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RESEARCH REPORT |
Department of Cariology Endodontology Pedodontology, Academic Center for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066 EA, Amsterdam, Netherlands;
* corresponding author, r.ozok{at}acta.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: dentin demineralization • dentin perfusion • dentinal fluid • remaining dentin thickness
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Root caries is becoming more common in communities with lower prevalence of coronal caries and longer life-expectancy (Katz et al., 1982). After gingival recession and the loss of the cementum, there is a continuous outward flow of the dentinal fluid (Ciucchi et al., 1995), which may intervene in dentin demineralization. The effect of perfusing pulp chamber with a supersaturated fluid at reducing lesion depth has been reported (Shellis, 1994).
The tubule diameters and density increase toward the pulp (Mjör and Nordahl, 1996), as does the perfusion rate (Reeder et al., 1978). During demineralization, although a large amount of mineral dissolves and the peritubular dentin is partly lost, the tubules remain predominantly intact (Arends et al., 1989). In contrast, it has been reported that tubule diameters are changed by in vitro demineralization (Arends et al., 1995). Any alteration in tubule diameters should have important consequences on the lesion progression in dentin, and the perfusion rate that varies with the fourth power of the tubule radius (Pashley, 1990).
The effects of dentinal fluid flow and the proximity to the pulp on the degree of dentin demineralization have not been tested in a systematic way. The aim of this study was to evaluate the effect of dentin perfusion on the in vitro demineralization of human root dentin and the effect of demineralization on the perfusion rate through dentin. The secondary aim was to analyze the correlation between the proximity to the pulp and the degree of demineralization.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Specimen Preparation
We prepared transverse tooth segments using two parallel horizontal cuts at approximately 4 mm above and 3 mm below the CEJ. After the pulp was removed, these slices were embedded in Vertex 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. We took mesiodistal radiographs to estimate the remaining dentin thickness. 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. 1
). This window was etched with 3.5 mol/L phosphoric acid for 15 sec, and rinsed.
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). Briefly, 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). The perfusion rate varies considerably between and among different teeth, and different locations within a tooth (Pashley et al., 1987). Therefore, to increase the statistical power with a relatively low sample size (n = 5), we prepared a larger batch of specimens (n = 40) and used a pre-determined range of perfusion rate (28-60 nL/min) as a specimen inclusion criterion from this batch.
Demineralization Process
The 3x3-mm window of exposed buccal dentin was covered with 1 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) that was changed each day. The perfusion fluid in the reservoir tubing leading up to the specimen and pulp chamber was de-ionized water (Fig. 1). We created the perfusion pressure (3.14 kPa) by positioning the reservoir 32 cm above the level of the air bubble. In the non-perfused group, we blocked the pulpal access to ensure that, although the pulp chamber was hydrated, no hydrostatic pressure was created.
Transverse Microradiography (TMR)
At the end of the five-day experiment, approximately 200-µm-thick plano-parallel sections were cut perpendicular to the surface from each lesion. The sections were sealed in a moist medium on plate-holders bearing an aluminum step wedge of 12 25-µm steps. We took microradiographs using a Cu(K
) x-ray source (Philips, Eindhoven, Netherlands) on high-resolution plates (Type 1A, Microchrome Technology, San Jose, CA, USA). The exposure time was 15 min at 20 kV and 20 mA. The microradiographs were digitized by a microscope (Zeiss Axioplan, Jena, Germany) and XC-77CE CCD camera (Sony, Tokyo, Japan). A 650 x 500-µm area at each of the 3 levels that correspond to those determined for remaining dentin thickness measurements (see below) were scanned. Densitometric analysis of these scanned sites was carried out with use of the computer software dedicated to TMR (TMR 1.25e, Inspektor Research Systems, Amsterdam, Netherlands). Integrated mineral loss (in vol% µm) was calculated on the basis of the following established definitions: Mineral content of sound dentin is 50 vol%, and lesion depth is the distance from the outer surface of the specimen (0 vol% mineral content) to the position where the mineral content reaches 95% of that of the sound dentin (Arends and ten Bosch, 1992).
Remaining Dentin Thickness (RDT) Measurements
Fig. 2 shows a digital TMR image of a 200-µm-thick section that was divided into three quadrilaterals, which were outlined by tracing of the dentinal tubule orientation. The cross-sectional areas within these tracings were measured (in mm2) by means of Axiovision 3.0 image analysis software (Zeiss, Hallbergmoos, Germany), and used as RDT values. Since the outer surface of the lesion was divided into equal lengths, the distance from the outer surface to the pulp and the length of the pulp chamber wall were responsible for the differences in area among these tracings. Since the RDT in each specimen varied naturally, the corresponding lesion depth and mineral loss values were correlated with RDT.
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Variation in perfusion rate with time was analyzed by repeated-measures analysis of variance. All of the statistical analyses were performed with the use of SPSS 10.0 for Windows (SPSS International BV, Gorinchem, Netherlands).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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. While perfusion led to a 22 vol% reduction in mineral loss, the lesion depth was reduced by 8%.
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The mean initial dentin perfusion rate (in nL/min + SD) in the perfused and non-perfused groups was 43.2 + 11.8 and 36.8 + 8.6, respectively (p = 0.078); there was no significant effect in either group on mineral loss (p = 0.280) or lesion depth (p = 0.632).
In the perfused group, the mean perfusion rate throughout the demineralization process was 49 nL/min, and the perfusion rate profile for each specimen did not vary significantly over time (p = 0.982).
It was observed that the inner boundary of the perfused lesions was diffuse or blurred, whereas in the non-perfused group, it was sharply defined (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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For the first time, an inverse correlation between remaining dentin thickness and the degree of demineralization is shown. Although an important factor, the increasing diameter and density of the tubules toward the pulp cannot solely explain this finding. The presence of the tubules provides an easy pathway for penetration of the cariogenic acids. However, in the perfused lesions, diffusion along the tubules was probably reduced or prevented by the outward counter-flow of water (Pashley and Matthews, 1993). Diffusion of the acetate buffer is therefore assumed to have occurred through the intertubular matrix. Perfusion rate increases with decreasing dentin thickness (Reeder et al., 1978). Therefore, in the perfused lesions as the RDT decreased, one would actually expect an enhanced protective effect of fluid flow. Thus, the observation of increased demineralization closer to the pulp in both groups (perfused and non-perfused) needs explanation. The increased outward fluid flow might increase the rate at which de-ionized water rinsed away any reaction products of the pH 5 acetate buffer that may have slowly occluded the tubules in the absence of flow (Pashley and Matthews, 1993). Another possible explanation: The lower mineral content of the dentin at regions closer to the pulp probably surpassed the protective effect of perfusion with water. During demineralization, the acid must diffuse from the outer solution into the lesion, and dissolved mineral must diffuse from the inner part of the lesion to the outside medium. As the mineral content of the intertubular matrix and the thickness of peritubular dentin, which is highly mineralized, decrease toward the pulp (Kinney et al., 1996; Mjör and Nordahl, 1996), the transport processes are expected to be faster (Arends et al., 1987; ten Cate et al., 1995). This is also true for natural caries lesions; deep dentin lesions, compared with more superficial ones extending less than 0.5 mm into the dentin, progress at a much faster rate (Foster, 1998).
The blurred (or diffuse) appearance of the inner boundary of the perfused lesion, in comparison with that of the non-perfused one, which was sharply defined (Fig. 3), is in very good accord with the description of a natural carious dentin lesion (Jones and Boyde, 1987; McIntyre et al., 2000). For the defense reactions, dentinal tubules and their contents provide indispensable communication media between the external stimulus and the pulp. Unfortunately, a shortcoming of the studies of dentin from extracted teeth is the absence of the live odontoblasts and their processes. However, to approximate clinical reality, we conclude that in vitro studies on root (dentin) demineralization should not disregard the effect of dentinal fluid flow.
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ACKNOWLEDGMENTS |
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Received November 1, 2001; Last revision August 12, 2002; Accepted September 5, 2002
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REFERENCES |
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  A.R. Ozok, M.-K. Wu, J.M. Ten Cate, and P.R. Wesselink Effect of Dentinal Fluid Composition on Dentin Demineralization in vitro J. Dent. Res., November 1, 2004; 83(11): 849 - 853. [Abstract] [Full Text] [PDF]
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