Apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing on root canal disinfection in dog teeth Nestor Cohenca, DDS,a Carlos Heilborn, DDS,a,b James D. Johnson, DDS, MS,a Daniel Silva Herzog Flores, DDS,c Izabel Yoko Ito, PhD,d and Lea Assed Bezerra da Silva, DDS, PhD,c Seattle, Washington; Asunción, Paraguay; and Ribeirão Preto, Brazil UNIVERSITY OF WASHINGTON, UNIVERSIDAD DEL PACÍFICO, AND UNIVERSITY OF SÃO PAULO Objective. The aim of this study was to compare in vivo the efficacy of 2 root canal disinfection techniques (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dog teeth with apical periodontitis. Study design. Two groups of root canals with pulp necrosis and apical periodontitis were evaluated according to the disinfection technique: group 1: apical negative pressure irrigation (EndoVac system); and group 2: apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. The first sample (S1) was collected after lesions were radiographically visible, and the second sample (S2) was collected after apical negative pressure irrigation (group 1) or conventional irrigation/triantibiotic dressing (group 2). All samples were seeded in a culture medium for anaerobic bacteria. Colony-forming unit counts were analyzed statistically by the Mann-Whitney test (␣ ⫽ .05). Results. Microorganisms were present in 100% of canals of both groups in S1. In S2, microorganisms were absent in 88.6% of group 1’s canals and 78.28% of group 2’s canals. There was no significant difference between the groups in either S1 (P ⫽ .0963) or S2 (P ⫽ .0566). There was significant (P ⬍ .05) bacterial reduction from S1 to S2 in both groups. Conclusion. In immature teeth with apical periodontitis, use of the EndoVac system can be considered to be a promising disinfection protocol, because it provided similar bacterial reduction to that of apical positive pressure irrigation (conventional irrigation) plus intracanal dressing with the triantibiotic paste, and the use of intracanal antibiotics might not be necessary. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e42-e46) Exposure of the dental pulp to microorganisms results in the development of pulpal and periradicular pathosis. The role of these microorganisms as the main etiologic factor for pulpal pathology has been well established.1,2 One of the objectives of endodontic therapy in cases of teeth with pulp necrosis and apical periodontitis is to eliminate microorganisms and their products and byproducts.1-3 In immature teeth with apical periodontitis, the biomechanical preparation can be more difficult, owing to the anatomic conditions of these teeth, mainly a Department of Endodontics, Dental School, University of Washington. b Department of Endodontics, Universidad del Pacífico. c Department of Paediatric Dentistry, Preventive and Community Dentistry, Dental School of Ribeirão Preto, University of São Paulo. d Department of Clinical Analysis, Toxicology and Bromatology, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Received for publication Jun 30, 2009; returned for revision Aug 14, 2009; accepted for publication Aug 16, 2009. 1079-2104/$ - see front matter © 2010 Published by Mosby, Inc. doi:10.1016/j.tripleo.2009.08.029 e42 the presence of thin divergent or parallel dentinal walls.4-7 This clinical situation creates a challenge for disinfection and obturation of the root canal system, possibly affecting the long-term treatment outcome. In view of this and owing to the polymicrobial nature of root canal system infection, some authors have proposed the use of a mixture of antibiotics— ciprofloxacin, metronidazole, and minocycline—to treat the diverse endodontic microflora. Hoshino et al.8 investigated in vitro the antibacterial efficacy of these drugs alone and in combination against bacteria of infected dentin, infected pulps, and apical periodontitis and found that no drug alone could kill all the bacteria, but the bactericidal efficacy of the drug combination was sufficiently potent to eradicate the bacteria from all of the samples. Sato et al.9 found that the same drug combination was very effective in killing bacteria in the deep layers of root canal dentin. However, the use of this intracanal dressing may have potential clinical and biologic side effects,4,10 including crown discoloration,7,11 development of resistant bacterial strains,12-16 and allergic reaction.17-26 Moreover, mino- OOOOE Volume 109, Number 1 cycline, one of the active components of this triantibiotic dressing, has been previously associated with angiogenesis inhibition.27,28 Recently, an alternative protocol of root canals with open apices has been introduced. Apical negative pressure irrigation (EndoVac; Discus Dental, Culver City, CA) has been claimed to provide significantly better cleanliness,29 disinfection,30 and safety as measured by apical extrusion of the irrigant31,32 compared with conventional irrigation and should be considered as a potential new approach for immature teeth with apical periodontitis. The present in vivo study was designed to compare the efficacy of 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dog teeth with apical periodontitis. MATERIALS AND METHODS Seventy-two root canals from immature second and third maxillary premolars and second, third, and fourth mandibular premolars of 4 5-month-old mongrel dogs were selected for this study. All teeth were examined radiographically to confirm incomplete root formation and open apices before start of the study. The animals were preanesthetised with an endovenous injection of Neozine (1 mg/kg body wt.; Aventis Pharma, São Paulo, Brazil) 15 minutes before the operative procedures and then anesthetised with an endovenous injection of tiletamine hydrochloride : zolazepam hydrochloride (Zoletil 50, 0.1 mL/kg body wt.; Virbac do Brasil Ind. e Com., São Paulo, Brazil) to facilitate the passage of an endotracheal tube. Inhalation anesthesia with Isoflurane (Abbott Laboratories, St. Laurent, Canada) was delivered using an inhalation anesthesia apparatus (Takaoka KT-20; Takaoka Ind. e Com., São Paulo, Brazil). Throughout the duration of the operative procedures, the animals were maintained on isotonic saline solution (0.9% NaCl; Glicolabor Indústria Farmacêutica, Ribeirão Preto, Brazil). Coronal access was done with spherical diamond burs complemented with tapered diamond burs (K. G. Sorensen, São Paulo, Brazil) under copious water cooling. After pulp removal, the root canals were left exposed to the oral cavity for 7 days to allow microbial contamination. After this period, the coronal accesses were sealed with zinc oxide– eugenol cement (SS White, Rio de Janeiro, Brazil) with no canal treatment to induce apical periodontitis, according to Leonardo et al.3 In immature teeth, the development of apical periodontitis occurs within 15 to 25 days.3 For this reason, radiographs were taken initially at 15 days and thereafter at 5-day intervals until periapical radiolucencies were observed, indicating the development of apical Cohenca et al. e43 periodontitis. Once the lesions were radiographically visible, the 72 root canals were randomly assigned to 2 groups of 36 canals each, according to the intracanal disinfection technique: group 1: apical negative pressure irrigation; and group 2: apical positive pressure irrigation (conventional irrigation) followed by intracanal dressing with a triantibiotic paste consisting of metronidazole, ciprofloxacin, and minocycline. Both groups were tested in each animal, and the experimental protocols were performed in alternate quadrants in a randomized manner. All teeth were isolated with a rubber dam, and the operative field was disinfected with 30% hydrogen peroxide until no bubbling of the peroxide occurred. All surfaces were then coated with tincture of iodine and allowed to dry. The temporary restoration was removed and the root canals were irrigated with sterile saline. Sterile cotton pellets were then used to dry the pulp chamber before the placement of 0.5 mL liquid dental transport medium (LDT) (Anaerobe Systems, Morgan Hill, CA) into each canal using a sterile needle coupled to a tuberculin syringe inserted 1 mm short of the estimated root canal length. The fluid was then agitated with a size 20 sterile stainless steel file. Any excess of LDT in the chamber was removed so that only the root canals remained filled. The LDT was then soaked from the canals with a sterile fine paper point (Mynol; Block Drug Corp., Jersey City, NJ) placed 1 mm short of the estimated root canal length and transfered to the LDT vial. This constituted the first sample (S1) for groups 1 and 2. All samples were immediately forwarded to the laboratory. The working length (WL) was established 1 mm short of the radiographic apex, and the canals were instrumented. Group 1: apical negative pressure irrigation The recommended protocol for the use of apical negative pressure irrigation includes 2 main phases: macroirrigation and microirrigation.29-31 Because our research model was aimed at testing the disinfection of immature teeth with open apices, the EndoVac protocol recommended by the manufacturer needed to be modified. Canals were irrigated using the macrocannula only after being gauged to fit the apical size of the canal. The macroirrigation was performed to the WL as the open-ended macrocannula was moved up and down in the canal from WL to a point just below the coronal orifice of the canal. During macroirrigation, 10 mL 2.5% sodium hypochlorite (NaOCl) was delivered via the master delivery tip at the access opening. The macrocannula was withdrawn from the canal in the presence of sufficient irrigant in the pulp chamber to ensure that the canal remained totally filled with irrigant and no air was drawn into the canal space. The e44 Cohenca et al. canals were left filled with NaOCl for 60 seconds and then irrigated with sterile saline and dried with sterile paper points. Each canal was then flushed with 2 mL 5% sodium thiosulfate to neutralize the NaOCl and then irrigated again with sterile saline and dried with sterile paper points. Approximately 0.5 mL LDT was delivered into the canals using a sterile needle coupled to a tuberculin syringe inserted at the WL. Any excess of LDT in the chamber was removed so that only the root canals remained filled. The fluid was then agitated with a size 20 sterile stainless steel file. Any excess of LDT in the chamber was removed so that only the root canals remained filled. The LDT was then soaked from the canals with a sterile fine absorbent paper point (Mynol Block Professional Dental Products, Jersey City, NJ) placed at the WL and transfered to the LDT vial. This constituted the second sample (S2) for group 1. All samples were immediately forwarded to the laboratory. All canals received a final irrigation with 2.5% NaOCl and were dried with sterile paper points, and the coronal accesses were restored with a double seal of glass ionomer cement (Vitrabond; 3M/Espe, St. Paul, MN) and silver amalgam (Sybraloy; Kerr Corporation, Orange, CA). Group 2: apical positive pressure irrigation plus triantibiotic intracanal dressing In this group, apical positive pressure irrigation was performed using a sterile 30-gauge side-vented port needle (Max-i-Probe; Dentsply/Tulsa Dental, York, PA) connected to a syringe. The syringe was filled with 2.5% NaOCl, and the needle was introduced into the canal at the WL. Each canal was irrigated with light pressure with 10 mL of 2.5% NaOCl. The canals were left filled with NaOCl for 60 seconds and then irrigated with sterile saline and dried with sterile paper points. Each canal was then flushed with 2 mL 5% sodium thiosulfate to neutralize the NaOCl and then irrigated again with 2 mL of sterile saline and dried with sterile paper points. A triantibiotic paste was prepared immediately before the treatment by mixing ciprofloxacin, metronidazole, and minocycline with sterile distilled water, at a concentration of 20 mg of each antibiotic, according to Reynolds et al.11 The paste was delivered into the root canals with a 20-gauge needle set at the WL and used with a backfill approach up to the level of the cementoenamel junction. The coronal access was then restored with a double seal of Cavit (3M/Espe) and glass ionomer cement (Vitrabond). The intracanal dressing was left in the canal for a period of 2 weeks. At the second treatment session, all teeth from this group were isolated with a rubber dam as already described. The coronal seal was removed with sterile high-speed burs followed by flushing of the pulp cham- OOOOE January 2010 ber with sterile saline. The triantibiotic intracanal dressing was flushed off the canals with 10 mL sterile saline, and the canals were dried with sterile paper points. Approximately 0.5 mL LDT was delivered into the canals using a sterile needle coupled to a tuberculin syringe inserted at the WL. The fluid was then agitated with a size 20 sterile stainless steel file. Any excess of LDT in the chamber was removed so that only the root canals remained filled. The LDT was then soaked from the canals with a sterile fine paper point (Mynol) placed at the WL and transfered to the LDT vial. This constituted the second sample (S2) for group 2. All canals received a final irrigation with 2.5% NaOCl and were dried with paper points, and the teeth were permanently restored in the same way as described for those in group 1. Throughout the experimental phase, the dogs of groups 1 and 2 were daily monitored for signs of pain associated with the dental procedures. Microbiologic processing The S1 and S2 samples were diluted in saline until reaching 1/10, 1/100, 1/1,000, and 1/10,000 final concentrations. Next, 50 mL of each dilution were seeded using the Westergreen technique in the following culture medium: trypticase soy agar supplemented with 0.5% yeast extract, 0.5% sheep defibrinated blood, 0.0005% hemin, and 0.00005% menadione, for detection of anaerobic bacteria. The dishes were placed in an anaerobiosis jar containing an atmosphere generator (Probac; São Paulo, Brazil) and were incubated for 10 days. After the incubation period, the number of colonyforming units (cfu) was counted with a stereomicroscope (Nikon, Tokyo, Japan). Data were analyzed statistically by the Mann-Whitney test, and a significance level of 5% was set for all analyses. RESULTS Two roots were lost in group 2, reducing the sample size in this group to 34 root canals. Microorganisms were present in 100% of the canals of both groups in S1, with cfu counts ranging from 40 to 820,000 (median 550) in group 1 and from 40 to 54,000 (median 320) in group 2. In S2, microorganisms were absent in 88.6% of the canals of group 1 (range 0 to 180 cfu) and 78.28% of the canals of group 2 (range 0 to 940 cfu). There was no statistically significant difference between the groups in either S1 (P ⫽ .0963) or S2 (P ⫽ .0566). Table I presents the cfu counts in the first sample collection (S1) and second sample collection (S2) of group 1 (EndoVac) and group 2 (Apical positive pressure irrigation ⫹ triantibiotic dressing). There was OOOOE Volume 109, Number 1 Cohenca et al. e45 Table I. Colony-forming unit counts in the first sample collection (S1) and second sample collection (S2) of group 1 and group 2 Group 1 Group 2 Sample collection Mean Q1-Q3 Min-Max Mean Q1-Q3 Min-Max P value* S1 S2 550 0 240-4,800 0-0 40-820,000 0-180 320 0 170-890 0-0 40-54,000 0-940 .0963 .0566 Group 1, Apical negative pressure irrigation (EndoVac); Group 2, apical positive pressure irrigation ⫹ triantibiotic paste; Q1, first quartile; Q3, third quartile. *Mann-Whitney test. significant (P ⬍ .05) bacterial reduction from S1 to S2 in both groups. DISCUSSION Infection control is mandatory in periapical repair, and the primary goal should be to reduce the microbial load to a low enough level where tissue healing can occur. Current disinfection protocols may include conventional irrigation with or without dressing with a triantibiotic paste7 and apical negative pressure irrigation.29-32 In the present in vivo study, apical negative pressure (EndoVac) eliminated microorganisms in 88.6% of the canals. These results are in agreement with those of Hockett et al.,30 who evaluated in vitro whether irrigation with apical negative pressure was more effective than traditional positive pressure irrigation in eradicating E. faecalis from preshaped root canals. Those authors found that apical negative pressure irrigation has the potential to achieve better microbial control than traditional irrigation delivery systems. In the present investigation, apical negative pressure irrigation presented satisfactory results in reducing the bacterial content of root canals similarly to apical positive pressure irrigation (conventional irrigation) associated with the use of a triantibiotic intracanal dressing (P ⬎ .05). After the beginning of the present investigation, Desai and Himel31 found no irrigant extrusion while using the EndoVac’s macro- and microcannulae at full working length. For immature teeth with wide open apices, safety combined with an efficient irrigation offers a valuable therapeutic alternative to conventional positive pressure irrigation. Moreover, the protocol using the triantibiotic intracanal dressing has potential biologic and clinical complications, including the development of resistant bacterial strains12-16 and allergic reaction to the intracanal dressing17-26 as the most concerning side effects. As recently stated in a review article,33 the use of antibiotics is not different from any other medications in that the benefits of using them must outweigh the risks involved. In the present study, there was a significant bacterial reduction from S1 to S2, without significant difference between the groups. In contrast to the results reported by Windley et al.,7 the association of topical antibiotic dressing to conventional irrigation did not further reduce the cfu counts in the root canals. Further research is needed to evaluate the radiographic outcome and histologic reaction of the periradicular tissues to apical negative pressure irrigation technique using the EndoVac system. In immature teeth, the large diameter of the root canal and the great apical divergence of the canal walls pose additional difficulty to the elimination of bacteria. The results of the present study demonstrated that, from a clinical standpoint, it may represent an important technical advance for the endodontic treatment of these teeth. In immature teeth with apical periodontitis, the apical negative pressure irrigation technique using the EndoVac system can be considered to be a promising disinfection protocol, because it provided similar bacterial reduction as that of the apical positive pressure irrigation (conventional irrigation) associated with intracanal dressing with a triantibiotic paste. In summary, the present results demonstrated that reliable disinfection can be achievable with efficient and safer irrigation delivery systems, such as the EndoVac system, and that the use of intracanal antibiotics might not be necessary. 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 Oral Radiol Endod 1965;20:340-9. 2. Moller AJ, Fabricius L, Dahlen G, Ohman AE, Heyden G. Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. 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J Endod 2008;34: 1374-7. 31. Desai P, Himel V. Comparative safety of various intracanal irrigation systems. J Endod 2009;35:545-9. 32. Fukumoto Y, Kikuchi I, Yoshioka T, Kobayashi C, Suda H. An ex vivo evaluation of a new root canal irrigation technique with intracanal aspiration. Int Endod J 2006;39:93-9. 33. Mohammadi A. Antibiotics as intracanal medicaments: a review. CDA 2009;37:99-108. Reprint requests: Profa. Dra. Léa Assed Bezerra da Silva Departamento de Clínica Infantil, Odontologia Preventiva e Social Faculdade de Odontologia de Ribeirão Preto Universidade de São Paulo Av. do Café, s/n Monte Alegre, 14040-904 Ribeirão Preto—SP Brazil lea@forp.usp.br