Eric C. Reynolds

Remineralization of Enamel Subsurface Lesions by Casein Phosphopeptide-stabilized Calcium Phosphate Solutions

Casein phosphopeptides (CPP) stabilize amorphous calcium phosphate (ACP), localize ACP in dental plaque, and are anticariogenic in animal and in situ human caries models. In this in vitro study, CPP-stabilized calcium phosphate solutions were shown to remineralize subsurface lesions in human third-molar enamel. Solutions were used to examine the effect of CPP-calcium phosphate concentration on remineralization. Other solutions were used to examine the effect of increasing pH, which decreased the concentrations of free calcium and phosphate ions and increased the level of CPP-bound ACP. Although most of the remineralizing solutions were supersaturated with respect to the amorphous and crystalline calcium phosphate phases, the solutions were stabilized by the CPP such that spontaneous precipitation of calcium phosphate did not occur. After a ten-day remineralization period, enamel lesions were sectioned, subjected to microradiography, and the mineral content determined by microdensitometry. All solutions deposited mineral into the bodies of the lesions, with the 1.0% CPP-calcium phosphate (pH 7.0) solution replacing 63.9 ±20.1% of mineral lost at an averaged rate of 3.9 ± 0.8 x 10-8 mol hydroxyapatite/m2/s. The remineralizing capacity was greater for the solutions with the higher levels of CPP-stabilized free calcium and phosphate ions. Remineralization was not significantly correlated with either the CPP-bound ACP or the degrees of saturation for hydroxyapatite, octacalcium phosphate, or ACP. However, remineralization was significantly correlated with the degree of saturation for dicalcium phosphate dihydrate (CaHPO4.2H2O), but this was attributed to the significant correlation of remineralization with the activity gradients from the solution into the lesion of some calcium phosphate ions and ion pairs, in particular the neutral ion pair CaHPO4 0. The CPP, by stabilizing calcium phosphate in solution, maintain high-concentration gradients of calcium and phosphate ions and ion pairs into the subsurface lesion and thus effect high rates of enamel remineralization.

Retention in Plaque and Remineralization of Enamel Lesions by Various Forms of Calcium in a Mouthrinse or Sugar-free Chewing Gum

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes incorporated into sugar-free chewing gum have been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to compare the ability of CPP-ACP, with that of other forms of calcium, to be retained in supragingival plaque and remineralize enamel subsurface lesions in situ when delivered in a mouthrinse or sugar-free gum in randomized, double-blind trials. In the mouthrinse study, only the CPP-ACP-containing mouthrinse significantly increased plaque calcium and inorganic phosphate levels, and the CPP were immunolocalized to the surfaces of bacterial cells as well as the intercellular matrix. In the chewing gum studies, the gum containing the CPP-ACP, although not containing the most calcium per piece of gum, produced the highest level of enamel remineralization independent of gum-chewing frequency and duration. The CPP could be detected in plaque extracts 3 hrs after subjects chewed the CPP-ACP-containing gum. The results showed that CPP-ACP were superior to other forms of calcium in remineralizing enamel subsurface lesions.

Remineralization of Enamel Subsurface Lesions by Sugar-free Chewing Gum Containing Casein Phosphopeptide-Amorphous Calcium Phosphate:

Casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP) exhibit anticariogenic potential in laboratory, animal, and human in situ experiments. The aim of this study was to determine the ability of CPP-ACP in sugar-free chewing gum to remineralize enamel subsurface lesions in a human in situ model. Thirty subjects in randomized, cross-over, double-blind studies wore removable palatal appliances with six human-enamel half-slabs inset containing subsurface demineralized lesions. The appliances were inserted immediately before gum-chewing for 20 min and then retained for another 20 min. This was performed four times per day for 14 days. At the completion of each treatment, the enamel half-slabs were paired with their respective demineralized control half-slabs, embedded, sectioned, and subjected to microradiography and densitometric image analysis, for measurement of the level of remineralization. The addition of CPP-ACP to either sorbitol- or xylitol-based gum resulted in a dose-related increase in enamel remineralization, with 0.19, 10.0, 18.8, and 56.4 mg of CPP-ACP producing an increase in enamel remineralization of 9, 63, 102, and 152%, respectively, relative to the control gum, independent of gum weight or type.

New Approaches to Enhanced Remineralization of Tooth Enamel

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.

Fluoride and Casein Phosphopeptide-Amorphous Calcium Phosphate

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) slows the progression of caries and remineralizes enamel subsurface lesions. The aim of this study was to determine the ability of CPP-ACP to increase the incorporation of fluoride into plaque and to promote enamel remineralization in situ. Randomized, double-blind, cross-over studies involved mouthrinses and dentifrices containing CPP-ACP and fluoride. The mouthrinses were used for 60 sec, three times/day for 5 days, and supragingival plaque was collected and analyzed for F. The dentifrices were rinsed as a water slurry for 60 sec four times/day for 14 days in an in situ model. The addition of 2% CPP-ACP to the 450-ppm-F mouthrinse significantly increased the incorporation of fluoride into plaque. The dentifrice containing 2% CPP-ACP produced a level of remineralization similar to that achieved with a dentifrice containing 2800 ppm F. The dentifrice containing 2% CPP-ACP plus 1100 ppm F was superior to all other formulations.

Acid Resistance of Enamel Subsurface Lesions Remineralized by a Sugar-Free Chewing Gum Containing Casein Phosphopeptide-Amorphous Calcium Phosphate

The aim of this clinical study was to investigate the acid resistance of enamel lesions remineralized in situ by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP: Recaldent™). The study utilized a double-blind, randomized, crossover design with two treatments: (i) sugar-free gum containing 18.8 mg of CPP-ACP, and (ii) sugar-free gum not containing CPP-ACP as control. Subjects wore removable palatal appliances with insets of human enamel containing demineralized subsurface lesions and chewed the gum for 20 min 4 times per day for 14 days. After each treatment the enamel slabs were removed and half of each lesion challenged with acid in vitro for 8 or 16 h. The level of remineralization was determined using microradiography. The gum containing CPP-ACP produced approximately twice the level of remineralization as the control sugar-free gum. The 8- and 16-hour acid challenge of the lesions remineralized with the control gum resulted in 65.4 and 88.0% reductions, respectively, of deposited mineral, while for the CPP-ACP-remineralized lesions the corresponding reductions were 30.5 and 41.8%. The acid challenge after in situ remineralization for both control and CPP-ACP-treated lesions resulted in demineralization underneath the remineralized zone, indicating that the remineralized mineral was more resistant to subsequent acid challenge. The results show that sugar-free gum containing CPP-ACP is superior to an equivalent gum not containing CPP-ACP in remineralization of enamel subsurface lesions in situ with mineral that is more resistant to subsequent acid challenge.

Physicochemical Characterization of Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

The Prevention of Sub-surface Demineralization of Bovine Enamel and Change in Plaque Composition by Casein in an Intra-oral Model

The ability of bovine milk phosphoprotein (casein) to be incorporated into plaque, prevent enamel sub-surface demineralization, and affect bacterial composition was determined using a modified intra-oral caries model. The intra-oral model consisted of a removable appliance containing a left and right pair of bovine enamel slabs placed to simulate an approximal area. Supragingival plaque was collected and impacted into the left and right inter-enamel spaces. The left side of the appliance was exposed to various sugar and salt solutions, while the right side was exposed to sugar and casein solutions. Sodium caseinate, the major fraction αs1-casein, and a tryptic digest of αs1-casein (TD-casein) were studied. Sodium caseinate at a level of 2% w/v in a 3% sucrose-3% glucose-salt solution (pH 7.0) prevented sub-surface enamel demineralization over a ten-day period as shown by microradiography and microhardness. Two exposures of a 2% wlv sodium caseinate, αs1-casein, or TD-casein solution (pH 7.0) per day prevented sub-surface enamel demineralization caused by six exposures of a 3% sucrose-3% glucose-salt solution per day over a ten-day period. Intact αs1-casein and tryptic peptides were shown immunochemically to be incorporated into the inter-enamel plaque. The incorporation of casein and its breakdown in plaque did not produce a significant change in the amount or composition of plaque bacteria. The ability of casein and tryptic peptides to prevent enamel demineralization was related to their incorporation into plaque, thereby increasing plaque calcium phosphate and acid-buffering capacity by the phosphoseryl, histidyl, glutamyl, and aspartyl residues and indirectly through catabolism by plaque bacteria.

Kappacin, a Novel Antibacterial Peptide from Bovine Milk

ABSTRACT Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans andPorphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated κ-casein (residues 106 to 169) [κ-casein(106–169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)149κ-casein-A(138–158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)149κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)149 κ-casein-A(138–158) displayed growth-inhibitory activity against S. mutans(MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Calcium phosphate‐based remineralization systems: scientific evidence?

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 (Recaldent (CPP-ACP), CASRN691364-49-5), an unstabilized amorphous calcium phosphate (ACP or Enamelon) and a bioactive glass containing calcium sodium phosphosilicate (NovaMin). 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 Enamelon technology for root caries and for the Recaldent 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.