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Class II Antigen-presenting Dendritic Cell and Nerve Fiber Responses to Cavities, Caries, or Caries Treatment in Human Teeth

Division of Cariology, Department of Oral Health Science, Course for Oral Life Science, Niigata University Graduate School of Medical and Dental Sciences, 5274, Gakkocho-dori 2-bancho, Niigata 951-8514, Japan;

*corresponding author, yoshiba{at}dent.niigata-u.ac.jp

	ABSTRACT

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Major histocompatibility complex (MHC) class II molecule-expressing cells are distributed in human dental pulp, and have been shown to accumulate beneath caries lesions. The responses of these cells and nerve fibers were analyzed under 5 different clinical conditions: shallow and deep experimental cavities, active and slow untreated caries, and treated caries. Under deep cavities, class II molecule-expressing dendritic cells displaced the injured odontoblasts during a period of one month, while such a response was not observed in shallow cavities and untreated or treated carious teeth. The class II molecules seen in the neural elements under active caries were no longer detectable in treated carious teeth. However, six months after treatment, clusters consisting of dendritic cells, T-lymphocytes, and nerve fibers still remained locally in the subodontoblastic area. These results indicate that dental pulps respond differently to cavity preparation and restoration between normal and caries conditions, and that immunoresponses persist for many months, even after caries treatment.

KEY WORDS: human dental pulp • MHC class II molecule-expressing cells • caries treatment • cavity preparation • adhesive system

	INTRODUCTION

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Caries removal, cavity preparation, and restoration with adhesive systems are generally conducted in dental practices. While there has been no shortage of material published on the subject of histological evaluations of pulpal responses to cavity preparation and restoration in animals, there has been little attempt to evaluate those in human teeth.

Dental pulp is equipped with major histocompatibility complex (MHC) class II molecule-expressing cells, which are the most capable antigen-presenting cells for initiating immune responses to exogenous antigenic stimuli. In intact teeth, they are distributed mainly in and around the layer of odontoblasts with dendritic profiles and are called pulpal dendritic cells. Drastic changes in their localization are induced by human dental caries (Yoshiba et al., 1996, 1998; Sakurai et al., 1999), and by cavity preparation in rats (Ohshima et al., 1995; Kamal et al., 2000; Tanabe et al., 2002). Analysis of these data suggests that class II molecule-expressing cells are highly sensitive to antigenic stimuli penetrating dentinal tubules.

A caries attack also induces changes in the distribution of nerve fibers. They become concentrated beneath caries lesions in human teeth (Yoshiba et al., 1998; Rodd and Boissonade, 2001). Significant interactions between nerve fibers and MHC class II antigen-expressing cells have been shown in pulpal inflammation (Yoshiba et al., 1996, 1998; Sakurai et al., 1999). Beneath caries lesions, nerve fibers and dendritic cells co-aggregate (Yoshiba et al., 1998; Sakurai et al., 1999); furthermore, Schwann cells also express class II antigen under advanced and active caries (Yoshiba et al., 1998).

Thus, the present study focused on how operative procedures exert an influence on the distribution of MHC class II molecule-expressing cells and nerve fibers, as well as odontoblasts. We postulated that pulpal responses would differ for cavities compared with caries, that caries activity would affect antigen-presenting cell patterns, and that treated carious teeth would no longer have their responses after 6 months. To verify our hypotheses, we analyzed pulpal responses under 5 different clinical conditions: shallow and deep experimental cavities, active and slow untreated caries, and treated caries.

	MATERIALS & METHODS

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Operative Procedures
The protocol for this study was reviewed and approved by the Niigata University Dental Hospital Review Board, and informed consent was obtained from subjects after the proposed study was fully explained. Thirty volunteers ranging in age from 22 to 31 yrs, who had been scheduled to undergo extraction for various therapeutic reasons, were enrolled in the study. Thirteen intact premolars were used for evaluation of pulpal response to cavity preparation. After the subjects had received local anesthesia, Class V circular preparations were cut into buccal surfaces with diamond burs under water-cooling. The distance from the bottom of the cavity to the pulpal wall ranged from 100 µm to 1.3 mm. The Clearfil MEGA BOND dental adhesive system and Clearfil AP-X composite resin (Kuraray Co., Okayama, Japan) were applied to the prepared teeth. As controls, 5 intact premolar teeth were extracted without treatment.

Alternatively, 11 third molars with occlusal caries at various stages of decay were treated with a caries detector dye (Caries Detector, Kuraray Co.) and restored with the same materials. Carious teeth were classified on the basis of the parameter of lesion activity (Schüpbach et al., 1990); yellowish discolored dentin was classified as active, and the brownish was classified as slow-progressing. These molars exhibited no clinical symptoms, either before or after treatment. As controls, 8 third molars with caries and 10 intact third molars were extracted without any treatment. The depth of cavities, caries activity, depth of caries pigmentation, and time periods are summarized in the Table.

Quantitative Analysis
Clusters of HLA-DR-positive cells were evident beneath 10 of the 11 treated caries samples. To evaluate the effects of caries treatment, we compared the densities of HLA-DR immunolabeled cells under the treated caries regions with those under the untreated non-caries regions of occlusal surfaces of the same teeth, as well as with those in the corresponding regions of intact teeth. Representative images viewed by means of a confocal laser scanning microscopy, one picture per tooth, were used for data analysis. On the sections examined, vascular and neural elements were immunofluorescent-negative for HLA-DR. We used image-analysis software (Win Roof, Mitani Co., Fukui, Japan) to calculate the dimensions of the immunopositive area within the fixed field (500 x 300 µm), so that the long side lay parallel and tangential to the pulp-dentin border. We applied ANOVA and Tukey tests to examine the statistical differences. Significance levels were set at p < 0.05.

	RESULTS

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Negative control sections showed no specific labeling (data not shown). Pulpal responses to experimental cavities, caries, or caries treatment are summarized in the Table.

Intact Control Teeth
Double immunofluorescence staining showed that HLA-DR-positive dendritic cells distributed mainly around an odontoblastic layer, and also along the dentin-pulp border (Fig. 1a). The bead-like PGP 9.5-labeled nerve fibers appeared to pass through the odontoblastic layer (Fig. 1b). A moderate immunoperoxidase reaction was observed on odontoblasts (Fig. 1b).

View larger version (122K): [in this window] [in a new window]   Figure 1. Photomicrographs of an intact tooth (a,b) and cavity-prepared normal teeth at 1 mo (c-h) and 2 mos (i,j) post-operatively. (c-e) Tooth, with dentin drilled halfway; (f-h) and (i,j) deep cavities, with dentin drilled more than two-thirds of the way. (a) Double channels of confocal laser scanning microscope indicate HLA-DR-positive cells (shown in red and yellow), and neural elements (in green) and odontoblasts (in green). (b) Nerve fibers are distinguishable from odontoblasts, due to the intensity of their immunoperoxidase labeling for PGP 9.5. (c) A floating section under the light microscope. Its confocal laser scanning microscopic image is shown in (d). Arrows and an asterisk in (c,d) indicate corresponding areas. (d) The thickness of the odontoblast layer is reduced under the cavity of half of the dentinal thickness (arrows). No aggregation of HLA-DR-positive cells is noted under the cavity margin (asterisk). (e) In the affected-odontoblast layer corresponding to (d), thoroughly dispersed HLA-DR-positive dendritic cells can be seen. (f) A floating section is viewed by the light microscope. Its confocal laser scanning microscopic image marked by a line in (f), corresponding to a cavity of two-thirds depth, is shown in (g). (g) HLA-DR-positive cells (in red) displace the odontoblasts under a two-thirds depth of cavity. (h) Immunoperoxidase labeling of (g) indicates that the lining cells are HLA-DR-positive cells. No cellular infiltration is seen beneath the area. (i,j) After 2 mos, HLA-DR-negative cells are lying along the dentin, under cavities reaching a depth of more than two-thirds (i). These lining cells demonstrate positive immunoreactivity for DSP (j, arrows). Bar = 500 µm (c,f), 300 µm (d), 100 µm (a,g), and 50 µm (b,e,h,i,j).

One month post-operatively, under the cavities into which the dentin had been drilled halfway (Fig. 1c), the layer of PGP 9.5-positive odontoblasts had thinned noticeably (Fig. 1d). While HLA-DR-positive cells were observed to gather at the lesion, no aggregation was visible below the cavity margin (Fig. 1d), where pulp tissue was determined to be histologically normal (not shown). Immunoperoxidase labeling indicated that the HLA-DR-positive dendritic cells dispersed among affected odontoblasts (Fig. 1e). Under deeper cavities corresponding to more than two-thirds of depth (Fig. 1f), HLA-DR-positive cells displaced odontoblasts below the cavities (Fig. 1g). Immunoperoxidase labeling revealed the presence of HLA-DR-positive cells along the dentinal matrix, with their processes extending into dentinal tubules (Fig. 1h). There was no evidence of odontoblastic activity. No cellular infiltration, including CD45-positive cells, was recognized below this level (Fig. 1h, Table).

In follow-ups 2 mos post-operatively, the displacement by HLA-DR-positive dendritic cells had disappeared, even under cavities extending to more than two-thirds depth, and HLA-DR-negative cells lined the dentin (Fig. 1i). To identify the characteristics of those newly differentiated cells, we analyzed their expression patterns for DSP. On serial sections, HLA-DR-negative cells demonstrated DSP-positive immunoreactivity (Fig. 1j). Matrix formation was not evident (Fig. 1j).

Treated Carious Teeth
Treatment of caries lesions was carried out on occlusal surfaces. The distance between cavity floors and pulpal walls varied from 400 µm to 2.3 mm. Beneath 10 of the 11 cavities sampled, aggregations of HLA-DR-positive cells were recognized locally, and always followed the accumulation of neural elements and CD45-positive T-lymphocytes. Odontoblasts in the vicinity of the lesion were damaged.

After treatment of advanced-active caries (Fig. 2a), clusters of HLA-DR-positive cells were observed under the odontoblastic layer (Fig. 2b). Upon closer inspection, co-aggregations of HLA-DR-positive dendritic cells and PGP 9.5-immunopositive neural elements were evident, and dendritic cells lay in dense concentrations along nerve bundles (Fig. 2c). Just above the clusters were injured odontoblasts (Fig. 2d). As well, co-aggregations of HLA-DR-positive dendritic cells (Fig. 2e) and CD45-positive T-lymphocytes (Fig. 2f) appeared there. Near the inflammatory lesions, a layer of flat cells was seen lining the atubular reparative dentin, but no cellular infiltration was observed below the area (Fig. 2g). Even below the deepest area of the cavity, where residual dentin measured 400 µm thick, fibrous reparative dentin-producing odontoblasts were detected (Figs. 2h, 2j). They showed positive reactions for PGP 9.5, and their processes continued to a calciotraumatic line (Fig. 2j). Neural elements demonstrated faint staining for HLA-DR (Fig. 2i). Dendritic cells were seen to surround lymphocytes (Fig. 2k).

View larger version (93K): [in this window] [in a new window]   Figure 2. Photomicrographs of treated active caries at 3 mos post-operatively. Under confocal laser scanning microscopy, HLA-DR-positive cells are shown in red, nerve fibers and odontoblasts are shown in green. (a) A stereomicroscopic view of a floating section shows that the cavity floor is close to the pulp horn (asterisk). (b) Image of (a) obtained by confocal laser scanning microscopy indicates clusters of HLA-DR-positive cells under the odontoblastic layer. (c) A closer view of (b, arrowhead) indicates the aggregated-HLA-DR-positive cells accompanying neural elements (yellow spots). (d) Histological findings of (b, arrow) show the damaged odontoblasts, as well as cellular infiltration. (e, f) Serial sections corresponding to (d) immunolabeled with HLA-DR (e) and CD45 (f). They accumulate in the same spot. (g) Near the inflamed lesions, no cellular infiltration is noted (asterisk) below atubular dentin. (h) Histological findings of (a, asterisk) indicate the fibrous reparative dentin. (i,j) Serial sections corresponding to (h) immunolabeled with HLA-DR (i) and PGP 9.5 (j). Neural elements are stained very faintly for HLA-DR (i, arrows). The processes of PGP 9.5-immunopositive odontoblasts are reaching the calciotraumatic line (j). (k) HLA-DR-positive dendritic cells embrace lymphocytes (arrows). Bar = 1 mm (a), 300 µm (b), 100 µm (c,h), 50 µm (d,e,f,g,i,j), and 20 µm (k).

Untreated Carious Teeth
Below the active caries, where yellowish discolored dentin extended more than two-thirds in depth, fibrous reparative dentin was followed by a wide unmineralized layer (Fig. 3a). The neural elements accumulated there showed immunopositive reactions for HLA-DR (Fig. 3b), as did several dendritic cells. Similarly, intense immunoperoxidase reactivity for HLA-DR was recognized on both dendritic cells and neural elements (Fig. 3c).

View larger version (74K): [in this window] [in a new window]   Figure 3. Photomicrographs of untreated caries teeth of advanced-active (a-c) and slow-progressing (d-f). Under confocal laser scanning microscopy, HLA-DR-positive cells are shown in red, nerve fibers and odontoblasts are shown in green. (a) Histological findings show the fibrous reparative dentin with a wide unmineralized layer. (b) Double channels of confocal laser scanning microscopy reveal the accumulated neural elements are immunoreactive for HLA-DR (shown in yellow), besides HLA-DR positive cells (shown in red). Reparative odontoblasts are positively stained for PGP 9.5 (shown in green). (c) The level of immunoperoxidase reactivity for HLA-DR on dendritic cells (arrows) and neural elements (arrowheads) are nearly the same. See (Fig. 2i) for comparison. HLA-DR negative neural elements are seen on the left lower side. (d) One hundred-µm section embedded in Epon shows atubular and compact reparative dentin under slow-progressing caries. (e) Histological findings of (d) demonstrate atubular reparative dentin lined with a layer of flat cells. (f) A confocal laser scanning microscopic image of (d) demonstrates HLA-DR-positive elements (shown in red) are scant in number, and PGP 9.5-positive cells (arrows, shown in green) line the reparative dentin (RD). Bar = 500 µm (d), 100 µm (a,b, f), 50 µm (c,e).

	DISCUSSION

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In the present study, it was difficult to get longer experimental durations of the clinical treatments. However, each of the 5 different clinical conditions showed unique patterns for HLA-DR-positive cells and nerve fibers (Figs. 1a, 1d, 1g, 2b, 2c, 3b, 3f).

The present study provides evidence that the depth of prepared cavities in normal teeth has an influence not only on the underlying odontoblasts, but also on the distribution of HLA-DR-positive dendritic cells. It is noteworthy that a 50% reduction in the thickness of residual dentin had an impact on the distribution of dendritic cells, while a two-thirds decrease induced the replacement of odontoblasts by HLA-DR-positive cells during periods of 2 wks or 1 mo post-operatively (Table). These gradational changes are in remarkable agreement with findings from our recent study on the distribution of odontoblast processes in human dentin (Yoshiba et al., 2002), which demonstrated that the processes were limited to the pulpal third of the dentin. In an intact human tooth cavity from which more than two-thirds of the dentin had been removed, the activity of odontoblasts would be severely impaired. On the other hand, most components of adhesive systems and composite resins are able to diffuse through the dentinal tubules and reach the pulp tissue; and further, they are thought not only to produce noxious effects on odontoblast cell lines (Costa et al., 1999), but also to impair the function of pulpal immunocompetent cells (Jontell et al., 1995). When combined with the traumatic damage caused by cutting instruments, certain unpolymerized resin components might actually impede underlying pulpal healing. In the meanwhile, no aggregations of dendritic cells were recognized under marginal areas of prepared cavities, and no aggregations of CD45-positive lymphocytes were detected in any sample of the cavity prepared normal teeth (Table). Thus, the materials used here provided excellent sealing characteristics, and they effectively prevented the ingress of noxious substances to the dentin-pulp complex.

After 2 months, the lining cells observed under the cavities reaching to more than two-thirds depth shifted from dendritic to newly differentiated cuboidal cells (Fig. 1i), and those cells were identified as an odontoblastic phenotype (Fig. 1j, Table). We could not follow up after the formation of reparative dentin; however, the appearance of DSP-positive cells along the dentin should suggest that a calcified matrix might be formed in the future. On the other hand, a recent histomorphometric study conducted with human intact teeth has shown that the remaining dentin thickness of cavity preparations is the most important factor which has an impact on the numbers of surviving odontoblasts and dentinal repair (About et al., 2001). Analysis of these data suggests that cavity preparation beyond two-thirds in depth should be avoided in normal teeth.

In contrast to restored non-carious teeth, the replacement of odontoblasts by dendritic cells was not recognized under untreated or treated caries lesions at any time during the experiments, even though the depth of cavities reached more than two-thirds (Table). Caries-affected dentin, after excavation and removal of the smear layer, is known to have a very low permeability, in comparison with that of normal dentin (Pashley et al., 1991; Tagami et al., 1992). The limited presence of HLA-DR-positive elements detected beneath the compact reparative dentin would indicate its low permeability (Fig. 3f). Thus, sclerotic and/or reactionary dentin induced by caries stimuli can effectively protect the underlying odontoblasts from traumatic damage caused by cutting instruments or from chemical stimuli from the filling materials, even if the cavity exceeds two-thirds in depth.

Against our expectation, even after caries treatment, small aggregations of HLA-DR-positive dendritic cells, accompanied by neural elements and CD45-positive T-lymphocytes, were left behind in 10 out of 11 samples (Table). Statistical significance was found between caries-affected and unaffected areas, even after treatment. The inflamed lesions would be the result of activities by bacteria, which had existed locally deep in the dentinal tubules and survived even after the disclosing dye-aided removal of caries. Dendritic cells embraced lymphocytes, and thus it is obvious that local antigen presentations and cellular and/or humoral immunoresponses persist even after the careful treatment of caries.

In advanced-active caries, class II molecules were detected in neural elements in addition to dendritic cells (Figs. 3b, 3c, Table). In a previous immunoelectron microscopic study, we showed that Schwann cells could function as antigen-presenting cells (Yoshiba et al., 1998). After caries treatment, HLA-DR-immunoreactive neural elements were found in only one sample; however, the level of immunoreactivity was rather weaker than that seen in positive dendritic cells (Fig. 2i) as well as neural elements in the untreated carious teeth (Fig. 3c). Analysis of these data suggests that the immunoreactivity in neural elements for HLA-DR is reversible, and that the function of antigen presentation could be negated by caries treatment.

In conclusion, the present study demonstrated that dental pulps respond differently to cavity preparation and restoration in normal or in caries conditions, and that antigen presentation and immunoresponses persist for many months, even after caries treatment. Further investigations are needed to ascertain how to control the bacterial activities that possibly remaining deep in the dentinal tubules.

	ACKNOWLEDGMENTS

 
This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Kerry Greer for editing the manuscript.

Received December 21, 2001; Last revision March 3, 2003; Accepted March 5, 2003

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