Original Paper

Caries Res 2010;44:364371

DOI: 10.1159/000316090

Received: March 10, 2010

Accepted after revision: June 1, 2010
Published online: July 29, 2010

Effect of Slow-Release Fluoride Devices and

Casein Phosphopeptide/Amorphous Calcium
Phosphate Nanocomplexes on Enamel
Remineralization in vitro
A.M.Al-Mullahi a K.J.Toumba b

Oral Health Department, Sultan Qaboos University Hospital, Sultan Qaboos University, Muscat,
Sultanate of Oman; b Paediatric Dentistry, Leeds Dental Institute, University of Leeds, Leeds, UK

Key Words
Casein phosphopeptide/amorphous calcium phosphate
De- and remineralization pH cycling Slow-release
fluoride device

Abstract
To investigate the remineralization effect of slow-release
fluoride glass devices (SFGD), casein phosphopeptide/amorphous calcium phosphate nanocomplexes (CPP-ACP) and
SFGD and CPP-ACP together on enamel pH cycling in vitro.
Eighty bovine enamel slabs were allocated to each of 4
groups (20/group): SFGD; CPP-ACP; SFGD + CPP-ACP; control.
Baseline surface microhardness (SMH; Knoop number) was
measured for all slabs which were then subjected to a pH
cycling regime for 10 days, and SMH was then remeasured.
The pH cycling regime involved immersion 5 times daily
(each for 5 min) in demineralization solution with the slabs
immersed in artificial saliva between dipping. The treatment
for the enamel slabs in the SFGD group involved exposure all
the time to 2 SFGD devices per group during the cycling regime. In contrast, slabs in the CPP-ACP group were exposed
to CPP-ACP slurry once daily for 30 min after the last demineralization challenge of the day. The slabs in the SFGD + CPPACP group received both treatments as the SFGD and CPPACP groups. The control group received no treatment during
the cycling. There was a statistically significant difference in

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enamel SMH change among the groups (one-way ANOVA;

p ! 0.0001). Enamel SMH values in the SFGD, CPP-ACP and
SFGD + CPP-ACP groups were all significantly higher than in
the control group (Bonferroni; p ! 0.0001). There was no significant difference in enamel SMH between the SFGD and
CPP-ACP groups (p 1 0.05). The use of both SFGD + CPP-ACP
showed a non-significant trend for improvement in enamel
SMH in comparison to SFGD or CPP-ACP alone.
Copyright 2010 S. Karger AG, Basel

The process of dental caries is initiated by the bacterial biofilm on the tooth surface which metabolizes fermentable carbohydrates producing acids as a by-product.
These acids will drop the pH in the biofilm leading to
chemical dissolution of the mineralized hard tissue, a
process known as demineralization [Featherstone, 2007].
This process is reversible in its early stages as the biofilm
pH is restored by saliva. The bioavailable calcium, phosphate and fluoride will remineralize the crystalline structure of the tooth, a process known as remineralization
[Selwitz et al., 2007]. This process of mineral loss and gain
occurs throughout the day maintaining a status of equilibrium. However, if this equilibrium is shifted in favour
of further demineralization, then a carious lesion develops [Featherstone, 2004]. The caries lesion can arrest or
reverse if protective factors like calcium, phosphate, fluoDr. Ali Al-Mullahi
Oral Health Department, Sultan Qaboos University Hospital
Sultan Qaboos University, PO Box 50, PC 123
Muscat (Sultanate of Oman)
Tel. +968 2414 7272, Fax +968 2414 7277, E-Mail mullahi@squ.edu.om

ride, pellicle proteins, antibacterial agents, salivary fluoride and substances that stimulate salivary flow predominate over the pathological factors [Featherstone, 2004].
Although many of these protective factors are present in
the oral environment, an extrinsic supply of these factors
like fluoride and bioavailable calcium and phosphate will
favour an equilibrium shift towards enamel remineralization.
The anticariogenicity of dairy products has been investigated by a number of authors [Reynolds and Johnson, 1981; Harper et al., 1986; Silva et al., 1987]. The anticariogenic effect was attributed to the phosphoprotein
casein and calcium phosphate [Harper et al., 1986]. Casein phosphopeptide (CPP) containing the sequence
Ser(P)-Ser(P)-Ser(P)-Glu-Glu has the ability to stabilize
calcium phosphate in solution by binding amorphous
calcium phosphate (ACP) with their multiple phosphoserine residues [Reynolds, 1998]. The CPP is the carrier
molecule for the ACP localizing the highly soluble calcium phosphate phase at the tooth surface. This will elevate
the levels of calcium and phosphate ions within dental
plaque which will enhance remineralization and inhibit
demineralization of enamel [Reynolds et al., 2003]. The
anticariogenicity of the CPP-ACP nanocomplexes has
been demonstrated in animal and in vitro caries models
[Reynolds et al., 1995; Reynolds, 1997]. Further studies
using human in situ caries models have shown that the
CPP-ACP could prevent enamel demineralization and
promote remineralization [Reynolds, 1998; Reynolds et
al., 2003; Iijima et al., 2004].
The slow-release fluoride glass device (SFGD) was developed in Leeds [Toumba and Curzon, 1993]. Initially
the devices used to be 4 mm in diameter and attached
directly to the buccal surface of the upper first permanent
molar using acid etch composite. The new SFGD is in the
form of a disc placed within a circular plastic bracket
which facilitates easy replacement and handling of
the device. The average weight of the new glass beads is
0.035 g, and the weight of the SFGD system (bead and
bracket) is 0.052 g [Al-Ibrahim, 2007]. This is a reduction
of 50% of the weight of the device in comparison to the
device used by Andreadis et al. [2006]. There are a number of clinical studies which demonstrated the ability of
the slow-release fluoride device to maintain a sustained
release of low levels of fluoride intra-orally [Toumba and
Curzon, 2005; Andreadis et al., 2006]. The elevated salivary fluoride concentration will enhance enamel remineralization and inhibit enamel demineralization. Enamel
remineralization with a slow-release fluoride device has
been demonstrated by Cain et al. [1994]. The group reSlow-Release Fluoride Glass Devices and
CPP-ACP Nanocomplexes

ported that the enamel remineralization produced by the

slow-release fluoride device used in their study was comparable to the enamel remineralization produced by 1,100
ppm fluoride toothpaste. The SFGD is a highly effective
intervention with a reported reduction in new caries increments in children after 2 years of 67% fewer new carious teeth and 76% fewer new carious surfaces, and a reduction in occlusal caries increment by 55% was also
demonstrated [Toumba and Curzon, 2005].
The use of CPP-ACP with fluoride and its synergistic
effect on enamel remineralization have been attributed
to the formation of CPP-stabilized amorphous calcium
fluoride phosphate [Reynolds et al., 2008], resulting in the
increased incorporation of fluoride ions into plaque, together with increased concentrations of bioavailable calcium and phosphate ions. This synergistic effect of CPPACP and fluoride in reducing caries was investigated in
animal, in vitro and in situ studies [Reynolds et al., 1995,
2008; Cochrane et al., 2008]. Although a number of studies evaluated the effect of CPP-ACP and fluoride on enamel remineralization [Cochrane et al., 2008; Reynolds et al.,
2008], this was not investigated with sustained and slowrelease fluoride devices. Therefore, the aim of this study
was to investigate the remineralization effect of CPP-ACP
nanocomplexes and slow-release fluoride devices on
enamel after a pH cycling condition in vitro.

Materials and Methods

Preparation of Enamel Slabs
The enamel slabs used in this study were obtained from 20
bovine incisors. The teeth were obtained from animals slaughtered at an age of 30 months and above. The teeth were stored immediately in a solution of distilled water and 0.1% thymol (SigmaAldrich). Prior to their sectioning, the teeth were cleaned using a
spoon excavator and a toothbrush to remove any soft tissue. Teeth
were examined for any defects using transillumination and reflected-light low-power microscopy (Leitz, Wetzlar, Germany).
Each tooth was mounted on a plate using yellow stick impression
compound (Kerr, UK). In order to separate the tooth, a Well Diamond Wire Saw, water-cooled, cutting machine (Well, Walter
Ebner, Le Locle, Switzerland) was used. After separating the buccal surface, the buccal section was cut into 4 slabs, according to
the relevant standard operating procedure (about 4 mm wide, 4
mm long and 2 mm deep). Each slab was used for 1 of the 4 legs,
in order to standardize slabs with the same origin throughout the
entire study. Each of the enamel slabs was embedded in a circular
block of cold curing polymethylmethacrylate resin, and then polished using P600, P1200 and P2000 grit abrasive papers (Wetdry
paper, 3M) under water cooling so that at least 300 m of the outermost enamel was removed to achieve a flat surface. After that
the enamel specimens were polished using 5- and 1-m alumina
paste.

Caries Res 2010;44:364371

365

Baseline Microhardness Measurements

For all the enamel slabs, the baseline surface microhardness
(SMH) was measured using a computer-aided Duramin Indenter Machine (Struers A/S, Denmark). The indentations were
made using a Knoop diamond under a 50-gram load for 15 s
[Zero et al., 1990]. The length of the indentation was measured
in micrometres using computer software that calculated the indentation length (m) and microhardness values (Knoop hardness number, KHN) after identifying the border of the indentation. Five indentations, spaced 50 m apart, were made on different areas of the enamel slabs, and the mean values of the
enamel microhardness were calculated. Then the enamel slabs
in the resin were placed into a special plastic tray with 10 circular windows. The resin blocks were secured in position using
modelling wax. Each of the groups received 2 plastic trays with
20 enamel slabs.
Experimental and Control Groups
Group 1: SFGD. An SFGD (13.3% F; Ultradent Products Inc.,
USA) was attached to the middle of the plastic tray. The device
was fixed on the surface of the tray using yellow stick impression
compound (Kerr). The two plastic trays were placed together
during the pH cycling regime.
Group 2: CPP-ACP. The CPP-ACP used in the study was in the
form of a slurry. The slurry was prepared by mixing 1 g of CPPACP topical crme (Tooth MousseTM, GC Corp., Tokyo, Japan)
with 4 ml of distilled water [Reynolds et al., 2008] using a Whirlimixer (Fisons) for 1 min. The solution was prepared daily just
prior to application. The application of the CPP-ACP slurry was
performed once daily for 30 min after the last demineralization
challenge of the day.
Group 3: SFGD and CPP-ACP. The enamel slabs in this group
received both of the treatments (slow-release fluoride and CPPACP) as described earlier for groups 1 and 2.
Group 4: Control. The enamel slabs in this group were exposed
to the pH cycling regime without any treatment.
pH Cycling Regime
The enamel specimens were dipped in 50 ml of the demineralization solution (1.5 mM CaCl2, 0.9 mM KH2PO4, 50 mM acetic
acid, pH 4.8) [ten Cate et al., 2006] for 5 min. After the demineralization challenge, the enamel specimens were rinsed in distilled
water for 1 min, then placed in 50 ml of artificial saliva [daytime;
calcium carbonate 0.07 g/l, magnesium carbonate 0.019 g/l, potassium dihydrogen phosphate 0.554 g/l, HEPES buffer (acid
form) 4.77 g/l, potassium chloride 2.24 g/l, pH 6.8] for 90 min.
This process was repeated until the enamel specimens had been
subjected to 5 demineralization challenges. After the last demineralization challenge, the enamel specimens were rinsed in distilled water for 1 min, then immersed in 50 ml of artificial saliva
[nighttime; calcium carbonate 0.05 g/l, magnesium carbonate
0.019 g/l, potassium dihydrogen phosphate 0.068 g/l, HEPES
buffer (acid form) 4.77 g/l, potassium chloride 2.24 g/l, pH 6.8].
The demineralization solution and the artificial saliva (daytime
and nighttime) were changed daily. Enamel slabs in group 1
(SFGD) were exposed to 2 slow-release fluoride devices throughout the cycling regime and group 2 (CPP-ACP) received CPP-ACP
slurry once daily for 30 min after the last demineralization challenge of the day as described earlier (fig.1).

366

Caries Res 2010;44:364371

Group 3 (SFGD + CPP-ACP) received both treatments of

groups 1 and 2, whereas group 4 (control) received no intervention throughout the cycling regime. The enamel slabs were kept
in an incubator at 37 C at all times except when being immersed
in the demineralization solution. All the groups were subjected to
the pH cycling regime for a 10-day period. At the end of the 10
days of cycling, all the enamel specimens were rinsed with distilled water and then removed from the special plastic trays and
stored in tightly sealed plastic containers with wet gauze until
analysed.

Postcycling SMH
All the enamel specimens (n = 80) were measured for SMH using a computer-aided Duramin Indenter Machine as described
previously.
Blinding and Reproducibility
All of the enamel specimens (n = 80) had codes to identify the
slabs and groups which they belonged to [e.g. M15-M is group 1
(SFGD) and 15 is the number of the slab]. Following pH cycling,
all the codes for the slabs were changed with random numbers
(from a table with random numbers) to prevent the examiner
from knowing the origin of the slab. After the pH cycling regime,
15% of the enamel specimens were randomly remeasured to assess
the intra-examiner agreement.
Statistical Analyses
The Bland-Altman plot was used to assess the intra-examiner
agreement. The agreement is considered to be good on average if
the bias (mean of difference) is close to zero. The data were analysed using the SPSS statistical software package for Windows,
version 15.0 (SPSS Inc., 19892006; Lead Technologies Inc., 1991
2000). Descriptive statistics were used to calculate the range,
mean, median and standard deviation. The normality of the data
distribution was assessed using box-and-whisker plots. One-way
ANOVA was used to assess if there was any significant difference
in enamel microhardness between the 4 groups of the study. Furthermore, the Bonferroni test was used to assess if there was any
significant difference between each of the groups. The test also
calculated the 95% confidence intervals (CI). The significance
was set at a level of p ! 0.05.

Results

The means and standard deviations for the difference

in SMH (baseline SMH postcycling SMH) for the study
groups are presented in table 1. The box-and-whisker
plots for the data of all the groups in the study appear to
be normally distributed as the mean values for difference
in enamel microhardness are very close to the median
values (fig.2).
One-way ANOVA analysis showed a statistically significant difference in the mean difference of enamel SMH
between the groups (p ! 0.0001). Bonferroni analysis
(table2) showed that the enamel SMH of the SFGD, CPPAl-Mullahi /Toumba

80 enamel slabs

Baseline SMH for the slabs

Enamel slabs allocated

to 4 groups (20/group)

pH cycling
Conditioning regime
5 demineralization challenges (each for 5 min)/day
Stored in artificial saliva between dipping
Enamel slabs were kept in an incubator at 37C
10-day period
Treatment during cycling
SFGD group: slabs were exposed to 2 slow-release fluoride devices
during the cycling regime
CPP-ACP group: exposed to CPP-ACP slurry once daily for 30 min
after the last demineralization challenge of the day
SFGD and CPP-ACP group: both treatments as for SFGD and CPP-ACP groups
Control: no treatment during the cycling regime

Postcycling
SMH for all the slabs

Fig. 1. Flow chart illustrating the study

protocol.

Difference in mean SMH (read 1 read 2)

250

Difference in SMH (KHN)

200
39
150

100

50

0
SFGD

CPP-ACP

SFGD +
CPP-ACP
Group

Control

20

20

40
200

250

300

350

Mean SMH (KHN)

Fig. 2. Box-and-whisker plot of the mean difference in enamel

microhardness for each group.

Fig. 3. Bland-Altman plot to assess intra-examiner agreement.

Slow-Release Fluoride Glass Devices and

CPP-ACP Nanocomplexes

Caries Res 2010;44:364371

367

Table 1. Difference in SMH for the 4

groups in the study

Diff. SMH

Mean

Standard deviation

SFGD
CPP-ACP
SFGD + CPP-ACP
Control

20
20
20
20

75.1
71.4
55.2
171.2

38.5
34.5
21.2
31.2

Diff. SMH = Difference in SMH (baseline SMH postcycling SMH).

Table 2. Bonferroni test for the 4 groups in the study

I group

J group

Mean difference
(I J)

Standard
error

Significance

95% CI
upper limit

lower limit

SFGD

CPP-ACP
SFGD + CPP-ACP
Control

3.7
19.9
96.1*

10.1
10.1
10.1

1.000
0.316
0.000

23.7
7.5
123.5

31.1
47.3
68.7

CPP-ACP

SFGD
SFGD + CPP-ACP
Control

3.7
16.2
99.8*

10.1
10.1
10.1

1.000
0.677
0.000

31.1
11.2
127.1

23.7
43.6
72.4

SFGD + CPP-ACP

SFGD
CPP-ACP
Control

19.9
16.2
116.0*

10.1
10.1
10.1

0.316
0.677
0.000

47.3
43.6
143.3

7.5
11.2
88.6

Control

SFGD
CPP-ACP
SFGD + CPP-ACP

96.1*
99.8*
116.0*

10.1
10.1
10.1

0.000
0.000
0.000

68.7
72.4
88.6

123.5
127.1
143.4

* p ! 0.05: the mean difference is significant.

ACP and SFGD + CPP-ACP groups were all significantly

harder in comparison to the control group (p ! 0.0001).
However, there was no significant difference between the
SFGD group and the CPP-ACP group (p 1 0.05, 95% CI
23.7 to 31.1). The use of both SFGD + CPP-ACP showed
a trend for further improvements in mean SMH in comparison to SFGD or CPP-ACP alone. However, this difference was not significant (p 1 0.05); the 95% CI for the
difference in enamel SMH between the SFGD and SFGD
+ CPP-ACP groups was 7.5 to 47.3, the 95% CI for the
CPP-ACP and SFGD + CPP-ACP groups was 11.2 to
43.6. The interpretation of the Bland-Altman plot (fig.3)
indicates a fairly acceptable level of agreement with a reported bias for the pair of measurements (read 1 read 2)
of 3.4.

368

Caries Res 2010;44:364371

Discussion

The enamel used in this study was of bovine origin as

human enamel was difficult to obtain. The use of bovine
enamel has been verified in a number of in vitro studies
to evaluate the effect of anticariogenic agents on enhancing enamel remineralization and inhibiting enamel demineralization [Brighenti et al., 2006; Lennon et al., 2006;
ten Cate et al., 2006]. There are some reported variations
in the characteristics of bovine enamel in comparison to
human enamel; such as it provides a less variable response
to both the cariogenic challenge and the anticariogenic
agents, the composition of bovine enamel is less variable
than human enamel [Mellberg, 1992]. In addition, human and bovine enamel have shown similar characteristics in subsurface caries lesion formation [Edmunds et al.,
Al-Mullahi /Toumba

1988]; lesion progression, as measured by mineral loss,

was in the ratio of 2.0:1.0 for bovine:human permanent
enamel, and measured by lesion depth it was 1.7: 1.0
[Amaechi et al., 1999]. The high rate of lesion progression
and lesion depth in bovine enamel indicates softer enamel in comparison to human enamel. These variations
make the bovine enamel response to treatments in the
experimental groups relatively more consistent [Mellberg, 1992] than with human enamel. Hence, this should
be taken into consideration when interpreting any demineralization and remineralization study using bovine
enamel.
Few published studies looked into the remineralization potential of the slow-release fluoride device [Corpron et al., 1986; Cain et al., 1994]. Cain et al. [1994] using
an in situ design study have evaluated the effect of slowrelease fluoride (copolymer membrane device) on enamel remineralization using transverse microradiography
analysis. The reported level of enamel remineralization
in the group using the device (0.232 mg F/day) was higher (30%) than the recorded level (13.7%) in the group using sodium fluoride toothpaste (1,100 ppm F). The group
with the slow-release fluoride device (0.07 mg F/day)
reported a level of remineralization comparable to the
sodium fluoride toothpaste group. The results from the
present study were in agreement with the findings of
Cain et al. [1994] and other studies which assessed the
remineralization potential of slow-release fluoride devices [Corpron et al., 1986; Cain et al., 1994]. After pH cycling, the enamel was significantly harder (p ! 0.001) in
the group which received the slow-release fluoride device
(mean difference in enamel SMH was 75 KHN) in comparison to the control group (171 KHN).
Microhardness indentation measurements can provide indirect evidence of mineral loss or gain [Featherstone et al., 1983; Arends and ten Bosch, 1992]. The drawback with the technique (SMH) used in the study is that
it cannot quantify the amount of mineral loss or gain;
however, it can provide qualitative evidence on mineral
changes within the enamel. The findings from this study
have shown that using the slow-release fluoride device
was effective in increasing enamel SMH after pH cycling
and hence inhibiting demineralization and promoting
remineralization of enamel.
CPP-ACP has been used in vitro, in situ and in vivo
with different products such as mouthwashes [Reynolds
et al., 2003], lozenges [Cai et al., 2003], slurries [Reynolds
et al., 2008], pastes [Tantbirojn et al., 2008] and chewing
gums [Shen et al., 2001; Manton et al., 2008] to evaluate
its remineralization potential. The use of CPP-ACP in

this study was in the form of a slurry (1 g of CPP-ACP

topical crme, Tooth Mousse, GC Corp., with 4 ml of distilled water) [Reynolds et al., 2008] applied once daily for
30 min. The CPP localizes the highly soluble calcium
phosphate phase (ACP) at the tooth surface, which will
elevate the levels of calcium and phosphate ions within
dental plaque [Reynolds et al., 2003]. Thus, the presence
of plaque is crucial for the action of the CPP-ACP molecule, as it will act like a reservoir for a high concentration
of calcium and phosphate ions resulting in a high degree
of saturation with respect to enamel mineral in plaque
fluid which will enhance remineralization and inhibit
demineralization of enamel [Reynolds, 1999]. Based on
this we felt that applying CPP-ACP paste for 1 min in this
in vitro designed study would not be sufficient to express
the effect of this intervention, taking into consideration
that in the two in vitro studies conducted by the Reynolds
group [Reynolds, 1997; Cochrane et al., 2008] a CPP-ACP
solution was used to remineralize the artificial carious
lesion by suspending them in the solution for a 10-day
period. Hence we decided to use the CPP-ACP as a slurry
for 30 min rather than direct application of the paste to
the tooth surface for 1 min. Following pH cycling in the
CPP-ACP group, the enamel was significantly harder in
comparison to the control group. The reported mean difference of enamel SMH was 71 KHN in comparison to the
control group, which had a mean difference of 171 KHN
indicating that CPP-ACP had increased the enamel hardness in comparison to the control group. These findings
are in agreement with a number of studies, which evaluated the remineralization potential of CPP-ACP [Shen et
al., 2001; Cai et al., 2003; Reynolds et al., 2003; Iijima et
al., 2004; Cochrane et al., 2008; Reynolds et al., 2008;
Tantbirojn et al., 2008].
The use of CPP-ACP with fluoride and its effect on
enamel remineralization have been investigated in animal, in vitro and in situ studies [Reynolds et al., 1995,
2008; Cochrane et al., 2008]. In the rat caries model, the
use of CPP-ACP and fluoride had significantly reduced
caries activity in comparison to the rats that either received CPP-ACP or fluoride alone [Reynolds et al., 1995].
This synergistic activity has been demonstrated in laboratory-based studies designed to investigate the remineralization of enamel subsurface lesions using CPP-ACP
and fluoride [Cochrane et al., 2008]. Reynolds et al. [2008]
demonstrated in their in situ study a significant increase
in enamel remineralization in the group that received 2%
CPP-ACP and fluoride toothpaste (1,100 ppm) in comparison to the group that received either 2% CPP-ACP or
the fluoride toothpaste (1,100 ppm). However, there are

Slow-Release Fluoride Glass Devices and

CPP-ACP Nanocomplexes

Caries Res 2010;44:364371

369

other studies, which failed to show the synergistic activity of CPP-ACP and fluoride. The in vitro study by Kumar et al. [2008] showed a 13% reduction in lesion depth
in the group which received the CPP-ACP and fluoride
tooth paste (1,100 ppm) treatment in comparison with
the 10% reduction in lesion depth in the group receiving
CPP-ACP alone or the 7% reduction in the group treated
with fluoride toothpaste alone. This study did not explore
if the reported synergistic effect of CPP-ACP and fluoride
in reducing lesion depth was statistically significant in
comparison to the reduction in lesion depth produced
by using either CPP-ACP or fluoride toothpaste alone.
Tantbirojn et al. [2008] did not find any difference in the
change in surface hardness of softened enamel between
the group exposed to CPP-ACP treatment and fluoridefree artificial saliva and the group which received CPPACP treatment and fluoride-containing (1 ppm F) artificial saliva. However, the cycling condition was carried
out for a relatively short period (48 h). The results from
this study showed a significant difference in the reduction (p ! 0.001) in enamel hardness in the group that received the CPP-ACP and the fluoride device (55 KHN) in
comparison with the control group (171 KHN). The combined use of slow-release fluoride devices and CPP-ACP
showed a trend towards further improvement in enamel
hardness in comparison to the use of either SFGD (75
KHN) or CPP-ACP (71 KHN) alone; however, this failed
to reach significance. There are a number of possible

causes for this finding, which includes having a larger

sample size in order to demonstrate this effect. Another
possible explanation could be the interaction between
calcium and the slow-release fluoride device and its subsequent effect on fluoride diffusion, phenomena reported
by different groups [Whitford et al., 1986; Adair et al.,
1993, 1994]. The authors hypothesized that the observed
reduction in fluoride release from the slow-release devices was due to precipitation of calcium fluoride and possibly other fluoride salts on the surface of the device impeding fluoride diffusion. However, this result was reported in the copolymer device and this effect was not
investigated in the slow-release fluoride device. The presence of dental plaque is crucial for the synergistic effect
of CPP-ACP with fluoride. The formation of CPP-stabilized amorphous calcium fluoride phosphate [Reynolds
et al., 2008], results in an increased incorporation of fluoride ions into plaque, together with increased concentrations of bioavailable calcium and phosphate ions. The environment of the present in vitro study has compromised
the effect of the CPP-ACP with fluoride due to the lack of
dental plaque, which has resulted in lower levels of enamel remineralization than anticipated.
In conclusion, SFGD and CPP-ACP nanocomplexes
were effective in increasing enamel hardness following
pH cycling. The combined use of SFGD + CPP-ACP
showed a trend for improvement in enamel hardness in
comparison to using either of these interventions alone.

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