Mineralogical Magazine, October 2005, Vol. 69(5), pp.

643652

Acidic demineralization of apatites studied by

scanning X-ray microradiography and microtomography

J. C. ELLIOTT*, F. R. G. BOLLET-QUIVOGNE, P. ANDERSON, S. E. P. DOWKER, R. M. WILSON AND G. R. DAVIS

Institute of Dentistry, Queen Mary, University of London, Francis Bancroft Building, Mile End Rd,
London E1 4NS, UK

ABS TR AC T

The mineral in bones and teeth is an impure form of hydroxylapatite (HAP), the principal impurity
being 25 wt.% carbonate. This mineral dissolves during remodelling of bone and also in dental caries
as a result of the action of acids produced by osteoclasts and by bacteria, respectively. In enamel,
demineralization proceeds with preferential loss of carbonate relative to phosphate. Surprisingly, in the
early stages, the demineralization is subsurface. In order to facilitate the understanding of physical
chemical aspects of these processes, we have undertaken studies of demineralization in model systems.
We give three examples here. The first two used scanning microradiography in which the specimen is
stepped across a 1030 mm diameter X-ray beam. Intensity measurements allow calculation of the
mineral mass per unit area in the X-ray path through the specimen. In the first experiment, porous HAP
sections were separated from a reservoir of acidic buffer by a column initially filled with water (the
diffusion length) and scanned with the X-ray beam perpendicular to the axis of the diffusion length.
The rate of total loss of mineral along each profile was calculated from the scans. The rate of
demineralization fell as the diffusion length increased. We believe the explanation is that the rate-
controlling step is the diffusion of dissolved HAP away from the solid to the buffer reservoir. In the
second experiment, demineralizing solution and water were pumped alternately, for equal lengths of
time, past blocks of porous HAP or enamel. The X-ray beam was perpendicular to the exposed surface.
As the rate of switching between solutions decreased, the mean rate of demineralization also fell. We
propose that this effect is due to retention of acid in the pores of the HAP during the time when water
flows, allowing further demineralization to take place during this time. The third study used X-ray
microtomography, a form of 3D microscopy, to study the loss of mineral in compacted carbonate
apatite powders. The powders were packed in six 10 mm internal diameter acrylic cylinders to a depth
of 4 mm (after pressing). One end was covered with a porous polyethylene disc and each tube placed in
acidic buffer for 70 days. Periodic examination by microtomography showed the development of
subsurface demineralization. Infrared spectroscopy of the dissected-out surface layers showed
preferential loss of carbonate over phosphate by comparison with deeper layers. Rietveld analysis of
X-ray powder diffraction data showed changes in the crystallographic structures of the apatites between
the initial and dissected-out apatite.

K EY WORDS :dental enamel, hydroxylapatite, teeth, demineralization, X-ray microtomography, scanning

microradiography.
Introduction
DENTAL enamel, the most mineralized tissue in the
body, is made up of ~95 wt.% of an impure
carbonate-containing apatite (CO3Ap) closely
related to hydroxylapatite (Ca10(PO4)6(OH)2,
HAP). The vol.% of mineral is usually quoted
as 86 vol.%, but a recent recalculation that used
an estimated density of the enamel apatite
(3.0 g cm 3) rather than of HAP (3.16 g cm 3)

yielded 96 vol.% (Elliott, 1997). Enamel apatite is

* E-mail: j.c.elliott@qmul.ac.uk microcrystalline with attened hexagons ~30 nm
DOI: 10.1180/0026461056950276 across and 0.1 to 5 mm or more in length. Most of
# 2005 The Mineralogical Society
 J. C. ELLIOTT ET AL.
the CO23 ions replace PO34 ions, but about 10%
are thought to replace OH ions in the axis
c changes is the preferential loss of a Mg- and CO3-
channels (Elliott, 1994). A recent Rietveld
renement of the structure has been published
(Wilson et ., 1999). Although there was
al
evidence that CO23 ions replaced PO34 ions, it
was not possible to determine the details of the
substitution. However, it2 seems probable that the
normal to the plane CO3 ion makes an angle of
~30 to the axis, as reported for a synthetic
c (Weatherell ., 1977).
sodium-containing CO3Ap that was studied by
Rietveld analysis of neutron diffraction powder
data (Wilson ., 2004). This orientation is also
et al
consistent with polarized infrared (IR) measure-
ments (Elliott, 1994).
Dental caries is an extremely prevalent disease
that is immensely costly to health services. The
main chemical change in the formation of enamel
lesions is the net dissolution of mineral by acids
of bacterial origin. An unusual and perplexing
feature of the dissolution process is that it takes
place below the surface of the enamel with the
retention of a relatively intact surface layer
(Fig. 1). Thus, in enamel lesions, loss of mineral
may be halted or even reversed through
reprecipitation of `mineral' from the saliva. In
the early stages of dental caries, there is a
preferential loss of Mg (Hallsworth ., 1972)
et al
and carbonate (CO3) (Hallsworth ., 1973). It
et al
FIG. 1. Slice (~15 mm) through a 3-dimensional micro-
tomography image of a human molar tooth. Highly
mineralized enamel (light) overlies less well mineralized
dentine which surrounds the unmineralized pulp (dark).
Caries lesions in enamel showing subsurface mineral
loss are seen as darker regions adjacent to ssures. To
the right, the slice cuts through a ssure; to the left, the
slice cuts through regions of mineral loss that extend in
three dimensions from a ssure which terminates in an
adjacent slice.
644
is not known if the process that results in these
rich phase in the enamel, or dissolution of a
homogeneous phase, followed by precipitation of
a less soluble apatite with less Mg and CO3.
However, there is no convincing independent
evidence that the apatite in enamel contains a
second, more soluble phase. There is also an
increased uoride concentration in carious lesions
et al
In order to try to understand the complex
chemical changes that take place in enamel during
dental caries, many investigators have studied de-
and remineralization in model systems that used
either sections or blocks of enamel or HAP
pellets. We present here three such demineraliza-
tion studies. They have in common the fact that
they all make use of X-ray attenuation measure-
ments. Dowker . (1999) gave an account of
et al
related, earlier experiments. Remineralization
processes can also be investigated by X-ray
attenuation methods, but generally the effects
are much more subtle and more difcult to
quantify (Gao ., 1993).
et al
The studies to be presented concern: (1) the
effect of changes in the diffusion path-length
between an acidic buffer reservoir and the surface
of a dissolving apatite pellet. This study
investigated the importance of diffusion processes
in the external solution of a solid dissolving in
acid; (2) the effect of switching periodically
between buffer and water on the mean rate of
dissolution of an HAP porous pellet. A depen-
dence on switching period might be expected if
there was inertia in the system, for example, the
demineralizing solution was retained in the pores
of the solid, so that demineralization continued
after the solution was switched from buffer to
water; and (3) the spatial distribution of the
apatite lost during demineralization of a pressed
pellet of carbonate apatite, including an IR and
powder X-ray diffraction (XRD) study of the
apatite at the end of the demineralization.
Experimental
Apatite and pellet preparation
The HAP was prepared by addition of solutions of
(NH4)2HPO4 to Ca(NO3)2.4H2O with a Ca/P mole
ratio of 1.67 at 8090C and pH 1011
(maintained by addition of ammonia solution).
The precipitate was ltered off, washed, dried at
80C, then 900C and ground to a ne powder.
Pellets were prepared by compacting the HAP
 ACIDIC DEMINERALIZATION OF APATITE

powder in the nger of a surgical glove,
isostatically pressing in oil (70 MPa for the
external diffusion study and 275 MPa for the pH
cycling experiments) followed by sintering in
steam at 900C for 24 h. The porosity for the
lower-pressure pellets was ~50% and ~20% for
the high-pressure ones.
Seven samples of CO3Ap were prepared by
adding 20 g of CaHPO4 to 150 g of ammonium
carbonate dissolved in 10 l of deionized water.
Stirring at 70C was maintained for 3 days with a
nal pH of 910. The apatites were separated by
ltration, washed well, and dried at 100C for
24 h. Powder XRD data were collected and
analysed by the Rietveld method as is described
later. This showed that two of the samples
contained 1.6 and 0.6 wt.% calcite, but the
others contained only well crystallized apatite.
The sample with 0.6 wt.% calcite was used in the
present study. This CO3Ap contained 11.7 wt.%
CO3 (see Wilson ., 2004 for a description of
et al
the analytical method) and the lattice parameters
determined in the Rietveld analysis were: =
9.4021 A and = 6.8963 A.
a
c diffusion of acid (and outward diffusion of the
Scanning microradiography
Detailed accounts of scanning microradiography
(SMR) have been published (Anderson .,
et al
2003; Elliott ., 1994). In this technique, the
et al
X-ray transmission of a narrow X-ray beam
through the specimen is measured with a
photon-counting detector (Fig. 2) rather than
the conventional photographic emulsion. This
has the advantages of avoiding the non-linearity
of emulsions and in particular, allows a much
larger dynamic range without problems of
background noise and saturation inherent to
photographic emulsions. A particular advantage
of using photon-counting methods rather than an
emulsion is that the specimen can be some
distance from the detector, so it can be placed in
a cell with a controlled environment, e.g.
temperature. In the present studies, permeable
apatite blocks or sections are mounted in cells
and immersed in aqueous acidic buffers. The
sections or blocks are varnished with nail
varnish all over, except for a window on one
face or edge so that the acid can diffuse through
this exposed face into the solid and cause
dissolution. The X-ray beam may be oriented
either perpendicular to the direction of inward
products of dissolution), or parallel to the
direction of diffusion (Anderson ., 1998).
et al
With the X-ray beam perpendicular, the pattern
of demineralization through the depth of a
permeable solid can be observed. On the other

FIG. 2. Schematic of scanning microradiographic stage with horizontal and vertical motions. Several cells containing
mineral sections are mounted on the stage so that they can be scanned in any predetermined sequence. Reacting
solutions can be pumped through the cells so that the course of the reaction with the mineral can be studied.
645
 J. C. ELLIOTT ET AL.
hand, if the beam is parallel to the direction of
diffusion, changes in the total apatite mineral
along the X-ray path through the depth of the
solid are measured, irrespective of the depth at
which the mineral is lost. Measurements can be
made at a single point, a series of points along a
line or over a 2-dimensional array of points.
Whatever arrangement of points is chosen,
measurements can be repeated sequentially in
order to study local changes in the specimen
with time. If the changes with time are relatively
slow, as they usually are, many cells can be
mounted on the scanning stage (Fig. 2),
allowing multiple experiments to be undertaken.
A Hilger & Watts Y33 X-ray generator with a
1006100 mm source (after foreshortening) and
Ag target was used at 40 kV and 1 mA tube
current. The aperture comprised two perpendi-
cular tantalum crossed slits (15 mm gap), posi-
tioned 3 cm from the source. X-ray photons were
counted with a high purity Ge solid state detector
coupled with a DSPEC plus (a combined digital
amplier and multichannel analyser), both from
EG&G Ortec (now AMETEK Inc). AgK a
photons were selected from the spectrum and
counted for xed time periods. A PC was used to
control both the counting and movement of the
SMR stage.
X-ray microtomography
A fourth generation X-ray microtomography
(XMT) system (Davis and Elliott, 2003) with a
laboratory X-ray generator (Ultrafocus HMX 160,
X-Tek Systems Ltd, UK, 5 mm source, tungsten
target, 160 kV max) was used at 90 kV and
256 mA. This system used a cooled slow-scan
CCD camera with a 1:1 lens coupling to a 70 mm
thick columnar CsI scintillator (Applied
Technologies Ltd, UK). The rotation axis was
vertical. In order to eliminate ring artefacts, time-
delay integration CCD readout was employed. In
this, the CCD is read out in synchrony with a
horizontal mechanical motion of the camera
across the X-ray beam. Thus the sensitivities of
all the recorded image pixels in the same CCD
column were identical as each was an average of
every physical detector element within that
column. In order to correct for beam hardening,
the detector was calibrated with a seven-step
aluminium (99.98 wt.%) wedge so that the data
appeared to have been collected with a nominally
monochromatic beam of 40 keV energy. In the
present study, 600 projections were collected over
646
360 (data collection time ~4 h) and recon-
structed using a cone-beam version of the
Cartesian axes preprojection algorithm (Davis,
1998). The nal reconstructed image comprised
36063506190 cubic voxels of side length
29.55 mm and represented the 3-dimensional
distribution of the linear attenuation coefcient
at the nominal monochromatic beam energy of
40 keV.TMThese images were analysed using the
Amira software package (TGS Template
Graphics Software Inc., USA).
For the XMT study, specimens were xed on
the lids of closed plastic bottles to prevent drying
out during the X-ray exposure. The bottles were
mounted kinematically on the XMT rotation stage
so they could be removed, the specimen (still
attached to the lids) placed in buffer for
demineralization, and then repositioned on the
stage for re-imaging. Successive scans give a set
of registered 3-dimensional images of the process
of mineral loss.
IR spectroscopy
Fourier transform infrared (FTIR) spectra were
recorded (Perkin-Elmer Spectrum GX FTIR
spectrometer) for specimens in KBr discs (2 mg/
300 mg KBr). Relative areas of the n3 CO3 and n4
PO4 bands were determined with the GRAMS/32
software (Galactic Industries Ltd, Salem, NH,
USA).
XRD and Rietveld analysis
Powder XRD patterns were collected following
the procedure described earlier (Wilson .,
et al
1999; Morgan ., 2000). A Ge 111 mono-
chromator giving Cu- a 1 radiation ( l =
et al
1.5406 A) and an INEL CPS-120 curved posi-
K
tion-sensitive detector were used. Samples (typi-
cally 5 mg) were spread on a at (711) cut Si
single crystal rotating specimen holder, with the
incoming monochromatic beam striking its
surface at an angle of ~3.5, to minimize
background. Data sets were collected for at least
1000 min and were calibrated using curves
produced from lead nitrate (99.999 wt.% purity)
usually run before and after each sample. The 29
most intense lead nitrate peaks between 19 and
1172y and = 7.8586 A (Nowotny and Heger,
a
1986) were used for the calibration. Rietveld
structure renements were made using the
computer program, GSAS (Larson and Von
Dreele, 1986).
 ACIDIC DEMINERALIZATION OF APATITE
Effect of external diffusion on the rate of demineralization
Ten 500 mm thick sections were cut from the
HAP block and varnished except for one edge.
These were mounted in polymethylmethacrylate
(PMMA) cells (the cross-section of one is shown
in Fig. 3), with the unvarnished edge uppermost
and thus exposed. The PMMA was used for
making the SMR cells as it is essentially X-ray
transparent. The sections were positioned at
increasing distances from 0 to 9 mm (these
distances will be referred to as diffusion
lengths, ) from the cell opening. All the cells
l The total mass of HAP lost per unit area of the
were lled with deionized water and then placed
in the same large PMMA reservoir as seen in
cross-section in Fig. 3. The reservoir was then
lled with1 demineralizing buffer (1 l of
0.1 mol l acetic acid adjusted to pH 4 with
NaOH). Physical mixing of the solution in the
reservoir and in the diffusion length were
minimized as the reservoir solution was not
stirred, and thermal gradients were minimized
by conducting the study in a temperature-
controlled room at 252C.
Microradiographic scans were made from
outside to within the section in a vertical direction
(Fig. 3) along two lines. These lines were
perpendicular to the exposed surface and sepa-
FIG. 3. Cross-section of cell with 500 mm thick section
of HAP in place. The diffusion length, , is the distance
l
between the HAP surface and the cell opening into the
buffer reservoir. The rate of demineralization can be
measured by repeated vertical scanning of the HAP
section through the X-ray beam. Ten such cells were
mounted in the 1 litre reservoir.
647
rated by 2 to 4 mm, depending on the cell. Each
line comprised 61 points separated by 40 mm
(10 s counting time per point, typically 20,000
photons at points in the solid). Standard
measurements (30 s counting time, typically
130,000 photons) were made outside each
section before and after each line scan for
correction of X-ray generator and detector
instabilities. The measurement sequence for the
ten sections took ~6 h and was repeated for
1680 h, except between 600 and 950 h when the
driving mechanism malfunctioned.
section exposed to buffer was calculated for each
time of measurement and for each section. For
these calculations, the mass attenuation coef-
cient of HAP for 2Ag- 1 a radiation (22.1 keV) was
K
taken as 4.69 cm g . From this data, the rate of
loss of mineral with time was calculated for each
section, and hence each diffusion length. The
rates were calculated by linear least-squares
tting a straight line to the data between 300 h
to the end of the experiment (1680 h). As there
were two line scans per section, there were two
determinations of the rate of loss of mineral for
each diffusion length.
Demineralization under pH cycling conditions
Five ~1.3 mm thick sections were cut from the
same HAP pellet and varnished except for one
surface. Similarly, ve enamel blocks, again
~1.3 mm thick, were cut from a human molar
tooth, and varnished except for the natural
surfaces. Five SMR cells were made from
PMMA. An enamel section and HAP block
were mounted in each cell so that the X-ray
beam was normal to the unvarnished surface, that
is to say, so that changes in the total apatite
mineral along the X-ray path through the depth of
the solid would be measured. A reservoir was
lled with 50 l of 0.1 mol l 1 acetic acid adjusted
to pH 4 with NaOH. A second reservoir was lled
with 50 l of deionized water. The reservoirs were
connected via four 2-way valves and peristaltic
pumps to each input of four SMR cells. Each cell
had two outputs (emptying and overow) which
were connected via a further four 2-way valves
and peristaltic pumps to waste. The valves were
computer controlled and programmed so that
buffer and water owed alternately for equal
times /2, where is the switching period, with a
T T
30 s emptying after each switching to prevent
contamination of one reservoir solution by the
 J. C. ELLIOTT ET AL.
other. The values of for the four cells were: 0.5,
T above, this enabled a sequence of XMT images to
1, 2 and 4 h. A constant ow of buffer was
pumped through the fth cell to act as a control.
X-ray attenuation measurements through the
specimens were made at ve points, immediately
preceded and followed by measurements in the
solution near the sample for normalization
purposes. The counting time was 20 s for all
measurements and the scanning sequence over all
specimens (~1 h) was repeated continuously until
all of the buffer solution had been used up
(~235 h).
Microtomography study of demineralization in compacted
carbonate apatite
Sleeves (10 mm bore61.6 mm wall627 mm
length) and pistons (10 mm diameter 6 45 mm
length) were made from PMMA. Six sleeves were
lled with ~0.6 g of CO3Ap each. For loading, a
sleeve had a piston put in one end, lled with
CO3Ap, then another piston pressed in from the
other end. One end of the sleeve was then put in
distilled water so that water was drawn up through
the powder without occluding air. Pressing was
continued to the limit of the strength of the sleeve.
The pressed pellet of CO3Ap was then pushed to
one end and its exposed surface covered with a
disc of porous polyethylene (Fig. 4), which was
retained with a ne nylon thread wound several
times over the length of the sleeve. The
protruding end of the piston was then cemented
to the inside surface of the lid of a plastic bottle.
As described in the microtomography section
FIG. 4. Polymethylmethacrylate sleeve (10 mm internal
diameter) with compressed disc of CO3Ap (11.7 wt.%
CO3) in buffer at pH 4.5. The exposed surface of the disc
has been covered with a porous polyethylene cylinder
retained in place with nylon thread (not shown).
648
be made of the process of demineralization.
The six CO3Ap pellets were demineralized, as
illustrated in Fig. 4, in acidic buffer1 (100 ml
sodium acetate/acetic acid, ~0.1 mol l , pH 4.5,
with a thymol crystal) for 70 days with weekly
replacement of the buffer. The XMT images were
collected at intervals of 1 to 3 weeks. At the end
of the experiment (70 days), the surface,
intermediate, deep intermediate and deep layers
were dissected out from ve of the pellets (the
other pellet was lost during microtomography) for
FTIR spectroscopy and XRD analysis for the
determination of lattice parameters.
Results and discussion
Effect of external diffusion on rate of demineralization
Fifteen of the 20 scans showed subsurface
demineralization, a characteristic of natural and
model caries lesions (Dowker ., 1999). An
et al
example is shown in Fig. 5. The other ve scans
showed no subsurface demineralization, although
there was some evidence of erosive loss of HAP.
The most likely explanation for this was the
trapping of a small air bubble on the deminer-
alizing surface which is known to be an
occasional problem. This explanation is consistent
with the observation that the rates of loss of HAP
for these ve scans (marked with crosses in
Fig. 6) were much less than those showing
subsurface demineralization. As the conditions
of demineralization for these ve scans were
clearly different from the remaining scans, they
were excluded from the data analysis.
The rates of HAP loss per unit area exposed to
buffer for the different diffusion lengths (in the
range 0.3610 8 to 1.8610 8 g cm 2 s 1)
FIG. 5. Scans of a HAP section (only every ~100 h
shown) for a diffusion length of 0.2 cm showing
subsurface demineralization, a feature typical of natural
caries lesions in dental enamel (see Fig. 1). Points are
separated by 40 mm.
 ACIDIC DEMINERALIZATION OF APATITE
FIG. 6. Rates of total mineral loss for different diffusion
lengths. (*) results with, and (6) results without,
subsurface demineralization. The line is a3 plot of
equation6 3 2with1 s = 0.00224 g cm
, p =
4.2610 cm s and = 7.4610 6 cm s 1, the
C D
K
latter two parameters being determined by least-squares
tting of equation 3 to the * data points (from Bollet-
Quivogne ., 2005).
et al
decrease as the diffusion length increases (Fig. 6).
This suggests that diffusion in the solution
external to the dissolving solid controls the rate
of dissolution.
A simple model has been used to derive a
quantitative relationship between the steady state
rate of dissolution, , and the diffusion length
R marked with crosses. The values for p and
(Bollet-Quivogne ., 2005). The assumptions
et al
made in its derivation were: (1) that the ionization
of the HAP and buffer could be ignored, so they
diffused as single species; and (2) that the buffer
concentration at the solid-liquid interface was
effectively equal to the reservoir concentration
soon after the start of, and throughout, the
experiment. This second assumption was based
on two propositions: the rst that the buffer
diffuses quickly by comparison with the time-
frame of the experiment primarily because its
substantial concentration in the reservoir provides
a signicant gradient for its inward+diffusion; and
second, that the consumption of H ions required
for the dissolution of HAP at the interface has a
negligible effect on the buffer concentration.
These assumptions are discussed further by
Bollet-Quivogne et. (2005) and at http://
al
www.smd.qmul.ac.uk/dental/oralgrowdev/
biophysics/publications/appendix1.doc.
For diffusion-controlled dissolution (Berner,
1978)
649
( s i)
R = K C C (1)
where is the rate of dissolution, is the
R K
dissolution rate constant, s the HAP concentra-
C
tion in the demineralizing solution at equilibrium,
and i the concentration of HAP at the solid-
C
interface. At steady state, the rate of dissolution
will equal the rate of diffusion of dissolved HAP
out of the diffusion length ( ). The latter, from
l
Fick's First Law is p i/ , where p is the
D C l D
diffusion coefcient of HAP, and taking into
account: (1) the concentration gradient of HAP in
the diffusion length is constant; and (2) the
concentration of HAP in the reservoir is zero.
Hence
Dp
K (Cs Ci ) = Ci 2
l
Eliminating i between equations 1 and 2 gives
C
Dp
R=
l + Dp / K
Cs 3
C s, the HAP concentration in the deminer-
alizing solution at equilibrium, was 2.24 g l 1,
calculated from the computer program Chemist
(MicroMath Research, Salt Lake City, USA).
Using this value, equation 3 was least-squares
tted to the data in Fig. 6, excluding results
D K
(with standard deviations) were p =
( 4 . 2 0 . 8 ) 661 0 6 c1m 2 s 1 a n d
D
=
(7.40.6)610 cm s . The value of p is of
K
D
the expected order of magnitude, though the
signicance of the value of is difcult to judge
K
because the surface area of the dissolving HAP
crystals in the face of the HAP aggregate is
unknown.
Demineralization under pH cycling conditions
The mass of HAP per unit area integrated along
the X-ray path as a function of time for various
switching periods is given in Fig. 7. The
intercepts with the vertical axis vary somewhat
because of the different initial thicknesses of the
samples. The slope of each plot, hence the rate of
loss of mineral with time, decreases as the
switching period increases, even though the
mean time of ow of buffer is the same for all
switching periods. The control has a higher rate of
mineral loss because the buffer ows for twice the
 J. C. ELLIOTT ET AL.

FIG. 7. Mass of HAP per unit area integrated along the X-ray path in hydroxylapatite as a function of time and for
different switching periods ( ). Water and pH 4 buffer owed alternately for equal times with = 0.5, 1, 2 and 4 h.
The plot for continuous demineralization in pH 4 buffer (labelled ?) is also given and crosses over the plot for =
T T
T
at ~140 h.
time. The results for enamel were similar to those transient phenomena that are responsible for this.
for HAP, but the rates of demineralization were The exact nature of these are still to be elucidated,
greater. This is not surprising as, unlike dental but are likely to involve diffusion and volume
enamel, the HAP had been heated to 900C and effects within the subsurface lesion so that buffer
was free of CO3, both of which would reduce the is retained in the pores.
rate of dissolution of HAP in acids.
The results show clearly that the rate of
demineralization increases as the frequency of Microtomography study of demineralization in compacted

switching increases. This implies that deminer- carbonate apatite

alization lasts for a time longer than the actual The volume percent of apatite in the pellets before
time of ow of buffer. There clearly must be some demineralization was ~60%, as determined from

FIG. 8. Left: axial slice (29.5 mm thick) through 3-dimensional microtomography data set of compressed CO3Ap disc
(10 mm diameter) after 46 days in buffer (darker regions correspond to lower concentrations of apatite). Right:
prole of apatite concentration along the white line in the axial slice.
650
 ACIDIC DEMINERALIZATION OF APATITE

the mass of apatite used to make the pellet and its

dimensions. Apart from the initial images before
the start of the experiment, all pellets showed
substantial subsurface demineralization and reten-
tion of a well-mineralized surface zone, as
illustrated in Fig. 8 for a pellet after 46 days in
buffer. As demineralization progressed, the
advancing front of the demineralized zone
moved deeper into the pellet, but there was
always some mineral left in the demineralized
zone, as illustrated by the plot in Fig. 8. These
results show that the model system described here
has many of the characteristics of natural caries
lesions, though the details, e.g. the thickness of FIG. 9. FTIR spectrum of apatite from the deep layer
the surface layer, differ. (lower trace) compared with the surface layer (upper
Figure 9 shows the FTIR spectra of the surface trace). Note the lower intensity of the n3 CO3 band at
and deep layers with the major bands identied. ~14001500 cm 1 in the surface layer material.

The n3 CO3 absorption at ~14001500 cm 1 is
clearly of lower intensity in the surface layer
compared with the deep layer, whereas the1
intensity of the n4 PO4 absorption at ~600 cm
is similar. Plots of the area ratio of the
n3 CO3/n4 PO4 bands decreased from the original,
outwards to the surface for all the pellets. This
mirrors the preferential loss of carbonate over
phosphate found in early caries lesions in enamel
(Hallsworth ., 1973).
et al
Figure 10 shows a plot of the axis the -
a- vs.
axis parameters for the unit cells of the original
CO3Ap, deep, intermediate and surface layers.
This clearly shows a linear increase in both the
and axes as the sample locus moves from the
c of apatitic material.
Samples collected from CO3Ap pellet after 70 days in
buffer at pH 4.5.
deep towards the surface layer. Such changes in
lattice parameters are consistent with there being
a structural change associated with the prefer-
ential loss of CO3, rather than dissolution of a
CO3-rich phase. The actual surface samples
themselves occupy a completely different position
in the gure indicating that they are not the end-
c member of a progressive alteration. Either there
has been some sort of collapse in the unit cell
leading to a reduction in the unit-cell parameters,
a or the surface layer is the result of reprecipitation

FIG. 10. Plot of axis . axis for the original CO3Ap and samples dissected out after 70 days in buffer at pH 4.5.
a vs c
There are only four `Deep' samples because, accidentally, one was not collected.
651
 J. C. ELLIOTT ET AL.
Conclusions
The investigations reported above demonstrate
that X-ray attenuation methods can, or could be,
used in a wide variety of novel studies of
dissolution processes in apatite-containing
systems (synthetic and biological apatites
including bone and dental enamel, whole tissue
or pressed powders). Such studies should give
detailed quantitative information about how the
dissolution of apatites is affected by chemical
composition, porosity and crystallinity. This
should lead to a much better understanding of
the dissolution behaviour of biological apatites,
including the physical chemistry of dental caries.
Studies of remineralization are also possible, but
much more difcult. Although this work was
restricted to apatites, the experimental approach
might be extended to a wide range of materials of
geological and mineralogical interest, and need
not even be restricted to acidic dissolution
processes.
Acknowledgements
This research was supported by the Medical
Research Council programme grant G9824467
and subsequently by the St Bartholomew's and
the Royal London Charitable Foundation grant
RAB 03/PJ/18. The microtomography was
supported by the Engineering and Physical
Sciences Research Council grant GR/R28911/01.
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[ Manuscript received 13 January 2005:
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