A.J.P. van Strijp

Host-Derived Proteinases and Degradation of Dentine Collagen in situ

Dentine root caries is a process of demineralization and degradation of the organic matrix by proteinases. In this in situ study, the presence and activity of the matrix metalloproteinases 1, 2 and 9 (MMP-1, MMP-2, MMP-9) in saliva and in completely demineralized dentine specimens were investigated. Furthermore, the activity of cathepsin B was determined in saliva. A correlation between these enzymes and the level of degraded collagen was investigated. Demineralized dentine specimens were mounted in the partial prosthesis of 17 volunteers. Saliva samples were taken at 0, 2 and 4 weeks. After 4 weeks, the enzymes were extracted from the dentine specimens and the collagen loss was assessed. The collagen loss varied between 0 and 40.3%. Zymography of the saliva and the dentine extract samples showed that (pro-)MMP-2 and (pro-)MMP-9 were present. The levels of active MMPs were assessed, using fluorogenic MMP-specific substrates. All but 3 of the 51 saliva samples showed MMP-1 activity ranging from 1.5 to 101.1 relative fluorescence units (RFU)/s. Forty-eight saliva samples showed gelatinolytic MMP-2/MMP-9 activity (1.7–141.1 RFU/s). MMP-1 activity was shown in all dentine extracts varying between 3.5 and 295.0 RFU/s. From the dentine extracts, 15 showed MMP-2/MMP-9 activity (0.2–13.7 RFU/s). The MMP activity from both saliva and dentine extracts did not correlate with the collagen loss. The activity of salivary cathepsin B varied from 4.8 to 42.2 arbitrary units/min. A positive correlation was found between salivary MMP activity and cathepsin B activity. This study revealed that gelatinolytic enzyme activity was present both in saliva and dentine collagen. No correlation could be observed, however, between the level of enzyme activity and the collagen loss of the dentine specimens.

Bacterial Colonization of Mineralized and Completely Demineralized Dentine in situ

The changing environment in a developing root lesion may result in a succession of the microbial flora in the dentine. As demineralization proceeds, the collagenous matrix is exposed, which could be c

Bacterial colonization and degradation of demineralized dentin matrix in situ.

Lesion formation in dentin involves demineralization followed by degradation of the exposed organic matrix. Proteinases from microorganisms present in the dentin are believed to play an important role in the breakdown of the dentinal collagen. In this study, the microflora colonizing decalcified dentin matrix, placed in situ, was identified. The gelatinolytic activity of the isolated strains was assessed and related to the degradation of the dentin matrix. The predominant species found were Streptococcus mitis, Peptostreptococcus productus, Lactobacillus casei, Propionibacterium species and Veillonella parvula. Marked interindividual variation in the composition of the microflora was observed. The microflora possessed gelatinolytic activity, although no correlation was found with the severity of dentin matrix degradation. The chemically determined loss of collagen varied between 0 and 67 wt% per participant and corresponded with the extent of collagen degradation observed by transmission electron microscopy.

Contact Microradiography of Dentine under Wet Conditions to Prevent Lesion Shrinkage

A considerable shrinkage of dentinal lesions is the result of desiccation of dentine sections during contact microradiography. We introduce a simple modification which allows the dentine to be wet during the microradiographic procedure. The volume stability of both sound and demineralized dentine sections microradiographed under wet conditions is compared to sections microradiographed while being exposed to the air. Under wet conditions no shrinkage could be detected of both sound and demineralized sections. Within 15 min exposure to the air, demineralized sections showed their lesion depth to be significantly reduced by 21%, resulting in an underestimation of the mineral loss of 44%. No shrinkage was observed in the air-dried sound dentine. Especially when high-resolution plates are used, which require an extended exposure time, microradiography of dentine sections under wet conditions is recommended. In longitudinal de- and remineralization studies, the use of water instead of impregnation with low-volatility liquids is to be preferred because of the possible effects of these liquids on the mineralization processes.

In situ Fluoride Retention in Enamel and Dentine after the Use of an Amine Fluoride Dentifrice and Amine Fluoride/Sodium Fluoride Mouthrinse

The aim of this in situ investigation was to study the effect of an amine fluoride/sodium fluoride mouthrinse (total F = 250 ppm) in addition to an amine fluoride dentifrice (F = 1,250 ppm) on the amount of acquired fluoride in enamel and dentine. In the partial prosthesis of 12 participants a combined specimen of slightly demineralized enamel and dentine was placed. During two consecutive experiments, each lasting 3 weeks, the participants used an amine fluoride dentifrice alone or in combination with a fluoride mouthrinse. After the in situ period the specimens were retrieved and both the enamel and the dentine specimens were analysed for the amount of KOH–soluble fluoride and structurally bound fluoride. The results showed a significant increase in both KOH–soluble and structurally bound fluoride in enamel and dentine when a fluoride mouthrinse was used. Whether the rinsing procedure was performed immediately after toothbrushing or with a delay of 2 h did not influence the results. Furthermore it was shown that dentine acquired substantially more fluoride than enamel during the experimental period. The results indicate that a fluoride mouthrinse used in addition to a fluoride dentifrice may have a beneficial effect on the protection of enamel and dentine against caries.

Chlorhexidine efficacy in preventing lesion formation in enamel and dentine: an in situ study

Background: Clinical studies on the caries-preventive properties of chlorhexidine mouthrinses are limited and the results are inconclusive. Aim: The aim of this study was to elucidate the contribution of a 0.2% chlorhexidine mouthrinse to the protection of enamel and dentine against demineralization. Methods: In this randomized two-treatment, two-leg study 14 individuals wearing partial prostheses were enrolled. Sound enamel and dentine specimens were placed in situ for 4 weeks. Twice daily, a mouthrinse was performed with either chlorhexidine or saline (control) depending on the experimental group the participant was allocated to. After the experimental period, plaque samples were collected from the surface of the specimens and from natural tooth surfaces to assess the organic acid production upon a sucrose challenge. The specimens were analyzed for mineral loss by transversal microradiography. Results: This study could not demonstrate a significantly better protection of enamel and dentine against demineralization by the chlorhexidine treatment compared to saline. No differences in acid production of plaque samples from the chlorhexidine-treated and control groups were observed. This result was also found for plaque samples originating from the natural tooth surfaces. Conclusion: Mouth rinsing with 0.2% chlorhexidine did not prevent demineralization of dentine and enamel in situ.

Solubilization of Dentin Matrix Collagen in situ

The effects of the oral environment on dentin matrix collagen were studied. In the partial prostheses of 12 participants, two completely demineralized dentin specimens were mounted covered by a Dacron gauze. After an experimental period of seven weeks, the specimens were transferred to a trypsin-containing buffer for determination of the amount of denatured collagen. Subsequently, the specimens were incubated with a bacterial collagenase for assessment of the amount of collagen. After the intra-oral exposure, there was a collagen loss varying between 1 and 47 wt%. This variation might be due to differences in proteolytic activity of the colonizing microflora. After exposure to the oral environment, only about 0.5 wt% of the available collagen was trypsin-degradable. This indicates a rapid solubilization of the denatured collagen from the specimens into the oral cavity. A separate group of specimens was examined by light microscopy and transmission electron microscopy. Various degrees of breakdown could be discerned. Some experimental specimens showed loss of surface integrity and tubules heavily infected with different types of micro-organisms. The lumens of the tubules were enlarged, sometimes creating caverns as a result of the loss of the intertubular collagenous matrix.

In situ Remineralisation of Enamel and Dentin after the Use of an Amine Fluoride Mouthrinse in Addition to Twice Daily Brushings with Amine Fluoride Toothpaste

It is often claimed that 3 fluoride moments a day significantly reduce the caries risk compared to 2 daily fluoride moments. However, previous research is not conclusive. Therefore, the aim of this study was to compare the effect on lesion progression of 2 versus 3 fluoride moments a day. A double-blind, randomized, cross-over in situ experiment was designed. The experiment comprised 2 in situ periods of 3 weeks with a washout period of 3 weeks in between. Sixteen participants wore an enamel and a dentine specimen with a preformed lesion placed buccally in their partial prosthesis. The participants brushed twice a day with a 1,400 ppm F (amine fluoride) toothpaste and rinsed once a day with either 250 ppm F (amine F/NaF) or a placebo rinse. At the end of the experiment the specimens were retrieved for fluoride analysis and the assessment of integrated mineral loss with transversal microradiography. The fluoride analysis showed a statistically significant increase in structurally bound fluoride in dentine, but not in enamel, when comparing the fluoride mouthrinse group with the placebo rinse group. The amounts of loosely bound, KOH-soluble fluoride were not different between both groups neither for enamel nor for dentine. In dentine IML gain was significantly (p < 0.05) higher for the fluoride mouthrinse group than for the placebo mouthrinse group. In enamel no statistically significant differences in IML gain were found. For dentine a third fluoride moment may be beneficial in enhancing remineralisation, even under the remineralising conditions as in this study.

In vitro Fluoride Uptake by Intra–Orally Aged and Contaminated Glass Ionomer Cement

release can be elevated by repeated exposure of the glass ionomer restorations to high doses of fluoride. Several reports have described such ‘recharging’ in vitro by treatment of glass ionomer samples with NaF solutions of various concentrations [Forsten, 1991, 1995; Takahashi et al., 1993; Creanor et al., 1994; Hatibovic-Kofman et al., 1997b] or with a fluoridated gel [Seppä et al., 1995]. In a previous in vitro study [Damen et al., 1996], we found that the uptake of fluoride by aged glass ionomer cement is partly inhibited following pre-incubation in saliva. It is not unlikely that during a long period in the mouth, salivary and plaque-derived compounds adsorb to the surface of glass ionomer restorations and block the inward (and outward) diffusion pathways. The aim of this study was to determine the effect of such contamination on the uptake of fluoride. To this end, discs of glass ionomer cement with one surface exposed were placed in the partial prostheses of a group of panelists during a period of 3 months. After retrieval from the mouth, the discs were incubated in a concentrated NaF solution and the subsequent fluoride release was determined and compared with fluoride release from control discs exactly as described previously [Damen et al., 1996]. Fuji Lining LC (GC International Corp., Tokyo, Japan) was chosen as the test material, because its easy handling facilitated reproducible experimental conditions. Similar fluoride uptake and release characteristics have been reported for this and restorative (type II) glass ionomer cements [Takahashi et al., 1993]. Discs 5.0 mm in diameter and about Short Communication

Influence of the Temperature of Extraoral Immersions in the Intraoral Cariogenicity Test Model

In an intraoral cariogenicity test model the influence of the temperature of the extraoral immersion solutions on both the demineralization of enamel and the pH drop of the plaque was studied. Ten volunteers wore the appliance for 3 weeks, except during 3 daily extraoral immersions of 15 min in a 3% sucrose solution. One side of the appliance was immersed in a solution at 37 °C, the other side in the same solution at room temperature (approximately 20 °C). The pH drop caused by plaque in situ as well as by plaque collected and suspended after the experiment was significantly faster at 37 °C. The lesions produced at this temperature were deeper and more demineralized. The rate of demineralization was almost twice as high at 37 than at 20 °C. Extraoral immersions at 37 °C are recommended to simulate in vivo conditions during immersion; in addition this procedure may shorten the experimental period.