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The Effects of the Solubility of Artificial Fissures on Plaque pH

Department of Cariology, Endodontology, Pedodontology, Academic Centre for Dentistry Amsterdam (ACTA), Louwesweg 1, NL-1066 EA Amsterdam, the Netherlands;

* corresponding author, e.zaura{at}acta.nl

	ABSTRACT

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Dissolution of the fissure walls may buffer acids formed in plaque and thus prevent the penetration of acids into the fissure. To test this, five volunteers wore dentin, enamel, and polyacrylate specimens with narrow grooves for 7 days to accumulate plaque. Temporal (pre- and post-glucose) and spatial (0-0.7 mm) pH profiles were recorded in the grooves in a flow-through reactor with pH microsensors. Mineral loss was assessed by transverse microradiography. We observed that resting pH did not differ among substrata. The median pH 1 hr post-glucose at the bottoms of dentin, enamel, and polyacrylate grooves was 6.7, 6.2, and 5.7, respectively (p < 0.01). On subject level, lesions formed in dentin correlated with pH changes in polyacrylate, where no buffering of acids due to mineral dissolution occurred. We conclude that fluoride-deficient tissue at the bottom of a fissure is at increased risk for caries, if acids are not buffered near the entrance to the fissure.

KEY WORDS: plaque • pH • demineralization • microsensors • fissure

	INTRODUCTION

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Since the introduction of fluoridated dentifrices in the 1970s, the extent and the severity of cavitations have diminished, and caries prevalence and incidence have declined (Fejerskov and Baelum, 1998). Today, the most caries is found in plaque stagnation sites shielded from toothbrushing and access of saliva, suggesting a limited effectiveness of fluoride at these sites.

In our groove model, developed to simulate and study plaque stagnation sites, fluoride dentifrices were most effective at the top of the groove (Lagerweij et al., 1996, 1997). An in vitro attempt to show that a high fluoride concentration would protect dentin more successfully (Zaura-Arite et al., 1999) revealed interesting findings: Although fluoride did inhibit mineral loss, it also changed the demineralization pattern along the depths of the grooves. Lesions were less demineralized but extended deeper into the groove than in the control group. We proposed that, due to the fluoride-induced inhibition of demineralization, fewer hydrogen ions were neutralized at the entrance to the groove, exposing the fluoride-deficient deeper parts of the groove to low pH and consequently resulting in the observed changes in the demineralization pattern.

One way to test this hypothesis would be to perform pH measurements along the depths of grooves of different solubilities. The dimensions of the grooves (0.2 mm wide) preclude the use of conventional pH electrodes, requiring a microsensor-approach where highly localized measurements are possible due to the micrometer scale sensing tip and the three-dimensional micro-scale positioning of the sensor. For almost two decades, microsensors have been used for the in situ detection of concentration gradients in industrial and environmental biofilms, and they are considered the best choice for direct measurements (Revsbech and Jørgensen, 1986; de Beer, 2000).

The overall aim of this research was to test the hypothesis that increased acid resistance of the fissure walls would lead to high acid challenge at the bottom of the fissure. We assessed this by microprofiling the plaque pH within artificial fissures of different solubilities. Additionally, we aimed to assess the relationship between the fissure pH and the demineralization potential of an individual.

	MATERIALS & METHODS

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Subjects and Specimens
Five healthy volunteers with no signs of active caries or periodontal disease participated in this study. The study protocol was approved by the institutional review board, and informed consent was obtained from all subjects. The previously developed in situ groove model was used (Lagerweij et al., 1997). This involved 3 0.2-mm-wide and 0.7- to 0.8-mm-deep grooves cut in 6-mm-diameter discs made from bovine enamel, bovine dentin, and polyacrylate (Perspex). For plaque accumulation, the discs from all 3 substrata were fixed to the buccal flanges of a removable mandibular appliance. During the experiment, the subjects were asked to maintain their regular diet, and to brush the dentition and the appliance with a fluoride-containing toothpaste twice a day, avoiding brushing over the specimens. The discs were removed after 7 days in situ. The experiment was repeated 2 or 3 times with each subject, with the various discs placed in different positions.

The Equipment for pH Measurements
For extra-oral plaque pH measurements, freshly prepared H⁺- selective membrane microelectrodes (de Beer, 2000) were used. Borosilicate glass microcapillaries were pulled with a micropipette puller (Pul-1, World Precision Instruments, Sarasota, FL, USA). Breaking the tips resulted in a tip with a diameter of 7 to 15 µm. The capillaries were subsequently dried for 15 min at 150°C, silanized at 200°C for 2 hrs in a glass container (175 mL) with 15 µL of N, N-dimethyl-trimethyl-silyl amine (Fluka), and filled with electrolyte solution (300 mM KCl in 50 mM KH₂PO₄ [pH 7]). The tip of the microcapillary was filled with liquid and polyvinyl chloride (PVC)-gelled membrane to improve the stability and performance of the sensor (de Beer et al., 1997). The liquid membrane consisted of 10% H-ionophore II, and 1% potassium tetrakis (4-chlorophenyl) borate in 2-nitrophenyl octal ether. For the PVC membrane, 10% PVC was added to the liquid membrane solution and mixed with 3 volumes of tetrahydrofuran (THF). All components were obtained from Fluka (Fluka Chemi, Zwijndrecht, the Netherlands). Microsensors were left for 2 hrs in air at room temperature for THF to evaporate, resulting in a solid H⁺-selective membrane at the tip of the sensor.

For microelectrode positioning, an XYZ computer-controlled microtranslator was used, consisting of 3 linear actuators (850 G, Newport Corporation, Evry, France). The microelectrode and the Ag/AgCl half-cell reference electrode (Radiometer Analytical S.A., Copenhagen, Denmark) were connected to an amplifier and a Data Acquisition Card (National Instruments, Le Blanc, France). Software developed in LabView (National Instruments) was used for data acquisition and positioning of the microelectrode. Electrodes were calibrated in standard pH 4 and pH 7 calibration buffers against the Ag/AgCl reference electrode before and after the experiments.

pH Measurements
Subjects were asked to refrain from any food and drink intake for at least 2 hrs before samples were taken. From each disc, we obtained 2 samples by breaking the disc into halves along the middle groove. The measurements were performed in a flow-through reactor, placed in a Faraday cage to minimize electrical disturbances. Fifty mL of 1 mM KH₂PO₄ (‘outside’ buffer) at pH 7 was passed over the samples at a constant flow rate of 16 mL/min. The reference electrode was placed in the ‘outside’ buffer, not in direct contact with the sample. The circulation of the buffer provided rinsing of the electrodes and prevented stagnation of reaction products above the specimen.

Under microscopic guidance (Stereomicroscope Stemi SV6, Zeiss, Göttingen, Germany), the microelectrode was positioned at the intersection of the specimen surface and the middle of the groove, just above the plaque surface. Next, the electrode was positioned 700 µm into the groove (‘bottom’). Measurement cycles were as follows: Average sensor potentials (125 readings/sec) were recorded at 10-µm intervals from the ‘bottom’ until 0.3 to 0.4 mm above the specimen in the ‘outside’ buffer (pH 7), after which the electrode was returned to the ‘bottom’. The readings in the ‘outside’ buffer served as the internal standard.

The resting plaque was probed at 2 to 4 sites along the groove until comparable profiles from 2 sites were recorded. Occasionally, after the first contact with the plaque, the electrodes gave false signals, as noted by the readings in the ‘outside’ buffer. In such a case, the electrode was replaced with a new one, and probing was re-started. Only when a value corresponding to the pH 7 of the ‘outside’ buffer was obtained within a few sec was the electrode used for further measurements. Then the microelectrode was left at the ‘bottom’, and the buffer solution was removed from the reactor. One drop (approximately 10 µL) of 10% glucose (pH 7, made in ‘outside’ buffer solution) was applied to the groove. After 2 min, the reactor was refilled with ‘outside’ buffer solution, the flow re-started, and 15-18 consecutive measurement cycles were recorded during 60 min. Then, to evaluate the effect of repeated probing on pH profiles, we placed the microsensor at 2 or 3 previously undisturbed sites in the groove and recorded additional profiles.

Microradiography
For the analysis of mineral loss, the dentin specimens were sectioned, and 2 sections per specimen were radiographed according to the transverse microradiography (TMR) procedure (Lagerweij et al., 1994; van Strijp et al., 1995). To quantitate the mineral loss in the groove, we determined the integrated mineral loss (IML, vol% x µm) at 4 equidistant positions throughout the depth of the groove and averaged the findings.

Data Analysis
All statistical analyses were performed with SPSS (Version 9.0). Variables included in the analysis were: substratum, resting pH, minimum pH (pH_min) following glucose application, time at which pH_min was reached (T_min), and pH 60 min after glucose application (pH₆₀). From each measured pH profile, 2 values were analyzed: at about 0.2-mm depth into the groove (‘top’) and at 0.7 mm into the groove (‘bottom’). The data were not normally distributed. The Wilcoxon Signed-ranks Test (p < 0.001) was used to compare the ‘top’ and ‘bottom’ variables of the same groove, and the effect of consecutive probing; the Kruskal-Wallis test (p < 0.001) was used to reveal differences among all 3 substrata; and the Mann-Whitney test (p < 0.01) was used to compare 2 independent pairs.

To correlate the mineral loss with the pH data, we ranked the subjects by average mineral loss in the dentin grooves, and calculated Spearman’s correlation.

	RESULTS

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Visual Inspection
In the grooves from subjects A and D, the plaque had accumulated flush with the surface of the disc. Few holes were visible in the samples from subjects C and E. During the positioning of the electrode, these areas were avoided. In the samples from subject B, a gap (approximately 20-60 µm wide) divided the plaque into 2 layers, both of which reached the surface of the disc.

Resting pH
The resting pH at the ‘bottom’ of one groove varied between 0 and 1.25 (median 0.2) pH units. The corresponding variation at the ‘top’ was less: from 0 to 0.88 (median 0.1) pH units.

The variability in the resting pH among samples was considerable (Table 1), with median pH at the ‘top’ and at the ‘bottom’ being 6.7 and 6.9, respectively. No statistical difference was found in the resting pH of plaque formed on the 3 substrata (Table 2).

View larger version (48K): [in this window] [in a new window]   Figure 1. Examples of plaque pH profiles by depth and by time in grooves from 3 substrata: (A) dentin, (B) enamel, and (C) polyacrylate (Perspex) in seven-day-old in situ-grown plaque (subject A). Specimens were placed in pH 7 buffer. The pH was measured at 10-µm intervals throughout the buffer (depth, -0.3 to 0 mm) and plaque (depth, 0 to 0.7 mm) in the groove, and at regular time points before (time, -2 to 0 min) and after (time, 2 to 50 min) 10% glucose application (arrow).

TMR Findings and Correlations with the pH Data
Fig. 2 shows a microradiograph from a dentin specimen after 1 wk in situ (subject A). The extent of demineralization of dentin specimens was variable among the subjects (Table 1), with mineral loss values up to 1200 vol% x µm. The maximum IML was found 150-250 µm deep into the groove (data not shown).

View larger version (96K): [in this window] [in a new window]   Figure 2. Microradiograph of a section cut from a dentin groove specimen after a seven-day in situ period (subject A), showing demineralization along the walls of the groove (arrow). Bar indicates 0.1 mm.

	DISCUSSION

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The pH of in situ grown plaque was assessed in vitro throughout the depth (0.7 mm) of narrow grooves in dentin, enamel, and polyacrylate. It was shown that, following a brief exposure to glucose, the pH at the bottom of the groove was related to the solubility of the substratum. The lowest pH was measured in polyacrylate, i.e., non-soluble grooves. By subject, the mineral loss in dentin correlated negatively with the minimum pH at the bottom of the polyacrylate grooves.

In dentin and in enamel, the lowest post-glucose pH was recorded in the tops of the grooves. The highest mineral loss (in dentin) was measured at the same position in the groove. This suggests the production of organic acids and also the buffering of acids near the entrance to the soluble groove. This is in line with the clinical situation, where early signs of fissure caries are usually found at the fissure entrance rather than in the fissure proper (Nyvad and Fejerskov, 1994). Both higher numbers of vital micro-organisms and more initial enamel lesions were found at the fissure entrance than at the deeper parts (Ekstrand and Bjørndal, 1997), when not fully erupted third molars were studied.

In polyacrylate, the post-glucose pH was low throughout the depth of the groove. If the acid production in polyacrylate is similar to that in dentin and enamel (i.e., the highest at the top of the groove), then the pH at the bottom of the groove decreased due to the diffusion of hydrogen ions. Consequently, in plaque stagnation sites, the deeper parts will be exposed to high acid challenge if the enamel at the top part of the fissure is fluoride-enriched i.e., less soluble in an acid environment. This will lead to increased caries risk. Clinically, this might explain the phenomenon known as ‘hidden caries’ (Ricketts et al., 1997)—radiologically detected lesions in dentin at the bottoms of fissures with sound enamel at the entrances.

An interesting finding was the negative correlation between the mineral loss in dentin and the minimum pH in the polyacrylate, though not in the enamel or dentin grooves. Since the 1940s (Stephan, 1944), plaque response to sugar has been linked to the caries activity of the individual. More recent studies find this correlation only when surfaces depleted or devoid of mineral—such as carious fissures and tongue (Fejerskov et al., 1992), white-spot lesions (Margolis and Moreno, 1992; Margolis et al., 1993), or samples pooled from whole-mouth with numerous white-spot lesions (Gao et al., 2001)—are compared with surfaces of caries-resistant individuals. If sound, thus mineral-rich, surfaces of caries-active individuals are considered, no or poor correlations were demonstrated between different pH parameters of fermenting plaque and caries activity (Fejerskov et al., 1992; Dong et al., 1999). These and our findings suggest that any pH change measured on a, in principle, dissolving substratum is a conservative estimate of the number of hydrogen ions formed, and thus not a reliable indicator of an individual’s caries potential.

Regarding the reliability of the microsensor pH measurements, it is documented that signal stability and lifetime of liquid membrane microsensors are poor compared with those of full-glass, amperometric, and optical microsensors (de Beer, 2000). Typically, liquid membrane sensors drift. They can be used for a few days, after which the detection limit is too high or the calibration levels off. However, these shortcomings were overcome by the use of freshly prepared electrodes for each sample, and by the use of the ‘outside’ buffer (pH 7) to correct for the drift and to monitor stability of the signal.

This study demonstrated the relation of fissure substratum solubility with in situ-grown plaque pH response to sugar in vitro. Future studies on changes in plaque mineral content should address whether plaque acids were instantly buffered by dissolving mineral, or by a depot accumulated in plaque. The consequences of substratum solubility on plaque microbial composition and acidogenicity should also be studied. Our findings suggest that plaque pH at the bottoms of plaque-filled fissures might become low, thus resulting in caries in the deeper parts of the fissure. This phenomenon depends on the solubility of fissure walls and, presumably, the properties of saliva.

	ACKNOWLEDGMENTS

 
We thank the staff of our Department for volunteering to wear in situ appliances, Dr. Frank Roe (Center for Biofilm Engineering, Montana State University, Bozeman), Dr. Dirk de Beer, and Dr. Armin Gieseke (Department of Biogeochemistry, Max Planck Institute for Marine Microbiology, Bremen, Germany) for sharing their knowledge of pH microsensors, and Dr. Irene Aartman (Department of Social Dentistry and Dental Health Education, Academic Centre for Dentistry Amsterdam, the Netherlands) for helping with statistical analysis of the data. The study was performed with financial support from the Netherlands Institute for Dental Sciences. A preliminary report was presented at the 48th ORCA Congress, July 4–8, 2001, in Graz, Austria. The work has been published as a chapter in a PhD thesis entitled "Plaque stagnation sites and dental caries. Studies on dental biofilm and dentin demineralization in narrow grooves", defended at the University of Amsterdam, The Netherlands.

Received September 25, 2001; Last revision May 17, 2002; Accepted June 13, 2002

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