journal of dentistry 38 (2010) 584590

available at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

The clinical application of surface pH measurements to

longitudinally assess white spot enamel lesions

Yuichi Kitasako a,*, Nathan J. Cochrane b, Matin Khairul c, Kanako Shida a,c,
Geoffrey G. Adams b, Michael F. Burrow b, Eric C. Reynolds b, Junji Tagami a,c,d
a
Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University,
5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113-8549, Japan
b
Cooperative Research Centre for Oral Health Sciences, Melbourne Dental School, Bio21 Institute of Molecular Science and Biotechnology,
University of Melbourne, Parkville, Victoria, Australia
c
Support Program for Improving Graduate School Education at Tokyo Medical and Dental University, Tokyo, Japan
d
Global Center of Excellence Program; International Research Center for Molecular Science in Tooth and Bone Diseases,
Tokyo Medical and Dental University, Tokyo, Japan

article info abstract

Article history: Objectives: Means of objectively assessing white spot enamel lesions (WSEL) are critical for
Received 2 February 2010 determining their potential activity and monitoring the success of preventive treatments.
Received in revised form The aim of this study was to determine whether surface pH measurements of WSEL changed
20 April 2010 during a preventive course of care designed to remineralize the lesions.
Accepted 21 April 2010 Methods: Eight healthy subjects (1 male and 7 females) with at least one WSEL were recruited
(1964 years). Each subject was placed on a preventive treatment program including the
daily application of a CPP-ACP paste (MI paste, GC Corp., Japan) with custom fitted trays for
Keywords: more than 6 months. The surface pH values of sound enamel and WSEL were monitored for
Casein phosphopeptide-stabilized up to 2 years using a micro-pH sensor. The visual appearance of the WSEL was monitored via
amorphous calcium phosphate digital photography, and images were analyzed qualitatively on a 5-point scale to assess the
White spot success of the remineralization preventive program. The relationship between the qualita-
pH tive assessment of WSEL appearance and the WSEL pH was investigated using a Spearmans
Enamel rho non-parametric correlation.
Saliva Results: The surface pH of the WSEL was different to that of the sound enamel surrounding it
in all patients at all times. All lesions showed visual improvement as the treatment period
progressed. The pH of the WSEL increased towards that of sound enamel over the course of
treatment significantly correlating with the visual improvement of the lesion (rho = 0.63,
p < 0.0001).
Conclusions: The clinical assessment of WSEL surface pH changes with time may have utility
as an additional objective measure for the assessment of WSEL activity.
# 2010 Elsevier Ltd. All rights reserved.

1. Introduction material. Recently, a new approach has been adopted called

the Minimal Intervention concept which aims to treat lesions
Conventionally dental caries has been treated by removing the in a non-invasive manner where possible.1 Non-cavitated
carious hard tissues and the placement of a restorative white spot enamel lesions (WSEL) can be arrested or reversed

* Corresponding author. Tel.: +81 3 5803 5483; fax: +81 3 5803 0195.
E-mail address: kitasako.ope@tmd.ac.jp (Y. Kitasako).
0300-5712/$ see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2010.04.010
 journal of dentistry 38 (2010) 584590
if the cariogenic challenge is sufficiently controlled or
therapeutic agents are applied to promote enamel reminer-
alization.2 One such agent is MI Paste that contains casein
phosphopeptide-stabilized amorphous calcium phosphate
(CPP-ACP) that has been shown in clinical trials to promote
the regression of early lesions.25 Means of objectively
assessing WSEL are critical for determining their potential
activity and monitoring the success of preventive treatments.
Current methods for assessing the activity of WSEL include
visual criteria,6,7 and lactic acid sensitive alginate impression
materials.8 Simple objective chair-side techniques for assist-
ing with lesion activity assessment would be an invaluable aid
for dental care providers for the management of early carious
lesions. Historically, the pH of a range of intraoral fluids have
been measured such as saliva9 and plaque.10 However, few of
these pH measurement techniques have become routinely
adopted in clinical practice.
One problem with measuring plaque is that often patients
will remove it immediately prior to appointments by thorough
brushing. This study sought to use an Ion Sensitive Field Effect
Transistor (ISFET) pH probe1114 to measure the surface pH of
WSEL and the sound enamel surrounding it with the prior
removal of the overlying plaque. This pH probe has been used
previously to examine the pH of arrested and active dentine
caries in vitro and validated against a pH imaging micro-
scope.14 To the authors knowledge this is the first time that a
pH sensor has been used to measure surface pH as a means of
monitoring WSEL. Therefore, the aim of this study was to
assess longitudinally the pH of WSEL and the sound enamel
surrounding it following the institution of a preventive
program to determine whether differences existed and
whether they would correlate to any observed changes in
lesion appearance.
2. Materials and methods
2.1. Study design and subject recruitment
Eight healthy subjects were recruited after ethical approval
was obtained from the Ethics Committee of the Tokyo Medical
and Dental University and informed consent forms were
signed. Each volunteer completed a medical history, and was
examined to assess their caries experience. For inclusion in
the study subjects were required to have at least one WSEL.
Exclusion criteria included smoking, evidence of poor oral
health including periodontal disease, recent professional
fluoride therapy (<2 weeks), consumption of fluoridated water
or any medication that could affect oral flora, and pregnancy.
Eight subjects participated in the study, one male and seven
females of 33 years mean age. Each subject had between one
and four WSEL with a total of 22 WSEL examined throughout
the course of this study.
2.2. Preventive program
Each subject was placed on a preventive program designed to
promote remineralization of their white spot lesions. This
consisted of applying 10% CPP-ACP paste (MI paste, GC
International, Itabashi-ku, Tokyo, Japan) to WSEL with custom
585
made trays for 30 min after evening tooth brushing. At the end
of the application time the patient removed the tray but did
not rinse or expectorate thereby leaving the residual paste
around the teeth. The intraoral tray was not worn during
consumption of food or drink or oral hygiene procedures, and
when the tray was removed, it was stored in a sealed moist
plastic bag at room temperature. Subjects were instructed to
rinse and clean their trays using tap water. Each subject
followed this treatment regime for at least 9 months up to 24
months and attended 3 monthly review appointments. No
alterations were made to the subjects diet and oral hygiene
procedures for the duration of the study. All subjects lived in a
city which did not have a fluoridated reticulated water supply
and used non-fluoride-containing toothpaste after breakfast
and before retiring at night. Patients were supplied with MI
Paste at each visit after returning their previously used tube
which was weighed to determine compliance.
2.3. Clinical digital imaging capturing
Intraoral digital photographs were taken at baseline and at each
recall appointment with a digital camera (Nikon D80, Tokyo,
Japan). Since WSEL are usually associated with plaque deposits,
these were removed using a cotton pellet and gently air-dried
for 10 s prior to taking the photographs. The following camera
settings were used: fine image quality; ISO 200, and the white
balance was set to speed light mode. The camera was held
approximately 45 cm from the subject perpendicular to the
buccal surface of the teeth. A commercially available colour
chart (colour and size matching sticker; CasMaTCHTM Bear
Medic, Japan) was imaged with the dentition to allow adjust-
ment of the colour values to a standardized level.15 The digital
images were analyzed qualitatively on a five point scale to
assess the success of the remineralization preventive program.
The five WSEL categories were: 1 original appearance of WSEL;
2 WSEL has reduced in size but was greater than half the
original size; 3 WSEL reduced to approximately half of initial
size; 4 WSEL less than half of original size but remained visible;
5 WSEL no longer visible.
2.4. Intraoral surface pH measurement
The intraoral surface pH of sound enamel or WSEL were
measured directly with the ISFET pH sensor of dimensions
0.8 mm wide by 7 mm long (sensing area; 0.015 mm wide by
0.75 mm long) (experimental manufacture, Horiba Ltd, Kyoto,
Japan) at each visit (Fig. 1ac). The small sensor size allowed
accurate positioning of the probe in the middle of the lesion to
record the pH at that site. These visits were scheduled at least
2 h after eating and at least 1 h after oral hygiene procedures
usually between 9:30 am and 11:30 am. Before measurement,
the pH values of the sensor were calibrated by standard
solutions of pH 4 and pH 7. The ISFET sensor was placed on the
surface of WSEL or sound enamel with a drop of water and the
reference electrode (Fig. 1d) was placed on a wet cotton roll
(10 mm  10 mm  30 mm: F3 Co., Nagoya, Japan) that was
located in the buccal sulcus to complete the electrical circuit
for measuring the pH. The pH value shown on the pH meter (F-
53, Horiba Ltd, Kyoto, Japan) was then recorded to one decimal
place.
586 journal of dentistry 38 (2010) 584590
Fig. 1 pH measurement system consisting of pH meter, signal converter and micro-pH sensor shown in (a). Dimensions of
the ISFET micro-pH sensor and the reference electrode shown in (b) and (c). Micro-pH sensor measuring the pH of a WSEL
shown in (d).
2.5. Saliva sampling, flow rate and buffering capacity
Whole stimulated saliva samples were collected from each
subject at each visit. Subjects were seated and relaxed for
several minutes prior to saliva collection. A 1 g piece of
unflavoured paraffin wax was chewed for 30 s and the saliva
was collected and discarded. The test sample was then
continuously collected into an ice-cooled vial for 5 min to
determine the saliva flow rate. Saliva collection was taken at
least 2 h after meals and at least 1 h after brushing to minimize
effects of the diurnal variability in salivary composition.16
Saliva pH and acidified pH was measured directly using the
hand-held electronic pH meter (checkbuff, Horiba Ltd, Kyoto,
Japan).17 After calibration using the supplied standard solu-
tions of pH 4.0 and 7.0, 0.25 mL of saliva was placed onto the
pH-sensitive electrode to measure the initial pH value within
30 s. Two hundred and fifty microlitres of lactic acid (pH 3.0)
was then titrated into the test saliva and mixed for 30 s using
the manufacturers auto-mixer and the pH recorded to
measure the buffering capacity.17
2.6. Statistical analysis
Differences between the measured parameters and time were
tested using linear mixed effects models.18 For parameters
measured at the tooth level (sound enamel pH, WSEL pH)
multilevel models were used with teeth nested inside
subjects. The relationship between the qualitative assess-
ment of WSEL appearance and the WSEL pH was investigated
using a Spearmans rho non-parametric correlation. Post hoc
comparisons of differences between time points were
performed using the Sidak adjustment for multiple compar-
isons.19 p-Values less than 0.05 were regarded as being
statistically significant. Statistical analyses were performed
using either SPSS (version 17, SPSS Inc, Chicago, IL, USA) or
Stata (version 10; StataCorp, College Station, TX, USA)
statistical software. Since only 4 subjects (7 WSEL) and 2
subjects (3 WSEL) were assessed at 18 and 24 months,
respectively, these time points were excluded from the
statistical analyses.
3. Results
The sound enamel pH values were not statistically different in
the same subject at the various time points and between
subjects. The mean pH of all sound enamel measurements
was 6.83 (95% CI 6.826.84, n = 135). The pH of the WSEL was
significantly different from the surrounding sound enamel at
all time points ( p < 0.001). The differences with treatment
time are shown in Table 1. At the initial visit the mean pH of
the WSEL (5.94  0.17) was significantly lower than at all the
later time points once the preventive program had been
instituted ( p < 0.001). The mean pH at 1 month (6.22  0.18)
was also significantly lower than at 12 months (6.49  0.20,
p = 0.004). Over the course of the preventive program the
difference between the pH of the WSEL and the sound enamel
reduced in magnitude although this was only significant
between baseline and 1 month (Table 1).
The mean flow rate, pH of the stimulated saliva and
acidified saliva throughout treatment is shown in Table 2.
There were differences in the flow rate with time ( p < 0.001).
The flow rate at baseline was significantly different compared
to the later time points ( p < 0.02) except for the 1-month value
( p = 0.06). There were significant differences in the stimulated
saliva pH with time ( p < 0.001). Post hoc comparisons showed
no significant difference between baseline and the 1-month
measurements ( p = 0.09), but the baseline measurements
were significantly lower than all other time points. From 1
month onwards, no statistically significant differences were
found between the pH measurements of the stimulated saliva.
Overall there were differences in the acidified saliva pH
 journal of dentistry 38 (2010) 584590 587

Table 1 Longitudinal pH values (mean W SD) of sound enamel and WSEL before and after the institution of the preventive
program.
Time (months)

0 1 3 6 9 12 18a 24a

Sound enamel 6.80  0.07 a 6.80  0.07 a 6.84  0.05 a 6.85  0.05 a 6.85  0.05 a 6.85  0.05 a 6.84  0.05 6.83  0.05
WSEL 5.94  0.17 6.22  0.18 b 6.38  0.18 bc 6.38  0.20 bc 6.44  0.20 bc 6.49  0.16 c 6.66  0.16 6.70  0.08
Paired differenceb 0.86  0.15 0.58  0.17 d 0.46  0.17 d 0.47  0.19 d 0.41  0.20 d 0.35  0.18 d 0.19  0.13 0.13  0.05

Similarly marked means in the same row are not significantly different ( p > 0.05).
a
18-month data based on 4 subjects (7 WSEL) and 24-month data based on 2 subjects (3 WSEL) this data was not used in the statistical
analysis due to missing data points.
b
All paired differences were significantly different from 0 ( p < 0.001).

Table 2 Longitudinal flow rate and pH values (mean W SD) of stimulated saliva and acidified saliva before and after the
institution of the preventive program.
Time (months)

0 1 3 6 9 12 18a 24a,b

Flow rate 0.66  0.35 a 0.94  0.37 ab 1.04  0.42 b 1.13  0.42 b 1.11  0.47 b 0.97  0.42 b 1.28  0.77 1.00
Stimulated pH 6.93  0.38 c 7.25  0.46 cd 7.35  0.32 d 7.35  0.23 d 7.29  0.25 d 7.50  0.23 d 7.45  0.24 7.45
Acidified pH 5.12  0.76 ef 5.08  0.75 e 5.86  0.66 fg 6.01  0.6 g 5.71  1.00 efg 5.87  0.85 efg 6.18  0.75 5.90

Similarly marked means in the same row are not significantly different ( p > 0.05).
a
18-month data based on 4 subjects and 24-month data based on 2 subjects this data was not used in the statistical analysis due to missing
data points.
b
Mean without standard deviation presented as only two data points.

between time points ( p = 0.001) although statistically there
was no clear trend.
The visual appearance of the WSEL was monitored via
digital photography throughout the course of care. The
response of a typical subject is shown in Fig. 2. At baseline
the WSEL was clearly visible on the mesial surface of tooth 13
yet after nightly application of CPP-ACP paste for 2 years the
appearance of the WSEL had greatly improved with translu-
cency of the enamel being recovered. Generally, the colour of
the WSEL gradually changed from pure white to less white or

Fig. 2 Clinical photograph of WSEL on the mesial of tooth 13 (arrow) and its response to treatment with time.
588 journal of dentistry 38 (2010) 584590
Fig. 3 Frequency of qualitative assessment scores of WSEL
with time following the institution of the preventive
program.
towards the natural tooth colour across the treatment period
studied (Fig. 2). The size and appearance of the WSEL improved
as measured using a 5-point scale during the course of the
preventive program is shown in Fig. 3. All lesions showed
improvement after 1 month with a greater frequency of higher
scores as the treatment period progressed. The weighed tubes
of returned CPP-ACP paste suggested good compliance with
the study protocol. There was a significant correlation
between the qualitative assessment of WSEL scores and the
WSEL pH (rho = 0.63, p < 0.0001).
4. Discussion
Dental caries is a result of pH fluctuations within a biofilm on
the dental hard tissues and hence considerable research has
been conducted on the pH of saliva and plaque fluid. Lesion
fluid, being more difficult to measure, has not been studied as
extensively. Additionally, the surface pH of WSEL and the
sound enamel surrounding it has not been examined. Previous
studies have demonstrated that intraoral pH fluctuates
through the day due to consumption of fermentable carbohy-
drates or acidic foods and beverages.9 Therefore, in this study
pH measurements were not made if the patient had eaten or
undertaken oral hygiene procedures in the last 2 or 1 h
respectively. In this way the recording of the pH was done
under resting conditions and at a similar time of day. This
study found that a pH sensor with ISFET was able to measure
the pH on the surface of sound and demineralized enamel and
monitor their changes with time.
Sound enamel is composed of 9% water by volume termed
the enamel fluid which will be in equilibrium with the plaque/
pellicle fluid overlying it.20 This in turn will be in equilibrium
with the saliva fluid. By placing a solid state electrode in a drop
of water on the surface of the tooth it is hypothesized that the
enamel fluid composition is reflected in the environment
around the micro-pH sensor. It was interesting to find that
sound enamel had a similar pH value between patients and
with time in the same patient. This value was in agreement
with the pH of sound enamel (6.7  0.2) determined by Shida
et al. using the same pH sensor.13 This may be due to the small
fluid volume in sound enamel being unable to influence the
surface pH.
Differences were found between the pH of WSEL and the
sound enamel surrounding it and may reflect differences in
the enamel fluid composition between these two areas. Little
research has been conducted on the pH of enamel fluid
however a number of authors have examined plaque fluid. The
plaque fluid of caries-free individuals was found to have a pH
of 7.02  0.05 and was significantly different to caries-positive
individuals that had a pH of 6.79  0.12.21 One hour after
sucrose rinses the plaque fluid in the caries-positive indivi-
duals fell to 5.88  0.37.21 Other authors have studied plaque
fluid pH and obtained values of 5.73  0.22,22 6.54  0.30,23
5.73  0.7924 and 6.76  0.04.25 The surface pH of sound
enamel and WSEL reported in this study are similar to the
plaque fluid measurements reported by Margolis and Mor-
eno21 and within the range reported by the other authors.2225
Therefore, the surface pH measurements may reflect the pH of
that environment prior to plaque removal and may relate to
the pH of the enamel fluid.
After the preventive program was instituted it was found
that the pH of the WSEL improved towards that of the sound
enamel surrounding it with time. This may reflect the success
of the preventive program prescribed to the patient. The
preventive program was designed to inhibit demineralization
and promote remineralization by using a CPP-ACP containing
product. CPP-ACP has been shown in in vitro,2628 in situ29,30 and
in clinical trials2-5,31 to have anticariogenic properties and to
restore translucency of WSEL. This has been attributed to their
ability to stabilize calcium and phosphate on the tooth surface,
thereby maintaining high activity gradients of calcium and
phosphate ions into the lesion to promote remineralization of
hard tissues. Additionally, CPP-ACP has been shown to
localize in plaque,30 buffer acid32,33 and potentially alter
plaque microbial composition.34,35 The improvement in WSEL
pH may be attributed to improved plaque control allowing
better access of saliva to the lesion or may result from an
accumulation of CPP-ACP in the plaque and WSEL acting to
buffer the pH or remineralization reducing the fluid volume in
the enamel.
The pH values of the stimulated saliva were all under the
upper pH limit of 7.8 described by Edgar et al.36 The pH of the
stimulated saliva marginally improved after institution of the
preventive program and this may again be due to the known
buffering effects of CPP-ACP.32,33 The patients all tended to
exhibit a low resting salivary flow rate. However, an increase
in flow rate was noted after baseline and this again may be
attributed to the regular use of MI paste.
None of the 22 WSEL monitored in this study progressed
to cavitation. Instead, the majority of lesions showed
improvement in appearance when monitored with clinical
photography and qualitative assessment. The results of the
current study are consistent with previous studies on CPP-
ACP25 as it was found that the visual appearance of the
lesions improved throughout the course of care. This may be
explained by the in vitro work of Cochrane et al.28 who found
that high levels of remineralization with CPP-ACP or CPP-
ACFP could return the translucency to enamel and improve
the aesthetics of WSEL. Therefore, the visual improvement
of the monitored WSEL in this study may be attributed to the
 journal of dentistry 38 (2010) 584590
remineralization effect of the CPP-ACP in MI Paste as shown
in previous studies.25
The qualitative assessment of the changes in WSEL
appearance significantly correlated with the change in surface
pH of the WSEL. While the surface pH measurement is not a
measure of mineral change it may indicate that the environ-
ment around the lesion has changed sufficiently so that the
balance of remineralization has been favoured compared to
demineralization. This may indicate that measuring the pH of
the WSEL can be used to assess changes in the lesion activity
with time. This may provide a means of objectively monitoring
the response of lesions and individualizing caries treatment
plans to patients and lesions. Additionally, it may be of use as
an educational or motivational tool. Further clinical study of
this pH sensor appears warranted.
In conclusion, this study found that micro-pH sensor could
be used to measure the pH of WSEL and sound enamel
surrounding it and that its longitudinal measurement corre-
lated with an observed improvement in the WSEL appearance.
The measurement of the surface pH of WSEL may provide an
additional means of assessing WSEL over time.
Acknowledgments
This project was supported by Grant #21592413 from the Japan
Society for the Promotion of Science, and for Global Center of
Excellence Program for International Research Center for
Molecular Science in Tooth and Bone Diseases at Tokyo
Medical and Dental University. Dr. Glenn Walker and Mr.
Masaomi Ikeda are acknowledged for assistance with statisti-
cal analysis.
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