Calcium phosphate-based remineralization systems: scientific evidence?

Authors


Professor EC Reynolds
Centre for Oral Health Science
School of Dental Science
The University of Melbourne
720 Swanston Street
Melbourne, Victoria 3010
Email: e.reynolds@unimelb.edu.au

Abstract

Dental caries remains a major public health problem in most communities even though the prevalence of disease has decreased since the introduction of fluorides. The focus in caries research has recently shifted to the development of methodologies for the detection of the early stages of caries lesions and the non-invasive treatment of these lesions. Topical fluoride ions, in the presence of calcium and phosphate ions, promote the formation of fluorapatite in tooth enamel by a process referred to as remineralization. The non-invasive treatment of early caries lesions by remineralization has the potential to be a major advance in the clinical management of the disease. However, for net remineralization to occur adequate levels of calcium and phosphate ions must be available and this process is normally calcium phosphate limited. In recent times three calcium phosphate-based remineralization systems have been developed and are now commercially available: a casein phosphopeptide stabilized amorphous calcium phosphate (RecaldentTM (CPP-ACP), CASRN691364-49-5), an unstabilized amorphous calcium phosphate (ACP or EnamelonTM) and a bioactive glass containing calcium sodium phosphosilicate (NovaMinTM). The purpose of this review was to determine the scientific evidence to support a role for these remineralization systems in the non-invasive treatment of early caries lesions. The review has revealed that there is evidence for an anticariogenic efficacy of the EnamelonTM technology for root caries and for the RecaldentTM technology in significantly slowing the progression of coronal caries and promoting the regression of lesions in randomized, controlled clinical trials. Hence the calcium phosphate-based remineralization technologies show promise as adjunctive treatments to fluoride therapy in the non-invasive management of early caries lesions.

Abbreviations and acronyms:
ACP

amorphous calcium phosphate

ACFP

amorphous calcium fluoride phosphate

CPP

casein phosphopeptides

FA

fluorapatite

Introduction

Dental caries is a pathological process of localized destruction of tooth tissue by micro-organisms. The disease is initiated via the demineralization of tooth hard tissue by organic acids produced from fermentable carbohydrate by dental plaque cariogenic bacteria. Even though in most developed countries the prevalence of dental caries has decreased through the use of fluorides, the disease remains a major public health problem.1

Fluoride ions promote the formation of fluorapatite in enamel in the presence of calcium and phosphate ions produced during enamel demineralization by plaque bacterial organic acids.2 This is now believed to be the major mechanism of fluoride ion’s action in preventing enamel demineralization.2,3 Fluoride ions can also drive the remineralization of previously demineralized enamel if enough salivary or plaque calcium and phosphate ions are available when the fluoride is applied. The non-invasive treatment of early caries lesions by remineralization has the potential to be a major advance in the clinical management of the disease. However, for every two fluoride ions, 10 calcium ions and six phosphate ions are required to form one unit cell of fluorapatite (Ca10(PO4)6F2). Hence on topical application of fluoride ions, the availability of calcium and phosphate ions can be the limiting factor for net enamel remineralization to occur and this is highly exacerbated under xerostomic conditions.

The clinical use of calcium and phosphate ions for remineralization has not been successful in the past due to the low solubility of calcium phosphates, particularly in the presence of fluoride ions. Insoluble calcium phosphates are not easily applied, do not localize effectively at the tooth surface and require acid for solubility to produce ions capable of diffusing into enamel subsurface lesions. On the other hand, soluble calcium and phosphate ions can only be used at very low concentrations due to the intrinsic insolubility of the calcium phosphates, in particular the calcium fluoride phosphates. Soluble calcium and phosphate ions do not substantially incorporate into dental plaque or localize at the tooth surface to produce effective concentration gradients to drive diffusion into the subsurface enamel. Three calcium phosphate-based remineralization systems have now been commercialized where the manufacturers claim the specific form of the calcium phosphate helps overcome the limited bioavailability of calcium and phosphate ions for the remineralization process (Table 1).

Table 1.   Commercially available calcium phosphate-based remineralization technologies
TechnologyCommercial productRemineralization claim
Casein phosphopeptide stabilized calcium phosphate (RecaldentTM, CPP-ACP)Trident White sugar-free gum, Recaldent sugar-free gum, Tooth Mousse, MI pasteRecaldentTM (CPP-ACP) a remineralizing ingredient that strengthens teeth by delivering calcium and phosphate to the tooth’s surface.
Unstabilized amorphous calcium phosphate (ACP, EnamelonTM)Enamel Care with liquid calcium, Nite White ACP, Day White ACP, Mentadent replenishing whiteRebuilds enamel. The deposition of hydroxyapatite onto teeth rebuilds enamel through a process called remineralization.
Bioactive glass containing calcium sodium phosphosilicate (NovaMinTM)Oravive toothpasteNourishes the teeth with essential calcium and phosphorous ions needed for the natural self-repair process of the teeth.

The first technology involves casein phosphopeptide stabilized amorphous calcium phosphate (RecaldentTM (CPP-ACP), CASRN691364-49-5) where it is claimed that the casein phosphopeptides (CPP) stabilize high concentrations of calcium and phosphate ions, together with fluoride ions, at the tooth surface by binding to pellicle and plaque. Although the calcium, phosphate and fluoride ions are stabilized by the CPP from promoting dental calculus, the ions are freely bioavailable to diffuse down concentration gradients into enamel subsurface lesions thereby effectively promoting remineralization in vivo.

The second technology is an unstabilized amorphous calcium phosphate (ACP or EnamelonTM). This technology applies calcium ions (e.g., calcium sulphate) and phosphate ions (e.g., ammonium phosphate, sometimes in the presence of fluoride ions) separately (e.g., from a dual chamber device) so that amorphous calcium phosphate or amorphous calcium fluoride phosphate forms intra-orally. More recently, this technology is also being used in bleach-based whitening products (Table 1). The manufacturers claim that the formation of amorphous calcium phosphate intra-orally helps rebuild tooth enamel through remineralization.

The third technology is a bioactive glass containing calcium sodium phosphosilicate (NovaMinTM) where the manufacturers claim that the glass particles release calcium and phosphate ions intra-orally to promote remineralization (Table 1).

In this paper the scientific evidence for each technology is reviewed. The scientific evidence was based on published original studies (papers and abstracts) reporting the testing of the technologies in various caries model systems and in randomized, controlled caries trials (Table 2).

Table 2.   Scientific evidence for anticaries activity and enamel subsurface lesion remineralization
TechnologyInhibition of caries in an animal modelInhibition of enamel demineralization in vitroPromotion of enamel subsurface lesion remineralization in vitroInhibition of enamel demineralization in situPromotion of enamel subsurface lesion remineralization in situInhibition of caries progression and promotion of regression of caries in a randomized controlled clinical trial
  1. ‡Faller RV, Pfarrer AM. Effects on remineralization and acid resistance from conventional and ‘remineralizing’ toothpastes. J Dent Res Sp Iss Abstract 1998;77:188.

RecaldentTM/CPP-ACPYes
(Reynolds et al., 1995)11
Yes
(Reynolds, 1998)23
(Yamaguchi et al., 2006)24
Yes
(Reynolds, 1997)22
Yes
(Reynolds, 1998)23
(Reynolds, 1987)21
Yes
(Shen et al., 2001)16
(Iijima et al., 2004)15
Yes
(Morgan et al., 2008)19
ACP/EnamelonTMYes
(Mundorff-Shrestha et al., 1999)4
(Grant et al., 1999)20
Yes
(Schemehorn et al., 1999b)7
(Hicks and Flaitz, 2000)8
Yes
(Schemehorn et al., 1999a)6
No references found Yes
Inhibition of root caries in a radiation therapy population but no reduction in coronal caries relative to control (1150 ppmF) (Papas et al., 1999)9
No
(Landrigan et al., 1998)
(Best et al., 1998)
No
(Eversole et al., 1998)
No
(Eversole et al., 1998)
(Faller and Pfarrer, 1998)‡
 No
(Faller et al., 1998)
 
NovaMinTMNo references foundNo references foundNo references foundNo references foundNo references foundNo references found

NovaMinTM technology

The NovaMinTM technology is based on calcium sodium phosphosilicate bioactive glass which is claimed to release calcium and phosphate ions intra-orally to help the self-repair process of teeth. As shown in Table 2, no published studies could be found supporting the remineralization of enamel subsurface lesions in vitro or in situ. Furthermore, no published studies could be found showing an anticariogenic efficacy of NovaMinTM in animal models or other caries model systems or randomized, controlled caries clinical trials. This technology appears to be at a very early stage of development.

EnamelonTM (ACP) technology

The EnamelonTM technology is based on unstabilized ACP, where a calcium salt (e.g., calcium sulphate) and a phosphate salt (e.g., ammonium phosphate) are delivered separately (e.g., from a dual chamber device) intra-orally. 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 ACP which 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 very unstable and rapidly transform to a more thermodynamically stable, insoluble crystalline phase (e.g., hydroxyapatite and fluorhydroxyapatite). However, before the phases transform calcium and phosphate ions should be transiently bioavailable to inhibit demineralization of enamel and promote enamel subsurface lesion remineralization.

Several papers have been published providing some evidence of efficacy of the EnamelonTM technology in model systems (Table 2). Two studies using the rat caries model have shown a superior efficacy of a dentifrice containing the EnamelonTM technology plus fluoride over a standard fluoride-alone dentifrice.4,5 However, two other reports (abstracts) suggest that the EnamelonTM technology plus fluoride was in fact inferior to the standard fluoride-alone dentifrice in the rat caries model.* This discrepancy may relate to how the EnamelonTM technology was delivered to the teeth of the animals as once the calcium salts and phosphate salts are mixed from the dual chamber device the ACP would immediately start to transform to an insoluble phase with little efficacy. Hence, a greater time between mixing the salts and the application to the teeth would result in a lower potential efficacy. Further, efficacy would be model sensitive and these different outcomes reported could also relate to differences in diet, particularly the level of calcium, phosphate and fluoride of the cariogenic diet.

A superior efficacy of the EnamelonTM technology plus fluoride over fluoride alone has also been suggested from in vitro cyclic demineralization assays by Schemehorn et al.,6,7 and by Hicks and Flaitz.8 However, two other reports (abstracts) from Eversole et al. and Faller et al. indicated that the EnamelonTM technology plus fluoride was in fact inferior to a standard fluoride dentifrice in in vitro cyclic demineralization assays. These authors suggested that the fluoride was less available in the EnamelonTM product. Only one in situ study on the EnamelonTM technology could be found. This study by Faller et al. compared the EnamelonTM technology plus fluoride with fluoride alone in an in situ remineralization model and concluded that the fluoride alone was superior to the EnamelonTM technology plus fluoride in promoting fluoride uptake into enamel subsurface lesions in situ. However, in this study subsurface remineralization using microradiography was not determined.

No published papers could be found demonstrating that the EnamelonTM technology could slow progression of coronal caries or promote regression of coronal caries in a randomized, controlled caries clinical trial. However, one small clinical trial of the technology in a group of high caries risk, head and neck radiation patients has been published.9 In this study no significant difference was found in coronal caries increment with the EnamelonTM plus fluoride dentifrice compared with that of the standard fluoride dentifrice. However, a significant difference in root caries increment was observed with the EnamelonTM plus fluoride dentifrice producing a lower increment than the standard fluoride dentifrice.

Although some of these published papers suggest that the EnamelonTM technology may have efficacy in preventing caries progression several authors have expressed concern with the unstabilized nature of the ACP that forms intra-orally with this technology. The unstabilized ACP rapidly transforms to crystalline phases in the mouth and in so doing may act to promote dental calculus. In the presence of fluoride ions the unstabilized ACP may produce fluorapatite. The formation of fluorapatite intra-orally would sequester available fluoride ions thereby reducing their ability to remineralize subsurface enamel during acid challenge.

Casein phosphopeptide amorphous calcium phosphate technology

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (RecaldentTM (CPP-ACP)) is a technology based on ACP stabilized by casein phosphopeptides (CPP). CPP containing the cluster sequence –Ser(P)-Ser(P)-Ser(P)-Glu-Glu- stabilize ACP in metastable solution.10 Through the cluster sequence the CPP bind to forming nanoclusters of ACP preventing their growth to the critical size required for nucleation and phase transformation.10 The CPP-ACP technology has been demonstrated to have anticariogenic activity in laboratory, animal and human in situ experiments as well as a randomized, controlled caries clinical trial (Table 2).

The CPP-ACP nanocomplexes have been shown to reduce caries activity in the rat caries model.11 Solutions (100 μl) containing different concentrations of CPP-ACP were applied to the animals’ molar teeth twice daily. Other groups of animals received 100 μl of either 500 ppm fluoride ions (positive control) or distilled water (negative control). The animals consumed a highly cariogenic sucrose/gluten diet that did not contain dairy products. The CPP-ACP significantly reduced caries activity in a dose-response fashion with 0.1% w/v CPP-ACP producing a 14% reduction and 1.0 % w/v CPP-ACP a 55% reduction relative to the distilled water control. CPP-ACP at 0.5% w/v produced a reduction in caries activity similar to that of 500 ppm fluoride. A solution containing both 0.5% w/v CPP-ACP and 500 ppm fluoride produced a significantly greater reduction in caries activity than either CPP-ACP or fluoride alone at the same concentrations.

The CPP-ACP technology has also been demonstrated to significantly increase the levels of calcium and phosphate ions in supragingival plaque when delivered in a mouthrinse and to promote the remineralization of enamel subsurface lesions in situ.12 In fact, in a mouthrinse clinical study, the CPP-ACP technology was shown to be superior to other forms of calcium phosphate including unstabilized ACP.12 These studies highlight the importance of the CPP in stabilizing the high levels of calcium and phosphate ions but also in delivering the ions to the tooth surface. Electron microscopic analysis of immunocytochemically stained thin sections of supragingival plaque samples12 showed that the CPP-ACP nanocomplexes were localized in the plaque matrix and on the surface of bacterial cells (Fig 1) confirming the work of Rose13,14 who showed the CPP-ACP nanocomplexes bound tightly to Streptococcus mutans and model plaque to produce a reservoir of bioavailable calcium ions.

Figure 1.

 The incorporation of the CPP-ACP nanocomplexes into supragingival plaque from a mouthrinse. (Reproduced with permission from 12.)

The ability of the CPP-ACP technology added to sugar-free chewing gum to remineralize enamel subsurface lesions has been demonstrated in several randomized, controlled, double-blind in situ clinical studies.12,15,16 The sugar-free gums (control and CPP-ACP containing gums) were chewed for either 20-minute periods, four times a day or for 5-minute periods, seven times a day. Microradiography and computer-assisted densitometric image analysis demonstrated that, independent of gum type and chewing duration (e.g., 20 minutes or 5 minutes), the CPP-ACP nanocomplexes produced a dose-related remineralization of enamel subsurface lesions in situ. Gum containing 18.8 mg and 56.4 mg of the CPP-ACP nanocomplexes, chewed for 20 minutes, four times per day for 14 days, increased enamel subsurface remineralization by 101 per cent and 151 per cent, respectively, relative to the control sugar-free gum. Microradiographs of the enamel lesions before and after remineralization showed that the CPP-ACP nanocomplexes promoted remineralization throughout the body of the lesion. Electron microprobe wavelength dispersive spectrometric analyses of sections of the remineralized enamel indicated that the mineral deposited was hydroxyapatite with a higher Ca:P ratio than normal apatite. Acid challenge of the enamel remineralized by the CPP-ACP nanocomplexes in situ showed that the remineralized apatite was more resistant to acid challenge than the normal calcium-deficient carbonated tooth enamel.15

It has been reported that the CPP-ACP nanocomplexes interact with fluoride ions to produce a novel ACFP phase.17,18 The identification of this novel ACFP phase is consistent with the observed additive anticariogenic effect of the CPP-ACP nanocomplexes and F.11,18 The anticariogenic mechanism of fluoride is the localization of the fluoride ion at the tooth surface, particularly in plaque in the presence of calcium and phosphate ions. This localization increases the degree of saturation with respect to fluorapatite (FA), thus promoting remineralization of enamel with FA during an acid challenge. It is clear that for the formation of FA (Ca10(PO4)6F2), calcium and phosphate ions must also be present with the fluoride ions. The reported additive anticariogenic effect of the CPP-ACP nanocomplexes and F therefore may be attributable to the localization of ACFP at the tooth surface by the CPP, which co-localizes calcium, phosphate and fluoride as bioavailable ions in the correct molar ratio to form fluorapatite. In a randomized, controlled, mouthrinse trial, a rinse containing 2.0% CPP-ACP nanocomplexes plus 450 ppm fluoride significantly increased supragingival plaque fluoride ion content to 33.0 ± 17.6 nM F/mg dry wt of plaque when compared to 14.4 ± 6.7 nM F/mg dry wt of plaque attained by use of a rinse containing the equivalent concentration of fluoride ions as sodium fluoride.18 Although marked increases in plaque calcium, phosphate and fluoride were found, calculus was not observed in any of the subjects, indicating that the plaque calcium fluoride phosphate remained stabilized at the tooth surface by the CPP as bioavailable ions and did not transform into a crystalline phase. These results indicate that the CPP act as a delivery vehicle to co-localize bioavailable calcium, fluoride and phosphate ions at the tooth surface. A dentifrice formulation containing 2% CPP-ACP nanocomplexes plus 1100 ppm F has been shown to be superior (2.6 times) to a dentifrice containing only 1100 ppm F in remineralization of enamel subsurface lesions with mineral that was more resistant to acid challenge (Fig 2).18 The CPP-ACP nanocomplexes plus fluoride dentifrice resulted in significantly greater incorporation of the fluoride into the subsurface enamel as fluorapatite as shown by electron microprobe wavelength dispersive spectrometry.18

Figure 2.

 Representative microradiographs of enamel subsurface lesions after remineralization in situ by various dentifrice formulations and acid challenge (AC) in vitro. The percentage enamel remineralization is shown in each panel as %R. (Modified from 18 and reproduced with permission.)

A randomized, controlled caries clinical trial of CPP-ACP-containing sugar-free chewing gum demonstrated that the CPP-ACP gum significantly slowed progression of caries and enhanced regression of caries compared with the control sugar-free gum.19 In the two-year study, 2720 school children were randomly assigned to either a test or control sugar-free gum. All subjects received accepted preventive procedures, including fluoridated water, fluoridated dentifrice and access to professional care. Subjects were instructed to chew their assigned gum for 10 minutes, 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 dentine level. Analysis of caries progression or regression was undertaken using a transition matrix. The CPP-ACP gum effected a significant 18 per cent reduction in caries progression after 24 months at the subject level and a 53 per cent greater regression (remineralization) of baseline lesions when compared with the control gum.19 These results are consistent with the proposed anticariogenic mechanism of the CPP-ACP technology being the inhibition of enamel demineralization and enhancement of remineralization through the localization of bioavailable calcium and phosphate ions at the tooth surface.

Conclusions

The RecaldentTM (CPP-ACP) technology has been shown to remineralize enamel subsurface lesions in situ and to significantly slow the progression of coronal caries and promote the regression of caries in a randomized, controlled clinical trial. Furthermore, the EnamelonTM (ACP) technology has been shown to decrease root caries increment in a clinical trial of high caries risk, head and neck radiation patients. Hence the new calcium phosphate-based remineralization technologies look promising as adjunctive treatments to topical fluorides in the non-invasive management of early caries lesions.

Footnotes

  • *

    Best JM, Eversole SL, Faller RV. Remineralization potential of conventional and novel toothpastes: rat model testing. J Dent Res Sp Iss Abstract 1998;77:246.
    Landrigan WF, Eversole SL, Best JM, Faller RV. Animal caries efficacy of conventional and ‘remineralizing’ toothpastes. J Dent Res Sp Iss Abstract 1998;77:843.

  • Eversole SL, Faller RV, Bitten ER, Featherstone JDB. Conventional and ‘remineralizing’ toothpastes compared in two pH cycling models. J Dent Res Sp Iss Abstract 1998;77:843.
    Faller RV, Eversole SL, Kelly SA, Lukantsova L, Dunipace AJ. In situ comparison of conventional fluoride and ‘remineralizing’ toothpastes. J Dent Res Sp Iss Abstract 1998;77:1017.

Ancillary