Basic Research—Biology
Periapical Inflammation Subsequent to Coronal Inoculation of Dog Teeth Root Filled with Resilon/Epiphany in 1 or 2 Treatment Sessions with Chlorhexidine Medication Jo~ ao M. Santos, DDS, PhD,* Paulo J. Palma, DDS,* Jo~ ao C. Ramos, DDS, PhD,* Ant onio S. Cabrita, MD, PhD,† and Shimon Friedman, DMD‡ Abstract Introduction: Therapeutic methods that inhibit microbial ingress into filled root canals are desirable. This in vivo study assessed the inhibition of periapical inflammation subsequent to coronal inoculation in canals medicated with 2% chlorhexidine gel and filled with Resilon/Epiphany (Pentron Clinical Technologies, Wallingford, CT). Methods: Six Beagle dogs each had 10 two-rooted premolars treated. In group 1 (n = 36 roots), 1 root/tooth had the canal conditioned with Primer Epiphany, filled with Epiphany sealer and Resilon core in 1 session, and coronally sealed with PhotacFil. In group 2 (n = 36 roots), the second root/tooth had the canal medicated with 2% chlorhexidine gel for 1 week and then filled and coronally sealed as in group 1. After 3 weeks, canals were exposed to the oral environment for 7 days, inoculated with isologous plaque, and coronally sealed. Negative controls treated as groups 1 and 2 remained sealed. Positive controls had canals unfilled and exposed. Seven months after inoculation, dogs were euthanized; jaw blocks processed for histologic examination; and periapical inflammation (PI) recorded as none, mild, or severe. Results: In groups 1 and 2, severe PI occurred in 5 of 65 roots (8%) and mild PI in 18 of 65 roots (28%) with a significantly higher (P = .031) PI incidence in group 2 than in group 1. Negative controls had only mild PI in 9 of 29 roots (31%). Roots medicated with 2% chlorhexidine gel had mild PI significantly more (P = .009) than roots filled in 1 session (more than 2-fold). Conclusions: Intracanal medication with 2% chlorhexidine gel and root filling with Resilon/Epiphany did not effectively inhibit apical periodontitis subsequent to coronal inoculation. (J Endod 2014;-:1–5)
Key Words 1-session treatment, chlorhexidine, coronal leakage, Resilon, root canal filling
A
pical periodontitis comprises the host’s response to pathogenic microorganisms colonizing the root canal system of the tooth (1). To maintain or regenerate the host tissues, the root canals are disinfected and filled, and the access cavity is restored to prevent microbial ingress; however, microorganisms can invade the filled canals over time via the coronal cavity, lateral canals, and dentinal tubules (2). They can propagate and elicit post-treatment apical periodontitis, undermining the prognosis (3). To improve the long-term prognosis, therapeutic methods need to be explored that can effectively prevent microbial ingress, including medications with sustained antimicrobial activity and root-filling materials offering improved sealing properties. Chlorhexidine (CHX) can be applied as an intracanal medication using different vehicles (4–7). The CHX molecule acts to increase the permeability of the microbial cell wall, penetrates the cell’s cytoplasm, and affects coagulation of the cytoplasmic content and cell death (8). CHX is effective against a wide range of gram-positive and gram-negative microorganisms as well as yeasts, facultative anaerobes, and aerobes (9, 10). Moreover, when used in root canals at a sufficient concentration and exposure time, CHX adsorbs onto root dentin and imparts substantive antimicrobial activity (4, 11–13), which could inhibit coronal microbial ingress (4). Furthermore, by inhibiting endogenous metalloproteinases, the substantive activity of CHX can moderate adhesive failures of dentin-bonding root-filling materials and maintain stability of the hybrid layer (14). All of the previously mentioned attributes notwithstanding, clinical data have not consistently supported the antimicrobial benefits of CHX applied as an intracanal medication (5–7, 15, 16). Additional experimental in vivo data were deemed necessary to explore the possible benefits of intracanal medication with CHX. Resilon (Pentron Clinical Technologies, Wallingford, CT) is a synthetic polymerbased root-filling material designed for application with a methacrylate-resin sealer (Epiphany, Pentron Clinical Technologies). Although bonded interfaces are expected between the Resilon core, sealer, and root canal wall (17), the resulting seal of Resilon/Epiphany has been questioned; when assessed in vitro, it has been superior (18), similar (19), and inferior (20) to that of conventional root fillings. Moreover, the bonded interface is susceptible to biofilm proliferation (21). Friedman et al (3), in an animal model, challenged filled root canals with an isologous inoculum within the pulp chamber to assess their resistance to microbial ingress in vivo. After periods of 4–10 months, 0%–20% of roots developed severe periapical inflammation attributed to infection, and 21%–33% developed mild inflammation in response to the treatment procedures (3, 12, 22). When tested in a similar model, roots filled with Resilon/Epiphany developed varying degrees of periapical inflammation (23–25), precluding definitive conclusions regarding the ability of this system to prevent microbial ingress. Additional experimental in vivo data were
From the *Department of Dentistry and †Institute of Experimental Pathology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; and ‡Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Jo~ao Miguel Marques dos Santos, Av Bissaya Barreto, Blocos de celas, 3000-075 Coimbra, Portugal. 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.2013.10.023
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Periapical Inflammation after Coronal Inoculation
1
Basic Research—Biology TABLE 1. Summary of the Experimental Protocol in the 2-rooted Mandibular and Maxillary Premolars of Dogs Assigned to the Test and Control Groups Groups
Roots treated (n)
Test Group 1
36
Group 2
36
Negative control Group 1A Group 2A Positive control
18 18 12
Treatment protocol
Inoculation
1-session treatment Resilon/Epiphany 2-session treatment with CHX Resilon/Epiphany
Yes
1-session treatment Resilon/Epiphany 2-session treatment with CHX Resilon/Epiphany Canal prepared without root filling
No
Yes
CHX, chlorhexidine.
deemed necessary to explore the possible benefits of root filling with the Resilon/Epiphany system. This in vivo study assessed the efficacy of intracanal medication with 2% CHX and root filling with Resilon/Epiphany in inhibiting periapical inflammation subsequent to coronal inoculation to explore the potential benefits of both therapeutic methods. Our null hypothesis was that roots medicated with 2% CHX and those filled in 1 session would have a similar incidence of periapical inflammation.
Material and Methods Animal and Tooth Preparation The study protocol was approved by the Animal Welfare Committee of the Direcc¸~ao-Geral de Veterinaria and complied with the International Guiding Principles for Biomedical Research Involving Animals (Geneva, 1985). For the first 2 interventions, dogs were anesthetized with 10 mg/kg thiopental (B. Braun Medical, Melsungen, Germany) and the inhalation of O2 and 2% isoflurane (Abbott Laboratories, Abbott Park, IL). For subsequent interventions, dogs were sedated with intramuscular administration of 7 mg/kg tiletamine/zolazepam (Virbac, Carros, France). Based on previous studies (3), six 2-year-old female Beagle dogs had treatment performed in 10 two-rooted premolars each for a total of 120 roots. Before treatment, teeth were radiographed, cleaned with pumice (Produits Dentaires, Vevey, Switzerland), isolated with a rubber dam (Hygienic, Cuyahoga Falls, OH), and thoroughly wiped with 10% povidone-iodine solution (Labesfal, Besteiros, Portugal) to ensure aseptic conditions. The access cavity was prepared with a sterile tapered high-speed bur (Dentsply Maillefer, Ballaigues, Switzerland) under saline irrigation. Canals were prepared with Hero642 instruments (Micro-Mega, Besancon, France) in a crown-down sequence and apically enlarged with K-type hand files to size 40–60 at the working length based on the canal’s size. They were intermittently irrigated with 10 mL 2.5% NaOCl, rinsed for 30 seconds with 3 mL 17% EDTA (Produits
Dentaires), flushed with 3 mL sterile saline, and dried with paper points (Dentsply Maillefer).
Groups Roots with prepared canals were assigned to test and control groups (Table 1). Test groups comprised the 6 two-rooted mandibular premolars. In group 1 (n = 36 roots), in 1 root/tooth, the canal was conditioned (Primer Epiphany) and filled (Root Canal Sealant Epiphany and Resilon) using warm compaction (SystemB; Sybron Endo, Orange, CA). The filling was photopolymerized for 20 seconds (Optilux 501; Demetron/Kerr Co, Karlsruhe, Germany), and the access was sealed (PhotacFil; 3M-ESPE, Seefeld, Germany). In group 2 (n = 36 roots), in the second root/tooth, 2% CHX gel was placed with a Lentulo spiral (Dentsply Maillefer), and the access was sealed with PhotacFil. After 1 week, the medicated roots were reaccessed, CHX gel was flushed out with 3 mL sterile saline, and canals were filled and resealed as in group 1. In the contralateral premolars, grouping of the canals in each tooth was reversed to equalize the distribution of mesial and distal roots in each group. After 3 weeks, the pulp chambers of teeth in groups 1 and 2 were reaccessed, left exposed to the oral environment for 1 week, inoculated with isologous plaque, and resealed with PhotacFil. Three maxillary premolars/dog were allocated to negative control groups 1A and 2A (n = 18 roots/group). They were treated as in groups 1 and 2, respectively, but their pulp chambers were left undisturbed. One maxillary premolar/dog served as a positive control (n = 12 roots) by leaving the prepared canals unfilled and exposed to the oral environment. Outcome Measures Periapical radiographs were exposed before treatment, immediately after treatment, and 4 and 7 months after treatment. At 7 months, animals were anesthetized with thiopental and euthanized by anesthetic overdose followed by bilateral perfusion with 10% phosphate-buffered formalin. Jaw and maxillary blocks were separated, dissected from the
Figure 1. Photomicrographs of mandibular premolar roots and periradicular tissues of dogs 7 months after experimental procedures showing the histologic classification of PI used in the study. (A) No PI: normal appearance of the root surface and surrounding tissues; (B) mild PI: localized inflammatory cell infiltrate without evident bone and root resorption; and (C) severe PI: diffuse infiltrate with evident bone or root resorption (hematoxylin-eosin; original magnification: 20).
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Santos et al.
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Basic Research—Biology
Figure 2. Photomicrographs of a mandibular premolar root and periradicular tissues of a dog 7 months after root filling and coronal inoculation. (A) An overview of root, periodontal ligament, extruded sealer, and bone. (B and C) Magnified views of the extruded sealer. Epiphany sealer expressed periapically is surrounded by a fibrous capsule, mononuclear inflammatory infiltrate, and foreign body giant cells (hematoxylin-eosin; original magnification: [A] 20, [B] 40, and [C] 80).
first premolar to the first molar, postfixed in phosphate-buffered formalin, and decalcified with Morse solution. Each block was divided between the roots of the third premolar and trimmed, embedded in paraffin, and serially sectioned every 6 mm along the mesiodistal plane, parallel to the long axis of canals. One in every 10 sections was stained with hematoxylin-eosin, and periradicular tissues were examined histologically under a light microscope (Nikon Eclipse E600; Nikon, Tokyo, Japan). One observer examined all specimens blinded to group allocation. Periradicular inflammation (PI) was rated in accordance with previous studies (3, 22, 26) as follows: 1. None: Normal appearance of root surface and surrounding tissues 2. Mild: Localized inflammatory infiltrate without bone and root resorption, and 3. Severe: Diffuse infiltrate with evident bone or root resorption (Fig. 1)
Analysis SPSS 14.0 (SPSS Inc, Chicago, IL) was used to compare the incidence of PI in groups 1 and 2 with the chi-square test. Significance was established at the 5% level.
Results Clinically, there was no evidence of swelling or a sinus tract associated with any of the treated teeth. During the follow-up, 1 mandibular tooth lost the coronal restoration without necessitating exclusion from analysis. One root from group 1A (negative control) was lost during histologic processing. Thirteen roots were excluded from analysis because of extruded Epiphany sealer (small amounts detected histologically in 12 roots and larger overfilling in 1 root). The excess Epiphany material was surrounded with fibrous capsules associated with mononuclear inflammatory infiltrate with evidence of phagocytosis (Fig. 2A–C). In the positive control group, all 12 roots had radiographic evidence of periapical pathosis and histologically confirmed severe PI (Table 2). In the negative control groups 1A and 2A, none of the roots had severe PI, and 9 of 29 roots (31%) had mild PI, without statistically significant differences between groups 2A and 1A (P = .184). In the test groups, 5 of 65 roots (8%) had severe PI, and 18 of 65 roots (28%) had mild PI. The incidence of PI was significantly higher (Pearson chi-square test, P = .031) in group 2 than in group 1; thus, the null hypothesis was rejected. Mild PI was observed in 19 of 46 roots (41%) treated in 2 sessions (groups 2 and 2A) compared with 8 of 48 roots (17%) treated in 1 session (groups 2 and 2A). The difference was statistically significant (chi-square test, P = .009).
Discussion An in vivo model that challenges root-filled canals with coronally inoculated microorganisms, simulating a clinical condition that can lead to post-treatment apical periodontitis (3, 22, 26), was used to JOE — Volume -, Number -, - 2014
assess the inhibition of microbial ingress by root canal medication with 2% CHX and root filling with the Resilon/Epiphany system. Canals were inoculated after 3 weeks to allow sufficient time for the Epiphany sealer to set. They were exposed to saliva for 1 week before inoculation to exploit possible gaps along the sealer-dentin interface (27) and to expose the root filling to possible enzymatic hydrolysis (28). The inoculated access cavities were sealed to support the selection of anaerobic microflora (29) as found in teeth with posttreatment apical periodontitis (30). A period of 7 months was allowed before outcome assessment, which was longer than in several studies based on the same model (3, 23–26) but shorter than in another such study (22). A short period might not be sufficient to infect the filled canals, whereas a long period could increase the risk of coronal breakdown of the teeth with undesirable exposure of the inoculated pulp chambers (22, 26). The 7-month observation interval used herein appeared to strike a good balance in which severe PI developed in some of the roots and coronal breakdown occurred in only 1 tooth. Severe PI was observed in all the positive control roots and in none of the negative controls, validating that coronal inoculation was its cause (3, 22). It occurred in 8% of the roots filled with Resilon/Epiphany, which was in agreement with previous reports (24, 26). The 8% incidence of severe PI after 7 months of inoculation was in line with the 11% increase in severe PI observed after a 3-month period of coronal exposure of roots filled with Resilon/Epiphany compared with roots that were not exposed (24). It also was similar to the 8% severe PI reported after a 6-month period of inoculation in roots filled with gutta-percha and a glass-ionomer cement-based sealer but lower than the 21% in roots in which a zinc oxide–eugenol sealer was used (26). In this regard, our results supported the suitability of the study model for in vivo evaluation of the resistance of root fillings to microbial ingress in contrast to the absence of severe PI previously reported for Resilon/Epiphany after 14 weeks of inoculation (23) and for guttapercha and a zinc oxide–eugenol–based sealer after 4- and 10-month inoculation periods (3, 22). TABLE 2. Scores of Periapical Inflammation Assessed Histologically 7 Months after Experimental Treatment Procedures in 2-rooted Premolars of Dogs
Groups Positive control Negative control Group 1A Group 2A Test Group 1 Group 2
Level of PI Roots assessed (n) None (%) Mild Severe 12
0
0
12 (100)
15 14
12 (80) 8 (57)
3 (20) 6 (43)
0 0
33 32
25 (76) 17 (53)
5 (15) 13 (41)
3 (9) 2 (6)
PI, periapical inflammation. One root was lost during processing, and 13 were excluded from analysis because of sealer extrusion.
Periapical Inflammation after Coronal Inoculation
3
Basic Research—Biology The very development of severe PI in 6% of roots medicated with CHX and in 8% of roots filled with Resilon/Epiphany suggested that both therapeutic methods only partially inhibited coronal microbial ingress and the development of post-treatment apical periodontitis. Although the antimicrobial mechanism and activity of CHX have been well established (8–10), its use as an intracanal medication, its substantive antimicrobial activity, and its potential stabilization of sealer-dentin interfaces have been supported only by in vitro research data (4, 11, 13, 14, 20, 27). Clinical assessments of the antimicrobial efficacy of CHX in teeth with infected root canals have failed to convincingly show benefits (5–7, 15, 16). The results of this study suggested the incapacity of medication with CHX to effectively inhibit microbial ingress and, thus, undermined the rationale for its clinical application. The system comprised of methacrylate resin–based sealer (Epiphany) and polymer-based core (Resilon) has been introduced to address a perceived weak link in conventional root fillings (17). Nevertheless, when assessed in vitro, this system’s sealing ability has been disputed (18–20), and its susceptibility to biofilm proliferation within the canal wall interface was highlighted (21). The results of this study cast further doubt on the system’s capacity to effectively inhibit microbial ingress. Integrating the available research data, root filings with the Resilon/ Epiphany system appeared to be pervious to microbial ingress. This might be caused by incomplete infiltration or polymerization of the resin within the hybrid layer and dentin substrate (31) and biodegradation and dissolution that undermine the resin’s bond to root dentin over time (14, 32). Mild PI was observed in 31% of the negative control roots within the range reported for root fillings with gutta-percha and a zinc oxide– eugenol–based sealer (3, 22, 24, 26). The comparable incidence of mild PI in roots that were treated under well-controlled conditions, filled with different materials, and not inoculated suggested that the mild inflammation represented a persistent tissue response to the endodontic treatment procedure. This suggestion was supported by the comparable incidence of mild PI in the test and negative control groups. The moderate incidence of mild PI could explain why it was not observed in studies in which the negative controls included 4 roots or less (23, 25). The incidence of mild PI was over 2-fold higher in roots medicated with 2% CHX gel than in roots filled in 1 session in both the test and control groups. This finding might be attributed to the toxicity of CHX coupled with the lengthy exposure of the periapical tissues (33) or inadvertent contamination during the period between treatment sessions. The likelihood of tissue irritation from parachloroaniline precipitate resulting from the interaction of NaOCl with CHX (34) was low because in this study NaOCl was followed by EDTA and saline and the canals were dried with paper points before CHX gel was applied. Nevertheless, the persistent PI echoed the recent report of a compromised healing outcome in roots in which canals were irrigated with 0.2% CHX (35). Taken together, the previous report and our results further undermined the rationale for the clinical application of CHX in root canal therapy.
Conclusions Within the limitation of the in vivo experimental method used, it was concluded that (1) intracanal medication with 2% CHX gel did not effectively inhibit the development of apical periodontitis in roots subjected to coronal microbial challenge, (2) root filling with the Resilon/ Epiphany system did not effectively inhibit the development of apical periodontitis when subjected to coronal microbial challenge, and (3) roots treated in 2 sessions and medicated with 2% CHX gel had over 4
Santos et al.
2-fold more periapical inflammation than roots treated in 1 session 7 months after treatment. The findings of this study should be combined with those of other in vivo and clinical outcome assessments to inform clinicians about the clinical application of CHX as an intracanal medication and the use of Resilon/Epiphany as a root filling system.
Acknowledgments Supported by grants from Fundac¸~ao para a Ci^encia e Tecnologia and Pentron Clinical Technologies. The authors deny any conflicts of interest related to this study.
References 1. Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340–9. 2. Saunders WP, Saunders EM. Coronal leakage as a cause of failure in root-canal therapy: a review. Endod Dent Traumatol 1994;10:105–8. 3. Friedman S, Torneck CD, Komorowski R, et al. In vivo model for assessing the functional efficacy of endodontic filling materials and techniques. J Endod 1997;23: 557–61. 4. Basrani B, Santos JM, Tjaderhane L, et al. Substantive antimicrobial activity in chlorhexidine-treated human root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:240–5. 5. Paquette L, Legner M, Fillery ED, et al. Antibacterial efficacy of chlorhexidine gluconate intracanal medication in vivo. J Endod 2007;33:788–95. 6. Malkhassian G, Manzur AJ, Legner M, et al. Antibacterial efficacy of MTAD final rinse and two percent chlorhexidine gel medication in teeth with apical periodontitis: a randomized double-blinded clinical trial. J Endod 2009;35:1483–90. 7. Manzur A, Gonzalez AM, Pozos A, et al. Bacterial quantification in teeth with apical periodontitis related to instrumentation and different intracanal medications: a randomized clinical trial. J Endod 2007;33:114–8. 8. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288–302. 9. Waltimo TM, Orstavik D, Siren EK, et al. In vitro susceptibility of Candida albicans to four disinfectants and their combinations. Int Endod J 1999;32:421–9. 10. Siqueira JF Jr, Batista MM, Fraga RC, et al. Antibacterial effects of endodontic irrigants on black-pigmented gram-negative anaerobes and facultative bacteria. J Endod 1998;24:414–6. 11. Parsons GJ, Patterson SS, Miller CH, et al. Uptake and release of chlorhexidine by bovine pulp and dentin specimens and their subsequent acquisition of antibacterial properties. Oral Surg Oral Med Oral Pathol 1980;49:455–9. 12. Komorowski R, Grad H, Wu XY, et al. Antimicrobial substantivity of chlorhexidinetreated bovine root dentin. J Endod 2000;26:315–7. 13. Rosenthal S, Spangberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488–92. 14. Breschi L, Mazzoni A, Nato F, et al. Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dent Mater 2010;26:320–5. 15. Wang CS, Arnold RR, Trope M, et al. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283–9. 16. Siqueira JF Jr, Paiva SS, Rocas IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7. 17. Shipper G, Orstavik D, Teixeira FB, et al. An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon). J Endod 2004;30:342–7. 18. Kqiku L, Stadtler P, Gruber HJ, et al. Active versus passive microleakage of Resilon/ Epiphany and gutta-percha/AH Plus. Aust Endod J 2011;37:141–6. 19. Fransen JN, He J, Glickman GN, et al. Comparative assessment of ActiV GP/glass ionomer sealer, Resilon/Epiphany, and gutta-percha/AH plus obturation: a bacterial leakage study. J Endod 2008;34:725–7. 20. Santos J, Tjaderhane L, Ferraz C, et al. Long-term sealing ability of resin-based root canal fillings. Int Endod J 2010;43:455–60. 21. Roth KA, Friedman S, Levesque CM, et al. Microbial biofilm proliferation within sealer-root dentin interfaces is affected by sealer type and aging period. J Endod 2012;38:1253–6. 22. Mah T, Basrani B, Santos JM, et al. Periapical inflammation affecting coronallyinoculated dog teeth with root fillings augmented by white MTA orifice plugs. J Endod 2003;29:442–6.
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Basic Research—Biology 23. Shipper G, Teixeira FB, Arnold RR, et al. Periapical inflammation after coronal microbial inoculation of dog roots filled with gutta-percha or resilon. J Endod 2005; 31:91–6. 24. Leonardo MR, Barnett F, Debelian GJ, et al. Root canal adhesive filling in dogs’ teeth with or without coronal restoration: a histopathological evaluation. J Endod 2007; 33:1299–303. 25. Duggan D, Arnold RR, Teixeira FB, et al. Periapical inflammation and bacterial penetration after coronal inoculation of dog roots filled with RealSeal 1 or Thermafil. J Endod 2009;35:852–7. 26. Friedman S, Komorowski R, Maillet W, et al. In vivo resistance of coronally induced bacterial ingress by an experimental glass ionomer cement root canal sealer. J Endod 2000;26:1–5. 27. Tay FR, Loushine RJ, Weller RN, et al. Ultrastructural evaluation of the apical seal in roots filled with a polycaprolactone-based root canal filling material. J Endod 2005; 31:514–9. 28. Hiraishi N, Yau JY, Loushine RJ, et al. Susceptibility of a polycaprolactone-based root canal-filling material to degradation. III. Turbidimetric evaluation of enzymatic hydrolysis. J Endod 2007;33:952–6.
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29. Ferreira FB, Campos Rabang HR, Pinheiro ET, et al. Root canal microbiota of dogs’ teeth with periapical lesions induced by two different methods. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:564–70. 30. Sundqvist G. Bacteriological studies of necrotic dental pulps. Umea, Sweden: University of Umea; 1976. 31. Hashimoto MA. review—micromorphological evidence of degradation in resindentin bonds and potential preventional solutions. J Biomed Mater Res B Appl Biomater 2010;92:268–80. 32. Marin-Bauza GA, Rached-Junior FJ, Souza-Gabriel AE, et al. Physicochemical properties of methacrylate resin-based root canal sealers. J Endod 2010;36:1531–6. 33. Babich H, Wurzburger BJ, Rubin YL, et al. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11: 79–88. 34. Basrani BR, Manek S, Sodhi RN, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 35. Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44: 583–609.
Periapical Inflammation after Coronal Inoculation
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Periapical Inflammation Subsequent to Coronal Inoculation of Dog Teeth Root Filled with Resilon/Epiphany in 1 or 2 Treatment Sessions with Chlorhexidine Medication Jo~ ao M. Santos, DDS, PhD,* Paulo J. Palma, DDS,* Jo~ ao C. Ramos, DDS, PhD,* Ant onio S. Cabrita, MD, PhD,† and Shimon Friedman, DMD‡ Abstract Introduction: Therapeutic methods that inhibit microbial ingress into filled root canals are desirable. This in vivo study assessed the inhibition of periapical inflammation subsequent to coronal inoculation in canals medicated with 2% chlorhexidine gel and filled with Resilon/Epiphany (Pentron Clinical Technologies, Wallingford, CT). Methods: Six Beagle dogs each had 10 two-rooted premolars treated. In group 1 (n = 36 roots), 1 root/tooth had the canal conditioned with Primer Epiphany, filled with Epiphany sealer and Resilon core in 1 session, and coronally sealed with PhotacFil. In group 2 (n = 36 roots), the second root/tooth had the canal medicated with 2% chlorhexidine gel for 1 week and then filled and coronally sealed as in group 1. After 3 weeks, canals were exposed to the oral environment for 7 days, inoculated with isologous plaque, and coronally sealed. Negative controls treated as groups 1 and 2 remained sealed. Positive controls had canals unfilled and exposed. Seven months after inoculation, dogs were euthanized; jaw blocks processed for histologic examination; and periapical inflammation (PI) recorded as none, mild, or severe. Results: In groups 1 and 2, severe PI occurred in 5 of 65 roots (8%) and mild PI in 18 of 65 roots (28%) with a significantly higher (P = .031) PI incidence in group 2 than in group 1. Negative controls had only mild PI in 9 of 29 roots (31%). Roots medicated with 2% chlorhexidine gel had mild PI significantly more (P = .009) than roots filled in 1 session (more than 2-fold). Conclusions: Intracanal medication with 2% chlorhexidine gel and root filling with Resilon/Epiphany did not effectively inhibit apical periodontitis subsequent to coronal inoculation. (J Endod 2014;-:1–5)
Key Words 1-session treatment, chlorhexidine, coronal leakage, Resilon, root canal filling
A
pical periodontitis comprises the host’s response to pathogenic microorganisms colonizing the root canal system of the tooth (1). To maintain or regenerate the host tissues, the root canals are disinfected and filled, and the access cavity is restored to prevent microbial ingress; however, microorganisms can invade the filled canals over time via the coronal cavity, lateral canals, and dentinal tubules (2). They can propagate and elicit post-treatment apical periodontitis, undermining the prognosis (3). To improve the long-term prognosis, therapeutic methods need to be explored that can effectively prevent microbial ingress, including medications with sustained antimicrobial activity and root-filling materials offering improved sealing properties. Chlorhexidine (CHX) can be applied as an intracanal medication using different vehicles (4–7). The CHX molecule acts to increase the permeability of the microbial cell wall, penetrates the cell’s cytoplasm, and affects coagulation of the cytoplasmic content and cell death (8). CHX is effective against a wide range of gram-positive and gram-negative microorganisms as well as yeasts, facultative anaerobes, and aerobes (9, 10). Moreover, when used in root canals at a sufficient concentration and exposure time, CHX adsorbs onto root dentin and imparts substantive antimicrobial activity (4, 11–13), which could inhibit coronal microbial ingress (4). Furthermore, by inhibiting endogenous metalloproteinases, the substantive activity of CHX can moderate adhesive failures of dentin-bonding root-filling materials and maintain stability of the hybrid layer (14). All of the previously mentioned attributes notwithstanding, clinical data have not consistently supported the antimicrobial benefits of CHX applied as an intracanal medication (5–7, 15, 16). Additional experimental in vivo data were deemed necessary to explore the possible benefits of intracanal medication with CHX. Resilon (Pentron Clinical Technologies, Wallingford, CT) is a synthetic polymerbased root-filling material designed for application with a methacrylate-resin sealer (Epiphany, Pentron Clinical Technologies). Although bonded interfaces are expected between the Resilon core, sealer, and root canal wall (17), the resulting seal of Resilon/Epiphany has been questioned; when assessed in vitro, it has been superior (18), similar (19), and inferior (20) to that of conventional root fillings. Moreover, the bonded interface is susceptible to biofilm proliferation (21). Friedman et al (3), in an animal model, challenged filled root canals with an isologous inoculum within the pulp chamber to assess their resistance to microbial ingress in vivo. After periods of 4–10 months, 0%–20% of roots developed severe periapical inflammation attributed to infection, and 21%–33% developed mild inflammation in response to the treatment procedures (3, 12, 22). When tested in a similar model, roots filled with Resilon/Epiphany developed varying degrees of periapical inflammation (23–25), precluding definitive conclusions regarding the ability of this system to prevent microbial ingress. Additional experimental in vivo data were
From the *Department of Dentistry and †Institute of Experimental Pathology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; and ‡Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Jo~ao Miguel Marques dos Santos, Av Bissaya Barreto, Blocos de celas, 3000-075 Coimbra, Portugal. 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.2013.10.023
JOE — Volume -, Number -, - 2014
Periapical Inflammation after Coronal Inoculation
1
Basic Research—Biology TABLE 1. Summary of the Experimental Protocol in the 2-rooted Mandibular and Maxillary Premolars of Dogs Assigned to the Test and Control Groups Groups
Roots treated (n)
Test Group 1
36
Group 2
36
Negative control Group 1A Group 2A Positive control
18 18 12
Treatment protocol
Inoculation
1-session treatment Resilon/Epiphany 2-session treatment with CHX Resilon/Epiphany
Yes
1-session treatment Resilon/Epiphany 2-session treatment with CHX Resilon/Epiphany Canal prepared without root filling
No
Yes
CHX, chlorhexidine.
deemed necessary to explore the possible benefits of root filling with the Resilon/Epiphany system. This in vivo study assessed the efficacy of intracanal medication with 2% CHX and root filling with Resilon/Epiphany in inhibiting periapical inflammation subsequent to coronal inoculation to explore the potential benefits of both therapeutic methods. Our null hypothesis was that roots medicated with 2% CHX and those filled in 1 session would have a similar incidence of periapical inflammation.
Material and Methods Animal and Tooth Preparation The study protocol was approved by the Animal Welfare Committee of the Direcc¸~ao-Geral de Veterinaria and complied with the International Guiding Principles for Biomedical Research Involving Animals (Geneva, 1985). For the first 2 interventions, dogs were anesthetized with 10 mg/kg thiopental (B. Braun Medical, Melsungen, Germany) and the inhalation of O2 and 2% isoflurane (Abbott Laboratories, Abbott Park, IL). For subsequent interventions, dogs were sedated with intramuscular administration of 7 mg/kg tiletamine/zolazepam (Virbac, Carros, France). Based on previous studies (3), six 2-year-old female Beagle dogs had treatment performed in 10 two-rooted premolars each for a total of 120 roots. Before treatment, teeth were radiographed, cleaned with pumice (Produits Dentaires, Vevey, Switzerland), isolated with a rubber dam (Hygienic, Cuyahoga Falls, OH), and thoroughly wiped with 10% povidone-iodine solution (Labesfal, Besteiros, Portugal) to ensure aseptic conditions. The access cavity was prepared with a sterile tapered high-speed bur (Dentsply Maillefer, Ballaigues, Switzerland) under saline irrigation. Canals were prepared with Hero642 instruments (Micro-Mega, Besancon, France) in a crown-down sequence and apically enlarged with K-type hand files to size 40–60 at the working length based on the canal’s size. They were intermittently irrigated with 10 mL 2.5% NaOCl, rinsed for 30 seconds with 3 mL 17% EDTA (Produits
Dentaires), flushed with 3 mL sterile saline, and dried with paper points (Dentsply Maillefer).
Groups Roots with prepared canals were assigned to test and control groups (Table 1). Test groups comprised the 6 two-rooted mandibular premolars. In group 1 (n = 36 roots), in 1 root/tooth, the canal was conditioned (Primer Epiphany) and filled (Root Canal Sealant Epiphany and Resilon) using warm compaction (SystemB; Sybron Endo, Orange, CA). The filling was photopolymerized for 20 seconds (Optilux 501; Demetron/Kerr Co, Karlsruhe, Germany), and the access was sealed (PhotacFil; 3M-ESPE, Seefeld, Germany). In group 2 (n = 36 roots), in the second root/tooth, 2% CHX gel was placed with a Lentulo spiral (Dentsply Maillefer), and the access was sealed with PhotacFil. After 1 week, the medicated roots were reaccessed, CHX gel was flushed out with 3 mL sterile saline, and canals were filled and resealed as in group 1. In the contralateral premolars, grouping of the canals in each tooth was reversed to equalize the distribution of mesial and distal roots in each group. After 3 weeks, the pulp chambers of teeth in groups 1 and 2 were reaccessed, left exposed to the oral environment for 1 week, inoculated with isologous plaque, and resealed with PhotacFil. Three maxillary premolars/dog were allocated to negative control groups 1A and 2A (n = 18 roots/group). They were treated as in groups 1 and 2, respectively, but their pulp chambers were left undisturbed. One maxillary premolar/dog served as a positive control (n = 12 roots) by leaving the prepared canals unfilled and exposed to the oral environment. Outcome Measures Periapical radiographs were exposed before treatment, immediately after treatment, and 4 and 7 months after treatment. At 7 months, animals were anesthetized with thiopental and euthanized by anesthetic overdose followed by bilateral perfusion with 10% phosphate-buffered formalin. Jaw and maxillary blocks were separated, dissected from the
Figure 1. Photomicrographs of mandibular premolar roots and periradicular tissues of dogs 7 months after experimental procedures showing the histologic classification of PI used in the study. (A) No PI: normal appearance of the root surface and surrounding tissues; (B) mild PI: localized inflammatory cell infiltrate without evident bone and root resorption; and (C) severe PI: diffuse infiltrate with evident bone or root resorption (hematoxylin-eosin; original magnification: 20).
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Basic Research—Biology
Figure 2. Photomicrographs of a mandibular premolar root and periradicular tissues of a dog 7 months after root filling and coronal inoculation. (A) An overview of root, periodontal ligament, extruded sealer, and bone. (B and C) Magnified views of the extruded sealer. Epiphany sealer expressed periapically is surrounded by a fibrous capsule, mononuclear inflammatory infiltrate, and foreign body giant cells (hematoxylin-eosin; original magnification: [A] 20, [B] 40, and [C] 80).
first premolar to the first molar, postfixed in phosphate-buffered formalin, and decalcified with Morse solution. Each block was divided between the roots of the third premolar and trimmed, embedded in paraffin, and serially sectioned every 6 mm along the mesiodistal plane, parallel to the long axis of canals. One in every 10 sections was stained with hematoxylin-eosin, and periradicular tissues were examined histologically under a light microscope (Nikon Eclipse E600; Nikon, Tokyo, Japan). One observer examined all specimens blinded to group allocation. Periradicular inflammation (PI) was rated in accordance with previous studies (3, 22, 26) as follows: 1. None: Normal appearance of root surface and surrounding tissues 2. Mild: Localized inflammatory infiltrate without bone and root resorption, and 3. Severe: Diffuse infiltrate with evident bone or root resorption (Fig. 1)
Analysis SPSS 14.0 (SPSS Inc, Chicago, IL) was used to compare the incidence of PI in groups 1 and 2 with the chi-square test. Significance was established at the 5% level.
Results Clinically, there was no evidence of swelling or a sinus tract associated with any of the treated teeth. During the follow-up, 1 mandibular tooth lost the coronal restoration without necessitating exclusion from analysis. One root from group 1A (negative control) was lost during histologic processing. Thirteen roots were excluded from analysis because of extruded Epiphany sealer (small amounts detected histologically in 12 roots and larger overfilling in 1 root). The excess Epiphany material was surrounded with fibrous capsules associated with mononuclear inflammatory infiltrate with evidence of phagocytosis (Fig. 2A–C). In the positive control group, all 12 roots had radiographic evidence of periapical pathosis and histologically confirmed severe PI (Table 2). In the negative control groups 1A and 2A, none of the roots had severe PI, and 9 of 29 roots (31%) had mild PI, without statistically significant differences between groups 2A and 1A (P = .184). In the test groups, 5 of 65 roots (8%) had severe PI, and 18 of 65 roots (28%) had mild PI. The incidence of PI was significantly higher (Pearson chi-square test, P = .031) in group 2 than in group 1; thus, the null hypothesis was rejected. Mild PI was observed in 19 of 46 roots (41%) treated in 2 sessions (groups 2 and 2A) compared with 8 of 48 roots (17%) treated in 1 session (groups 2 and 2A). The difference was statistically significant (chi-square test, P = .009).
Discussion An in vivo model that challenges root-filled canals with coronally inoculated microorganisms, simulating a clinical condition that can lead to post-treatment apical periodontitis (3, 22, 26), was used to JOE — Volume -, Number -, - 2014
assess the inhibition of microbial ingress by root canal medication with 2% CHX and root filling with the Resilon/Epiphany system. Canals were inoculated after 3 weeks to allow sufficient time for the Epiphany sealer to set. They were exposed to saliva for 1 week before inoculation to exploit possible gaps along the sealer-dentin interface (27) and to expose the root filling to possible enzymatic hydrolysis (28). The inoculated access cavities were sealed to support the selection of anaerobic microflora (29) as found in teeth with posttreatment apical periodontitis (30). A period of 7 months was allowed before outcome assessment, which was longer than in several studies based on the same model (3, 23–26) but shorter than in another such study (22). A short period might not be sufficient to infect the filled canals, whereas a long period could increase the risk of coronal breakdown of the teeth with undesirable exposure of the inoculated pulp chambers (22, 26). The 7-month observation interval used herein appeared to strike a good balance in which severe PI developed in some of the roots and coronal breakdown occurred in only 1 tooth. Severe PI was observed in all the positive control roots and in none of the negative controls, validating that coronal inoculation was its cause (3, 22). It occurred in 8% of the roots filled with Resilon/Epiphany, which was in agreement with previous reports (24, 26). The 8% incidence of severe PI after 7 months of inoculation was in line with the 11% increase in severe PI observed after a 3-month period of coronal exposure of roots filled with Resilon/Epiphany compared with roots that were not exposed (24). It also was similar to the 8% severe PI reported after a 6-month period of inoculation in roots filled with gutta-percha and a glass-ionomer cement-based sealer but lower than the 21% in roots in which a zinc oxide–eugenol sealer was used (26). In this regard, our results supported the suitability of the study model for in vivo evaluation of the resistance of root fillings to microbial ingress in contrast to the absence of severe PI previously reported for Resilon/Epiphany after 14 weeks of inoculation (23) and for guttapercha and a zinc oxide–eugenol–based sealer after 4- and 10-month inoculation periods (3, 22). TABLE 2. Scores of Periapical Inflammation Assessed Histologically 7 Months after Experimental Treatment Procedures in 2-rooted Premolars of Dogs
Groups Positive control Negative control Group 1A Group 2A Test Group 1 Group 2
Level of PI Roots assessed (n) None (%) Mild Severe 12
0
0
12 (100)
15 14
12 (80) 8 (57)
3 (20) 6 (43)
0 0
33 32
25 (76) 17 (53)
5 (15) 13 (41)
3 (9) 2 (6)
PI, periapical inflammation. One root was lost during processing, and 13 were excluded from analysis because of sealer extrusion.
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Basic Research—Biology The very development of severe PI in 6% of roots medicated with CHX and in 8% of roots filled with Resilon/Epiphany suggested that both therapeutic methods only partially inhibited coronal microbial ingress and the development of post-treatment apical periodontitis. Although the antimicrobial mechanism and activity of CHX have been well established (8–10), its use as an intracanal medication, its substantive antimicrobial activity, and its potential stabilization of sealer-dentin interfaces have been supported only by in vitro research data (4, 11, 13, 14, 20, 27). Clinical assessments of the antimicrobial efficacy of CHX in teeth with infected root canals have failed to convincingly show benefits (5–7, 15, 16). The results of this study suggested the incapacity of medication with CHX to effectively inhibit microbial ingress and, thus, undermined the rationale for its clinical application. The system comprised of methacrylate resin–based sealer (Epiphany) and polymer-based core (Resilon) has been introduced to address a perceived weak link in conventional root fillings (17). Nevertheless, when assessed in vitro, this system’s sealing ability has been disputed (18–20), and its susceptibility to biofilm proliferation within the canal wall interface was highlighted (21). The results of this study cast further doubt on the system’s capacity to effectively inhibit microbial ingress. Integrating the available research data, root filings with the Resilon/ Epiphany system appeared to be pervious to microbial ingress. This might be caused by incomplete infiltration or polymerization of the resin within the hybrid layer and dentin substrate (31) and biodegradation and dissolution that undermine the resin’s bond to root dentin over time (14, 32). Mild PI was observed in 31% of the negative control roots within the range reported for root fillings with gutta-percha and a zinc oxide– eugenol–based sealer (3, 22, 24, 26). The comparable incidence of mild PI in roots that were treated under well-controlled conditions, filled with different materials, and not inoculated suggested that the mild inflammation represented a persistent tissue response to the endodontic treatment procedure. This suggestion was supported by the comparable incidence of mild PI in the test and negative control groups. The moderate incidence of mild PI could explain why it was not observed in studies in which the negative controls included 4 roots or less (23, 25). The incidence of mild PI was over 2-fold higher in roots medicated with 2% CHX gel than in roots filled in 1 session in both the test and control groups. This finding might be attributed to the toxicity of CHX coupled with the lengthy exposure of the periapical tissues (33) or inadvertent contamination during the period between treatment sessions. The likelihood of tissue irritation from parachloroaniline precipitate resulting from the interaction of NaOCl with CHX (34) was low because in this study NaOCl was followed by EDTA and saline and the canals were dried with paper points before CHX gel was applied. Nevertheless, the persistent PI echoed the recent report of a compromised healing outcome in roots in which canals were irrigated with 0.2% CHX (35). Taken together, the previous report and our results further undermined the rationale for the clinical application of CHX in root canal therapy.
Conclusions Within the limitation of the in vivo experimental method used, it was concluded that (1) intracanal medication with 2% CHX gel did not effectively inhibit the development of apical periodontitis in roots subjected to coronal microbial challenge, (2) root filling with the Resilon/ Epiphany system did not effectively inhibit the development of apical periodontitis when subjected to coronal microbial challenge, and (3) roots treated in 2 sessions and medicated with 2% CHX gel had over 4
Santos et al.
2-fold more periapical inflammation than roots treated in 1 session 7 months after treatment. The findings of this study should be combined with those of other in vivo and clinical outcome assessments to inform clinicians about the clinical application of CHX as an intracanal medication and the use of Resilon/Epiphany as a root filling system.
Acknowledgments Supported by grants from Fundac¸~ao para a Ci^encia e Tecnologia and Pentron Clinical Technologies. The authors deny any conflicts of interest related to this study.
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Basic Research—Biology 23. Shipper G, Teixeira FB, Arnold RR, et al. Periapical inflammation after coronal microbial inoculation of dog roots filled with gutta-percha or resilon. J Endod 2005; 31:91–6. 24. Leonardo MR, Barnett F, Debelian GJ, et al. Root canal adhesive filling in dogs’ teeth with or without coronal restoration: a histopathological evaluation. J Endod 2007; 33:1299–303. 25. Duggan D, Arnold RR, Teixeira FB, et al. Periapical inflammation and bacterial penetration after coronal inoculation of dog roots filled with RealSeal 1 or Thermafil. J Endod 2009;35:852–7. 26. Friedman S, Komorowski R, Maillet W, et al. In vivo resistance of coronally induced bacterial ingress by an experimental glass ionomer cement root canal sealer. J Endod 2000;26:1–5. 27. Tay FR, Loushine RJ, Weller RN, et al. Ultrastructural evaluation of the apical seal in roots filled with a polycaprolactone-based root canal filling material. J Endod 2005; 31:514–9. 28. Hiraishi N, Yau JY, Loushine RJ, et al. Susceptibility of a polycaprolactone-based root canal-filling material to degradation. III. Turbidimetric evaluation of enzymatic hydrolysis. J Endod 2007;33:952–6.
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29. Ferreira FB, Campos Rabang HR, Pinheiro ET, et al. Root canal microbiota of dogs’ teeth with periapical lesions induced by two different methods. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:564–70. 30. Sundqvist G. Bacteriological studies of necrotic dental pulps. Umea, Sweden: University of Umea; 1976. 31. Hashimoto MA. review—micromorphological evidence of degradation in resindentin bonds and potential preventional solutions. J Biomed Mater Res B Appl Biomater 2010;92:268–80. 32. Marin-Bauza GA, Rached-Junior FJ, Souza-Gabriel AE, et al. Physicochemical properties of methacrylate resin-based root canal sealers. J Endod 2010;36:1531–6. 33. Babich H, Wurzburger BJ, Rubin YL, et al. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11: 79–88. 34. Basrani BR, Manek S, Sodhi RN, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 35. Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44: 583–609.
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