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journal of dentistry 38 (2010) 584590

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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
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
Cooperative Research Centre for Oral Health Sciences, Melbourne Dental School, Bio21 Institute of Molecular Science and Biotechnology,
University of Melbourne, Parkville, Victoria, Australia
Support Program for Improving Graduate School Education at Tokyo Medical and Dental University, Tokyo, Japan
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: (Y. Kitasako).
0300-5712/$ see front matter # 2010 Elsevier Ltd. All rights reserved.
journal of dentistry 38 (2010) 584590 585

if the cariogenic challenge is sufficiently controlled or made trays for 30 min after evening tooth brushing. At the end
therapeutic agents are applied to promote enamel reminer- of the application time the patient removed the tray but did
alization.2 One such agent is MI Paste that contains casein not rinse or expectorate thereby leaving the residual paste
phosphopeptide-stabilized amorphous calcium phosphate around the teeth. The intraoral tray was not worn during
(CPP-ACP) that has been shown in clinical trials to promote consumption of food or drink or oral hygiene procedures, and
the regression of early lesions.25 Means of objectively when the tray was removed, it was stored in a sealed moist
assessing WSEL are critical for determining their potential plastic bag at room temperature. Subjects were instructed to
activity and monitoring the success of preventive treatments. rinse and clean their trays using tap water. Each subject
Current methods for assessing the activity of WSEL include followed this treatment regime for at least 9 months up to 24
visual criteria,6,7 and lactic acid sensitive alginate impression months and attended 3 monthly review appointments. No
materials.8 Simple objective chair-side techniques for assist- alterations were made to the subjects diet and oral hygiene
ing with lesion activity assessment would be an invaluable aid procedures for the duration of the study. All subjects lived in a
for dental care providers for the management of early carious city which did not have a fluoridated reticulated water supply
lesions. Historically, the pH of a range of intraoral fluids have and used non-fluoride-containing toothpaste after breakfast
been measured such as saliva9 and plaque.10 However, few of and before retiring at night. Patients were supplied with MI
these pH measurement techniques have become routinely Paste at each visit after returning their previously used tube
adopted in clinical practice. which was weighed to determine compliance.
One problem with measuring plaque is that often patients
will remove it immediately prior to appointments by thorough 2.3. Clinical digital imaging capturing
brushing. This study sought to use an Ion Sensitive Field Effect
Transistor (ISFET) pH probe1114 to measure the surface pH of Intraoral digital photographs were taken at baseline and at each
WSEL and the sound enamel surrounding it with the prior recall appointment with a digital camera (Nikon D80, Tokyo,
removal of the overlying plaque. This pH probe has been used Japan). Since WSEL are usually associated with plaque deposits,
previously to examine the pH of arrested and active dentine these were removed using a cotton pellet and gently air-dried
caries in vitro and validated against a pH imaging micro- for 10 s prior to taking the photographs. The following camera
scope.14 To the authors knowledge this is the first time that a settings were used: fine image quality; ISO 200, and the white
pH sensor has been used to measure surface pH as a means of balance was set to speed light mode. The camera was held
monitoring WSEL. Therefore, the aim of this study was to approximately 45 cm from the subject perpendicular to the
assess longitudinally the pH of WSEL and the sound enamel buccal surface of the teeth. A commercially available colour
surrounding it following the institution of a preventive chart (colour and size matching sticker; CasMaTCHTM Bear
program to determine whether differences existed and Medic, Japan) was imaged with the dentition to allow adjust-
whether they would correlate to any observed changes in ment of the colour values to a standardized level.15 The digital
lesion appearance. 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. Materials and methods 2 WSEL has reduced in size but was greater than half the
original size; 3 WSEL reduced to approximately half of initial
2.1. Study design and subject recruitment size; 4 WSEL less than half of original size but remained visible;
5 WSEL no longer visible.
Eight healthy subjects were recruited after ethical approval
was obtained from the Ethics Committee of the Tokyo Medical 2.4. Intraoral surface pH measurement
and Dental University and informed consent forms were
signed. Each volunteer completed a medical history, and was The intraoral surface pH of sound enamel or WSEL were
examined to assess their caries experience. For inclusion in measured directly with the ISFET pH sensor of dimensions
the study subjects were required to have at least one WSEL. 0.8 mm wide by 7 mm long (sensing area; 0.015 mm wide by
Exclusion criteria included smoking, evidence of poor oral 0.75 mm long) (experimental manufacture, Horiba Ltd, Kyoto,
health including periodontal disease, recent professional Japan) at each visit (Fig. 1ac). The small sensor size allowed
fluoride therapy (<2 weeks), consumption of fluoridated water accurate positioning of the probe in the middle of the lesion to
or any medication that could affect oral flora, and pregnancy. record the pH at that site. These visits were scheduled at least
Eight subjects participated in the study, one male and seven 2 h after eating and at least 1 h after oral hygiene procedures
females of 33 years mean age. Each subject had between one usually between 9:30 am and 11:30 am. Before measurement,
and four WSEL with a total of 22 WSEL examined throughout the pH values of the sensor were calibrated by standard
the course of this study. 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
2.2. Preventive program reference electrode (Fig. 1d) was placed on a wet cotton roll
(10 mm  10 mm  30 mm: F3 Co., Nagoya, Japan) that was
Each subject was placed on a preventive program designed to located in the buccal sulcus to complete the electrical circuit
promote remineralization of their white spot lesions. This for measuring the pH. The pH value shown on the pH meter (F-
consisted of applying 10% CPP-ACP paste (MI paste, GC 53, Horiba Ltd, Kyoto, Japan) was then recorded to one decimal
International, Itabashi-ku, Tokyo, Japan) to WSEL with custom 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 statistical software. Since only 4 subjects (7 WSEL) and 2
subjects (3 WSEL) were assessed at 18 and 24 months,
Whole stimulated saliva samples were collected from each respectively, these time points were excluded from the
subject at each visit. Subjects were seated and relaxed for statistical analyses.
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 3. Results
continuously collected into an ice-cooled vial for 5 min to
determine the saliva flow rate. Saliva collection was taken at The sound enamel pH values were not statistically different in
least 2 h after meals and at least 1 h after brushing to minimize the same subject at the various time points and between
effects of the diurnal variability in salivary composition.16 subjects. The mean pH of all sound enamel measurements
Saliva pH and acidified pH was measured directly using the was 6.83 (95% CI 6.826.84, n = 135). The pH of the WSEL was
hand-held electronic pH meter (checkbuff, Horiba Ltd, Kyoto, significantly different from the surrounding sound enamel at
Japan).17 After calibration using the supplied standard solu- all time points ( p < 0.001). The differences with treatment
tions of pH 4.0 and 7.0, 0.25 mL of saliva was placed onto the time are shown in Table 1. At the initial visit the mean pH of
pH-sensitive electrode to measure the initial pH value within the WSEL (5.94  0.17) was significantly lower than at all the
30 s. Two hundred and fifty microlitres of lactic acid (pH 3.0) later time points once the preventive program had been
was then titrated into the test saliva and mixed for 30 s using instituted ( p < 0.001). The mean pH at 1 month (6.22  0.18)
the manufacturers auto-mixer and the pH recorded to was also significantly lower than at 12 months (6.49  0.20,
measure the buffering capacity.17 p = 0.004). Over the course of the preventive program the
difference between the pH of the WSEL and the sound enamel
2.6. Statistical analysis reduced in magnitude although this was only significant
between baseline and 1 month (Table 1).
Differences between the measured parameters and time were The mean flow rate, pH of the stimulated saliva and
tested using linear mixed effects models.18 For parameters acidified saliva throughout treatment is shown in Table 2.
measured at the tooth level (sound enamel pH, WSEL pH) There were differences in the flow rate with time ( p < 0.001).
multilevel models were used with teeth nested inside The flow rate at baseline was significantly different compared
subjects. The relationship between the qualitative assess- to the later time points ( p < 0.02) except for the 1-month value
ment of WSEL appearance and the WSEL pH was investigated ( p = 0.06). There were significant differences in the stimulated
using a Spearmans rho non-parametric correlation. Post hoc saliva pH with time ( p < 0.001). Post hoc comparisons showed
comparisons of differences between time points were no significant difference between baseline and the 1-month
performed using the Sidak adjustment for multiple compar- measurements ( p = 0.09), but the baseline measurements
isons.19 p-Values less than 0.05 were regarded as being were significantly lower than all other time points. From 1
statistically significant. Statistical analyses were performed month onwards, no statistically significant differences were
using either SPSS (version 17, SPSS Inc, Chicago, IL, USA) or found between the pH measurements of the stimulated saliva.
Stata (version 10; StataCorp, College Station, TX, USA) 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
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).
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.
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).
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.
Mean without standard deviation presented as only two data points.

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

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
Fig. 3 Frequency of qualitative assessment scores of WSEL enamel and WSEL reported in this study are similar to the
with time following the institution of the preventive plaque fluid measurements reported by Margolis and Mor-
program. 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
towards the natural tooth colour across the treatment period the pH of the enamel fluid.
studied (Fig. 2). The size and appearance of the WSEL improved After the preventive program was instituted it was found
as measured using a 5-point scale during the course of the that the pH of the WSEL improved towards that of the sound
preventive program is shown in Fig. 3. All lesions showed enamel surrounding it with time. This may reflect the success
improvement after 1 month with a greater frequency of higher of the preventive program prescribed to the patient. The
scores as the treatment period progressed. The weighed tubes preventive program was designed to inhibit demineralization
of returned CPP-ACP paste suggested good compliance with and promote remineralization by using a CPP-ACP containing
the study protocol. There was a significant correlation product. CPP-ACP has been shown in in vitro,2628 in situ29,30 and
between the qualitative assessment of WSEL scores and the in clinical trials2-5,31 to have anticariogenic properties and to
WSEL pH (rho = 0.63, p < 0.0001). 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
4. Discussion phosphate ions into the lesion to promote remineralization of
hard tissues. Additionally, CPP-ACP has been shown to
Dental caries is a result of pH fluctuations within a biofilm on localize in plaque,30 buffer acid32,33 and potentially alter
the dental hard tissues and hence considerable research has plaque microbial composition.34,35 The improvement in WSEL
been conducted on the pH of saliva and plaque fluid. Lesion pH may be attributed to improved plaque control allowing
fluid, being more difficult to measure, has not been studied as better access of saliva to the lesion or may result from an
extensively. Additionally, the surface pH of WSEL and the accumulation of CPP-ACP in the plaque and WSEL acting to
sound enamel surrounding it has not been examined. Previous buffer the pH or remineralization reducing the fluid volume in
studies have demonstrated that intraoral pH fluctuates the enamel.
through the day due to consumption of fermentable carbohy- The pH values of the stimulated saliva were all under the
drates or acidic foods and beverages.9 Therefore, in this study upper pH limit of 7.8 described by Edgar et al.36 The pH of the
pH measurements were not made if the patient had eaten or stimulated saliva marginally improved after institution of the
undertaken oral hygiene procedures in the last 2 or 1 h preventive program and this may again be due to the known
respectively. In this way the recording of the pH was done buffering effects of CPP-ACP.32,33 The patients all tended to
under resting conditions and at a similar time of day. This exhibit a low resting salivary flow rate. However, an increase
study found that a pH sensor with ISFET was able to measure in flow rate was noted after baseline and this again may be
the pH on the surface of sound and demineralized enamel and attributed to the regular use of MI paste.
monitor their changes with time. None of the 22 WSEL monitored in this study progressed
Sound enamel is composed of 9% water by volume termed to cavitation. Instead, the majority of lesions showed
the enamel fluid which will be in equilibrium with the plaque/ improvement in appearance when monitored with clinical
pellicle fluid overlying it.20 This in turn will be in equilibrium photography and qualitative assessment. The results of the
with the saliva fluid. By placing a solid state electrode in a drop current study are consistent with previous studies on CPP-
of water on the surface of the tooth it is hypothesized that the ACP25 as it was found that the visual appearance of the
enamel fluid composition is reflected in the environment lesions improved throughout the course of care. This may be
around the micro-pH sensor. It was interesting to find that explained by the in vitro work of Cochrane et al.28 who found
sound enamel had a similar pH value between patients and that high levels of remineralization with CPP-ACP or CPP-
with time in the same patient. This value was in agreement ACFP could return the translucency to enamel and improve
with the pH of sound enamel (6.7  0.2) determined by Shida the aesthetics of WSEL. Therefore, the visual improvement
et al. using the same pH sensor.13 This may be due to the small of the monitored WSEL in this study may be attributed to the
journal of dentistry 38 (2010) 584590 589

remineralization effect of the CPP-ACP in MI Paste as shown 7. Ekstrand KR, Ricketts DNJ, Longbottom C, Pitts NB. Visual
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