Acceleration of Enamel Subsurface Lesion Remineralisation by Intralesion pH Modulation.

Remineralisation of demineralised enamel subsurface lesions can be enhanced by pretreatment of the lesions with base (NaOH). The aim of this study was to test the effect of intralesion pH modulation on remineralisation of demineralised enamel subsurface lesions by casein phosphopeptide-stabilised amorphous calcium fluoride phosphate (CPP-ACFP) in vitro. Two remineralisation models were utilised, the first involving 60-min cyclic pH modulation for 105 h and the second involved short-term cyclic pH modulation (12-min cycle, 240 min total duration) compared with the equivalent time of continuous treatment (200 min total duration). The intralesion pH modulation was achieved by cyclic exposure to a pH 12.9 NaOH solution and a CPP-ACFP remineralisation solution at pH 5.5. Percent remineralisation was assessed using transverse microradiography with data statistically analysed using a 2-sample Student t test. For the first model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (43.8 ± 6.9%) than the control group (28.2 ± 5.8%) cycled with water. For the second model, the intralesion pH modulation group had significantly (p < 0.001) higher remineralisation (23.1 ± 3.4%) than the group with continuous equivalent CPP-ACFP treatment time (1.9 ± 1.3%). In both models, intralesion pH modulation significantly accelerated remineralisation, and this was attributed to the effect pH modulation had on the diffusion gradients of ions/ion pairs and the degree of saturation with respect to apatite phases within the lesion fluid.

Effects of Bovine Serum Albumin and High pH Pre-Treatment on the Remineralisation of Enamel Subsurface Lesions in vitro.

Accumulated intra-lesion protein such as serum albumin has been speculated to impede remineralisation of carious enamel lesions. The aim of this study was to assess whether intra-lesion bovine serum albumin (BSA) affected subsequent remineralisation of enamel subsurface lesions. Confocal microscopy was used to confirm localisation of BSA in artificial enamel subsurface lesions and its subsequent degradation by a high pH sodium hypochlorite treatment. An in vitro remineralisation experiment tested the effect of intra-lesion BSA, and its degradation by sodium hypochlorite, on remineralisation of subsurface lesions by casein phosphopeptide stabilised amorphous calcium fluoride phosphate. In addition, lesions without BSA were pre-treated with one of 2 high pH solutions (sodium hypochlorite or sodium hydroxide) prior to remineralisation to test whether the high pH pre-treatment influenced remineralisation. Data were obtained on remineralisation using transverse microradiography and were analysed with a one-way ANOVA. Intra-lesion BSA had no significant effect on remineralisation compared with that of control lesions. Pre-treatment of BSA-containing lesions with sodium hypochlorite significantly increased remineralisation. The lesions without BSA that were pre-treated with either sodium hypochlorite or sodium hydroxide also showed the same level of remineralisation as the BSA-containing lesions pre-treated with sodium hypochlorite indicating that the increased remineralisation was pH related. Hence, it was concluded that intra-lesion BSA did not affect remineralisation of artificial enamel subsurface lesions in this model system and that a high pH pre-treatment enhanced remineralisation.

Effects of soy and bovine milk beverages on enamel mineral content in a randomized, double-blind in situ clinical study.

Soy beverages are promoted as healthy alternatives to bovine milk even though they can contain added sugar. OBJECTIVES: To compare enamel mineral content after consumption of bovine milk or a soy beverage in a double-blind, randomized, cross-over in situ clinical study. MATERIALS AND METHODS: Human enamel slabs with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers who consumed 200 ml of either bovine milk or a soy beverage over a 60 s period once a day for 15 days. Enamel lesion depth and mineral content were measured using transverse microradiography. Saliva samples were collected immediately after consuming the beverages and calcium, inorganic phosphate and fluoride levels analysed. Data were statistically analysed using a linear mixed model. RESULTS: Depth of the enamel subsurface lesions increased by 7.1 ±â€¯2.0 µm and mineral content decreased by 47 ±â€¯22 vol% min.µm after consumption of the soy beverage indicating demineralization. However, after consumption of bovine milk the depth of the lesions decreased by 7.6 ±â€¯3.5 µm and mineral content increased by 202 ±â€¯43 vol% min.µm indicating remineralization. The changes were significantly different (p < 0.001) between the two beverages. Fluoride levels were similar in the saliva samples for both beverages, however the calcium and inorganic phosphate levels for the bovine milk group were significantly higher (p < 0.02) than those for the soy beverage group. CONCLUSIONS: In this randomized, double-blind in situ clinical trial consumption of a soy beverage demineralized enamel whereas bovine milk produced remineralization. CLINICAL SIGNIFICANCE: Although soy beverages are promoted as healthy alternatives to bovine milk the added sugar and low calcium bioavailability of the soy drink makes frequent consumption a caries risk. (Trial registration no. ISRCTN19137849).

Self-assembly of dental surface nanofilaments and remineralisation by SnF2 and CPP-ACP nanocomplexes.

Dental caries, erosion and hypersensitivity are major public health problems. SnF2 is used widely in oral care products to help prevent/treat these conditions. Casein phosphopeptide-stabilised amorphous calcium phosphate nanocomplexes (CPP-ACP) are a biomimetic nanotechnology of salivary phosphopeptide-ACP complexes that deliver bioavailable calcium and phosphate ions to promote dental remineralisation (repair). We show here using in vitro studies and a double-blind, randomised controlled, cross-over design in situ clinical trial that SnF2 and CPP-ACP interact to form a nanofilament coating on the tooth surface and that together they are superior in their ability to promote dental remineralisation. Sn(II) by cross-linking the CPP-ACP helps to stabilise the complexes which improves delivery to the tooth surface and enhances binding and ion incorporation into tooth mineral. The combination of SnF2 and CPP-ACP in oral care products may significantly improve their efficacy in prevention/treatment of dental caries/erosion and hypersensitivity.

Importance of bioavailable calcium in fluoride dentifrices for enamel remineralization.

OBJECTIVES: To compare remineralization of enamel subsurface lesions by fluoride dentifrices with added calcium in a double-blind, randomized, cross-over, in situ study. METHODS: Human enamel with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1 g toothpaste/4 ml H2O) was rinsed for 60 s, 4 times per day for 14 days. Seven toothpastes were tested: (i) 1450 ppm F (NaF), (ii) 5000 ppm F (NaF), (iii) 1450 ppm F (MFP) with calcium sodium phosphosilicate (CSP), (iv) 1450 ppm F (MFP) with CaCO3/Arg, (v) 1150 ppm F (SnF2) with amorphous calcium phosphate (ACP), (vi) 1100 ppm F (NaF) with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and (vii) 5000 ppm F (NaF) with functionalized tri-calcium phosphate (TCP). Total (acid soluble) and bioavailable (water soluble) calcium, inorganic phosphate and fluoride levels of the dentifrices were measured using ion chromatography (F/MFP) and spectrophotometry (Ca and inorganic phosphate). Enamel lesion mineral content was measured using transverse microradiography. Data were statistically analysed using a linear mixed model. RESULTS: All calcium and fluoride containing toothpastes released > 90% of bioavailable fluoride and were superior to the respective fluoride alone toothpastes in remineralization of enamel subsurface lesions. The level of remineralization followed the order: CPP-ACP/1l00 ppm F > ACP/1150 ppm F = TCP/5000 ppm F > 5000 ppm F = CaCO3/Arg/1450 ppm F = CSP/1450 ppm F > 1450 ppm F. Bioavailable calcium levels significantly correlated with enhanced remineralization of enamel subsurface lesions. CONCLUSIONS: Bioavailable calcium in fluoride dentifrices enhanced remineralization of enamel subsurface lesions.

The Interactions of CPP-ACP with Saliva.

The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP-ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP-ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP-ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP-ACP complexes are localised at the tooth surface to promote remineralisation.

The potential acidogenicity of liquid breakfasts.

OBJECTIVES: To determine the potential acidogenicy of liquid breakfasts. METHODS: In vitro acid production by Streptococcus mutans was measured in the beverages at a pH of 5.5, as was the fall in pH over 10min. The buffering capacity was determined, as well as the calcium, inorganic phosphate and fluoride concentrations (total and soluble) of the beverages. Bovine milk (UHT) was used for comparison. RESULTS: The rate of acid production by S. mutans, and pH fall over 10min was greater in liquid breakfasts compared to bovine milk. All beverages except one demonstrated a significantly lower buffering capacity than bovine milk. All beverages contained significantly greater concentrations of soluble calcium than bovine milk, and all except two contained significantly more soluble inorganic phosphate. CONCLUSIONS: S. mutans was able to generate significantly more acid in the liquid breakfasts than in bovine milk, indicating these drinks may contribute to a cariogenic diet. In general, the liquid breakfasts required significantly less acid than bovine milk to reduce their pH to the approximate critical pH for enamel demineralisation. However, the liquid breakfasts also tended to contain significantly more soluble calcium and inorganic phosphate than bovine milk. CLINICAL SIGNIFICANCE: The substantial amounts and various types of sugars found within liquid breakfast beverages may result in a significant pH drop in dental plaque following consumption of these products.

Streptococcus mutans biofilm disruption by κ-casein glycopeptide.

UNLABELLED: Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities. OBJECTIVES: To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions. METHODS: Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn). RESULTS: CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37µm and a biovolume of 3.88µm(3)/µm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity. CONCLUSIONS: KCG and KCG-Zn may have potential as natural biofilm disruptive agents.

Porphyromonas gingivalis cysteine proteinase inhibition by kappa-casein peptides.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study κ-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide κ-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as κ-casein(117-137) using synthetic peptides. Kinetic studies revealed that κ-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with κ-casein(109-137) significantly reduced lesion development in a murine model of infection.