Copyright Eur J Oral Sci 2003
Eur J Oral Sci 2003; 111: 258–262
Printed in UK. All rights reserved
European Journal of
Oral Sciences
ISSN 0909-8836
Human enamel dissolution in citric acid
as a function of pH in the range
2.30 £ pH £ 6.30 – a nanoindentation
study
Michele E. Barbour1,2, David M.
Parker3, Geoff C. Allen2, Klaus D.
Jandt1,4
1
University of Bristol, Department of Oral and
Dental Science, Biomedical Engineering and
Biomaterials Science Section, Bristol, UK,
2
University of Bristol, Interface Analysis Center,
Bristol, UK; 3GlaxoSmithKline, Consumer
Healthcare, Coleford, UK; 4Institut f'r
Materialwissenschaft und
Werkstofftechnologie, Friedrich-SchillerUniversity Jena, Jena, Germany
Barbour ME, Parker DM, Allen GC, Jandt KD. Human enamel dissolution in citric acid
as a function of pH in the range 2.30 £ pH £ 6.30 – a nanoindentation study. Eur J Oral
Sci 2003; 111: 258–262. Eur J Oral Sci, 2003
The objective of this study was to investigate the dissolution of human enamel in citric
acid solutions over a wide range of pH. The in vitro conditions are considered to be
relevant to soft drink-induced enamel erosion. Nanoindentation was used to investigate changes in the nanomechanical properties of polished enamel surfaces after
exposure to citric acid solutions. Solutions used had 38.1 mmol l)1 citric acid and pH
greater than 2.3 but less than 6.3 (2.30 6 pH 6 6.30). Samples were exposed to
rapidly stirred, constant composition solutions for 120 s. Statistically significant
changes in enamel hardness and reduced elastic modulus were observed after exposure
to all solutions. There was an approximately linear dependence of enamel hardness on
solution pH for 2.90 6 pH 6 6.30. Below pH 2.90, enamel is thought to have
reached the lowest possible hardness value. The reduction in enamel dissolution caused
by an increase in pH of a soft drink is likely to be small. Product modification to
reduce the erosive potential of drinks may require additional methods such as addition
of calcium salts.
Soft drinks are well known to cause enamel erosion
in situ and in vitro (1). Citric acid is the main acid in
many fruit drinks and juices, with typical concentrations
of 15–45 mmol l)1 (2), and is also present in confectionery (3). As a result, enamel dissolution in citric acid
solutions has been the subject of many in vitro investigations (2, 4, 5, 6). Factors such as pH (2, 4, 5), exposure
time (5) and degree of saturation with respect to
hydroxyapatite (6) have been investigated. In contrast,
citrate has been added to soft drinks to reduce the cariogenic potential of the drink (7), and has been shown to
reduce the rate of enamel dissolution when compared to
other organic ions at pH 3.5 in vitro (8).
It has long been recognized that rate of enamel dissolution increases with decreasing pH for all acids.
However, to the authors’ knowledge, no studies have
been carried out to investigate thoroughly the dependence of enamel dissolution on pH over a wide range of
pH values. This is particularly important in the case of
citric acid. Citric acid is a tribasic carboxylic acid and
causes enamel dissolution by reaction of hydroxyapatite
with acid: Ca10(PO4)6(OH)2 + 2H+ 10Ca2+ +
6PO43– + 2H2O (9).
The citrate ion is a chelating ligand, and forms a soluble complex with the calcium ion, promoting further
dissolution (8). Chelation is not expected to occur at low
Klaus D. Jandt, Institut f'r Materialwissenschaft
und Werkstofftechnologie, Friedrich-SchillerUniversity Jena, L1bdergraben 32, D)07743
Jena, Germany
Telefax: +49–36–41947732
E-mail: [email protected]
Key words: atomic force microscopy; calcium;
enamel; hardness; tooth erosion
Accepted for publication February 2003
pH, but has been shown to cause an increase in enamel
dissolution rate above pH 3.9 (2).
The relationship between pH and enamel dissolution
has been employed in the design of soft drinks with a
reduced erosive potential (10). An increase in pH, often
accompanied by addition of calcium and/or phosphate
salts, has been shown to reduce the erosive potential of
soft drinks in situ and in vitro (11–17). The addition of
calcium and, to a lesser extent an increase in pH, however, are associated with a less appealing drink taste (10).
A balance between good drink taste and reduced erosive
potential is desirable. It is not clear whether the erosive
potential of a soft drink could be reduced by a slight
increase in pH, sufficiently small that it does not
adversely affect the taste of the drink, without addition
of the calcium and/or phosphate salts. Alternatively,
addition of calcium and/or phosphate may prove sufficient to reduce the erosive potential and an increase in
pH may be unnecessary. A study of the reduction in
enamel dissolution rate afforded by a particular increase
in pH of citric acid solutions is therefore desirable.
Nanoindentation has recently been used to investigate
enamel dissolution rate in situ (14) and in vitro (6).
Nanoindentation is very sensitive to changes in the
nanomechanical properties of the enamel surface, and
has been shown to detect enamel softening due to 40 min
Enamel dissolution and pH
of exposure to orange juice in situ (14) and 120 s of
exposure to citric acid (pH 3.30) in vitro (6).
The aim of this study was to investigate the dependence of enamel dissolution rate on pH in citric acid over
a wide pH range. The solution compositions and exposure times are considered to have strong relevance for
soft drink related erosion (2, 18–21). The objective of this
study was to use nanoindentation to determine the
hardness and reduced elastic modulus of enamel surfaces
after exposure to constant composition citric acid solutions with pH between 2.30 and 6.30.
Material and methods
Thirty erupted human molars were stored in tap water with
thymol after removal of the roots and pulp. Enamel samples
were cut from the buccal and lingual sides using a diamond
saw. Samples were embedded in epoxy resin (Stycast; Hitek
Electronic Materials, Scunthorpe, UK) and the natural
surface was polished using 1200 grit silicon carbide paper
and 0.25 lm aluminum oxide powder. Care was taken to
remove only sufficient enamel to provide a surface large
enough for analysis. This resulted in samples with an average area for exposure of 3–4 mm2. Samples were cleaned by
ultrasonication in ethanol for 2–3 min before and after
polishing.
Sixteen solutions were prepared with 38.1 mmol l)1
(8.00 g l)1) citric acid monohydrate (Sigma-Aldrich, Poole,
UK) and distilled water. The pH of the solutions was
adjusted to values between 2.30 and 6.30 using NaOH as
shown in Table 1. The pH was measured using a pH meter
(HI 9321 Microprocessor pH meter; Hanna Instruments,
Leighton Buzzard, UK) calibrated using standard calibration solutions at pH 4.01 and pH 7.01 (Fisher Scientific,
Loughborough, UK). The accuracy of the pH detector was
estimated by recording the pH of the calibration solutions
between each measurement. The titratable acidity was
determined by calculating the charge imbalance of each
solution and subtracting the value from that for a solution
with pH 7, hence calculating the moles of OH– necessary to
Table 1
The pH and titratable acidity values of the citric acid solutions
investigated in this study
pH
Titratable acidity mmol l)1
2.30
2.50
2.70
2.90
3.10
3.30
3.50
3.70
3.90
4.10
4.30
4.70
5.10
5.50
5.90
6.30
112
108
103
97.8
92.3
86.7
81.3
76.1
70.9
65.4
59.6
47.7
36.8
26.3
15.5
6.92
259
adjust the pH of each solution to 7. Titratable acidity is
shown in Table 1. Charge imbalance was calculated using a
computer program (22).
Eight samples were assigned to each solution, and to a
control group of samples, giving a total of 136 samples.
Samples were randomly assigned to one of the solutions
using random numbers generated using atmospheric noise
(www.random.org). The control samples were not exposed
to any solution prior to nanoindentation, and therefore
represented the condition of the enamel before exposure to
acid. Each other sample was exposed to 50 ml of the
appropriate solution, stirred using a magnetic stirrer operated at 500 r.p.m., for 120 s. After exposure the sample was
rinsed in running distilled water for 10 s to remove any
citrate, calcium and phosphate ions and stop the dissolution
process. Excess water was removed from the surface of the
enamel by touching absorbent paper to the edge of the
epoxy resin, without touching the enamel surface. The
enamel samples were then allowed to dry thoroughly in air
before analysis. The study was performed at room temperature, 24.2 ± 0.1C.
Solutions were analysed chemically in order to demonstrate that the study was performed under constant composition conditions. Eight enamel samples were exposed to
the solution at pH 2.30, which, with the lowest pH, was
expected to cause the greatest enamel dissolution, for 120 s
at 500 r.p.m. The initial and final pH of the citric acid
solution was measured. The citric acid solution was analysed for phosphate content after exposure to enamel (23).
The degree of saturation with respect to all phases of
calcium phosphate of all solutions was initially zero as the
solutions contained no calcium or phosphate. The maximum possible change in degree of saturation was estimated
using a computer program (22).
Nanoindentation was used to measure the hardness and
reduced elastic modulus of the enamel samples, and was
performed in air under ambient conditions. A Hysitron
Triboscope nanoindenter (Hysitron, Minneapolis, MN,
USA) on a Nanoscope IIIa atomic force microscope (AFM)
(Digital Instruments, Santa Barbara, CA, USA) was used to
perform nanoindentation. A Berkovich tip with a tip angle
of 142 and a tip radius of approximately 100–200 nm was
used. The enamel surface was imaged using the nanoindenter tip before each indentation to ensure that the surface
was clean and free from damage. Each sample was indented
five times in different areas. Hardness and reduced elastic
modulus were calculated by the Hysitron software using the
method of Oliver & Pharr (24).
For each sample, five values of hardness and reduced
elastic modulus were obtained. These values were not
independent as they were obtained from a single sample.
The mean of the five was therefore taken as a single value
for each sample. Since the data were found not to be normally distributed, a Kruskal–Wallis test and box-whisker
plots were used to identify statistically significant differences
within the data at a 95% confidence level.
Results
The citric acid solution with an initial pH of 2.29 ± 0.01
had a final pH of 2.30 ± 0.01 after 120 s contact with an
enamel sample. The accuracy of the pH detector was
estimated to be ± 0.01 pH units. The phosphate content
after 300 s contact with an enamel sample was below the
minimum detection limit of the test and was estimated to
260
Barbour et al.
5
4
Hardness (GPa)
hardness (P < 0.00001) and the reduced elastic modulus
(P < 0.00001) of samples treated with different solutions
and those of untreated enamel at a 95% confidence level.
There was no statistically significant difference
between the hardness of samples exposed to solutions
with 2.30 6 pH 6 2.90. Above this pH there was a
linear dependence of hardness on pH, as shown in Fig. 1
(H ¼ 1.08 · pH ) 2.78; R2 ¼ 0.99).
There was an increase in reduced elastic modulus with
pH for all solutions. The shape of the reduced elastic
modulus graph was rather different from that of hardness, however, and fits well to a polynomial, as shown
in Fig. 2 (Er ¼ )4.29 · (pH)2 + 50.8 · pH ) 54.0;
R2 ¼ 0.93).
Untreated enamel
Linear fit for pH 3.1 – 6.3
R2 = 0.99
3
2
1
0
2
3
4
5
6
pH
Fig. 1. Median hardness of enamel samples exposed to citric
acid solutions with 2.30 < pH < 6.30. A linear fit to the data
for 3.1 £ pH £ 6.3 is also shown. The triangular point
represents the hardness for enamel samples not exposed to any
solution.
Reduced elastic modulus (GPa)
110
100
90
80
70
Untreated enamel
2nd order polynomial
fit for pH 2.3 – 6.3
R2 = 0.97
60
50
40
30
2
3
4
5
6
pH
Fig. 2. Median reduced elastic modulus of enamel samples
exposed to solutions with 2.30 < pH < 6.30. A second-order
polynomial fit to the data for 2.3 £ pH £ 6.3 is also shown. The
triangular point represents the reduced elastic modulus for
enamel samples not exposed to any solution.
be 0 ± 2 lm l)1. The maximum possible change in
degree of saturation with respect to hydroxyapatite,
assuming approximately stoichiometric dissolution, was
therefore £ 0.00005.
The hardness (H) and reduced elastic modulus (Er) of
untreated enamel were found to be 4.74 ± 0.14 GPa and
104.8 ± 2.8 GPa, respectively.
Enamel hardness and reduced elastic modulus as a
function of solution pH are shown in Figs 1 and 2. The
95% confidence intervals are indicated by error bars.
Each point represents a total of 40 measurements with
five measurements from each of eight samples. There was
a statistically significant difference between both the
Discussion
Changes in enamel hardness and reduced elastic modulus
as a result of exposure to citric acid solutions were
investigated over the pH range 2.30 6 pH 6 6.30 using
nanoindentation. Nanoindentation was used for this
study because it is extremely sensitive to the early stages
of enamel dissolution in situ (14) and in vitro (6). Using
this technique it is therefore possible to distinguish
enamel dissolution after short exposure times that have a
high clinical relevance. The solutions used in this study
had citric acid concentrations typical of soft drinks (2).
There was no significant change in the pH or phosphate
concentration of the solutions over the exposure time.
The maximum possible change in degree of saturation
with respect to hydroxyapatite was therefore less than
0.00005, which is not expected to cause any change in
enamel dissolution (6, 25). The solutions can therefore be
considered to have had constant compositions during the
exposure time. The solutions were also highly undersaturated with respect to all other phases of calcium
phosphate during the exposure time, and precipitation of
these phases on the surface was therefore not thermodynamically favored.
There is some disagreement in the literature with
regard to baseline values of hardness and reduced elastic
modulus of sound enamel. This may be explained by the
recent demonstration that hardness and reduced elastic
modulus of molar enamel vary from H < 3 GPa and
Er < 70 GPa close to the enamel–dentin junction to
H > 6 GPa and Er > 115 GPa close to the enamel
surface (26). The baseline hardness and reduced elastic
modulus of enamel found in this study were
4.74 ± 0.14 GPa and 104.8 ± 2.8 GPa, respectively.
Although the information given in previous publications
as to the types of teeth used is in some cases scarce, the
value for hardness is in agreement with values reported
for unerupted molars (6), incisors (27) and primary
molars (28) but is higher than other values reported for
unerupted molars (14), third molars (29), and molars
(30). The value for reduced elastic modulus is in agreement with values reported for unerupted molars (6, 14),
incisors (27) and molars (30) but is higher than other
values reported for third molars (29), and primary
molars (28). The values found in this study are thought
Enamel dissolution and pH
to be at the higher end of the scale because efforts were
made to remove as little enamel as possible from the
surface during the polishing procedure. The baseline
values for the mechanical properties are therefore close
to those for surface enamel.
The plateau in hardness of samples exposed to solutions with 2.30 6 pH 6 2.90 seen in Fig. 1 is unlikely to
indicate that these solutions do not differ in their erosive
potential. It is suggested that there is a minimum possible
value of the hardness of softened enamel. If the enamel
structure is further weakened by continued exposure to
dissolving solutions after this minimum hardness value
has been reached, the softened enamel at the surface is
removed. The underlying enamel is then exposed to the
dissolving solution. As the dissolution proceeds, it is
suggested that the hardness approaches a constant value,
independent of exposure time, which corresponds to the
minimum possible enamel hardness. A similar result was
observed in a previous study (6) and also in a microhardness study of enamel dissolution (31). It is suggested,
therefore, that the samples exposed to solutions with
2.30 6 pH 6 2.90 had the minimum value of enamel
hardness before the net material loss that is observed
under these experimental conditions.
This assertion is supported by the reduced elastic
modulus data. The reduced elastic modulus increased
with increasing pH over the range 2.30 6 pH 6 2.90,
indicating that there was a difference in the nanomechanical properties of the enamel surfaces exposed to
these solutions. It has been shown in investigations of
thin films that reduced elastic modulus is sensitive to
underlying materials at greater depths than hardness
(32, 33). Although these materials have different
mechanical properties from enamel, the results may be
applicable to the system, as there is a soft layer of
enamel overlying hard, sound enamel. Hence, if the
enamel close to the surface had the same mechanical
properties but the thickness of the softened region was
greater for solutions with lower pH, one could expect
that the reduced elastic modulus was lower for solutions
with lower pH. As the hardness is less sensitive to
underlying material, one would expect to see a constant
hardness in this region. This greater sensitivity of
reduced elastic modulus to underlying material (32, 33)
may also explain the difference between the shapes of
the hardness and reduced elastic modulus graphs
(Figs 1 and 2).
An approximately linear dependence of enamel
hardness on solution pH for pH values > 2.90 can be
seen in Fig. 1. Some studies of the relationship between
enamel material loss and pH have indicated a different
relationship, including an exponential (5, 12) and other
functions (4, 34), in citric and other acids. Comparison
of these results is complicated, owing to the different
experimental conditions and different parameters
measured. The studies described above used exposure
times of 30 min to 4 h, compared with the 120 s used in
this study. The exposure time used in this study is
considered to have a high clinical relevance, as it is
comparable to the clearance time for citric acid in vivo
(19–21). In addition, the stirring or agitation rate is
261
lower in some of the above studies than in the present
study (5, 34), and is unknown in others (2, 4, 12);
hence, diffusion of ions close to the enamel surface may
play a greater role in these studies than in the present
study. Some studies compared solutions with different
acids and acid concentrations (12, 34), further complicating interpretation.
Some in vitro studies of enamel dissolution have indicated that no enamel or hydroxyapatite dissolution can
be detected above certain pH values in both citric and
other acids. This pH is reported as 3.8 (34) and 4.4 (4) for
enamel, and 5.5 (12) for hydroxyapatite. Other studies,
however, indicate that dissolution takes place at pH 6
(35, 36) for enamel, pH 6.5 (37) for fluorapatite, and at
pH 7 (38) for hydroxyapatite. All of these studies took
place over prolonged exposure times compared with the
study presented here. In some studies, pH was kept
constant during the experiments (37, 38). This was not
done in other studies (2, 4, 5, 12, 34).
According to the solubility isotherm of calcium
hydroxyapatite, dissolution is thermodynamically
favored in the absence of calcium at pH values as high
as 8 (39). In addition, chelation by the citrate ion is
expected to cause additional dissolution above pH 3.9
(2). It is possible that lack of dissolution above a pH
of approximately 4 observed in some previous studies
results from the longer exposure times and lower
agitation rates (2, 4, 34). Substantial calcium accumulation at the enamel surface or in the dissolving
solution and an increase in pH may therefore have
prevented or reduced enamel dissolution at these
higher initial pH values. This problem is avoided in
the present study, as the solution was rapidly stirred
(500 r.p.m), had a constant composition, and the
exposure time was short (120 s).
In terms of soft drink modification, this study indicates
that the reduction in enamel dissolution rate arising from a
microbiologically and organoleptically acceptable
increase in pH of a soft drink is likely to be small, at least
in vitro. For example, a pH of 3.8 has been used in a experimental drink with substantially reduced erosive potential (13–17). This experimental soft drink also had an
increased calcium concentration relative to typical soft
drinks. The results presented here show that an increase
from pH 3.30, a typical pH of a soft drink (18), to pH 3.80
would result in an increase in hardness of only about 15%
over 120 s exposure, indicating a small reduction in erosive potential. It must be noted that the exposure times in
this study are much shorter than the previous studies
described (13–17). However, this demonstrates that the
addition of calcium and the reduction in titratable acidity
may have been largely responsible for the reduction in
erosive potential of the experimental drink. This is supported by other studies which have demonstrated a
marked reduction in enamel dissolution rate due to addition of calcium and phosphate to acidic solution (25, 40).
Acknowledgments – M.E.B. and K.D.J. gratefully acknowledge GlaxoSmithKline for financial support in the form of a
PhD studentship. M.E.B. thanks Peter Shellis and Keith Rose
for stimulating discussion.
262
Barbour et al.
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