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.

BackgroundA large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues.FindingsWe compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit.Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG.ConclusionsWe conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.

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The promotion and the inhibition of hydroxyapatite formation by various substances were determined by measurement of the induction time of spontaneous precipitation (ti) from supersaturated solutions. Silica was found to decrease ti 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.

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.

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, carboxymefhylcellulose, 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 carboxymefhylcellulose 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 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 µmolCa/cm² after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 µmol/cm²). All polymers largely inhibited further demineralization (1.2–12.3 µmol/cm²) except polyacrylic acid which, because of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 µmol/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 adsorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.

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.