Original Paper
Caries Res 2012;46:147–155
DOI: 10.1159/000337240

Received: October 4, 2011
Accepted after revision: January 26, 2012
Published online: April 5, 2012

Remineralisation by Chewing Sugar-Free Gums
in a Randomised, Controlled in situ Trial Including
Dietary Intake and Gauze to Promote PlaqueFormation
N.J. Cochrane P. Shen S.J. Byrne G.D. Walker G.G. Adams Y. Yuan
C. Reynolds B. Hoffmann S.G. Dashper E.C. Reynolds
Oral Health CRC, Melbourne Dental School, Bio21 Molecular Science and Biotechnology Institute, University of
Melbourne, Melbourne, Vic., Australia

Key Words
Caries in situ models ⴢ Casein phosphopeptide
amorphous calcium phosphate ⴢ Enamel subsurface lesion
remineralisation ⴢ Gauze ⴢ Plaque composition ⴢ Sugar-freechewing gum

Abstract
Remineralisation has been shown to be an effective mechanism of preventing the progression of enamel caries. The aimof this double-blind, randomised, cross-over in situ study
was to compare enamel remineralisation by chewing sugarfree gum with or without casein phosphopeptide amorphous calcium phosphate (CPP-ACP) where the enamel lesions were exposed to dietary intake and some were covered
with gauze to promote plaque formation. Participants wore
removable palatal appliances containing 3 recessed enamel
half-slabs with subsurface lesions covered with gauze and 3
without gauze. Mineral content was measured by transverse
microradiography, and plaque composition was analysed byreal-time polymerase chain reaction. For both the gauzefree and gauze-covered lesions, the greatest amount of remineralisation was produced by the CPP-ACP sugar-freegum, followed by the gum without CPP-ACP and then the© 2012 S. Karger AG, Basel
0008–6568/12/0462–0147$38.00/0
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E-Mail karger@karger.ch
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Accessible online at:www.karger.com/cre

no-gum control. Recessing the enamel in the appliance allowed plaque accumulation without the need for gauze.
There was a trend of less remineralisation and greater variation in mineral content for the gauze-covered lesions. The
cell numbers of total bacteria and streptococci were slightly
higher in the plaque from the gauze-covered enamel for 2 ofthe 3 treatment legs; however, there was no significant difference in Streptococcus mutans cell numbers. In conclusion,chewing sugar-free gum containing CPP-ACP promoted
greater levels of remineralisation than a sugar-free gum
without CPP-ACP or a no-gum control using an in situ remineralisation model including dietary intake irrespective ofwhether gauze was used to promote plaque formation ornot.
Copyright © 2012 S. Karger AG, Basel

The chewing of sugar-free gum has been shown to promote the remineralisation of enamel subsurface lesions insitu [Leach et al., 1989] and to reduce the incidence ofdental caries in clinical trials [Kandelman and Gagnon,
1990; Makinen et al., 1995; Beiswanger et al., 1998]. The
anticariogenic effect of chewing sugar-free gum has been
attributed to salivary flow stimulation [Jenkins and EdProf. E.C. Reynolds
Melbourne Dental School, University of Melbourne
720 Swanston Street
Melbourne, VIC 3010 (Australia)
Tel. +61 3 9341 1547, E-Mail e.reynolds @ unimelb.edu.augar, 1989; Dawes and Dong, 1995], resulting in an increase in plaque pH [Yankell and Emling, 1989; Macpherson and Dawes, 1993], an increase in the availability ofcalcium and phosphate ions [Leach et al., 1989] and theclearance of food debris [Addy et al., 1982]. Casein phosphopeptide amorphous calcium phosphate (CPP-ACP), astabilised source of bioavailable calcium and phosphateions, has been added to sugar-free gum to enhance its anticariogenic properties [Shen et al., 2001]. The ability of
CPP-ACP to enhance the remineralisation of enamel subsurface lesions has been shown in several in situ studies[Shen et al., 2001; Reynolds et al., 2003; Cai et al., 2007;
Manton et al., 2008]. Additionally, a sugar-free gum containing CPP-ACP significantly slowed the progression
and enhanced the regression of approximal caries relative
to a control sugar-free gum in a 24-month randomised
controlled clinical trial [Morgan et al., 2008].
Most of the in situ studies investigating the remineralisation effect of CPP-ACP in sugar-free gum have
utilised a removable palatal appliance that contained demineralised subsurface lesions in slabs recessed in the appliance to encourage plaque accumulation. In this model,
the appliance was removed during eating or drinking to
avoid the confounding effect of participants’ diet on remineralisation [Shen et al., 2001; Reynolds et al., 2003;
Cai et al., 2007; Manton et al., 2008]. This in situ model
has shown a fluoride dose response [Reynolds et al.,2008], and the remineralisation results for a CPP-ACPcontaining sugar-free gum [Shen et al., 2001] were foundto be predictive of the efficacy obtained for the gum in arandomised, controlled clinical trial [Morgan et al.,2008]. Alternative in situ remineralisation models havebeen used in which the enamel slabs are retained in themouth during normal dietary intake and the slabs arecovered with a layer of gauze to encourage plaque accumulation. Although there has not been a direct comparison of results obtained with each of these in situ models,
it has been claimed that exposure to normal dietary intake and inclusion of a gauze covering are necessary components of any in situ model used to assess the remineralisation potential, in spite of the expected increased variance and decreased power of the study [Leach et al., 1989].
Therefore, the aim of this study was to compare theremineralisation effect of a CPP-ACP-containing sugarfree chewing gum with that of a regular sugar-free chewing gum and a no-gum control group in an in situ model
where the appliance was exposed to the normal diet toprovide a regular acid challenge and some of the enamelslabs were covered with gauze to promote plaque formation.
148

Caries Res 2012;46:147–155

Subjects and Methods
Participants
Nine healthy adults living in a fluoridated community participated in this double-blind, randomised, crossover study. The
participants were recruited from staff and students of the Melbourne Dental School, University of Melbourne. Study inclusion
criteria were: age 18–60 years; at least 22 natural teeth; gum-stimulated salivary flow rate 61.0 ml/min and unstimulated whole
salivary flow rate 60.2 ml/min. Subjects were additionally excluded if: their occlusion precluded fabrication of an in situ appliance; they were currently using antibiotics or medications that
may affect salivary flow rate; they had current caries activity or
periodontal disease, a history of rampant caries or any gross oral
pathology. This study protocol was approved by the University of
Melbourne Human Research Ethics Committee (No. 0932529).
The number of participants required for this study was based on
the results of a pilot study. The required sample size was calculated using the G*Power version 3.1 (http://www.psycho.uniduesseldorf.de/abteilungen/aap/gpower3/) sample size package
and was based on a repeated measures analysis of variance with 1
within-subject factor with 3 levels, an effect size of 0.97, a correlation, ␳, between any pair of treatment means of 0.5 and a nonsphericity correction, ␧, of 0.5. The effect size of 0.97 was basedon detecting differences between ⌬Zd – ⌬Zr means of 50 (nogum control), 225 (sugar-free gum without CPP-ACP) and 500(sugar-free gum with CPP-ACP), and a common standard deviation of 200 within groups. These values were obtained from thepilot study. The non-sphericity correction adjusts for heterogeneity in the variances of the repeated measures. With a 5% significance level and a power of 90%, at least 6 subjects were required.
To allow for subject non-compliance and drop-out, and since it ispreferable to have a balanced design for the statistical analysis(equal numbers of subjects should be given each treatment in eachtime period) 9 subjects (3 per treatment sequence) were recruited
to participate in the study.
Intra-Oral Appliances
A removable palatal acrylic appliance covering the first premolars to the most posterior teeth and retained by 4 narrow-gauge
stainless-steel circumferential clasps was fabricated for each subject. Three windows on each side of the appliance accommodated
3 enamel half-slabs containing demineralised subsurface lesions(6 half-slabs per appliance). The enamel half-slabs were inset intolarger windows in the undersurface of the appliance to expose thesubsurface lesions in the smaller windows on the exposed surfaceof the appliance. Three of the 6 half-slabs on one side were coveredby multifilament polyester surgical gauze (PETKM3002, SurgicalMeshTM, Brookfield, Conn., USA). The side containing the 3half-slabs covered by gauze was randomly chosen for each subject
but remained the same for each treatment. The half-slabs withand without overlying gauze were recessed into the appliance by
1.5 mm to protect the dental plaque accumulating on the slab/
gauze. The slabs and gauze were securely attached to the undersurface of the appliance with heavy body polyvinyl siloxane impression material (Kerr, North Ryde, N.S.W., Australia).
Preparation of Enamel Subsurface Lesions
Extracted third molars were obtained from oral surgeons andgeneral practitioners in the Royal Dental Hospital of Melbourne

Cochrane et al.

or private practice. The teeth were sterilised and stored in a 10%(v/v) neutral phosphate-buffered formalin solution for 2 weeks.
Sound relatively planar buccal and lingual surfaces with minimal
cracking, staining and fluorosis (as viewed under a dissecting microscope) were selected, then polished wet to a mirror finish using
Soflex (3M) polishing discs on a slow-speed contra-angle dental
handpiece. Each polished surface was then sawn from the toothas an approximately 8 ! 4 mm slab, using a water-cooled diamond blade saw, and the whole slab was covered with acid-resistant nail varnish except for 2 (occlusal and gingival) mesiodistal
windows (approx. 1 ! 7 mm) separated from each other by about
1 mm. Lesions were created in the enamel windows by mounting
each slab onto the end of a 3- to 4-cm stick of yellow dental sticky
wax and immersing in 40 ml of unagitated demineralisation buffer [White, 1987], consisting of 80 ml/l Noverite K-702 (polycacrylate, Lubrizol Corporation, Wickliffe, Ohio, USA), 500 mg/l hydroxyapatite (Bio-Gel쏐 HTP, Bio-Rad Laboratories, Richmond,
Calif., USA) and 0.1 mol/l lactic acid (Ajax Chemicals, Auburn,
N.S.W., Australia), pH 4.8, for 4 days at 37 ° C with solutions
changed after 2 days. This demineralisation procedure produced
consistent subsurface lesions with intact surface layers, as evaluated by microradiography of sections of the artificial lesions. After demineralisation, each enamel slab was sawn through the
midline of each window into two 4 ! 4 mm slabs, and the cut
surface of each slab was covered with nail varnish. One half ofeach slab was retained as the demineralisation control (control
half-slab) and stored in a humidified environment. The other halfof the enamel slab was inset into the intra-oral appliance (test halfslab) with polyvinyl siloxane impression material as describedabove. The enamel half-slabs and gauze were removed and replaced with new enamel half-slabs and sterile gauze at the beginning of every test period, thus bringing the total number of enamel half-slabs used to 162. After each treatment period, the test
enamel half-slabs with and without overlying gauze were removed. All half-slabs from each side were used for plaque analysis
using real-time polymerase chain reaction (RT-PCR) to determine total bacterial count, total streptococcal count, Streptococcus sanguinis and Streptococcus mutans count and afterwards
were paired with their control half-slabs and embedded, sectioned and analysed to determine mineral content changes.
Study Products and Randomisation
Subjects were randomly assigned to one of the following
groups: (1) sugar-free gum containing 18.8 mg/g CPP-ACP; (2)regular sugar-free gum without CPP-ACP, and (3) no-gum chewing control. The sugar-free chewing gums with and without CPPACP were prepared by the Clinical and Consumer Group of KraftAdams Worldwide Research and Development (N.J., USA). The
gums were provided as coded products in sealed packages andstored at room temperature.
The treatment sequences were generated by the Statistics andAnalytical Sciences Group of Kraft-Adams Worldwide Researchand Development and assigned to subject codes. The study teamthen randomly assigned participants to a subject code. The gum
allocation was only divulged by the study sponsor after all thedata had been acquired.
Study Protocol
The study was conducted at the Royal Dental Hospital of Melbourne, Melbourne, Australia, over a 12-month period. Prior to

In situ Remineralisation by Chewing
Gums

the first treatment period, all subjects wore their appliance without the enamel slabs or gauze attached for 24 h a day for 3 consecutive days to ensure that their appliance fitted comfortably.
Three days later, the study commenced. Participants were allocated to one of the three groups according to the random code andwore their appliance with enamel slabs and polyester gauze attached for 24 h a day (except during oral hygiene procedures) for
14 days. At the end of this 14-day period, they rested from thestudy for 1 week while new enamel slabs and new polyester gauze
were attached to the appliance. Participants were then allocatedtheir next randomly assigned treatment, and the process was repeated until each participant completed each of the 3 regimes.
Participants chewed 1 slab of their allocated gum product for
20 min, 5 times a day on each of the 14 days after the following
meals and snacks: breakfast; morning tea; lunch; afternoon tea;dinner. Participants did not eat or drink anything when chewing
their allocated gum and kept a diary of times and duration of gum
chewing and times when appliances were removed for oral hygiene procedures. Participants were instructed to maintain theirusual diet and oral hygiene procedures for the duration of thestudy. The appliances were only removed briefly by the participants twice daily when they brushed their own teeth, and at oneof these times they also cleaned their appliance. All participants
used a standard fluoride toothpaste (0.245% w/v NaF, Crest Cavity Protection, Procter and Gamble, Cincinnati, Ohio, USA) toclean their own teeth for the duration of the study. Participants
were instructed to clean only the palatal fitting surface (undersurface) of their appliance using a fluoride-free denture cleanser
paste (Dentu-Creme, Polident, GlaxoSmithKline, Moon Township, Pa., USA) and toothbrush and not to brush the exposed sideof the appliance. After cleaning the appliance, they gently rinsed
both the exposed surface and the undersurface areas with a plastic squeeze bottle containing distilled de-ionised water. When theparticipants returned to the clinical site with their appliances atthe conclusion of each 14-day treatment period, the research teamremoved the half-slabs for plaque and mineral content analysis.
Sectioning, Microradiography and Microdensitometric Image
Analysis
Each pair of enamel half-slabs (remineralisation half-slab
paired with its demineralisation control half-slab) had the nailvarnish carefully removed and was embedded in transparent
cold-curing methacrylate resin (Paladur, Heraeus Kulzer, Germany). Sections 200–300 ␮m thick were cut from the embedded halfslabs perpendicularly to the lesion surface through the midline ofboth half-lesions using an internal annulus saw microtome (Leitz
1600, Ernst Leitz, Wetzlar, Germany). The sections were thenlapped down to 90 8 5 ␮m using a RotoPol21/RotoForce4 lapping instrument (Struers, Denmark) with 1,200-grit lapping paper. Each section, which contained the remineralised half-lesion
and the paired demineralised control half-lesion from the same
enamel slab, was radiographed along with an aluminium step
wedge of increments 7 ! 37.5 ␮m thick using Microchrome High
Resolution glass plates (HTA Enterprises, Microchrome Technology Products, San Jose, Calif., USA) and nickel-filtered copper K␣
radiation as described previously [Shen et al., 2011].
Radiographic images of the lesions were viewed via transmitted light through a Leica DM 5500B microscope (Leica, Germany). The images were acquired by a Progres쏐 MF scan digital camera (Jentopik, Jena, Germany) under the control of Image-Pro

Caries Res 2012;46:147–155

149

Plus version 7.0 imaging software on a Sci-Tech Imaging Workstation (Sci-Tech, Preston, Vic., Australia). Images of the lesions andthe neighbouring areas of sound enamel were scanned using the
program’s line luminance function that gave readings in grey values. An area free of artefacts or cracks was selected for analysis.
Each scan comprised 200 readings taken from the tooth surface
to sound enamel. The start and end of the lesion were defined as
the points where the mineral density was 20 and 95% that of thesound enamel [ten Cate et al., 1996]. The step wedge image oneach slide was scanned, and the averaged step grey value readings
were plotted against aluminium thickness. The readings of the
tooth section images lay within the linear portion of the step
wedge curve, and linear regression was used to convert the grey
value data into values of equivalent thicknesses of aluminium.
The section thicknesses were measured and the percent mineraldata computed using the equation of Angmar et al. [1963] and thelinear absorption coefficients of aluminium, organic matter plus
water and apatite mineral (131.5, 11.3 and 260.5, respectively). The
image of the median strip between the two lesions was scanned 6
times and averaged to give a control sound-enamel densitometricprofile. The lesion images (remineralisation windows and demineralisation 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 but avoiding any irregularities commonly found at the lesion edges, and the volume percent mineral content profiles were computed.
RT-PCR Analysis of PlaqueThe enamel half-slabs (including gauze when used) were removed from the appliance and immediately placed into 500 ␮l of
20 mM Tris buffer and subjected to sonication in a water bath for
10 min to remove the plaque biofilm. The enamel half-slab wasthen removed, and the plaque sample frozen in liquid N2 andstored at –80 ° C prior to analysis. DNA was extracted from plaqueusing the Power Biofilm DNA isolation kit (Geneworks, Thebarton, S.A., Australia) and a bead beater to ensure DNA extraction
from Gram-positive bacteria. DNA concentration was determined using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Scoresby, Vic., Australia). DNA concentration in nanograms per microlitre was then converted to the equivalent number of cells per microlitre for use as RT-PCR standards using
chromosome size (online suppl. table 1; for all online suppl. material, see www.karger.com/doi/10.1159/000337240). DNA fromplaque samples was diluted to 10 ng/␮l prior to RT-PCR in order
to limit inhibition of the PCR reaction due to high concentrationsof DNA. Four sets of oligonucleotide primers were used targeting
the glucosyltransferase B gene of S. mutans, the D -alanine:D -alanine ligase gene of S. sanguinis, and the 16S rRNA gene for all
streptococci and total bacteria (suppl. table 1). RT-PCR conditions
were optimised for the Corbett Rotor Gene P (Corbett Research,
Mortlake, N.S.W., Australia). Reactions were carried out in triplicate, in a 25-␮l reaction volume consisting of 12.5 ␮l of Platinum
L SYBR Green qPCR Supermix UDG (Invitrogen, Mt Waverley,Vic., Australia), 9.5 ␮l DNase-free deionised water and 200 n M
final concentration of forward and reverse primer and 2 ␮l template DNA. RT-PCR reaction conditions for primer pairs consisted of an initial heating step at 50 ° C for 2 min, initial denaturation
at 95 ° C for 2 min followed by 35 cycles of denaturation at 95 ° C
for 15 s, annealing at 58 ° C for 30 s and extension at 72 ° C for 30 s.
Fluorescence data were collected immediately following the ex-

150

Caries Res 2012;46:147–155

tension step of each cycle. Specificity of the primer pairs was confirmed by melt curve analysis by heating from 72 to 95 ° C in 0.2 ° C
increments. Melting peaks were compared to the bands obtained
following agarose gel electrophoresis, where 10 ␮l of the PCR
product was run on a 1.5% agarose gel with a 100-bp DNA ladder.
The amplification products were visualised and photographed
under a UV light transilluminator. Reactions for which melting
peaks indicated the presence of non-specific amplification product were excluded from analysis. Numbers of bacterial cells were
determined by reference to a standard curve created using a 10fold serial dilution of a mix of DNA from equivalent cell numbersof S. mutans and S. sanguinis grown in batch culture. When determining the total number of streptococcal cells, it was assumed
that the average number of 16S rRNA genes per streptococcus genome was 5. This was determined by averaging the 16S rRNA genecopy number for streptococci listed in the Ribosomal RNA Database (accessed October 28, 2010) [Lee et al., 2009]. When determining the total cell number of bacteria, it was assumed that the
average number of 16S rRNA genes per genome (cell) in bacteria
from dental plaque was also 5. This was determined by cross-referencing organisms listed in both the Human Oral Microbiome
Database [Chen et al., 2010] (accessed October 28, 2010) and the
Ribosomal RNA Database and then averaging the 16S rRNA genecopy numbers obtained. All data analysis was carried out using
Corbett Rotor Gene application software, version 6.1, build 93(Qiagen, Doncaster, Vic., Australia).
RT-PCR analysis was also conducted on supragingival plaquecollected from all participants after the 3 treatment legs. All participants abstained from all oral hygiene procedures for 4 days. At
the end of this period, plaque was collected from 4 sites on the
buccal and lingual surfaces of the maxillary molars with a sterilised curette and analysed as described above.
Data Analysis and Statistical Considerations
The mineral content profile of each enamel slab’s demineralised and remineralised lesion was compared with the mediansound enamel mineral content profile of the same section. The
difference between the areas under the densitometric profile ofthe demineralised lesion and the median sound enamel, calculated by trapezoidal integration, was represented by ⌬Zd. The difference between the areas under the densitometric profile of theremineralised lesion and the median sound enamel, calculated bytrapezoidal integration, was represented by ⌬Zr. These ⌬Z values
were then used to calculate total mineral loss or gain (⌬Zd – ⌬Zr)
and percent mineral change (%R), i.e. %R = 100 ! (⌬Zd –⌬Zr)/⌬Zd. A negative ⌬Zd – ⌬Zr (or %R) value indicated mineral had been lost and therefore the lesion had progressed, whereas a positive value indicated mineral had been gained and therefore the lesion had regressed. Lesion depth (LD) was also measured
for each lesion image and the change after treatment calculated as⌬LD = LDd – LDr. A negative ⌬LD indicated an increase in LD.
The effects of treatment and gauze on enamel remineralisation
were compared between slabs with and without gauze using the
integrated mineral gain/loss, ⌬Zd – ⌬Zr, as the primary outcome
measure. Secondary outcome variables were %R and ⌬LD.
The outcome measures from the RT-PCR analysis of theplaque samples were cell counts of total bacteria, all streptococci,
S. sanguinis and S. mutans. The relative amounts of all streptococci, S. sanguinis and S. mutans as a percentage of total bacterial
counts and S. sanguinis and S. mutans as a percentage of all strep-

Cochrane et al.

Table 1. Enamel subsurface lesion parameters after exposure to sugar-free chewing gum containing CPP-ACP, sugar-free chewing gum
without CPP-ACP or a no-gum control (means 8 SD)

Treatment

Gauze
⌬Zd

No-gum control (n = 9)
Sugar-free gum without
CPP-ACP (n = 9)
Sugar-free gum with
CPP-ACP (n = 8)No gauze⌬Zd – ⌬Zr

⌬LD

%R

3,8548427 –1368218d –11.2810.6e –3.685.4d⌬Zd⌬Zd – ⌬Zr

⌬LD

%R

3,7698272 478107d –1.587.4e

1.382.5d
6.582.2a3,9138345

1528201b–1.986.5a

3.785.3b

3,9428257 270865a

4,2308373

5958207b, c

5.383.7b

13.584.8b, c

4,1458595 7098239b, c 8.184.3a 14.686.0b, c

0.183.7⌬LD = LDd – LDr; the mean LDd (initial lesion depth) was 125 8 7 ␮m. a p < 0.05, b p < 0.005: significantly different from no-gum
control after Sidak adjustment for multiple comparisons; c p < 0.005: significantly different from sugar-free gum without CPP-ACP
after Sidak adjustment for multiple comparisons; d p < 0.05, e p < 0.001: significantly different between gauze and no gauze for no-gum
control.

tococcal counts were also calculated for each plaque sample. log10transformations of the bacterial count data and complementarylog-log transformations of the relative abundance data were usedprior to analysis.
The unit of analysis was the participant. The outcome measures
were determined for the demineralised and remineralised lesions
from each enamel slab, and the resultant values averaged by participant, treatment and presence of gauze. Descriptive statistics(mean and standard deviation) were calculated for all outcome
measures, and the data were analysed using a linear mixed modelling approach [Verbeke and Molenberghs, 2000]. Fixed effects included in the models were treatment, gauze, treatment by gauzeinteraction and treatment period; participant was included as arandom effect. Gauze was also specified as a repeated measures effect with compound symmetry covariance structure to account forthe correlated data as the appliances included enamel slabs bothwith and without gauze at each treatment period. Post hoc comparisons of treatment differences were performed on the marginalmeans using the Sidak adjustment for multiple comparisons. Modelling assumptions were checked using residual and normal probability plots. p values less than 0.05 were regarded as being statistically significant. All analyses were conducted using either SPSS(version 18.0; SPSS Inc., Chicago, Ill., USA) or Stata (version 10.1;
Stata Corp. LP, College Station, Tex., USA) statistical software.

Results

pant in 1 treatment leg was not included in the analysis
as the treated enamel half-slabs were damaged (online
suppl. fig. 1).
Remineralisation Data
The mean values for the enamel subsurface lesion parameters and their relative change after exposure to thesugar-free gum with and without CPP-ACP and the nogum control are shown in table 1. The demineralised control lesions all had a similar initial integrated mineral loss(⌬Zd) prior to exposure to the oral environment. For both
the gauze-free and gauze-covered lesions, the greatest
amount of remineralisation was provided by chewing thesugar-free gum containing CPP-ACP, followed by the sugar-free gum without CPP-ACP and then the no-gum
chewing control. Gauze-covered lesions in the no-gum
chewing group were found to undergo further demineralisation during the treatment period as shown by a significant decrease in total mineral content (⌬Zd – ⌬Zr) andincrease in lesion depth (table 1). The gauze-covered lesions all tended to undergo less remineralisation than thegauze-free lesions though this difference was only statistically significant for the no-gum control group (table 1).

Participants
Thirteen eligible individuals were screened of which 4
participants were excluded because their occlusion precluded a comfortably fitting palatal appliance. The mean
participant age was 38 years (range 21–52 years) with 4
females and 5 males. The mean unstimulated and stimulated whole salivary flow rates of the participants were0.57 8 0.31 and 3.05 8 1.48 ml/min, respectively. All
participants completed the study. The data for 1 partici-

Microbiological Data
Plaque was seen to develop in the appliance recesses
on both the gauze-free and gauze-covered enamel halfslabs, which was confirmed by RT-PCR analysis (table 2).
The RT-PCR analysis of the plaque samples overlying theenamel half-slabs found no differences in numbers of total bacteria, total streptococci, S. sanguinis and S. mutans
between the various treatments (table 2). However, thepresence of gauze tended to increase the total cell number

In situ Remineralisation by Chewing
Gums

Caries Res 2012;46:147–155

151

Table 2. Bacterial composition of supragingival plaque and plaque overlying the enamel half-slabs in the appliance exposed to sugarfree chewing gum containing CPP-ACP, sugar-free chewing gum without CPP-ACP or a no-gum control (means 8 SD)

Bacterial parameterlog10 (number of total bacterial cells)
log10 (number of streptococcal cells)
Percent streptococci/total bacteria
log10 (number of S. sanguinis cells)
Percent S. sanguinis/total bacteria
Percent S. sanguinis/streptococci
log10 (number of S. mutans cells)
Percent S. mutans/total bacteria
Percent S. mutans/streptococcia, b, cNo gum (n = 9)

Sugar-free gum without
CPP-ACP (n = 9)

Sugar-free gum with
CPP-ACP (n = 8)

no gauze

no gauzegauze

no gauze

7.980.4
7.480.4
31.4811.5
5.380.9
1.181.8b3.686.4
5.581.8
6.0810.1
21.0830.7

8.080.4
7.580.5
33.0818.8
5.081.3
2.486.2b4.6810.5
5.581.9
4.486.8
21.8836.7

7.880.5c
8.280.7c
7.480.5c
7.980.5c
50.9822.8 57.2822.6
5.081.3c
5.281.4c
4.487.5c
1.482.8cc
2.986.2c
7.389.6
5.281.7
5.482.5
2.984.6
2.082.1
6.989.2
7.3810.8gauze

8.380.3a
7.780.5a7.280.5a
7.780.3a43.1825.6 32.9816.3
5.081.1
5.181.0
0.680.7a
0.581.1a
1.581.7
1.382.2
4.782.1
5.481.8
4.4811.3 2.986.9
5.9812.1 10.4818.9

8.480.4
7.880.5
22.6819.5
6.880.8
8.7817.0
19.5824.7
4.082.6
0.280.2
1.181.9

p < 0.05: similarly marked values in the same row are significantly different.

of bacteria and streptococci with this reaching significance in the no-gum and CPP-ACP sugar-free gum
groups (table 2). There were no significant differences in
the cell numbers of S. mutans and the percentage ofS. mutans of total bacteria and total streptococci in theplaque with or without gauze.
The 4-day-old supragingival plaque obtained from theparticipants had a lower percentage of streptococci compared with the 14-day-old plaque from the appliance. Additionally, the percentage of S. sanguinis per total bacteria
or total streptococci in the supragingival plaque was significantly higher than that in the plaque from the appliance. The percentage of S. mutans per total bacteria or
total streptococci in the supragingival plaque was significantly lower than that in the plaque from the appliance(table 2).
Adverse Events
A number of non-serious adverse events (treatmentrelated or unrelated) were recorded for 5 of the 9 participants. The majority were related to the appliance and included minor discomfort or ulceration. No serious adverse events were recorded, and no participants withdrew
due to adverse events.

Discussion

Intra-oral remineralisation models are important forthe development and testing of novel anticariogenic
agents prior to conducting expensive and time-consum152

gauze

Supragingival
plaqueCaries Res 2012;46:147–155

ing clinical trials. This study tested the remineralisation
efficacy of a CPP-ACP-containing sugar-free gum compared with a sugar-free gum without CPP-ACP and a nogum control in a palatal appliance in situ model including
normal dietary intake and gauze to promote plaque formation. A gauze covering over enamel subsurface lesions
in an in situ model has been recommended as a methodof encouraging plaque formation [Leach et al., 1989];
therefore, in the current study we included 3 enamel halfslabs with a gauze covering and 3 without to determine
whether there was a promotion of plaque formation by
the gauze. All enamel half-slabs with or without gauze
were recessed in the appliance to allow the developmentof a uniform biofilm [Walker et al., 2009].
For both the gauze-free and gauze-covered lesions, thegreatest amount of remineralisation was provided bychewing the sugar-free gum containing CPP-ACP, followed by the sugar-free gum without CPP-ACP and then
the no-gum chewing control (table 1). This is consistent
with previous studies that have shown that a sugar-freegum containing CPP-ACP is superior to a sugar-free gum
without CPP-ACP in the remineralisation of enamel subsurface lesions [Shen et al., 2001; Morgan et al., 2008].
This has been attributed to the localisation of bio-available calcium and phosphate ions at the tooth surface thatcan depress enamel demineralisation and promote enamel remineralisation [Cochrane et al., 2010].
The palatal appliance used in this study was similar indesign to a palatal appliance used in previous in situ studies [Shen et al., 2001; Reynolds et al., 2003; Cai et al., 2007;
Manton et al., 2008; Walker et al., 2009] but different in
Cochrane et al.

that the enamel half-slabs were fixed in place from theundersurface of the appliance using polyvinyl siloxane.
Furthermore 3 of the 6 enamel half-slabs in the appliancewere covered with polyester gauze, and the appliance wasworn at all times including meals and removed only during oral hygiene procedures twice per day. In one of theprevious in situ studies [Shen et al., 2001], a sugar-freegum containing 18.8 mg CPP-ACP was shown to promote a significantly greater remineralisation of subsurface lesions (18.2 8 1.7% remineralisation) comparedwith a normal sugar-free gum (9.0 8 1.4% remineralisation) and no-gum chewing (3.3 8 1.0%) when chewed 4
times per day for 20 min. The levels of remineralisation
obtained in the study of Shen et al. [2001] were slightly
higher and the variances lower than that of the current
study. The major difference between these two studies
was that the appliances were removed at meal times in the
study of Shen et al. [2001]. In the current study, participants maintained their usual diet, and therefore lesions
were subjected to the different cycles of de- and remineralisation based on the form and frequency of the participants’ dietary choices. This highlights the increased variance introduced into a study by exposing the enamel halfslabs to the participants’ diet. An in vitro acid challenge
after a treatment phase can be incorporated to test theacid resistance of deposited mineral in a controlled fashion [Iijima et al., 2004; Cai et al., 2007]. Notwithstanding
the influence of the diet, significant differences were stillfound between no-gum chewing, the sugar-free gum
without CPP-ACP and the CPP-ACP-containing sugarfree gum in agreement with the results of previous in situ
models not including normal dietary intakes [Shen et al.,
2001; Cai et al., 2007].
The gauze-covered lesions all tended to undergo lessremineralisation than the gauze-free lesions; however,the difference was only statistically significant for the
no-gum control group (table 1). Zero [1995] found thatgauze-free lesions consistently had a greater remineralisation response than gauze-covered lesions. In the model of Zero [1995], the bovine enamel was mounted flushwith the buccal flange so that no recess was present toprotect and allow the development of plaque. The appliance in the current study utilised a 1.5-mm-deep recess
to allow plaque formation on the enamel surface protected from the abrasive forces of food and the tongue. A
difference in remineralisation between gauze-covered
and gauze-free lesions can either relate to increased demineralisation, decreased remineralisation or a combination of both. The RT-PCR analysis of the plaque indicated that gauze-covered lesions did exhibit slightly

higher total bacterial cell numbers for 2 of the 3 treatments but no differences in levels of S. mutans were detected between gauze and no-gauze plaques. It is possiblethat the presence of an unnatural covering over theenamel lesions acts to impede diffusion of saliva hence
lowering its ability to prevent demineralisation and promote remineralisation [Dawes et al., 1989; Mellberg,
1992]. This may explain why the gauze-covered lesions
in the no-gum chewing group underwent further demineralisation during the treatment period with an increase in lesion depth.
The gauze covering of enamel subsurface lesions has
been proposed as a method of encouraging plaque formation [Leach et al., 1989] and controlling plaque thickness[Zero, 1995]. The primary contention is that gauze-freelesions may be plaque free, thereby resembling smoothsurfaces and not resembling an at-risk site [Zero, 1995].
Other authors have recessed the enamel slabs below theacrylic flange as in the current study to protect the plaque[Corpron et al., 1986; Dijkman et al., 1986; Cai et al., 2007;
Walker et al., 2009]. The current study clearly shows thata recess can be used to protect plaque as RT-PCR analysis
confirmed that plaque was present and it could be visualised on the enamel half-slabs. The non-gauze plaquealso developed the same level of S. mutans as the gauzerelated plaque. The gauze may offer the plaque protection
from the abrasive forces in the mouth if there is no otherprotection for the plaque but it has been postulated that
this may lead to the formation of a less natural plaque[Zero, 1995]. Hence, allowing the development of a thickness-controlled biofilm by the use of a recess in the appliance may be preferable to the use of an unnatural covering to promote plaque formation.
To test whether the composition of the plaque on theappliance was similar to natural plaque, 4-day-old supragingival plaque was obtained from the participants. It
was found that the supragingival plaque had a higher percentage of S. sanguinis and a lower percentage of S. mutans compared with the 14-day-old plaque from the appliance (table 2). Due to ethical reasons supragingival
plaque was collected after 4 days rather than 14 days
which would have corresponded to the age of the plaqueon the appliance enamel. Therefore, differences in thecomposition of the supragingival plaque compared withthe appliance enamel plaque may be related to biofilm
maturation changes with time. CPP-ACP has been suggested to alter biofilm composition [Neeser et al., 1994;
Rose, 2000; Rahiotis et al., 2008] but in the current study
no significant differences in total bacterial cell numbers
or composition were found between treatments. Howev-

In situ Remineralisation by Chewing
Gums

Caries Res 2012;46:147–155

153

er, this may relate to the protection of the plaque by therecess/gauze in the appliance design and the low level of
CPP-ACP in the sugar-free gum.
Ultimately, an in situ model is used to serve as a bridge
between in vitro experiments and clinical trials [Zero,1995]. Therefore, an in situ model should be predictive ofclinical trial results. Many clinical studies have now been
conducted demonstrating the benefits of sugar-free gum
compared with no-gum chewing [Kandelman and Gagnon, 1990; Makinen et al., 1995; Beiswanger et al., 1998].
Additionally, the benefits of a CPP-ACP sugar-free gum
compared with a normal sugar-free gum has recently
been shown [Morgan et al., 2008]. The results of the current in situ study for gauze-covered and gauze-free enamel lesions are in agreement with these clinical trial results
as the CPP-ACP-containing sugar-free gum was found tobe superior to the sugar-free gum without CPP-ACP
which was in turn superior to no-gum chewing. Given
that plaque was present on the recessed gauze-free enamel pieces and that the remineralisation values tended tobe higher and the variance lower for the gauze-free data,then the gauze-free model should allow fewer participants to be used to differentiate remineralisation treatment effects. Furthermore as the gauze-free model does
not involve an unnatural covering over the enamel lesion

surface being studied, then the gauze-free model may be
the preferred in situ model for screening novel remineralisation agents.

Conclusions

Sugar-free chewing gum containing CPP-ACP wasfound to promote higher levels of remineralisation thana sugar-free gum without CPP-ACP or a no-gum control
using an intra-oral remineralisation model including dietary intake irrespective of whether gauze was used topromote plaque formation.
Acknowledgements
The support of Kraft-Adams Worldwide Research and Development and the technical assistance of Greg Hanson are gratefully acknowledged.

Disclosure Statement
Kraft-Adams provided the chewing gum products and thepayments for the trial participants. None of the investigators received payments from Kraft-Adams for the study.

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