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Journal of Dental Research

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 DOI: 10.1177/0022034510376046

published online 25 August 2010J DENT RESN. J. Cochrane, F. Cai, N. L. Huq, M. F. Burrow and E. C. Reynolds

New Approaches to Enhanced Remineralization of Tooth Enamel  

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1

CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE

DOI: 10.1177/0022034510376046

Received December 11, 2009; Last revision March 18, 2010; Accepted May 9, 2010

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

J Dent Res X(X):xx-xx, XXXX

ABSTRACTDental 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 remineral-ization in an attempt to prevent disease progres-sion 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 deposi-tion 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 crys-talline, 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 bio-available 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 phospho-peptide-amorphous calcium phosphate, fluoride.

INTRODuCTION

Dental caries is a highly prevalent disease, and although, in most developed countries, its prevalence has declined, the disease remains a major pub-

lic 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 pre-vent 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 supersatu-rated 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 demineral-ized 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, submandibu-lar, and whole saliva are supersaturated with respect to most solid calcium phases (Larsen and Pearce, 2003) (Table 1). However, precipitation of cal-cium 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 mecha-nism 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 phos-phoproteins ensures that the ions remain bioavailable to diffuse into mineral-deficient lesions to allow for remineralization of demineralized crystals, while preventing surface deposition in the form of calculus. However, net reminer-alization 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

New Approaches to Enhanced Remineralization of Tooth Enamel

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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 light-induced fluorescence following removal of orthodontic appli-ances. The majority of the lesions were stable, with no measurable signs of regression even after two years. It was con-cluded 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 fluor-hydroxyapatite 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 con-ditions (Reynolds et al., 2008). When adequate levels of cal-cium and phosphate ions are together with the fluoride ions, it has been shown in vitro that this combination can produce sub-stantial 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 con-centrations) has been shown to give rise to predominantly sur-face remineralization (Arends and Ten Cate, 1981; ten Cate et al., 1981; Ögaard et al., 1988; Willmot, 2004). Surface-only remineralization does little to improve the aesthetics and struc-tural properties of the deeper lesion. Ideally, a remineralization system should supply stabilized bioavailable calcium, phos-phate, 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 solu-bility of the calcium phosphates, particularly in the presence of fluoride ions. Numerous authors have investigated various

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 6.6 Gregory et al., 1970

β-tricalcium phosphateb TCP β 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

Enamel apatitec 104.3-114.4 Patel and Brown, 1975

Amorphous calcium phosphate ACP Ca3(PO4)1.87(HPO4)0.2 24.8 Meyer and Eanes, 1978

aKsp = 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 (PO4

3-)6 (OH-)2.bPure ß-TCP is not found in biological systems, but is present with a Mg substitution.cCalculated in terms of ion activity product for hydroxyapatite.

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 -Asp1-Ser(P)-Ser(P)-Glu-Glu5- [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 αs1 (59–79) -Glu63-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu70 [7] Reynolds et al., 1995

CPP β (1–25) -Glu14-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu [7] Reynolds et al., 1995

CPP αs2 (46–70) -Ser(P)56-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala-Glu63 [7] Reynolds, 1998

CPP αs2 (1–21) -Ser(P)8-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln-Glu18 [7] Reynolds, 1998

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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 addi-tion of 1 ppm fluoride ions. Higher concentrations have not been used due to the instability of the solutions. No clinical reminer-alization systems have been developed using a single low-con-centration 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 types—crystalline, unstabilized amorphous, or stabilized amorphous formulations. New prod-ucts 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 non-cavitated caries lesions.

CRYSTALLINE CALCIuM PhOSPhATE REMINERALIzING SYSTEMS

Calcium phosphate can exist in one of numerous crystalline phases (Table 1). Each of these crystalline phases has different solubili-ties, 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 cal-cium 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 under-saturated 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 cal-cium 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 denti-frices (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,

2009). One paper has reported in vitro findings that this material improved surface microhardness of demineralized enamel com-pared 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 phospho-silicate 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 physi-cal 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.

uNSTABILIzED AMORPhOuS CALCIuM PhOSPhATE SYSTEMS

The Amorphous Calcium Phosphate (ACP) technology is an unstabilized calcium and phosphate system that has been devel-oped 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 prod-uct results in the immediate precipitation of ACP or, in the pres-ence 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 irradia-tion. 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 solu-tions 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 cal-cium 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 solu-tion 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 “calcium-fluoride-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 reminer-alization 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 signifi-cantly enhanced plaque fluoride retention from a separate fluoride rinse, confirms the important role of externally applied bioavail-able 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 pro-mote 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 phosphopro-teins in saliva, pellicle, and plaque that are not at full stabili-zation 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 technolo-gies need to be conducted to demonstrate efficacy in prevent-ing 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 stabiliz-ing 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;

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 remineral-ize 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 approxi-mately 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 phos-phopeptides) by partial enzymic digestion. This has led to the development of a remineralization technology based on casein phosphopeptide-stabilized amorphous calcium phosphate com-plexes (CPP-ACP) (Reynolds et al., 1995; Cross et al., 2005) [Recaldent® CASRN691364–49–5] and casein phosphopeptide-stabilized 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 com-mercial 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 taste-less (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 incor-porated 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)–Glu–Glu–, indicating that other acidic residues of the phosphopeptide sequence contribute to the stabilization of calcium phosphate (Cross et al., 2005). This inter-action prevents growth of the calcium and phosphate ion clusters to the critical size required for nucleation and phase transforma-tions (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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In solution, an equilibrium exists between free and CPP-bound calcium, phosphate, and fluoride ions. This equilibrium is dependent on environmental factors such as pH, ion concentra-tion, 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 demineraliza-tion. 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 inhibi-tion of enamel demineralization by CPP-ACP can be found in Cochrane and Reynolds (2009). The evidence for remineral-ization efficacy has been shown with CPP-ACP and CPP-ACFP 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 evi-dence demonstrating enamel remineralization and caries pre-vention 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 CPP-ACP gum significantly slowed progression and enhanced regres-sion 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 transi-tion 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-orthodon-tic 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 imme-diately after orthodontic debonding. After bracket removal, professional tooth cleaning and drying, visual scoring (0–4) 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 fluo-ride 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 measure-ments. 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 com-mented 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 ther-apy, who were randomly assigned to either a dental cream con-taining 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 pla-cebo. 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 sig-nificantly reduced caries experience relative to the placebo dentifrice, with a slightly better efficacy than the 1190-ppm-fluoride dentifrice. Over 70% (72.3%) of the children remained caries-free using the CPP/calcium carbonate dentifrice com-pared with 53.2% using the fluoride paste and 31.1% using the placebo at 24 months. Since CPP/calcium carbonate in the pres-ence of salivary phosphate would spontaneously form CPP-ACP, it is likely that the efficacy of this dentifrice was at least in

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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).

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, con-sumption of the control sugar confection resulted in significant demineralization (progression) of the enamel subsurface lesions. However, consumption of the sugar confections containing CPP-ACP did not result in lesion progression, but in fact in significant remineralization (regression) of the lesions. Remineralization by consumption of the sugar + 1.0% CPP-ACP confection was significantly greater than that obtained with the sugar-free confection; however, the sugar-free confec-tion 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

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 elec-troneutrality 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 apa-tite 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 amelogen-esis (Huq et al., 2000). Hence, the CPPs, once bound to apatite crystals in the enamel subsurface lesion, may have an important

Figure 1. A confocal microscope image showing CPP inside a CPP-ACP 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, Göttingen, 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.

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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 fluo-ride, 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 flu-orapatite 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

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 color-coded 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.

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significantly increase the level of bioavailable calcium and phosphate ions (Reynolds et al., 2003). A randomized, double-blind, cross-over in situ study was conducted to measure cal-cium and phosphate incorporation into plaque after 5 days of rinsing with either water, 2% CPP-ACP, 6% CPP-ACP, or unsta-bilized calcium and phosphate. The plaque calcium and phos-phate levels following rinsing with the water and the unstabilized calcium and phosphate rinses were similar, whereas both CPP-ACP rinses resulted in significantly higher incorporation of calcium and phosphate ions (Reynolds et al., 2003). When the plaque CPP levels were determined with competitive ELISA, it was found that 3 hours post-exposure to CPP-ACP, there remained a 4.6-fold higher peptide content than baseline levels. Electron microscopic analysis of immunocytochemically stained thin sections of supragingival plaque samples (Reynolds et al., 2003) showed that the CPP-ACP nanocomplexes were localized in the plaque matrix and on the surfaces of bacterial cells, con-firming the work of Rose (2000a,b), who showed the CPP-ACP nanocomplexes bound to Streptococcus mutans and model plaque to produce a reservoir of bioavailable calcium ions. These results are also consistent with those of Schüpbach et al. (1996), who showed the CPP to inhibit binding of mutans strep-tococci to saliva-coated enamel in vitro and in animal studies. These authors suggested that CPP-ACP incorporates into pelli-cle and plaque and results in an ecological transition of the bacterial population, which, together with the remineralizing capacity of the CPP-ACP, modifies the plaque’s cariogenic potential.

The method of binding CPP-ACP into plaque has been hypoth-esized to be due to calcium cross-linking (Rose, 2000a,b; Reynolds et al., 2003), and/or hydrophobic and hydrogen-bond-mediated interactions (Reynolds et al., 2003). Using acid and alkali extrac-tion, to help distinguish between these mechanisms of binding, investigators found that the majority of the bonds localizing CPP in the plaque were hydrophobic and/or hydrogen-bond-mediated

interactions between the CPP and bacterial cell/pellicle surfaces, since the peptides were released predomi-nantly by alkaline extraction (Reynolds et al., 2003). These results are consistent with the proposed 3D structure of the CPP-ACP nanocom-plexes showing calcium and phos-phate ion clusters encapsulated by the surface-bound CPP (Cross et al., 2007b). The surface-bound CPP mol-ecules display a hydrophobic patch on the surface of the nanocomplex that may be responsible for the bind-ing and localization of the complexes at the tooth surface.

In a randomized, controlled, mouthrinse trial, a rinse containing 2.0% CPP-ACP nanocomplexes plus 450 ppm fluoride significantly increased supragingival plaque flu-oride 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 con-taining the equivalent concentration of fluoride ions (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 cal-cium, fluoride, and phosphate remained stabilized at the tooth surface by the CPP as bioavailable ions and did not transform into a crystalline phase.

The release of calcium, phosphate, and fluoride ions by the CPP localized in plaque would be driven thermodynamically, as described above. However, this process in dental plaque would be promoted by low pH. As the pH of plaque decreased by bac-terial acid production, then this would facilitate the release of calcium, phosphate, and fluoride ions from the complexes. The CPP in plaque could act as a sink for salivary calcium, phos-phate, and fluoride ions to increase the ionic content of plaque when the plaque pH again rises if the peptides remain intact. However, plaque peptidases and phosphatases can degrade the phosphopeptides. The dephosphorylation of the phosphoserines of the CPP by phosphatases substantially reduces the ability of the peptides to bind calcium and phosphate ions. From the immunolocalization time-course study of CPP in plaque (Reynolds et al., 2003), the half-life of CPP in plaque was cal-culated to be 124.8 min. Furthermore, casein has been shown to be hydrolyzed by salivary sediment bacteria (Reynolds and Riley, 1989) in a similar timeframe; hence the decrease in detec-tion of the CPP in plaque is likely to be attributable to the enzy-mic digestion of the peptides to fragments not recognized by the antibodies used for the assay (Reynolds et al., 2003). Cross et al. (2005) have shown that the full-length CPPs are required for maximal stabilization of calcium and phosphate ions; therefore, enzymic hydrolysis of the CPP in plaque would reduce their stabilization capacity. It should be noted that the enzymic break-down of the CPP has been shown to produce a plaque pH rise

Figure 3. Bright-field transmission electron micrographs of demineralized enamel showing the central defect of a demineralized crystal (A) and CPP-ACFP-remineralized enamel crystal (B), where the central defect (marked with an arrow) has been substantially restored. The images were taken from the body of the lesion 40 µm below the surface.

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through the production of ammonia (Reynolds and Riley, 1989). Hence, this process may contribute to the inhibition of deminer-alization 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, CaHPO4

0, 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 CaH2FPO4

0 and HF0 to diffuse down activity gradients to dis-sociate 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 CPP-ACP-produced mineral was more acid-resistant than the non-CPP-ACP-treated lesions. The CPP-ACP-treated lesions tended to lose mineral only upon acid challenge below the remineral-ized zone compared with the control lesions, which lost mineral throughout the lesion. These results are consistent with the pro-duction 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 phos-phoseryl 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 random-ized, controlled clinical trials.

FuTuRE DIRECTIONS

Active white-spot lesions have been shown to have a greater likelihood of regression (remineralization) by CPP-ACP treat-ment 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: micro-abrasion (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 sys-tems 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 phosphos-eryl 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 syn-thetic approaches (Attard et al., 2009), it is possible to incorpo-rate 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 tem-plates 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 peptide-stabilized 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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remineralization with bioavailable calcium, phosphate, and flu-oride 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 remineraliza-tion treatments. Of the remineralization technologies currently commercially available, the CPP-ACP and CPP-ACFP technol-ogy has the most evidence to support its use.

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