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Effect of Dentinal Fluid Composition on Dentin Demineralization in vitro

Department of Cariology Endodontology Pedodontology, Academic Center for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066 EA, Amsterdam, Netherlands;

^* corresponding author, r.ozok{at}acta.nl

	ABSTRACT

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Dentin demineralization is reduced by perfusion with water. We hypothesized that a simulated dentinal fluid (SDF) that contains albumin, in addition to electrolytes, would be more effective in reducing dentin demineralization than water alone, and this effect would increase with increasing flow rate of SDF. Perfusion rate in tooth segments that carried buccal cervical dentin windows was measured in a fluid transport set-up. These windows were then demineralized under perfusion with water, or SDF at 1.47 kPa for 31 days. We analyzed integrated mineral loss and lesion depth with the use of transverse microradiography (TMR), which revealed that 38% more mineral dissolved from dentin lesions perfused with water than from those perfused with SDF. The former were also 18% deeper. Flow rate of dentinal fluid showed no correlation with demineralization. We concluded that composition of dentinal fluid is an important determinant of the rate of lesion formation and progression in dentin.

KEY WORDS: dentin demineralization • dentinal fluid composition • dentin perfusion

	INTRODUCTION

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The destruction caused by the carious process is either accelerated or restricted by the responses of the pulp-dentin complex (Larmas, 2001). Dentinal fluid, an important component of the pulp-dentin complex, forms a communication medium between the pulp (via the odontoblastic layer) and remote regions of the dentin.

Caries-like dentin lesions formed in vitro with perfusion of the pulp with a simulated dentinal fluid (SDF), which contained electrolytes, were less deep than lesions in non-perfused teeth (Shellis, 1994). Dentinal fluid flow (perfusion with water) reduces mineral dissolution from in vitro dentin lesions in comparison with non-perfused ones (Özok et al., 2002a). Thus, the reduction in mineral dissolution is expected to be more pronounced as the flow rate increases. Flow rate increases closer to the pulp, and as the dentin gets thinner (Pashley, 1985). However, in the study by Özok et al.(2002a), lesions formed closer to the pulp were deeper than those formed in superficial dentin, and contained less mineral. This required further analysis.

Since demineralization is essential for caries progress in dentin, the composition of dentinal fluid is presumably of primary importance in maintaining the equilibrium between the peritubular dentin and the dentinal fluid (Larmas, 2001). During the caries process, in contrast, calcium and phosphate levels do not increase as expected (Larmas et al., 1986). In normal function, the composition of dentinal fluid is controlled by the odontoblasts (Bishop, 1992). However, a disturbance, such as dentinal exposure or dental caries, may lead to a change in the composition of dentinal fluid (Turner et al., 1989), which is then more likely to be a transudate from pulpal capillaries (Maita et al., 1991). Dentinal fluid transuding from exposed dentin in vivo contains large amounts of albumin (Knutsson et al., 1994). The effect of a SDF, containing albumin in addition to electrolytes, on the changes in the mineral content of in vitro dentin lesions has not been studied.

We tested the null hypotheses: (1) that perfusing dentin with a simulated dentinal fluid (SDF) containing 1% albumin and electrolytes has no additional protective effect on the use of water alone as a perfusate on in vitro demineralization of human cervical root dentin; and (2) that the flow rate of dentinal fluid has no effect on demineralization.

	MATERIALS & METHODS

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Third molars used in this study were extracted as a part of normal treatment at the Department of Oral Surgery at the Academic Medical Center (AMC) of the University of Amsterdam, and stored in 1.5 mmol/L NaN₃ solution for at least 4 wks (Özok et al., 2002b). The Institutional Review Board of the Academic Center for Dentistry Amsterdam (ACTA) approved all procedures regarding the use of human tissues.

Specimen Preparation
Transverse tooth segments were cut at the cervical level [4 mm above and 3 mm below the cementum-enamel junction (CEJ)] (Fig.). After the pulp was removed, these slices were embedded in Vertex self-polymerizing methyl methacrylate polymer (Dentimex, Zeist, Netherlands). Access into the pulp chamber was obtained on the lingual side. The buccal dentin was exposed with the use of 240-, 400-, and 600-grit abrasive paper. The margins adjoining the tooth and Vertex were sealed with cyanoacrylate glue (Permacol, Ede, Netherlands). The buccal surface was coated with nail varnish, leaving a 3x3-mm dentin window exposed, with equal halves on either side of the CEJ (Fig.). This window was etched with 3.5 mol/L phosphoric acid for 15 sec, then rinsed.

View larger version (36K): [in this window] [in a new window]   Figure. The location of the segment in the tooth, and the fluid transport set-up, and the set-up used to create dentin lesions under perfusion.

Flow Rate Measurements
We used a fluid transport set-up (Özok et al, 2002a), working at 1.47-kPa pulpal pressure (Ciucchi et al., 1995), to measure flow rate through the dentin (Fig.). The set-up consisted of a silicon-rubber tube bearing the specimen, with one end open to atmospheric pressure via the fluid reservoir, and the other connected to a glass capillary in which an air bubble was introduced. The computerized infrared detector (Flodec, De Marco Eng., Geneva, Switzerland) traced the displacement of the air bubble to a minimum of 5 µm (2.7 nL).

Dentin permeability varies considerably among different teeth, and among different locations within a tooth (Pashley, 1985; Pashley et al., 1987). Therefore, we distributed the specimens between the experimental groups not randomly, but taking care that all groups had similar mean ± SD values. Several specimens were screened (n = 28), and were then divided into two groups, A and B. The mean flow rate (in nL/min ± SD) in groups A and B was 10.1 ± 11.6 and 10.4 ± 11.6, respectively. There was no significant difference between groups (Mann-Whitney U test, p = 0.943).

Demineralization Process
The 3x3-mm window of exposed buccal dentin was covered with 3 mL of demineralization solution (2.2 mmol/L CaCl₂•2H₂O; 2.2 mmol/L KH₂PO₄, 50 mmol/L CH₃COOH, and 1.5 mmol/L NaN₃ at pH 5.0) (ten Cate et al., 1998).

Throughout the 31-day demineralization period, the specimens in group A were perfused with de-ionized water, and those in group B with a simulated dentinal fluid (SDF). The SDF contained: 30.0 mmol/L HEPES; 107.8 mmol/L H₃PO₄; 0.93 mmol/L CaO; 0.60 mmol/L MgO; 77.6 mmol/L NaCl (Shellis, 1994); 1.5 mmol/L NaN₃, and 1% bovine serum albumin (BSA) fraction V (Sigma-Aldrich Chemie, Steinheim, Germany). The pH was adjusted to 7.4 by the addition of KOH pellets at room temperature. Both demineralizing buffer and perfusates were changed every 3 days.

We created the perfusion pressure (1.47 kPa) by positioning the reservoir leading up to the specimen 15 cm above the pulp chamber.

Transverse Microradiography (TMR)
We rinsed 31-day-demineralized specimens and cut 200-µm-thick plano-parallel sections perpendicular to the lesion surface using a Well 3242 diamond wire saw (Le Locle, Switzerland). Microradiographs of these sections were taken as described earlier (Özok et al., 2002a). These microradiographs were digitized, and on each digital image a 2500 x 1550-µm area at 3 levels (occlusal, middle, and cervical) was scanned for densitometric analysis (TMR 1.25e software, Inspektor Research Systems, Amsterdam, Netherlands). Integrated mineral loss (IML) (vol% µm) and lesion depth (µm) were calculated as described (Arends and ten Bosch, 1992), based on the mean of these 3 scans, for each specimen.

Statistics
Statistical analyses were done with SPSS 10.0 for Windows (SPSS International, Gorinchem, Netherlands). We used a one-way ANOVA test to assess overall differences between RDT means, and the effect of perfusate on IML and lesion depth means. Once overall differences among the means were observed, post hoc tests of LSD (least significant difference) were performed for multiple comparisons. The Spearman correlation between flow rate and demineralization was calculated. We considered values of p 0.05 as significant.

	RESULTS

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We examined the mean ± SD IML and lesion depth values for 31-day lesions formed in dentin perfused with water or SDF (Table 1). Dentin lesions that formed under perfusion with water showed 38% more mineral loss than those formed in SDF-perfused dentin (p = 0.0001). The former were also 18% deeper (p = 0.005).

We dialyzed the BSA-containing SDF (2.50 mmol/L calcium) against BSA-free SDF (0.92 mmol/L calcium), and analyzed the amount of albumin-bound or dissolved calcium concentration by the use of atomic absorption spectrometry. Approximately 0.62 mmol/L calcium was dissolved from the internal solution (BSA-containing SDF). When the same measurement was repeated after the pH of the external solution (BSA-free SDF) was adjusted to 5 with the addition of acetic acid buffer, the amount of calcium ions dissolving from the internal solution increased to 1.02 mmol/L.

Effect of Flow Rate on Demineralization
We took advantage of natural variation in permeability of teeth to test the effect of flow rate of dentinal fluid on demineralization. The teeth in groups A and B were further divided into 2 subgroups of low and high dentin permeability (n = 7). The mean flow rate (in nL/min ± SD) for water- or SDF-perfused low- or high-permeability specimens was 1.6 ± 1.3, 1.6 ± 1.4, 18.7 ± 10.8, and 19.3 ± 10.4, respectively. A Mann-Whitney U test confirmed a significant difference between low- and high-permeability means in both water- and SDF-perfused groups (p = 0.002 and p = 0.002, respectively). The mean RDT (in mm² ± SD) was 4.29 ± 0.61 and 4.28 ± 0.84 for water-perfused low- and high-permeability groups, respectively. The corresponding values for SDF-perfused low- and high-permeability groups were 4.22 ± 0.81 and 4.80 ± 0.63, respectively. A one-way ANOVA test revealed no significant difference between the groups regarding RDT (p = 0.421). We examined the means ± SD of IML and lesion depth in water- or SDF-perfused lesions formed in low- or high-permeability dentin (Table 2). Lesions formed in water-perfused low-permeability dentin contained 50% less mineral (p = 0.001) and were 23% deeper (p = 0.016) than those formed in SDF-perfused low-permeability dentin. Lesions formed in water-perfused high-permeability dentin contained 28% less mineral (p = 0.021) and were 14% (p = 0.103) deeper than those formed in SDF-perfused low-permeability dentin.

A perfusate containing electrolytes and BSA may have reduced the flow rate. Two extra specimens were prepared and perfused with a 1% solution of BSA, BODIPY FL conjugate (Molecular Probes, Leiden, Netherlands), for the measurement of BSA concentration in the filtrate (data not shown), which was only 12% of the expected concentration.

	DISCUSSION

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We must reject the first null hypothesis, because the mineral content of the in vitro dentin lesions perfused with SDF, containing albumin and electrolytes, was significantly higher than that in those perfused with water only. The reason for this may be either reduced mineral dissolution from the dentin, or adsorption of the perfusate-derived calcium and phosphates to the tubule walls, and perhaps their diffusion into demineralized intertubular matrix.

Although SDF-perfused lesions were less deep than those perfused with water, when the specimens were divided into subgroups in relation to their permeability, only in lesions of low-permeability dentin was this difference significant. We assume that interdiffusion of SDF and demineralizing buffer within dentinal tubules caused precipitation of minerals derived from SDF and/or dissolving peritubular dentin. No microscopy was performed in this study to confirm this assumption. However, there is evidence that interaction between the demineralizing buffer and SDF or dissolved dentin mineral could initiate mineral precipitation in dentinal tubules, which mimics sclerotic dentin-like formation (Daculsi et al., 1987; Shellis, 1994). The possible effect of the degree of saturation of dentinal fluid on demineralization needs further analysis.

Additionally, mineral and albumin adsorption to peritubular dentin might form an increased resistance in the tubule, which would decrease the inward diffusion of the acidic buffer. Our finding that a lower-than-expected concentration of BSA filtered across the dentin specimens suggests that the rest of the BSA presumably remained in the perfusate in the pulp chamber, and/or adsorbed to peritubular dentin during perfusion. Albumin retention in dentinal tubules and up to 43% reduction in flow rate have been reported (Pashley et al., 1982; Hahn and Overton, 1997). This phenomenon would more easily occur in narrower tubules than in larger ones. The similarity of RDT between the low- and high-permeability groups suggests that the difference in dentin permeability was determined by the differences in tubule density and diameters (Pashley, 1985). Thus, low-permeability dentin contained probably fewer tubules per unit area and/or narrower tubules. However, the lack of a significant difference in IML and lesion depth between the low- and high-dentin-permeability groups of water-perfused teeth suggests that differences in flow rate do not regulate dentin demineralization. We conclude therefore that, although dentinal fluid flow protects dentin against demineralization, the protective effect falls short as tubular density and diameters increase.

The BSA-bound calcium was sufficiently high that some of it was released on dissolution, thereby increasing the dissolved calcium concentration of the SDF. Albumin releases calcium in exchange for free hydrogen when pH drops. Our findings have shown the same. It is most probable that, with the addition of the calcium from dissolved dentin apatite after acid challenge, the saturation at the dentin front increased to a level to favor remineralization. Another possibility is that the growth of calcium phosphate crystals may be promoted by the albumin bound to peritubular dentin surfaces. Although many proteins, when present in a supersaturated solution, may act as inhibitors of calcium phosphate, nucleation of octacalcium-phosphate-like seed crystals around immobilized albumin has been shown (Nancollas et al., 1989).

Reports on the ionic composition of pulpal, predentinal, and dentinal fluid are scarce and varying (Larmas et al., 1986; Larsson et al., 1988). Regarding the stability of the solution over 31 days, we used a modified SDF (Shellis, 1994), which is mainly based on the in vivo electrolyte content of predentin in developing rat molar teeth (Larsson et al., 1988). Serum albumin constitutes approximately half of the total protein content of dentinal fluid, which is about one-fifth of that of plasma (Maita et al., 1991). Therefore, we added 1% BSA in the SDF.

The possible functions of albumin, which is a principal transport and depot protein for fatty acids and divalent cations in plasma (McGilvery and Goldstein, 1983), in dentinal fluid have not been fully identified. Although in much lower concentrations than those of dentin (Thomas and Leaver, 1975; Okamura et al., 1979), caries lesions of enamel contain serum albumin (Robinson et al., 1998), which was shown to inhibit in vitro demineralization of enamel up to 50% (Arends et al., 1986).

We must accept the second null hypothesis, because we found no significant correlation between the flow rate of dentinal fluid and demineralization. In accord with this finding, although perfusing the pulp with a SDF reduced the depth of in vitro lesions formed in cementum-covered root dentin (Shellis, 1994), the application of pulpal pressures (1.34 or 3.14 kPa) had no further effect.

Since in vitro models supply improved control over experimental conditions that are difficult or impossible to establish in vivo, they provide preliminary information for the clarification of clinical phenomena. A major shortcoming of the laboratory studies of the dentin is the lack of vitality of the tissue. Demineralization of the dentin in vitro has been studied extensively. However, such findings seem to be of limited value unless the dentinal fluid is taken into consideration.

	ACKNOWLEDGMENTS

 
We thank the staff of the Department of Maxillofacial Surgery of the Academic Medical Center of the University of Amsterdam (AMC) for providing the teeth used in the present study. The technical assistance of M.J. Buijs and R.A.M. Exterkate is also gratefully acknowledged. This study was financially supported by the Netherlands Institute for Dental Research (IOT). This paper is based on a thesis submitted to the University of Amsterdam, in partial fulfillment of the requirements for the PhD degree.

Received August 1, 2003; Last revision September 3, 2004; Accepted September 7, 2004

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