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nanomaterials
Article
Hierarchical Microstructure of Tooth Enameloid in Two
Lamniform Shark Species, Carcharias taurus
and Isurus oxyrinchus
Jana Wilmers 1,* , Miranda Waldron 2and Swantje Bargmann 1,3
Citation: Wilmers, J.; Waldron, M.;
Bargmann, S. Hierarchical
Microstructure of Tooth Enameloid in
Two Lamniform Shark Species,
Carcharias taurus and Isurus oxyrinchus.
Nanomaterials 2021,11, 969. https://
doi.org/10.3390/nano11040969
Academic Editor: Daniel Kiener
Received: 16 March 2021
Accepted: 6 April 2021
Published: 9 April 2021
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1
Chair of Solid Mechanics, University of Wuppertal, 42119 Wuppertal, Germany; bargmann@uni-wuppertal.de
2Electron Microscope Unit, University of Cape Town, Cape Town 7701, South Africa;
miranda.waldron@uct.ac.za
3Wuppertal Center for Smart Materials, University of Wuppertal, 42119 Wuppertal, Germany
*Correspondence: wilmers@uni-wuppertal.de; Tel.: +49-202-439-2086
Abstract:
Shark tooth enameloid is a hard tissue made up of nanoscale fluorapatite crystallites
arranged in a unique hierarchical pattern. This microstructural design results in a macroscopic
material that is stiff, strong, and tough, despite consisting almost completely of brittle mineral. In this
contribution, we characterize and compare the enameloid microstructure of two modern lamniform
sharks, Isurus oxyrinchus (shortfin mako shark) and Carcharias taurus (spotted ragged-tooth shark),
based on scanning electron microscopy images. The hierarchical microstructure of shark enameloid
is discussed in comparison with amniote enamel. Striking similarities in the microstructures of the
two hard tissues are found. Identical structural motifs have developed on different levels of the
hierarchy in response to similar biomechanical requirements in enameloid and enamel. Analyzing
these structural patterns allows the identification of general microstructural design principles and
their biomechanical function, thus paving the way for the design of bioinspired composite materials
with superior properties such as high strength combined with high fracture resistance.
Keywords: enameloid; microstructure; shark; teeth
1. Introduction
Shark teeth have always exerted a special kind of fascination on humans as they are
the perfectly designed, highly efficient natural weapons of a deadly hunter. The teeth are
arranged in multiple rows behind each other in the shark’s jaws
(Figure 1)
and exhibit a
variety of shapes and sizes among different species, ranging from flattened domes over
needles to triangular cutting tools with sharp, serrated edges. Even between closely
related species, a wide variety of tooth shapes can be found [
1
] and may in fact be used to
identify species [
2
]. These morphological variations have been attributed to differences
in feeding behavior and, thus, mechanical loads [
3
–
5
]. Optimal functionality is further
guaranteed by regular shedding and replacement of the teeth. The replacement rate varies
between species and with age and water temperature. For spotted ragged-tooth sharks
(
Carcharias taurus
), an average tooth loss rate of 1.06 teeth per day was identified [
6
], which
means an individual spotted ragged-tooth shark will shed over 13,500 teeth in a lifetime.
For other species, even more rapid replacement can be found [7,8].
Based on mechanical studies [
3
,
9
], it has been proposed that the frequent tooth re-
placement of shark teeth is due to wear rather than failure by tooth fracture. Wear on a
small scale would not impact the structural strength of the tooth but would reduce its
efficiency as a cutting or piercing tool. Studies aimed at understanding the biomechanics
of shark teeth have found remarkably similar stress patterns even for drastically different
tooth morphologies under static loading [
9
]. Dynamic tests show the increased cutting
efficiency of serrated edges compared to smoother teeth, as well as their drastically faster
Nanomaterials 2021,11, 969. https://doi.org/10.3390/nano11040969 https://www.mdpi.com/journal/nanomaterials
Nanomaterials 2021,11, 969 2 of 16
mechanical wear [
3
]. In many shark species, however, tooth-on-prey contact leading to
wear is comparatively rare as only large prey is manipulated with teeth [10].
(a)jaw of I. oxyrinchus (b)C. taurus at Two Oceans Aquarium, Cape Town
Figure 1.
Shark teeth are arranged in series. Multiple rows of teeth grow behind each other and are
continuously replaced over a lifetime. The primary function of the teeth of I. oxyrinchus (
a
,
b
) is to
pierce (not cut) prey. The jaws of both shark species can be opened very wide to swallow prey in
one piece.
It is well known that the mechanical properties of biological materials strongly depend
on their complex microstructures. Hypermineralized tissues like shark tooth enameloid
consist almost exclusively of nanoscale brittle mineral crystallites [
4
], yet exhibit consider-
able strength and exceptionally high fracture toughness way beyond that of the individual
constituents. The relationship between the sophisticated microstructural hierarchy and the
mechanical performance of other highly mineralized tissues such as amniote enamel or mol-
lusc nacre has attracted considerable research interest, e.g., [
11
–
15
]. Shark enameloid, while
similar in appearance and function to amniote enamel, is considerably less understood.
Enameloid consists of elongated fluorapatite (Ca2(PO4)F) crystallites with a roughly
hexagonal cross-section visible in TEM images in different shark species [
16
,
17
]. The crys-
tallites have a width of
50 nm
to
80 nm
and a length exceeding
1000 nm
[
17
,
18
]. They are
densely packed and arranged in a complex hierarchical structure. In all neoselachian
sharks, the majority of the enameloid cover consists of bundled crystallite enameloid
(BCE) and the tooth’s outer surface is covered in single crystal enameloid(SCE) (Single
crystal enameloid is also commonly referred to as ‘shiny-layered enameloid’ reflecting
its optical appearance) [
19
–
21
]. In the BCE, fluorapatite nanocrystallites are arranged in
bundles that may be oriented longitudinally, radially, or circumferentially within the tooth,
with slight variations occurring between different shark
species [20,22].
In modern sharks
(
selachimorpha
), bundles close to the dentine-enameloid junction are three-dimensionally
interwoven, a structure type referred to as tangled bundled enameloid (TBE). Further from
the dentine, the bundle arrangement becomes more regular, with the bundles aligned
parallel to each other and to the tooth’s longitudinal axis, a structural motif referred to as
parallel bundled enameloid (PBE).
This characteristic layered structure, is often referred to as a ‘triple layered structure’,
with TBE, PBE and SCE each constituting one layer. However, the microstructure of
shark enameloid has been found to be more complex than this description implies as
it does not take into account, e.g., radial structural elements described in [
21
]. Instead,
a nomenclature discerning the inner enameloid consisting of parallel bundled and tangled
bundled enameloid, jointly referred to as the BCE unit, and an outer enameloid layer
referred to as the ridge/cutting edge layer (RCEL) has been suggested in [
19
]. The RCEL
itself consists of an external layer of single crystal enameloid and an internal layer of
circumferential bundles. The inner and the outer enameloid are separated clearly as
visible in micrographs, while transitions between the substructures within each layer are
generally smooth. Elemental analysis of shark tooth enameloid shows that the crystallite
Nanomaterials 2021,11, 969 3 of 16
composition varies slightly between the inner and the outer enameloid, with the outer
enameloid being richer in the substituting ions magnesium and sodium than the inner,
bundled enameloid [18].
The enameloid microstructures in other neoselachian species vary more drastically from
the patterns identified in modern sharks. Batoids (rays and skates), for instance, exhibit a less
complex microstructure, with some species even losing bundles completely [23,24].
This study characterizes and compares the enameloid microstructure of two modern
lamniform sharks, Isurus oxyrinchus (shortfin mako shark) and Carcharias taurus (spotted
ragged-tooth shark). A description of enameloid hierarchical microstructure in the style of
the established description of amniote enamel [
14
,
25
,
26
] is proposed. As amniote enamel
serves the same protective function as enameloid, structural similarities may be indicative
of biomechanical function. Such similarities between enameloid structure and the enamel
of different mammalian species are identified and discussed here. By focusing on structural
motifs and patterns in shark enameloid, the foundation for microstructure design in syn-
thetic composite materials is laid. As biological materials exhibit sophisticated structures
on multiple length scales, they commonly combine highly desirable properties such as
high fracture toughness and strength which are difficult to obtain in typical engineering
materials [27].
Thus, mimicking some of nature’s microstructural design principles may be
the key in designing high performance composites.
2. Materials and Methods
Teeth of two species of the order Lamniformes, the spotted ragged-tooth or sand tiger
shark (Carcharias taurus) and the shortfin mako shark (Isurus oxyrinchus), were studied.
Both species feed mainly on bony fish and occasionally other elasmobranches or even sea
turtles. Their teeth are used primarily to incapacitate prey before ingestion or, less often,
to rip pieces from larger prey.
Samples of I. oxyrinchus teeth were acquired from Hout Bay, South Africa. The
C. taurus
teeth samples had been shed naturally and were kindly provided by Two Oceans Aquarium,
Cape Town, South Africa. The teeth originate from one or more of the 5 adult females
living in captivity (but born free) at the aquarium, weighing
80 kg
to
170 kg
. Teeth samples
were stored at ambient conditions before specimen preparation and imaging. Teeth of both
species were placed in a mold, covered in Spurrs resin and put in an oven at
60 °C
for 12 h
to allow the resin to set. The teeth were then ground in the longitudinal and transverse
sections
(Figure 2) and
the exposed areas were polished with
0.2 µm
aluminum powder.
The samples were sonicated at
50 Hz
in distilled water for 2 min and then etched with
10 % hydrochloric acid for 2 min and rinsed in distilled water for 5 min. They were carbon
coated in an evaporation coater and viewed with a Tescan MIRA SEM at
5 kV
. The SEM
images for both species at different magnifications are gathered in the Supplementary
Material Figures S1–S15.
Nanomaterials 2021,11, 969 4 of 16
(a)tooth of I. oxyrinchus (b)tooth of C. taurus
Figure 2.
Tooth morphology of the two studied species. Both species have slender, dagger-like teeth without serrated
edges. The teeth are curved slightly to improve the grip on prey. Sketches on the left show the tooth and its transversal
cross-section. Arrows indicate the facial direction. The shaded planes correspond to the section planes used in imaging.
The enameloid cover consists of inner and outer enameloid, marked in the cross-section with BCE and RCLE, respectively.
I. oxyrinchus
teeth (
a
) are monocuspid while C. taurus teeth (
b
) are tricuspid with a large main cusp and small outer cusplets
indicated by white arrowheads. The I. oxyrinchus tooth (
a
) exhibits a small amount of wear on the lingual side of the tip.
The C. taurus tooth depicted in (b) is fractured at the tip.
3. Results: Microstructure Description of Enameloid in I. oxyrinchus and C. taurus
The teeth of both studied species, I. oxyrinchus and C. taurus, have remarkably similar
morphologies (Figure 2). The teeth are slender and needle-like and possess no serrated
edges. This morphology is commonly associated with piercing of prey [
5
]. Both are curved
in an S-shape with the tooth protruding forward and then curving backward into the oral
cavity and only the tip curving forward again. The anterior side especially of I. oxyrinchus
teeth is flattened. The most pronounced difference between teeth of the two species is the
presence of small tricuspids in C. taurus not found in I. oxyrinchus.
The teeth imaged in this work were approximately
10 mm
in length and had a lenticu-
lar cross-section with a width of
2.5 mm
in the upper region where the teeth were sectioned.
Other samples were larger, with lengths reaching
15 mm
for I. oxyrinchus and
20 mm
for
C. taurus. The enameloid cover in the studied transversal sections had a thickness of
ca.
0.5 mm
for I. oxyrinchus and ca.
0.4 mm
for C. taurus. The enameloid thickness var-
ied slightly between different teeth; in the longitudinally sectioned tooth of C. taurus,
the thickness on the anterior side of the tooth was
0.4 mm
and on the posterior side it was
0.7 mm.
3.1. Enameloid Microstructure of Isurus oxyrinchus
The enameloid cover of I. oxyrinchus teeth exhibits the typical layered structure in
longitudinal section as seen in Figure 3a,b. The outermost layer with a thickness of
30 µm
to
35 µm
consists of densely packed crystals (Figure 3c) in the upper region of the tooth. In
the lower region, no single crystal enameloid is visible and the outer enameloid consists
solely of a single layer of circumferentially oriented bundles as seen in Figure 3f.
The outer enamel covers a layer of parallel bundled enameloid (Figure 3d,e) with a
thickness of ca.
170 µm
. In this region, crystal bundles are aligned parallel to each other and
oriented along the tooth’s longitudinal axis. Closer to the dentine, this clear arrangement
becomes less regular and transitions smoothly into entangled bundles. In the longitudinal
section depicted in Figure 3, the transition from dentine in the tooth’s core to the enameloid
Nanomaterials 2021,11, 969 5 of 16
cover appears smooth (Figure 3b) due to only minor etching. In the transversal section
Figure 4a, a sharp junction between enameloid and dentine is apparent.
Figure 3.
I. oxyrinchus tooth in longitudinal section. The enameloid cover is characteristically layered as seen in (
a
,
b
).
Zooming into the outer enameloid, a densely packed outer layer is found in the top part of the tooth (
c
) while in the lower
part, distinct circumferential bundles are apparent (
f
). (
d
) The outer enameloid surrounds a layer of parallel aligned crystal
bundles. (e) Magnified view of the broken tip of a bundle from (d).
Nanomaterials 2021,11, 969 6 of 16
Figure 4.
Transversal section of I. oxyrinchus tooth. The tooth’s enameloid cover is built up in layers (
a
). The outer enameloid
is designated RCLE and clearly separated (dashed line, arrow heads in zoomed-in view) from the inner enameloid consisting
of PBE and TBE. (
b
,
c
) show the outer edge of the enameloid cover and radial elements (arrow heads) running towards the
outer enameloid. In between the radial elements, bundles are oriented parallel to the tooth’s longitudinal axis. (
d
,
e
) show
three-dimensional entanglement of bundles close to the dentine-enameloid junction.
The bundles in the inner enameloid have a diameter of ca.
4 µm
to
9 µm
and consist of
densely packed crystallites with an average width of
66 nm
visible in Figure 5a. This size is
in good agreement with the range of
50 nm
to
80 nm
found for I. oxyrinchus in [
17
,
18
]. The
crystallites are several micrometers in length, but an exact measurement is impossible in the
micrographs as the visible length exceeds the field of view. Within a bundle, the elongated
crystallites are aligned parallel to each other without visible pores or gaps between them
(Figure 5c). Neighboring crystallites remain in contact in the TBE when the bundle curves
and changes direction (Figure 5b).
Figure 5.
Fluorapatite crystallites in I. oxyrinchus tooth. (
a
) Fracture surface showing individual crystallites. (
b
) The thin,
rod-like crystallites change orientation in the TBE (transversal section). Neighboring crystal rods remain parallel. (
c
) Densely
packed crystallites within a crystallite bundle (transversal section). The crystallites are aligned parallel to each other and in
close contact with each other.
Nanomaterials 2021,11, 969 7 of 16
Inner, bundled enameloid and outer enameloid are clearly separated with a dis-
tinct boundary clearly visible in the medial and distal tooth edges in transversal section
Figure 3a.
In these edges, the outer enameloid is thicker while in the middle of the lin-
gual and labial faces, the outer enameloid’s thickness is
30 µm
to
35 µm
as found in the
longitudinal section.
In the transversal section, the different regions of the inner enameloid are more distinct
due to stronger etching. Adjacent to the enameloid-dentine junction, crystallite bundles are
three-dimensionally interwoven (Figure 4d,e). The TBE is approximately
270 µm
to
290 µm
thick. The surrounding PBE region is only marginally thinner. The inner enameloid, thus,
appears to be made up of equal amounts of TBE and PBE. Exact measurements, however,
are difficult as the transition between the regions is smooth.
The parallel bundles of the inner enameloid are interspersed with regularly arranged
radial elements with a thickness of ca.
1.5 µm
(Figure 4b,c). The distance between two radial
elements ranges from
6 µm
to
10 µm
. This corresponds to the diameter of an individual
bundle, thus, the radial elements appear to separate individual rows of parallel aligned
bundles. The crystallites within the radial elements are oriented at almost a 90
◦
angle to the
bundle direction (i.e., parallel to the imaging plane) and run from the enameloid-dentine
junction radially outwards to the outer tooth surface. As shown in Figure 4b, the radial
elements merge into the outer enameloid layer which consists of densely packed, randomly
oriented crystallites.
3.2. Enameloid Microstructure of Carcharias taurus
Figure 6depicts the longitudinal section of a C. taurus tooth. In this section, no outer
enameloid is visible and the PBE appears to extend right to the tooth edge (Figure 6a,d)
with no outer enameloid visible. A thin layer of outer enameloid or remainders of one
could possibly be obscured by the embedding material covering the tooth’s edge. The PBE
transitions smoothly into a region of TBE with three-dimensionally interwoven crystallite
bundles as visible in Figure 6b,c. The enemaloid-dentine junction is sharp and clearly
visible with no dentine reaching into the enameloid.
In the transversal section in Figure 7a, the typical layered structure of modern shark’s
enameloid is readily identifiable. The outer enameloid is clearly visible in the medial and
distal tooth edges and narrows drastically towards the middle of the lingual and the labial
faces. In the middle of these tooth faces, the outer enameloid appears to vanish completely
(Figure 7b).
The transition from parallel aligned crystallite bundles (PBE) to the entangled, inter-
woven bundles (TBE) in the inner enameloid is smooth without any sharp boundaries. The
PBE region has a thickness of about
115 µm
measured in the transversal section Figure 7.
The TBE with a thickness of ca. 215 µm is significantly thicker.
The crystal bundles in the inner enameloid have a diameter of
5 µm
with the crystals’
long axes running parallel to the bundle’s long axis, as seen in Figure 6e,f. Figure 8
shows close-up micrographs of fluorapatite crystallites. The crystallites have an average
thickness of
60 nm
and are aligned along their elongated longitudinal axes. The crystallites
are densely packed within a bundle and appear to be in direct contact along the whole
crystallite length. This contact and the alignment are maintained even when the crystallites
curve and change direction in the TBE (Figure 8b).
Close to the edge, radial elements intersecting the transversal plane at approximately
90
◦
can be seen in Figure 7b,c. Each of these radial elements has a thickness of ca.
1.5 µm
and the distance between two elements is approximately
5 µm
or slightly higher (up to
7.5 µm
) which correlates to one bundle diameter. The crystallites within the radial elements
are aligned in parallel with each other and their long axes are oriented at almost a 90
◦
angle
to the long axes of the bundles as seen in Figure 9a which shows two radial elements with
the crystallite bundles between them.
Nanomaterials 2021,11, 969 8 of 16
Figure 6.
C. taurus tooth in longitudinal section. In the area imaged in (
a
), parallel bundle enameloid (PBE) reaches all the
way to the outer surface which is obscured by embedding material. (
b
,
c
) show three-dimensionally entangled bundles
belonging to the TBE close to the dentine. (
d
–
f
) show a sequence of images with increasing magnification into the PBE in the
outer range of the inner enameloid. Between the bundles, remnants of layers between the bundles are visible. An individual
bundle (f) consists of densely packed, parallel aligned crystallites.
Nanomaterials 2021,11, 969 9 of 16
Figure 7.
Transversal section of C. taurus tooth. (
a
) The enameloid cover is made up of an outer enameloid (RCLE) clearly
separated (dashed line, arrow heads in zoomed-in view) from the inner enameloid which itself consists of TBE and PBE.
(
b
,
c
) In the middle of the tooth edge, the outer enameloid appears to be worn away and parallel aligned crystal bundles
(PBE) intersected by evenly spaced thin radial elements (arrow heads) are visible. (
d
,
e
) In the inner enameloid close to the
dentine, the bundles are interwoven and change orientation frequently in all directions.
The crack apparent in (a,b) likely occurred during specimen preparation.
Figure 8.
Fluorapatite crystallites in C. taurus tooth. (
a
) Individual crystallites at the polished sectioning plane. (
b
) Crystal-
lites curve and change orientation in the TBE while remaining parallel to their neighbors (transversal section). (
c
) Within a
bundle in the inner enameloid, crystallites are in close contact with each other and aligned along their long axis (transver-
sal section).
Nanomaterials 2021,11, 969 10 of 16
(a)enameloid of C. taurus (b)enamel of M. rufogriseus
Figure 9.
Transversal sections of (
a
) the outer enameloid of C. taurus and (
b
) the modified radial enamel in a tooth of
Macropus rufogriseus (red-necked wallaby). Both species exhibit radial sheets of densely packed crystallites separating
rows of bundled crystallites. The hydroxyapatite crystallites of the mammalian enamel are thinner than the fluorapatite of
enameloid but the higher-level bundles and radial elements have the same size in both tissues. Despite the significant size
difference between the two species, the imaged teeth are of similar size with a crown height of
10 mm
in C. taurus and ca.
7 mm in M. rufogriseus.
4. Microstructural Features of Modern Shark Enameloid
4.1. Lamniform Shark Enameloid Structure
Isurus oxyrinchus and Carcharias taurus exhibit a remarkably similar enameloid mi-
crostructure corresponding to the typical layered structure known for modern
sharks [19,20].
The enameloid of non-selachian species shows drastically varying levels of complexity with
some exhibiting crystallite bundles [
24
] while the fossilized teeth of some Ctenacanthiformes
are so highly mineralized that individual crystallites cannot be distinguished [28].
The microstructural complexity in modern sharks has been suggested to be a functional
adaptation to the specialized feeding behavior in comparison to other chondrichthyes [
28
].
The enameloid microstructure of I. oxyrinchus and C. taurus enameloid described here
exhibits a clear hierarchical set-up. It consists of thin, needle-like crystallites with a length
exceeding a few micrometers. The crystallites of both species have an average width of ca.
60 nm
to
65 nm
, which falls perfectly into the range found in [
18
] for
I. oxyrinchus
and is in
the same range as the
50 nm
identified in [
17
]. Table 1summarizes the sizes of all structural
features in both species.
The majority of the enameloid cover is composed of these crystallites, arranged in
densely packed crystallite bundles. The enameloid region adjacent to the dentine is in
both species characterized by intertwining bundles, corresponding to the TBE. In C. taurus,
the boundary between dentine and TBE is well-defined while in I. oxyrinchus, especially in
longitudinal section (Figure 3), the transition is smooth and some dentine extends into the
enameloid cover. A similarly smooth transition has been found for I. oxyrinchus in [
4
] and
for C. taurus in [29].
The TBE is covered by PBE in which the crystallite bundles are aligned parallel to each
other and the tooth’s longitudinal axis in both studied species. In I. oxyrinchus, TBE and PBE
regions are of similar thickness while in C. taurus, the TBE layer makes up approximately
two thirds of the bundled enameloid unit (Table 1).
Nanomaterials 2021,11, 969 11 of 16
Table 1. Size of structural features in modern shark enameloid and mammalian enamel.
I. oxyrinchus
Enameloid
C. taurus
Enameloid
Wallaby
Enamel
Human
Enamel
thickness TBE [µm] 270– 290 215 - -
thickness PBE [µm] 250 115 - -
thickness outer enameloid [µm] 30–35 10–30 - -
crystal width [nm] 50–75 55–70 45–50 50–70 [30,31]
bundle diameter [µm] 4–9 5 3–4 4–8 [32]
thickness of radial elements [µm] 1.5 1.5 1–1.5 -
distance radial elements [µm] 6–10 5 4 -
The outermost layer of a modern shark’s tooth typically consists of single crystal
enameloid in which crystals are oriented randomly. In the studied teeth of I. oxyrinchus, this
outer layer is visible and in the lower regions of the tooth, a single layer of circumferential
bundles as also found in [
18
] can be identified. The outer enameloid in I. oxyrinchus here
is with ca.
30 µm
thickness significantly thicker than the one described in [
18
] for the
same species.
For C. taurus, no outer enameloid layer could be found in longitudinal section. The
outer enameloid is thick in the tooth’s medial and distal edges and thins over the tooth’s
circumference until it vanishes in the middle of the lingual and labial faces (Figure 7).
The
C. taurus
tooth studied in [
29
] exhibits an outer enameloid layer of approximately
uniform thickness. This suggests that a thin outer layer might have been worn away,
either during normal function, after shedding due to environmental conditions [
10
] or
during etching. The outer enameloid of
C. taurus
does not exhibit circumferential bun-
dles as are found in
I. oxyrinchus.
This is the most significant difference in the enameloid
microstructures of the two species.
From the outer enameloid, thin radial elements reach into the inner enameloid where
they intersperse individual rows of parallel bundles. The presence of such radial elements
has been documented for I. oxyrinchus [
18
], C. taurus [
29
] and numerous other lamniform
shark species [19,20,33].
4.2. Radial Elements in Enameloid
Generally, the radial elements found in the outer enameloid and PBE are referred
to as ‘radial bundles’ [
19
,
20
]. Their geometry is described as ‘ribbon-like’ bundles in
I. oxyrinchus
[
18
]. FIB-SEM tomography of the enameloid of two carcharhiniform sharks [
33
]
shows the three-dimensional arrangement of radial elements close to the SCE. The crystal-
lites are arranged within a thin layer between the bundles with frequent gaps in the layer
and transversal connections between adjacent layers apparent.
In the studied section of I. oxyrinchus, the crystals within the radial elements are
aligned parallel to the transversal section (Figure 4) while in the transversal section of
C. taurus
(Figure 7), the crystallites in the radial elements intersect the imaging plane at an
angle, compare also Figure 9a. From these micrographs, the radial element layers appear
to be continuous thin sheets with two directions (the radial and the axial one) being much
larger than the thickness and have no gaps readily apparent. The sheets separate rows of
crystal bundles in which the crystals are oriented at an angle to the sheet crystals. In both
species, this angle appears to be close to 90◦.
This structural motif of thin sheets separating rows of crystal bundles from each
other can also be found in the dental enamel of some herbivorous mammals [
34
,
35
] and is
there referred to as modified radial enamel. Figure 9shows the radial elements in C. taurus
enameloid and the modified radial enamel in a molar of Macropus rufogriseus, the red-
necked wallaby, showing the striking similarity between the structures. The crystallites
in the marsupial’s enamel are hydroxyapatite and slightly thinner than the fluorapatite
crystals of C. taurus’s enameloid. Similarly, the bundles are thinner than in the enameloid,
but the sheets have the same thickness of
1.5 µm
(Table 1). The similar length scale of
Nanomaterials 2021,11, 969 12 of 16
both structures is remarkable when considering the large size difference between the two
species. The investigated teeth are of similar size with a crown height of
10 mm
in C. taurus
and ca.
7 mm
in M. rufogriseus. The diameter of the shark tooth is with
2.5 mm
of the same
order as an individual cusp of M. rufogriseus’s molar. For mammalian enamel, the size of
microstructural elements is independent of the size of a single tooth or the animal [14].
Modified radial enamel in mammals is generally interpreted as an adaptation to
large axial stresses arising due to horizontal mastication movements that are especially
common in grazing species. These axial stresses favor crack propagation in radial di-
rection, i.e., cracks travelling perpendicular to the tooth’s longitudinal axis from the sur-
face towards the softer dentine. Modified radial enamel introduces vertical decussation
planes, i.e., planes of abrupt crystal orientation discontinuities, within the crack path.
This results in deflection of the crack and twisting of the crack surface, thus, effectively
slowing or arresting cracks before reaching the dentine and protecting the tooth from
catastrophic fracture [14].
Slender shark teeth like the ones studied here are loaded in
bending during holding and shaking of prey [
9
]. This loading case results in dominant
axial tensile stresses which would cause radial crack growth. The decussation planes
introduced by the presence of radial sheets, thus, can distort the crack path. The crystallite
bundles are oriented axially and, thus, are stiff under bending and do not exhibit significant
weak propagation paths for radial cracks. The radial elements, therefore, provide tough-
ening against chipping of the tooth, i.e., crack propagation parallel to the tooth surface,
without introducing additional weakness against crack propagation under bending.
5. Structural Hierarchy in Dental Materials: Similarities between Enameloid
and Enamel
Shark enameloid and amniote enamel consist of different minerals and have been
shown to differ in evolutionary origin [
19
,
36
] but both dental tissues serve the same function
as the outermost layer of the tooth that protects the softer dentine. Thus, identification
of common microstructure patterns—such as radial elements—can give valuable insight
into biomechanical function, as seen in Section 4.2, and may form the basis for the design
of bioinspired composite materials. Therefore, the hierarchical microstructure of shark
enameloid is in the following described in comparison with amniote enamel for which a
well-established terminology exists ([
26
], see also [
14
]). Remarkably, both tissues exhibit
the same five levels of hierarchy, depicted in Figure 10.
The smallest building block in each tissue is the individual nanocrystal which is
designated as Level 0 of the hierarchy. Level I describes the local arrangement of crys-
tallites. In the shark enameloid studied here, individual crystallites are either aligned in
parallel or—in the shiny layer enameloid—fully randomly arranged. The parallel aligned
enameloid crystals are densely packed into crystallite bundles on Level II which in the
enamel nomenclature is also referred to as the module level. In dental enamel, different
modules can be identified [
14
] but the most prominent are the ‘rods’ or ‘prisms’ typical for
mammalian enamel. These generally have a rounded cross-section with diameters of
2 µm
to
10 µm
and, within the rods, enamel crystallites are predominantly arranged parallel to
each other and to the rod’s length axis. This arrangement is identical to the bundles in
shark enameloid.
The assembly of these modules forms Level III of the hierarchy. Different assem-
blies such as TBE and PBE can be referred to as enameloid types, in analogy to ‘enamel
types’. Much of the functional adaptation of mammalian enamel occurs on this third
level of the hierarchy that is characterized by locally varying arrangements of modules
and single crystallites as is the case in the discussed parallel bundled enameloid inter-
spersed with radial elements. Over the thickness of the enameloid cap, different enameloid
types occur, and some enameloid types may only be found in certain regions of the tooth.
For
I. oxyrinchus
and C. taurus, the enameloid consists of an innermost layer of TBE, sur-
rounded by PBE which in turn is covered by the outer enameloid. This varying pattern
constitutes Level IV of the structural hierarchy and is the equivalent to the schmelzmuster
(from the German ‘Zahnschmelz’ for tooth enamel) of enamel.
Nanomaterials 2021,11, 969 13 of 16
In many evolved mammalian species, the enamel consists of an inner layer of de-
cussated enamel types in which rods are interwoven or arranged at sharp angles to each
other [14].
This inner layer is commonly covered by radial enamel in which the rods aligned
parallel and the whole tooth is covered in a thin layer of “prism-less”, i.e., single crystallite
enamel. This typical schmelzmuster is interpreted to be an adaptation for fracture resis-
tance, as cracks can easily travel along the boundaries between parallel aligned bundles but
the presence of decussation deflects and bifurcates the crack path, resulting in toughening
and arrest of the crack before critical failure. The layered structure of a shark tooth is
a clear match for this set-up, suggesting that the parallel bundled enameloid increases
stiffness of the tooth necessary for piercing while the tangled bundled enameloid increases
fracture resistance. Strikingly, the relative thickness of the TBE layer found in I. oxyrinchus
and
C. taurus
in this study (Table 1) corresponds exactly to the
50 %
to
65 %
of decussated
enamel found in mammalian enamel [14].
Remark: The structural hierarchy of Isurus oxyrinchus teeth is also discussed in [
18
].
A six-level hierarchy description is proposed in which the fluorapatite unit cell consti-
tutes Level 1 and, thus, the smallest building block and Level 6 describes the whole
tooth. The higher-level structural features identified in [
18
] correspond to Levels II–IV
described above.
Figure 10.
Structural hierarchy of shark teeth enameloid. The enameloid cover of an individual shark tooth exhibits a
five-level hierarchical microstructure. On Level IV, the schmelzmuster, different structural patterns are combined to improve
the biomechanical function of the tooth. These Level III patterns are the different enameloid types and consist of the Level II
enameloid modules. In shark enameloid, the most common module is a bundle of parallel aligned crystallites (Level I).
The lowest level of the hierarchy, Level 0, is the individual nanoscale crystallite.
6. Conclusions
Hard tissues such as shark enameloid are biological composites of nanoscale mineral
crystals arranged in intricate hierarchical patterns interspersed with only minor amounts
of remnant protein. The microstructural design in these tissues results in a macroscopic ma-
terial that is stiff, strong and tough despite consisting almost completely of brittle mineral.
Analysis of micrographs of two lamniform shark species, the shortfin mako (Isurus
oxyrinchus) and the spotted ragged-tooth shark (Carcharias taurus), reveals the hierarchical
structure of enameloid. Fluorapatite nanocrystallites (Level 0) are arranged in bundles
(Level II) that themselves are arranged in a layered pattern over the enameloid cover
(Level IV). The microstructural arrangements found in both species are remarkably similar,
both contain parallel aligned bundles (PBE) interspersed with radial elements and an inner
layer of three-dimensionally interwoven TBE. In this work, the hierarchy of enameloid
microstructure is discussed in comparison to the well-established nomenclature used to
describe the microstructure of amniote enamel [
26
]. The microstructures of both tissues
exhibit the same five hierarchical levels, despite their different evolutionary origin.
Nanomaterials 2021,11, 969 14 of 16
Remarkably, on different hierarchical levels, direct structural analogues for shark
enameloid microstructure patterns could be identified in mammalian enamel. Shark enam-
eloid contains radial elements separating rows of crystal bundles on Level III, a structural
motif strikingly similar to the mammalian modified radial enamel. Another Level III enam-
eloid type, the tangled bundle enameloid, is structurally identical to the decussated irregular
enamel of marsupials and proboscideans [
35
,
37
]. On Level IV, these different enameloid
types are arranged in a layered structure that corresponds to the typical schmelzmusters of
mammalian enamel.
These striking structural similarities allow one to draw conclusions on enameloid
microstructure function based on the wealth of information available for enamel [
14
].
Modified radial enamel, for instance, has been found to increase fracture resistance while
maintaining high stiffness. Likewise, the radial elements in enameloid are likely to increase
fracture toughness by providing ‘easy’ crack propagation paths along the discontinuities
in crystallite orientation. These deliberately introduced crack paths result in energy dis-
sipation through crack deflection and can guide fracture away from sensitive parts of
the tooth. Similarly, the characteristic layered structure of lamniform shark enameloid is
functionally identical to mammalian enamel with an inner decussated enamel providing
fracture toughness and protection of the soft dentine from cracks.
By transferring such biological design principles to synthetic materials, unique prop-
erty combinations such as high strength combined with high fracture resistance may be
achieved. Mimicking the full hierarchical structure of a biological tissue over all involved
length scales may still be unachievable with modern fabrication techniques, but adopt-
ing individual structural motifs has been used successfully in the development of ‘self-
sharpening’ knives inspired by rat teeth [
38
] or impact resistant glass based on nacre [
39
].
As the described structural motifs of radial elements and the layered structure have de-
veloped independently in the different tissues, they should be investigated further with
regards to the toughening mechanisms they provide and to derive design principles to be
applied in bioinspired composites and metamaterials.
Supplementary Materials:
The SEM micrographs of the studied teeth are available online at https:
//www.mdpi.com/2079-4991/11/4/969/s1, Figures S1–S15.
Author Contributions:
Conceptualization, J.W. and S.B.; SEM imaging, M.W.; formal analysis, J.W.
and S.B.; investigation, J.W.; resources, S.B.; writing—original draft preparation, J.W.; writing—review
and editing, J.W., S.B. and M.W.; visualization, J.W. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Informed Consent Statement: Not applicable.
Acknowledgments:
Specimen of Carcharias taurus (spotted ragged-tooth shark) kindly provided
by Two Oceans Aquarium, South Africa. Specimen of Macropus rufogriseus (red-necked wallaby,
Figure 9b
) kindly provided by Zoo Duisburg, Germany. Micrograph Figure 9b courtesy of M.
Wurmshuber, Chair of Materials Physics, Montanuniversität Leoben, Austria.
Conflicts of Interest: The authors declare no conflict of interest.
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