J.J.M. Damen

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.

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.

Silica-induced Precipitation of Calcium Phosphate in the Presence of Inhibitors of Hydroxyapatite Formation

The promotion and the inhibition of hydroxyapatite formation by various substances were determined by measurement of the induction time of spontaneous precipitation (t) from supersaturated solutions. Silica was found to decrease t. in Hepes-buffered (pH 7.2) supersaturated solutions with a wide range of calcium-to-phosphate ratios and concentrations. Also, in suspensions of the oral bacteria S. mutans or C. matruchotii in 1 mmol/L calcium, 7.5 mmol/ L phosphate, and 50 mmol/L Hepes (pH 7.2), silica was capable of stimulating precipitation. Macromolecules derived from these bacteria by freezing and thawing appeared to be strong inhibitors of calcium phosphate precipitation. In the presence of silica, the effects of these bacterial inhibitors could be partially overcome, which supports the idea that silica in dental plaque is a promoter of calculus formation. In contrast, inhibition of calcium phosphate precipitation by a low-molecular-weight inhibitor, pyrophosphate, could not be counteracted by silica.

The Effect of Silicic Acid on Calcium Phosphate Precipitation

So that a possible involvement in the mineralization of dental plaque could be investigated, the effects of silicic acid on calcium phosphate precipitation were assessed in vitro. By measuring the decrease in Ca2+ concentration (by means of ion-selective electrodes), we determined both spontaneous precipitation and seeded crystal growth from solutions that contained 1 mmol/L calcium, 7.5 mmol/L phosphate, 50 mmol/L Hepes pH 7.2, and various amounts of silicic acid. Polymerized silicic acid, but not its monomer, was found both to cause a 60% reduction in the lag period that precedes spontaneous precipitation and to enhance the growth rate of seeded hydroxyapatite crystals. Silica suspensions showed effects similar to those of polysilicic acid. In all cases, the precipitated material was found to be hydroxyapatite. Whereas seeded brushite crystals grew slowly without silicic acid, hydroxyapatite was the only mineral detected after crystal growth in the presence of silicic acid. Apparently, polysilicic acid acted as a substrate for hydroxyapatite nucleation, inducing secondary nuclei on both hydroxyapatite and brushite crystals. The finding that polysilicic acid could overcome part of the inhibitory effect of a phosphoprotein on calcium phosphate precipitation gave additional support for the idea that polysilicic acid and silica may promote the formation of dental calculus.

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.

A Single-section Model for Enamel De- and Remineralization Studies. 1. The Effects of Different Ca/P Ratios in Remineralization Solutions

A prerequisite for the accurate measurement of differences between pre- and post-experimental mineral profiles in single sections by quantitative microradiography is a high degree of reproducibility of the analytical procedures. We have determined the reproducibility of both the production and analyses of microradiographs. Lesions were made in seven single bovine enamel sections and radiographed three times. Each microradiograph was then analyzed on three different occasions by use of an image analysis system. This resulted in only small standard deviations in the lesion parameters. The method was used to determine the sites of mineral deposition in lesions in single sections during remineralization in three different solutions, one of which contained 1.5 mmol/L Ca and 0.9 mmol/L PO4 (standard solution); the other two solutions were low in either Ca or PO4, but all three had the same degree of supersaturation. The mineral profiles and lesion parameters were determined after lesion formation and after remineralization for 4 days, 1, 2, and 3 weeks. The decrease in IML (integrated mineral loss) in the lesions remineralized in the standard solution was greatest during the first week. Lesion profiles revealed that, under non-standard conditions, mineral deposition was retarded in the deeper part of the lesion, in contrast to the even distribution of mineral deposition from the standard solution. Differences in IML changes and lesion profiles between these bovine enamel experimental groups diminished when remineralization continued for 2 and 3 weeks. Possibly, the reactivity of mineral surfaces in newly made lesions affected the diffusion ofremineralizing ions from low concentration solutions to the lesion front.

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 fluore-scence 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 crosslinks 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 ulti-mately to caries arrestment.

Induction of Calcium Phosphate Precipitation by Titanium Dioxide

Titanium powder and various titanium dioxides were tested for their capacity to reduce the induction time for calcium phosphate precipitation from supersaturated solutions. Only after a pretreatment aimed at increasing its oxide surface layer was titanium powder found to accelerate the precipitation from solutions containing 2 mmol/L CaCl2, 2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, and to induce precipitation from metastable solutions containing 1.2 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, at 37°C. Even stronger effects were found when suspensions of the titanium dioxides anatase or rutile (10-50 μg/mL) were added to these solutions. TiO2 appeared to serve as a reactive substrate for secondary nucleation at a wide range of calcium-to-phosphate ratios and concentrations, even in the presence of 40 mg/mL bovine serum albumin, which completely inhibited precipitation in control incubations. These results suggest that the oxide surface layer of titanium implants may induce calcium phosphate precipitation in the metal-to-bone interface, which may play a role in the integration of such implants in bone.

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.

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 µm 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.