Letter

pubs.acs.org/NanoLett

Compressive Residual Strains in Mineral Nanoparticles as a Possible

Origin of Enhanced Crack Resistance in Human Tooth Dentin
Jean-Baptiste Forien,† Claudia Fleck,‡ Peter Cloetens,§ Georg Duda,† Peter Fratzl,⊥ Emil Zolotoyabko,¶
and Paul Zaslansky*,†
†
Julius Wolff Institute, Charité−Universitätsmedizin, 13353 Berlin, Germany
‡
Materials Engineering, Berlin Institute of Technology, 10623 Berlin, Germany
§
European Synchrotron Radiation Facility, 38043 Grenoble, France
⊥
Department of Biomaterials, Max-Planck-Institute of Colloids and Interfaces, 14424 Potsdam, Germany
¶
Department of Materials Science and Engineering, Technion−Israel Institute of Technology, 32000 Haifa, Israel
*
S Supporting Information

ABSTRACT: The tough bulk of dentin in teeth supports enamel, creating cutting and
grinding biostructures with superior failure resistance that is not fully understood.
Synchrotron-based diffraction methods, utilizing micro- and nanofocused X-ray beams,
reveal that the nm-sized mineral particles aligned with collagen are precompressed and
that the residual strains vanish upon mild annealing. We show the link between the
mineral nanoparticles and known damage propagation trajectories in dentin,
suggesting a previously overlooked compression-mediated toughening mechanism.

KEYWORDS: X-ray diffraction, apatite, residual strain, mineralized-collagen-fibers, diffraction nanotomography

P ermanent human teeth, once fully erupted, serve for entire

lifetimes despite repeated exposure to stresses during
mastication. Teeth endure largely due to the impressive fracture
Tooth dentin is thus a compound bionano-composite, and it is
fascinating because it is a bone-like tissue that lacks
remodeling/regeneration capacity. Consequently, accumulated
toughness of dentin, a bone-like material1 that forms the bulk of damage is not removed biologically as is the case in bones, yet
the crown and roots. Unlike bone, dentin does not remodel nor dentin structures exhibit remarkable life-long endurance even
does it heal as other tissues do. Dentin contains no living cells, without healing. Dentin must therefore be exceptionally well-
only cell extensions that are confined to characteristic channels, designed, possessing damage tolerance and ample toughness.
so-called dental tubules, (Figure 1) which perforate the entire A variety of mechanisms jointly contribute to fracture
structure. The tubules radiate outward from the pulp, encasing toughness8 and crack-growth resistance9 in dentin. For
the cellular outgrowths that extend from the sensitive dental- example, uncracked ligament bridging shows discontinuous
pulp odontoblast cells. Many tubules in dentin, especially in the segments of cracks, separated by unbroken tissue that reduces
crown, are lined by a 1−2 μm thick mineral sheath (peritubular stress concentration at the crack tip, slowing down crack
dentin, PTD) devoid of collagen protein.2 The mineral tablets propagation. At smaller length scales, microcracks create
in PTD have no overall preferred orientations,3 although some energy-consuming damage-resisting zones.10 Fracture tough-
local order is observed when examined with electron or X-ray ness further increases with fiber pull-out and local deflections of
beams at the nanometer length-scale.3,4 The matrix surrounding crack paths, for example, at ITD/PTD interfaces. Overall,
tubules and encasing the pulp is a fibrous mass of intertubular similar to other bone-like tissues, dentinal toughening
dentin (ITD). A typical fracture surface of ITD (Figure 1a) mechanisms largely depend on structural inhomogeneity and
reveals mineralized collagen fibers5 arranged in layers oriented anisotropy. Fracture toughness is significantly higher along
predominantly orthogonal to the tubules6 (Figure 1b). Like all dental-tubule trajectories and orthogonal to the fiber layers, a
biological nanocomposites of the bone family of materials,7 design that hinders damage from advancing toward the pulp.
dentin comprises ∼48 vol % dahllite mineral, a carbonate-rich Cracks thus propagate more easily sideways across tubules
hydroxyapatite in the form of tablet-shaped nanoparticles, because such cracks advance between the layers of mineralized
which in this work we refer to simply as “apatite”. An additional collagen fibrils. In this orientation, a lower fracture toughness
20 vol % of dentin is occupied by water, whereas the rest are KIC ≈ 1.8 MPa m1/2 was reported,11 while in the orthogonal
proteins, mainly collagen fibers randomly oriented within
incremental growth layers in the ITD. Many of the mineral Received: January 13, 2015
particles are embedded in these collagen fibers with a preferred Revised: April 28, 2015
orientation of the crystal c-axes along the collagen fiber axis.

© XXXX American Chemical Society A DOI: 10.1021/acs.nanolett.5b00143

Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters Letter

Figure 1. Typical human dentin microstructure: (a) scanning electron micrograph taken from a fractured root surface; mineralized collagen fibers
(∗) form a matrix in which tubules (black arrow) are located; scale bar = 2 μm; (b) schematic illustration of idealized dentin microstructure, in which
tubules (yellow hollow cylinders, marked with rrows), radiating outward from the pulp (not shown), are surrounded by layers of mineralized
collagen fibers (brown rods, ∗), oriented mostly orthogonally to the tubule axis. PTD mineral surrounding the tubules has no overall preferred
crystallographic orientation,3 whereas the apatite c-axis of mineral crystallites associated with the fibers is aligned with the fiber axis.

Figure 2. X-ray diffraction nanotomography reveals the spatial separation of apatite orientations with respect to the axis of dental tubules: (a)
schematic illustration of a sample with longitudinally oriented tubules mounted on edge, parallel to the azimuthal rotation axis, ω. The tubules,
marked by the green arrows and depicted in yellow/white, run along the sample longitudinal axis. Diffraction patterns of the mineral (002) reflection
were collected by scanning lines across the rotated sample on the gray marked plane. (b, c) Reconstructed nanotomography slices showing the
spatial distribution of diffraction intensities “in plane” and “out of plane” (see plane in panel a). The in-plane integrated diffraction signal shown in
panel b arises from apatite crystals predominantly oriented with their c-axes aligned with the collagen fibrils layers. A rather homogeneous
distribution of diffraction intensities corresponds to the overall felt-like pattern of ITD. The spatial distribution of the integrated diffraction signal of
apatite crystals, with the c-axes oriented out of the plane of the collagen fibrils layers (c), exhibits high brightness spots (green arrows). The size and
spacing of these spots suggest that they correspond to PTD surrounding tubules that are observed in cross-section. Scale bar = 50 μm.

orientation parallel to the tubules, values as high as KIC ≈ 3.4 Zentrum-Berlin, Germany, and ESRF, Grenoble, France). We
MPa m1/2 were found.12,13 A similar relationship to structure is found apatite crystallites to be compressed along the c-axis; the
known for the fatigue behavior of dentin14 where the endurance compression was significantly larger in crystallites aligned with
strength is only 24 MPa in orientations across the tubules, as collagen, as compared to those having the c-axis in other
compared with 44 MPa reported for orientations along the orientations. Mineral compression impedes crack propagation
tubules. Though it is difficult to associate specific dentin across the fibers, and stops cracks from running between the
microstructures to the toughness contributions that they tubules, because excessive tensile stress is needed to allow such
entail,8 collagen fiber-orientations appear to be particularly crack opening/propagation. We thus propose that a previously
important for the overall tissue strength and damage resistance. unidentified toughness mechanism exists in dentin, directly
To shed additional light on the link between the micro- arising from the nanocomposite ultrastructure of ITD, a
architecture of dentin and its remarkable longevity and mechanism that probably exists in other collagen-based
resistance to damage propagation, we studied the material members of the bone family of materials.7
properties of the embedded mineral nanoparticles. We By means of X-ray diffraction nanotomography15,16 (see
measured the c-lattice parameters of apatite in differently Supporting Information), we studied the spatial arrangement
treated human-tooth dentin samples by using focused X-ray and orientation of apatite crystallites in human root dentin
beams at synchrotron beamlines (BESSY II, Helmholtz- (Figure 2a, Figure S2) where collagen fibrils in the ITD17 have
B DOI: 10.1021/acs.nanolett.5b00143
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Nano Letters Letter

well-known orientations, predominantly orthogonal to the

tubules. We observe a clear spatial separation between crystal
orientations in the ITD and in the PTD, known to have no
preferred orientation of the mineral.3 Complementary cross-
sectional diffraction-tomography (Figure 2a) reconstructed
slices reveal dissimilar spatial distributions of the diffraction
intensities when integrating counts either across (Figure 2b) or
along (Figure 2c) tubule trajectories. For comparison, Figure
S3 shows the same tubules as visualized by phase contrast-
enhanced nanotomography. The reconstructed (002) diffrac-
tion intensities across the tubules (Figure 2b) are relatively
homogeneous, mainly revealing intensity contributions from
mineral in the uniform in-plane fibers of the ITD matrix
surrounding the tubules. In contrast, diffracted intensities from
apatite crystallites with the c-axes oriented parallel to the
tubules, that is, orthogonal to the ITD collagen layers, reveal
dispersed white spots, manifesting higher local intensities. The
periodic appearance of these spots in the cross-section suggests
that their source is the PTD (Figure 2c), where the average
random apatite orientation differs from those in the collagen
fiber layers.3 Thus, the different groups of crystallites in ITD
and PTD are distinguishable, and it is therefore possible to
differentiate between them when mapping (002) diffraction
intensities.
We determined the c-lattice parameters of apatite in human
tooth dentin when analyzing diffraction patterns taken from
specimens with well visible tubules (Figure 3a,b) and
consequently for which the collagen fiber orientation was
known.17 Furthermore, we measured the azimuthal variations
of the (002) Debye-ring radii (Figure 3c). Six samples were
mapped in both hydrated and air-dried states. For comparison,
additional samples, annealed at 220 °C for ∼1 h (Figure S2
shows typical temperature ramping), were also measured. We
compared the c-lattice parameter in crystallites residing on the
collagen−fibril axis with those in other orientations. The
crystals attached to collagen in dentin have the c-axis along the
collagen fibers,5 and we term them “on-axis” (highlighted in
orange, Figures 2b and 3c). Crystallites having the c-axis in
other orientations are termed “off-axis” (highlighted in green,
Figures 2c and 3c). In hydrated (subscript h) samples, we find a
substantial negative difference, Δch = con − coff = −0.0054 ±
0.0016 Å (mean ± standard deviation), between the “on-axis”
(con) and “off-axis” (coff) lattice parameters extracted from the
very same diffraction patterns. In the same samples, air-dried
(subscript d), this difference is smaller, Δcd = −0.0028 ±
0.0018 Å, and the difference tends to zero, Δc = 0.0002 ±
0.0008 Å, in samples annealed at 220 °C. Transforming these
data into a relative lattice parameter difference, δ,
con − coff
δ=
(con + coff )/2 (1)
we find that in the untreated hydrated samples, “on-axis”
mineral crystallites are compressed along their c-axis by δh ≈
−0.08%, as compared with “off-axis” crystallites. After air-
drying, which reduces the water content of dentin (by exposure
to ambient 33−35 RH%), this relative lattice parameter
difference drops to δd ≈ −0.04%. Upon annealing at 220 °C,
Figure 3. Schematic illustration of X-ray diffraction measurements and
which destroys the organic molecules, the difference δ vanishes their analysis: (a) spatially resolved nonoverlapping diffraction patterns
(Figure 4a). were collected when scanning across and along dentin samples in the
By using published estimates for the elastic modulus of x- and z-directions using slender X-ray beams. (b) The tubule
apatite E = 114 GPa,18 we convert the native relative difference, silhouettes, visible by optical microscopy, run in parallel (the
δh, into compressive stress of σh = E δh ≈ 90 MPa. Interestingly, approximate orientation indicated by the green arrow) and serve to
this value is well above typical mastication stresses, which do identify the predominant orientation of the collagen fibers (orange

C DOI: 10.1021/acs.nanolett.5b00143
Nano Lett. XXXX, XXX, XXX−XXX
Nano Letters Letter

Figure 3. continued not affected by variations in the absolute values of the apatite
lattice parameters, known to have a considerable spread in
arrow), most of which are arranged in layers orthogonal to the tubule teeth22 due to local variations in chemical composition.23,24
axes. Red dots depict approximate beam size and position on the The in situ measurements reveal relative changes of the c-lattice
sample. Scale bar = 100 μm. (c) In typical diffraction patterns, the parameter in the same areas of each sample before and after
apatite (002) peak intensities and positions vary due to preferred annealing.
orientations of the crystals, many of which are orientated “on-axis”, Typically, heating carbonated hydroxyapatite results in a
along the collagen long axis (orange outlines). A difference in the c-axis decrease of the c-lattice parameter due to the discharge of
lattice parameters can be seen, as compared with the “off-axis” crystals gaseous products.23 However, this occurs only at temperatures
that are not aligned with collagen (green outlines). Variation of the that are much higher than those that we used, and it causes an
peak positions (2θ) as a function of the azimuthal angle (plotted
beneath the diffraction pattern) reveals a different strain state of apatite
opposite effect on the c-lattice parameter compared to what we
crystals aligned with the collagen fibrils (orange) as compared with observe in dentin, namely further lattice shrinkage. Interest-
other orientations, for example, along the tubules (green). ingly, from the difference between the strain values in air-dried
samples in both experiments (Figure 4a,b), we deduce that the
not normally exceed 40 MPa.19 Thus, normal “working “off-axis” mineral in air-dried dentin is also compressed by
conditions” of teeth do not usually exceed the in-built residual about 0.1% compared to the annealed state.
stress of mineral in dentin suggesting that teeth have an in-built Our experimental findings allow us to draw two important
“safety margin” of stress. For damage to propagate through conclusions: (1) “on-axis” mineral crystals, associated with
dentin, tensile loads exceeding this value are needed. This collagen, are under compressive stress as a result of strong
suggests that generally this tissue is not overloaded during food mineral/collagen interactions; (2) “off-axis” mineral crystals,
processing and therefore should not break under “normal” not directly associated with collagen, experience far less stress.
conditions of use. We suggest that compressive stresses arise at early stages of
To verify the overall compressive state of the “on-axis” dentin biomineral formation, when mineral particles take the place of
crystallites, we performed in situ annealing-diffraction experi- water.25 When water is expelled from the system (for example
ments with air-dried samples. Diffraction patterns were following drying), collagen fibrils experience contraction,26
collected from several root−dentin samples initially at room which is transferred to the tightly attached apatite particles.
temperature, then in situ heated to 125 and 250 °C, followed Correspondingly, we expect that collagen in dentin experiences
by postannealing room-temperature measurements. We used a tensile residual strain/stress, as indirectly predicted by other
thin gold coating as a calibration standard to correct for subtle studies.27
but significant variations in the sample−detector distance We are not able to directly verify this claim since collagen
incurred due to thermal expansion (Supporting Information). does not sustain heating at 250 °C, and it significantly changes
The “on-axis” c-lattice parameters calculated from the (002) its dimensions due to dehydration and denaturation. However,
Debye-ring radii were corrected by accounting for apatite we found corroborative evidence supporting the notion that
thermal expansion20 (α) at each measured temperature (αapatite tensile stresses exist in dentin collagen fibrils, even under
= 10.6 × 10−6/°C). Mean relative differences in the “on-axis” c- unloaded conditions.27 We compared collagen diffraction
lattice parameters Δc/c of three investigated samples, measured patterns taken at room temperature and at 125 °C (Figure
in the original and in situ heated states and compared to their S4), considering that collagen fibrils still remain essentially
postannealing room temperature values, are plotted in Figure 4, undamaged at this temperature.21 Examination of the Debye
panel b. We find that prior to annealing, the “on-axis” rings showed that the lateral spacing between collagen fibrils in
(collagen-associated) mineral is under compressive strain, ϵ1 the collagen fiber bundles decreases from 11.43 ± 0.06 Å at
= Δc/c = −0.15%, and it endures mild annealing at 125 °C. room temperature down to 10.38 ± 0.19 Å at 125 °C. A similar
Indeed, collagen in dentin is known to remain stable at these lateral shrinkage was previously reported in dentin21 and for
temperatures.21 The “on-axis” strain drops to zero (ϵ3 = 0) other sources of collagen.25 It is known that shrinkage in the
upon annealing at 250 °C, as soon as the collagen molecules are lateral direction is accompanied by the respective contraction of
destroyed. We note that strains determined in this manner are the collagen molecules along the fibrils, though an order of

Figure 4. Relative differences (δ) and “on-axis” strain in c-lattice parameters obtained in two types of experiments: (a) comparing diffraction signals
within the same Debye ring in each diffraction pattern, collected from “on-axis” and “off-axis” apatite crystallites with respect to the collagen fibrils;
relative difference (δ) for hydrated and dried samples were obtained by averaging over six specimens, while data for annealed samples were found by
averaging over four specimens; (b) comparing only “on-axis” c-lattice parameters in the same samples measured in situ at different temperatures with
the postannealed state; experimental points are obtained by averaging over three specimens; error bars represent standard deviations.

D DOI: 10.1021/acs.nanolett.5b00143
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Nano Letters Letter

magnitude smaller, as observed in tendon samples under mineralized collagen fibers in dentin presumably exist also in
controlled drying.26 Thus, collagen fibers in hydrated dentin are bone (see, for example, ref 31) and must serve to enhance
elongated as compared with fibers subjected to annealing at 125 strength and fracture toughness at the most fundamental
°C, and a significant part of this elongation is due to the structural length-scale. We propose that this route is used in
presence of water. Other studies report lower dentin strength nature in addition to well-known toughening recipes such as
following heat-treatments exceeding 150 °C21 presumably due hierarchical architectural designs on different length-scales37 or
to a decoupling between the organic phase and mineral and are periodic arrangements of stiff and compliant layers.38
related to the removal of the tightly bound water layer.28
The strong collagen/mineral interactions in native tooth
dentin and forces acting across the collagen/mineral interfaces
■
*
ASSOCIATED CONTENT
S Supporting Information
require that collagen fibrils experience tensile stress to balance The Supporting Information section contains detailed
the mineral compressive state. Collagen fibrils in dentin thus act descriptions of the materials and methods used. These include
as tensed strings resisting buckling during mastication, while sample preparation steps, diffraction nanotomography, diffrac-
the apatite mineral particles serve as compressed fillers that tion mapping and in situ annealing procedures and phase-
stiffen the fibers. Tensed mineralized fibrils entail stiffness to contrast enhanced nanotomography imaging of tubules in
dentin, supporting the tubular microstructure against collapse dentin. Additionally, details of the data analysis procedures are
under externally applied bending or compressive loads (Figure presented, together with results of collagen small angle
S5). At the same time, the mineral phase, which is typically scattering. A schematic representation of typical bending forces
brittle in tension, is protected against tensile loads due to its and stresses acting on teeth is also shown. The Supporting
compressive residual-stress state. This structural nanocompo- Information is available free of charge on the ACS Publications
site design thus hinders crack propagation, preferentially in website at DOI: 10.1021/acs.nanolett.5b00143.

■
directions orthogonal to the mineralized collagen fibril layers,
and results in a tougher tooth structure, specifically along AUTHOR INFORMATION
tubules, that is, in the general direction of the pulp, which has
Corresponding Author
great evolutionary advantages for the survival of teeth.
*E-mail: [email protected]. Phone: +49 30 450 55 95
Various man-made engineered structures, exhibiting im-
89. Fax: +49 30 450 55 99 69.
proved strength and endurance, benefit from incorporating
residual compressive strains/stresses; tempered glass, rein- Notes
forced concrete, and gear wheels are well-known examples. The authors declare no competing financial interest.
Typically, compression stresses are intentionally induced
beneath outer surfaces aimed at preventing crack formation
and propagation into the component interior.29 In dentin, we
■ ACKNOWLEDGMENTS
The authors acknowledge Stefan Siegel, Chenghao Li, and
find mineral compression widely distributed as a damage- Rémi Tucoulou for their technical support at the beamlines.
prevention mechanism within the collagen-fiber nanostructures, The Helmholtz-Zentrum Berlin (Bessy II) and the ESRF are
which offers protection against crack initiation due to gratefully acknowledged for beamtime allocation. P.Z., C.F.,
mastication loads. The notion that compressive forces imposed and J-B.F. acknowledge funding by the DFG through SPP1420.
on the mineral cause tensile forces in collagen implies that the E.Z. thanks Shore Research Fund in Advanced Composites
magnitude of the compressive residual stress in the mineral will (Technion) for partial financial support.
strongly depend on the mineral/collagen ratio. Thus, regions in
teeth with lower mineral content (e.g., the root) will exhibit
different residual stress and strain than regions with higher
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F DOI: 10.1021/acs.nanolett.5b00143
Nano Lett. XXXX, XXX, XXX−XXX