-
Biomaterials 28 (2007) 2
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J H
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Abstract
1. Introduction
5 mm in diameter and may be up to several millimetres long,and
the majority are arranged with their long axes atapproximately 901
to the enamel-dentine junction (EDJ).
The orientation of prisms in enamel has been studied in
the growth of the HA crystallites was lost. It has beenreported
from grazing-incidence synchrotron X-ray dif-fraction experiments
that there is a higher degree of
ARTICLE IN PRESScrystallite alignment in surface enamel compared
to enamelclose to the EDJ [6]. However, only linear slices from
EDJto surface were probed in these experiments. In an earlier
0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights
reserved.
doi:10.1016/j.biomaterials.2007.02.019
Corresponding author. Tel.: +441133438331.E-mail address:
[email protected] (M. Al-Jawad).Dental enamel is the most
highly mineralised andhardest biological tissue. It is comprised of
approximately96% mineral, 3% water, and 1% organic matter
(non-collagenous protein) by weight [1]. The mineral is
non-stoichiometric calcium hydroxyapatite (Ca10(PO4)6OH2)with
carbonate, uoride, sodium, and magnesium ionsfrequently found
within the structure. These hydroxyapa-tite (HA) crystallites are
laid down as nanorods with cross-sectional dimensions of 50 nm 25
nm and up to 1mmlong [2]. Clusters of these nanorods, known as
prisms,contain around 1000 crystallites. They are approximately
the past using electron microscopy. Although this is avaluable
tool for nding the prism shape and size in aparticular plane of
enamel, it is a qualitative technique anddoes not give detailed,
quantitative information on thedegree of alignment in different
parts of a tooth. Previouswork using X-ray diffraction on human
dental enamel hasestablished the space group and lattice parameters
as P63/m(hexagonal) and a 9.441(2) A and c 6.878(1) A respec-tively
[35]. However, these values were obtained frommeasurements of
powdered enamel collected from severalteeth, and as a result any
information on the spatialvariation of the lattice parameters and
texture relating toWe have used synchrotron X-ray diffraction to
study the texture and the change in lattice parameter as a function
of position in a cross
section of human dental enamel. Our study is the rst to map
changes in preferred orientation and lattice parameter as a
function of
position within enamel across a whole tooth section with such
high resolution. Synchrotron X-ray diffraction with a micro-focused
beam
spot was used to collect two-dimensional (2D) diffraction images
at 150mm spatial resolution over the entire tooth crown. Contour
mapsof the texture and lattice parameter distribution of the
hydroxyapatite phase were produced from Rietveld renement of
diffraction
patterns generated by azimuthally sectioning and integrating the
2D images. The 002 Debye ring showed the largest variation in
intensity. This variation is indicative of preferred
orientation. Areas of high crystallite alignment on the tooth cusps
match the expected
biting surfaces. Additionally we found a large variation in
lattice parameter when travelling from the enamel surface to the
enamel-
dentine junction. We believe this to be due to a change in the
chemical composition within the tooth. The results provide a new
insight on
the texture and lattice parameter proles within enamel.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Enamel; Hydroxyapatite; Apatite structure; Synchrotron
X-ray diffraction; Texture; Preferred orientation2D mapping of
texture and latt
Maisoon Al-Jawada,, Axel Steuwerb,Robert Cywinski
aLeeds Dental Institute, UniversbFaME38 at the ILL-ESRF, 6
rue
cInstitute for Materials Research, UnivdSchool of Physics and
Astronomy, U
Received 19 December 200
Available online9082914
e parameters of dental enamel
usan H. Kilcoynec, Roger C. Shorea,David J. Wooda
of Leeds, Leeds, LS2 9LU, UK
orowitz, 38042 Grenoble, France
ity of Salford, Salford, M5 4WT, UK
rsity of Leeds, Leeds, LS2 9JT, UK
ccepted 16 February 2007
February 2007
www.elsevier.com/locate/biomaterials
-
study Hirota examined the tilting of the enamel-prismorientation
in a human canine using laboratory two-dimensional (2D) X-ray
diffraction [7], however only 12points within the tooth were
measured and therefore theinformation obtained about the prism
orientation cannotbe for the whole tooth. In this paper we aim to
show for therst time how synchrotron X-ray diffraction can be used
todetermine the basic crystallographic parameters of the HAphase
across a whole intact tooth section, allowing us toexplore
composition and texture on the sub-millimetrelength-scale.
Characterising the orientation distribution ofthe anisotropic
apatite crystallites of dental enamel aids thefundamental
understanding of the natural growth andformation of dental enamel,
and provides insights into howsynthetic enamel-like materials may
be developed.
lattice parameters distribution maps. The 2y angle-range for
thisexperiment was 2y 5301for comparison this corresponds to a
2y-range of 9581 on a conventional lab-based X-ray diffraction
apparatuswith CuKa radiation of wavelength l 1.54 A. For our
experimentalsetup, the main diffraction peak of the HA phase (2 1
1) was located in the
centre of our 2y range at 16.771. Vacuum tube slits were used to
focus theX-ray beam to a diameter of 150mm on the sample. A 500mm
thick toothsection was mounted in transmission geometry onto a
travelling sample
platform such that the tooth could be scanned in two
orthogonal
directions perpendicular to the beam. A charge-coupled device
(CCD) 2D
ARTICLE IN PRESSM. Al-Jawad et al. / Biomaterials 28 (2007)
29082914 29092. Materials and methods
2.1. Specimen preparation
The sample used in this study was a section of an adult
mandibular
second premolar (LR5). The tooth was collected with informed
consent
from a patient undergoing routine orthodontic extraction at the
Leeds
Dental Institute. The extracted tooth had its pulp removed and
was
sterilised by autoclaving prior to storage at 4 1C in a thymol
solution toprevent bacterial growth. A precision diamond blade
cutter was used to
cut the tooth into 500mm thick longitudinal sections
perpendicular to thebuccal and lingual surfaces. The sections were
then polished by hand to
remove any surface roughness. A photograph of the section used
for this
study is given in Fig. 1. The four arrows mark the tracks
plotted in Fig. 10.
2.2. Synchrotron X-ray diffraction
The measurements were taken on the XMaS beamline [8] at the
European Synchrotron Radiation Facility (ESRF) using an
X-ray
wavelength of l 0.82 A (equivalent to X-ray energies of 15 keV)
and asample to detector distance of 163.09mm. The 2y angle-range
for ourexperiment was limited by our experimental setup and the
sample to
detector distance. We compromised on the very high-angle HA
reections
in order to position the 2D detector closer to the sample and
therefore
greatly reduced our counting times. This allowed us to collect
more
diffraction patterns per tooth and obtain higher resolution
texture and
Fig. 1. Photograph of 500mm thick tooth section from a human
adult
lower right premolar. The arrows mark the tracks through the
tooth
plotted in Fig. 10.detector with 2048 2048 pixel resolution was
mounted behind the sampleand perpendicular to the incident beam for
the collection of 2D diffraction
images. The cross-hairs of a telescope were positioned in line
with the
beam centre in order to align the beam position on the tooth. A
schematic
of the experimental setup with an example 2D diffraction image
is shown
in Fig. 2. A single diffraction image had an exposure time of 5
s and
therefore a 150mm-resolution map of the tooth on a grid of 10mm
7mmcould be collected in approximately 8 h by moving the sample
relative to
the beam in an x and y direction.
2.3. Data analysis
2D diffraction images were pre-processed with the ESRF
software
Fit2D [9]. Each image was sectioned into 51 slices [10] and
these integratedslices were then used to create Intensity versus 2y
patterns for Rietveldrenement [11], i.e. 72 diffraction patterns
per 2D image. Conventionally
only one Bragg reection in the diffraction pattern is used in
texture
analysis therefore any slight changes in sample volume as a
function
of position would affect the sample absorption and could affect
the texture
coefcient obtained. However, using the Rietveld method minimises
the
effect of variations in sample volume since all reections are
used to
obtain the t. In addition, in our study, we used a scaling
factor in the
renement procedure and found that changes in the scale factor
were small
as a function of position within the tooth, indicating that
variations in
section thickness were negligible. A total of 1095 diffraction
patterns were
rened and therefore an in-house automated batching procedure
was
written and used to input the patterns into the GSAS Rietveld
renement
software [12]. The instrument parameters such as X-ray
wavelength,
sample to detector distance, and peak-shape prole were
determined using
a LaB6 standard sample. These parameters were then kept xed
for
renements of the data. The scale parameter and background
parameters
(four terms) were rened rst. The lattice parameters and
crystallite
size (Lorentzian particle broadening term) were rened next,
starting from
the values for HA taken from Young [3]. Finally the texture was
rened
using a spherical harmonics function [13] and the preferred
orientation
values for the 002 reection were extracted from this. The values
of
preferred orientation ranged from 0 (randomly oriented) to 3.5
(strongly
textured). The quality of the renement was determined by least
squares
methods where the goodness of t increased as w2 approached
unitywhereby:
w2 R2wp
R2e,
where Rwp is the weighted R-factor and Re the expected
R-factor.Fig. 2. Experimental setup at the XMaS beamline, ESRF,
with an
example 2D diffraction image.
-
3. Results
3.1. Preferred orientation in enamel
Preferred orientation has both a magnitude and adirection.
Through our analyses of the X-ray diffractiondata we have been able
to quantify both these parameters.In order to obtain an overview of
the preferred orientationin a tooth section the 2D X-ray scans were
arranged toform a composite map of CCD images of the tooth, asshown
in Fig. 3. Each small square in the image is one 2Ddiffraction
pattern. The centres of adjacent diffractionpatterns are 150 mm
apart. The shape of the tooth canclearly be seen from this
composite image. The darkerpatterns in the middle of the tooth are
from dentine and thelighter patterns covering these are from the
enamel. At thesurface the enamel is thinner, therefore there is a
halo ofdarker images along the edge of the tooth where there is
textured than the enamel.The intensity pattern around the Debye
ring of the 002
reection was used to evaluate the texture direction.
Theintensity was integrated over 3601 in a narrow bandcontaining
the 002 reection and plotted versus theazimuthal angle. Fig. 5
shows a typical example of theresulting curve for one diffraction
scan where there are twopronounced peaks separated by approximately
1801. Bytting these peaks to a Gaussian peak shape, the
deviationangle, f, of the crystallite axis relative to vertical
wasdetermined. By applying this procedure to each of thediffraction
images, a map of the local orientation of thetexture in the 002
direction in the enamel is obtained.Fig. 6 shows a map of the
orientation angles overlaid
onto the composite image of the tooth. In Fig. 6, forclarity,
only every fourth value of f has been drawn. It canbe seen from
this that the texture direction in the 002reection is approximately
perpendicular to, and followsthe contour of the EDJ. From our
knowledge of thestructure of dental enamel it would appear that
thepreferred orientation in the 002 direction approximatelyfollows
the direction of the enamel prism arrangement.
3.2. Rietveld refinement
The extent of preferred orientation in the tooth sectionhas been
quantied using Rietveld renement. An example
ARTICLE IN PRESSM. Al-Jawad et al. / Biomater2910partial air
scattering. This can also be seen in the ssurewhere there is a gap
between the two cusps. Additionally, itcan be seen that below the
ssure there is a circular regionof enamel which is darker than the
surrounding patterns. Itis likely that this is caused by a ssure
caries lesion whichhas partially demineralised the enamel in that
area.In Figs. 4ad four individual diffraction scans from
different parts of the tooth are shown. Figs. 4a, c and
dillustrate the change in texture direction in the 002 plane
atdifferent positions within the enamel. Variations inintensity
around diffraction rings are indicative of texturein the tooth
enamel. The strongest texture (the mostextreme variation in
intensity) was found in the 002reection (2y 13.71)labelled in Fig.
4a. A line throughzero degree has also been marked in Fig. 4a. Fig.
4b showsthe diffraction pattern from dentine. Here the peaks
aremuch broader indicating that the crystallites are smaller.There
is also much less variation in intensity around theFig. 3.
Composite of 2714 diffraction images arranged spatially to show
the outline of the tooth specimen.Debye rings indicating that
the dentine is much less
Fig. 4. (a), (c) and (d) Illustrate the change in texture
direction at
difference positions within the enamel. (b) Shows the poorly
crystalline
nature of dentine.
ials 28 (2007) 29082914of a typical 1D Intensity versus 2y
diffraction patterntogether with the calculated pattern is shown in
Fig. 7. The
-
open circles are the observed data points, and the solid lineis
the calculated diffraction pattern. Below the pattern is aplot of
the difference (observedcalculated), and beneathare the tick marks
for the 2y peak positions for thecalculated diffraction pattern of
HA. The difference plotshows that the agreement between observed
and calculateddata is generally very good with a typical value for
w2 of1.5. The nal parameters obtained from this renementare given
in Table 1. Similar renements were carried outon all diffraction
patterns.A contour map showing the change in magnitude of
preferred orientation in the 002 diffraction peak has
beenplotted in Fig. 8. The 002 preferred orientation parametershave
been extracted for the zero degree slice of each 2D
ARTICLE IN PRESS
Fig. 6. Texture direction of the 002 reection of hydroxyapatite
crystal-
lites in enamel, calculated using 2D diffraction images.
Fig. 5. Typical intensity versus Azimuthal angle curve for the
002
reection showing the pronounced texture in this sample. The left
hand
peak has been tted to a Gaussian.
Fig. 7. Typical diffraction pattern including the raw data
(circles), the calculate
2y peak positions for the calculated diffraction pattern of
hydroxyapatite.
M. Al-Jawad et al. / Biomaterials 28 (2007) 29082914
2911diffraction image (see Fig. 4a). Areas with higher values
ofpreferred orientation parameter are more strongly texturedi.e.
the crystallites are more aligned to the zero degreedirection in
these areas. Areas with low texture coefcienthave less well-aligned
crystallites.
3.3. Change in lattice parameters
Both the a- and c-lattice parameters of HA were renedin each
diffraction pattern and after carrying out Rietveldrenements of
1095 data sets it was noticed that neither thea- nor c-lattice
parameters were constant as a function ofposition. Note that the
variation was not due to X-raywavelength drift as a function of
time, but was clearlydependent on the position within the tooth.
Trends in thelattice-parameter changes have been plotted in Fig.
9relative to the average lattice parameters. The relativepercentage
change in the a-lattice parameter (Ra) across thetooth section was
calculated using:
Ra an a0
a0
100%,
where an is the rened lattice parameter for diffractionpattern
n, and a0 is the overall average lattice parameter.The same type of
equation was used for dening thec-lattice parameter variation, Rc.
Both Ra and Rc have beend diffraction pattern (solid line), the
difference, and the tick marks for the
-
ARTICLE IN PRESS
el
a b 901, g 1201535.49(8)
7.83(9)
1.9(1)
1.5
(SH) is the 002 spherical harmonic preferred orientation
term.
aterTable 1
Rened structural parameters for typical diffraction pattern of
dental enam
Space group
a (A)
c (A)
a, b, gV (A3)
Y(particle)002(SH)w2
Y(particle) is the coefcient for Lorentzian particle size
broadening, and 002
M. Al-Jawad et al. / Biom2912plotted as contour plots in Figs.
9a and b. It can be seenfrom these gures that the a- and c-lattice
parameters varybetween 0.6% and +0.3% of their average values. In
allcases the uncertainty in the lattice parameters did notexceed 1
103, and the values for the average latticeparameters were a0
9.5165(6) A and c0 6.9394(2) A.These plots clearly reveal that
there is a systematicvariation of the lattice parameters as a
function of positionwithin the enamel.Figs. 10ad shows plots of Ra
and Rc as a function of
distance from the surface for the four tracks through theenamel
indicated in Fig. 1. In all regions, Ra (lledsymbols) decreases
with increasing distance from the toothsurface, especially around
the cusps. In Figs. 10b and cthere is second peak in the Ra curve
at around 600 mm fromthe surface indicating a region of enamel
which has ahigher a-lattice parameter than the surrounding
enamel.This region can be seen clearly in the contour plot in
Fig.9a. Along each track, the c-lattice parameter curves
(opensymbols) are much atter. This indicates that Rc is
lessdependent on the distance from the enamel surface thanRa. In
both the a- and c-lattice parameter values, there isalso a
difference between the lingual and the buccal sides ofthe
tooth.
Fig. 8. Texture distribution map generated from the calculated
texture
coefcient via Rietveld renement.(HA)
Phase hydroxyapatite
P63/m (#176)
9.4660(9)
6.9018(3)
ials 28 (2007) 290829144. Discussion
It has been seen previously that there is a higher degreeof
crystallite alignment in surface enamel compared toenamel close to
the EDJ [6]. However, in that work, onlylinear slices from EDJ to
surface were probed. The resultsfrom our study show that the
texture distribution ismuch more complex than previously thought.
We can see
Fig. 9. (a) a-lattice parameter and (b) c-lattice parameter
contour maps
showing the change in lattice parameter value at different
positions
around the tooth.
-
ARTICLE IN PRESSaterin Fig. 8 that HA crystallites are most
aligned in the cuspalregions: on both sides of the buccal cusp and
on the innerside of the lingual cusp HA crystallites are highly
aligned.Conversely, along the sides of the tooth away from thecusps
generally the crystallites are less ordered. It isinteresting to
note that the areas of high crystallitealignment match the expected
occlusal surfaces of a lowersecond premolar [14]. This may be an
evolutionarydevelopment of enamel so that the regions of
enamelwhich are exposed to the largest load are the strongest. It
isinteresting to note that a strong correlation
betweenfunctionality and texture in human bone has been reportedby
Bacon [15] where he observed that the living conditions
Fig. 10. Four tracks through the tooth section going from enamel
surface
to EDJ showing the change in lattice parameter as a function of
distance
from the enamel surface. The tracks are indicated in Fig. 1.
M. Al-Jawad et al. / Biom(either on a steep hillside, or on the
at) of two Neolithictribes radically affected the HA crystallite
growth andalignment on the lower front edge of the tibia.
Althoughdental enamel cannot regenerate itself as bone can, it
islikely that through evolution the degree of crystallitealignment
in different regions of a tooth has beenoptimised for the function
of the tooth.Changes in lattice parameter can be indicative of
changes
in enamel crystal chemistry as well as changes in the
stress/strain-state of a material. Separating these two
possibleeffects or even using the lattice parameters to
determinecompositional changes in biological apatites is
notstraightforward as changes in crystal chemistry can bethe result
of several ionic substitutions, such as Na, Mg, Clor F, as well as
variations in the carbonate content and theCa/P ratio. However, the
magnitude of the changes seenacross our tooth suggest that the
changes in latticeparameters plotted in Figs. 9 and 10 arise
predominantlyfrom changes in the chemical composition of the enamel
indifferent regions of the tooth. Chemical analysis of
thedistribution of uoride, carbonate and magnesium inenamel has
been carried out previously by Robinson etal. By dissecting tooth
sections into pieces weighing2050 mg, the amount of uoridated
apatite was determinedby etching the tooth sections and analysing
the uorideconcentration in the post-etched buffer solution [16],
theconcentration of carbonate was determined by dissolvingeach
piece in acid and measuring the volume of CO2emitted [17], and
concentration of magnesium was foundusing an atomic absorption
spectrophotometer [18]. Theyfound that uoride concentrations
decreased in going fromthe enamel surface to the EDJ, while
carbonate andmagnesium concentrations increased (from 2% to 46%,and
from 0.2% to 0.5% respectively) across the samedistance [19]. In
dentine, changes in the a-lattice parameterof up to 0.5% have been
reported in going from the EDJinto the centre of the dentine [20].
This trend has beenexplained principally as a result of the
increased substitu-tion of CO3
2 for PO43 associated with the more immature
dentine crystallites. Our results are of a comparable orderof
magnitude and we believe them to be the result ofcompositional
change. Comparing Figs. 9a and b it can beseen that there is more
variation in the a-lattice parameterthan in c. This trend has also
been seen in dentine [20]where the a-lattice parameter decreased by
0.5% withincreasing distance from the EDJ into the dentine,
whereasthe c-lattice parameter only decreased by 0.1% over thesame
distance. In addition to a decrease in latticeparameters going from
the surface enamel to the EDJ,there is a difference in the lattice
parameters on the buccaland lingual sides of the tooth indicating a
change in crystalchemistry on the different sides of the tooth.
This couldeither be due to the different functions of the two sides
ofthe tooth or due to their slightly different oral environ-ments,
or a combination of both.Cuy et al. have generated 2D distribution
maps for the
hardness (H) and Youngs modulus (E) of enamel
usingnanoindendation [21]. In the molars they investigated,
theyfound that the cuspal regions had higher hardness andYoungs
modulus. They report values ranging fromH46GPa to Ho3GPa and
E4115GPa and Eo70Gpa,respectively, going from the enamel surface to
the EDJ.The contour maps they generated of H and E show
similarfeatures to our lattice-parameter distribution maps,
in-dicating that the crystallographic and mechanical proper-ties of
enamel are closely linked, therefore anunderstanding of both is
necessary in order to fullyunderstand the function of enamel in
different parts of atooth.
5. Conclusions
Using spatially resolved synchrotron X-ray diffractionwe have
quantied the changes in texture and latticeparameters in dental
enamel as a function of positionwithin the tooth. With this
technique, in a few hours ofdata collection, we have generated 2D
distribution maps ofboth texture and lattice-parameter changes in
enamel with
ials 28 (2007) 29082914 2913150 mm resolution. This has given
detailed quantitativeinformation on the degree of crystallite
alignment in
-
different regions of tooth enamel not previously reported.It has
also shown that the lattice parameters maps, relatedto changes in
crystal chemistry, are more complicated thatpreviously thought
indicating that understanding hetero-geneities within a single
tooth is as important as realisingthe differences between teeth. We
have shown thatcharacterising the crystallographic properties of
dentalenamel is crucial in order to design optimised
dentalrestorative materials. Finally, we have shown through
thiswork that synchrotron X-ray diffraction is a powerfultechnique
in the study of the crystallography and micro-structure of dental
enamel and it could be equallysuccessful in the study of other
biological hard tissues, inthe study of synthetic biomaterials, and
in the study of bio-synthetic complexes.
[5] Wilson RM, Elliott JC, Dowker SEP, Smith RI. Rietveld
structure
renement of precipitated carbonate apatite using neutron
diffraction
data. Biomaterials 2004;25(11):220513.
[6] Low IM. Depth-proling of crystal structure, texture, and
micro-
hardness in a functionally graded tooth enamel. J Am Ceram
Soc
2004;87(11):212531.
[7] Hirota F. Prism arrangement in human cusp enamel deduced
by
X-ray diffraction. Arch Oral Biol 1982;27(11):9317.
[8] Brown SD, Bouchenoire L, Bowyer D, Kervin J, Laundy D,
Longeld MJ, et al. The XMaS beamline at ESRF: instrumental
developments and high resolution diffraction studies. J
Synchrotron
Radiat 2001;8(6):117281.
[9] Hammersley AP. FIT2D: An Introduction and Overview. ESRF
Internal Report 1997; ESRF97HA02T.
[10] Hammersley AP, Svensson SO, Hanand M, Fitch AN,
Hausermann
D. Two-dimensional detector software: from real detector to
idealised image or two-theta scan. High Pressure Res
1996;14(46):
23548.
ARTICLE IN PRESSM. Al-Jawad et al. / Biomaterials 28 (2007)
290829142914Acknowledgments
This work was performed on the EPSRC-funded CRGbeamline (XMaS
BM28) at the ESRF. We are grateful toL. Bouchenoire and J. Wright
(ESRF) for their invaluableassistance and to S. Beaufoy for
additional administrativesupport. Also, we would like to thank the
FaME38 facilityfor providing the VAMAS approved precise
samplemounting system. Thanks to C. Sullivan at Leeds
DentalInstitute for producing the photograph in Fig. 1.
Thisresearch was funded by the UK Medical Research Council.
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2D mapping of texture and lattice parameters of dental
enamelIntroductionMaterials and methodsSpecimen
preparationSynchrotron X-ray diffractionData analysis
ResultsPreferred orientation in enamelRietveld refinementChange
in lattice parameters
DiscussionConclusionsAcknowledgmentsReferences