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Dental Gum

Casein Phosphopeptide-Amorphous Calcium Phosphate and the

Remineralization of Enamel
a report by
Eric C Reynolds, AO

Professor of Dental Science and Head, School of Dental Science, The University of Melbourne

Casein phosphopeptide-amorphous calcium Reynolds and colleagues, using an in situ animal caries Eric C Reynolds, AO is Professor of
Dental Science and Head of the
phosphate (CPP-ACP) nanocomplexes are derived model, showed that exposure of enamel plaque to
School of Dental Science at The
from bovine milk protein, casein and calcium and solutions of tryptic peptides of casein significantly University of Melbourne. Professor
phosphate. The concept of CPP-ACP as a reduced enamel subsurface demineralization. Casein Reynolds is also Associate Dean of
the Faculty of Medicine, Dentistry
remineralizing agent was first postulated in 1998.1 A peptides were incorporated into enamel plaque and
and Health Sciences at The
number of subsequent studies have demonstrated were associated with increases in the plaques content University of Melbourne and Chief
CPP-ACP to have anticariogenic activity in of calcium and phosphate.The investigators concluded Executive Officer of the Cooperative
Research Centre for Oral Health
laboratory, animal, and human in situ experiments.2-4 that the tryptic peptides responsible for caseinates
Science. He has been researching
This has led to the incorporation of CPP-ACP into anticariogenic activity were the calcium phosphate- and teaching for over 20 years on
food products and dental products as a new tool in the stabilizing CPPs (casein phosphopeptides).1 These the etiology and prevention of oral
diseases. He has over 200 scientific
fight against caries. CPPs contain a specific sequence, which markedly
publications and 16 patents.
increases the apparent solubility of calcium phosphate Professor Reynolds is on the
CPP-ACPA New Adjunctive Agent for by stabilizing ACP, forming solutions that are editorial board for the Australian
Dental Journal and has also been a
Caries Control supersaturated with respect to the calcium phosphates.
member of the editorial board for
the Journal of Dental Research. He
Caries initiation is associated with demineralization of In an animal caries study, a 0.5% weight per volume has received numerous awards and
distinctions, with the most
subsurface tooth enamel. Calcium and phosphate are (w/v) solution of CPP-ACP nanocomplexes was
significant being the Clunies Ross
lost from the subsurface enamel, resulting in the demonstrated to be comparable with a 500ppm National Science and Technology
formation of a subsurface lesion. At this early stage, the fluoride solution in reducing caries activity.10 CPP- Award in 2002 , which honours
those who make important
caries lesion is reversible via a remineralization process ACP solutions, applied twice daily to the teeth of
contributions to science for the
involving the diffusion of calcium and phosphate ions specific-pathogen-free rats orally infected with economic, social and environmental
into the subsurface lesion to restore lost structure. Since Streptococcus sobrinus, a bacterium that causes tooth decay benefit of Australia, and the Victoria
Prize in 2005, which is awarded to
several studies had demonstrated that milk-based in humans, significantly reduced caries activity, with
an individual whose scientific
products appeared to have anticariogenic properties in 0.1% w/v CPP-ACP producing a 14% reduction, and discovery or technological
animal models,5-8 attention was focused on identifying 1.0% w/v CPP-ACP producing a 55% reduction innovation has significantly
advanced the State of Victorias
the specific milk-based agents that were responsible for relative to the distilled water control. The
knowledge base and has potential
the anticaries effect. anticariogenicity of CPP-ACP and fluoride was to lead to a commercial outcome.
In 2005, he was appointed an
Officer of the Order of Australia
1. Reynolds EC,Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review, Spec
(AO) for service to community
Care Dentist (1998);8: pp. 816. dental health through research and
2. Reynolds EC, et al., Advances in enamel remineralization: Anticariogenic casein phosphopeptide - amorphous calcium development of preventive and
restorative products, to dental
phosphate, J Clin Dent (1999);10: pp. 8688.
education through curriculum
3. Reynolds EC, et al.,Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or development, and as an
sugar-free chewing gum, J Dent Res (2003);82: pp. 206211. administrator and teacher.
4. Shen F, et al., Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-
amorphous calcium phosphate, J Dent Res (2001);80: pp. 20662070.
5. Reynolds EC, Black CL, Confectionery composition and rat caries, Caries Res (1987);21: pp. 538545.
6. Navia JM, Lopez H,Rat caries assay of reference foods and sugar-containing snacks, J Dent Res (1983); 62: pp. 893898.
7. Bowen WH, et al., A method to assess cariogenic potential of food-stuffs, J Am Dent Assoc (1980); 100: pp. 677681.
8. Harper DS, et al., Cariostatic evaluation of cheeses with diverse physical and compositional characteristics, Caries Res
(1986);20: pp. 123130.
9. Reynolds EC, et al.,Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat, J Dent Res
(1995)74: pp. 12721279.
10. Moller IJ, Poulsen S, The effect of sorbitol containing chewing gum on the incidence of dental caries, plaque and gingivitis in
Danish schoolchildren, Community Dent Oral Epidemiol (1973);1: pp. 5867.

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Dental Gum
additive, as animals receiving 0.5% CPP-ACP plus
500ppm fluoride had significantly lower caries activity
than those animals receiving either CPP-ACP or
fluoride alone.10
A human in situ caries model study evaluated the ability
of a 1.0% CPP-ACP solution as a mouthrinse, twice
daily, to prevent enamel demineralization. The enamel
plaque that developed in this model was
bacteriologically similar to normal supragingival plaque
on frequent exposure to sucrose solutions. The twice-
daily use of the 1.0% CPP-ACP solution resulted in a
144% increase in calcium level and a 160% increase in
inorganic phosphate level in the interenamel plaque
recovered from the removable intraoral appliance used
in the study. Moreover, CPP-ACP produced a 51
19% reduction in enamel mineral loss caused by
frequent sugar-solution exposure.1
The probable anticariogenic mechanism for CPP-
ACP is the localization of calcium and phosphate ions
at the tooth surface, which buffers the free calcium
and phosphate ion activities, thereby helping to
maintain a state of supersaturation with respect to
tooth enamel, depressing demineralization, and
enhancing remineralization.1-2
CPP-ACP in Chewing Gum
Clinical trials of sugar-free chewing gum have shown
that the gum is non-cariogenic and, in fact, can have an
anticariogenic effect through the stimulation of saliva.11-
13
Therefore, it was proposed that sugar-free gum may
be an excellent delivery vehicle for CPP-APP to
remineralize enamel subsurface lesions.
The effect of incorporating CPP-ACP into sugar-free
gum on enamel remineralization was evaluated in
three randomized, double-blind, crossover studies in
30 patients.4 The subjects in the studies wore
removable palatal acrylic appliances with six human-
enamel half-slabs inset containing subsurface
demineralized lesions. The protocols of the three
studies were identical except for the specific sweetener
(sorbitol or xylitol), weight, and type (slab or pellet) of
sugar-free gum, CPP-ACP dose, and number of
treatments.The gums were provided as coded products
and were stored at room temperature. All chewing
gum treatments were double-blinded and randomized.
11. Kandelman D, Gagnon G,A 24-month clinical study of the incidence and progression of dental caries in relation to consumption
of chewing gum containing xylitol in school preventive programs, J Dent Res (1990);69: pp. 17711775.
12. Beiswanger BB, et al., The effect of chewing sugar-free gum after meals on clinical caries incidence, J Am Dent Assoc
(1998);129: pp. 16231626.
13. Reynolds EC, et al.,Retention in plaque and remineralization of enamel lesions by various forms of calcium in a mouthrinse or
sugar-free chewing gum, J Dent Res (2003);82(3): pp. 206211.
52
For each study, all subjects crossed over to each
randomly assigned treatment, with at least one week
between treatments.
All subjects used standard fluoride dentifrice for the
duration of each study and chewed the gums at their
natural chewing frequency for 20 minutes, four times
daily for 14 days.The appliances were worn for the 20
minutes of gum chewing and for 20 minutes
following. In the no-treatment control, the appliances
were worn for 40 minutes. Subjects were instructed
not to eat or drink while wearing the appliance and to
rinse and clean their appliances using the fluoride-free
denture cleanser plate and toothbrush provided. They
were informed not to brush the area containing the
enamel blocks. No alterations were made to the
subjects diet and oral hygiene procedures for the
duration of each study. After completion of each
treatment period, the enamel blocks were removed
from the appliances for processing. Microradiographic
analyses of the enamel lesions were evaluated using
appropriate computerized imaging software. The
chewing of sugar-free gum produced a four- to seven-
fold increase in salivary flow rate over the two-minute
period in all subjects. No significant difference was
observed between the sorbitol-based and xylitol-based
gums in their ability to stimulate saliva or remineralize
subsurface enamel lesions. The addition of CPP-ACP
to either the sorbitol- or xylitol-based gums at 10.0,
18.8, or 56.4mg produced a significant (p>0.001)
increase in enamel remineralization, with a 63%, 102%,
and 152% average increase, respectively, relative to the
sugar-free gum not containing CPP-ACP. No
significant correlation was found between individual
unstimulated or stimulated salivary flow rates and
percentage enamel remineralization values obtained for
any of the treatments. These results indicated that the
addition of CPP-ACP to sugar-free chewing gum
significantly enhanced remineralization of enamel
subsurface lesions in a dose-related manner,
independent of gum weight or type.
The ability of CPP-ACP to be retained in
supragingival plaque and remineralize enamel
subsurface lesions in situ when delivered in a
mouthrinse or sugar-free gum was studied in
randomized, double-blind trials involving 30 adult
subjects.13 The mouthrinse study was a randomized,
double-blind, cross-over design involving four coded
U S D E N T I S T RY 2 0 0 6
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REFERENCES: 1. Data on file. 2. 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. 3. Reynolds EC, Cai F, Shen P, Walker GD. Retention in plaque and remineralization of enamel lesions by
various forms of calcium in a mouthrinse or sugar-free chewing gum. J Dent Res. 2003; 82: 206-211. RECALDENT is milk derived and, therefore, not recommended for your patients with milk
allergies. RECALDENT is digestible by patients with lactose intolerance. RECALDENT is a registered trademark of Recaldent Pty. Ltd.
Reynolds_edit.qxp 23/10/06 2:58 pm Page 54
Dental Gum
mouthrinses.Two of the mouthrinses contained CPP-
ACP (2% w/v and 6% w/v, respectively), the third
mouthrinse contained an unstabilized slurry of 60mM
CaCl2 and 40mM sodium phosphate. The fourth
mouthrinse was de-ionized water and acted as the
control. In the mouthrinse study, only the CPP-ACP-
containing mouthrinses significantly increased plaque
calcium and inorganic phosphate levels, and the CPP
were immunolocalized to the surfaces of bacterial
cells, as well as to the intercellular plaque matrix.
In the chewing gum studies, two randomized,
double-blind cross over remineralization trials were
conducted with three pellet and three slab sugar-free
gums containing different forms of calcium, including
CPP-ACP. According to the results, the gum
containing the CPP-ACP, although not containing
the most calcium per piece of gum, produced the
highest level of enamel remineralization independent
of gum-chewing frequency and duration. The CPP
could be detected in plaque extracts three hours after
subjects chewed the CPP-ACP-containing gum.
These results highlight the importance of CPP in
delivering ACP to the tooth surface. Moreover, the
results also demonstrate the importance of the CPP in
stabilizing ACP and producing a highly water-soluble
calcium phosphate phase. The CPP-ACP gums were
superior to the other sugar-free gums in
remineralizing enamel subsurface lesions in situ, even
though the other gums contained from five to 13
times the level of total calcium.
In a recent double-blind, randomized, cross-over study,
designed to investigate the acid resistance of enamel
lesions remineralized in situ by a sugar-free chewing
gum containing CPP-ACP, the active treatment group
demonstrated superior resistance to a subsequent acid
challenge.14 The study compared sugar-free gum
containing 18.8mg of CPP-ACP and sugar-free gum
not containing CPP-ACP. Subjects wore removable
palatal appliances with insets of human enamel
containing demineralized subsurface lesions, and
chewed the gum for 20 minutes four times daily for 14
days. After each treatment, the enamel slabs were
removed and half of each lesion challenged with acid in
vitro for eight or 16 hours.The level of remineralization
was determined using microradiography. The gum
containing CPP-ACP produced approximately twice
the level of remineralization as the control sugar-free
gum. The eight- and 16-hour acid challenge of the
lesions remineralized with the control gum resulted in
14. Iijima Y, et al., Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein
phosphopeptide-amorphous calcium phosphate, Caries Res (2004);38(6): pp. 551556.
15. Morgan MV, et al.,CPP-ACP gum slows progression and enhances regression of dental caries, 84th IADR General Session
and Exhibition, (2006) June 28 July 1: Abstract 2445.
54
65.4% and 88.0% reductions, respectively, of deposited
mineral, while for the CPP-ACP-remineralized lesions
the corresponding reductions were 30.5% and 41.8%.
The acid challenge after in situ remineralization for
both control and CPP-ACP-treated lesions resulted in
demineralization underneath the remineralized zone,
indicating that the remineralized mineral was more
resistant to subsequent acid challenge.
CPP-ACP gum has also been shown to slow the
progression and enhance the regression of dental caries
in adolescents. 2,720 subjects were randomly assigned
to either a test or control group. All subjects received
accepted preventive procedures and optimum systemic
fluoride exposure. The test group received a sugar-free
gum containing 54.4mg CPP-ACP, while the control
group received an identical gum without CPP-ACP.
Subjects were instructed to chew their assigned gum for
10 minutes, three times per day, with one session
supervised on school days, over the two-year study
period. Standardized digital bitewing radiographs were
taken at the baseline and at the completion of the
clinical trial. Analysis of caries progression or regression
was undertaken using a transition matrix. There was a
statistically significant difference in the distributions of
the transition scores between the two groups
(p<0.001). The CPP-ACP gum slowed progression of
caries compared with the control gum. Nineteen
percent more of the approximal surfaces experienced
caries progression in the control gum group compared
to the same surfaces in the CPP-ACP gum group.The
CPP-ACP gum also enhanced regression of caries
compared with the control gum. Fifty-six percent more
of the approximal surfaces experienced caries regression
in the CPP-ACP gum group compared to those in the
control gum group.15
Summary
The enamel remineralizing properties of CPP-ACP have
been determined from many years of basic and evidence-
based clinical research. The incorporation of CPP-ACP
into chewing gum has been shown to increase enamel
remineralization.This represents a significant advance in
treatment of early carious lesions and prevention in the
21st century. Dental practitioners should recommend
CPP-ACP-containing products to their patients,
particularly children, as well as adults who are at high risk
for caries. Remineralization of early carious lesions may
continue to emerge in importance as fluoride did in the
past for caries prevention and reduction.
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