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RESEARCH REPORTS |
1 Institute of Dentistry, PO Box 41, 00014 University of Helsinki, Finland;
2 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital (HUCH), Helsinki, Finland;
3 Department of Physiology and Experimental Pathophysiology, University of Erlangen/Nuernberg, Germany; and
4 Finnish Student Health Service;
* corresponding author, pentti.kemppainen{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: pulpal pain • matrix metalloproteinase-8 • gingival crevicular fluid • neurogenic inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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At local sites of various tissues, sensory nerves are closely apposed to immuno-effector cells, including neutrophils, monocytes/macrophages, and fibroblasts (Stead et al., 1989; McGillis and Fernandez, 1999). Clinically, the pro-inflammatory neuropeptides released by sensory nerves co-ordinate the activities of the immune-effector cells (McGillis and Fernandez, 1999), and may play an important role in the pathogenesis of periodontitis (Luthman et al., 1989). Painful teeth with pulpitis have been shown to be associated with elevated levels in GCF of pro-inflammatory peptides, such as substance P (SP) and neurokinin A (NKA) (Awawdeh et al., 2002a). Matrix metalloproteinase (MMP)-8, released by neutrophils, monocytes/ macrophages, and fibroblasts, is the major pivotal tissue-destructive protease, especially in the periodontitis-affected gingiva and gingival crevicular fluid (GCF) (Sorsa et al., 1988, 2004; Kiili et al., 2002; Mäntylä et al., 2003). Recently, research has shown that MMP-8, in addition to its surrogate catalytic action (Uitto et al., 2003; Sorsa et al., 2004), can also exert anti-inflammatory or defensive characteristics (Owen et al., 2004).
With this background, we studied whether painful tooth stimulation can induce changes in GCF MMP-8 levels in humans. To evaluate the spatial spread of pulpal-pain-evoked effects, we analyzed the MMP-8 levels in GCF collected from the crevices of the stimulated upper incisors and of non-stimulated upper and lower incisors.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Stimulation Techniques
The dental stimulation was generated with a constant-current tooth stimulator as previously described (Kemppainen et al., 1985). During the experimental sessions, the permanent upper right central incisor (tooth 11) was electrically stimulated at an intensity of 3x the individual threshold. The stimulation period lasted 90 sec. The stimulator featured a built-in circuit for measuring electrode resistance. The resistances of the stimulated teeth were monitored throughout the experiment in monopolar coupling, and they ranged from 2 to 4 M
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GCF Samples
GCF samples were collected from the mesiobuccal and distobuccal aspects of each tooth prior to tooth stimulation, during stimulation, and after stimulation ended. Gingival tissue around the stimulated and non-stimulated control teeth underwent careful periodontal examination, which included measurement of plaque index, gingival bleeding, probing depths, and a panoramic radiographic examination. None of the volunteers had used antibiotics within the preceding month. To avoid blood contamination and possible stimulation of GCF flow during clinical measurements (probing, etc.), we collected GCF samples prior to other clinical recordings. The sampling site was isolated with cotton rolls, and supragingival plaque was carefully removed. The region was dried with a gentle air stream, and GCF was then collected by means of standardized filter paper strips. The strip was placed into the crevice until mild resistance was felt, and left there for 30 sec. After that, the strip was immediately placed in a polypropylene tube. We measured the GCF volume with a standardized weighing method (Mettler AJ 100/GWB) by weighing the polypropylene tube with the standardized filter paper strip inside, before and immediately after the sample collection. Thereafter, each strip was eluted into 300 µL of 20 mM phosphate buffer, pH 6.0, containing 0.15 M NaCl and 0.1% Tween 20 (Apajalahti et al., 2003). The eluates were stored at –20°C prior to analysis. Our recent study (Mäntylä et al., 2003) has showed the average comparative GCF MMP-8 levels in adult periodontitis to be about 2500 µg/L, in gingivitis, 750 µg/L, and in healthy sites, 100 µg/L.
Western Immunoblot and Immunofluorometric Assay (IFMA)
We analyzed the molecular forms of MMP-8 in GCF from the stimulated and control teeth by the Western immunoblot method, with specific polyclonal antibody for MMP-8 used at 2 µg/mL final concentrations, and by quantitated computer image scanning as previously described (Hanemaaijer et al., 1997; Lindy et al., 1997; Kiili et al., 2002; Prikk et al., 2002; Apajalahti et al., 2003). Human PMN and rheumatoid synovial culture media (Hanemaaijer et al., 1997; Lindy et al., 1997; Kiili et al., 2002) served as positive controls for MMP-8.
MMP-8 concentrations in the GCF were determined by a time-resolved immunofluorometric assay (IFMA), and the amounts of monoclonal antibodies 8708 and 8706 for MMP-8 were 1.5 µg and 0.5 µg per assay, respectively (Hanemaaijer et al., 1997; Apajalahti et al., 2003; Mäntylä et al., 2003).
Course of the Experiments
The subjects were comfortably seated in a dental chair. The investigation consisted of two sessions with a one-week interval. In the first session, eight human volunteers underwent painful tooth pulp stimulation (3x pain threshold) of the upper right central incisor. The subjective pain levels were evaluated by a visual analogue scale, VAS (0 = no pain, 100 = the worst imaginable pain intensity). A total of 8 GCF samples was collected from the upper right central incisor (tooth 11 = stimulated tooth), from the upper left lateral incisor (tooth 22), and from the lower right central incisor (tooth 41). Samples 1 and 2 were taken before the stimulation began, and their average value served as a baseline. Samples 3 and 4 were taken during stimulation, followed by samples 5 to 8, taken after the end of tooth stimulation. In the second control session, GCF samples were similarly collected, without tooth stimulation, from six subjects who had participated in the first session. During tests, each subject’s heart rate (HR) and mean arterial blood pressure (MAP) responses were semi-automatically recorded (Omron, Digital blood pressure monitor, HEM-705C, Osaka, Japan) from the left upper arm at the same time as the GCF samples were taken. HR and MAP measurements served to clarify whether pulpal pain may have induced some systemic stress reactions, as indicated by possible changes in cardiovascular parameters (Kemppainen et al., 2001).
Data Analysis
MMP-8 values were normalized due to large inter-individual variability. The grand mean of all MMP-8 values from each measurement site was calculated separately. For ongoing statistical analysis, the means of baseline, stimulation, 4 min after, and 8 min after the end of stimulation values were extracted and normalized with this grand mean (percent change as compared with grand mean). A Friedman non-parametric ANOVA was then used to compare baseline, stimulation, and post-stimulation periods. In case of significance, a Wilcoxon’s matched-pair test was performed for post hoc comparisons. A p-value of less than 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The average intensity of this stimulation was 47 ± 2.2 µA (± SEM, n = 8), and the respective average pain magnitude estimate on VAS scores was 74.4 ± 3.8 (± SEM, n = 8).
Stimulation of tooth 11 significantly raised MMP-8 levels (IFMA analysis) in adjacent GCF. These pulpal-pain-evoked elevations in MMP-8 levels occurred during stimulation and remained elevated several minutes after the end of stimulation. Simultaneously, no marked changes in GCF MMP-8 levels could be detected at tooth 22 or tooth 41 (Fig. 1
). The control session without stimulation showed that the repeated measurements themselves did not modulate GCF MMP-8 levels (data not shown). The elevated MMP-8 levels in the GCF of the stimulated tooth were clearly lower than those of the gingivitis and periodontitis sites in our earlier study (Mäntylä et al., 2003).
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) shows that the samples contained bands at 60 to 80 kDa, corresponding to PMN-type active and pro-enzymes, and from 45 to 55 kDa, corresponding to mesenchymal-type (non-PMN) active and pro-enzymes. We found that the PMN-type isoform was slightly elevated during tooth stimulation (lane 5), and the non-PMN-type MMP-8 was elevated after the end of tooth stimulation (lanes 6, 7). No marked changes appeared in blots for MMP-8 of the non-stimulated teeth (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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MMPs are known to be produced by various immuno-effector cells, such as neutrophils, monocytes/macrophages, and fibroblast-like cells (Uitto et al., 2003; Sorsa et al., 2004), often influenced by the pro-inflammatory neuropeptides released from the closely apposed sensory nerves in various tissues (McGillis and Fernandez, 1999), including gingiva (Luthman et al., 1989). Animal studies have shown the existence of branched nerves innervating both intrapulpal and periodontal tissues (Foster and Robinson, 1994). Activation of nociceptive afferents induces a local axon-reflex-mediated neurogenic inflammatory reaction (Wårdell et al., 1993), which results from the release of pro-inflammatory neuropeptides such as substance P (SP) and calcitonin-gene-related-peptide (CGRP) from the peripheral nerve terminals. Interestingly, recent clinical findings in patients suffering from pulpal pain and inflammation confirm that pulpal pain can cause an axon-reflex-mediated elevation of these pro-inflammatory mediators in the GCF of the painful tooth (Awawdeh et al., 2002a). Thus, this pulpal stimulation-evoked increase in the GCF MMP-8 levels of the stimulated tooth (described above) could result from a local neurogenic, possibly axon-reflex-mediated, inflammatory mechanism.
Evidence suggests that the release of neuropeptides from the peripheral terminals of afferent nerve fibers can modulate inflammatory mediators, cytokines, and proteolytic enzymes in inflammatory diseases (Scott et al., 1994). SP and related neuropeptides exert potent and extensive pro-inflammatory actions, such as increased capillary permeability, vasodilatation, prostaglandin E2, MMP, interleukin (Il)-1, and tumor necrosis factor (TNF)-
secretion by resident cells at the sites of inflammation (Scott et al., 1994). Il-1, TNF-, and PGE2 can trigger neutrophils, gingival fibroblasts, and epithelial cells to express MMPs, including MMP-8 (Uitto et al., 2003; Sorsa et al., 2004). Overall, neuropeptides, either directly or through action on cytokines, prostaglandins, and TNF-, can induce the secretion of MMP-8 in the periodontium (Scott et al., 1994; Hanemaaijer et al., 1997; Tervahartiala et al., 2001; Uitto et al., 2003; Sorsa et al., 2004).
In this study, the resistances of stimulated teeth varied between subjects from 2 to 4 M, indicating that no short-circuiting to periodontal tissues occurred (Närhi et al., 1982). Moreover, electrophysiological evidence indicates that the maximal strength (below 60 µA) of the electrical current used in this study for dental stimulation, even when applied to the periodontium, does not activate extrapulpal nerve fibers (Närhi et al., 1982). These findings indicate that the activation of intrapulpal nociceptive afferent fibers triggered MMP-8 changes in GCF.
Clinical evidence indicates that, in comparison with healthy teeth, teeth with symptomatic pulpitis have increased concentrations of pro-inflammatory neuropeptides in pulp tissue and in GCF (Awawdeh et al., 2002a,b), and of MMP-8 (Wahlgren et al., 2002) in pulp tissue of these teeth. Furthermore, a majority of orthodontic patients report tooth pain at 4 to 24 hrs after the insertion of fixed appliances (Scheurer et al., 1996); this pain is associated with elevated GCF MMP-8 levels from periodontally healthy teeth (Apajalahti et al., 2003). Overall, such elevated levels of pro-inflammatory neuropeptides and MMP-8 in painful teeth occur both in pulp tissue and in adjacent GCF.
Pulpal pain and inflammation lead to an increase in intrapulpal pressure (Byers and Närhi, 1999). Thus, increased fluid flow from the root canal system through the apical foramen and dentinal tubules may cause delivery of MMP-8 to periodontal ligament space. In our study, the subjects’ gingival health was excellent and without gingival recession, which contradicts the idea that increased GCF MMP-8 levels resulted from direct migration from tooth pulp to GCF.
Stress has been shown to modulate some periodontitis-relevant immune parameters in GCF (Deinzer et al., 1999). Since pulpal pain evoked only local elevations in GCF MMP-8 levels and caused no marked modulations in systemic cardiovascular parameters, systemic stress mechanisms probably did not significantly contribute to the present results.
The present investigation indicates that experimental pulpal pain can produce local elevations in GCF levels of the potent host-tissue-destructive protease, MMP-8. Analysis of these data supports the possibility of a local neurogenic spread of inflammation from intrapulpal to surrounding periodontal tissues. Moreover, this pulp-based neurogenic process may reflect the phenomenon observed in normal tissue turnover, and, perhaps, predispose such sites to the progression of periodontal destruction (Sorsa et al., 2004). Alternatively, depending on the role of MMP-8, this reaction may also be, at least in part, anti-inflammatory or defensive (Owen et al., 2004).
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ACKNOWLEDGMENTS |
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Received March 22, 2004; Last revision December 9, 2004; Accepted January 18, 2005
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REFERENCES |
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