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Characterization of crocodile teeth: Correlation of composition,
microstructure, and hardness
Joachim Enax
, Helge-Otto Fabritius
, Alexander Rack
, Oleg Prymak
, Dierk Raabe
Matthias Epple
Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen
(CeNIDE), University of Duisburg-Essen, Universitaetsstr. 5-7, 45117 Essen, Germany
Microstructure Physics and Alloy Design, Max-Planck-Institut für Eisenforschung, Max-
Planck-Str. 1, 40237 Düsseldorf, Germany
European Synchrotron Radiation Facility (ESRF), 6 Rue Jules Horowitz, 38000
Grenoble Cedex, France
* Correspondence to:
Matthias Epple, Institute of Inorganic Chemistry and Center for Nanointegration
Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaetsstr. 5-7, 45117
Essen, Germany
Telephone: + 49 (0) 201 183-2413
Fax: + 49 (0) 201 183-2621
Structure and composition of teeth of the saltwater crocodile Crocodylus porosus were
characterized by several high-resolution analytical techniques. X-ray diffraction in
combination with elemental analysis and infrared spectroscopy showed that the mineral
phase of the teeth is a carbonated calcium-deficient nanocrystalline hydroxyapatite in all
three tooth-constituting tissues: Dentin, enamel, and cementum. The fluoride content in
the three tissues is very low (<0.1 wt%) and comparable to that in human teeth. The
mineral content of dentin, enamel, and cementum as determined by thermogravimetry is
71.3, 80.5, and 66.8 wt%, respectively. Synchrotron X-ray microtomography showed the
internal structure and allowed to visualize the degree of mineralization in dentin, enamel,
and cementum. Virtual sections through the tooth and scanning electron micrographs
showed that the enamel layer is comparably thin (100-200 µm). The crystallites in the
enamel are oriented perpendicularly to the tooth surface. At the dentin-enamel-junction,
the packing density of crystallites decreases, and the crystallites do not display an
ordered structure as in the enamel. The microhardness was 0.60±0.05 GPa for dentin,
3.15±0.15 GPa for enamel, 0.26±0.08 GPa for cementum close to the crown, and
0.31±0.04 GPa for cementum close to the root margin. This can be explained with the
different degree of mineralization of the different tissue types and is comparable with
human teeth.
Biomineralization; Teeth; Calcium phosphate; Mechanical properties; Synchrotron X-ray
microtomography; Crocodiles
1. Introduction
Crocodiles belong to a very old phylogenetic group that has prevailed for millions of
years (Erickson et al., 2012; Janke et al., 2005). Unlike human teeth, reptile teeth
including crocodile teeth are continuously replaced (Kieser et al., 1993; Osborn, 1974;
Poole, 1961). For an approximately 4 m long crocodile (13 ft), it was estimated that each
tooth was replaced 45 times during the lifetime of the animal (Poole, 1961). Crocodiles
possess so-called thecodont teeth which are attached in sockets in the jaw (Dauphin
and Williams, 2008). Compared with other animals, crocodiles exert extraordinarily high
bite-forces and tooth pressures (Erickson et al., 2012). Like all vertebrate teeth,
crocodile teeth consist of a crown and a root.
In general, the interior bulk of the tooth crown consists of softer, less mineralized bone-
like dentin covered by an external layer of harder, highly mineralized enamel. The root,
however, consists of dentin (interior) that is covered by an external layer of cementum.
The inorganic mineral of human enamel is a calcium-deficient carbonated
hydroxyapatite, simplified: Ca
(OH), with small amounts of an organic matrix
(Busch et al., 2001; Dorozhkin and Epple, 2002; Fincham et al., 1999; Lowenstam and
Weiner, 1989). For details on teeth in general see, e.g., Teaford et al. (2000).
In contrast to human teeth and shark teeth (which are fully replaced upon loss) (Marks
Jr. and Schroeder, 1996; Smith et al., 2012), the root of a crocodile tooth is hollow. Each
mature functional tooth is accompanied by a small initial replacement tooth on the
lingual side of the root that grows from a bud formed by a specialized dental lamina.
Together, they form a tooth family unit. Crocodylian teeth cycle continuously. While the
new tooth grows, it is moving outward and induces the resorption of the root of the old
tooth which is then shed (Wu et al., 2013). The human tooth eruption follows a similar
pattern. During the change from deciduous tooth to permanent tooth, the root of the old
tooth is resorbed by osteoclasts and the crown erupts as a compact object (Marks Jr.
and Schroeder, 1996).
Studies of the structure of reptile enamel were reported by Dauphin (1987), Sahni
(1987), and Sander (1999). In general, reptile teeth have not been as thoroughly
investigated as the teeth of other large animals; one reason is that their enamel does not
consist of defined prisms such as mammalian teeth which can be more easily analyzed
(Sander, 2000). Because reptile enamel is lacking prisms, it is typically denoted as
"prismless enamel", a feature which is common to most non-mammalian amniotes
(reptiles) (Sander, 2000). Currently, there are only a few reports about the structures of
crocodile and alligator teeth (Dauphin and Williams, 2007; Dauphin and Williams, 2008;
Erickson et al., 2012; Osborn, 1998; Sander, 1999; Sato et al., 1993; Sato et al., 1990;
Shimada et al., 1992).
To close this gap, we have analyzed the chemical and crystallographic composition, the
ultrastructure, and the microhardness of dentin, enamel, and cementum, of teeth of the
saltwater crocodile Crocodylus porosus with the aim to correlate all parameters, namely,
structure, hardness, and biological function, to gain an integral view. Additionally, we
compare these properties with human teeth.
2. Materials and methods
2.1 Sample preparation and analytical methods
Teeth of the recent crocodile species C. porosus were stored in dry state at room
temperature. We used five different teeth to produce fine powders of dentin, enamel,
and cementum (several mg per sample) by mechanical abrasion with a Proxxon fine
drilling and polishing tool FBS 230/E, equipped with a diamond-coated drill. The mineral
phase and the size of the crystalline domains of these powder samples were determined
by infrared (IR) spectroscopy and X-ray powder diffraction (XRD) measurements.
Fourier-transform infrared spectroscopy (FTIR) was carried out with a Bruker Vertex 70
instrument in potassium bromide (KBr) pellets (range 400-4000 cm
and 2 cm
resolution). XRD measurements were carried out with a Panalytical Empyrean
diffractometer equipped with a furnace (XRK 900, Anton Paar) using a silicon single
crystal as sample holder to minimize scattering. First, a diffractogram was measured at
30 °C. Then, the sample was heated to 750 °C and he ld at this temperature for 2 h
before another diffractogram was measured. The measurements at 750 °C were
performed to identify the conversion products of the mineral phase after thermal
treatment. Rietveld refinement for the calculation of the lattice parameters and the size
of the crystalline domains was performed using the Bruker software TOPAS 4.2. The
correction for instrumental peak broadening as determined with an LaB
powder sample,
National Institute of Standards and Technology (NIST), as standard reference material
(SRM 660b), was included. As reference, we used the pattern of hydroxyapatite (#9-
0432) from the International Centre for Diffraction Data (ICDD) database.
A part of the remaining powdered sample material was used to perform elemental
analysis to determine the overall chemical composition and to confirm the identity of the
mineral phases. Calcium, magnesium, and sodium were determined with atomic
absorption spectroscopy (AAS), fluoride with ion-selective potentiometry, and phosphate
with ultraviolet (UV) spectroscopy. All measurements were carried out using several mg
of powdered dentin, enamel, and cementum which were dissolved in concentrated
hydrochloric acid. For fluoride analysis we used ion-selective potentiometry (ion-
selective electrode, ISE; pH/ION 735, WTW; the measurements were performed by
Analytische Laboratorien GmbH, Lindlar, Germany). Atomic absorption spectroscopy
was performed with a Thermo Electron M-Series instrument. Phosphate was determined
with a Varian Cary 300 UV-Vis spectrophotometer as phosphate-molybdenum blue
Thermogravimetry (TG) was used to determine the contents of water, organic matrix,
and carbonated apatite in the remaining powder samples of dentin, enamel, and
cementum from five different teeth. The experiments were carried out in a Netzsch STA
449 F3 Jupiter instrument in dynamic oxygen atmosphere at a heating rate of 2 K min
from 25 to 1200 °C in open alumina crucibles. For V icker’s microhardness tests, the
teeth were axially cut with a jeweler’s saw (for the convention of axial and transversal
denomination see Figure 1). Subsequently, the samples were embedded in one-
component UV-curable methyl methacrylate CEM 4000 Lightfix resin (Cloeren
Technology GmbH, Wegburg) that was cured in a Struers UV-Box using the bottom
source only for 3 min and with bottom and top source together for 6 min. The surfaces of
interest were polished using successively abrasive papers with decreasing grit sizes
(120, 220, 400, 600, 1000, 2500, and 4000; Hermes) followed by polishing with a 3 µm
diamond suspension (Struers), and finally with a 0.1 µm silica suspension (Buehler;
Saphir 320/330 instrument, ATM). In addition to polished samples, parts of teeth
fractured to expose either cross sections or axial sections were also prepared for
scanning electron microscopy (SEM). All SEM samples were mounted on standard
aluminum holders, rotary shadowed with 4 nm of platinum using a Gatan PECS 682
sputter coater, and observed in a high resolution scanning electron microscope (Zeiss
Gemini 1540XB) at acceleration voltages of 5-10 kV using a small aperture (30 µm) and
either an in-lens secondary electron (SE) detector or a backscattered electron (BSE)
detector for compositional contrast. For a clearer view on the microstructure, selected
samples were superficially etched using aqueous EDTA solution (0.15 M and 2.5%
glutaraldehyde for 20 min) followed by a quick rinse by double-distilled H
O and 100%
methanol for 1 s each. Where necessary, contrast and brightness of the digital images
were adjusted using Adobe Photoshop CS3 (Adobe Inc.).
Synchrotron X-ray microtomography (SRµCT) is a very useful technique for the
visualization of microstructures because it provides 3D data sets in a widely non-
destructive manner. This technique was already successfully used to study biological
materials, e.g., bone microstructures (Bonse et al., 1994; Larrue et al., 2011; Sanchez et
al., 2012) and human teeth (Dowker et al., 2004; Neues et al., 2009; Sanchez et al.,
SRµCT analysis was used to evaluate the gray values as indication of the local density
of the material and thus the degree of mineralization as well as to create virtual 3D
sections of the tooth. SRµCT analyses were carried out at beamline ID19 of the
European Synchrotron Radiation Facility (ESRF), Grenoble, France. Experimental
details of the beamline and on the evaluation procedure can be found in Weitkamp et al.
(2010). The 3D images and virtual sections were rendered with the software VGStudio
MAX 2.1. The gray values were identified by the graphic software ImageJ 1.45s
(Schneider et al., 2012). For the measurements, the sample was placed 935 mm
upstream of an indirect X-ray imaging detector operating with an effective pixel size of
5 µm (ESRF inhouse CCD camera FReLoN type F_7899 in combination with a 125 µm-
thick LuAG:Ce single-crystal scintillator and a 2.8× magnifying optical system,
2048·2048 pixels). Polychromatic radiation was chosen, i.e. the beamline U17.6
undulator was combined with only 2.8 mm Al and 0.4 mm Cu absorbers at a gap of 15
mm in order to gain a sufficient photon flux density (the ESRF operated in 4-bunch
mode). The resulting spectrum was dominated by the 2
harmonic of the undulator
around an X-ray photon energy of 35 keV. 2000 projection images were acquired over a
tomographic scan range of 180 degrees with an exposure time of 2 s each. Single-
distance phase retrieval was applied in order to establish a direct correlation between
the gray levels of the voxels and the materials associated with them before the
tomographic reconstruction by means of filtered-back projection was performed
(Weitkamp et al., 2011). The chosen approach is robust and allows to work with a single
propagation distance only but as well only for a single component: The dentin-enamel
junction (dej) was fringe-free after this phase-retrieval while other interfaces such as
dentin-air remained with fringes (Paganin et al., 2002).
Vicker’s microhardness tests were carried out on the exposed axial sections of two teeth
in four designated areas of interest: The crown enamel, crown dentin, the distal
cementum layer close to the crown and the cementum layer close to the proximal
margin of the tooth. In every area of interest on each sample, 20 indentations were
made with a Leco M-400-H1 microhardness testing device. The location of each
indentation was manually chosen and a weight of 10 g (HV0.01) was applied for 15 s.
These experiments, including the distance between two indentations, were performed
according to DIN EN ISO 6507-1 and DIN EN ISO 6507-4 and the indentations were
manually controlled and evaluated. Vicker’s hardness HV0.01 was converted into
Berkovich hardness H according to HV0.01/kg mm
= 92.65 s
3. Results
A typical tooth of the crocodile species C. porosus has a cone-shaped crown and a
hollow, roughly cylindrical root (Figure 1). The absolute size of each tooth varies
depending on the age and size of individual animals.
The transition between dentin and enamel is marked by the so-called dentin-enamel-
junction (dej) and is clearly visible on scanning electron micrographs of axially polished
cross sections through the teeth (Figures 2A, 2B, and 3A). The crocodile enamel has a
thickness of about 100-200 µm at the crown (Figure 3A), becomes thinner towards the
root and disappears completely just before the onset of the hollow part (Figure 2A). In
this region, an enamel-cementum interface can be observed where a thin cementum
layer is present that covers the enamel layer (Figure 2A). The uniform contrasts
observed in composition-sensitive backscattered electron (BSE) contrast micrographs
(Figure 2) indicate a homogeneously distributed mineral content in all three layers.
Surfaces of fractured enamel show that it consists of small elongated mineral crystallites
that are mainly oriented with their long axes perpendicular to the tooth surface (Figures
3C and 4). These crystallites are very densely packed and therefore the shape and
dimensions of individual crystallites are difficult to determine. Superficial etching of
polished enamel surfaces revealed the needle-like shape of the mineral structures. The
crystallites in the enamel have a length of a few µm. No defined crystallite bundles as
they occur in prismatic enamel of e.g., mammalians were visible (Figure 4). Throughout
the enamel, a fine horizontal (parallel to the tooth surface) striation can be observed
(Figure 4A). At the dentin-enamel-junction (dej) the packing density of the crystallites
decreases and their orientation becomes more random (Figures 3B, 4B). The dej is
further characterized by a relatively high content of randomly arranged organic fibers
(Figures 2B, 3B). The mineral phase of the cementum is not as clearly structured as in
the enamel in terms of regularly arranged crystallites. The cementum contains a loose
network of randomly arranged organic fibers (Figures 2C, 3D). These fibers disappear in
the transition between cementum and dentin (dentin-cementum-junction, dcj), which is
not as sharp as the dej (Figures 2C, 3D). The dentin of crocodile teeth is pervaded by
numerous µm-sized dentin tubuli (Figure 2D). The SEM-micrographs of fractured teeth
indicate that the mineral phase of dentin also consists of needle-shaped crystallites
(Figure 2B). However, a well-defined structural organization as found in enamel could
not be observed with the techniques used in our study.
Elemental analysis of the crocodile teeth shows that the enamel contains more calcium,
phosphate, and sodium but less magnesium than dentin (Table 1). Sodium is present in
dentin and enamel but absent in the cementum. The fluoride concentration in crocodile
teeth altogether is very low (<0.1 wt%). The cementum contains significantly more
fluoride than dentin and enamel. The magnesium content is very high, especially in
dentin, and it was included to compute the (Ca+Mg)/P molar ratio in the apatite mineral
phase. In all cases, the stoichiometry of a calcium-deficient hydroxyapatite was found,
i.e. around n(Ca+Mg):n(P)=1.67:1 (Dorozhkin and Epple, 2002).
The mineral phase of the teeth was analyzed by X-ray powder diffraction in combination
with Rietveld analyses (Figure 5 and Table 2). The diffractograms of dentin, enamel, and
cementum show the typical peak pattern of hydroxyapatite. While the diffractograms of
dentin and cementum show broad diffraction peaks, those of the enamel are slightly
sharper. The average size of the crystalline domains is 8-9 nm, and the lattice
parameters and cell volumes differ just a little between dentin, enamel, and cementum.
Heating of the samples to 750 °C in vacuum resulted in a mixture of hydroxyapatite and
β-tricalcium phosphate, β-Ca
, in comparable amounts indicating the presence of
a calcium-deficient hydroxyapatite in the initial phase (i.e. before calcination) (Dorozhkin
and Epple, 2002).
The IR-spectra recorded for dentin, enamel, and cementum (Figure 6) show the
absorption bands specific for phosphate (490-640 cm
and 900-1220 cm
), carbonate
(875 cm
and 1360-1590 cm
), and water (3010-3660 cm
). Additional bands that
indicate the organic matrix (mainly proteins) appear in all three layers at 2940 cm
and 1600-1700 cm
(amide I band of proteins) (Preston et al., 2011).
Thermogravimetry of dentin, enamel, and cementum shows three main regions of mass
loss: Release of water (<200 °C), the combustion of the organic matrix (200-500 °C),
and finally the release of CO
from carbonated apatite (>500 °C) (LeGeros, 1981; Peters
et al., 2000) (Figure 7). The residue is the decarboxylated mineral phase, i.e. pure
calcium phosphate. Enamel has a much higher mineral content than dentin and
cementum with a low content of organic matrix (Table 3). Cementum has a lower
mineral content than dentin. The content of water is comparable in all three tissues. Note
that the content of water may be variable, depending on the storage conditions.
SRµCT analyses show the exact geometry of the crocodile teeth and the spatial
arrangement of the constituting layers (Figure 8). The enamel layer (whitish color) is
confined to the crown and generally very thin with the thickest regions located at the
tooth tip. From the 3D images, it is difficult to distinguish between dentin and the very
thin cementum layer that covers the root due to a very similar X-ray absorption contrast.
In axial sections, it becomes obvious that the hollow root takes up over two thirds of the
total tooth size, and that the crown is comparatively small. Moreover, the cavity formed
by the root has the shape of a distally pointed cone, and the root’s wall thickness is
decreasing in proximal direction.
In the SRµCT-3D images, the enamel has a white color, and the dentin has a gray color.
Evaluation of the gray values using line scans through the tooth crown (Figures 9A and
10A) shows that the enamel has a much higher gray value (~2.8) than the dentin (~1.8).
The root (Figures 9B and 10B), however, shows a lower gray value for the dentin (~1.3)
compared to the crown dentin. Cementum has a slightly higher gray value than the
dentin in the root.
Representative Vicker’s microhardness tests were performed at different positions of
polished tooth samples of C. porosus. For better comparability, the results were
converted into Berkovich hardness. We found 0.60±0.05 GPa for dentin, 3.15±0.15 GPa
for enamel, 0.26±0.08 GPa for cementum close to the crown, and 0.31±0.04 GPa for
cementum close to the margin of the root (Table 4).
4. Discussion
The teeth of the saltwater crocodile C. porosus are mainly characterized by their specific
shape, a small crown with a comparably large root, and the three constituting tissues
dentin, enamel, and cementum that differ in microstructure, composition, and resulting
mechanical properties. The major part of both the crown and the root consists of dentin
which contains dentin tubuli that are also present in human teeth (Marten et al., 2010).
Both exterior layers, the cementum covering the root and the enamel covering the
crown, are separated from the dentin by a structurally distinct layer, the dentin-
cementum-junction (dcj) and the dentin-enamel-junction (dej), respectively. The dentin-
enamel-junction is also present in mammalian teeth (Line and Novaes, 2005; Walker
and Fricke, 2006). Within the enamel, the mineral crystallites are all oriented parallel,
hence, this type of enamel is typically denoted as "parallel crystallite enamel" (Sander,
2000). A similar enamel microstructure was found for teeth of Alligator mississippiensis
(Sato et al., 1990). The horizontal striation observed on etched enamel surfaces
indicates the presence of incremental growth lines that have also been described in
teeth of other ectotherm animals (Line and Novaes, 2005).
X-ray diffractograms of dentin and enamel showed broad diffraction peaks which
indicate a comparable size of crystalline domains between these tissues. That is
remarkable because in mammalian enamel and shark enameloid the apatite rods have a
larger size of crystalline domains than the apatite nanocrystals in dentin (LeGeros, 1994;
Xue et al., 2008). The lattice parameters of the mineral in crocodile teeth are very similar
to those of human teeth and of geological hydroxyapatite and confirm the presence of
apatite. The small differences in the lattice parameters of dentin, enamel, and cementum
can be ascribed to different amounts of incorporated ions, e.g., magnesium, sodium, and
carbonate into the apatite lattice, as it is well known for biological apatite (Dauphin and
Williams, 2007; LeGeros, 1981). Magnesium as substituent is known to reduce the
crystallinity of apatites while sodium has no significant influence (Elstnerova et al., 2010;
LeGeros, 1994). Especially magnesium strongly influences the lattice parameters.
Apatites with high contents of magnesium become amorphous (Masayuki et al., 1986).
The fluoride content of crocodile teeth is very low like in human teeth (Aoba, 1997;
LeGeros, 1981). Overall, the results of elemental analysis agree well with microprobe
analyses of recent reptile teeth (Dauphin and Williams, 2007; Dauphin and Williams,
2008). Our results indicate that dentin, enamel, and cementum all contain
nanocrystalline hydroxyapatite. The size of the crystalline domains of the different tooth
layers is comparable.
The amide I band of proteins in the IR-spectra in enamel is less intense compared to
dentin and cementum, probably due to the lower content of organic matrix. This is
supported by thermogravimetry. Human dentin and cementum are known to have a high
content of organic matrix which consists mainly of collagen (Wiesmann et al., 2005). The
thermogravimetric analysis of human dentin showed similar results as found here for
crocodile dentin (Lim and Liboff, 1972). In general, the IR spectra of dentin, enamel, and
cementum are all very similar to each other and confirm the results of X-ray diffraction.
They are also very similar to human teeth (LeGeros, 1981), synthetic hydroxyapatite
(Zhou et al., 1993)
and also shark teeth which do not consist of hydroxyapatite but of
fluoroapatite, Ca
F (Enax et al., 2012).
The results of X-ray diffraction correspond well with the microstructure analysis by
scanning electron microscopy. The prismless enamel of crocodile teeth contains no
crystallite bundles and no complex microstructures but only needle-like mineral
structures which are ordered perpendicularly to the tooth surface. Human teeth show a
especially high fracture toughness (Padmanabhan et al., 2010; Yilmaz et al., 2013) due
to the presence of crystallite bundles (enamel prisms) which are hierarchically ordered in
a mm-thick enamel layer (Ang et al., 2010; Dunlop and Fratzl, 2010; He and Swain,
2008). In contrast, the enamel of crocodile teeth is very thin compared to mammalian
teeth, which is consistent with their specific function. Crocodiles do not use their teeth for
cutting and chewing, but only for gripping and securing their prey, which is then pulled
into the water and killed by drowning. Generally, their teeth are not used to dismember
prey animals; instead crocodiles use violent body movements to tear pieces off which
are then swallowed. The enamel cap on mammalian teeth can be up to 5 mm thick
(Lucas et al., 2008). Here, SRµCT showed clearly that the enamel thickness of crocodile
teeth reaches its maximum at the tooth tip and is rapidly thinning towards the root. This
indicates that crocodile teeth must be very resistant at the tip area, probably optimized
for snapping their prey. Crocodiles are ambush hunters which attack with a very fast bite
where they exert extraordinarily high bite-forces and tooth pressures (Erickson et al.,
2012). The thick enamel at the tip of the teeth might help to prevent damage upon
impact on hard parts of the prey, e.g. bones, during the attack. Interestingly, the
arrangement of teeth in the upper and lower jaw is such that opposing teeth will not get
in contact if the bite misses the target. This is also corroborated by the fact that the root
dentin shows a lower gray value than the crown dentin, corresponding to a lower mineral
content and thus a higher potential for deformation without suffering a brittle fracture.
The comparably higher deformability and lower hardness together with the large size of
the root and thus large contact area with the jawbone via the teeth-ridge further helps to
dissipate the pressure and thus the kinetic energy acting on individual teeth during the
impact on prey animals.
Thus, the structural and compositional organization of the crocodile tooth is ideally
suited for its function. Nevertheless, the hunting mode using very fast and powerful bites
presumably increases the risk of teeth getting damaged compared to other predatory
animals like mammalian carnivores that use similarly shaped canine teeth for killing their
prey. However, these animals use their teeth in a much more controlled, almost surgical
mode by biting into specific neuralgic points of their prey. The constant replacement of
crocodile teeth may thus represent an additional adaptation to their predation technique
that was necessary to compensate the higher risk of damage. Vicker’s microhardness
tests of crocodile teeth gave a hardness close to the hardness of human teeth and shark
teeth. This is surprising because crocodile teeth do not have hierarchically organized
crystallite bundles like human teeth. The different hardness values of the different
tissues found in crocodile teeth can be explained by different degrees of mineralization
and thus by different mineral contents which was shown by SRµCT, SEM, elemental
analysis, and thermogravimetry. This is in good agreement with results for human teeth.
It was shown by spatially resolved studies that an increasing calcium content (and thus
mineral content) leads to a higher hardness in human enamel (Cuy et al., 2002; Jeng et
al., 2011). Note that the microhardness of teeth is lower than that of pure (geological)
hydroxyapatite which amounts to 5.4±1.3 GPa (White et al., 2001). This is because a
geological hydroxyapatite crystal does not contain an organic matrix and is therefore
much more brittle (and less elastic).
5. Conclusions
The hardness of human teeth and crocodile teeth is comparable, although their
microstructures are significantly different. The prismless enamel layer of crocodile teeth
is very thin, with a maximum thickness at tooth tip and consists of nanocrystalline
hydroxyapatite, in striking contrast to mammalian enamel and shark tooth enameloid.
Structure and composition of crocodile teeth are well suited for their biological function.
In contrast to most mammals, crocodiles do no not need their teeth for cutting but mainly
for holding their prey. Crocodiles are changing their teeth continuously during their
lifetime, possibly because of their way of hunting, i.e. very fast and powerful bites. As
consequence, crocodiles have a higher risk of tooth-damage than other animals like
mammalian carnivores. The construction of their teeth is well adapted to their biological
We thank C. Fischer, Essen, Germany, for help with the tooth preparation, M. Ruiz,
Grenoble, France, for support during the beamtime at the ESRF, and A. Gillis, Halifax,
Canada, for helpful discussions. We thank the Deutsche Forschungsgemeinschaft for
support within the priority program SPP 1420.
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Figure captions
Figure 1: Image of a crocodile tooth (C. porosus), including the convention of axial and
transversal directions, with an additional view into the hollow root (insert).
Figure 2: Scanning electron micrographs in back-scattered electron mode (BSE) of a
polished tooth surface of C. porosus, showing the dentin-enamel-junction and also the
dentin-cementum-junction (A), the dentin-enamel-junction in higher magnification (B),
the dentin-cementum-junction in higher magnification (C), and the dentin with its tubuli
(D). (d dentin, e enamel, c cementum, dej dentin-enamel-junction, dcj dentin-
Figure 3: Scanning electron micrographs of fractured tooth samples of C. porosus. (A)
The overview of an axially fractured tooth tip shows a clear border between dentin and
enamel. (B) The organization of the dentin-enamel-junction at higher magnification. (C)
Area at the tooth surface where the outermost layer of enamel was chipped off,
revealing the constituting small elongated mineral crystallites that are mainly oriented
with their long axes perpendicular to the tooth surface. (D) The organization of the
dentin-cementum-junction at higher magnification. (d dentin, e enamel, c cementum, dej
dentin-enamel-junction, dcj dentin-cementum-junction).
Figure 4: Scanning electron micrographs recorded in BSE contrast of the exposed axial
surface of enamel from teeth of C. porosus that was gently etched using EDTA to
expose the shapes and arrangement of the mineral crystallites. (A) The outermost
enamel consists of horizontal layers of parallel needle-shaped crystallites that are all
oriented with their long axes perpendicular to the tooth surface. The different contrasts,
especially of the horizontal bands, originates from slight differences in composition. (B)
Close to the dentin-enamel-junction, the horizontal layers of needle-shaped crystallites
are still visible, but frequently interrupted by the cavities of small canaliculae. (e enamel,
dej dentin-enamel-junction).
Figure 5: X-ray powder diffractograms of dentin (A), enamel (B), and cementum (C) of
teeth of C. porosus in comparison to pure hydroxyapatite (#9-0432 from the ICDD
database; computed as nanocrystalline phase) (D).
Figure 6: IR spectra obtained from dentin, enamel, and cementum of teeth of C.
Figure 7: Thermogravimetric analysis of dentin, enamel, and cementum from teeth of C.
porosus: <200 °C: Release of water (1), 200-500 °C: Combustion of the organic matrix
(2), and >500 °C: Release of CO
from carbonated apatite (3).
Figure 8: SRµCT-3D images of the complete tooth (A) and of two virtual axial sections
(B, C) through a tooth of C. porosus.
Figure 9: Line scans on transverse sections through the crocodile tooth obtained by
SRµCT to generate the gray value profiles shown in Figure 10. A representative virtual
section of the crown (A) and a section through the root just below the altitude where the
enamel has been replaced by cementum (B) are shown. Interference fringes between
enamel and air as well as dentin and air remain due to the phase-retrieval chosen
algorithm (Paganin et al., 2002).
Figure 10: Comparison of the gray values of two virtual transversal sections through a
tooth of C. porosus. Linescan of the tooth crown (A) and linescan of the tooth root (B).
Table 1: The chemical composition of dentin, enamel, and cementum of teeth of C.
porosus (in wt%), in comparison to human teeth.
C. porosus
Human teeth
LeGeros (1981)
Dentin Enamel Cementum
Dentin Enamel
20.56 28.56 23.01 27.0 36.0
38.80 44.05 32.95 39.9
Ca/P molar
ratio 1.26:1 1.55:1 1.65:1 1.60:1 1.57:1
molar ratio 1.51:1 1.63:1 1.74:1 1.72:1 1.60:1
0.78 1.01 -- 0.3 0.5
2.47 1.10 0.75 1.1 0.44
0.06 0.09 0.21 0.05 0.01
Calculated from the content of phosphorus.
Table 2: Crystallographic properties of the mineral phase in teeth of C. porosus
compared to geological hydroxyapatite crystals and to human teeth.
Saenger and Kuhs
LeGeros (1981) and
Enax et al. (2012). The standard deviation is given in
/ Å
/ Å
/ Å
Size of
/ nm
Crocodile dentin
Crocodile enamel
Crocodile cementum
Geological hydroxyapatite
Human dentin
Human enamel
Shark dentin
Shark enameloid (fluoroapatite)
Table 3: Results of the thermogravimetric analysis of dentin, enamel, and cementum of
teeth of C. porosus.
/ wt%
/ wt%
/ wt%
Release of water
(<200 °C) 2.7
Combustion of the organic matrix
(200-500 °C) 24.2
Release of CO
from carbonated apatite
(>500 °C) 1.8
Mineral content (residual) 71.3
Table 4: Results of Vicker’s microhardness tests (HV0.01) of C. porosus teeth
compared to values obtained for human teeth and shark teeth from literature. The values
are averages of all tested specimens including the standard deviation. They were
converted into Berkovich hardness in GPa.
del Pilar Gutierrez-Salazar and Reyes-
Gasga (2003),
Malek et al. (2001), and
Enax et al. (2012).
Crocodile t
/ GPa
Human teeth
/ GPa
Shark teeth
/ GPa
Dentin 0.60±0.05 0.5..0.6
Enamel 3.15±0.15 2.9..3.9
(distal, close to crown) 0.26±0.08
(proximal, close to root margin) 0.31±0.04
... Furthermore, the labiolingually flattened tooth morphology is also reflected in this radicular pulp (Figure 2a). Unlike the hollow root with a large opened radicular pulp in Crocodylus (Enax et al., 2013), the conical-shaped root in Hamipterus retains only a very small foramen (60 μm) for connection of the radicular pulp (Figure 5i). ...
... This layer extends from the root to the partial tooth crown (Figure 6b,d) and is surrounded by a thick layer of cellular cementum at the beginning of the root area. Similar to that in crocodylians (Berkovitz & Shellis, 2017;Enax et al., 2013), this acellular layer in Hamipterus is likely the acellular cementum layer, considering the typical arrangement of dentin structure. From the outer surface inward, the mineral phase of this acellular layer is not clearly structured in terms of irregularly arranged crystallites (Figures 2c and 4g), which conforms to the loose network of randomly arranged organic fibers in the cementum (Berkovitz & Shellis, 2017). ...
... Although some dental calculus may also cover the root area and preserve a variety of biomolecules or their lacunae (Velsko & Warinner, 2017;Warinner et al., 2014), it lacks the presence of regular parallel growth lines in the same anatomical position across different teeth, as observed in the cellular cementum. The thick, cellular cementum, found mostly on the lingual side of the crown-root area of the tooth in Hamipterus, has hardly been noted in other pterosaurs, dinosaurs, and crocodylians (e.g., Enax et al., 2013;Heckeberg & Rauhut, 2020;Vidovic, 2010). This cellular cementum responds to tooth wear and movement (Fehrenbach & Popowics, 2016), so its development indicates that the teeth of Hamipterus are well stabilized in the alveoli. ...
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The toothed members of Pterosauria display an extremely wide range of tooth morphologies that supported a variety of feeding habits. Histological studies on the teeth of different pterosaur clades are potentially valuable in understanding the development of their tooth diversity. In this study, we used histological sections and scanning electron microscopy to describe and interpret the tooth microstructure of Hamipterus (Pterodactyloidea). Our analysis is based on seven teeth of Hamipterus (six isolated and one from a skull) from the Lower Cretaceous collected in Hami, China. Our results show that the enamel on the tooth crown is thin (~25 μm) in Hamipterus and covers only approximately half of the tooth crown. This thin enamel of the Hamipterus tooth makes it vulnerable and often becomes damaged during taphonomic and diagenetic processes. The radicular pulp inside the conical-shaped root shows a spindle space with a small foramen at the bottom, while the coronal pulp shows a small tunnel (100-140 μm in diameter). We estimate that the small teeth of Hamipterus likely took approximately 80 days to form. Furthermore, the tooth has Andresen lines, which represent 7-15 days period. For stable articulation of the tooth in the alveolus, the thick cellular cementum is concentrated on the lingual side of the root. The acellular cementum (~40 μm thick) layer runs from the root to the partial tooth crown.
... Despite all the studies concerning dentition of extant and extinct crocodilians, there is little literature on the detailed histology and histochemistry of modern and fossil crocodilian teeth (Westergaard & Ferguson 1986, 1987Dauphin 1987;Erickson 1992Erickson , 1996Dauphin & Williams 2008;Fruchard 2012;Enax et al. 2013;Gren & Lindgren 2014;Thomadakis 2015). The three dental tissues, enamel, dentine and cementum, differ in microanatomy, microstructure and chemical composition (Hillson 2005), as well as their two interphases (enamel-dentine junction, EDJ, and cementum-dentine junction, CDJ). ...
... Enamel (light and dark green in Suppl. tab. 1) in extant and extinct crocodilians is thin, around 100-200 μm (Enax et al. 2013), being thicker at the crown apex and finer towards the limit with the root (Figs. 1C-H, 2A-B). ...
... In other words, the lines that are observed at the optical level (which reflect the daily increments with a light and dark line representing a day) are also observed in the microanatomy of some parts of the fossil dentine. This alternate presence of dentinal tubules in short-period incremental lines has not been described previously either in living or fossil archosaurs, which usually present regular distribution in dentine (Dauphin & Williams 2008;Enax et al. 2013). It could be related with crocodilian development, which is very dependent of environmental conditions (Cott 1961;Revol 1995;Fruchard 2012). ...
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Teeth provide information about the evolutionary pathway of an organism, its biology and habitat. This is the case even of fossilized teeth, since they have perdurable biomineralized structures, as biological apatite. The material that has been selected for this study comprises teeth from modern crocodilian individuals and extinct Cretaceous crocodylomorphs from Lo Hueco site. Microanatomy, histochemistry and crystallographic nature of enamel, dentine and cementum have been characterized by Polarized Light Microscopy, SEM-EDS, Confocal Raman Spectroscopy and SR-µXRD. A focus has been made on dentine lamination. In the fossil sample short-period incremental lines show alternate presence of dentinal tubules that has not been described previously either in living or fossil archosaur. This could be related to influence of environmental circadian rhythms in the abundance, size and/or activity of cells depositing dentine in the day-night cycle. Regarding histochemical and crystallographic compositions, the major and mostly unique phase is HA, but in the case of fossil teeth, a secondary phase identified as hematite appears locally between discontinuities of the material. Incremental lines would not be related to variation in chemical composition and furthermore do not present different HA crystallographic nature (different directions of HA or different crystallite sizes) either. Only small intensity oscillations are observed in the fossil sample by SR-µXRD which are compatible with the alternating abundance of dentinal tubules. Crystallinity differences between modern and fossil material, as crystallite size and presence of CO32- groups could be explained by postdepositional processes.
... This phenomenon occurs in fish, amphibians and some reptiles [25]. In the crocodile, the inside of the tooth base is empty [26,27]. ...
... It can be said that the repair processes in such a tooth are impossible; however, the damaged tooth can be replaced by another one. A crocodile 4 m long was found to have had its teeth replaced 45 times [27]. A similar process appears to be relevant in Komodo Dragons. ...
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Simple Summary The goal of this study was a detailed analysis of the mandibular teeth structure of the Komodo dragon (Varanus komodoensis), based on macroscopic, histological and computed microtomography examinations. The samples were collected post mortem from two adult female Komodo dragons from Wroclaw Zoo. Macroscopically, each mandibular tooth is laterally flattened and its dental crown has an arcuate shape with the typical denticles of a tooth’s caudal margin. The base of the tooth is filled with abundant plicidentine with secondary lamellae. The cavity in the central part of the tooth is well visible, while collagen fibers fill the spaces between the inner lamellae. In the epiphyseal part, the tooth wall is thin and contains numerous dentin trabeculae that grow into the tooth cavity. The dentine is most developed in the mid-tooth, where numerous tubules are observed. The presence of plicidentine, a small number of odontoblasts and a relatively large amount of adipose tissue cells are typical of the mandibular teeth of V. komodoensis. Abstract The present study aimed to characterize the macrostructure and microstructure of the mandibular teeth of the Komodo dragon (Varanus komodoensis) and the methods it uses to obtain food. Examinations were performed using a stereoscopic microscope, autofluorescence method, histological method and computed microtomography. A detailed macro- and micro-structural description of V. komodoensis mandibular teeth were made. The mandibular teeth are laterally flattened along their entire length and the dental crown is hooked caudally. The part of the nasal margin of the tooth crown is irregular, while the caudal margin of the tooth is characteristically serrated, except for the tooth base area. There are longitudinal grooves on the lingual and vestibular surfaces up to the lower third of the tooth height. The mandibular tooth is surrounded by a cuff made of the oral mucosa, containing the opening of the venom gland. In the histological structure of the tooth, the enamel covering the tooth crown and the dentin under the enamel are distinguished. The inside of the tooth, except its basal part, is filled with the tooth chamber, while the inside of the lower part of the tooth is filled with plicidentine, which corresponds to external furrows on the enamel. The plicidentine arrangement resembles a honeycomb. A small amount of dentine folds reach up to the tooth apex. Characteristic features of the structure of the mandibular teeth in V. komodoensis may indicate their significant role, in addition to the venom glands, in obtaining food in the natural environment of this species.
... This phenomenon occurs in fish, amphibians and some reptiles [25]. In the crocodile, the inside of the tooth base is empty [26,27]. ...
... It can be said that the repair processes in such a tooth are impossible; however, the damaged tooth can be replaced by another one. A crocodile 4 m long was found to have had its teeth replaced 45 times [27]. A similar process appears to be relevant in Komodo Dragons. ...
... Enamel does not contain collagenous proteins, and it is characterized by a higher degree of mineralization and a more complex structure when compared to enameloid of fishes [92]. Although tooth enamel in reptiles and amphibians is, with some exceptions [96], structurally simple and aprismatic [72,74,89,97,98], enamel in mammalian teeth is prismatic and characterized by the presence of organized bundles of hydroxyapatite crystals that confer it exceptional hardness and resistance to stresses [89,[99][100][101]. ...
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The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.
... It was also already shown mostly in mammals (e.g., Kay 1981;Shellis et al. 1998;Lucas et al. 2008;Thiery et al .2017) but also in fish (e.g., Delaunois et al. 2020;Velasco-Hogan et al. 2021) that a thicker enamel/enameloid layer is related to harder diet Despite the importance of understanding tooth enamel, this tissue remains relatively poorly investigated in reptiles, with mostly quantitative comparisons having been performed for crocodiles and extinct species (e.g., Olejniczak and Grine 2006;Enax et al. 2013;Sellers et al.2019). Compared to mammals, reptiles replace their teeth continuously (Edmund 1969;Zaher and Rieppel 1999;Richman and Handrigan. ...
Synopsis Teeth are composed of the hardest tissues in the vertebrate body and have been studied extensively to infer diet in vertebrates. The morphology and structure of enamel is thought to reflect feeding ecology. Snakes have a diversified diet, some species feed on armored lizards, others on soft invertebrates. Yet, little is known about how tooth enamel, and specifically its thickness, is impacted by diet. In this study, we first describe the different patterns of enamel distribution and thickness in snakes. Then, we investigate the link between prey hardness and enamel thickness and morphology by comparing the dentary teeth of 63 species of snakes. We observed that the enamel is deposited asymmetrically at the antero-labial side of the tooth. Both enamel coverage and thickness vary a lot in snakes, from species with thin enamel, only at the tip of the tooth to a full facet covered with enamel. There variations are related with prey hardness: snakes feeding on hard prey have a thicker enamel and a lager enamel coverage while species. Snakes feeding on softer prey have a thin enamel layer confined to the tip of the tooth.
... The prevalence of tooth crown preservation has been attributed to the presence of a harder component, enamel, combined with the fact that they are less hollow than the roots, which have no enamel and thinner walls, with a larger internal cavity. Those features make the tooth crown more resistant to breakage, increasing its potential for preservation (Enax et al., 2013). In addition, all material described here have sharp broken edges without sign of abrasion, expected if long to moderate transport in a fluvial system had occurred. ...
The Feliz Deserto Formation (Berriasian–Valanginian, Sergipe-Alagoas Basin, NE Brazil) preserved some of the earliest South American fossil records of the rifting stages which resulted in the Gondwana supercontinent break-up during the Lower Cretaceous. Recently, the first spinosaurid theropod record of this formation was described, based on a tooth recovered from Canafístula 01 fossil locality in Sergipe State. We add herein twenty-seven isolated specimens to the fossil record of the Lower Cretaceous Feliz Deserto Formation. The new material includes seven isolated spinosaurid theropod teeth of the spinosaurine clade, as well as an indeterminate theropod preungual pedal phalanx. In addition, we describe an isolated crocodyliform osteoderm, as well as eighteen isolated teeth, some of which were taxonomically identified in three distinct morphotypes of neosuchian crocodyliforms. These findings expand the Gondwanan fossil record of both spinosaurine theropods and neosuchian crocodyliforms. Despite the fragmented nature of the specimens, these new fossils allowed the characterization of their general taphonomic features with low fluvial transport of bioclast prior to the burial. The depositional paleoenvironment of the Canafístula 01 locality is compatible with the deltaic system unit, that characterizes part of the Feliz Deserto Formation during the Lower Cretaceous. These fossil findings exemplify the co-occurrence of spinosaurids and more than one taxon of crocodyliforms in the deltaic-lacustrine paleoenvironment represented by the Feliz Deserto Formation. These new occurrences reinforce the fossiliferous potential of the Canafístula 01 locality, especially related to the paleovertebrates from the Lower Cretaceous of Brazil.
... As in other Crocodyliformes, the dental attachment of Notosuchus terrestris is a gomphosis, characterized by the presence of three attachment tissues: cementum, alveolar bone and PDL (Miller 1968;Berkovitz & Sloan 1979;McIntosh et al. 2002;Enax et al. 2013;Mestriner et al. 2021). The identification of these attachment tissues presented here are the first for a notosuchian crocodyliform and allows comparisons with other toothed archosaurs, including non-avian dinosaurs and other pseudosuchians. ...
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Notosuchia represents a highly diverse lineage of Crocodyliformes usually characterized by the presence of a heterodont dentition. Although the dental anatomy has been thoroughly analysed in some taxa, information regarding tooth microstructure and dental attachment tissue are still poorly explored. With the purpose to obtain new data regarding feeding habits, tooth growth rates and nature of the dental attachment in notosuchians, here we describe and interpret the microstructure of the tooth and tooth attachment tissues of Notosuchus terrestris from the Late Cretaceous of Argentina (MPCA-PV 250). We found that the relative and absolute enamel thickness of Notosuchus is more similar to the one reported for carnivorous notosuchians (e.g. Baurusuchidae). The dentine microstructure allowed us to infer that the tooth growth rates of Notosuchus are reduced in comparison with other notosuchians. The tooth attachment of Notosuchus corresponds with a typical gomphosis, which involves the presence of cementum, alveolar bone, and periodontal ligamentum. The spatial arrangement of these tissues exhibits the plesiomorphic condition for archosaurs and the new data supports a high degree of conservatism of this feature. The data available on the absolute and relative cementum thickness reveals that Notosuchus exhibits the highest values of absolute and relative cementum thickness among the analysed crocodyliforms. Different from other archosaurs, in which alveolar bone is only formed by woven fibred bone, the alveolar bone of our specimen is formed by both woven and parallel fibred bone, which indicates local variations in the rates of the alveolar bone formation. □ Dental growth rate, dental histology, attachment tissues, Notosuchia, feeding habits.
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Large theropod dinosaurs are often reconstructed with their marginal dentition exposed because of the enormous size of their teeth and their phylogenetic association to crocodylians. We tested this hypothesis using a multiproxy approach. Regressions of skull length and tooth size for a range of theropods and extant varanid lizards confirm that complete coverage of theropod dinosaur teeth with extraoral tissues (gingiva and labial scales) is both plausible and consistent with patterns observed in living ziphodont amniotes. Analyses of dental histology from crocodylians and theropod dinosaurs, including Tyrannosaurus rex, further indicate that the most likely condition was complete coverage of the marginal dentition with extraoral tissue when the mouth was closed. This changes our perceptions about the appearance and oral configuration of these iconic predators and has broad implications for our interpretations of other terrestrial animals with large teeth.
Depending on the mineralization pattern of dentine, teeth can be divided into three histological types (orthodont, osteodont, and pseudoosteodont type). However, the development and structural characteristics of pseudoosteodentine has not been systematically investigated yet. Here, the teeth of Trichiurus Lepturus were selected for revealing a maturation process during pseudoosteodentine formation and describing ultrastructural details of pseudoosteodentine architecture. Micro-computed tomography, scanning electron microscopy, hematoxylin-eosin, Masson staining and immunohistochemistry using a dentine sialophosphoprotein (DSPP) antibody were used to analyze the microstructure and the development of the dentine. Compared with Muraenesox cinereus orthodentine, the ultrastructure of pseudoosteodentine, dentine development, the localization and migration of odontoblasts during odontogenesis in T. lepturus were observed in detail. In pseudoosteodentine, orthodentine and osteodentine all contain similar tubule-like structures and tubule openings. Labeled by DSPP immunohistochemistry for secretory odontoblasts, the organic matrix of pseudoosteodentine forms in two stages: secreting matrix by orthodentine odontoblasts with inverted nuclear polarity and the formation of osteodentine by stellate odontoblasts throughout the dental papilla. Our findings increase the understanding of the odontogenesis and structure of pseudoosteodentine and might provide a new model for the study of biomineralization and tooth development.
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Arthropoda, which represent nearly 80% of all known animal species, are protected by an exoskeleton formed by their cuticle. The cuticle represents a hierarchically structured multifunctional biocomposite based on chitin and proteins. Some groups, such as Crustacea, reinforce the load-bearing parts of their cuticle with calcite. As the calcite sometimes contains Mg it was speculated that Mg may have a stiffening impact on the mechanical properties of the cuticle (Becker et al., Dalton Trans. (2005) 1814). Motivated by these facts, we present a theoretical parameter-free quantum-mechanical study of the phase stability and structural and elastic properties of Mg-substituted calcite crystals. The Mg-substitutions were chosen as examples of states that occur in complex chemical environments typical for biological systems in which calcite crystals contain impurities, the role of which is still the topic of debate. Density functional theory calculations of bulk (Ca,Mg)CO3 were performed employing 30-atom supercells within the generalized gradient approximation as implemented in the Vienna Ab-initio Simulation Package. Based on the calculated thermodynamic results, low concentrations of Mg atoms are predicted to be stable in calcite crystals in agreement with experimental findings. Examining the structural characteristics, Mg additions nearly linearly reduce the volume of substituted crystals. The predicted elastic bulk modulus results reveal that the Mg substitution nearly linearly stiffens the calcite crystals. Due to the quite large size-mismatch of Mg and Ca atoms, Mg substitution results in local distortions such as off-planar tilting of the CO2� group.
The room-temperature unit-cell volumes of synthetic hydroxylapatite, Ca5(PO4)3OH, fluorapatite, Ca5(PO4)3(F(1-x),OH(x)) with x = 0.025, and chlorapatite, Ca5(PO4)3(Cl0.7,OH0.3), have been measured by high-pressure (diamond anvil-cells) synchrotron X-ray powder diffraction to maximum pressures of 19.9 GPa, 18.3 GPa, and 51.9 GPa, respectively. Fits of the data with a second-order Birch-Murnaghan EOS (i.e. (dK/dP)(P=0) = 4) yield bulk moduli of K0 = 97.5 (1.8) GPa, K0 = 97.9 (1.9) GPa and K0 = 93.1(4.2) GPa, respectively. The room-pressure volume variation with temperature was measured on the same hydroxyl- and fluoropatite synthetic samples using a Huber Guinier camera up to 962 and 907°C, respectively. For hydroxyl- and fluorapatite, the volume data were fitted to a second-order polynomial: V(T)/V293 = 1 + α1 (T-293) + α2 (T-293)2 with T expressed in K leading to α1(OH) = 2.4(±0.1) x 10-5 K-1, α2(OH) = 2.7(±0.1) x 10-8 K-2 and α1(F) = 3.4(±0.1) x 10-5 K-1, α2(F) = 1.6(±0.1) x 10-8 K-2, respectively. A significant increase is observed in hydroxylapatite thermal expansion above ca. 550 °C and extra reflections start to clearly appear on the X-ray film above 790 °C. These features are interpreted as the progressive dehydration of slightly Ca-deficient hydroxylapatite (i.e. with Ca/P < 1.67). Phase relation calculations, taking these new volume data for apatite into account, show that at 1200 °C, in the presence of kyanite + SiO2, hydroxylapatite should dehydrate to form γ-Ca3(PO4)2 + Ca3Al2Si3O12 below 12 GPa, i.e. below the upper-pressure stability-limit of apatite that was previously determined experimentally.