Original Paper
Caries Res 2010;44:33–40
DOI: 10.1159/000275572

Received: April 14, 2009
Accepted after revision: November 9, 2009
Published online: January 15, 2010

Casein Phosphopeptide-Amorphous Calcium
Phosphate Incorporated into Sugar Confections
Inhibits the Progression of Enamel Subsurface
Lesions in situ
G.D. Walker F. Cai P. Shen G.G. Adams C. Reynolds E.C. Reynolds
Cooperative Research Centre for Oral Health Science, Melbourne Dental School, Bio21 Molecular Science andBiotechnology Institute, University of Melbourne, Melbourne, Vic., Australia

Key Words
Casein phosphopeptide-amorphous calcium phosphate Enamel demineralization, prevention  Recaldent 
Sugared confection

Abstract
Casein phosphopeptide-amorphous calcium phosphate(CPP-ACP) has been demonstrated to exhibit anticariogenic
activity in randomized, controlled clinical trials of sugar-free
gum and a tooth cream. Two randomized, double-blind,crossover studies were conducted to investigate the potential of CPP-ACP added to hard candy confections to slow the
progression of enamel subsurface lesions in an in situ model.
The confections studied were: (1) control sugar (65% sucrose+ 33% glucose syrup); (2) control sugar-free; (3) sugar + 0.5%(w/w) CPP-ACP; (4) sugar + 1.0% (w/w) CPP-ACP; (5) sugarfree + 0.5% (w/w) CPP-ACP. Participants (10 and 14 in study 1
and 2) wore a removable palatal appliance containing enamel half-slabs with subsurface lesions, except for meals andoral hygiene procedures, and consumed 1 confection 6times a day for 10 days. The enamel half-slabs were inset toallow the development of plaque on the enamel surface.
Participants rested for 1 week before crossing over to another confection. The appliances were stored in a humid
container at 37 ° C when not in the mouth. After each treat-© 2010 S. Karger AG, Basel
0008–6568/10/0441–0033$26.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com

Accessible online at:www.karger.com/cre

ment period, the enamel half-slabs were removed, pairedwith their demineralized control half-slabs, embedded, sectioned and then analysed using transverse microradiography. In both studies consumption of the control sugar confection resulted in significant demineralization (progression) of the enamel subsurface lesions. However, consumption of the sugar confections containing CPP-ACP did not
result in lesion progression, but in fact in significant remineralization (regression) of the lesions. Remineralization by
consumption of the sugar + 1.0% CPP-ACP confection was
significantly greater than that obtained with the sugar-freeconfection.
Copyright © 2010 S. Karger AG, Basel

Casein phosphopeptides (CPP), containing the cluster
sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, have a remarkable ability to stabilize amorphous calcium phosphate(ACP) in metastable solution [Cross et al., 2004, 2005].
Through the multiple phosphoseryl residues, the CPPbind to embryonic clusters of ACP preventing their
growth to the critical size required for nucleation andphase transformation; hence, the calcium and phosphateions of the ACP remain highly soluble and bio-available[Reynolds, 1998]. CPP-ACP complexes display anticariogenic activity in vitro, in animal models and in humanProf. Eric C. Reynolds
Melbourne Dental School, University of Melbourne
720 Swanston Street
Melbourne, Vic. 3010 (Australia)
Tel. +61 3 9341 1547, Fax +61 3 9341 1596, E-Mail e.reynolds @ unimelb.edu.auin situ studies [Reynolds et al., 1995; Iijima et al., 2004;
Oshiro et al., 2007; Rahiotis and Vougiouklakis, 2007;
Cochrane et al., 2008].
The CPP-ACP complexes have been shown to reduce
caries activity in specific-pathogen-free rats orally infected with Streptococcus sobrinus when CPP-ACP was applied twice daily in solution to the animals’ molar teeth[Reynolds et al., 1995]. CPP-ACP has also been reported
to significantly prevent enamel and dentine demineralization and promote remineralization in vitro [Reynolds,
1997; Oshiro et al., 2007; Rahiotis and Vougiouklakis,
2007]. Furthermore CPP-ACP added to various products
such as sugar-free chewing gum, mouthwashes, milk andsugar-free confections significantly enhanced remineralization of enamel subsurface lesions in situ [Shen et al.,
2001; Cai et al., 2003; Reynolds et al., 2003; Iijima et al.,
2004; Walker et al., 2006; Cai et al., 2007]. Enamel subsurface lesions remineralized in situ by chewing sugarfree gum containing CPP-ACP were more acid resistant
than normal tooth enamel [Iijima et al., 2004]. Significant
remineralization of enamel subsurface lesions has also
been observed in a human in situ study in which subjects
consumed sugar-free pressed mint confections containing CPP-ACP 4 times a day for 14 days [Cai et al., 2003].
Recently, a randomized controlled clinical trial (RCT)
demonstrated that chewing sugar-free gum containing
CPP-ACP, relative to a normal sugar-free gum, reduced
progression and enhanced regression of approximal caries in a population of schoolchildren in Australia [Morgan et al., 2008]. Other clinical RCTs have recently shownthat CPP-ACP can regress white-spot lesions in postorthodontic populations [Andersson et al., 2007; Bailey
et al., 2009]. As CPP-ACP is an approved food ingredient,
it may have the potential to be added to sugar confections
to help lower their cariogenicity. The aim of this study
was to investigate the effect of incorporating CPP-ACP
into prototype sugar hard candy (boiled confections) on
the progression and/or regression of enamel subsurface
lesions in a clinical RCT utilizing an in situ model.
Materials and Methods
Study Design and Subject Recruitment
Two double-blind, randomized, crossover in situ studies wereconducted to assess the capacity of CPP-ACP in sugar-containing
hard candy (boiled confections) to prevent the progression ofenamel subsurface lesions in situ. Approval for the studies was
obtained from the University of Melbourne Human Research
Ethics Committee. Healthy adult participants were recruitedfrom staff and postgraduate students of the Melbourne Dental
School, University of Melbourne. Ten participants (5 males and 5

34

Caries Res 2010;44:33–40

females) aged 23–47 years were recruited for study No. 1, and 14
participants (8 males and 6 females) aged 21–47 years were recruited for study No. 2. Many of the participants were involved inboth studies. The number of participants (10–14) required for thestudies was based on our previous studies using the same in situ
model and crossover design [Reynolds et al., 2008; Cai et al.,2009]. All participants had at least 22 natural teeth with no active
caries, periodontal disease or other oral pathology, and none ofthem were using antibiotics or medications known to affect thesalivary flow rate. Unstimulated saliva was collected for 2 minfrom subjects at rest whilst leaning forward with their heads tilted downwards, allowing saliva to flow into a preweighed centrifuge tube. Stimulated saliva was similarly collected for 2 min fromsubjects whilst they consumed each confection. All subjects had
unstimulated and stimulated salivary flow rates in excess of 0.2
and 1.0 ml/min, respectively.
Intra-Oral Appliances
Removable mid-palatal acrylic appliances covering the palate
from the first premolar to the last tooth in the arch were produced
for each subject as described previously [Iijima et al., 2004]. Thebase of the appliance was retained in the mouth by 4 narrowgauge stainless-steel circumferential clasps. The appliances were
designed with bilateral troughs (15 mm long, 7 mm wide, 3 mmdeep) cut into the base and designed to house the enamel slabs.
The enamel slabs were retained by sticky wax as described by
Iijima et al. [2004] to produce a 1-mm-deep trough above theenamel surface to allow plaque to establish and be retained.
Preparation of Enamel Subsurface Lesions
Subsurface enamel lesions were created using the techniqueemployed by Iijima et al. [2004]. Sound relatively planar buccal
and lingual surfaces free of cracks, stains and fluorotic lesions (asviewed under a dissecting microscope) were selected from extracted human third molars obtained from the Royal Dental Hospital of Melbourne and previously treated for at least 2 weeks in
10% (w/v) buffered formal-saline. The teeth were thrice rinsed
with Milli-Q water, and the outer enamel surface was removed
and polished wet to a mirror finish using Soflex (3M) discs on aslow-speed contra-angle dental handpiece. Each polished surface
was then sawn from the tooth as a slab measuring approximately
8 ! 4 mm, using a water-cooled diamond blade saw. The wholeslab was then covered with acid-resistant nail varnish except for
2 (occlusal and gingival) mesiodistal windows as described by
Iijima et al. [2004], each measuring 1 ! 7 mm, separated fromeach other by 1 mm. Subsurface demineralized lesions were created in the enamel windows by immersing each slab in 40 ml unagitated demineralization buffer containing 20 g/l Carbopol 907(carboxypolymethylene; BF Goodrich, Cleveland, Ohio, USA)
[White, 1987], 500 mg/l hydroxyapatite (Bio-Gel HTP, Bio-Rad
Laboratories, Richmond, Calif., USA), 0.1 mol/l lactic acid (Ajax
Chemicals, Auburn, N.S.W., Australia), pH 4.8, for 4 days at 37 ° C.
After 2 days, the slabs were removed from the buffer, rinsed thricewith Milli-Q water, blotted dry and placed into fresh demineralization buffer for another 2 days, then again rinsed and dried.
After demineralization, each enamel slab was sawn through the
midline of each window into two 4 ! 4 mm half-slabs, and thecut surface of each half-slab was covered with nail varnish. One
half-slab of each pair was retained as the demineralization control
and stored in a labelled 1.7-ml microcentrifuge tube together with

Walker/Cai/Shen/Adams/Reynolds/
Reynolds

a drop of Milli-Q water, to create a humidified environment. The
other enamel half-slab of the pair was inset into an intra-oral appliance as described above with the subsurface enamel lesions exposed but recessed 1 mm below the surface of the appliance tocreate a plaque trap. Care was taken to keep the windows free ofwax. Each two half-slab pairs were randomly assigned to either
control or treatment. Four enamel half-slabs were inset into eachappliance, two on each side in the bilateral troughs [Iijima et al.,2004].
Study Products and Randomization
For the two in situ studies, boiled confections (hard candy;
3.8 g each) were prepared by the Clinical and Consumer Group
Cadbury-Adams Worldwide Research and Development (N.J.,
USA), some containing CPP-ACP (Recaldent CASRN 691364-495) obtained from Recaldent Pty Ltd. (Melbourne, Australia). For
study 1, the 4 products tested were: (1) control sugar, (2) sugar +0.5% w/w CPP-ACP, (3) sugar + 1.0% w/w CPP-ACP and (4) control sugar-free. For study 2, the 4 products tested were: (1) control
sugar, (2) sugar-free + 0.5% w/w CPP-ACP, (3) sugar + 1.0% CPPACP and (4) control sugar-free. The confections were identical inappearance and taste and were provided as coded products insealed packages and stored at room temperature. The sugar confections were 65% sucrose, 33% glucose syrup (corn syrup) andthe remainder colouring and flavouring. The sugar-free confections were 95% w/w isomalt (palatinit) with the remainder colouring, flavouring and intense sweeteners (acesulfame K and aspartame).
For each study, the treatment sequences were generated by theStatistics and Analytical Sciences group of Cadbury-Adams
Worldwide Research and Development and assigned to subject
codes (fig. 1). Participants were then randomly assigned to subject
codes by the study team (F.C. and P.S.). For each clinical study, theconfection allocation was only divulged by the study sponsor after all data for that study had been acquired, data queries resolved
and the database locked.
Study Protocols
Both studies were conducted at the Royal Dental Hospital of
Melbourne, Melbourne, Australia, with subjects recruited andboth studies completed within a 12-month period. Participants
wore the appliances except during meals and oral hygiene procedures, and consumed 1 of the confections 6 times a day at 10.00
a.m., 11.00 a.m., 12.00 p.m., 2.00 p.m., 3.00 p.m. and 4.00 p.m., byallowing it to dissolve in the mouth, for 10 consecutive days (treatment period). The average time for the confection to dissolve wasapproximately 10 min. Following each treatment period, participants rested for 1 week (washout period), then crossed over toanother confection. This was repeated until all 4 confection products for each study had been consumed by each participant. Whenthe appliance was removed from the mouth, it was briefly rinsed
with deionized water, kept in a sealed moist plastic bag and stored
at 37 ° C. When wearing the appliance, participants were instructed not to eat anything except the study confections and not todrink anything except water. Participants were instructed to rinseand clean their appliances using a fluoride-free denture cleanser
paste and toothbrush provided by the sponsor. They were also
informed not to brush the area containing the enamel blocks. Participants kept a diary of confection consumption, brushing times
and times the appliance was out of the mouth. At the beginning

CPP-ACP Prevents Demineralizationin situRandomized
n = 10Randomized
n = 14

A: n = 3
B: n = 3
C: n = 2
D: n = 2

A: n = 3
C: n = 4
D: n = 3
E: n = 4

AB: n = 3
BC: n = 3
CD: n = 2
DA: n = 2

ABC: n = 3
BCD: n = 3
CDA: n = 2
DAB: n = 2

ABCD: n = 3
BCDA: n = 3
CDAB: n = 2
DABC: n = 2

Period 1

Period 2

Period 3

Period 4

AC: n = 1
AD: n = 2
CE: n = 4
DA: n = 1
DC: n = 2
EA: n = 2
ED: n = 2

ACD: n = 1
ADE: n = 2
CEA: n = 2
CED: n = 2
DAE: n = 1
DCA: n = 2
EAC: n = 2
EDC: n = 2

ACDE: n = 1
ADEC: n = 2
CEAD: n = 2
CEDA: n = 2
DAEC: n = 1
DCAE: n = 2
EACD: n = 2
EDCA: n = 2

Analysed
A: n = 10
B: n = 10
C: n = 10
D: n = 10

Analysed
A: n = 14
C: n = 14
D: n = 14
E: n = 14

Study 1

Study 2

Fig. 1. Participant disposition for the two clinical studies. A =Sugar-free control; B = sugar + 0.5% CPP-ACP; C = sugar + 1.0%
CPP-ACP; D = sugar control; E = sugar-free + 0.5% CPP-ACP.

Caries Res 2010;44:33–40

35and end of each day of each treatment period, participants brushedtheir teeth with a standard fluoride dentifrice (Colgate Cavity
Protection) containing 1,000 ␮g/g F supplied by the sponsor andotherwise maintained their normal oral hygiene practices and dietary practices except for the restrictions during appliance wear
times. After completion of each treatment period, the enamelslabs were visually inspected for plaque coverage and then removed from the appliances, rinsed with Milli-Q water and stored
in moist, labelled microcentrifuge tubes.
Sectioning, Microradiography and Microdensitometric Image
Analysis
After each treatment, the enamel half-slabs were paired with
their respective control half-slabs and then dehydrated in absolute alcohol. Each pair of half-slabs was embedded, sectioned,
lapped to 80 8 5 ␮m and subjected to microradiography andcomputer-assisted densitometric analysis [Iijima et al., 2004].
Each section was radiographed along with an aluminium step
wedge of increments 10 ! 14 ␮m thick using Microchrome highresolution glass plates (type 1A; Microchrome, USA) and nickelfiltered copper K␣ radiation of 20 kV, 10 mA for 5 min. The radiographic apparatus used has been described previously by Malcolm [1972]. Radiographic images of the lesions were viewed
using a Dialux 20 microscope (Leitz, Wetzlar, Germany). The images were acquired by a digital camera (Insight) using imaging
software (Optimate version 5.2). Images of the lesions and theneighbouring areas of sound enamel were scanned using the programme’s Ln line and Ln depth functions that give readings ingrey values between 0 and 256 and the length of the scan, respectively. Each lesion was scanned 6 times through an area free ofartefacts or cracks with each scan comprising 200 readings takenfrom the tooth surface through the lesion to sound enamel. An
aluminium step wedge image on each slide was scanned and theaveraged step grey value readings were plotted against aluminium
thickness. The readings of the tooth section image within the linear portion of the step wedge curve and linear regression wereused to convert the grey value data into values of equivalent thickness of aluminium. The section thickness was measured using a
micrometre to 85 ␮m and the mineral content (vol%) computed
using the equation of Angmar et al. [1963] and the linear absorption coefficients of aluminium, organic matter plus water and apatitic mineral (131.5, 11.3 and 260.5, respectively). The image ofthe median strip of sound enamel between the two lesions wasscanned 6 times and averaged to give a control densitometric profile of sound enamel. The lesion images (treated windows anddemineralization control windows) to the gingival and occlusal
side of the median strip of sound enamel were similarly scanned,
as closely as possible to the median strip avoiding any irregularities commonly found at the lesion edges, and mineral content/
depth profiles were computed.
Outcomes and Data Analysis
The mineral content/depth profile of each treated and control
demineralized lesion was compared with the same profile of themedian sound enamel in the same section. The difference between the areas under the densitometric profile of the demineralized control lesion and the median sound enamel, calculated bytrapezoidal integration, is represented by ⌬Zc (integrated mineral loss). The difference between the areas under the densitometric profile of the treated lesion and the median sound enamel is

36

Caries Res 2010;44:33–40

represented by ⌬Zt. These ⌬Z values were then used to calculate
total mineral loss or gain (⌬Zc – ⌬Zt) and percent mineral change(%MC), i.e. %MC = (⌬Zc – ⌬Zt)/⌬Zc ! 100. A negative ⌬Zc – ⌬Zt
or %MC value indicates mineral has been lost and therefore thelesion has progressed, whereas a positive value indicates mineral
has been gained and therefore the lesion has regressed. The maximum mineral content of the surface layer (SL) and the minimum
mineral content of the body of the lesion (BL) for each treated (t)and control (c) lesion were also measured and the percent change
after treatment was calculated as %⌬SL = (SLt – SLc)/SLc ! 100
and %⌬BL = (BLt – BLc)/BLc ! 100. A negative %⌬SL or %⌬BL
indicates there has been a loss of mineral from the SL or BL, respectively. A positive value indicates a gain in mineral content.
Lesion depth (LD) was also measured for each lesion image andthe percent change after treatment was calculated as %⌬LD. A
negative %⌬LD indicates an increase in LD.
For each study, the primary outcome measure was total mineral loss or gain (⌬Zc – ⌬Zt). This was compared across confection
products using a repeated-measures analysis of variance (ANOVA) model with factors for treatment sequence, treatment period
and confection type [Jones and Kenward, 2003]. The secondary
outcome measures, SLt, BLt and LDt were compared across confection products using repeated-measures ANOVA models withfactors for treatment sequence, treatment period and confection
type and covariate, SLc, BLc and LDc, respectively. Repeated-measures ANOVA was also used to compare the control lesions between confection type. Post hoc comparisons of treatment differences were performed on the marginal means using the Sidak
adjustment for multiple comparisons [SPSS, 2008]. ANOVA assumptions were checked using residual and normal probability
plots. The unit of analysis was the participant. The outcome measures were determined for the treatment and control lesions foreach enamel slab, and the resultant values averaged by participant
and treatment period. p values less than 0.05 were regarded as being statistically significant. All analyses were conducted using either SPSS (version 17, SPSS Inc., Chicago, Ill., USA) or Stata (version 10, Stata Corp LP, College Station, Tex., USA) statistical software.

Results

All randomized participants completed both in situ
studies with 100% reported compliance (fig. 1). No adverse events were reported in either study. The mean values for the enamel subsurface lesion parameters after exposure to the different confections in situ and their relative change compared with their original (control) lesions
for both studies are presented in table 1. Analysis of thecontrol lesions showed no statistically significant differences between confection types for any of the lesion parameters ⌬Zc, SLc or BLc. For the participants of bothstudies, their mean unstimulated salivary flow rate was
1.17 8 0.71 ml/min, and their mean confection-stimulated salivary flow rate was 4.25 8 1.78 ml/min. There
was no significant difference in stimulation of salivary
Walker/Cai/Shen/Adams/Reynolds/
Reynolds

A

B

C

D

E

F

Fig. 2. Representative microradiographic

images of enamel sections after consumption of the 5 confections in situ. A Representative image of lesions before treatment. B Representative image of lesions
treated with control sugar confections.
C Representative image of lesions treatedwith sugar confections containing 0.5%
CPP-ACP. D Representative image of lesions treated with the control sugar-freeconfections. E Representative image of lesions treated with the sugar confections
containing 1.0% w/w CPP-ACP. F Representative image of lesions treated with sugar-free confections containing 0.5% CPPACP.

flow rate by any of the confections used for the two studies.
Plaque developed in the troughs above the enamel
half-slabs in the appliances by the end of each treatment
period, such that it completely covered the surface of theenamel. In both studies consumption of the sugar control
confection over the treatment period resulted in progression of the enamel subsurface lesions as shown by a significant decrease in total mineral content (⌬Zc – ⌬Zt), an
increase in LD and significant decreases in mineral content of the SL and of the BL (table 1). In contrast to thesugar control confections, the sugar confections containing CPP-ACP did not result in progression of the enamel
subsurface lesions in either study (table 1). Consumptionof the sugar + 0.5% CPP-ACP and sugar + 1.0% CPP-ACP
confections did not result in a significant increase in LD(table 1) and in fact produced a significant increase in total mineral content and a significant increase in mineralcontent of the SL and the BL (table 1). Consumption of thesugar + 0.5% CPP-ACP and sugar + 1.0% CPP-ACP confections exhibited a clear CPP-ACP dose-related remineralization (regression) of the original enamel subsurface
lesions. The increase in total mineral content producedby the sugar + 1.0% CPP-ACP confection was significantly greater than that produced by the sugar-free confection
CPP-ACP Prevents Demineralizationin situin both studies (table 1). The effects of the sugar in progressing the enamel subsurface lesions and the role of the
CPP-ACP in negating the effects of the sugar and remineralizing (regressing) the subsurface lesions can be clearly seen in representative microradiographs of the lesions(fig. 2).

Discussion

Dental caries is a dynamic process involving periodsof demineralization and remineralization. When demineralization is greater than remineralization, the lesion
progresses. An in situ model used in this study allowed
plaque to form over an enamel window already containing a defined and uniform subsurface demineralized lesion. Frequent consumption (6 times per day) of sugar
confections using this model resulted in significant progression of the subsurface enamel lesions in the 10-day
period confirming the cariogenicity of the confections.
The same sugar confections with 0.5% (w/w) or 1.0%(w/w) CPP-ACP added did not result in lesion progression but in fact produced significant remineralization(regression) of the lesions in the same time period. The
progression of the subsurface lesions following exposure
Caries Res 2010;44:33–40

37

Table 1. Effect of CPP-ACP on enamel subsurface lesion parameters (means 8 SD)

Confection type

Study 1 (n = 10)
Sugar control
Sugar + 0.5% CPP-ACP
Sugar-free control
Sugar + 1.0% CPP-ACP
Study 2 (n = 14)
Sugar control
Sugar-free control
Sugar + 1.0% CPP-ACP
Sugar-free + 0.5% CPP-ACP

Total mineral change⌬Zc – ⌬Zt (%MC)
vol%min  ␮m

Surface layer
SLt (%⌬SL)
vol%min

Body of lesion
BLt (%⌬BL)
vol%minLesion depth
LDt (%⌬LD)␮m–209.6865.3 (–6.1)
118.5839.0 (3.5)a150.4836.2 (4.8)a318.1874.4 (8.6)a, b
p < 0.0001

48.089.1 (–12.7)
63.984.0 (15.9)a65.188.7 (9.1)a68.785.8 (11.1)ap = 0.001

35.3811.6 (–18.5)
51.883.8 (14.7)c
52.089.2 (14.4)c
55.086.9 (14.6)ap = 0.002

108.883.3 (–11.6)
101.983.0 (NC)a97.583.9 (NC)c
99.081.9 (NC)ap = 0.0001–228.38128.7 (–7.5)
163.1854.6 (4.9)a357.1890.8 (11.0)a, b
414.9880.5 (12.7)a, b
p < 0.0001

51.384.9 (–7.0)
65.0810.3 (13.4)a70.785.9 (20.9)a68.988.8 (20.8)ap < 0.0001

41.486.4 (–10.4)
52.2812.6 (12.0)c
57.984.9 (25.1)a, b
56.489.7 (27.6)a, d
p < 0.0001

104.985.1 (–7.6)
96.887.9 (NC)a99.686.6 (NC)a98.883.8 (NC)ap = 0.0001

Figures in parentheses are percentages.
Total mineral change analysed using repeated-measures
ANOVA models which included factors for treatment sequence,treatment period and confection type.
Surface layer analysed using repeated-measures ANOVA models which included factors for treatment sequence, treatment period and confection type, and a covariate SLc; overall and multiple-comparisons test of confection type performed on marginalmeans for SLt (evaluated at SLc = 58.3 in study 1 and SLc = 58.1 instudy 2).
Body of lesion analysed using repeated-measures ANOVA
models which included factors for treatment sequence, treatment
period and confection type, and a covariate BLc; overall and multiple-comparisons test of confection type performed on marginalmeans for BLt (evaluated at BLc = 47.0 in study 1 and BLc = 46.8 instudy 2).

Lesion depth analysed using repeated-measures ANOVA
models which included factors for treatment sequence, treatment
period and confection type, and a covariate LDc; overall and multiple-comparisons test of confection type performed on marginalmeans for LDt (evaluated aT LDc = 98.0 ␮m in study 1 and 2).
NC = No change in lesion depth.
a p ≤ 0.005: significantly different from sugar control after Sidak adjustment for multiple comparisons; b p ≤ 0.005: significantly different from sugar-free control after Sidak adjustment for
multiple comparisons; c p ≤ 0.05: significantly different from sugar control after Sidak adjustment for multiple comparisons; d p ≤0.05: significantly different from sugar-free control after Sidak
adjustment for multiple comparisons.

to the sugar control confections is consistent with thesugar being metabolized by the plaque bacteria, present
on the enamel slabs, to acid which then enhanced enamel subsurface demineralization (fig. 2). The presence ofdental plaque on the enamel slabs was clearly visible, andsignificant subsurface enamel demineralization in 10day exposure to the sugar control confections confirmed
the plaque’s viability and cariogenicity. The study was designed to encourage growth and maintenance of a viableplaque on the enamel lesions by appliance design andlong intra-oral exposure times. Furthermore, when theappliances were out of the mouth, they were kept at 37 ° C
in a humid environment to maintain plaque viability.
Subjects were also instructed not to brush the enamelslabs on the appliance or eat anything but the confections
when wearing the appliance.

CPP-ACP when incorporated into the sugar confections not only prevented the lesion progression observedwith the control sugar confections, but also managed toremineralize (regress) the subsurface lesions that were
initially present (fig. 2; table 1). The level of net demineralization that occurred with the control sugar confections highlights the ability of CPP-ACP to remineralize
subsurface lesions notwithstanding the concomitant exposure to sugar and consequent acid challenge. Interestingly, Cochrane et al. [2008] have shown that CPP-ACP
solutions could still produce net remineralization ofenamel subsurface lesions in vitro even at pH 5.5. It was
hypothesized that the slightly acidic environment helped
to maintain the porosity of the lesion SL to allow the calcium and phosphate ions from the CPP-ACP to diffuse
into the subsurface lesion. However, in that study remin-38

Caries Res 2010;44:33–40

Walker/Cai/Shen/Adams/Reynolds/
Reynolds

eralization declined rapidly below pH 5.5 [Cochrane etal., 2008], and this would explain why the sugar confection containing 0.5% CPP-ACP did not remineralize aswell as the sugar-free confection containing CPP-ACP at
the same level. The efficacy of CPP-ACP in the sugar confections may also be partly explained by the capacity of
CPP-ACP to bind to plaque as demonstrated by immunohistochemical staining of supragingival plaque in vivo
[Reynolds et al., 2003]. CPP-ACP bound to the plaqueoverlying the enamel lesions would localize calcium andphosphate ions to help prevent demineralization and promote remineralization of the underlying lesion.
Consumption of the sugar-free control confection
without CPP-ACP resulted in a small but significant level of remineralization through the natural remineralizing ability of saliva. Consumption of the sugar-free confection containing 0.5% (w/w) CPP-ACP resulted in a significantly higher level of remineralization than thesugar-free control. This is consistent with the alreadywell-demonstrated efficacy of CPP-ACP as a remineralizing agent in sugar-free products including confectionery [Cai et al., 2003] and other sugar-free products suchas chewing gum, mouthwashes and dentifrices in a variety of in situ studies [Shen et al., 2001; Reynolds et al.,
2003; Iijima et al., 2004; Walker et al., 2006; Cai et al.,2007; Reynolds et al., 2008]. Recently these in situ studies
were criticized because the stability of the mineral deposited in the subsurface lesions to acid challenge was notestablished [Zero, 2009]. However, in 3 of the publishedin situ studies, the remineralized enamel pieces were acidchallenged in vitro after treatment to determine the stability of the mineral deposited in the lesions. These substantial posttreatment acid challenges showed that theenamel subsurface lesions remineralized with CPP-ACP
were more acid resistant than normal tooth enamel [Iijima et al., 2004; Cai et al., 2007; Reynolds et al., 2008]. The
increased acid resistance was attributed to the replacement of carbonated apatite with more acid-resistant apatite and also to the inhibition of enamel demineralization
by adsorbed CPP-ACP. Zero [2009] has also criticizedthese studies stating that this posttreatment acid challenge, although substantive, is not equivalent to the demineralization/remineralization cycling conditions of
the human mouth. He further criticized the in situ model used in the studies stating that in his opinion it was notconducive to oral biofilm formation [Zero, 2009]. The
current study goes some way to address these criticisms
by the frequent consumption (6 times per day) of sugar
which produced a frequent acid challenge in the mouthand by the demonstration of the effects of a cariogenic

biofilm that formed on the protected surface above theenamel pieces inset into the removable appliances. The
incorporation of these changes into the in situ model(i.e. frequent sugar consumption and cariogenic biofilm
development) produced results that confirmed thosepublished previously showing net remineralization by
the CPP-ACP after posttreatment in vitro acid challenge[Iijima et al., 2004; Cai et al., 2007; Reynolds et al.,2008].
The aim of any in situ model is to predict the outcomeof a larger, clinical RCT whilst minimizing the risk to thesubjects, controlling as many of the product-unrelated
variables as possible and being mindful of subject comfort and compliance. The ultimate test of any in situ model is therefore its ability to predict the outcome of an RCT.
As stated above, many published in situ studies on the
CPP-ACP technology have demonstrated a significant
remineralization of enamel subsurface lesions predicting
a slowing of caries progression and promotion of lesion
regression in an RCT. This prediction has now been validated in 4 published RCTs [Andersson et al., 2007; Morgan et al., 2008; Bailey et al., 2009; Rao et al., 2009]. One
RCT demonstrated that CPP-ACP in a sugar-free chewing gum, relative to a normal sugar-free gum, significantly slowed progression and enhanced regression of approximal caries in a population of schoolchildren [Morgan et al., 2008]. Another 2 RCTs demonstrated that
CPP-ACP delivered in a tooth cream significantly regressed white-spot lesions in postorthodontic populations [Andersson et al., 2007; Bailey et al., 2009], andrecently another RCT showed that CPP and calcium carbonate in a toothpaste formulation, which would spontaneously form CPP-ACP in saliva, significantly reduced
caries experience in children compared with a placebo
toothpaste [Rao et al., 2009].
In conclusion, this in situ study showed that consumption of sugar confections containing CPP-ACP significantly remineralized (regressed) subsurface enamel lesions. The results suggest that incorporation of CPP-ACP
into sugar confections may reduce their cariogenicity.

CPP-ACP Prevents Demineralizationin situ

Caries Res 2010;44:33–40

Acknowledgment
The support of Cadbury-Adams Worldwide Research and Development is gratefully acknowledged.

39

References
Andersson A, Skold-Larsson K, Hallgren A, Petersson LG, Twetman S: Effect of a dental
cream containing amorphous calcium phosphate complexes on white spot lesion regression assessed by laser fluorescence. Oral
Health Prev Dent 2007;5:229–233.
Angmar B, Carlstrom D, Glas J: Studies on theultrastructure of dental enamel. J Ultrastruct Res 1963;8:12–23.
Bailey DL, Adams GG, Tsao CE, Hyslop A, Escobar K, Manton DJ, Reynolds EC, Morgan
MV: Regression of post-orthodontic lesions
by a remineralizing cream. J Dent Res 2009;88:1148–1153.
Cai F, Manton DJ, Shen P, Walker GD, Cross KJ,
Yuan Y, Reynolds C, Reynolds EC: Effect ofaddition of citric acid and casein phosphopeptide-amorphous calcium phosphate to asugar-free chewing gum on enamel remineralization in situ. Caries Res 2007; 41: 377–
383.
Cai F, Shen P, Morgan MV, Reynolds EC: Remineralization of enamel subsurface lesions
in situ by sugar-free lozenges containing casein phosphopeptide-amorphous calcium
phosphate. Aust Dent J 2003; 48:240–243.
Cai F, Shen P, Walker GD, Reynolds C, Yuan Y,
Reynolds EC: Remineralization of enamel
subsurface lesions by chewing gum withadded calcium. J Dent 2009;37:763–768.
Cochrane NJ, Saranathan S, Cai F, Cross KJ,
Reynolds EC: Enamel subsurface lesion remineralisation with casein phosphopeptide
stabilised solutions of calcium, phosphateand fluoride. Caries Res 2008;42:88–97.
Cross KJ, Huq NL, Palamara JE, Perich JW,
Reynolds EC: Physicochemical characterization of casein phosphopeptide-amorphous
calcium phosphate nanocomplexes. J Biol
Chem 2005;280:15362–15369.

40

Caries Res 2010;44:33–40

Cross KJ, Huq NL, Stanton DP, Sum M, Reynolds
EC: NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59–79) by stabilized amorphous
calcium fluoride phosphate nanocomplexes.
Biomaterials 2004; 25:5061–5069.
Iijima Y, Cai F, Shen P, Walker G, Reynolds C,
Reynolds EC: Acid resistance of enamel subsurface lesions remineralized by a sugar-freechewing gum containing casein phosphopeptide-amorphous calcium phosphate.
Caries Res 2004;38:551–556.
Jones B, Kenward MG: Design and Analysis of
Crossover Trials, ed 2. Boca Raton, Chapman & Hall/CRC, 2003.
Malcolm AS: A microradiographic apparatus.
Aust Dent J 1972;17:454–460.
Morgan MV, Adams GG, Bailey DL, Tsao CE,
Fischman SL, Reynolds EC: The anticariogenic effect of sugar-free gum containing
CPP-ACP nanocomplexes on approximal
caries determined using digital bitewing radiography. Caries Res 2008;42:171–184.
Oshiro M, Yamaguchi K, Takamizawa T, Inage
H, Watanabe T, Irokawa A, Ando S, Miyazaki M: Effect of CPP-ACP paste on tooth mineralization: an FE-SEM study. J Oral Sci
2007;49:115–120.
Rahiotis C, Vougiouklakis G: Effect of a CPPACP agent on the demineralization and remineralization of dentine in vitro. J Dent
2007;35:695–698.
Rao SK, Bhat GS, Aradhya S, Devi A, Bhat M:
Study of the efficacy of toothpaste containing casein phosphopeptide in the preventionof dental caries: a randomized controlled trial in 12- to 15-year-old high caries risk children in Bangalore, India. Caries Res 2009;43:
430–435.

Reynolds EC: Remineralization of enamel subsurface lesions by casein phosphopeptidestabilized calcium phosphate solutions. J
Dent Res 1997;76:1587–1595.
Reynolds EC: Anticariogenic complexes ofamorphous calcium phosphate stabilized bycasein phosphopeptides: a review. Spec Care
Dentist 1998;18:8–16.
Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker GD, Morgan MV, Reynolds C: Fluoride
and casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2008; 87: 344–
348.
Reynolds EC, Cai F, Shen P, Walker GD: Retention in plaque and remineralization of enamel lesions by various forms of calcium in amouthrinse or sugar-free chewing gum. J
Dent Res 2003;82: 206–211.
Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF, Johnson IH, Perich JW: Anticariogenicity of calcium phosphate complexes oftryptic casein phosphopeptides in the rat. J
Dent Res 1995;74:1272–1279.
Shen P, Cai F, Nowicki A, Vincent J, Reynolds
EC: Remineralization of enamel subsurface
lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate. J Dent Res 2001; 80: 2066–
2070.
SPSS: Statistics Base 17.0 User’s Guide. Chicago,
SPSS Inc, 2008.
Walker G, Cai F, Shen P, Reynolds C, Ward B,
Fone C, Honda S, Koganei M, Oda M, Reynolds EC: Increased remineralization of tooth
enamel by milk containing added casein
phosphopeptide-amorphous calcium phosphate. J Dairy Res 2006;73:74–78.
White DJ: Use of synthetic polymer gels for artificial carious lesion preparation. Caries Res
1987;21:228–242.
Zero DT: Recaldent – evidence for clinical activity. Adv Dent Res 2009;21:30–34.

Walker/Cai/Shen/Adams/Reynolds/
Reynolds