N.J. Cochrane

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.

Enamel Subsurface Lesion Remineralisation with Casein Phosphopeptide Stabilised Solutions of Calcium, Phosphate and Fluoride

Casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) and amorphous calcium fluoride phosphate (CPP-ACFP) solutions have been shown to remineralise enamel subsurface lesions. The aim of this study was to determine the effect of ion composition of CPP-ACP and CPP-ACFP solutions on enamel subsurface lesion remineralisation in vitro. CPP-bound and free calcium, phosphate and fluoride ion concentrations in the solutions were determined after ultrafiltration. The ion activities of the free ion species present were calculated using an iterative computational program. The mineral deposited in the subsurface lesions was analysed using transverse microradiography and electron microprobe. CPP was found to stabilise high concentrations of calcium, phosphate and fluoride ions at all pH values (7.0–4.5). Remineralisation of the subsurface lesions was observed at all pH values tested with a maximum at pH 5.5. The CPP-ACFP solutions produced greater remineralisation than the CPP-ACP solutions at pH 5.5 and below. The mineral formed in the subsurface lesions was consistent with hydroxyapatite and fluorapatite for remineralisation with CPP-ACP and CPP-ACFP, respectively. The activity gradient of the neutral ion pair CaHPO4⁰ into the lesion was significantly correlated with remineralisation and together with HF⁰ were identified as important species for diffusion.

Remineralization of enamel subsurface lesions in situ by the use of three commercially available sugar‐free gums

BACKGROUND Commercially available sugar-free chewing gums have been claimed to provide oral health benefits. AIM The aim of this randomized, double-blind crossover in situ study was to compare the efficacy of three commercially available sugar-free chewing gums: Trident White, Orbit, and Orbit Professional, in remineralizing enamel subsurface lesions in situ. DESIGN Specimens containing enamel subsurface lesions were sectioned into test and control half-slabs with the test half-slabs inserted into removable palatal appliances. For each test chewing period, subjects were randomly allocated one of three test gums. Subjects (n = 10) chewed the randomly allocated gum for a 20-min period four times per day for 14 days. Each subject chewed all three test gums, with a 7-day washout period between crossovers. After each 14-day cycle, test and control half-slabs were paired, embedded in resin, sectioned, and subjected to microradiography to determine remineralization. RESULTS The gum TW produced significantly greater remineralization (18.4 +/- 0.9%) than Orbit (8.9 +/- 0.5%) and Orbit Professional (10.5 +/- 0.9%). CONCLUSION The superior remineralization activity of the TW gum in situ was attributed to the presence of casein phosphopeptide-amorphous calcium phosphate nanocomplexes.

Calcium phosphopeptides -- mechanisms of action and evidence for clinical efficacy.

Phosphoproteins/phosphopeptides with clusters of acidic residues are found throughout nature, where they aid in the prevention of unwanted precipitation of solid calcium phosphates. The acidic residues, particularly phosphoserine, interact with calcium and stabilize clusters of calcium and phosphate. Saliva and milk are two examples of biological fluids that contain such phosphoprotein/phosphopeptide-stabilized calcium phosphates, and both share a similar evolutionary pathway. Saliva has been shown to have remineralization potential and is of critical importance in maintaining the mineral content of teeth in the oral environment. Milk can be enzymatically modified to release casein phosphopeptides that contain the clusters of residues that allow milk to stabilize high concentrations of calcium and phosphate. These casein phosphopeptide-stabilized amorphous calcium phosphate nanocomplexes (CPP-ACP) can stabilize even higher concentrations of calcium and phosphate than milk and can be considered a salivary biomimetic, since they share many similarities to statherin. The mechanisms of action and the growing body of scientific evidence that supports the use of CPP-ACP to augment fluoride in inhibiting demineralization and enhancing the remineralization of white-spot lesions are reviewed.

Effect of added calcium phosphate on enamel remineralization by fluoride in a randomized controlled in situ trial.

UNLABELLED Dental products containing calcium phosphate and fluoride are claimed to enhance enamel remineralization over fluoride products. OBJECTIVES To compare remineralization of enamel subsurface lesions by dental products with added calcium phosphate in a double-blind, randomized, cross-over in situ study. METHODS Human enamel specimens with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1g product plus 4 ml H(2)O) of each product was rinsed for 60s, 4 times per day for 10 days. Six products were tested (i) placebo, (ii) 1000 ppm F, (iii) 5000 ppm F, (iv) Tooth Mousse (TM), (v) TM plus 900 ppm F (TMP) and (vi) Clinpro with 950 ppm F. Calcium, inorganic phosphate and fluoride levels were measured in post-rinse/saliva samples using ion chromatography. Mineral content was measured using transverse microradiography. RESULTS Only TM and TMP significantly increased salivary calcium and phosphate levels. The products produced remineralization in the following order from lowest to highest: placebo<1000 ppm F=Clinpro<5000 ppm F<TM<TMP. CONCLUSION Clinpro was not significantly different to 1000 ppm F whereas TM and TMP were superior to 5000 ppm F with TMP producing the highest level of enamel lesion remineralization.

Consumption of milk with added casein phosphopeptide-amorphous calcium phosphate remineralizes enamel subsurface lesions in situ.

BACKGROUND Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) is an anticariogenic agent that is suitable to be added to foods. The aim of this double-blind, three-way crossover randomized study was to investigate the capacity of CPP-ACP, when added to bovine milk, to remineralize enamel subsurface lesions in situ. METHODS Ten subjects drank 100 mL of bovine milk containing no added CPP-ACP (control milk), 0.2% (w/v) CPP-ACP or 0.3% (w/v) CPP-ACP, for 30 seconds once daily for 15 days, whilst wearing removable appliances with attached slabs of enamel containing subsurface enamel lesions. After each treatment and a one-week washout period, subjects crossed over to another treatment and this was repeated until they had consumed each of the three milk products. At the completion of each treatment the enamel slabs were removed and remineralization was determined using microradiography. RESULTS The results demonstrated that all three milk samples remineralized enamel subsurface lesions in situ. However, the two milk samples containing added CPP-ACP each produced significantly greater remineralization than the control milk. CONCLUSIONS 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.

Effect of casein phosphopeptide-amorphous calcium phosphate added to acidic beverages on enamel erosion in vitro.

BACKGROUND To investigate, in vitro, the effect on enamel erosion of the addition of 0.2% w/v casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) to four commercially-available soft drinks, two of which were carbonated. METHODS Enamel specimens (n=27) were sectioned from sound extracted human third molar teeth and polished to a mirror finish. Exposed enamel windows of 1 mm2 were created by painting the surface with acid-resistant nail varnish. Four citric flavoured soft drinks (pH range 2.2 to 2.4) and distilled deionized water (DDW) were tested. Each drink was tested with and without 0.2% CPP-ACP w/v. The specimens were placed into 50 mL of solution at 37 °C for 30 minutes, rinsed and varnish removed. The samples were profiled with a white light profilometer and erosive depths recorded. RESULTS All soft drinks tested caused enamel erosion but adding 0.2% w/v CPP-ACP significantly reduced (p<.05) erosive depth in all test solutions in comparison with the solutions without CPP-ACP. The erosive depths for all solutions with 0.2% CPP-ACP did not differ significantly from those of DDW. CONCLUSIONS Adding CPP-ACP at 0.2% w/v significantly decreased the erosivity of all four soft drinks. The erosivity of the soft drinks with 0.2% CPP-ACP added did not differ significantly from that of distilled water.

An X-ray Microtomographic Study of Natural White-spot Enamel Lesions

White-spot enamel lesions are an early presentation of dental caries and are ideally managed by non-invasive procedures. The aim of this study was to characterize white-spot enamel lesions by x-ray microtomography. In particular, mineral content across the lesion from the surface to the base of the lesion was measured and surface layers defined. Molars with long buccal white-spot enamel lesions were collected, photographed, and each sectioned to produce 3 500-µm-thick sections. The sections were mounted and imaged by quantitative x-ray microtomography at a 15-µm voxel size. We analyzed line profiles through the middle of each 3D image to determine mineral content and depth. The surface layer thickness of the lesions ranged from 35 to 130 µm, with the maximum mineral content in this layer being 74% to 100% of that of sound enamel. The average mineral content across the lesions ranged from 1.73 to 2.48 g/cm3. No significant differences could be found between lesions clinically categorized as active and those categorized as inactive. However, for depth-matched active and inactive lesions, the active lesions exhibited a more porous surface layer than the inactive lesions. White-spot enamel lesions are highly variable, with surface layers of considerable thickness.

Remineralisation by Chewing Sugar-Free Gums in a Randomised, Controlled in situ Trial Including Dietary Intake and Gauze to Promote Plaque Formation

Remineralisation has been shown to be an effective mechanism of preventing the progression of enamel caries. The aim of this double-blind, randomised, cross-over in situ study was to compare enamel remineralisation by chewing sugar-free gum with or without casein phosphopeptide amorphous calcium phosphate (CPP-ACP) where the enamel lesions were exposed to dietary intake and some were covered with gauze to promote plaque formation. Participants wore removable palatal appliances containing 3 recessed enamel half-slabs with subsurface lesions covered with gauze and 3 without gauze. Mineral content was measured by transverse microradiography, and plaque composition was analysed by real-time polymerase chain reaction. For both the gauze-free and gauze-covered lesions, the greatest amount of remineralisation was produced by the CPP-ACP sugar-free gum, followed by the gum without CPP-ACP and then the no-gum control. Recessing the enamel in the appliance allowed plaque accumulation without the need for gauze. There was a trend of less remineralisation and greater variation in mineral content for the gauze-covered lesions. The cell numbers of total bacteria and streptococci were slightly higher in the plaque from the gauze-covered enamel for 2 of the 3 treatment legs; however, there was no significant difference in Streptococcus mutans cell numbers. In conclusion, chewing sugar-free gum containing CPP-ACP promoted greater levels of remineralisation than a sugar-free gum without CPP-ACP or a no-gum control using an in situ remineralisation model including dietary intake irrespective of whether gauze was used to promote plaque formation or not.