Casein Phosphopeptide-Amorphous Calcium
Phosphate: The Scientific Evidence
E.C. Reynolds
Cooperative Research Centre for Oral Health Science, School of
Dental Science, Bio21 Institute of Molecu­lar Science and
Biotechnology, The University of Melbourne, 720 Swanston Street,
Victoria 3010, Australia; e.reynolds@unimelb.edu.au
Adv Dent Res 21:25-29, August, 2009
A novel calcium phosphate remineralization technology
has been developed based on casein phosphopeptide-
amorphous calcium phosphate (CPP-ACP) [RecaldentTM]. The
purpose of this review was to determine the scientific evidence
to support a role for this new remineralization technology as an
adjunct to fluoride treatment in the non-invasive management
of early caries lesions. CPP-ACP has been shown to reduce
caries development in the rat caries model. The technology has
also been demonstrated to inhibit enamel and dentin demineral-
ization and to promote remineralization in several independent
in vitro and in vivo studies. Further, CPP-ACP has been shown
to slow the progression of caries significantly and to promote
the regression of early lesions in randomized, controlled clini-
cal trials. Therefore, evidence exists to support the clinical use
of the CPP-ACP technology as an adjunct to fluoride treatment
in the non-invasive management of early caries lesions.
Introduction
Dental caries is initiated via the demineralization of tooth hard
tissue by organic acids produced from fermentable carbohy-
drate by dental plaque cariogenic bacteria. Fluoride ions, in the
presence of calcium and phosphate ions, can help replace the
lost mineral of early caries lesions by remineralization. The non-
invasive treatment of early caries lesions by remineralization
has the potential to be a major advance in the clinical manage-
ment of the disease. However, for every 2 fluoride ions, 10
calcium ions and 6 phosphate ions are required to form one unit
cell of fluorapatite [Ca10(PO4)6F2]. Hence, on topical applica-
tion of fluoride ions, the availability of calcium and phosphate
ions can be the limiting factor for net enamel remineraliza­
tion to occur, and this is highly exacerbated under xerostomic
conditions. A delivery system for bioavailable calcium and
phosphate ions therefore may have a role as an adjunct to fluo-
ride treatment in the management of early caries lesions.
DOI: 10.1177/0895937409335619
The clinical use of calcium and phosphate ions for reminer-
alization has not been successful in the past, due to the low
solubility of calcium phosphates, particularly in the presence of
fluoride ions. Insoluble calcium phosphates are not easily
applied, do not localize effectively at the tooth surface, and
require acid for solubility to produce ions capable of diffusing
into enamel subsurface lesions. In contrast, soluble calcium and
phosphate ions can be used only at very low concentrations,
due to the intrinsic insolubility of the calcium phosphates,
particularly the calcium fluorophosphates. Soluble calcium and
phosphate ions do not substantially incorporate into dental
plaque or localize at the tooth surface to produce effective concen-
tration gradients to drive diffusion into the subsurface enamel.
A new calcium phosphate remineralization technology has now
been developed based on casein phosphopeptide-amorphous
calcium phosphate (CPP-ACP) [RecaldentTM CASRN691364-
49-5], where it is claimed that the CPP stabilizes high con­
centrations of calcium and phosphate ions, together with
fluoride ions, at the tooth surface by binding to pellicle and
plaque. Although the calcium, phosphate, and fluoride ions are
stabilized by the CPP from promoting dental calculus, the ions
are freely bioavailable to diffuse down concentration gradients
into enamel subsurface lesions, thereby effectively promoting
remineralization in vivo.
In this paper, the scientific evidence for the CPP-ACP tech-
nology is reviewed. The scientific evidence was based on pub-
lished original studies of the CPP-ACP technology in various
caries model systems and in randomized, controlled clinical
trials (Table).
CPP-ACP Structure
CPP containing the active sequence –Ser(P)-Ser(P)-Ser(P)-Glu-
Glu- has a remarkable ability to stabilize calcium and phosphate
as nanoclusters of ions in metastable solution (Cochrane et al.,
2008). Through the active sequence, the CPP binds to forming
nanoclusters of calcium and phosphate ions to form nanocom-
plexes of around 1.5 nm radius, preventing the growth of the
nanoclusters to the critical size required for nucleation and
phase transformation.
Key Words
CPP-ACP, remineralization, evidence, efficacy.
Presented at the International Conference on Novel Anti-caries and
Remineralizing Agents, held in Vina del Mar, Chile, January 10-12,
2008
The authors declare no conflict of interest.
25
26  Reynolds Adv Dent Res 21: 2009

Table. Scientific Evidence for Anti-caries Activity and Enamel Subsurface Lesion Remineralization by CPP-ACP Nanocomplexes

Clinical Inhibition of Caries

Inhibition of Promotion of Promotion of Reports of Progression and/or
Inhibition of Mutans Enamel Enamel White-spot Promotion of Regression
Inhibition of Enamel/Dentin Streptococci Subsurface Inhibition of Subsurface Lesion of Caries (white spots)
Caries in Deminerali­ Adherence Lesion Enamel Lesion Regression in Randomized
Animal zation and Plaque Reminerali­ Deminerali­ Reminerali­ and Caries Controlled Clinical
Models in vitro Formation zation in vitro zation in situ zation in situ Stabilization Trials

YES YES   YES   YES   YES YES YES YES

Guggenheim Reynolds, 1998   Neeser   Reynolds,   Reynolds, Cai et al., Ardu et al., Andersson et al., 2007
et al., 1999 Mazzaoui   et   al.,   1998   1998 2003 2007 Morgan et al., 2008
Reynolds et al., 2003   1994   Oshiro et al., Reynolds Milnar, Bailey et al., 2009
et al., 1995 Yamaguchi   Schüpbach   2007 et al., 2003 2007
et al., 2006   et al.,   Cochrane Walker et al., Ng and
Nasab et al.,   1996   et al., 2006 Manton,
2007   Guggenheim   2008 Cai et al., 2007
Oshiro et al.,   et al., 2007 Vlacic et al.,
2007   1999 Reynolds 2007
Rahiotis and et al., 2008
Vougiouklakis, Manton et al.,
2007 2008
Rees et al.,
2007
Sudjalim et al.,
2007
Tantbirojn et al.,
2008
Yamaguchi
et al., 2007

Anti-cariogenicity of CPP-ACP and promoting remineralization of enamel and dentin in vitro

(Table). Other in vitro studies have suggested that the peptides
in the Rat Caries Model
of CPP-ACP inhibit binding of mutans streptococci to saliva-
The CPP-ACP nanocomplexes, in purified form (Reynolds et al., coated apatite by 75-83% (Table).
1995) or in a crude micellar form (Guggenheim et al., 1999),
have been shown to reduce caries activity in the rat caries model.
In a study by Reynolds et al. (1995), solutions (100 µL) containing Efficacy of CPP-ACP in situ
different concentrations of CPP-ACP were applied to the animals’
In several different studies, the CPP-ACP technology has been
molar teeth twice daily. Other groups of animals received 100 µL
demonstrated to increase the levels of calcium and phosphate
of either 500-ppm-fluoride ions (positive control) or distilled
ions in supragingival plaque significantly when delivered in a
water (negative control). The animals consumed a highly cario-
mouthrinse, and to promote the remineralization of enamel
genic sucrose/gluten diet that did not contain dairy products. The
subsurface lesions in situ (Table). In fact, in one of the studies,
CPP-ACP significantly reduced caries activity in a dose-response
the CPP-ACP technology was shown to be superior to other
fashion, with 0.1% w/v CPP-ACP producing a 14% reduction
forms of calcium phosphate in its ability to remineralize enamel
and 1.0 % w/v CPP-ACP a 55% reduction relative to the distilled
subsurface lesions (Reynolds et al., 2003). These studies high-
water control. CPP-ACP at 0.5% w/v produced a reduction in
light the importance of the CPP not only in stabilizing high
caries activity similar to that of 500 ppm fluoride. A solution
levels of calcium and phosphate ions, but also in delivering the
containing both 0.5% w/v CPP-ACP and 500 ppm fluoride
ions to the tooth surface (Fig. 1). These results demonstrating
produced a significantly greater reduction in caries activity than
CPP-ACP bound to bacterial cells confirmed the work of Rose
either CPP-ACP or fluoride alone at the same concentrations.
(2000a,b), who showed that CPP-ACP bound to Streptococcus
mutans and model plaque produced a reservoir of bioavailable
Inhibition of Demineralization calcium ions.
and Promotion of Remineralization The ability of the CPP-ACP technology added to sugar-free
chewing gum to remineralize enamel subsurface lesions has been
by CPP-ACP in vitro
demonstrated in several randomized, controlled, double-blind
Many independent studies now published have demonstrated the in situ clinical studies (Table). The sugar-free gums (control and
efficacy of the CPP-ACP technology in inhibiting demineralization CPP-ACP-containing gums) were chewed for either 20-minute
Adv Dent Res 21: 2009 CPP-ACP: Scientific Evidence   27
periods, four times a day, or for five-minute periods, seven
times a day. Microradiography and densitometric image analysis
demonstrated that, independent of gum type and chewing dura-
tion (e.g., 20 min or 5 min), the CPP-ACP nanocomplexes pro-
duced a dose-related remineralization of enamel subsurface
lesions in situ. Gum containing 18.8 mg and 56.4 mg of the
CPP-ACP nanocomplexes, chewed for 20 min four times per
day for 14 days, increased enamel subsurface remineralization
by 102% and 152%, respectively, relative to the control sugar-
free gum. Micro­radiographs of the enamel lesions before and
after remineralization showed that the CPP-ACP promoted
remineralization throughout the body of the lesion. Electron
microprobe analyses of sections of the remineralized enamel
indicated that the mineral deposited was hydroxyapatite with a
higher Ca:P ratio than normal apatite. Acid challenge of the
enamel remineralized by the CPP-ACP nanocomplexes in situ
showed that the remineralized apatite was more resistant to acid
challenge than the normal calcium-deficient carbonated tooth
enamel (Cai et al., 2007).
Recently, Schirrmeister et al. (2007) tested the ability of
sugar-free gum to remineralize enamel subsurface lesions in an
in situ model and reported no differences in remineralization
between the gum chewing groups and the no-gum group. The
authors concluded that chewing sugar-free gum had no signifi-
cant effect on remineralization in their model. The standard
deviations for the measured remineralization in this study ranged
from 143% to 737%, which were far too large for the detection
of differences between and among groups in a 14-day in situ
remineralization study. These results are in conflict with those of
the above in situ studies, as well as with those of Leach et al.
(1989) and others who have shown significant remineralization
by sugar-free gum in situ. These in situ findings have been con-
firmed in several long-term clinical caries trials (Kandelman and
Gagnon, 1990; Beiswanger et al., 1998), which showed that
chewing sugar-free gum significantly reduced clinical caries
incidence relative to incidence in a no-gum control group. The
Schirrmeister et al. (2007) study also involved chewing gums
containing casein/hydrolyzed casein and calcium phosphate,
sometimes incorrectly described as CPP-ACP. This material has
also been used in other studies, with mixed results (Itthagarun
et al., 2005). Although this material has been mistakenly described
as CPP-ACP, it does not contain bioavailable calcium and phos-
phate ions complexed with CPP in an amorphous form, and does
not exhibit the very high solubility and other properties of the
CPP-ACP nanocomplex technology (RecaldentTM).
Interaction of CPP-ACP with Fluoride
It has been reported that CPP-ACP interacts with fluoride ions
to produce novel nanoclusters of calcium, fluoride, and phos-
phate ions (Reynolds et al., 2008). The identification of this
novel CPP-stabilized form of calcium, fluoride, and phosphate
ions is consistent with the observed additive anti-cariogenic
effects of the CPP-ACP nanocomplexes and fluoride (Reynolds
et al., 2008). The anti-cariogenic mechanism of fluoride is the
localization of the fluoride ion at the tooth surface. This local-
ization promotes remineralization of enamel with fluorapatite
(FA). It is clear that, for the formation of FA [Ca10(PO4)6F2],
Figure 1. The incorporation of the CPP-ACP nanocomplexes into
supragingival plaque from a mouthrinse. Electron histochemistry of a
supragingival plaque sample showing the CPP-ACP nanocomplexes
localized in the plaque matrix and on the surface of bacterial cells
(from Reynolds et al., 2003).
calcium and phosphate ions must also be present with the fluoride
ions. The reported additive anti-cariogenic effect of CPP-ACP
and fluoride may therefore be attributable to the localization of
the novel calcium, fluoride, and phosphate ion nanoclusters at
the tooth surface by the CPP, which co-localizes calcium, phos-
phate, and fluoride as bioavailable ions in the correct molar ratio
to form fluorapatite (Reynolds et al., 2008). In a randomized,
controlled, mouthrinse trial, a rinse containing 2.0% CPP-ACP
nanocomplexes plus 450 ppm fluoride significantly increased
supragingival plaque fluoride ion content to 33.0 ± 17.6 nmol
F/mg dry wt of plaque, when compared with 14.4 ± 6.7 nmol
F/mg dry wt of plaque attained by the use of a rinse containing
the equivalent concentration of fluoride ions as sodium fluoride
(Reynolds et al., 2008). Although marked increases in plaque
calcium, phosphate, and fluoride were found, calculus was not
observed in any of the study participants, indicating that the
plaque calcium fluoride phosphate remained stabilized at the
tooth surface by the CPP as bioavailable ions and did not trans-
form into a crystalline phase. A dentifrice formulation contain-
ing 2% CPP-ACP nanocomplexes plus 1100 ppm F has been
shown to be superior (2.6 times) to a dentifrice containing only
1100 ppm F in remineralization of enamel subsurface lesions
in situ with mineral that was more resistant to acid challenge
(Fig. 2) (Reynolds et al., 2008). The CPP-ACP nanocomplexes-
plus-fluoride dentifrice resulted in significantly greater incorpo-
ration of fluoride into the subsurface enamel as fluorapatite, as
shown by electron microprobe analysis (Reynolds et al., 2008).
These results indicate that the CPP is an excellent delivery
vehicle to co-localize bioavailable calcium, fluoride, and phos-
phate ions at the tooth surface to remineralize subsurface enamel
lesions with fluorapatite.
Efficacy of CPP-ACP in Randomized
Controlled Clinical Trials
A randomized, controlled caries clinical trial of CPP-ACP-
containing sugar-free chewing gum demonstrated that the
28  Reynolds Adv Dent Res 21: 2009

three-month period of daily tooth-

brushing with a fluoride dentifrice.
The other treatment protocol
was daily topical application of a
0.05% sodium fluoride mouthwash
combined with use of a fluoride
dentifrice for 6 mos. Clinical
examinations were repeated after
1, 3, 6, and 12 mos, and data were
compared with baseline measure-
ments. The study showed that 64%
of white spots regressed in the
CPP-ACP group compared with
23% in the fluoride group, which
was significantly different (p <
0.01). The authors commented that
the visual assessment indicated a
more favorable outcome with
CPP-ACP treatment.
In another study (Bailey et al.,
2009), 45 participants with 408
white-spot lesions were examined
Figure 2. Representative microradiographs of enamel subsurface lesions after remineralization in situ by immediately after orthodontic ther-
various dentifrice formulations and acid challenge (AC) in vitro. The percentage enamel remineraliza- apy and were randomly assigned to
tion is shown in each panel as %R (from Reynolds et al., 2008). The scale bar in each image represents either a tooth cream containing
100 µm.
10% CPP-ACP treatment or a pla-
cebo cream treatment. Participants
CPP-ACP gum significantly slowed progression of caries and were instructed to apply the products twice daily for 12 wks after
enhanced regression of caries compared with the control sugar- normal hygiene procedures (participants were supplied with
free gum (Morgan et al., 2008). In the 24-month study, 2720 toothpaste containing 1000 ppm F as NaF). Following initial
schoolchildren were randomly assigned to either a CPP-ACP or assessment, lesions were assessed at 4, 8, and 12 wks. The lesions
control sugar-free gum. All children received accepted preventive were scored for lesion severity and activity according to the
procedures, including fluoridated water, fluoridated dentifrice, ICDAS II criteria. At 12 wks, 31% more of the ICDAS code 2
and access to professional care. They were instructed to chew (white spot visible when wet) and 3 (loss of enamel surface integ-
their assigned gum for 10 min three times per day, with one ses- rity) lesions had regressed with the CPP-ACP compared with the
sion supervised on school days. Standardized digital radiographs placebo treatment (Odds Ratio = 2.3, p = 0.04). It was concluded
were taken at baseline and at the completion of the trial. The that significantly more post-orthodontic white-spot lesions
radiographs, scored by a single examiner, were assessed for regressed with the CPP-ACP treatment.
approximal caries at both the enamel and dentin levels. Analysis
of caries was undertaken at a participant level, and progression
Clinical Reports on CPP-ACP
or regression at a surface level, according to a transition matrix.
The CPP-ACP gum produced a significant 18% reduction in A topical cream called Tooth MousseTM (Europe and Australasia)
caries progression after 24 mos on a participant basis, and the or MI PasteTM (USA and Japan) contains 10% w/w CPP-ACP
CPP-ACP gum also produced a 53% greater regression (remin- nanocomplexes, and this product has been used clinically in
eralization) of baseline lesions when compared with the control several published reports for the successful non-invasive treat-
gum (Morgan et al., 2008). ment of mild to moderate fluorotic lesions and for the reversal
Results from a randomized, controlled clinical trial of post- of early caries lesions and for caries stabilization (Table). The
orthodontic white-spot regression by a CPP-ACP tooth cream published results are consistent with the proposed anti-cariogenic
have been published (Andersson et al., 2007). The study mechanism of the CPP-ACP technology being the inhibition of
involved 26 participants with 152 visible white-spot lesions on enamel demineralization and enhancement of remineralization
60 incisors and canines immediately after orthodontic debond- through the localization of bioavailable calcium and phosphate
ing. After bracket removal, professional tooth cleaning and ions at the tooth surface.
drying, a visual scoring (0-4) and laser fluorescence assessment
were performed. The participants were randomly assigned to
Conclusion
two different treatment protocols with the aim of remineralizing
the lesions. One treatment was a daily topical application of a The CPP-ACP (RecaldentTM) technology has been shown to
tooth cream containing CPP-ACP for 3 mos, followed by a remineralize enamel subsurface lesions in vivo and to slow the
Adv Dent Res 21: 2009 CPP-ACP: Scientific Evidence   29
progression of caries significantly and promote the regression of
early caries in randomized, controlled clinical trials. Therefore,
evidence exists to support the clinical use of the CPP-ACP tech-
nology as an adjunct to fluoride treatment in the non-invasive
management of early caries lesions.
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