Fig. 11c and d. Direct crack propagation for vertical samples can

    be found in Fig. 11c, which is in accordance with the relativelybrittle failure shown in the ss curve. Horizontal samples, on

    Fig. 10. Nanoindentation tests along the cross-section of an osteoderm show difference in (a) hardness and (b) reduced modulus in various regions. Note the gradien

    transition between the two regions. (c) Representative loaddepth curves for dorsal and ventral regions show distinct difference in mechanical properties at the maximumload of 1000lN.

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    the other hand, exhibit wavy and complex fracture paths and re-

    veal evidence of lamellae buckling and fiber bridging, as shown inFig. 11d. These toughening mechanisms can prevent catastrophic

    collapse of the whole structure and correspond to the resisting

    steps before ultimate fracture observed in the ss curve

    (Fig. 11b). The observation further illustrates the anisotropy in

    mechanical properties for alligator osteoderm.

    To establish a relationship connecting the macroscopic com-

    pressive behaviors and the localized nanoindentation results, the

    elastic modulus from the two tests were compared. To begin with,

    the elastic modulus was first calculated from the reduced modulus

    reported from the nanoindentation test. The relationship between

    reduced modulus and elastic modulus can be presented as

    1

    Er1 m2s

    Es1 m2i

    Ei

    where Eris the reduced modulus, Es is the elastic modulus of the

    specimen andEiis the elastic modulus of the indenter tip.msrepre-sents the Poissons ratio of specimen andmimeans the Poissons ra-tio of the indenter tip. For a Berkovich diamond tip, Ei is 1140 GPa

    andm i is 0.07[35]. Since we are unable to determine the Poissonsratio of the osteoderm, the Poissons ratio of a typical compact bone,

    which is 0.18 for bovine femur [46], is used to calculate the elastic

    modulus. From the above equation, the elastic moduli of alligator

    osteoderm from nanoindentation tests are 13.6 GPa in the ventral

    region and 20.0 GPa in the dorsal region (using the average reduced

    Table 2

    Elastic modulus measured from nanoindentation on dorsal and ventral regions of alligator osteoderm, with comparison to bovine and human femurs, and the porosity effect on

    the elastic modulus calculated by the Bonfield and Clark equation.

    Nanoindentation modulus (GPa) 10% porosity (GPa) 20% porosity (GPa) 30% porosity (GPa) 40% porosity (GPa)

    Alligator Osteoderm (Dorsal) 20.0 16.4 10.7 5.5 2.1

    Alligator Osteoderm (Ventral) 13.6 11.2 7.3 3.7 1.4

    Human Femur 20.1[47]

    Bovine Femur 23.1[48]

    Fig. 11. Representative compressive stressstrain curves for (a) vertical and (b) horizontal samples under dry and rehydrated conditions. Stereoscopic images showing typical

    failure mechanisms in (c) vertical and (d) horizontal samples after compressive deformation.

    Table 1

    Mechanical properties of dry and rehydrated alligator osteoderm samples in

    horizontal and vertical directions (40 samples for each condition).

    Direction Hydration

    state

    Ultimate compressive

    strength ravg(MPa)Compressive elastic

    modulus E(GPa)

    Vertical Dry 142.1 21.4 1.04 0.11

    Vertical Rehydrated 124.2 21.8 0.94 0.15

    Horizontal Dry 117.5 15.9 1.19 0.22

    Horizontal Rehydrated 88.6 12.5 1.07 0.17

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    Fig. 12. Experimental demonstrations of two proposed deformation mechanisms: (a) Flexibility provided by sutures and connective fibers allows limited bending. The

    neighboring plates could be bent upward to 10 and downward to 20. (b) The major porosity in the center core of the osteoderm is able to absorb some energy a

    deformations lower than 10% before cracks start to propagate in the cortex. Large channels and small voids (circled) were found to be squeezed and distorted from 0% and4% to 7% deformation.

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    moduli), which showed comparable values with the elastic moduli

    of human[47]and bovine[48] femur. However, it is significantly

    higher and cannot relate to the compressive modulus. We sug-

    gested that it should be a result of porosity effect for macroscopic

    mechanical tests. By applying the Bonfield and Clark[49]equation,

    which is a modified version of the Mackenzie [50]equation, we can

    estimate the contribution of porosity to the elastic modulus:

    E E01 1:9p 0:9p2

    whereEis the elastic modulus with porosity, E0 is the elastic mod-

    ulus of the solid phase only and p represents porosity. Table 2

    gives the elastic modulus of bulk material at different porosities

    where the elastic modulus from the nanoindentation test is usedas E0. As the result showed, the elastic modulus of compressive

    tests clearly cannot reach the calculated value, even when an over-

    estimated 40% porosity (the average porosity was 13% as esti-

    mated from the l-CT scans) is taken into account, implying thatthe alligator osteoderm is a complex composite and may incorpo-

    rate factors other than porosity upon mechanical responses at the

    macroscale.

    4.6. Deformation mechanisms

    Apart from the localized and global mechanical responses of the

    mineralized bony tissues within the osteoderm, two additional

    deformation mechanisms were proposed: flexibility and sandwich

    structure. Thus, simple tests were conducted to demonstrate these

    mechanisms, as shown inFig. 12. The sutures and non-mineralizedconnective fibers at the lateral edge of osteoderms provide a

    Fig. 13. Schematic illustrations of three deformation mechanisms of alligator dermal armor: (a) Sutures and non-mineralized collagen fibers provide flexibility; (b) sandwich

    structure absorbs energy; (c) graded mechanical property from dorsal to ventral regions offers optimization in load redistribution and energy dissipation.

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    limited flexibility that allows bending to some extent. As illus-

    trated inFig. 12a, the connection device of alligator armor is able

    to be bent upward to 10, while the bending downward can go

    up to 20. This implied that the osteoderm is designed to suffer

    more flexure downward, corresponding to the main function of

    load dissipation for protection. On the other hand, a whole piece

    of osteoderm with a cross-sectional surface revealed was subjected

    to a large-scale compressive test to demonstrate the function of thesandwich structure. We focused on the deformation of the major

    channel in the center core and the surrounding small voids before

    cracks started to propagate in the dense cortex, which is 10%

    deformation. The three successive images at the bottom half of

    Fig. 12b showed the cross-section at 0%, 4% and 7% strain, where

    the major channel was found to be squeezed and distorted, and

    were thus able to absorb additional energy. Also, the void at the

    lower right became smaller as the bony plate is being compressed,

    possibly resulting from plastic buckling and wall collapsing of the

    cellular foam. These simple demonstrations illustrated that flexi-

    bility and sandwich structure are both incorporated in the protec-

    tion mechanisms of the dorsal shield of alligator.

    Based on the experimental results, we summarized three defor-

    mation mechanisms of the alligator armor under external loads

    (Fig. 13). Whenan externalcompressiveforce actson theosteoderm,

    sutures and non-mineralized collagenous fibers connecting adja-

    cent bony plates can dissipate loads by providing a limited amount

    of flexibility (Fig. 13a)[15]. The movements between neighboring

    osteoderms can avoid further deformation under small loads. As

    the load increases, the sandwich structure of a bony plate functions

    as a preliminary route for energy absorbance (Fig. 13b). The porous

    interior acts as a cellular foam, undergoing deformations such as

    elastic bending, plastic bucklingor wall breakageto absorb a certain

    amount of energy. This mechanism may prevent cracking and thus

    maintainstructural integrityof thecortex at small strains. The third

    mechanism incorporates the structural and mechanical properties

    of the material itself by combining the dorsal and ventral cortex

    (Fig. 13c).The hard andhighly mineralized dorsal regioncan sustain

    higher vertical stresses as the mechanical testing indicates, and be-cause of the unique shape of the osteoderm, the loads then transfer

    down to the less-mineralized, more compliant ventral region. The

    ventral region thus suffers from stresses oriented preferentially in

    thehorizontal direction. Thebasal regionconsists mainlyof parallel

    oriented collagen fibrils (lamellar-zonal bone) and possesses better

    capability to absorb energy horizontally through various toughen-

    ing mechanisms. In addition, the dorsal and ventral region is joined

    through a mechanical property gradient, where interfacial tough-

    ness is enhanced and stress is better redistributed. The integration

    of these three deformation mechanisms mayleadto a synergistic ef-

    fect and therefore an optimized dermal armor for alligator.

    5. Conclusions

    Dermal armors developed in reptiles as well as some mammals

    and fish are considered to be optimized for both protection and

    flexibility through millions of years of evolution. In the present

    study, we investigated the structure and mechanical behaviors of

    the American alligator (Alligator mississippiensis) osteoderms at

    multiple length scales. The dermal armor of alligator is a hierarchi-

    cally structured composite consisting of mineralized rigid bony

    plates connected by non-mineralized collagen fibers. Through the

    experiments, we established the structuralmechanical property

    relationships and deformation mechanisms of the osteoderm, and

    eventually proposed how the dorsal shield of alligator protects

    against external threats from the whole armor system to individual

    plates. The major discoveries of this study are concluded asfollows:

    (1) Complex 3-D sutures and the non-mineralized connective

    fibers between neighboring osteoderms provide flexibility

    for the whole armor. Strategies such as bridging and stretch

    ing of the collagen fibers and interlocking of the sutures are

    utilized, which are similar to those observed in armadillo

    carapace and turtle shell.

    (2) The sandwich structure of the osteoderm shows a compact

    cortex surrounding the porous core, enhancing bending stiffness and energy absorption ability with reduced weight. The

    intricate 3-D network of the neurovascular system is respon

    sible for the spongy interior.

    (3) The osteoderm is composed of four different bone morphol

    ogies, vertically from the outmost surface to the ventra

    region: outer sheath (woven bone with porosity), woven

    and lamellar bone, woven bone with major neurovascular

    cavities and lamellar-zonal bone. The varying mineral con-

    tents and porosity result in different localized hardnes

    and reduced modulus values across the osteoderm: from a

    hard and stiff dorsal cortex gradually transform to a more

    compliant ventral base. Similar design strategies have been

    applied in various natural armors as well, implying evolu-

    tionary convergence for defensive functionality.

    (4) Cross-sectional fracture surfaces of osteoderm through kee

    region indicate various degrees of mineralization and thus

    different microstructures in the dorsal and the ventra

    regions: the dorsal region is highly mineralized, showing

    granular morphology and flat fracture surface while the ven-

    tral region is less mineralized, showing flexible and twisted

    fibrils. Incorporating the two regions with mild mechanica

    gradient leads to the anisotropy in compressive behaviors

    the vertical orientation is able to bear higher loads, while

    the horizontal orientation can absorb more energy through

    multiple toughening mechanisms including lamellae buck-

    ling and fiber bridging.

    (5) Three deformation mechanisms are proposed for the derma

    armor of alligator: (1) the flexibility provided by sutures and

    non-mineralized collagen fibers can dissipate energy undersmall loads; (2) deformations of the cellular foam interior

    absorb impact energy without cortex cracking; (3) a combi

    nation of the hard dorsal region and the compliant ventra

    region with graded mechanical properties offers optimiza-

    tion in load re-distribution and energy absorbance.

    Acknowledgements

    The authors gratefully thankYu-ChenChan, Hsien-WeiChen, Su

    Yueh Tsai and Prof. Jenq-Gong Duh (MSE Department, Nationa

    Tsing Hua University) for their support and assistant with the FE

    SEM, FE-EPMA and ash-content measurements. We acknowledgeChia-Chi Chien, Bai-Hong Ke, Tsung-Tse Lee and Prof. Yeu-Kuang

    Hwu (Institute of Physics, Academia Sinica) forhelp with thetechni

    calwork andadvices onl-CT scans and3-D image reconstruction athe National Synchrotron Radiation Research Center (NSRRC). We

    would also thank Hsi-Ming Yang, Li-Chi Hsu and Prof. Jyh-Wei Lee

    (MSE Department, Ming ChiUniversity of Technology) for their help

    with nanoindentation measurements. This research is supported by

    National Science Council, Taiwan (NSC100-2218-E-007-016-MY3

    and NSC101-2628-E-007-017-MY3).

    Appendix A. Figures with essential colour discrimination

    Certain figures in this article, particularly Figs. 113, are diffi-cult to interpret in black and white. The full colour images can

    C.-Y. Sun, P.-Y. Chen/ Acta Biomaterialia 9 (2013) 90499064 9063

    http://-/?-http://-/?-http://-/?-
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    be found in the on-line version, at doi:http://dx.doi.org/10.1016/

    j.actbio.2013.07.016.

    References

    [1] Meyers MA, Chen P-Y, Lin AY-M, Seki Y. Biological materials: structure andmechanical properties. Prog Mater Sci 2008;53:1206.

    [2] Chen P-Y, McKittrick J, Meyers MA. Biological materials: functional adaptationsand bioinspired designs. Prog Mater Sci 2012;57:1492704.[3] Dunlop JWC, Fratzl P. Biological composites. Ann Rev Mater Res 2010;40:124.[4] Yang W, Chen IH, Gludovatz B, Zimmermann EA, Ritchie RO, Meyers MA.

    Natural flexible dermal armor. Adv Mater 2013;25:3148.[5] Currey JD. Mechanical properties and adaptations of some less familiar bony

    tissues. J Mech Behav Biomed Mater 2010;3:35772.[6] Ikoma T, Kobayashi H, Tanaka J, Walsh D, Mann S. Microstructure, mechanical,

    and biomimetic properties of fish scales from Pagrus major. J Struct Biol2003;142:32733.

    [7] Bruet BJ, Song J, Boyce MC, Ortiz C. Materials design principles of ancient fisharmour. Nat Mater 2008;7:74856.

    [8] Lin YS, Wei CT, Olevsky EA, Meyers MA. Mechanical properties and thelaminate structure of Arapaima gigas scales. J Mech Behav Biomed Mater2011;4:114556.

    [9] Marino Cugno Garrano A, La Rosa G, Zhang D, Niu LN, Tay FR, Majd H, et al. Onthe mechanical behavior of scales from Cyprinus carpio. J Mech Behav BiomedMater 2012;7:1729.

    [10] Yang W, Gludovatz B, Zimmermann EA, Bale HA, Ritchie RO, Meyers MA.

    Structure and fracture resistance of alligator gar (Atractosteus spatula) armoredfish scales. Acta Biomater 2013;9:587689.

    [11]Song J, Reichert S, Kallai I, Gazit D, Wund M, Boyce MC, et al. Quantitativemicrostructural studies of the armor of the marine threespine stickleback(Gasterosteus aculeatus). J Struct Biol 2010;171:31831.

    [12] Allison PG, Chandler MQ, Rodriguez RI, Williams BA, Moser RD, Weiss Jr CA,et al. Mechanical properties and structure of the biological multilayeredmaterial system,Atractosteus spatula scales. Acta Biomater 2013;9:528996.

    [13]Rhee H, Horstemeyer MF, Hwang Y, Lim H, El Kadiri H, Trim W. A study on thestructure and mechanical behavior of the Terrapene carolina carapace: apathway to design bio-inspired synthetic composites. Mater Sci Eng C2009;29:23339.

    [14]Achrai B, Wagner HD. Micro-structure and mechanical properties of the turtlecarapace as a biological composite shield. Acta Biomater 2013;9:5890902.

    [15] Krauss S, Monsonego-Ornan E, Zelzer E, Fratzl P, Shahar R. Mechanical functionof a complex three-dimensional suture joining the bony elements in the shellof the red-eared slider turtle. Adv Mater 2009;21:40712.

    [16]Balani K, Patel RR, Keshri AK, Lahiri D, Agarwal A. Multi-scale hierarchy ofChelydra serpentina: microstructure and mechanical properties of turtle shell. JMech Behav Biomed Mater 2011;4:144051.

    [17]Chen IH, Kiang JH, Correa V, Lopez MI, Chen PY, McKittrick J, et al. Armadilloarmor: mechanical testing and micro-structural evaluation. J Mech BehavBiomed Mater 2011;4:71322.

    [18]Rhee H, Horstemeyer MF, Ramsay A. A study on the structure and mechanicalbehavior of theDasypus novemcinctusshell. Mater Sci Eng C 2011;31:3639.

    [19]Erickson GM, Lappin AK, Vliet KA. The ontogeny of bite-force performance inAmerican alligator (Alligator mississippiensis). J Zool 2003;260:31727.

    [20]Scheyer TM, Sander PM. Histology of ankylosaur osteoderms: implications forsystematics and function. J Vertebr Paleontol 2004;24:87493.

    [21] Alibardi L, Thompson MB. Fine structure of the developing epidermis in theembryo of the American alligator (Alligator mississippiensis,Crocodilia,Reptilia).

    J Anat 2001;198:26582.[22]Grigg G, Gans C. Morphology and physiology of the Crocodylia. In: Glasby CG,

    Ross GJB, Beesley PL, editors. Fauna of Australia, vol. 2A. Amphibia andReptilia. Canberra: Australian Government Publishing Service; 1993.

    [23] Vickaryous MK, Sire JY. The integumentary skeleton of tetrapods: origin,evolution, and development. J Anat 2009;214:44164.

    [24] Vickaryous MK, Hall BK. Development of the dermal skeleton in Alligatormississippiensis(Archosauria, Crocodylia) with comments on the homology ofosteoderms. J Morphol 2008;269:398422.

    [25]Farlow JO, Hayashi S, Tattersall GJ. Internal vascularity of the dermal plates ofStegosaurus(Ornithischia, Thyreophora). Swiss J Geosci 2010;103:17385.

    [26] Buffrenil V, Sire JY, Rage JC. The histological structure of glyptosaurineosteoderms (Squamata: Anguidae), and the problem of osteodermdevelopment in squamates. J Morphol 2010;271:72937.

    [27] Cerda IA, Desojo JB. Dermal armour histology of aetosaurs (Archosauria:Pseudosuchia), from the Upper Triassic of Argentina and Brazil. Lethaia

    2011;44:41728.[28] Farlow JO, Thompson CV, Rosner DE. Plates of the dinosaur stegosaurus: forced

    convection heat loss fins? Science 1976;192:11235.[29] Curry Rogers K, DEmic M, Rogers R, Vickaryous M, Cagan A. Sauropod dinosaur

    osteoderms from the Late Cretaceous of Madagascar. Nat Commun2011;2:564.

    [30] Seidel MR. The osteoderms of the American alligator and their functionalsignificance. Herpetologica 1979;35:37580.

    [31] Hutton JM. Age determination of living nile crocodiles from the corticalstratification of bone. Copeia 1986:33241.

    [32] Tucker AD. Validation of skeletochronology to determine age of freshwatercrocodiles (Crocodylus johnstoni). Mar Freshw Res 1997;48:34351.

    [33] Saalfeld DT, Webb KK, Conway WC, Calkins GE, Duguay JP. Growth andcondition of American alligators (Alligator mississippiensis) in an InlandWetland of East Texas. Southeast Nat 2008;7:54150.

    [34] Song YF, Chang CH, Liu CY, Chang SH, Jeng US, Lai YH, et al. X-ray beamlines forstructural studies at the NSRRC superconducting wavelength shifter. JSynchrotron Radiat 2007;14:3205.

    [35] Rho JY, Zioupos P, Currey JD, Pharr GM. Variations in the individual thicklamellar properties within osteons by nanoindentation. Bone 1999;25:295300.

    [36] Currey JD. Mechanical properties of bone tissues with greatly differingfunctions. J Biomech 1979;12:3139.

    [37]Kienzle E, Kopsch G, Koelle P, Clauss M. Chemical composition of turtles andtortoises. J Nutr 2006;136:2053S4S.

    [38]Chen PY, Stokes AG, McKittrick J. Comparison of the structure and mechanicalproperties of bovine femur bone and antler of the North American elk (Cervuselaphus canadensis). Acta Biomater 2009;5:693706.

    [39]Gibson LJ. Biomechanics of cellular solids. J Biomech 2005;38:37799.[40] Seki Y, Kad B, Benson D, Meyers MA. The toucan beak: structure and

    mechanical response. Mater Sci Eng C 2006;26:141220.[41] Bodde SG, Meyers MA, McKittrick J. Correlation of the mechanical and

    structural properties of cortical rachis keratin of rectrices of the Toco Toucan(Ramphastos toco). J Mech Behav Biomed Mater 2011;4:72332.

    [42] Currey JD. The structure of bone tissue. Bones: structure andmechanics. Princeton, NJ: Princeton University Press; 2002. pp. 3-26.

    [43] Oliver WC, Pharr GM. An improved technique for determining hardness and

    elastic modulus using load and displacement sensing indentationexperiments. J Mater Res 1992;7:156483.

    [44] Chen PY, Lin AY, McKittrick J, Meyers MA. Structure and mechanical propertiesof crab exoskeletons. Acta Biomater 2008;4:58796.

    [45]Chen P-Y, Schirer J, Simpson A, Nay R, Lin Y-S, Yang W, et al. Predation versusprotection: fish teeth and scales evaluated by nanoindentation. J Mater Res2011;27:10012.

    [46]Pithioux M, Lasaygues P, Chabrand P. An alternative ultrasonic method formeasuring the elastic properties of cortical bone. J Biomech 2002;35:9618.

    [47] Zysset PK, Edward Guo X, Edward Hoffler C, Moore KE, Goldstein SA. Elasticmodulus and hardness of cortical and trabecular bone lamellae measured bynanoindentation in the human femur. J Biomech 1999;32:100512.

    [48]Rho JY, Pharr GM. Effects of drying on the mechanical properties of bovinefemur measured by nanoindentation. J Mater Sci Mater Med 1999;10:4858.

    [49]Bonfield W, Clark EA. Elastic deformation of compact bone. J Mater Sci 1973;8:15904.

    [50]Mackenzie JK. The elastic constants of a solid containing spherical holes. ProcPhys Soc B 1950;63:211.

    9064 C.-Y. Sun, P.-Y. Chen / Acta Biomaterialia 9 (2013) 90499064

    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