article WS

R E V I E W Australian Dental Journal 2012; 57: 404–408

doi: 10.1111/adj.12006

Casein phosphopeptide-amorphous calcium phosphate: a remineralizing agent of enamel

D Gurunathan,* S Somasundaram,* SA Kumar�

*Department of Pedodontics, Saveetha Dental College, Chennai, India. �Department of Orthodontics, Saveetha Dental College Chennai, India.

ABSTRACT

The pathogenesis of enamel caries involves a succession of demineralization and remineralization activities. The net effect can result in lesion consolidation when the redeposited minerals improve the resistance of the surface to the extent that the usual level of cariogenic activity cannot cause mineral dissolution. Since it has been established that the caries process is a continuum, albeit one that is interrupted numerous times daily, it is therefore possible to intervene at any stage with a therapeutic product or an intervention methodology. Incorporation of remineralizing treatments into routine dental care programmes will have a strong impact on the public health aspects of caries control. A recent advancement in the phenomenon of remineralization is the casein phosphopeptides. These casein phosphopeptides localize the amorphous calcium phosphate and are proved to be anticariogenic. This review discusses the mechanism of remineralization by casein phosphopeptide-amorphous calcium phosphate and its incorporation into various products.

Keywords: Dental caries, enamel remineralization, preventive tool.

Abbreviations and acronyms: ACP = amorphous calcium phosphate; CD-CP = casein derivatives coupled with calcium phosphate; CPP = casein phosphopeptide; CPP-ACP = casein phosphopeptide-amorphous calcium phosphate; CSMH = cross-sectional microhardness; HA = hydroxyapatite; NaF = sodium fluoride; SFGD = slow-release fluoride glass device.

(Accepted for publication 13 May 2012.)

INTRODUCTION

Dental caries covers the continuum from the first atomic level of demineralization, through the initial enamel followed by dentinal involvement, to eventual cavitation. The dynamic balance between demineral- ization and remineralization determines the end result. At its most basic level, dental caries is the result of a process whereby the mineral constituents of the tooth, principally calcium and phosphate, are demineralized by organic acids produced by bacteria dwelling in a biofilm adhering to the tooth surface.1 Calcium and phosphate are lost from the subsurface enamel, result- ing in the formation of subsurface lesion. At this early stage, the caries lesion is reversible via a remineraliza- tion process involving the diffusion of calcium and phosphate ions into the subsurface lesion to restore lost molecular structure.

Milk is an excellent protein food that provides essential amino acids and organic nitrogen for humans and animals of all ages. Milk also contains factors that have anticariogenic properties such as calcium,

phosphate, casein and lipids. Dairy products were recognized in the late 1950s as a food group that is effective in preventing dental caries. Epidemiological studies indicate children and adolescents with a low incidence of dental caries drank more milk than those with a high caries incidence.2

Casein is the predominant phosphoprotein in bovine milk present primarily as calcium phosphate stabilized micellular complexes and accounts for almost 80% of its total protein.3 A group of peptides, known as casein phosphopeptides (CPP), have been shown to stabilize calcium and phosphate, preserving them in an amor- phous or soluble form termed as amorphous calcium phosphate (ACP). CPP containing the active sequence – Ser(P)-Ser(P)-Ser(P)-Glu-Glu- has a remarkable ability to stabilize calcium and phosphate as nanoclusters of ions in a metastable solution.4 ACP, (Ca3(PO4)

2· 3H2O), is a postulated precursor in the formation of hydroxyapatite (HA). The ACPs exhibit a very high solubility and are readily converted to hydroxyapatite, which makes them suitable mineralizing agents.5 The main advantage of ACP is its facile, single solid phase

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formulation and its biocompatibility with both hard and soft tissues, which is equal to that of hydroxyap- atite and various di-, tri- and tetracalcium phosphates.6

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 and do not localize effectively at the tooth surface. In addition, acid is required to produce ions capable of diffusing into enamel subsurface lesions. In contrast, soluble calcium and phosphate ions can be used only at very low concentrations due to the intrinsic insoluble nature of the calcium phosphates. Hence, 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. To overcome these difficulties a new calcium phosphate remineralization technology has been developed based on casein phosphopeptide- amorphous calcium phosphate (CPP-ACP), where CPP stabilizes high concentrations of calcium and phosphate ions, together with fluoride ions, at the tooth surface by binding to pellicle and plaque.7

Though the concept of using CPP-ACP as a remin- eralizing agent was introduced in 1998,8 using casein for caries prevention was addressed as early as the 1980s.9 ACP technology was introduced in the early 1990s7 and has demonstrated anticariogenic activity in laboratory, animal and human in situ experiments.10–12

It is established that remineralization by consumption of the sugar confection with 1.0% CPP-ACP is greater than that obtained with the sugar-free confection.13

This has led to the incorporation of CPP-ACP into food products and dental products as a new tool in the fight against caries.

Mechanism of anticariogenic activity

Prevention of dental caries by milk-derived bioactive peptides is a complex physical and chemical sequence of cascading events. In general, bioactive peptides with anticariogenic activity have multiple functions to pre- vent dental lesions including bacterial inhibition, com- petitive exclusion to enamel binding sites, improved buffering capacity in the pellicle surrounding teeth, reduced enamel demineralization and enhanced enamel remineralization.12,14

Mechanism of action in remineralization

CPP-bound ACP acts as a reservoir of calcium and phosphate ions including the neutral ion pair CaHPO4. Under acidic conditions, the CPP-bound ACP would buffer plaque pH and in doing so would dissociate to calcium phosphate ions including CaHPO4. The acid is

generated by dental plaque bacteria or during forma- tion of HA in the enamel lesion during remineraliza- tion. The increase in plaque calcium and phosphate ions and ion pairs would offset any fall in pH, thereby preventing enamel demineralization. CPP-ACP solu- tions are efficient remineralizing solutions as they would consume the acid generated during enamel lesion remineralization, leading to more CaHPO4 and thus maintaining its concentration gradient into the lesion.9

The CPP, by stabilizing calcium phosphate in a metastable solution, facilitates high concentrations of calcium and phosphate ions, including CaHPO4. The CPP will also maintain the high activities of the free calcium and phosphate ions during remineralization through the reservoir of bound ACP. The bound ACP, by being in dynamic equilibrium with free calcium and phosphate ions, will maintain the concentrations of the species involved in diffusion into the lesion.9

The mineralization process involves diffusion of CaHPO4 and associated calcium and phosphate ions through the protein and water filled pores of carious surface enamel into the body of the enamel lesion. Once in the body of the enamel lesion, these calcium and phosphate species, by dissociation, would increase the activities of Ca2+ and PO4

3- and thereby increase the degree of saturation with respect to HA. The formation of HA in the lesion would lead to the generation of acid and phosphate, including the neutral phosphoric acid, which would diffuse out of the lesion down a concen- tration gradient.9

Reynolds and colleagues15,16 reported that CPP-ACP binds readily to the surface of the tooth, as well as to the bacteria in the plaque10 surrounding the tooth. In this way, CPP-ACP deposits a high concentration of ACP in close proximity to the tooth surface. Therefore under acidic conditions, this localized CPP-ACP buffers the free calcium and phosphate ions, substantially increasing the level of calcium phosphate in plaque and maintaining a state of supersaturation that inhibits enamel demineralization and enhances remineraliza- tion. 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.17 CPP-ACP showed significant reminer- alization of artificial caries under scanning electron microscope18,19 and has been proved to be more effective than 500 ppm of NaF.20 This explains the effectiveness of CPP-ACP as an efficient remineralizing agent.

Casein and plaque

A lower susceptibility of the mandibular anterior teeth to caries is related to the higher calcium and inorganic

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phosphate levels in plaque taken from these teeth compared with plaque taken from other areas of the mouth. Indeed, there is an inverse association of plaque calcium and inorganic phosphate levels with caries experience.21 CPP-ACP have been found to increase the levels of calcium and phosphate in plaque up to five- fold in human in situ caries models, thereby acting as a calcium-phosphate reservoir. Hence, a state of super- saturation with respect to enamel mineral is main- tained, thereby depressing enamel demineralization and enhancing remineralization mechanism.22 An in situ caries model showed that exposure of inset-enamel plaque to solutions containing tryptic peptides of casein significantly reduced enamel subsurface demineraliza- tion.14 As low as 0.1% of CPP-ACP prevented mineral loss and provided a potential source of calcium for subsequent remineralization, thereby restricting the caries process.23 Marchisio et al.25 in an in vivo evaluation of CPP-ACP (Recaldent molecule) con- cluded that 48% of patients had an increase in plaque and salivary pH level.24

CPP-ACP in chewing gum

The results of various in vivo studies indicated that the addition of CPP-ACP to sugar-free chewing gum significantly enhanced remineralization of enamel sub- surface lesions in a dose-related manner, independent of gum weight or type.22,25–27 A xylitol-containing gum with CPP-ACP was shown to produce a dose-related increase in enamel remineralization.27

Moreover, the addition of CPP-ACP to citric acid flavoured gum negated the effect of the citric acid and produced a remineralizing effect greater than that of neutral sugar-free gum without citric acid.8,17 In a randomized, double-blind crossover in situ study superior remineralization activity was attributed to the presence of CPP-ACP nanocomplexes in sugar-free chewing gum.26

CPP-ACP in mouthrinse

CPP-ACP containing mouthrinse significantly increased plaque calcium and inorganic phosphate levels, and the CPP were found to be immunolocalized to the surfaces of bacterial cells as well as the intercellular matrix.22

In a randomized, controlled mouthrinse trial, a rinse containing 2.0% CPP-ACP plus 450 ppm fluoride significantly increased supragingival plaque fluoride ion content than that attained by the use of a rinse containing the equivalent concentration of fluoride ions. Calculus was not observed in any of the study participants, even with an increase in plaque calcium, phosphate and fluoride, 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.28 When a mouthrinse that contained casein derivatives coupled with calcium phosphate (CD-CP) was compared with a sodium fluoride (NaF) mouthrinse in subjects with salivary gland dysfunction, no difference was found between the two mouthrinses, with regard to caries preventive efficacy.29

CPP-ACP in sealants

Sealants based on ACP-filled methacrylate composites have the potential to remineralize carious enamel lesions.30 Incorporation of 1.56% w ⁄ w CPP-ACP into the glass ionomer cement showed an enhanced protec- tion of the adjacent dentine during acid challenge in vitro.31

CPP-ACP in milk

Addition of 2.0–5.0 g CPP-ACP to milk substantially increases its ability to remineralize enamel subsurface lesions.32 The remineralizing effect of CPP-ACP in milk was dose-dependent with milk containing 0.2% CPP- ACP and 0.3% CPP-ACP producing an increase in mineral content of 81% and 164%, respectively, relative to the control milk.33

Interaction of CPP-ACP with fluoride

It has been reported that CPP-ACP interacts with fluoride ions to produce novel nanoclusters of calcium, fluoride and phosphate ions. The identification of this novel CPP-stabilized form of calcium, fluoride and phosphate ions is consistent with the observed additive anticariogenic effects of the CPP-ACP nanocom- plexes.34 A dentifrice containing 2% CPP-ACP nano- complexes plus 1100 ppm F has been shown to be superior (2.6 times) to a dentifrice containing only 1100 ppm F in remineralization of enamel subsurface lesions in situ with mineral that was more resistant to acid challenge.28 CPP-ACP containing tooth mousse remineralized initial enamel lesions and it has also shown a higher remineralizing potential when applied as a topical coating after the use of a fluoridated toothpaste.35

A combination of slow-release fluoride glass device (SFGD) and CPP-ACP nanocomplexes has shown a significant improvement of surface microhardness of demineralized enamel unlike individual components.36

The results indicate that the CPP is an excellent delivery vehicle to localize bioavailable calcium, fluoride and phosphate ions at the tooth surface to remineralize subsurface enamel lesions with fluorapatite.28

However, Lata et al.37 evaluated the cross-sectional microhardness (CSMH) of subsurface remineralization using fluoride, ACP-CPP and a combination of both

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and observed that combination does not provide any additive remineralization potential compared to fluo- ride alone.

CONCLUSIONS

This review has revealed CPP-ACP as a safe and novel carrier for calcium, phosphate and fluoride ions to promote enamel remineralization. The calcium phos- phate-based remineralization technologies show prom- ise as adjunctive treatment to fluoride therapy in the management of early caries lesions. 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. Hence, CPP-ACP has application in oral care products, dental professional products and foodstuffs.

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Address for correspondence: Dr G Deepa

Reader Department of Pedodontics

Saveetha Dental College 162 Poonamalle High Road

Chennai 77 India

Email: [email protected]

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