Effect of fluoride-releasing filling materials on underlying dentinal lesions in vitro.

Fluoride-releasing materials placed over carious tissue are assumed to enhance remineralisation of the underlying lesion. This remineralisation, however, also depends on the availability of calcium and phosphate, which may be supplied by the pulpal fluid. The aim of this study was to measure the fluoride release of glass ionomer cements (GICs) into underlying dentin and to measure the effect of the released fluoride on the remineralisation of the underlying dentinal lesions using transversal microradiography. Discs of fluoride-releasing GIC were placed on top of dentinal lesions in an in vitro model. The discs and the dentin slabs were covered completely by a protective layer of nail varnish, leaving only the pulpal side of the dentin slab open, and hence the dentinal tubules as the pathway for the incubation fluid to the GIC disc. Specimens were incubated in a remineralisation buffer. The materials tested were a conventional GIC, an experimental GIC that was designed to have a high fluoride release, and an inert material. Fluoride was found to penetrate through the dentin slab into the surrounding fluid. Fluoride uptake from the experimental GIC was higher than from the conventional GIC. Mineral content-depth profiles after 10 weeks' remineralisation revealed that in the outer 30 microm of the lesion a higher mineral deposition occurred for the experimental GIC than in both other groups. No differences in the overall change of integrated mineral loss were found for the tested materials. We conclude that high fluoride release from filling materials only results in superficially increased remineralisation of underlying demineralised dentin.

Acidogenicity of buccal plaque after a single rinse with amine fluoride - stannous fluoride mouthrinse solution.

Caries and gingivitis prevention may benefit from chemotherapeutic plaque control, therefore we compared in a cross-over study with 5 subjects the anti-acidogenic effects of a single use of AmF-SnF2 mouthrinse solutions (Meridol with and without 5% alcohol) with baseline and with the effects of a placebo and a chlorhexidine mouthrinse (CHX). Buccal plaque was collected 0.5, 3 and 8 h after the subjects used one of the mouthrinses, each time before and after a rinse with 10% sucrose to induce lactic acid production. Samples were analysed for acid anions by capillary electrophoresis and for protein. At 0.5 h after the use of AmF-SnF2 or CHX, the concentration of acetate in resting plaque was 70% lower than at baseline or after using the placebo. Average post-sucrose acetate and lactate concentrations in the placebo group were 30-80% higher than at baseline; up to 3 h this difference was significant. 8 h after using AmF-SnF2 or CHX, the post-sucrose acetate and lactate concentrations were still 30-50% lower than after the placebo, and up to 40% lower than at baseline. To conclude, AmF-SnF2 in both Meridol formulations and CHX were shown to have a similar potency to inhibit acid production after a single rinse.

Fluoride-dependent formation of mineralized layers in bovine dentin during demineralization in vitro.

Demineralization of dentin in the presence of fluoride produces lesions with a mineralized surface layer which becomes thicker and more mineralized with higher fluoride concentrations whereas the lesion depth is hardly affected. The aim of this study was to investigate the effects of the time of fluoride treatment and the amount of fluoride taken up on the properties of the mineralized layer. Discs of bovine dentin embedded in methylmethacrylate with one surface exposed were demineralized in 50 mM acetic acid, 2.2 mM CaCl2, 2.2 mM KH2PO4, pH 5.0. At the start and/or later during the demineralization period, the specimens were incubated individually for 1 or 2 days in 10 ml of the same demineralization solution supplemented with 0.5, 2.0 or 5.0 ppm fluoride, which was then assessed for changes in calcium and fluoride concentrations. After 2, 5 and 8 days, specimens were sectioned for microradiographic analysis so as to follow development of the lesions and the mineralized layers. The results were the following: While demineralization with fluoride present at the first day led to the formation of a surface layer, fluoride present only at a later day produced a subsurface layer, not at the lesion front but closer to the surface. This layer resulted from (re)precipitation and not from preservation of the original mineral. The 'integrated mineral content' of the surface layer increased linearly with the uptake of fluoride, which resulted in an apparent fluorapatite content of about 20 vol%. The profiles of the surface layers remained unchanged during continued demineralization in the absence of fluoride. It was concluded that in the presence of fluoride mineral loss is reduced as a result of the reprecipitation of dissolved mineral ions as a layer of fluoride-enriched apatite. This layer does not offer protection of underlying dentin against continued demineralization.

A novel pyrroleninone cross-link from bovine dentine.

The aim was to identify suspect collagen cross-links in dentine, eluting close to known cross-links in ion-exchange HPLC. Bovine tooth roots as source of dentine were powdered, demineralised, reduced, and acid-hydrolysed. Cross-linking amino acids were isolated from the acid hydrolysate by size exclusion, adsorption, and sequential ion exchange chromatography. In addition to dihydroxylysinonorleucine and hydroxylysylpyridinoline, an unknown cross-link was isolated (V-2). The ultraviolet, mass, and nuclear magnetic resonance spectra support the proposed structure of V-2, a trimeric amino acid with a pyrroleninone nucleus.

The effect of intrinsic fluoride in cows' milk on in vitro enamel demineralization.

The fluoride concentration in cows' milk has been reported to vary with the fluoride levels in drinking water but it seldom exceeds 0.5 microg/ml. This raised a question as to whether any caries-protective effect could be attributed to the intrinsic fluoride of milk. Two samples of cows' milk with intrinsic fluoride concentrations of 0.03 and 0.3 microg/ml, respectively, were assessed for their protective effect on enamel in an in vitro demineralization model at relatively severe and mild acidic challenges (pH 4.6 and 5.0, respectively). Polished enamel discs were incubated individually in 5.0 ml of demineralization solution for 20 h per day alternated with 1-hour incubations in 1.0 ml of milk or control buffers: group 1, demineralization solution only (negative control); group 2, milk with 0.03 microg/ml fluoride; group 3, milk with 0.03 microg/ml fluoride; supplemented with NaF to 0.3 microg/ml fluoride; group 4, milk with 0.3 microg/ml fluoride; group 5, 0.3 microg/ml fluoride in 20 mM HEPES, pH 6.7; group 6, milk with 0.03 microg/ml fluoride supplemented with NaF to 5.0 microg/ml fluoride (positive control). The solutions were renewed each day and the calcium concentration in the demineralization solutions was followed during 4 days. The results showed that the protective effect of intrinsic milk fluoride on enamel is limited by the severity of the acidic challenge: There was a significant inhibition of the demineralization in groups 3-6 compared to groups 1 and 2, but only at pH 5.0 (p<0.0001) and not at pH 4.6 (p = 0.2). The organic components of milk had limited protection against demineralization because milk and HEPES with the same fluoride concentration gave similar results. The 36% reduction in calcium loss at pH 5.0 by treatment with milk with only 0.3 microg/ml fluoride is an indication that intrinsic milk fluoride has some caries-protective properties.

Inhibition of dentin demineralization by fluoride in vitro.

Compared with the knowledge accumulated on enamel-fluoride interactions, relatively little data is available regarding fluoride effects on dentin. This applies to both laboratory and clinical studies into the efficacy of fluoride schemes for the prevention of root surface caries. This study aimed to determine the effects of fluoride and pH on the demineralization of dentin, such as to provide information necessary to develop preventive programmes. Bovine dentin blocks were subjected to undersaturated calcium- and phosphate-containing solutions in the pH range 4.0-6.0 with fluoride added at concentrations between 0.5 and 10 ppm. Non-fluoride solutions served as controls. Mineral loss was assessed chemically and by transversal microradiography. Comparisons were made with similar studies on enamel demineralization. The results showed that demineralization of dentin depends on both pH and fluoride concentration in the demineralizing solution. Inhibition of demineralization that could be relevant from a clinical point of view was found at fluoride values 5-10 times the corresponding values for enamel. Also rapid depletion of fluoride from the solutions was observed, indicating the high uptake capacity of dentin for fluoride. Lesion depth depended on pH of the solution while the fluoride levels were associated with the surface layer, both in mineral content and depth. For dentin we propose a demineralization mechanism where acid penetrates rapidly into the tissue, presumably through the tubules, after which the released calcium and phosphate is partly trapped by the inward diffusing fluoride. This leads to the formation of a surface layer, which may even be hypermineralized compared to sound dentin.

Modification of amino acid residues in carious dentin matrix.

The Maillard reaction between sugar and protein has been postulated as the cause for the browning and arrestment of caries lesions. This reaction has been implicated as the cause for decreased degradability of collagen in vivo. The aim of the present study was to verify the occurrence of the reaction in vivo. Carious and sound dentin samples were taken from extracted human teeth and analyzed for the fluorescence characteristic of the Maillard reaction and oxidation and, by HPLC, for Maillard products. In addition, physiological cross-links were analyzed by HPLC. Oxidation- and Maillard reaction-related fluorescence increased in collagenase digests from carious dentin. Advanced Maillard products (carboxymethyllysine and pentosidine) increased, whereas furosine, a marker for the initial reaction, was not observed consistently. This implies no direct addition of sugars to protein, but rather the addi-tion of smaller metabolites and glycoxidation products. In addition, the physiological cross-links hydroxylysinonorleucine and dihydroxylysinonorleucine decreased in carious dentin. Also for hydroxylysylpyridinoline, a decrease was observed, but not consistently. In conclusion, the caries process modifies amino acids in dentin collagen, which can lead to increased resistance against proteolysis and ultimately to caries arrestment.

Enzymatic release of sequestered cows' milk fluoride for analysis by the hexamethyldisiloxane microdiffusion method.

The concentrations of diffusible and total fluoride in cows' milk samples from areas with widely different fluoride levels in drinking water were determined using a fluoride electrode. The diffusible fluoride was determined by direct hexamethyldisiloxane microdiffusion while for total fluoride, samples were subjected to either open ashing or digestion with proteolytic enzymes before microdiffusion. Magnesium nitrate was studied as a new fixative for milk during open ashing and compared with magnesium acetate. Diffusible fluoride ranged from 0.024 to 0.28 microgram ml-1 while total fluoride ranged from 0.05 to 0.31 microgram ml-1. The use of proteolytic enzymes before microdiffusion resulted in total fluoride measurement. It was concluded that all fluoride in milk is inorganic in nature with the bound fluoride being physically or chemically sequestered in the milk proteins. The proposed method is convenient for total fluoride analysis in milk.

The Maillard reaction in demineralized dentin in vitro.

The Maillard reaction between carbohydrate and protein has been proposed as a cause of the browning of carious lesions. The aim of the present investigation was to determine the occurrence of this reaction in bovine dentin collagen in vitro and to establish the effect of the reaction on the proteolytic degradation of bovine dentin collagen in vitro. Slices of demineralized bovine dentin were incubated with 0.2 M glucose or buffer for 10 weeks at 37 degrees C. The formation of initial (furosine) and advanced (pentosidine) products of the Maillard reaction in dentin exposed to glucose was confirmed by HPLC. After reduction with NaBH4 to prevent intermediate Maillard products from further reaction, slices were either degraded with collagenase for fluorescence measurement or incubated with trypsin or pepsin to assess enzymatic degradation. Fluorescence characteristic for the Maillard reaction increased in glucose-exposed slices. Degradation of collagen by pepsin, but not by trypsin, was greatly depressed following glucose pretreatment. This may indicate an altered sensitivity to proteolytic degradation; the Maillard reaction thus has a potential role in caries arrestment.

Influence of polymers for use in saliva substitutes on de- and remineralization of enamel in vitro.

A number of polymers which have previously been tested for their applicability as thickening agents in saliva substitutes were studied in vitro for their caries-protective properties. These were: polyacrylic acid, carboxymethylcellulose, xanthan gum, guar gum, hydroxyethylcellulose and porcine gastric mucin. The polymers were tested for their effects on: (1) growth of hydroxyapatite crystals in a supersaturated calcium phosphate solution, (2) dissolution of hydroxyapatite crystals in 50 mM acetic acid, pH 5.2 and (3) demineralization and remineralization of bovine enamel in a pH-cycling model. Growth of hydroxyapatite crystals was strongly inhibited by polyacrylic acid and carboxymethylcellulose at very low concentrations (0.005% w/v). Other polymers displayed lower inhibition of hydroxyapatite crystal growth. Hydroxyapatite dissolution was inhibited by all polymers except by hydroxymethylcellulose and xanthan gum. This occurred both in the presence of the polymers as well as after a 30-min preincubation. In the pH-cycling experiment, bovine enamel specimens with preformed lesions were alternately exposed to a demineralization buffer and a remineralization buffer containing the polymers hydroxyethylcellulose, carboxymethylcellulose, xanthan gum, polyacrylic acid, or porcine gastric mucin. A remineralization buffer containing 1 ppm NaF was used as a positive control. Under the experimental conditions, the control experiment without additives resulted in a net mineral loss (30.6 mumol Ca/cm2 after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 mumol/cm2). All polymers largely inhibited further demineralization (1.2-12.3 mumol/cm2) except polyacrylic acid which, inhibited of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 mumol/cm2). In conclusion, polymers tested in this study, except the polyacrylic acid, reduced the demineralization of enamel in vitro. The precise mechanism of the protective effect is not clear but it is speculated that formation of an absorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.

Effect of a fluoridated toothpaste on lesion development in plaque-filled dentine grooves: an intra-oral study.

Fluoride can inhibit caries at plaque-retention sites, but some studies indicate that fluoride is less effective in fissures than on smooth surfaces. To study the efficacy of fluoridated toothpastes at plaque-retention sites, an intra-oral model was used with bovine coronal dentine discs, in which grooves of two different widths were sawn. The discs were mounted in the partial prostheses of 31 participants divided into two groups. One group brushed with a non-fluoridated toothpaste and a second with a paste containing 1,000 ppm fluoride as NaF. After 3 months, the specimens were retrieved and from each a thin section was taken for microradiographic analysis. Lesions which developed in the grooves resembled natural lesions in terms of the presence of a surface layer and the mineral content profiles. Extensive lesions followed the direction of the dentinal tubules. The mineral loss was quantified half-way into and at the base of the grooves and ranged from 0 to 20,000 vol% x microns. Analysis of variance showed that the mineral loss was significantly influenced by the treatment and the width of the grooves (p < 0.001). In the broad grooves the average mineral loss was 19% smaller in the fluoride group than in the non-fluoride group, in the narrow grooves this value was 7%. Taking the two treatment groups together, the average mineral loss was largest half-way into the broad grooves (4,921 vol% x microns) and smallest at the base of the narrow grooves (2,289 vol% x microns). The results with this new intra-oral model indicate that the dimensions of small grooves in dentine, and thus their accessibility, determine not only their susceptibility to caries but also the protective effect of a fluoridated toothpaste.

Reproducibility of TMR for the determination of longitudinal mineral changes in dental hard tissues.

Transverse microradiography (TMR) of thin sections is the principal method for determining mineral changes in experimental lesions. Because it has become also the standard by which newly-developed methods are validated, demands on all steps of the procedure are high. Thin sections of enamel and dentin require high-precision cutting or lapping to preserve surface structures, but their different brittleness and elasticity preclude a uniform procedure. Image analysis permits the measurement of fine details at a micron scale, but because most studies are aimed at overall mineral changes, they are usually lost on broadening of the scan areas and averaging of experimental groups. Small errors in repositioning of the scan areas for repetitive measurements may yield distorted lesion profiles, while quantitative data such as the integrated mineral loss are hardly influenced. A major reason for "irreproducibility" of TMR are the different definitions of the lesion parameters as used by various research groups. We recommend that these definitions be agreed on if the full potential of TMR as an international standard is to be utilized.

The effect of a fluoridated toothpaste on dentinal lesions in plaque-filled grooves: an intra-oral crossover study.

Recently, we introduced an intra-oral dentin demineralization model to evaluate the efficacy of a fluoridated toothpaste at plaque-retention sites with limited access to salivary components and fluoride. Usage of the fluoridated toothpaste reduced mineral loss from grooves in sound dentin by 15% compared with a non-fluoridated toothpaste (Lagerweij et al., 1997). In the current study, preformed dentinal lesions were subjected to similar toothpaste treatments. To improve the power of the model in discriminating between toothpaste groups, we changed the design from monadic to crossover to reduce the influence of variation between the participants. Two sets of specimens were placed in the partial prostheses of 29 participants who used a toothpaste containing either 1000 ppm fluoride or no fluoride during two consecutive six-week periods. Changes in mineral content were analyzed by transverse microradiography. The average mineral loss after the non-fluoride period was 1785 vol% x micron (SD 2399), and 1335 vol% x micron (SD 2039) after the fluoride period, a 25% inhibition in demineralization. Analysis of variance showed that the demineralization was significantly influenced not only by the type of treatment (p < 0.001 level), but also by the participant (p < 0.001), the width of the groove (p < 0.001), and the depth into the groove where changes in mineral content were measured (p < 0.017). Twenty-one percent of all the lesions showed remineralization. No significant difference in the extent or the occurrence of remineralization was found between the treatments.

Demineralization of dentine grooves in vitro.

Caries mainly occurs at retention sites for plaque such as pits, fissures, margins of restorations and interproximal sites. An in vitro model was developed to investigate dentine caries in narrow gaps. Forty bovine coronal dentine discs were covered with bonding agent. In 20 discs, two grooves, 200 and 340 mu m wide and 500 mu m deep, were sawn to mimic fissures. In the 20 other discs, a 1-mm-wide groove was made and, after polyester sheets were placed against both walls, filled with composite. After curing of the composite and removal of the sheets, grooves of 22 mu m remained at the borders of the restoration. All grooves were exposed to 8% methylcellulose gel 0.1 M lactic acid at pH 4.8 for 1 week. Demineralization was determined by microradiography of sections sawn out of the center of the discs. The walls of the grooves showed subsurface lesions, which decreased in size towards the base of the grooves. The average mineral losses (vol % x mu m) at the entrance of the 22-, 200- and 340-mu m-wide grooves were 1,112 (SD 370), 1,277 (293) and 1,277 (255), halfway down the groove 218 (150), 659 (244) and 797 (207) and at the base of the groove 140 (88), 285 (145) and 504 (205), respectively. Analysis of variance and Tukey's B test showed that the average mineral losses from grooves of different width and from sites at different depth were all significantly different at p<0.05 level, but not for the mineral losses from the entrance of the two wider grooves and from the two lower sites in the narrowest groove. The reduced mineral loss in the two narrower groove widths is assumed to be caused by the more limited inward diffusion of acids and outward diffusion of mineral ions through the methylcellulose gel.

Uptake and release of fluoride by saliva-coated glass ionomer cement.

The 'recharging' of aged glass ionomer cement restorations with fluoride by exposing them to a concentrated NaF solution has been suggested to improve their performance as devices with slow fluoride release. We have studied the interference of salivary coatings on glass ionomer cement with the uptake of fluoride in vitro. Freshly prepared glass ionomer discs were leached in distilled water for 14 weeks, then incubated in human saliva for 2 h, 24 h or 1 week before exposure to 0.53 M NaF (1% fluoride) for 5 min. Fluoride uptake by the cement was measured indirectly as the subsequent increase in fluoride release. A 2-hour incubation in saliva reduced the fluoride uptake by half, a 24-hour incubation by 74%. The major part (93-95%) of the fluoride that was taken up was released on the first day. A 1-week salivary coating reduced the uptake by 49%, but also caused some retardation of the fluoride release: 80% of the total release was on the first day. It was concluded that despite the considerable interference by salivary coating, the amount of fluoride that can be taken up by aged glass ionomer cement remains significant. However, the fast release will limit the usefulness of such recharging in vivo.

pH-cycling of enamel and dentin lesions in the presence of low concentrations of fluoride.

F-dentifrice usage causes slightly elevated fluoride levels in saliva. Therefore, the effects of permanent low fluoride concentrations versus daily dentifrice treatments were studied on enamel and dentin lesions in a pH-cycling model of alternating demineralization and remineralization. Groups received 1) no fluoride treatment, 2) 3 mu M (0.06 ppm) F continuously present during re- and demineralization or 3) daily 5-min F-dentifrice treatments. Solutions were analyzed for changes in calcium and fluoride. Cumulative results (10 d) showed that for the non-fluoride group the dentin lesions increased, while for enamel lesions mineral uptake and loss were balanced. Addition of 3 mu M F caused small, non-significant, enhancement of remineralization (1-7%), while demineralization was significantly inhibited for both tissues (9-23%). The daily dentifrice treatments resulted in a balance between mineral uptake and loss of dentin, due to inhibited demineralization (-33%) and enhanced remineralization (+79%). For enamel, the F-dentifrice treatments resulted in 43% reduction of demineralization, with no significant effect on remineralization. Fluoride loss from the 3 mu M F cycling solutions was significant (up to 50%) and constant during the experimental period. Microradiographic analysis showed remineralization at the lesion front in enamel. In dentin, the lesion depth was increased in all groups, with concomitant mineral deposition in the surface region of the dentifrice group. Results indicate that slightly elevated fluoride levels may be considerably less effective in inhibiting lesion progression in dentin than in enamel, and suggest mineral uptake and loss to occur at similar depths for enamel lesions, while demineralization and remineralization occur at different depths in dentin.

Lipoteichoic acid inhibits remineralization of artificial subsurface lesions and surface-softened enamel.

Dental plaque produces not only acids by which underlying enamel is demineralized but also compounds which may inhibit repair of the lesions. The aim of this study was to determine how lipoteichoic acid, a bacterial compound that is abundant in dental plaque and inhibits calcium phosphate precipitation in vitro, affects the remineralization of incipient enamel lesions. Subsurface and surface-softened lesions were made in thin sections of bovine enamel, incubated with various amounts of lipoteichoic acid (isolated from Lactobacillus casei), and remineralized in 1.5 mmol/L CaCl2, 0.9 mmol/L KH2PO4, 130 mmol/L KCl, and 20 mmol/L Hepes, pH 7.0. Remineralization was followed during several weeks by repeated microradiography of the sections, and characterized by the changes in the integrated mineral loss of the lesions and the differential mineral profiles. The results showed that: (1) the effects of lipoteichoic acid on lesion remineralization were dose-dependent; (2) in subsurface lesions only the highest dose of lipoteichoic acid affected remineralization, which was delayed throughout the lesions; and (3) in surface-softened enamel, lipoteichoic acid did not affect the remineralization of the deeper parts, but remineralization of a surface zone of from 25 to 30 microns was increasingly inhibited with increasing doses. These effects were explained by different permeabilities of the surfaces of both types of lesions for the inhibitor: In subsurface lesions, lipoteichoic acid may have mainly clogged the porosities in the surface layer, whereas it could penetrate substantially into surface-softened enamel.

Adsorption of [3H]-lipoteichoic acid to hydroxyapatite and its effect on crystal growth.

3H-labelled lipoteichoic acid was isolated from Lactobacillus casei cells cultured in the presence of [2-3H]-glycerol. The adsorption characteristics of both native and deacylated [3H]-lipoteichoic acid on to hydroxyapatite crystals were determined in 2.0 mM CaCl2, 50 mM Hepes, pH 7.2. The maximum numbers of adsorption sites per unit surface area were 80 +/- 3 and 49 +/- 2 nmol/m2 for native and deacylated lipoteichoic acid, respectively. At low concentrations, which were previously found to stimulate hydroxyapatite growth in a supersaturated solution (1.0 mM CaCl2, 7.5 mM KH2PO4, 50 mM Hepes, pH 7.2), lipoteichoic acid appeared for a large part adsorbed to the crystals, while crystal growth was delayed when there was an excess of unbound lipoteichoic acid in solution. These results suggest that lipoteichoic acid released from plaque bacteria may have dual effects both on the precipitation of calcium phosphate in dental plaque and on the remineralization of lesions in the underlying hard tissues.

The influence of the organic matrix on demineralization of bovine root dentin in vitro.

The effect of matrix degradation on the rate of demineralization of dentin lesions was investigated. It was hypothesized that the demineralized matrix would inhibit the demineralization of the underlying mineralized dentin. Bovine root dentin specimens were alternately demineralized and incubated with either a bacterial collagenase or buffer (control). The demineralization was carried out under various conditions: Acetic acid solutions were used to form incipient and advanced erosive lesions, and lactic acid solutions containing a bisphosphonate were used to form incipient subsurface lesions. Under all conditions, the demineralization was found to be accelerated when the matrix was degraded by collagenase. This increase was more pronounced in advanced erosive lesions than in incipient lesions. Microscopic examination of collagenase-treated specimens revealed that the matrix of erosive lesions contained several layers of differently affected matrices, whereas the matrix of subsurface lesions appeared to be equally affected throughout the lesion. In conclusion, the matrix degradation was different in erosive and subsurface lesions but promoted the demineralization in both types of lesions.