Abstract #26 — Detection of IL-2 in healthy and inflamed dental pulps

AAE Abstracts of Papers

Vol. 19, No. 4, April 1993

191

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Abstract #22 - Concentration of CGRP in symptomatic and asymptomatic human pulp tissue. H.K. Kanning*, D.L. Carnes University of Texas, San Antonio, Texas Calcitonin gene-related peptide (CGRP) is a neuropeptide which is involved in inflammation, and which may have a stimulating effect on dentin production in responseto noxious insult_ CGRP immunoreactive nerve fibers have been shown to increase beneath the injured dentin of rat molars. Information on human subjects relating to CGRP is minimal however. The purpose of this study was to determine the concentration of CGRP in symptomatic and asymptomatic human dental pulp tissue. The control group consisted of pulp tissue collected from 20 asymptomatic third molars. The test group consisted of pulp tissue collected from 17 symptomatic teeth with deep caries having a clinical diagnosis of irreversible pulpitis. The teeth were extracted and immediately placed in liquid nitrogen. The teeth were split longitudinally, the pulp tissues were removed, homogenized in radioimmunoassay buffer and frozen at 4C. A radioimmu noassayfor CGRP was performed using rabbit antiserum to human CGRP and '2Sl-humanCGRP. CGRP values determined in pulp samples were normalized to total protein by the Pierce BCA method. CG RP was present in both symptomatic and asymptomatic human pulp tissue. Significantly higher levels of CGRP were found in pulp tissue samples from symptomatic teeth, as compared to CGRP levels found in asymptomatic pulp tissue specimens (p < 0.01). The results indicate that CGRP levels are elevated in human pulp tissue which is undergoing an inflammatory response due to deep carious lesions. This study was supported, in part, by the Research and Education Foundation of the AAE.

Abstract #23 - Pharmacological regulation of neuropeptide secretion from dental pulp. K.M. Hargreaves*, D. Jackson, M. Engelstad, M. Garry University of Minnesota, Minneapolis, Minnesota We have previously developed an in vitro method for measuring secretion of calcitonin gene-related peptide (CGRP) from dental pulp (J Endo in press), in the present study, we evaluated whether catecholamines alter the evoked secretion of immunoreactive CGRP (iCGRP) from capsaicinsensitive fibers in dental pulp. Pulp tissue from bovine mandibular incisors was extracted, chopped into 200 pm cubes, and placed in 2cc chambers for in vitro superfusion. Following a baseline recovery period, oxygenated Krebs buffer (pH 7.4 @ 37°C) containing norepinephrine, clonidine arterenol (0.01nM-100 pM) or vehicle (n=6-11/group) was superfusedthrough thetissue before and aftera stimulatingpulse ofcapsaicin 30 pM), a neurotoxinthat selectively stimulates nociceptors. Levels of iCGRP were analyzed by a validated radioimmunoassay. NE produced a dosedependentsuppression of capsaicin-evoked iCGRP secretion, with maximal effects observed at 10nM (100% suppression; p < 0.01 ). Additional studies demonstrated that pre-treatment with both the alpha agonist clonidine (p < 0.01 ) and the beta agonist arterenot(p < 0.01 ) significantly suppressed capsaicinevoked secretion of iCGRP. Collectively, these studies indicate that adrenergic drugs inhibit afferent nerve activity in dental pulp. This observation suggests that catecholamine, such as epinephrineincluded in local anesthetic cartridges, may have a direct neural target for suppressing activity of peripheral pain fibers (nociceptors) in dental pulp. This study was supported in part by NIDR grants DE10096, K16 DE00270 and F32-DE05606.

Abstract #24- Peripheral opioid receptor mediated analgesia in inflamed dental pulps.

R. Uhle*, A. Reader, R. Nist, M. Beck, W. Meyers, J. Weaver Ohio State University, Columbus, Ohio Analgesia produced by opiates has classically been thought of as a centrally mediated phenomena. However, recent studies have shown

that opiate receptors are present peripherally on primary afferent nerves and that activation of these receptors can produce analgesia. To determine whether a low dose of fentanyl would produce analgesia, we measuredthe degreeofpain reliefobtainedwith fentanyt,administered via the periodontal ligament injection, in symptomatic, inflamed teeth. All subjects presented for emergency treatment and had a posterior tooth with a clinical diagnosis of irreversible pulpitis. Twenty subjects randomly received either 10ug of fentanyl citrate or saline placebo via the periodontal ligament injection in a double-blind manner. The subjects rated their pain prior to injection and rated pain intensity and pain relief for 60 minutes post-injection. Low dose fentanyl injected via the periodontal ligament injectionin inflamed teeth provided significantly (p = 0.0186) greater pain relief than the salineplacebo, as measured by ANOVA analysis. Since the dose of fentanyl usedin this study was less than the dose required to provide analgesia by a central mechanism, the results are consistent with a peripheral opiate receptor mechanism of action. This study was supported by the Graduate Students' Research Fund, Department of Endodontics, Ohio State University.

Abstract #25 - Temporal and spatial characterization of the putpat response to injury. T.A. Bachman*, K.R. Baumgardner, M. Litz, W.T. Butler, R.N. D'Souza University of Texas, Houston, Texas, University of Iowa, Iowa City, Iowa Odo ntoblast differentiation du ring tooth developmentis controlled by a seriesof reciprocal interactionsbetweenthe ectodermal-derivedenamel organ and the neural crest-derived dental papilla. Although these events may be reiterated during repair, the biochemical and cellular interactions that lead to the formation of reparative dentin at the site of injury to the dentin-pulp (DP) complex are poorly understood. This study addresses the hypothesis that the odontoblast phenotype is involvedin the formation of reparativedentin. Cervicalcavitypreparations were placed within dentin of first molars in adult Sprague Dawley rats and tissue sections prepared from paraffin blocks at various intervals (days 0 to 14). Immunohistochemical methods were used to determine whether transforming growth factor-beta I(TGF-1), a growth factor know to play a central role in developmentand repair, localizedin cells and matrix during injury and repair. We also evaluatedwhether DSP, a dentin sialoprotein that marks odontoblast differentiation during development, was expressed by the replacement pool of cells subjacent to newly formed reparative dentin matrix. The temporal and spatial pattern of expression of TGF-1 during reparative dentin formation suggests a role for this factor as a biologic inducer of matrix formation in repair, immunoreactive £)SPwas also seen in the matrix and within replacement cells concomitant with the onset ot mineralization of reparative dentin. Our results suggest that cells related to the odontoblastic lineage synthesize and secrete reparative dentin matrix. Collectively, data from these studies will increase understanding of the DP complex and its reaction to the carious process and to restorative procedures. This study was supported by NIDR Grants R37 DE05092 and K16 DE00175.

Abstract #26 - Detection of IL-2 in healthy and inflamed dental pulps.

J. Bailey*, C. Rauschenberger University of Maryland, Baltimore, Maryland Hahn eta/., 1989, reported that irreversible pulpitis was associated with an increase in T tymphocytes (T-cells) responsible forthe regulativn of pulpal immunopathologicchanges under carious lesions. Interleukin2 (IL-2) is a growth factor responsible for T-cell proliferation and maturation, and may signal pathologic pulpal changes. The purpose of this study was to determine if IL-2 could be detected in dental pulps clinically diagnosed as heatthy or irreversibly inflamed. Healthy pulp tissues were obtained from 18 impacted third molars and the inflamed

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AAE Abstracts of Papers

Journal of EndodonUcs

(Abstract #26, continued)

(Abstract #28, continued)

samples were provided by 14 carious molars. The teeth in the inflamed group presented to the University of Maryland Dental School Clinic with spontaneous pain. Each tooth in the inflamed pulp group respondedto vitality tests, and radiographically exhibiteddeep coronal caries with no evidence of apical pathology. The teeth in the healthy group were free of pulpal symptoms, exhibited no radiographic caries or apical pathology. Immediatelyfollowing extraction all teeth were sectioned longitudinally u ndercoolant, fractured to obtain pulpal tissues, and samplesfrozen at -70°C. When sample collection was completed,the pulpal tissues were thawed for 30 min., macerated in PBS buffer, and the supernatant evaluated for the presence of IL-2 using a modified ELISA. IL-2 was detected in the healthy and inflamed pulpal tissues evaluated by this study. Additionally, a t-test revealed that significant differences existed between IL-2 levels in the healthy and inflamed pulpal tissues (p < O.05). These results suggest that IL-2 may be a suitable marker for analyzing inflammatory pulpal disease. This project was supported in part by a G raduate Student Research Award of the Research and Education Foundation of the AAE.

The results indicated statistically significantly less dye leakage in the pH 1 and 2 amalgam groups. All other amalgam groups showed no significant differences. TERM root-end filling groups demonstrated statistically significantly less apical leakage at each pH evaluated than the amalgam root-end filled groups. The most significant finding was in the groups at pH 5 and less which demonstrated dissolution of the exposed root apex. The root loss ranged from 0.06 to 0.62 mm and was greatest in the lowest pH groups. The group in which the dye was mixed with the deionized water produced a solution with a pH of 2.66. Measurement of the pH of the dye solutions after the 7 days found a significant buffering effect of the root structure on the dye solutions. If methylene blue dye solutions are to be used in leakage studies, the pH of these solutions should be adjusted to near neutrality to avoid adverse pH effects. This project was supported in part by N IDR Grant DE06427 and by the Medical College of Georgia Dental Research Center.

Abstract #27 - Hypoxia in pulpal inflammation demonstrated by 3H-Misonidazole retention.

K.R. Baumgardner*, R.E. Walton, J.W. Osborne, J.L. Born University of Iowa, Iowa City, Iowa, and University of New Mexico, Albuquerque, New Mexico Misonidazele(MISO) is a bioreductive]y-activated markerofceltular hypoxia which preferentially binds to cells with decreased oxygen tension. Inflammation in the pulp causes increased pressure which may create areas of hypoxic cells. The purpose of this study was to characterize the hypoxic-mediated binding of 3H-MISO in induced pulpal inflammation in rat molars. In 15 male rats, cavity preparations were performed on 3 of 41 st molars with the 4th molar as an unoperated control. On day 4 after cavity preparation, the rats were injected with 3H-MISO and sacrificed 24 h later. Molarswere prepared using routine histologic and autoradiographictechniques. Sampleswere evaluated under light microscopy for extent of inflammatory involvement and degree of 3H-MISO retention (grain counts per unit area, statistical comparisons using ANOVA). Pulpal inflammatorylesions, resulting with or without pulp exposure, that were larger than 0.35 mm (-+0.05)demonstrated cores of hypoxic cells. In teeth that had no pulp exposure, the pulp distal to an inflammatory lesion with a hypoxic core also had significantly greater 3H-MISO retention (p < 0.05). 3H-MISO retention in pulps with smaller inflammatory lesions, and in unoperated 2nd and 3rd molars, was not significantlydifferent from the 1st molar controls. Zones ofhypoxiccells occur in larger pulpal inflammatory lesions. When these larger lesions arose in teeth without a pulp exposure, the entire coronal pulp was also disrupted and retained more 3H-MfSO. This research was funded by NIH/NIDR Grant #K16-DE00175.

R e s e a r c h S e m i n a r III Friday, April 30, 9:00 am - 12:00 noon Graduate Student Section Abstract #28 - An evaluation of the effect of methylene blue dye pH on apical leakage. D.L. Starkey, R.W. Anderson, D.H. Pashley Medical College of Georgia, Augusta, Georgia The effect of varying the pH of 2% methylene blue dye on apical leakage of endodontic root end fillings was evaluated, Eighty-four roots of extracted human teeth were cleaned and shaped, obturated, apically resected, and amalgam or TERM root-end fillings were placed. The roots were immersed in dye solutions of pH 1,2,3,5,7 or 2% unbuffered deionized water solution of methylene blue under a vacuum for t5 minutes followed by storage at 37°C for 7 days. The roots were longitudinally sectioned and linear dye penetration was measured.

Abstract #29 - Effect of bleaching on coronal microleakage of endodontically treated teeth. G.R. Karren*, P.D. Primack, J.C. Kulild US Army Dental Activity, Fort Gordon, Augusta, Georgia The purpose of this investigation was to determine the effect of 30% hydrogen peroxide and sodium perborate on gutta-percha root canal obturation using two different sealers as measured by coronal dye leakage. Sixty maxillary anterior teeth were prepared using step-back technique to #50 master apical file size. The canals were obturated using lateral condensation of gutta-percha and sealer. Roth's 801 was used as a sealer in the first 30 teeth and AH26 was used in the remaining 30 teeth. Two additional teeth each were used as positive and negative controls. Following removal of gutta-percha to the radiographically verified CEJ level, the sealer was allowed to set for 5 days at 37°C in 100% humidity before placing experimental materials in to the chambers. The two sealers were examined using one of three treatment methods. A mixtu re of 30% hydrogen peroxide and sodium perboratewas placed in the first group, a mixture of saline and porcelain powder in the second, and the third received only a dry cotton pellet. The access opening was sealed with IRM. The bleaching agents or saline mixture was allowed to remain in the chamber for 5d, during which time the teeth were thermal cycled for 80 repetitions of 14s duration in alternating 60°C and 0°C water baths. At the end of the 5 days, the chamber contents were replaced with fresh materials and the same procedure repeated. The experimental agents were next removed from the chamber and after irrigation and drying, a layer of sticky wax was placed covering the entire tooth surface except for the access opening. Nail polish was placed over the sticky wax. All teeth were immersed in India ink for 5 days in a vacuum chamber. Demineralization in 5.0% nitric acid (5 days), dehydration in 99.8% methyl alcohol (3 days), and storage in methyl salicylate completed the clearing process. Results were assessed by measuring the greatest extent of linear dye leakage between the obturation material and root canal surface. The use of bleaching agents did not cause a significant increase in microleakage.

Abstract #30 - Coronal leakage: In vitro bacterial penetration through unsealed, unobturated canals. S. Gish*, D. Drake, L.R. Wilcox, R.E. Walton University of Iowa, Iowa City, Iowa It is currently unclear how quickly the root canal system, when exposed to the oral environment, becomes contaminated to the point that retreatment may be necessary. Data from extensive preliminary studies showed no bacterial penetration through obturated canals after 90 days when exposed to a single organism (S. anqinosus). The aim of this investigation was to delineate the conditions and time necessary for different oral bacterial species, in mixed cultures, to penetrate obturating materials occupying the apical 4-5mm of the canal. Thirty extracted, single-rootedteeth were prepared. Twenty-five of the canals were obturated using lateral condensation. A hot plugger