CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE

N.J. Cochrane, F. Cai, N.L. Huq,

M.F. Burrow, and E.C. Reynolds*
Cooperative Research Centre for Oral Health Science,
Melbourne Dental School, Bi021 Institute, The University of
Melbourne, 720 Swanston Street, Victoria 3000, Australia;
*corresponding author, e.reynolds@unimelb.edu.au

New Approaches to Enhanced

Remineralization of Tooth Enamel

J Dent Res 89(11):1187-1197, 2010

Abstract

Introduction

Dental caries is a highly prevalent diet-related

disease and is a major public health problem. A
goal of modern dentistry is to manage non-cavitated
caries lesions non-invasively through remineralization in an attempt to prevent disease progression and improve aesthetics, strength, and function.
Remineralization is defined as the process whereby
calcium and phosphate ions are supplied from a
source external to the tooth to promote ion deposition into crystal voids in demineralized enamel, to
produce net mineral gain. Recently, a range of
novel calcium-phosphate-based remineralization
delivery systems has been developed for clinical
application. These delivery systems include crystalline, unstabilized amorphous, or stabilized
amorphous formulations of calcium phosphate.
These systems are reviewed, and the technology
with the most scientific evidence to support its
clinical use is the remineralizing system utilizing
casein phosphopeptides to stabilize and deliver
bioavailable calcium, phosphate, and fluoride ions.
The recent clinical evidence for this technology is
presented and the mechanism of action discussed.
Biomimetic approaches to stabilization of bioavailable calcium, phosphate, and fluoride ions
and the localization of these ions to non-cavitated
caries lesions for controlled remineralization show
promise for the non-invasive management of
dental caries.

KEY WORDS: remineralization, casein phosphopeptide-amorphous calcium phosphate, fluoride.

DOI: 10.1177/0022034510376046
Received December 11, 2009; Last revision March 18, 2010;
Accepted May 9, 2010
International & American Associations for Dental Research

ental caries is a highly prevalent disease, and although, in most developed

countries, its prevalence has declined, the disease remains a major public health problem (Selwitz et al., 2007). Signs of the caries process cover a
continuum from the first molecular changes in the apatite crystals of the tooth,
to a visible white-spot lesion, through to dentin involvement and eventual
cavitation. Progression through these stages requires a continual imbalance
between pathological and protective factors that results in the dissolution of
apatite crystals and the net loss of calcium, phosphate, and other ions from the
tooth (demineralization). The chemistry of this process has been reviewed by
Robinson et al. (2000). A goal of modern dentistry is to manage non-cavitated
caries lesions non-invasively through remineralization in an attempt to prevent disease progression and improve aesthetics, strength, and function.
The term remineralization has been used previously by authors to
describe mineral gain, including precipitation of mineral onto enamel surfaces
(Tung and Eichmiller, 2004). Precipitation is ion clusters forming in a supersaturated solution as a solid phase. In this review, remineralization is defined as
the process whereby calcium and phosphate ions are supplied from a source
external to the tooth to promote ion deposition into crystal voids in demineralized enamel to produce net mineral gain. The term void is used to define any
accessible space in a crystal caused by ion loss from the demineralization
process. This definition of remineralization therefore includes any crystal
repair to bring about net mineral gain to an enamel subsurface lesion, but does
not extend to precipitation of solid phases onto enamel surfaces.
The ability of saliva to remineralize demineralized enamel crystals stems
from its ability to supply bioavailable calcium and phosphate ions to the
tooth. At physiological pH, unstimulated and stimulated parotid, submandibular, and whole saliva are supersaturated with respect to most solid calcium
phases (Larsen and Pearce, 2003) (Table 1). However, precipitation of calcium phosphate phases in saliva normally does not occur, due to the presence
of salivary proteins, particularly statherin (Schlesinger and Hay, 1977) and
proline-rich phosphoproteins (Oppenheim et al., 1971). The proposed mechanism of action is that the segments of the proteins containing phosphoseryl
residues, in particular the statherin sequence (Table 2), bind to calcium and
phosphate ion clusters, preventing growth of the ion cluster to the critical size
required for precipitation and transformation into a crystalline phase (Hay and
Moreno, 1989).
This critical stabilization of calcium and phosphate ions by salivary phosphoproteins ensures that the ions remain bioavailable to diffuse into mineraldeficient lesions to allow for remineralization of demineralized crystals, while
preventing surface deposition in the form of calculus. However, net remineralization produced by saliva is small and is a slow process, with a tendency
for the mineral gain to be in the surface layer of the lesion due to the low ion

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J Dent Res 89(11) 2010

Table 1. Biologically Relevant Crystalline and Amorphous Calcium Phosphate Phases and Their Solubility Products
Calcium Phosphate Phase

Abbrev.

Chemical Formula

Kspa (-log)

Reference

Crystalline

Brushite, dicalcium phosphate dihydrate

DCPD

CaHPO4.2H2O

-tricalcium phosphateb

TCP

Ca3(PO4)2

29.5

Gregory et al., 1974

Octacalcium phosphate

OCP

Ca8H2(PO4)6.5H2O

98.6

Shyu et al., 1983

Hydroxyapatite

HA

Ca10(PO4)6(OH)2

117.2

McDowell et al., 1972

Fluorapatite

FA

Ca10(PO4)6(F)2

120.3

McCann, 1968

104.3-114.4

Patel and Brown, 1975

ACP

Ca3(PO4)1.87(HPO4)0.2

Enamel apatitec
Amorphous calcium phosphate

6.6

24.8

Gregory et al., 1970

Meyer and Eanes, 1978

Ksp = solubility product. A solution is supersaturated with respect to a particular calcium phosphate phase when the ion activity product (IAP) is
greater than the Ksp. For example, where IAPHA/KspHA > 1, IAPHA is (Ca2+)10 (PO43-)6 (OH-)2.
Pure -TCP is not found in biological systems, but is present with a Mg substitution.
c
Calculated in terms of ion activity product for hydroxyapatite.
a
b

Table 2. Negatively Charged Segments of Proteins and Peptides Involved in Calcium Phosphate Stabilization
Peptide / Protein

Sequence (number of negative residues in brackets)

References

Statherin

-Asp -Ser(P)-Ser(P)-Glu-Glu - [5]

Schlesinger and Hay, 1977

Proline-rich proteins

-Asp2-Leu-Asp-Glu-Asp6- [4]
-Val7-Ser(P)-Gln-Glu-Asp11- [3]
-Asp21-Ser(P)-Glu-Gln-Phe25- [3]
-Asp27-Glu-Glu-Arg-Gln31- [3]

Hay and Moreno, 1989

CPP s1 (5979)

-Glu63-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu70 [7]

Reynolds et al., 1995

CPP (125)

-Glu -Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu [7]

Reynolds et al., 1995

CPP s2 (4670)

-Ser(P)56-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu63 [7]

Reynolds, 1998

CPP s2 (121)

-Ser(P)8-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu18 [7]

Reynolds, 1998

14

concentration gradient from saliva into the lesion (Silverstone,

1972). Recently, van der Veen et al. (2007) and Mattousch et al.
(2007) examined white-spot lesions with quantitative lightinduced fluorescence following removal of orthodontic appliances. The majority of the lesions were stable, with no
measurable signs of regression even after two years. It was concluded that new remineralization systems were necessary to
achieve effective lesion regression (Mattousch et al., 2007; van
der Veen et al., 2007).
Fluoride is the cornerstone of the non-invasive management
of non-cavitated caries lesions, but its ability to promote net
remineralization is limited by the availability of calcium and
phosphate ions (Reynolds et al., 2008). Fluoride ions can drive
the remineralization of extant non-cavitated caries lesions if
adequate salivary or plaque calcium and phosphate ions are
available when the fluoride is applied. For fluorapatite or fluorhydroxyapatite to form, calcium and phosphate ions are required,
as well as fluoride ions. Several authors have now shown that
enamel remineralization in situ and the retention of fluoride in
plaque are dependent on the availability of calcium ions (Chow
et al., 2000; Whitford et al., 2005; Reynolds et al., 2008; Vogel
et al., 2008). Hence, on topical application of fluoride ions, the
availability of calcium and phosphate ions can be the limiting
factor for fluoride retention and net enamel remineralization to

occur, and this is highly exacerbated under hyposalivation conditions (Reynolds et al., 2008). When adequate levels of calcium and phosphate ions are together with the fluoride ions, it
has been shown in vitro that this combination can produce substantial remineralization of lesions of enamel and even those
penetrating the underlying dentin in pH-cycling experiments
(ten Cate, 2001; ten Cate et al., 2008). Therefore, the challenge
now is to achieve this clinically, since salivary remineralization
of enamel promoted by topical fluoride (particularly high concentrations) has been shown to give rise to predominantly surface remineralization (Arends and Ten Cate, 1981; ten Cate
et al., 1981; gaard et al., 1988; Willmot, 2004). Surface-only
remineralization does little to improve the aesthetics and structural properties of the deeper lesion. Ideally, a remineralization
system should supply stabilized bioavailable calcium, phosphate, and fluoride ions that favor subsurface mineral gain
rather than deposition only in the surface layer.

Enamel Remineralizing Systems

The fundamental difficulty with the clinical application of
calcium and phosphate remineralization systems is the low solubility of the calcium phosphates, particularly in the presence
of fluoride ions. Numerous authors have investigated various

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Enamel Remineralization 1189

calcifying solutions in an attempt to remineralize demineralized

tooth structure. Normally, these solutions have contained
between 1 and 3 mM calcium ions with phosphate ions in the
ratio of 1:1 (Koulourides et al., 1961; Wefel and Harless, 1987)
or 1.66:1 (Koulourides et al., 1961; ten Cate and Arends, 1977;
Silverstone et al., 1981; Iijima et al., 1999), often with the addition of 1 ppm fluoride ions. Higher concentrations have not been
used due to the instability of the solutions. No clinical remineralization systems have been developed using a single low-concentration calcium and phosphate solution, since they are not
effective in localization of ions at the tooth surface in significant
concentrations to promote enamel subsurface remineralization
in vivo (Reynolds et al., 2003). This has led to the development
of novel calcium-phosphate-based delivery systems containing
high concentrations of calcium phosphate. These systems can be
categorized into one of three typescrystalline, unstabilized
amorphous, or stabilized amorphous formulations. New products utilizing these three types of delivery systems are now
commercially available, and the manufacturers claim that these
products provide new avenues for the remineralization of noncavitated caries lesions.

2009). One paper has reported in vitro findings that this material
improved surface microhardness of demineralized enamel compared with fluoride-alone products, possibly through an abrasive
effect (Karlinsey and Mackey, 2009). However, no studies of
this material have been published demonstrating its ability to
remineralize enamel subsurface lesions.
A variation on the use of crystalline calcium phosphates is
the use of solid calcium sodium phosphosilicates, referred to as
bioactive glasses. One of the first bioactive glasses developed
was 45S5 Bioglass, which contained 45% SiO2, 24.5% Na2O,
24.5% CaO, and 6% P2O5 (Cao and Hench, 1996). This glass
has been studied for its ability to assist osteogenesis (Hench
et al., 1971) and repair periodontal bone defects (Lovelace et al.,
1998). For dental applications, this calcium sodium phosphosilicate glass is marketed under the name of Novamin. It has
been studied in vitro and clinically as a treatment for dentin
hypersensitivity, with the proposed mechanism being the physical occlusion of dentin tubules (Du et al., 2008). Novamin has
also been claimed by the manufacturers to have applications in
enamel subsurface remineralization, although no publications
supporting this claim could be found.

Crystalline Calcium Phosphate

Remineralizing Systems

Unstabilized Amorphous Calcium

Phosphate Systems

Calcium phosphate can exist in one of numerous crystalline phases

(Table 1). Each of these crystalline phases has different solubilities, and many have been tested as potential methods of delivering
calcium and phosphate ions to subsurface enamel lesions. The
problem with applying crystalline material to the oral cavity to
promote enamel remineralization is the poor solubility of the calcium phosphate phases, such that the calcium and phosphate ions
are unavailable for remineralization. These crystalline calcium
phosphate phases must be released from the product on contact
with saliva and then dissolve in that fluid to liberate ions capable
of diffusing into the enamel subsurface lesion. The dissolution of
the calcium phosphate phase in saliva requires that saliva be undersaturated with respect to that crystalline phase. Based on some
typical concentrations of calcium, phosphate, and fluoride ions in
saliva, the pH at which the various crystalline phases will dissolve
has been calculated (Larsen and Pearce, 2003). These calculations
show that, at the normal pH range of saliva, these crystalline calcium phosphate phases would not dissolve (Larsen and Pearce,
2003). Furthermore, localization of significant quantities of solid
calcium phosphate phases at the tooth surface is problematic
(Reynolds et al., 2003).
Brushite (Table 1) has been added to products such as dentifrices (Zhang et al., 1995; Sullivan et al., 1997) in an attempt to
enhance the remineralization of enamel subsurface lesions. Brushite
is one of the more soluble crystalline calcium phosphate phases;
however, remineralization of subsurface lesions in vivo and slowing
of caries progression in clinical trials have not been shown.
Tricalcium phosphate (TCP) (Table 1) has recently been
added to dental products that are claimed by the manufacturers
to remineralize white-spot lesions. Interestingly, the TCP is
referred to as functionalized, since it has been altered by ball
milling with sodium lauryl sulfate (Karlinsey and Mackey,

The Amorphous Calcium Phosphate (ACP) technology is an

unstabilized calcium and phosphate system that has been developed and commercialized. It is based on unstabilized amorphous
calcium phosphate, where a calcium salt (e.g., calcium sulphate)
and a phosphate salt (e.g., potassium phosphate) are delivered
separately (e.g., from a dual-chamber device) intra-orally or
delivered in a product with a low water activity (Tung and
Eichmiller, 2004). As the salts mix with saliva, they dissolve,
releasing calcium and phosphate ions. The mixing of calcium
ions with phosphate ions to produce an ion activity product for
amorphous calcium phosphate that exceeds its solubility product results in the immediate precipitation of ACP or, in the presence of fluoride ions, amorphous calcium fluoride phosphate
(ACFP). In the intra-oral environment, these phases (ACP and
ACFP) are potentially very unstable and may rapidly transform
into a more thermodynamically stable, crystalline phase (e.g.,
hydroxyapatite and fluorhydroxyapatite). However, before
phase transformation, calcium and phosphate ions should be
transiently bioavailable to promote enamel subsurface lesion
remineralization.
An ACFP-forming dentifrice has been tested in a clinical trial
studying high-caries-risk patients post-head and neck irradiation. In this study, the dentifrice forming ACFP was superior to
the fluoride-alone dentifrice in lowering root caries increment;
however, there was no significant difference in coronal caries
increment between the two products (Papas et al., 2008).
Chow et al. (2000) have investigated the ability of two solutions immediately mixed together and then used as a mouthrinse
to promote remineralization of enamel subsurface lesions, using
an in situ remineralization model. One solution contained calcium ions and the other fluoride and phosphate ions, such that
when mixed together and with saliva they would immediately

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form an amorphous calcium fluoride phosphate phase. The solution used by Chow et al. (2000) contained higher concentrations
of calcium and fluoride ions than phosphate ions, and hence the
precipitate that formed has been referred to as a calciumfluoride-like phase. However, in the presence of phosphate in
the solution, as well as in saliva, it is likely that some phosphate
would have incorporated into the initial amorphous phase that
formed as previously described (Christoffersen et al., 1988;
LeGeros, 1991). The two-solution rinse promoted more remineralization of enamel subsurface lesions than the fluoride-alone
rinse in this study. This work, along with that of Vogel et al.
(2008), who showed that pre-rinsing with a Ca solution significantly enhanced plaque fluoride retention from a separate fluoride
rinse, confirms the important role of externally applied bioavailable calcium in enhancing the intra-oral retention of fluoride and
promoting enamel subsurface lesion remineralization.
Although some of these published papers suggest that the
unstabilized ACP/ACFP technology may have efficacy in
preventing caries progression, some authors have expressed
concern with the unstabilized nature of the product that forms
intra-orally with this technology. The unstabilized ACP/ACFP
may transform to poorly soluble phases in the mouth, and, in
so doing, may act to promote dental calculus. The formation
of fluoride-containing apatite intra-orally would sequester
available fluoride ions, thereby reducing their ability to promote remineralization of subsurface enamel lesions. It is
likely, though, that some of the ACP/ACFP phases that are
produced intra-orally may be stabilized by the phosphoproteins in saliva, pellicle, and plaque that are not at full stabilization capacity. This may explain the bioavailability of these
technologies in the presence of saliva and the positive in situ
model results. However, long-term randomized controlled
caries clinical trials of the unstabilized ACP/ACFP technologies need to be conducted to demonstrate efficacy in preventing coronal caries and safety by the lack of dental calculus
promotion with long-term use.

Stabilized Amorphous Calcium

Phosphate Systems
Calcium and phosphate ions are essential for human life, and as
such, their solubility in biological systems is tightly regulated by
proteins. The consequences of disruption of this regulation can
result in pathologic calcifications such as calculi. Biological
fluids containing high concentrations of calcium and phosphate
ions also contain inhibitory ions such as pyrophosphate (Feagin
et al., 1969) and proteins to ensure stabilization. These stabilizing proteins include the caseins in milk (Holt, 1992) and
statherin in saliva (Schlesinger and Hay, 1977) (Table 2). A
biomimetic remineralization system replicating the stabilization
properties of the milk caseins and salivary statherin has been
developed based on casein phosphopeptides.

Casein Phosphopeptides
Dairy products are linked with good oral health, since they have
been shown to have anticariogenic properties in numerous
model systems (Reynolds and Johnson, 1981; Rosen et al., 1984;

J Dent Res 89(11) 2010

Harper et al., 1986; Krobicka et al., 1987). These properties have

been attributed to calcium, phosphate, and casein (Harper et al.,
1986; Silva et al., 1987). The ability of bovine milk to remineralize enamel subsurface lesions has been demonstrated in vitro by
McDougall (1977) and Mor and Rodda (1983). Casein is the
major protein group found in milk and accounts for approximately 80% of the total protein (Aimutis, 2004). In milk, casein
exists in micelles that stabilize calcium and phosphate ions. The
ability of casein to stabilize calcium and phosphate ions resides
in sequences that can be released as small peptides (casein phosphopeptides) by partial enzymic digestion. This has led to the
development of a remineralization technology based on casein
phosphopeptide-stabilized amorphous calcium phosphate complexes (CPP-ACP) (Reynolds et al., 1995; Cross et al., 2005)
[Recaldent CASRN691364495] and casein phosphopeptidestabilized amorphous calcium fluoride phosphate complexes
(CPP-ACFP) (Cross et al., 2004; Cochrane et al., 2008; Reynolds
et al., 2008). These complexes have been incorporated into commercial sugar-free chewing gums [Trident Xtra Care (Americas),
Recaldent (Japan)] and dental cream [Tooth Mousse and Tooth
Mousse Plus (Europe and Australia), MI Paste and MI Paste Plus
(Japan and Americas)].

CPP-ACP and CPP-ACFP

The casein phosphopeptides (CPP) are approximately 10%
(w/w) of the protein casein (Swaisgood, 1982). They are tasteless (Swaisgood, 1982), have low antigenicity (Park and Allen,
2000), and can be purified as CPP-ACP complexes from a
casein enzymic digest by filtration (Reynolds, 1998). CPP-ACP
has been GRAS-affirmed (Generally Recognized as Safe) by the
Food and Drug Administration of the United States of America
and other regulatory bodies around the world and can be incorporated into oral care products and foods. Four major bovine
CPPs containing the sequence Ser(P)Ser(P)Ser(P)Glu
Glu, where Ser(P) represents a phosphoseryl residue (Table 2),
have been shown to stabilize high concentrations of calcium and
phosphate ions in metastable solution supersaturated with respect
to the calcium phosphate solid phases (Cross et al., 2005) at
acidic and basic pH (Reynolds, 1997; Cochrane et al., 2008). A
1% CPP solution at pH 7.0 can stabilize 60 mM calcium and 36
mM phosphate (Reynolds et al., 1995; Reynolds, 1997).
Additionally, stabilization of calcium phosphate phases by CPP
has been shown in the presence of fluoride ions (Cross et al.,
2004; Cochrane et al., 2008). Calcium on the surfaces of the
calcium and phosphate ion clusters primarily interacts with the
CPP through the negatively charged residues of the peptides
(Cross et al., 2005). However, CPPs bind more calcium and
phosphate ions than can be attributed to just the calcium-binding
motif Ser(P)Ser(P)Ser(P)GluGlu, indicating that other
acidic residues of the phosphopeptide sequence contribute to the
stabilization of calcium phosphate (Cross et al., 2005). This interaction prevents growth of the calcium and phosphate ion clusters
to the critical size required for nucleation and phase transformations (Cross et al., 2005). This is similar to the properties of
statherin; however, the capacity of the casein phosphopeptides
is significantly greater than that of statherin, due to the higher
content of phosphoseryl and other acidic residues (Table 2).

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Enamel Remineralization 1191

In solution, an equilibrium exists between free and CPPbound calcium, phosphate, and fluoride ions. This equilibrium is
dependent on environmental factors such as pH, ion concentration, and the presence of competing binding surfaces for the
CPP (Cochrane and Reynolds, 2009). The dissociation constants
characterizing the binding of calcium and phosphate ions to the
CPP have been determined to be in the millimolar range (Park
et al., 1998; Cross et al., 2005), indicating that CPPs only
weakly bind calcium and phosphate ions, thus allowing for a
dynamic equilibrium between free and CPP-bound ions. This
therefore provides a reservoir of bioavailable ions.

Scientific Evidence for Remineralization by

CPP-ACP and CPP-ACFP
There is now a large body of scientific evidence demonstrating
that CPP-ACP and CPP-ACFP can promote the remineralization
of enamel subsurface lesions. Additionally, there is a body of
work studying the ability of CPP-ACP to prevent demineralization. Although the prevention of demineralization is often due in
part to promotion of remineralization, these studies are not
reviewed in this paper. Further information regarding the inhibition of enamel demineralization by CPP-ACP can be found in
Cochrane and Reynolds (2009). The evidence for remineralization efficacy has been shown with CPP-ACP and CPPACFP in a variety of vehicles in laboratory (Reynolds, 1997;
Cochrane et al., 2008) and human in situ experiments (Shen
et al., 2001; Cai et al., 2007; Reynolds et al., 2008), as well
as in randomized, controlled clinical trials (Andersson et al.,
2007; Morgan et al., 2008; Bailey et al., 2009; Rao et al.,
2009). The CPP-ACP literature has been reviewed by several
authors (Reynolds, 1998; Llena et al., 2009; Yengopal and
Mickenautsch, 2009), with the most recent being a systematic
meta-analysis concluding that there is sufficient clinical evidence demonstrating enamel remineralization and caries prevention by regular use of products containing CPP-ACP
(Yengopal and Mickenautsch, 2009).
One randomized, controlled caries clinical trial of CPP-ACP
assessed the impact of CPP-ACP in sugar-free gum relative to a
control sugar-free gum. This trial demonstrated that the CPPACP gum significantly slowed progression and enhanced regression of caries compared with the control sugar-free gum (Morgan
et al., 2008). In the 24-month study, 2720 schoolchildren were
randomly assigned to either a CPP-ACP or a control sugar-free
gum. All children received accepted preventive procedures,
including fluoridated water, fluoridated dentifrice, and access to
professional care. Participants were instructed to chew their
assigned gum for 10 min three times per day, with one session
supervised on school days. Standardized digital radiographs
were taken at baseline and at the completion of the trial. The
radiographs, scored by a single examiner, were assessed for
approximal caries at both the enamel and dentin levels. Analysis
of caries progression or regression was undertaken with a transition matrix. The CPP-ACP gum effected an 18% reduction in
caries progression after 24 months at the participant level, with
a 53% greater regression (remineralization) of baseline lesions
when compared with the control gum (Morgan et al., 2008).

Two randomized, controlled clinical trials of post-orthodontic white-spot lesion regression by a CPP-ACP dental cream
have been reported by Andersson et al. (2007) and Bailey et al.
(2009). The study by Andersson involved 26 individuals with
152 visible white-spot lesions on 60 incisors and canines immediately after orthodontic debonding. After bracket removal,
professional tooth cleaning and drying, visual scoring (04) and
laser fluorescence assessment were performed. The participants
were randomly assigned to two different treatment protocols
with the aim of remineralizing the lesions. One treatment was a
daily topical application of a dental cream containing crude
CPP-ACP for 3 months, followed by a 3-month period of daily
toothbrushing with a fluoride dentifrice. The other treatment
protocol was daily topical application of a 0.05% sodium fluoride mouthwash combined with the use of a fluoride dentifrice
for 6 months. Clinical examinations were repeated after 1, 3, 6,
and 12 months, and data were compared with baseline measurements. The study showed that 63% of white spots regressed in
the CPP-ACP group compared with 25% 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.
The study by Bailey et al. (2009) examined 45 individuals,
with 408 white-spot lesions immediately after orthodontic therapy, who were randomly assigned to either a dental cream containing 10% CPP-ACP treatment or a placebo cream treatment.
They were instructed to apply the products twice daily for 12
weeks after normal oral hygiene procedures (they were supplied
with dentifrice containing 1000 ppm F as NaF). The participants
also received supervised fluoride mouthrinses. Following initial
assessment, lesions were assessed at 4, 8, and 12 weeks. The
lesions were scored for lesion severity and activity according to
the International Caries Diagnosis and Assessment System II
(ICDAS II) criteria (http//www.ICDAS.org). At 12 weeks, 31%
more of the ICDAS code 2 (white spot visible when wet) and
code 3 (loss of enamel surface integrity) lesions had regressed
with the CPP-ACP cream compared with the control treatment
(Odds Ratio = 2.3, p = 0.04). In both treatment groups, active
lesions were more likely to regress than inactive lesions (Odds
Ratio = 5.07, p < 0.001). It was concluded that significantly
more post-orthodontic white-spot lesions regressed with the
CPP-ACP cream treatment over a 12-week period (Bailey et al.,
2009).
A clinical trial by Rao et al. (2009) compared the efficacy of
three dentifrices: (1) 2% CPP and calcium carbonate, (2) 1190
ppm fluoride as sodium monofluorophosphate, and (3) a placebo. One hundred fifty schoolchildren were randomly assigned
to use one of the dentifrices for 2 years. At the end of the study,
it was found that the 2% CPP/calcium carbonate dentifrice significantly reduced caries experience relative to the placebo
dentifrice, with a slightly better efficacy than the 1190-ppmfluoride dentifrice. Over 70% (72.3%) of the children remained
caries-free using the CPP/calcium carbonate dentifrice compared with 53.2% using the fluoride paste and 31.1% using the
placebo at 24 months. Since CPP/calcium carbonate in the presence of salivary phosphate would spontaneously form CPPACP, it is likely that the efficacy of this dentifrice was at least in

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Recently, randomized, double-blind, crossover studies were

conducted to investigate the potential of CPP-ACP added to
sugar confections to slow the progression of enamel subsurface
lesions in an in situ model (Walker et al., 2010). The confections
studied were: (1) control sugar (65% sucrose + 33% glucose
syrup); (2) control sugar-free (98% isomalt + acesulfame K +
aspartame); (3) sugar + 0.5% (w/w) CPP-ACP; (4) sugar + 1.0%
(w/w) CPP-ACP; and (5) sugar-free + 0.5% (w/w) CPP-ACP.
Participants wore a removable palatal appliance containing
enamel half-slabs with subsurface lesions, except for meals and
oral hygiene procedures, and consumed one confection 6 times
a day for 10 days. The enamel half-slabs were inset to allow
plaque to develop on the enamel surface. 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% CPPACP confection was significantly greater than that obtained
with the sugar-free confection; however, the sugar-free confection containing 0.5% CPP-ACP produced the greatest level of
remineralization. It was concluded that the remineralization
ability of the CPP-ACP significantly contributed to the efficacy
in slowing the progression of the lesions upon frequent sugar
challenge.

Mechanism of Action for CPP-ACP

Figure 1. A confocal microscope image showing CPP inside a CPPACP remineralized enamel subsurface lesion. After a 10-day period of
remineralization with CPP-ACP, the enamel block containing a
subsurface lesion was cut from the dentin toward the surface of the
enamel, stopping just under the subsurface lesion. The enamel was
then broken through the lesion to produce a cross-sectional surface of
the lesion interior. This broken surface was rinsed with Tris-buffered
saline (TBS) and then blocked with 1% normal goat serum in TBS. The
surface was then exposed to 0.2% anti-CPP antibody in TBS and then
thoroughly rinsed with TBS. Following the rinsing, the secondary
antibody (FITC-conjugated goat anti-rabbit IgG) was applied, followed
by further rinsing with TBS. The image was taken with an Axiovert 200
M inverted microscope (Carl Zeiss, Gttingen, Germany) fitted with a
Zeiss LSM 510 META Confocal scan head with the 458/477/488 nm
Argon laser and a 10X plan apochromatic objective. The arrow points
to the enamel surface, and the intense red staining shows the CPP in
the interior of the CPP-ACP remineralized subsurface lesion.

part related to the remineralizing properties of CPP-ACP. The

efficacy may also be partly due to the ability of the CPP-ACP to
inhibit enamel demineralization (Reynolds et al., 1982;
Reynolds, 1998).

The mechanisms of action of CPP-ACP need to be considered at

a location inside the enamel subsurface lesion, as well as at the
surface of that lesion. The CPP-ACP and CPP-ACFP have been
determined to be amorphous electroneutral nanocomplexes with
a hydrodynamic radius of 1.53 0.04 nm and 2.12 0.26 nm,
respectively (Cross et al., 2004, 2005). From the size and electroneutrality of the nanocomplexes, it would be expected that
they would enter the porosities of an enamel subsurface lesion
and diffuse down concentration gradients into the body of the
subsurface lesion (Cochrane et al., 2008; Reynolds et al., 2008).
Recently, it has been shown, with confocal laser microscopy and
fluorescently labeled anti-CPP antibodies, that CPP was present
inside a CPP-ACP remineralized enamel subsurface lesion (Fig. 1).
Once present in the enamel subsurface lesion, the CPP-ACP
would release the weakly bound calcium and phosphate ions
(Park et al., 1998; Cross et al., 2005; Cochrane and Reynolds,
2009), which would then deposit into crystal voids. The release
of the calcium and phosphate ions would be thermodynamically
driven. The CPPs have a high binding affinity for apatite (Cross
et al., 2007a); hence, on entering the lesion, the CPPs would
bind to the more thermodynamically favored surface of an apatite crystal face. Interestingly, the CPPs have been shown to
prefer binding to the (100) and (010) faces of hydroxyapatite
crystals (Fig. 2), such that crystal growth would be allowed to
continue only at the hydroxyapatite (001) plane or along the
c-axis, which is the pattern of crystal growth during amelogenesis (Huq et al., 2000). Hence, the CPPs, once bound to apatite
crystals in the enamel subsurface lesion, may have an important

J Dent Res 89(11) 2010

Enamel Remineralization 1193

Figure 2. A molecular model of the -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- motif bound onto the (100) face of hydroxyapatite (HA). The atoms are colorcoded as follows: calcium (1) atoms are light-blue crosses, calcium (2) atoms are dark-blue crosses, oxygen atoms are red, phosphorus atoms are
magenta, carbon atoms are green, nitrogen atoms are blue, and hydrogen atoms are grey. The symbol X indicates a crystallographic axis
projecting into the paper. Four views are presented: (a) a side view along the c-axis, with the peptide rendered in CPK and the crystal atoms in
line form; (b) as in (a), but viewed from above, looking down on the HA (100) face; (c) as in (b), with the peptide displayed in stick form and
the atoms in the HA surface within 0.25 nm of the peptide rendered in CPK; and (d) as in (b), with the peptide displayed in stick form and the
atoms of the peptide within 0.25 nm of the HA surface rendered in CPK.

role in regulating anisotropic crystal growth and also inhibiting

crystal demineralization (Reynolds et al., 1982).
When CPP-ACP is provided with a low background of fluoride, electron microprobe analysis has shown that the mineral
formed in the enamel lesion is consistent with hydroxyapatite,
and when fluoride is present, the mineral is consistent with fluorapatite or fluorhydroxyapatite (Cochrane et al., 2008).
Transmission electron microscopic images of demineralized and

CPP-ACFP-remineralized crystals of an enamel subsurface

lesion are shown in Fig. 3. The demineralized enamel crystals
contained numerous voids (central defects), whereas the voids
(central defects) following remineralization with CPP-ACFP
showed substantial occlusion (Fig. 3). The diffraction patterns of
this newly formed mineral were consistent with those of apatite.
The CPP-ACP nanocomplexes have also been demonstrated
to bind onto the tooth surface and into supragingival plaque to

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Cochrane et al.

J Dent Res 89(11) 2010

interactions between the CPP and

bacterial cell/pellicle surfaces, since
the peptides were released predominantly by alkaline extraction
(Reynolds et al., 2003). These results
are consistent with the proposed 3D
structure of the CPP-ACP nanocomplexes showing calcium and phosphate ion clusters encapsulated by
the surface-bound CPP (Cross et al.,
2007b). The surface-bound CPP molecules display a hydrophobic patch
on the surface of the nanocomplex
that may be responsible for the binding and localization of the complexes
at the tooth surface.
In a randomized, controlled,
Figure 3. Bright-field transmission electron micrographs of demineralized enamel showing the central
mouthrinse
trial, a rinse containing
defect of a demineralized crystal (A) and CPP-ACFP-remineralized enamel crystal (B), where the
2.0% CPP-ACP nanocomplexes
central defect (marked with an arrow) has been substantially restored. The images were taken from
plus 450 ppm fluoride significantly
the body of the lesion 40 m below the surface.
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
significantly increase the level of bioavailable calcium and
nmol F/mg dry wt of plaque attained by the use of a rinse conphosphate ions (Reynolds et al., 2003). A randomized, doubletaining the equivalent concentration of fluoride ions (Reynolds
blind, cross-over in situ study was conducted to measure calet al., 2008). Although marked increases in plaque calcium,
cium and phosphate incorporation into plaque after 5 days of
phosphate, and fluoride were found, calculus was not observed
rinsing with either water, 2% CPP-ACP, 6% CPP-ACP, or unstain any of the study participants, indicating that the plaque calbilized calcium and phosphate. The plaque calcium and phoscium, fluoride, and phosphate remained stabilized at the tooth
phate levels following rinsing with the water and the unstabilized
surface by the CPP as bioavailable ions and did not transform
calcium and phosphate rinses were similar, whereas both CPPinto a crystalline phase.
ACP rinses resulted in significantly higher incorporation of
The release of calcium, phosphate, and fluoride ions by the
calcium and phosphate ions (Reynolds et al., 2003). When the
CPP localized in plaque would be driven thermodynamically, as
plaque CPP levels were determined with competitive ELISA, it
described above. However, this process in dental plaque would
was found that 3 hours post-exposure to CPP-ACP, there
be promoted by low pH. As the pH of plaque decreased by bacremained a 4.6-fold higher peptide content than baseline levels.
terial acid production, then this would facilitate the release of
Electron microscopic analysis of immunocytochemically stained
calcium, phosphate, and fluoride ions from the complexes. The
thin sections of supragingival plaque samples (Reynolds et al.,
CPP in plaque could act as a sink for salivary calcium, phos2003) showed that the CPP-ACP nanocomplexes were localized
phate, and fluoride ions to increase the ionic content of plaque
in the plaque matrix and on the surfaces of bacterial cells, conwhen the plaque pH again rises if the peptides remain intact.
firming the work of Rose (2000a,b), who showed the CPP-ACP
However, plaque peptidases and phosphatases can degrade the
nanocomplexes bound to Streptococcus mutans and model
phosphopeptides. The dephosphorylation of the phosphoserines
plaque to produce a reservoir of bioavailable calcium ions.
of the CPP by phosphatases substantially reduces the ability of
These results are also consistent with those of Schpbach et al.
the peptides to bind calcium and phosphate ions. From the
(1996), who showed the CPP to inhibit binding of mutans strepimmunolocalization time-course study of CPP in plaque
tococci to saliva-coated enamel in vitro and in animal studies.
(Reynolds et al., 2003), the half-life of CPP in plaque was calThese authors suggested that CPP-ACP incorporates into pelliculated to be 124.8 min. Furthermore, casein has been shown to
cle and plaque and results in an ecological transition of the
be hydrolyzed by salivary sediment bacteria (Reynolds and
bacterial population, which, together with the remineralizing
Riley, 1989) in a similar timeframe; hence the decrease in deteccapacity of the CPP-ACP, modifies the plaques cariogenic
tion of the CPP in plaque is likely to be attributable to the enzypotential.
mic digestion of the peptides to fragments not recognized by the
The method of binding CPP-ACP into plaque has been hypothantibodies used for the assay (Reynolds et al., 2003). Cross et al.
esized to be due to calcium cross-linking (Rose, 2000a,b; Reynolds
(2005) have shown that the full-length CPPs are required for
et al., 2003), and/or hydrophobic and hydrogen-bond-mediated
maximal stabilization of calcium and phosphate ions; therefore,
interactions (Reynolds et al., 2003). Using acid and alkali extracenzymic hydrolysis of the CPP in plaque would reduce their
tion, to help distinguish between these mechanisms of binding,
stabilization capacity. It should be noted that the enzymic breakinvestigators found that the majority of the bonds localizing CPP
down of the CPP has been shown to produce a plaque pH rise
in the plaque were hydrophobic and/or hydrogen-bond-mediated

J Dent Res 89(11) 2010

Enamel Remineralization 1195

through the production of ammonia (Reynolds and Riley, 1989).

Hence, this process may contribute to the inhibition of demineralization and promotion of remineralization observed in situ/
in vivo by the CPP-ACP.
The released calcium, phosphate, and fluoride ions at the
enamel surface will participate in a variety of equilibria to form
a range of calcium phosphate species, depending on the pH and
fluoride availability as described by Cochrane et al. (2008). The
process of diffusion into a subsurface lesion must be an overall
electroneutral process. Therefore, diffusion potential into an
enamel subsurface lesion can be characterized by the activity
gradient of the neutral ion pair, CaHPO40, into the lesion
(Reynolds, 1997; Cochrane et al., 2008). As the neutral ion pair
diffuses down its activity gradient into the lesion, unimpeded by
the charges in plaque, pellicle, and the enamel, it will dissociate
along its diffusion path to produce calcium and phosphate ions,
capable of depositing into crystal voids promoting crystal
growth. In the case of CPP-ACFP, fluoride would then also be
provided that would allow the formation of the neutral species
CaH2FPO40 and HF0 to diffuse down activity gradients to dissociate and accelerate growth of a fluoride-containing apatite
(Cochrane et al., 2008).
The mineral gained by enamel subsurface lesions during
in situ treatment with CPP-ACP has been acid-challenged to
determine its relative solubility (Iijima et al., 2004; Cai et al.,
2007; Reynolds et al., 2008). These studies found that the CPPACP-produced mineral was more acid-resistant than the nonCPP-ACP-treated lesions. The CPP-ACP-treated lesions tended
to lose mineral only upon acid challenge below the remineralized zone compared with the control lesions, which lost mineral
throughout the lesion. These results are consistent with the production of a more stable mineral phase (e.g., hydroxyapatite or
fluorapatite, as shown by electron microprobe analysis) that has
a lower solubility than a calcium-deficient carbonated apatite of
normal tooth enamel (Iijima et al., 2004).

Other Biomimetic Approaches to

Remineralization
The biomimetic approach to enamel remineralization has
recently been extended by the use of self-assembling peptide
scaffolds to promote remineralization of enamel subsurface
lesions (Kirkham et al., 2007). Anionic synthetic peptides that,
at neutral pH, self-assemble to form a beta-sheet structure were
used to pre-treat enamel subsurface lesions in vitro. The lesions
were then subjected to in vitro pH cycling with demineralization
and remineralization solutions, according to the method of
Robinson et al. (1992). The results suggested that the peptide
treatment significantly reduced enamel demineralization as
measured by phosphate content of the demineralization solution.
The authors speculated that the inhibition of demineralization
may have been attributed to mineral formation in the lesion
induced by peptide self-assembly. Other studies have also
shown that self-assembling polymers and biomimetic peptides
based on dentin phosphophorin, which contain multiple phosphoseryl residues like the CPP, are capable of nucleating
hydroxyapatite (Hartgerink et al., 2001; Chang et al., 2006).

However, these approaches with other biomimetic peptides need

to be validated by the demonstration of enamel subsurface
lesion remineralization in situ and then ultimately in randomized, controlled clinical trials.

Future Directions
Active white-spot lesions have been shown to have a greater
likelihood of regression (remineralization) by CPP-ACP treatment compared with inactive lesions (Bailey et al., 2009).
This is presumably due to active lesions having a more porous
surface layer that allows for better penetration of the ions
required for remineralization. Therefore, means of improving
the remineralization of inactive lesions should be investigated.
Possible approaches that have been suggested include: microabrasion (Ardu et al., 2007), acid etching (Flaitz and Hicks,
1994), bleaching/deproteination (Robinson et al., 1990; Ng and
Manton, 2007), or a combination approach such as bleaching
and etching (Milnar, 2007). Bleaching appears to be an effective
method of deproteinating the lesion surface to increase porosity
inter-prismatically without the need for acid etching. However,
there is considerable scope to develop an improved method of
deproteination of the lesion surface for substantial enhancement
of enamel remineralization treatments. Development of an
effective pre-treatment to increase the surface porosity of caries
lesions without acid etching will represent a significant advance
in clinical remineralization.
Another approach to improve current remineralization systems is to improve the biomimetic peptides used to stabilize,
deliver, and control remineralization. It has been shown that
the ability of the CPPs to stabilize calcium and phosphate ions
in supersaturated solution is associated with the length and
sequence of the peptides and the specific number of phosphoseryl residues (Reynolds et al., 1982; Cross et al., 2005, 2007b).
Furthermore, the binding of the CPPs to pellicle and plaque
appears to be associated in part with the hydrophobic residues
of the peptides (Cross et al., 2007b). With modern peptide synthetic approaches (Attard et al., 2009), it is possible to incorporate additional phosphoseryl residues and alter other residues to
test the ability of the modified peptide for better stabilization
and delivery of bioavailable calcium and phosphate ions, and for
control of enamel remineralization by forming scaffolds or templates or by binding and directing anisotropic crystal growth.
This approach, together with an effective lesion pre-treatment
method, should lead to the development of a superior peptidestabilized calcium, phosphate, and fluoride remineralization
system that will represent a major advance in the non-invasive
clinical management of non-cavitated caries lesions.

Concluding Remarks
A goal of modern dentistry is the non-invasive management of
non-cavitated caries lesions involving remineralization systems
to repair the enamel with fluorapatite or fluorhydroxyapatite. In
individuals at risk of disease, procedures should be instituted to
prevent the onset of disease, and in those in whom disease is
already evident, the lesions should be treated non-invasively by

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Cochrane et al.

remineralization with bioavailable calcium, phosphate, and fluoride ions to restore the strength and aesthetic appearance of the
lesion and to increase resistance to future acid challenge.
Further study of the biomimetic molecules involved in calcium
fluoride phosphate stabilization and nucleation may provide
further improvements in the development of novel remineralization treatments. Of the remineralization technologies currently
commercially available, the CPP-ACP and CPP-ACFP technology has the most evidence to support its use.

References
Aimutis WR (2004). Bioactive properties of milk proteins with particular
focus on anticariogenesis. J Nutr 134:989S-995S.
Andersson A, Skold-Larsson K, Hallgren A, Petersson LG, Twetman S
(2007). Effect of a dental cream containing amorphous calcium phosphate complexes on white spot lesion regression assessed by laser fluorescence. Oral Health Prev Dent 5:229-233.
Ardu S, Castioni NV, Benbachir N, Krejci I (2007). Minimally invasive
treatment of white spot enamel lesions. Quintessence Int 38:633-636.
Arends J, Ten Cate JM (1981). Tooth enamel remineralization. J Crystal
Growth 53:135-147.
Attard TJ, OBrien-Simpson NM, Reynolds EC (2009). Identification and
suppression of b-elimination byproducts arising from the use of FmocSer(PO3Bzl,H)-OH in peptide synthesis. Int J Pept Res Ther 15:69-79.
Bailey DL, Adams GG, Tsao CE, Hyslop A, Escobar K, Manton DJ, et al.
(2009). Regression of post-orthodontic lesions by a remineralizing
cream. J Dent Res 88:1148-1153.
Cai F, Manton DJ, Shen P, Walker GD, Cross KJ, Yuan Y, et al. (2007).
Effect of citric acid and casein phosphopeptide-amorphous calcium phosphate to a sugar-free chewing gum on enamel remineralization in situ.
Caries Res 41:377-383.
Cao W, Hench LL (1996). Bioactive materials. Ceramics International
22:493-507.
Chang S, Chen H, Liu J, Wood D, Bentley P, Clarkson B (2006). Synthesis
of a potentially bioactive, hydroxyapatite-nucleating molecule. Calcif
Tissue Int 78:55-61.
Chow LC, Takagi S, Carey CM, Sieck BA (2000). Remineralization effects of a
two-solution fluoride mouthrinse: an in situ study. J Dent Res 79:991-995.
Christoffersen J, Christoffersen MR, Kibalczyc W, Perdok WG (1988).
Kinetics of dissolution and growth of calcium fluoride and effects of
phosphate. Acta Odontol Scand 46:325-336.
Cochrane NJ, Reynolds EC (2009). Casein phosphopeptides in oral health.
In: Food constituents and oral health: current status and future prospects. Wilson M, editor. Cambridge: CRC Press.
Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC (2008). Enamel
subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride. Caries Res 42:88-97.
Cross KJ, Huq NL, Stanton DP, Sum M, Reynolds EC (2004). NMR studies
of a novel calcium, phosphate and fluoride delivery vehicle-(S1)casein(5979) by stabilized amorphous calcium fluoride phosphate
nanocomplexes. Biomaterials 25:5061-5069.
Cross KJ, Huq NL, Palamara JE, Perich JW, Reynolds EC (2005).
Physicochemical characterization of casein phosphopeptide-amorphous
calcium phosphate nanocomplexes. J Biol Chem 280:15362-15369.
Cross KJ, Huq NL, OBrien-Simpson NM, Perich JW, Attard TJ, Reynolds
EC (2007a). The role of multiphosphorylated peptides in mineralized
tissue regeneration. Int J Pept Res Ther 13:479-495.
Cross KJ, Huq NL, Reynolds EC (2007b). Casein phosphopeptides in oral
healthchemistry and clinical applications. Curr Pharm Des 13:793-800.
Du MQ, Bian Z, Jiang H, Greenspan DC, Burwell AK, Zhong J, et al.
(2008). Clinical evaluation of a dentifrice containing calcium sodium
phosphosilicate (novamin) for the treatment of dentin hypersensitivity.
Am J Dent 21:210-214.
Feagin FF, Walker AA, Pigman W (1969). Evaluation of the calcifying
characteristics of biological fluids and inhibitors of calcification. Calcif
Tissue Int 4:231-244.

J Dent Res 89(11) 2010

Flaitz CM, Hicks MJ (1994). Role of the acid-etch technique in remineralization of caries-like lesions of enamel: a polarized light and scanning
electron microscopic study. ASDC J Dent Child 61:21-28.
Gregory TM, Moreno EC, Brown WE (1970). Solubility of dicalcium phosphate dihydrate in the system calcium hydroxideorthophosphoric
acidwater at 5, 15, 25, and 37.5 degrees Celsius. J Res Natl Bur Stds
Sec A: Phys Chem 74(A):461-475.
Gregory TM, Moreno EC, Patel JM, Brown WE (1974). Solubility of
b-Ca3(PO4)2 in the system Ca(OH)2-H3PO4-H2O at 5, 15, 25, and 37C.
J Res Natl Bur Stand 78:667-674.
Harper DS, Osborn JC, Hefferren JJ, Clayton R (1986). Cariostatic evaluation of cheeses with diverse physical and compositional characteristics.
Caries Res 20:123-130.
Hartgerink JD, Beniash E, Stupp SI (2001). Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 294:1684-1688.
Hay DI, Moreno EC (1989). Statherin and the acidic proline-rich proteins.
In: Human saliva: clinical chemistry and microbiology. Tenovuo JO,
editor. Florida: CRC Press, pp. 131-150.
Hench LL, Splinter RJ, Allen JC, Greenlee TK (1971). Bonding mechanisms
at the interface of ceramic prosthetic materials. J Biomed Mater Res
5:117-141.
Holt C (1992). Structure and stability of bovine casein micelles. Adv Protein
Chem 43:63-151.
Huq NL, Cross KJ, Reynolds EC (2000). Molecular modelling of a multiphosphorylated sequence motif bound to hydroxyapatite surfaces. J
Mol Model 6:35-47.
Iijima Y, Takagi O, Ruben J, Arends J (1999). In vitro remineralization of
in vivo and in vitro formed enamel lesions. Caries Res 33:206-213.
Iijima Y, Cai F, Shen P, Walker G, Reynolds C, Reynolds EC (2004). Acid
resistance of enamel subsurface lesions remineralized by a sugar-free
chewing gum containing casein phosphopeptide-amorphous calcium
phosphate. Caries Res 38:551-556.
Karlinsey RL, Mackey AC (2009). Solid-state preparation and dental application of an organically modified calcium phosphate. J Mater Sci
44:346-349.
Kirkham J, Firth A, Vernals D, Boden N, Robinson C, Shore RC, et al.
(2007). Self-assembling peptide scaffolds promote enamel remineralization. J Dent Res 86:426-430.
Koulourides T, Cueto H, Pigman W (1961). Rehardening of softened enamel
surfaces of human teeth by solutions of calcium phosphates. Nature
189:226-227.
Krobicka A, Bowen WH, Pearson S, Yang DA (1987). The effects of cheese
snacks on caries in desalivated rats. J Dent Res 66:1116-1119.
Larsen MJ, Pearce EI (2003). Saturation of human saliva with respect to
calcium salts. Arch Oral Biol 48:317-322.
LeGeros RZ (1991). Calcium phosphates in oral biology and medicine. San
Francisco: Karger.
Llena C, Forner L, Baca P (2009). Anticariogenicity of casein phosphopeptideamorphous calcium phosphate: a review of the literature. J Contemp
Dent Prac 10:1-9.
Lovelace TB, Mellonig JT, Meffert RM, Jones AA, Nummikoski PV,
Cochran DL (1998). Clinical evaluation of bioactive glass in the treatment
of periodontal osseous defects in humans. J Periodontol 69:1027-1035.
Mattousch TJ, Van der Veen MH, Zentner A (2007). Caries lesions after
orthodontic treatment followed by quantitative light-induced fluorescence: a two year follow-up. Eur J Orthod 29:294-298.
McCann HG (1968). The solubility of fluorapatite and its relationship to that
of calcium fluoride. Arch Oral Biol 13:987-1001.
McDougall WA (1977). Effect of milk on enamel demineralization and
remineralization in vitro. Caries Res 11:166-172.
McDowell H, Gregory T, Brown W (1972). Solubility of Ca5(PO4)3OH in
the system Ca(OH)2-H3PO4-H2O at 5, 15, 25 and 37C. J Res Natl Bur
Stds 81(A):273-281.
Meyer JL, Eanes ED (1978). A thermodynamic analysis of the amorphous
to crystalline calcium phosphate transformation. Calcif Tissue Res
25:59-68.
Milnar FJ (2007). Considering biomodification and remineralization techniques as adjuncts to vital tooth-bleaching regimens. Compend Contin
Educ Dent 28:234-236, 238-240.

J Dent Res 89(11) 2010

Enamel Remineralization 1197

Mor BM, Rodda JC (1983). In vitro remineralisation of artificial caries-like

lesions with milk. NZ Dent J 79:10-15.
Morgan MV, Adams GG, Bailey DL, Tsao CE, Fischman SL, Reynolds EC
(2008). The anticariogenic effect of sugar-free gum containing CPPACP nanocomplexes on approximal caries determined using digital
bitewing radiography. Caries Res 42:171-184.
Ng F, Manton DJ (2007). Aesthetic management of severely fluorosed incisors in an adolescent female. Aust Dent J 52:243-248.
gaard B, Rlla G, Arends J, ten Cate JM (1988). Orthodontic appliances
and enamel demineralization. Part 2. Prevention and treatment of
lesions. Am J Orthod Dentofacial Orthop 94:123-128.
Oppenheim FG, Hay DI, Franzblau C (1971). Proline-rich proteins from
human parotid saliva. I. Isolation and partial characterization. Biochemistry
10:4233-4238.
Papas A, Russell D, Singh M, Kent R, Triol C, Winston A (2008). Caries
clinical trial of a remineralising toothpaste in radiation patients.
Gerodontology 25:76-88.
Park O, Swaisgood HE, Allen JC (1998). Calcium binding of phosphopeptides derived from hydrolysis of a [sic] alpha s-casein or beta-casein
using immobilized trypsin. J Dairy Sci 81:2850-2857.
Park OJ, Allen JC (2000). Antigenicity of casein phosphopeptides prepared
with immobilized glutamic acid-specific endopeptidase or trypsin. Nutr
Res 20:359-370.
Patel PR, Brown WE (1975). Thermodynamic solubility product of human
tooth enamel: powdered sample. J Dent Res 54:728-736.
Rao SK, Bhat GS, Aradhya S, Devi A, Bhat M (2009). Study of the efficacy
of toothpaste containing casein phosphopeptide in the prevention of
dental caries: a randomized controlled trial in 12- to 15-year-old high
caries risk children in Bangalore, India. Caries Res 43:430-435.
Reynolds EC (1997). Remineralization of enamel subsurface lesions by
casein phosphopeptide-stabilized calcium phosphate solutions. J Dent
Res 76:1587-1595.
Reynolds EC (1998). Anticariogenic complexes of amorphous calcium
phosphate stabilized by casein phosphopeptides: a review. Spec Care Dentist
18:8-16.
Reynolds EC, Johnson IH (1981). Effect of milk on caries incidence and bacterial composition of dental plaque in the rat. Arch Oral Biol 26:445-451.
Reynolds EC, Riley PF (1989). Protein dissimilation by human salivarysediment bacteria. J Dent Res 68:124-129.
Reynolds EC, Riley PF, Storey E (1982). Phosphoprotein inhibition of
hydroxyapatite dissolution. Calcif Tissue Int 34(Suppl 2):52-56.
Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF, Johnson IH, et al.
(1995). Anticariogenicity of calcium phosphate complexes of tryptic
casein phosphopeptides in the rat. J Dent Res 74:1272-1279.
Reynolds EC, Cai F, Shen P, Walker GD (2003). Retention in plaque and
remineralization of enamel lesions by various forms of calcium in a
mouthrinse or sugar-free chewing gum. J Dent Res 82:206-211.
Reynolds EC, Cai F, Cochrane NJ, Shen P, Walker G, Morgan MV, et al.
(2008). Fluoride and casein phosphopeptide-amorphous calcium phosphate. J Dent Res 87:344-348.
Robinson C, Hallsworth AS, Shore RC, Kirkham J (1990). Effect of surface
zone deproteinisation on the access of mineral ions into subsurface caries lesions of human enamel. Caries Res 24:226-230.
Robinson C, Kirkham J, Baverstock AC, Shore RC (1992). A flexible and
rapid pH cycling procedure for investigations into the remineralisation
and demineralisation behaviour of human enamel. Caries Res 26:14-17.
Robinson C, Shore RC, Brookes SJ, Strafford S, Wood SR, Kirkham J
(2000). The chemistry of enamel caries. Crit Rev Oral Biol Med
11:481-495.
Rose RK (2000a). Effects of an anticariogenic casein phosphopeptide on
calcium diffusion in streptococcal model dental plaques. Arch Oral Biol
45:569-575.
Rose RK (2000b). Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide. Caries Res 34:427-431.
Rosen S, Min DB, Harper DS, Harper WJ, Beck EX, Beck FM (1984).
Effect of cheese, with and without sucrose, on dental caries and recovery of Streptococcus mutans in rats. J Dent Res 63:894-896.

Schlesinger DH, Hay DI (1977). Complete covalent structure of statherin, a

tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. J Biol Chem 252:1689-1695.
Schpbach P, Neeser JR, Golliard M, Rouvet M, Guggenheim B (1996).
Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci.
J Dent Res 75:1779-1788.
Selwitz RH, Ismail AI, Pitts NB (2007). Dental caries. Lancet 369:
51-59.
Shen P, Cai F, Nowicki A, Vincent J, Reynolds EC (2001). Remineralization
of enamel subsurface lesions by sugar-free chewing gum containing
casein phosphopeptide-amorphous calcium phosphate. J Dent Res
80:2066-2070.
Shyu LJ, Perez L, Zawacki SJ, Heughebaert JC, Nancollas GH (1983). The
solubility of octacalcium phosphate at 37C in the system Ca(OH)2-H
3PO4-KNO3-H2O. J Dent Res 62:398-400.
Silva MF, Burgess RC, Sandham HJ, Jenkins GN (1987). Effects of watersoluble components of cheese on experimental caries in humans. J Dent
Res 66:38-41.
Silverstone LM (1972). Remineralization of human enamel in vitro. Proc R
Soc Med 65:906-908.
Silverstone LM, Wefel JS, Zimmerman BF, Clarkson BH, Featherstone MJ
(1981). Remineralization of natural and artificial lesions in human
dental enamel in vitro. Effect of calcium concentration of the calcifying
fluid. Caries Res 15:138-157.
Sullivan RJ, Charig A, Blake-Haskins J, Zhang YP, Miller SM, Strannick M,
et al. (1997). In vivo detection of calcium from dicalcium phosphate
dihydrate dentifrices in demineralized human enamel and plaque. Adv
Dent Res 11:380-387.
Swaisgood H (1982). Chemistry of milk protein. In: Developments in dairy
chemistry 1: proteins. Fox PF, editor. London, UK: Elsevier Science
Publishing Co.
ten Cate JM (2001). Remineralization of caries lesions extending into dentin. J Dent Res 80:1407-1411.
ten Cate JM, Arends J (1977). Remineralization of artificial enamel lesions
in vitro. Caries Res 11:277-286.
ten Cate JM, Jongebloed WL, Arends J (1981). Remineralization of artificial
enamel lesions in vitro. IV. Influence of fluorides and diphosphonates
on short- and long-term remineralization. Caries Res 15:60-69.
ten Cate JM, Buijs MJ, Miller CC, Exterkate RA (2008). Elevated fluoride
products enhance remineralization of advanced enamel lesions. J Dent
Res 87:943-947.
Tung MS, Eichmiller FC (2004). Amorphous calcium phosphates for tooth
mineralization. Compend Contin Educ Dent 25(9 Suppl 1):S9-S13.
van der Veen MH, Mattousch T, Boersma JG (2007). Longitudinal development
of caries lesions after orthodontic treatment evaluated by quantitative lightinduced fluorescence. Am J Orthod Dentofacial Orthop 131:223-228.
Vogel GL, Schumacher GE, Chow LC, Takagi S, Carey CM (2008).
Ca pre-rinse greatly increases plaque and plaque fluid F. J Dent Res
87:466-469.
Walker GD, Cai F, Shen P, Adams GG, Reynolds C, Reynolds EC (2010).
Casein phosphopeptide-amorphous calcium phosphate incorporated
into sugar confections inhibits the progression of enamel subsurface
lesions in situ. Caries Res 44:33-40.
Wefel JS, Harless JD (1987). The use of saturated DCPD in remineralization
of artificial caries lesions in vitro. J Dent Res 66:1640-1643.
Whitford GM, Buzalaf MA, Bijella MF, Waller JL (2005). Plaque fluoride
concentrations in a community without water fluoridation: effects of
calcium and use of a fluoride or placebo dentifrice. Caries Res 39:100-107.
Willmot DR (2004). White lesions after orthodontic treatment: does low
fluoride make a difference? J Orthod 31:235-242.
Yengopal V, Mickenautsch S (2009). Caries preventive effect of casein
phosphopeptide-amorphous calcium phosphate (CPP-ACP): a metaanalysis. Acta Odontol Scand, 21:1-12.
Zhang YP, Din CS, Miller S, Nathoo SA, Gaffar A (1995). Intra-oral remineralization of enamel with a MFP/DCPD and MFP/silica dentifrice
using surface microhardness. J Clin Dent 6:148-153.

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