Basic Research—Technology
Direct Effect of Endodontic Sealers on Trigeminal Neuronal Activity Nikita B. Ruparel, DDS, MS, PhD, Shivani B. Ruparel, PhD, Paul B. Chen, BS, Blake Ishikawa, DDS, and Anibal Diogenes, DDS, MS, PhD Abstract Introduction: Endodontic sealers are selected on the basis of their antimicrobial properties and ability to provide a tight seal. Sealer extrusions, whether intentional or unintentional, are common during obturation procedures. Such events have been correlated with increased postoperative discomfort and persistent pain states. However, the mechanisms underlying this phenomenon are largely unknown. Thus, we sought to evaluate the effect of commonly used endodontic sealers on peripheral nociceptors. We hypothesized that endodontic sealers can directly activate trigeminal nociceptors in a concentration-dependent manner, resulting in release of calcitonin gene-related peptide (CGRP), a potent modulator of neurogenic inflammation. Methods: Rat trigeminal sensory neurons were exposed in vitro to vehicle, zinc oxide-eugenol (ZOE)–based sealer, AH Plus, EndoSequence BC sealer, or RealSeal SE. Neuronal activation was measured by quantification of neuropeptide (CGRP) release. In addition, cultured neurons were also subjected to the set form of all 4 sealers. The concentration of CGRP released was quantified by using a radioimmunoassay. Data were analyzed by using oneway analysis of variance with Newman-Keuls multiple comparison post hoc test. Results: Both ZOE-based sealer and AH Plus in their fresh form evoked greater CGRP release than the control groups. Conversely, EndoSequence BC and RealSeal sealers both reduced basal GCRP release at all concentrations tested. Evaluation of the set sealers revealed that only ZOE-based sealer evoked significant CGRP release compared with its control group. Conclusions: Overall, our results suggest that sealers can directly activate trigeminal nociceptors, leading to a robust release of CGRP, and may therefore lead to pain and neurogenic inflammation. This direct activation along with the immunologic response may underlie the symptoms and flare-up occurrences often seen with sealer extrusions. (J Endod 2014;40:683–687)
Key Words AH Plus, CGRP, endodontic sealer, EndoSequence BC sealer, neuropeptide, neuroscience, obturation, pain, RealSeal SE, trigeminal neurons, zinc oxide eugenol
P
ersistent pain is an ongoing problem after endodontic treatment. A recent systematic review demonstrated that approximately 14% of patients who received endodontic treatment had persistent pain 1 week after obturation (1). Moreover, Nixdorf et al (2) demonstrated that approximately 5.3% of patients experience pain 6 months after endodontic therapy, and only about 3.4% of it is from a non-odontogenic source. The etiology of non-odontogenic pain can be attributed to myofascial pain, headaches, neuropathic pain, or referred pain, whereas persistent pain from odontogenic origin could be due to missed canal, incomplete debridement, tooth fracture, occlusion, neuronal plasticity from a prolonged inflammatory state, or overfill of obturation material (2). Overfill of obturation material into the periradicular tissue has been correlated with foreign body reactions, flare-ups, and increased postoperative discomfort (3–7). Endodontic sealers are an essential part of obturation; not only do they provide for an adequate seal between the core material and the dentinal walls, but they are also effective antimicrobials inside the canal system (8, 9). Sealers may be extruded into the periradicular tissue when compacting the obturation core material. Therefore, sealer extrusion may stimulate an inflammatory response and activate sensory neurons, perhaps causing persistent pain after endodontic therapy. The dental pulp and periradicular tissue are densely innervated with polymodal nociceptors capable of detecting noxious thermal, mechanical, and chemical stimuli (10–15). Similar to the pulp, the periradicular tissues are densely innervated and undergo changes in response to inflammation at a cellular and molecular level (10–12). Although the long-term or neurotoxic effect of sealers has been previously investigated (16–22), to the best of our knowledge, no study has evaluated the direct effect of sealers on nociceptor activation. We therefore hypothesized that endodontic sealers will cause activation of a specialized subclass of peptidergic nociceptors, leading to vesicular exocytosis and release of calcitonin gene-related peptide (CGRP). Importantly, several groups have evaluated levels of CGRP as a model for assessing neurogenic inflammation and nociceptor activation (13, 23). Therefore, by using an in vitro neuropeptide release assay, we sought to evaluate the direct effect of several sealer compounds on activation of nociceptors and possibly neurogenic inflammation.
From the Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas. Nikita Ruparel and Shivani Ruparel contributed equally to this study. Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.030
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Basic Research—Technology Methods Animals Adult male Sprague-Dawley rats (weight, 200–250 g each) (Charles River, Wilmington, MA) were used in this study. All animal study protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to the International Association for the Study of Pain and federal guidelines. Animals were housed for 1 week before the experiments with food and water available ad libitum. Rat Trigeminal Ganglia Primary Culture Rat trigeminal ganglia (TG) were quickly removed after decapitation, and neuronal cultures were prepared as previously described (15, 16). Cells were plated on 24-well poly-D-lysine– coated plates (for immunoreactive CGRP [iCGRP] experiments). The TG cultures were maintained at 37 C, 5% CO2 for 5 days in the presence of 100 ng/mL nerve growth factor (Harlan Laboratories, Indianapolis, IN). CGRP Release Assay Culture experiments were performed on day 5 at 37 C by using modified Hanks’ balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA) buffer (10.9 mmol/L HEPES, 4.2 mmol/L sodium bicarbonate, 10 mmol/L dextrose, and 0.1% bovine serum albumin were added to 1 HBSS). To evaluate the effects of freshly mixed sealers on TG neurons, two 20-minute baseline samples were collected, followed by application of freshly mixed zinc oxide-eugenol (ZOE)–based sealer (Roth Root; Pearson Dental, Sylmar, CA), AH Plus (Dentsply, Tulsa, OK), EndoSequence BC sealer (BC) (Brasseler USA, Savannah, GA), or RealSeal SE sealer (SybronEndo, Orange, CA) at 25 mg/mL and 50 mg/mL concentrations for 30 minutes. All sealers were weighed along with a preweighed cell insert (BD Biosciences, Bedford, MA) with a pore size of 1 mm. The inserts were then placed in wells of a 24-well culture plate. After stimulation with sealers, the inserts were removed, and the cells were washed twice in HBSS for 20 minutes each. To evaluate the effect of sealer in a set form, the same experimental design and protocol were used except that each sealer was mixed and allowed to set overnight at 37 C. All groups were subjected to a 20-minute 250 mmol/L KCl (potassium chloride) solution to determine the remainder of the releasable CGRP pool from the cultured neurons. iCGRP Radioimmunoassay A previously used (17) primary antibody against CGRP (final dilution of 1:1,000,000; donated by Dr M. J. Iadarola, National Institutes of Health, Bethesda, MD) was added in the tubes containing superfusate from cultured rat TG and incubated at 4 C for 24 hours. Then 100 mL [125I-]-Tyr0-CGRP28–37 (20,000 cpm) and 50 mL goat antirabbit antisera coupled to ferric beads (PerSeptive Diagnostics, Cambridge, MA) were added to these tubes. The tubes were incubated for another 24 hours at 4 C. The assay was stopped by using immunomagnetic separation of bound from free tracer. All compounds used in experiments were tested for interference with the radioimmunoassay. The minimum detectable levels for CGRP for this assay are 3 fmol and the 50% displacement at 28 fmol. Data Analysis All experiments were conducted in triplicate with n = 6 per group. Data were analyzed by using Prism software version 5 (GraphPad Software, San Diego, CA). The results were analyzed with significance set at 684
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P < .05 by using one-way analysis of variance with Newman-Keuls post hoc test for multiple comparison post hoc test or Student t test analysis for comparing 2 groups.
Results Freshly mixed ZOE-based sealer (25 mg/mL) evoked 18-fold increase in iCGRP levels compared with control group (Fig. 1). Moreover, 50 mg/mL of freshly mixed as well as 50 mg/mL of the set form of ZOEbased sealer evoked 28-fold increase in iCGRP compared with control group (Fig. 1). Interestingly, only the freshly mixed AH Plus at 25 mg/mL concentration but not at 50 mg/mL concentration evoked 2.7fold significant iCGRP release compared with control group (Fig. 2). In contrast to ZOE-based sealer and AH Plus, both BC sealer and RealSeal SE sealers at both concentrations and forms inhibited iCGRP release from TG neurons compared with control group (Figs. 3 and 4). It is noteworthy that the overall iCGRP release from ZOE-based sealer at both concentrations was dramatically greater than any sealers used. To evaluate whether eugenol in ZOE-based sealer was responsible for the increased release, we subjected a group of cells to undiluted liquid eugenol (100 wt% or 6.1 mmol/L). Application of eugenol alone evoked 7.5-fold increase in iCGRP (Fig. 5).
Discussion Factors contributing to postoperative endodontic pain are many, and identifying these factors is critical to minimizing pain experienced by patients between appointments or after treatment. Factors that have previously been identified are preoperative pain, overinstrumentation (24), obturation material such as gutta-percha (25) and silver points (26), and extrusion of debris (24), irrigants (27, 28), and intracanal medicaments such as calcium hydroxide (Ca(OH)2) (29–31). Moreover, extrusion of sealer has been shown to have cytotoxic effects on the periapical tissues, causing periapical inflammation, necrosis (25), and pain (32). However, to date no study has evaluated the direct effect of sealers on trigeminal nociceptor activation. The present study therefore sought to evaluate the effects of 4
Figure 1. Effect of ZOE-based (RR) sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of ZOE-based sealer (RR) on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. *P < .05, **P < .01, as tested by one-way analysis of variance compared with control (buffer).
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Figure 2. Effect of AH Plus sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/ mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of AH Plus on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by one-way analysis of variance compared with control (buffer).
Figure 4. Effect of RealSeal SE sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of RealSeal SE on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by one-way analysis of variance compared with control (buffer).
different sealers, ZOE-based, AH Plus, BC, and RealSeal SE sealer, on activation of trigeminal sensory neurons in vitro. Our results demonstrate that both ZOE-based and AH Plus sealers are direct activators of sensory neurons. All the sealers were tested as freshly mixed pastes and in their set form. ZOEbased sealer was the only sealer that demonstrated neuronal activation in both forms. Interestingly, previous studies have demonstrated increased cytotoxicity of freshly mixed ZOE sealer compared with the set mix (19, 33). Thus, all sealers were tested as freshly mixed pastes and in their set form. However, only ZOE-based sealer demonstrated significant neuronal activation and CGRP release in both forms. ZOE-
based endodontic sealers are eugenol-based sealers. Recent studies have demonstrated the direct activation of nociceptors by eugenol via transient receptor potential (TRP) receptors (34–36). Two most critical pain-sensing channels, namely the transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), have been extensively studied. These channels are both thermosensitive receptors that mediate peripheral pain caused by noxious heat, cold, or inflammation (37–39). In this study, control experiments were performed in which the eugenol used in the ZOE-based sealer evoked comparable levels of CGRP release compared with the mixed sealer. This experiment suggests that most of the neuronal activation seen with this sealer can be attributed to eugenol. Interestingly, eugenol has been widely used as a topic analgesic in dentistry. The mechanisms for eugenol-induced analgesia are likely related to desensitization of TRPV1 and inhibition of voltageactivated calcium channels with continued exposure (40, 41).
Figure 3. Effect of BC sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of BC sealer on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. *P < .05, **P < .01, as tested by one-way analysis of variance compared with control (buffer).
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Figure 5. Effect of eugenol sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle or undiluted eugenol liquid on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by Student t test.
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Basic Research—Technology Nonetheless, eugenol acutely activates trigeminal neurons, resulting in a robust release of CGRP, as observed in this study. Therefore, ZOEbased sealers likely activate peripheral nociceptors via agonistic effects of eugenol on TRPV1 and TRPA1 receptors, resulting in CGRP release and possibly neurogenic inflammation. Similarly to ZOE-based sealer, AH Plus also evoked significant iCGRP release compared with the control group. Interestingly, an increase in the concentration of the sealer did not result in increased CGRP release, but an inhibition was seen. A possible explanation for this could be neuronal desensitization where either higher concentration or repeated and prolonged exposure of a drug/compound can inhibit nociceptor activation. Epoxy resin–based sealers have been shown to have significant cytotoxicity in the periradicular tissues by inducing inflammatory mediators such as cyclooxygenase2, nitric oxide synthase, receptor activator of nuclear factor kappa B ligand, and reactive oxygen species (17, 20). Moreover, increased expression of proinflammatory cytokines such as interleukin-6 and interleukin-8 (18) and marked decrease in mitochondrial activity (22) have been shown to be associated with AH Plus. Importantly, a case report demonstrated sensory loss on extrusion of AH Plus sealer into the mandibular canal because of neurotoxicity (42). Although it is not known which compound in AH Plus is the causative element, it would be fair to assume that it is a combination of various compounds present in the epoxide and the amine paste that together produce proinflammatory changes. In addition to these effects, ours is the first report demonstrating the direct activation of nociceptors that can further contribute to neurogenic inflammation and post-obturation discomfort. Both BC and RealSeal SE sealers not only failed to activate nociceptors, leading to release of iCGRP, but they also inhibited the basal release of iCGRP from trigeminal neurons (Figs. 3 and 4). Importantly, a final stimulus of KCl (250 mmol/L) was applied to all groups at the end of the experiment. This important positive control causes global activation of nociceptors, leading to a robust release of CGRP (data not shown). Our data demonstrate that none of the sealers depleted the overall releasable pool from the neurons or caused immediate neuronal death. Therefore, it can be speculated that both BC and RealSeal SE may be transiently inhibiting neuronal depolarization via a mechanism that is currently unknown. Also, this mechanism appears to be dependent on the presence of the sealers because the KCl stimulus was applied after the sealers were removed from the neuronal culture. An intriguing aspect of both these sealers is that they are calcium hydroxide (Ca(OH)2)-containing sealers, even though RealSeal is also a resin-based sealer. Interestingly, Ca(OH)2-containing sealers have been demonstrated to be less cytotoxic when compared with AH26, AH Plus, ZOE-based sealers, and Sargenti (N2) paste (17, 33). Thus, these findings suggest that both BC sealer and RealSeal SE have an acute inhibitory activity in trigeminal activation, and the long-term effect of these products on neuronal physiology, which is largely unknown, deserves further investigation. Overall, this study highlights and reinforces the practice of prevention of material extrusion into the periradicular tissue during endodontic treatment. Although 2 of the 4 sealers tested appeared to be non-noxious to trigeminal neurons, the possibility of neurotoxicity that can occur during the prolonged direct contact with periapical neurons in a clinical situation (as compared with a short contact period in the in vitro assay) cannot be ruled out. Moreover, careful attention must be paid when using algogenic compounds such as ZOE-based and AH Plus sealers during obturation to perhaps minimize transient or permanent patient discomfort.
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Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Pak JG, White SN. Pain prevalence and severity before, during, and after root canal treatment: a systematic review. J Endod 2011;37:429–38. 2. Nixdorf DR, Moana-Filho EJ, Law AS, et al. Frequency of persistent tooth pain after root canal therapy: a systematic review and meta-analysis. J Endod 2010;36: 224–30. 3. Georgopoulou M, Sikaras S, Anastasiadis P. Pain after obturation of the root canals. Hell Stomatol Chron 1988;32:249–54. 4. Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med 2004;15:348–81. 5. Oliet S. Single-visit endodontics: a clinical study. J Endod 1983;9:147–52. 6. Seltzer S, Naidorf IJ. Flare-ups in endodontics: I—etiological factors. J Endod 2004; 30:476–81. discussion 475. 7. Siqueira JF Jr. Microbial causes of endodontic flare-ups. Int Endod J 2003;36: 453–63. 8. Faria-Junior NB, Tanomaru-Filho M, Berbert FL, Guerreiro-Tanomaru JM. Antibiofilm activity, pH and solubility of endodontic sealers. Int Endod J 2013;46:755–62. 9. Saleh IM, Ruyter IE, Haapasalo M, Orstavik D. Survival of Enterococcus faecalis in infected dentinal tubules after root canal filling with different root canal sealers in vitro. Int Endod J 2004;37:193–8. 10. Byers MR. Sensory innervation of periodontal ligament of rat molars consists of unencapsulated Ruffini-like mechanoreceptors and free nerve endings. J Comp Neurol 1985;231:500–18. 11. Byers MR. Dynamic plasticity of dental sensory nerve structure and cytochemistry. Arch Oral Biol 1994;39(Suppl):13S–21. 12. Kimberly CL, Byers MR. Inflammation of rat molar pulp and periodontium causes increased calcitonin gene-related peptide and axonal sprouting. Anat Rec 1988; 222:289–300. 13. Fehrenbacher JC, Sun XX, Locke EE, et al. Capsaicin-evoked iCGRP release from human dental pulp: a model system for the study of peripheral neuropeptide secretion in normal healthy tissue. Pain 2009;144:253–61. 14. Gibbs JL, Melnyk JL, Basbaum AI. Differential TRPV1 and TRPV2 channel expression in dental pulp. J Dent Res 2011;90:765–70. 15. Kim YS, Jung HK, Kwon TK, et al. Expression of transient receptor potential ankyrin 1 in human dental pulp. J Endod 2012;38:1087–92. 16. Araki K, Suda H, Barbosa SV, Spangberg LS. Reduced cytotoxicity of a root canal sealer through eugenol substitution. J Endod 1993;19:554–7. 17. Huang FM, Tai KW, Chou MY, Chang YC. Cytotoxicity of resin-, zinc oxide-eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells. Int Endod J 2002;35:153–8. 18. Huang FM, Tsai CH, Yang SF, Chang YC. Induction of interleukin-6 and interleukin-8 gene expression by root canal sealers in human osteoblastic cells. J Endod 2005;31: 679–83. 19. Hume WR. An analysis of the release and the diffusion through dentin of eugenol from zinc oxide-eugenol mixtures. J Dent Res 1984;63:881–4. 20. Kim TG, Lee YH, Bhattari G, et al. PPARgamma inhibits inflammation and RANKL expression in epoxy resin-based sealer-induced osteoblast precursor cells E1 cells. Arch Oral Biol 2013;58:28–34. 21. Meryon SD, Johnson SG, Smith AJ. Eugenol release and the cytotoxicity of different zinc oxide-eugenol combination. J Dent 1988;16:66–70. 22. Schwarze T, Fiedler I, Leyhausen G, Geurtsen W. The cellular compatibility of five endodontic sealers during the setting period. J Endod 2002;28:784–6. 23. Flores CM, Leong AS, Dussor GO, et al. Capsaicin-evoked CGRP release from rat buccal mucosa: development of a model system for studying trigeminal mechanisms of neurogenic inflammation. Eur J Neurosci 2001;14:1113–20. 24. Walton R, Fouad A. Endodontic interappointment flare-ups: a prospective study of incidence and related factors. J Endod 1992;18:172–7. 25. Gutierrez JH, Brizuela C, Villota E. Human teeth with periapical pathosis after overinstrumentation and overfilling of the root canals: a scanning electron microscopic study. Int Endod J 1999;32:40–8. 26. Seltzer S, Green DB, Weiner N, DeRenzis F. A scanning electron microscope examination of silver cones removed from endodontically treated teeth. Oral Surg Oral Med Oral Pathol 1972;33:589–605. 27. Mehra P, Clancy C, Wu J. Formation of a facial hematoma during endodontic therapy. J Am Dent Assoc 2000;131:67–71. 28. Sabala CL, Powell SE. Sodium hypochlorite injection into periapical tissues. J Endod 1989;15:490–2. 29. Bramante CM, Luna-Cruz SM, Sipert CR, et al. Alveolar mucosa necrosis induced by utilisation of calcium hydroxide as root canal dressing. Int Dent J 2008;58:81–5.
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Basic Research—Technology 30. Lindgren P, Eriksson KF, Ringberg A. Severe facial ischemia after endodontic treatment. J Oral Maxillofac Surg 2002;60:576–9. 31. Sharma S, Hackett R, Webb R, et al. Severe tissue necrosis following intra-arterial injection of endodontic calcium hydroxide: a case series. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:666–9. 32. Froes FG, Miranda AM, Abad Eda C, et al. Non-surgical management of paraesthesia and pain associated with endodontic sealer extrusion into the mandibular canal. Aust Endod J 2009;35:183–6. 33. Matsumoto K, Inoue K, Matsumoto A. The effect of newly developed root canal sealers on rat dental pulp cells in primary culture. J Endod 1989;15: 60–7. 34. Xu H, Delling M, Jun JC, Clapham DE. Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 2006;9: 628–35. 35. Yang BH, Piao ZG, Kim YB, et al. Activation of vanilloid receptor 1 (VR1) by eugenol. J Dent Res 2003;82:781–5.
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36. Chung G, Im S, Oh SB, Kim J. Activation of TRPA1 by eugenol. IADR Abstract 2012;1929. 37. Bautista DM, Jordt SE, Nikai T, et al. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell 2006;124:1269–82. 38. Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. 39. Obata K, Katsura H, Mizushima T, et al. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury. J Clin Invest 2005; 115:2393–401. 40. Park CK, Kim K, Jung SJ, et al. Molecular mechanism for local anesthetic action of eugenol in the rat trigeminal system. Pain 2009;144:84–94. 41. Seo H, Li HY, Perez-Reyes E, Lee JH. Effects of eugenol on T-type Ca2+ channel isoforms. J Pharmacol Exp Ther 2013;347:310–7. 42. Gonzalez-Martin M, Torres-Lagares D, Gutierrez-Perez JL, Segura-Egea JJ. Inferior alveolar nerve paresthesia after overfilling of endodontic sealer into the mandibular canal. J Endod 2010;36:1419–21.
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Direct Effect of Endodontic Sealers on Trigeminal Neuronal Activity Nikita B. Ruparel, DDS, MS, PhD, Shivani B. Ruparel, PhD, Paul B. Chen, BS, Blake Ishikawa, DDS, and Anibal Diogenes, DDS, MS, PhD Abstract Introduction: Endodontic sealers are selected on the basis of their antimicrobial properties and ability to provide a tight seal. Sealer extrusions, whether intentional or unintentional, are common during obturation procedures. Such events have been correlated with increased postoperative discomfort and persistent pain states. However, the mechanisms underlying this phenomenon are largely unknown. Thus, we sought to evaluate the effect of commonly used endodontic sealers on peripheral nociceptors. We hypothesized that endodontic sealers can directly activate trigeminal nociceptors in a concentration-dependent manner, resulting in release of calcitonin gene-related peptide (CGRP), a potent modulator of neurogenic inflammation. Methods: Rat trigeminal sensory neurons were exposed in vitro to vehicle, zinc oxide-eugenol (ZOE)–based sealer, AH Plus, EndoSequence BC sealer, or RealSeal SE. Neuronal activation was measured by quantification of neuropeptide (CGRP) release. In addition, cultured neurons were also subjected to the set form of all 4 sealers. The concentration of CGRP released was quantified by using a radioimmunoassay. Data were analyzed by using oneway analysis of variance with Newman-Keuls multiple comparison post hoc test. Results: Both ZOE-based sealer and AH Plus in their fresh form evoked greater CGRP release than the control groups. Conversely, EndoSequence BC and RealSeal sealers both reduced basal GCRP release at all concentrations tested. Evaluation of the set sealers revealed that only ZOE-based sealer evoked significant CGRP release compared with its control group. Conclusions: Overall, our results suggest that sealers can directly activate trigeminal nociceptors, leading to a robust release of CGRP, and may therefore lead to pain and neurogenic inflammation. This direct activation along with the immunologic response may underlie the symptoms and flare-up occurrences often seen with sealer extrusions. (J Endod 2014;40:683–687)
Key Words AH Plus, CGRP, endodontic sealer, EndoSequence BC sealer, neuropeptide, neuroscience, obturation, pain, RealSeal SE, trigeminal neurons, zinc oxide eugenol
P
ersistent pain is an ongoing problem after endodontic treatment. A recent systematic review demonstrated that approximately 14% of patients who received endodontic treatment had persistent pain 1 week after obturation (1). Moreover, Nixdorf et al (2) demonstrated that approximately 5.3% of patients experience pain 6 months after endodontic therapy, and only about 3.4% of it is from a non-odontogenic source. The etiology of non-odontogenic pain can be attributed to myofascial pain, headaches, neuropathic pain, or referred pain, whereas persistent pain from odontogenic origin could be due to missed canal, incomplete debridement, tooth fracture, occlusion, neuronal plasticity from a prolonged inflammatory state, or overfill of obturation material (2). Overfill of obturation material into the periradicular tissue has been correlated with foreign body reactions, flare-ups, and increased postoperative discomfort (3–7). Endodontic sealers are an essential part of obturation; not only do they provide for an adequate seal between the core material and the dentinal walls, but they are also effective antimicrobials inside the canal system (8, 9). Sealers may be extruded into the periradicular tissue when compacting the obturation core material. Therefore, sealer extrusion may stimulate an inflammatory response and activate sensory neurons, perhaps causing persistent pain after endodontic therapy. The dental pulp and periradicular tissue are densely innervated with polymodal nociceptors capable of detecting noxious thermal, mechanical, and chemical stimuli (10–15). Similar to the pulp, the periradicular tissues are densely innervated and undergo changes in response to inflammation at a cellular and molecular level (10–12). Although the long-term or neurotoxic effect of sealers has been previously investigated (16–22), to the best of our knowledge, no study has evaluated the direct effect of sealers on nociceptor activation. We therefore hypothesized that endodontic sealers will cause activation of a specialized subclass of peptidergic nociceptors, leading to vesicular exocytosis and release of calcitonin gene-related peptide (CGRP). Importantly, several groups have evaluated levels of CGRP as a model for assessing neurogenic inflammation and nociceptor activation (13, 23). Therefore, by using an in vitro neuropeptide release assay, we sought to evaluate the direct effect of several sealer compounds on activation of nociceptors and possibly neurogenic inflammation.
From the Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas. Nikita Ruparel and Shivani Ruparel contributed equally to this study. Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.030
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Basic Research—Technology Methods Animals Adult male Sprague-Dawley rats (weight, 200–250 g each) (Charles River, Wilmington, MA) were used in this study. All animal study protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and conformed to the International Association for the Study of Pain and federal guidelines. Animals were housed for 1 week before the experiments with food and water available ad libitum. Rat Trigeminal Ganglia Primary Culture Rat trigeminal ganglia (TG) were quickly removed after decapitation, and neuronal cultures were prepared as previously described (15, 16). Cells were plated on 24-well poly-D-lysine– coated plates (for immunoreactive CGRP [iCGRP] experiments). The TG cultures were maintained at 37 C, 5% CO2 for 5 days in the presence of 100 ng/mL nerve growth factor (Harlan Laboratories, Indianapolis, IN). CGRP Release Assay Culture experiments were performed on day 5 at 37 C by using modified Hanks’ balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA) buffer (10.9 mmol/L HEPES, 4.2 mmol/L sodium bicarbonate, 10 mmol/L dextrose, and 0.1% bovine serum albumin were added to 1 HBSS). To evaluate the effects of freshly mixed sealers on TG neurons, two 20-minute baseline samples were collected, followed by application of freshly mixed zinc oxide-eugenol (ZOE)–based sealer (Roth Root; Pearson Dental, Sylmar, CA), AH Plus (Dentsply, Tulsa, OK), EndoSequence BC sealer (BC) (Brasseler USA, Savannah, GA), or RealSeal SE sealer (SybronEndo, Orange, CA) at 25 mg/mL and 50 mg/mL concentrations for 30 minutes. All sealers were weighed along with a preweighed cell insert (BD Biosciences, Bedford, MA) with a pore size of 1 mm. The inserts were then placed in wells of a 24-well culture plate. After stimulation with sealers, the inserts were removed, and the cells were washed twice in HBSS for 20 minutes each. To evaluate the effect of sealer in a set form, the same experimental design and protocol were used except that each sealer was mixed and allowed to set overnight at 37 C. All groups were subjected to a 20-minute 250 mmol/L KCl (potassium chloride) solution to determine the remainder of the releasable CGRP pool from the cultured neurons. iCGRP Radioimmunoassay A previously used (17) primary antibody against CGRP (final dilution of 1:1,000,000; donated by Dr M. J. Iadarola, National Institutes of Health, Bethesda, MD) was added in the tubes containing superfusate from cultured rat TG and incubated at 4 C for 24 hours. Then 100 mL [125I-]-Tyr0-CGRP28–37 (20,000 cpm) and 50 mL goat antirabbit antisera coupled to ferric beads (PerSeptive Diagnostics, Cambridge, MA) were added to these tubes. The tubes were incubated for another 24 hours at 4 C. The assay was stopped by using immunomagnetic separation of bound from free tracer. All compounds used in experiments were tested for interference with the radioimmunoassay. The minimum detectable levels for CGRP for this assay are 3 fmol and the 50% displacement at 28 fmol. Data Analysis All experiments were conducted in triplicate with n = 6 per group. Data were analyzed by using Prism software version 5 (GraphPad Software, San Diego, CA). The results were analyzed with significance set at 684
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P < .05 by using one-way analysis of variance with Newman-Keuls post hoc test for multiple comparison post hoc test or Student t test analysis for comparing 2 groups.
Results Freshly mixed ZOE-based sealer (25 mg/mL) evoked 18-fold increase in iCGRP levels compared with control group (Fig. 1). Moreover, 50 mg/mL of freshly mixed as well as 50 mg/mL of the set form of ZOEbased sealer evoked 28-fold increase in iCGRP compared with control group (Fig. 1). Interestingly, only the freshly mixed AH Plus at 25 mg/mL concentration but not at 50 mg/mL concentration evoked 2.7fold significant iCGRP release compared with control group (Fig. 2). In contrast to ZOE-based sealer and AH Plus, both BC sealer and RealSeal SE sealers at both concentrations and forms inhibited iCGRP release from TG neurons compared with control group (Figs. 3 and 4). It is noteworthy that the overall iCGRP release from ZOE-based sealer at both concentrations was dramatically greater than any sealers used. To evaluate whether eugenol in ZOE-based sealer was responsible for the increased release, we subjected a group of cells to undiluted liquid eugenol (100 wt% or 6.1 mmol/L). Application of eugenol alone evoked 7.5-fold increase in iCGRP (Fig. 5).
Discussion Factors contributing to postoperative endodontic pain are many, and identifying these factors is critical to minimizing pain experienced by patients between appointments or after treatment. Factors that have previously been identified are preoperative pain, overinstrumentation (24), obturation material such as gutta-percha (25) and silver points (26), and extrusion of debris (24), irrigants (27, 28), and intracanal medicaments such as calcium hydroxide (Ca(OH)2) (29–31). Moreover, extrusion of sealer has been shown to have cytotoxic effects on the periapical tissues, causing periapical inflammation, necrosis (25), and pain (32). However, to date no study has evaluated the direct effect of sealers on trigeminal nociceptor activation. The present study therefore sought to evaluate the effects of 4
Figure 1. Effect of ZOE-based (RR) sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of ZOE-based sealer (RR) on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. *P < .05, **P < .01, as tested by one-way analysis of variance compared with control (buffer).
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Figure 2. Effect of AH Plus sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/ mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of AH Plus on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by one-way analysis of variance compared with control (buffer).
Figure 4. Effect of RealSeal SE sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of RealSeal SE on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by one-way analysis of variance compared with control (buffer).
different sealers, ZOE-based, AH Plus, BC, and RealSeal SE sealer, on activation of trigeminal sensory neurons in vitro. Our results demonstrate that both ZOE-based and AH Plus sealers are direct activators of sensory neurons. All the sealers were tested as freshly mixed pastes and in their set form. ZOEbased sealer was the only sealer that demonstrated neuronal activation in both forms. Interestingly, previous studies have demonstrated increased cytotoxicity of freshly mixed ZOE sealer compared with the set mix (19, 33). Thus, all sealers were tested as freshly mixed pastes and in their set form. However, only ZOE-based sealer demonstrated significant neuronal activation and CGRP release in both forms. ZOE-
based endodontic sealers are eugenol-based sealers. Recent studies have demonstrated the direct activation of nociceptors by eugenol via transient receptor potential (TRP) receptors (34–36). Two most critical pain-sensing channels, namely the transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), have been extensively studied. These channels are both thermosensitive receptors that mediate peripheral pain caused by noxious heat, cold, or inflammation (37–39). In this study, control experiments were performed in which the eugenol used in the ZOE-based sealer evoked comparable levels of CGRP release compared with the mixed sealer. This experiment suggests that most of the neuronal activation seen with this sealer can be attributed to eugenol. Interestingly, eugenol has been widely used as a topic analgesic in dentistry. The mechanisms for eugenol-induced analgesia are likely related to desensitization of TRPV1 and inhibition of voltageactivated calcium channels with continued exposure (40, 41).
Figure 3. Effect of BC sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle, freshly mixed 25 mg/mL, freshly mixed 50 mg/mL, or set mix 50 mg/mL of BC sealer on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. *P < .05, **P < .01, as tested by one-way analysis of variance compared with control (buffer).
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Figure 5. Effect of eugenol sealer on CGRP release from cultured trigeminal neurons. Evaluation of effect of treatment with vehicle or undiluted eugenol liquid on iCGRP release from cultured trigeminal neurons. Data are presented as the mean standard error of the mean. ***P < .001, as tested by Student t test.
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Basic Research—Technology Nonetheless, eugenol acutely activates trigeminal neurons, resulting in a robust release of CGRP, as observed in this study. Therefore, ZOEbased sealers likely activate peripheral nociceptors via agonistic effects of eugenol on TRPV1 and TRPA1 receptors, resulting in CGRP release and possibly neurogenic inflammation. Similarly to ZOE-based sealer, AH Plus also evoked significant iCGRP release compared with the control group. Interestingly, an increase in the concentration of the sealer did not result in increased CGRP release, but an inhibition was seen. A possible explanation for this could be neuronal desensitization where either higher concentration or repeated and prolonged exposure of a drug/compound can inhibit nociceptor activation. Epoxy resin–based sealers have been shown to have significant cytotoxicity in the periradicular tissues by inducing inflammatory mediators such as cyclooxygenase2, nitric oxide synthase, receptor activator of nuclear factor kappa B ligand, and reactive oxygen species (17, 20). Moreover, increased expression of proinflammatory cytokines such as interleukin-6 and interleukin-8 (18) and marked decrease in mitochondrial activity (22) have been shown to be associated with AH Plus. Importantly, a case report demonstrated sensory loss on extrusion of AH Plus sealer into the mandibular canal because of neurotoxicity (42). Although it is not known which compound in AH Plus is the causative element, it would be fair to assume that it is a combination of various compounds present in the epoxide and the amine paste that together produce proinflammatory changes. In addition to these effects, ours is the first report demonstrating the direct activation of nociceptors that can further contribute to neurogenic inflammation and post-obturation discomfort. Both BC and RealSeal SE sealers not only failed to activate nociceptors, leading to release of iCGRP, but they also inhibited the basal release of iCGRP from trigeminal neurons (Figs. 3 and 4). Importantly, a final stimulus of KCl (250 mmol/L) was applied to all groups at the end of the experiment. This important positive control causes global activation of nociceptors, leading to a robust release of CGRP (data not shown). Our data demonstrate that none of the sealers depleted the overall releasable pool from the neurons or caused immediate neuronal death. Therefore, it can be speculated that both BC and RealSeal SE may be transiently inhibiting neuronal depolarization via a mechanism that is currently unknown. Also, this mechanism appears to be dependent on the presence of the sealers because the KCl stimulus was applied after the sealers were removed from the neuronal culture. An intriguing aspect of both these sealers is that they are calcium hydroxide (Ca(OH)2)-containing sealers, even though RealSeal is also a resin-based sealer. Interestingly, Ca(OH)2-containing sealers have been demonstrated to be less cytotoxic when compared with AH26, AH Plus, ZOE-based sealers, and Sargenti (N2) paste (17, 33). Thus, these findings suggest that both BC sealer and RealSeal SE have an acute inhibitory activity in trigeminal activation, and the long-term effect of these products on neuronal physiology, which is largely unknown, deserves further investigation. Overall, this study highlights and reinforces the practice of prevention of material extrusion into the periradicular tissue during endodontic treatment. Although 2 of the 4 sealers tested appeared to be non-noxious to trigeminal neurons, the possibility of neurotoxicity that can occur during the prolonged direct contact with periapical neurons in a clinical situation (as compared with a short contact period in the in vitro assay) cannot be ruled out. Moreover, careful attention must be paid when using algogenic compounds such as ZOE-based and AH Plus sealers during obturation to perhaps minimize transient or permanent patient discomfort.
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Acknowledgments The authors deny any conflicts of interest related to this study.
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