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Effect of Perfusion with Water on Demineralization of Human Dentin in vitroDepartment of Cariology Endodontology Pedodontology, Academic Center for Dentistry Amsterdam (ACTA), Louwesweg 1, 1066 EA, Amsterdam, Netherlands; Correspondence: * corresponding author, r.ozok{at}acta.nl
Dentinal fluid rarely features in caries studies of dentin. The mutual effects of in vitro perfusion and dentin demineralization were investigated. The correlation between the remaining dentin thickness and demineralization was also analyzed. Buccal cervical dentin windows in human tooth segments were demineralized either with or without perfusion with water at 3.14 kPa. Transverse microradiography revealed that dentin perfusion reduced the amount of mineral loss from the lesions by 22vol%; the reduction in lesion depth was 8%. Perfusion rate, which was measured throughout the demineralization process by means of a fluid transport model, did not change significantly. Lesions formed closer to the pulp exhibited increased mineral loss and lesion depth. In conclusion, dentinal fluid flow offers some protection against demineralization. For a better approximation of clinical reality, therefore, in vitro studies on dentinal caries should consider the effect of dentinal fluid flow.
Key Words: dentin demineralization • dentin perfusion • dentinal fluid • remaining dentin thickness
Unlike enamel, dentin is a vital tissue capable of producing reactions to external stimuli (Thylstrup and Fejerskov, 1994). It is highly hydrated, and most of the water content is located in the dentinal tubules, which are filled with dentinal fluid (Knutsson et al., 1994). In normal function, the composition of dentinal fluid is controlled by the odontoblasts (Bishop, 1992). However, following a disturbance such as dental caries, attrition, or restorative procedures, it may be closer to the composition of the transudates from pulpal capillaries (Turner et al., 1989; Maita et al., 1991). 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.
Intact human third molars, extracted for reasons unrelated to this study, were used. The teeth were stored in 1.5 mmol/L NaN3 solution for at least 4 wks (Özok et al., 2002). The Institutional Review Board of the Academic Center for Dentistry Amsterdam (ACTA) approved all procedures regarding the use of human tissues.
Specimen Preparation
Perfusion Rate Measurements We used a modified fluid transport set-up (Wu et al., 1998) working at 3.14 kPa to measure the perfusion rate (Fig. 1  ). 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
Transverse Microradiography (TMR)
Remaining Dentin Thickness (RDT) Measurements
Statistics The difference between the two groups (perfused vs. non-perfused) regarding initial perfusion rate and RDT was analyzed by independent-samples t test. The Pearson correlation between RDT and demineralization was calculated. The effect of perfusion on demineralization was analyzed by MANOVA, with initial perfusion rate and RDT as co-factors. 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).
The mean mineral loss and lesion depth values in the perfused and non-perfused lesions are presented in the Table  . While perfusion led to a 22 vol% reduction in mineral loss, the lesion depth was reduced by 8%.
The mean RDT (in mm2 + SD) (total of the three quadrilaterals) in the perfused and non-perfused groups was 1.49 + 0.34 and 1.51 + 0.39, respectively, and there was no significant difference between the groups (p = 0.916). There was also no significant difference between the perfused and non-perfused groups when the mean areas of the corresponding top, middle, or bottom quadrilaterals were separately compared (data not shown). However, RDT influenced both the mineral loss and lesion depth significantly (p = 0.002 and p = 0.001, respectively). As the RDT decreased, the mineral loss and lesion depth increased (r = -0.391, p = 0.033, and r = -0.545, p = 0.002, respectively). 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
The present findings demonstrate that outward dentinal fluid flow diminishes mineral dissolution from the dentin, but it has less effect on lesion progression into the tissue. Using a simulated dentinal fluid that was supersaturated with respect to apatite, Shellis (1994) also found that perfusion of the pulp reduced the depth of demineralization. The application of hydrostatic pressure, however, had no further effect on the reduction of lesion depth. In the study by Shellis (1994), the cementum was not removed, and the perfusion rates were not measured; it is therefore probable that there was no perfusion through the dentin. Therefore, the current study took a more systematic approach, where perfusion rate was measured throughout the experiment. It is possible, however, that the use of water as a perfusion fluid in our study may have underestimated the protective effect of outward fluid flow, compared with the use of a perfusion fluid that was buffered to pH 7.4, and that contained calcium and phosphate in a manner similar to that used by Shellis (1994). Here we attempted to clarify one aspect, namely, the effect of flow of the dentinal fluid on dentin demineralization. For this purpose, an in vitro model of early demineralization events was developed, and de-ionized water was chosen as a perfusion fluid. Future studies will deal with other aspects of the dentinal fluid (e.g., electrolyte composition, degree of saturation, and protein concentration) that may play an essential role in lesion formation and progress into the dentin. 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
This study was supported by the Netherlands Institute for Dental Research (IOT). The assistance of R.A.M. Exterkate and M.J. Buijs in the accomplishment of this research is gratefully acknowledged. Received for publication November 1, 2001. Revision received August 12, 2002. Accepted for publication September 5, 2002.
Journal of Dental Research, Vol. 81, No. 11,
733-737 (2002)
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ABSTRACT
INTRODUCTION
) 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 (
