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CLINICAL DENTISTRY AND RESEARCH 2012; 36(2): 15-21 BACTERIAL REDUCTION IN INFECTED ROOT CANALS TREATED WITH CALCIUM HYDROXIDE USING HAND AND ROTARY INSTRUMENT: AN IN-VIVO STUDY M. Ömer Görduysus, DDS, PhD Professor, Department of Endodontics, Faculty of Dentistry, Hacettepe University, ABSTRACT Background and Aim: The aim of this in vivo study was Ankara, Turkey to evaluate the microbial flora in infected root canals after Zeliha Yılmaz, DDS, PhD the treatment with and without calcium hydroxide (Ca(OH)2) Associate Professor, Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey dressing using hand and rotary instruments. Subjects and Methods: Twenty-eight patients were selected. After access cavity preparation completed, the first culture Melahat Görduysus, DDS, PhD were taken from each root canal with sterile paper points. Professor, Department of Endodontics, Four groups were formed. Group I and II were instrumented Faculty of Dentistry, Hacettepe University, Ankara, Turkey Canan Kaya, DDS Department of Endodontics, Faculty of Dentistry, Hacettepe University, Ankara, Turkey Dolunay Gülmez, MD Assistant Professor, Department of Medical Microbiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey with ProTaper instruments, group III and IV were instrumented step-back technique with K files. Group I and III were medicated with Ca (OH)2 paste. One week after, the second culture was taken and the canals were filled. All samples were submitted to microbiological processing to evaluate anaerobe and aerobe microorganism. The results statistically analyzed with McNemar and Chi-Square tests at p<0.05. Results: There was a significant decrease in bacteria elimination in Group III compared to the other groups (p<0.05). Lindihana Emini, M.Sc Groups, in which Ca(OH)2 was applied, were found significantly Specialist of Dental Pathology and better to eliminate bacteria in root canals than the groups Endodontics Dental Clinic, Medident, Tetovo, Fyrom Veton Hoxha, DDS, PhD Professor, Department of Dental Pathology and Endodontics University Dental Clinic Center, without Ca(OH)2. Conclusion: Better root canal disinfection could be obtained by mechanical preparation with application of Ca(OH)2 for one week. Prishtina, Kosova Correspondence Zeliha Yılmaz DDS,PhD Department of Endodontics, Faculty of Dentistry, Hacettepe University, 06100 Sıhhiye Ankara/Turkey Phone: +90 312 3052260 Key words: Calcium Hydroxide, Microbiology, Root Canal Treatment, Rotary Instruments Fax: +90 312 3052832 Submitted for Publication: 10.05.2011 E-mail: [email protected], [email protected] Accepted for Publication : 06.21.2012 15 CLINICAL DENTISTRY AND RESEARCH INTRODUCTION It is well known that remaining bacteria, either in the root canal space or in parts of root canal system that are not filled, is the main cause for root canal treatment failure.1,2 Thus, removal of vital and necrotic pulp tissue, microorganisms and their toxins is an important part of endodontic treatment.3 Unfortunately, studies have shown that both stainless steel file systems which have been used for years and NiTi rotary instruments that have gained popularity recently have lead to inadequate disinfection of root canal space because of the complexity of root canal morphology, apical ramifications, isthmuses, other morphological challenges and bio-film form of root canal bacteria.4 As a result of technological developments, Ni-Ti rotary instruments have gained increased taper and different designs that can significantly improve the cleaning and shaping procedure. Many studies have been conducted to compare Ni-Ti rotary instruments with stainless steel instruments.5,6 However, there have been few in-vivo studies in which NiTi rotary instruments provided better results in reducing or eliminating intra-canal bacteria, compared with stainless steel instruments. During endodontic treatment, both mechanical preparation and intra-canal dressing are used to reduce or eliminate bacteria. Some studies have shown that mechanical preparation significantly decreases bacteria in the root canal. Yet, in approximately 20%-50% of the canals, remaining bacteria was observed.7,8 For this reason, in addition to mechanical preparation, different concentrations and types of irrigation solutions and various medicaments are used to remove microorganisms and to achieve disinfection.4 It has been reported that the use of sodium hypochlorite (NaOCl) in different concentrations during mechanical preparation decreases the number of bacteria. Despite the use of this solution, however, positive culture was obtained in 4060% of the root canals.1,9 In addition to irrigation solutions, intra-canal medicaments are used to remove bacteria and to disinfect the root canal systems. Calcium hydroxide (Ca(OH)2) has been the most frequently used intra-canal medicament due to its antimicrobial effect, osteoclastic activity and suitable tissue response.10-12 Whether or not there is an apical lesion, in cases with necrotic pulp, multiple-visit treatment and the use of Ca(OH)2 as medicament is recommended for a week to achieve the desired disinfection.10,13 This in-vivo study takes stock of results obtained in previous research highlighted above and aims to assess the amount 16 of microbial flora in infected root canals by using Ni-Ti rotary instruments and stainless steel instruments, once with and once without the use of Ca(OH)2. SUBJECTS AND METHODS Case selection Patient population consists of 28 people (18 female, 10 male) who frequented Hacettepe University, Faculty of Dentistry, Department of Endodontics. In total, 28 teeth (9 maxillary incisors, 2 maxillary premolars, 6 maxillary molars, 2 mandibular incisors, 1 mandibular premolar and 8 mandibular molar) have been treated. Patient selection criteria were as follows: 1- The teeth with intact pulp chamber walls, 2- Necrotic pulps, negative response to the pulp tests (electrical and cold), 3- Clinical and radiographic evidence of chronic apical periodontitis lesions, 4- Teeth from patients who have not received antibiotic therapy within the preceding month, 5- Patients without swelling or any symptoms or periodontal problems. Before initiation, the study was approved by the Ethics Committee of the Hacettepe University and informed consent was obtained from each patient. Endodontic Treatment and Sampling Procedures After the clinical and radiographic examination of the patients, each tooth was cleaned of plaque, calculus and attachments. All decay and unsupported tooth structure were removed and canal orifices were localized after access cavity preparation. Following the rubber-dam isolation, all tooth surfaces were cleaned with a cotton pellet immersed in 2.6% NaOCl. A #15 K file was inserted into canals for apical patency; then, the first root canal sample was taken. The sterile paper point was placed in each root canal to a level approximately 1 mm short of the apex, based on diagnostic radiographs, and was used to soak up the fluid in the canal. Each paper point was kept in the canal for 3 minutes. Paper points were then placed into aseptically tubes containing 1 mL tiyoglukonated buyyon. Four groups were formed, each with 7 teeth. After estimating the working length, root canals in Groups I and II were prepared with ProTaper Ni-Ti rotary instruments (DentsplyMaillefer, Ballaigues, Switzerland) to a master apical size of #30 using an endodontic micromotor (X-Smart, Dentsply, Infected Root Canals Treated with Calcium Hydroxide Maillefer). RC-Prep (RC-Prep; Premier Dental, Norristown, PA) chelating agent was used for lubrication. In Groups III and IV, root canals were prepared with the step-back technique using stainless steel files, with the master apical file being determined according to each tooth’s anatomy. The canals were irrigated with 0.5 ml 2.5% NaOCl after each file. In all cases, chemo-mechanical preparation was completed at the first appointment. After the instrumentation, 5 ml 2.5% NaOCl was applied as final flush; then each canal was dried up by using sterile paper point (Spident, Incheon, Korea). In Groups I and III, root canals were filled with Ca(OH)2 (SUREPaste, Korea). Ca(OH)2 paste was placed by using a lentulo spiral and packed with a cotton pellet at the level of canal orifice. A radiograph was taken to ensure proper placement of the paste in the canal. Then the access cavities were filled with at least 4 mm of a temporary filling material (Nucavfil; PSP Dental, Belvedere, Kent, UK). The second appointment was scheduled for the following week. At this time, the tooth was isolated with rubber-dam and the operative field was disinfected. In Groups I and III where Ca(OH)2 was applied, Ca(OH)2 paste was rinsed out of the canal by using 2 ml sterile saline solution and master apical files. In Groups II and IV, after temporary filling material was removed, each canal was irrigated with only 2 ml sterile saline solution. The second culture was taken from each canal as outlined above. Subsequently, the canals were filled with gutta-percha (Diadent, Seoul, Korea) and AH 26 root canal sealer (Dentsply Detrey, Konstanz, Germany) using cold lateral compaction technique. The tooth was temporized with glass ionomer cement and a permanent restoration was planned. Microbiologic Analysis Root canal samples were taken with sterile paper points which were left in the canal for at 3 minutes and placed in 1 ml of thioglycolate broth. Each sample was vortexed and 10μl of the sample was transferred onto each of the following media: Sheep blood agar, chocolate agar, MacConkey agar and Schaedler agar. All media were incubated overnight in 5-10% CO2 at 37 ºC (-/+ 2), except Schaedler agar which was incubated in anaerobic atmosphere for 48 hours. Colony counts were determined for each group of bacteria with different colony morphologies and subcultured for identification. Aerobic bacteria were identified to genus level by Gram stain morphology, catalase and coagulase tests, growth on bile-esculin agar, growth in 6.5% NaCl and CAMP reaction. Anaerobic bacteria were identified to species level by catalase test, indole reaction and Crystal System (Becton Dickinson, USA). Statistical Analysis A non-parametric test (McNemar) was used for determining the differences between first and second measurement in the intergroup and the Chi-Square test was used for evaluating the differences between the groups. A p value <0.05 considered as statistically significant. RESULTS Table 1 shows the total distribution of bacterial types at the beginning of access cavity preparation in the first visit and at the end of preparation in the second visit. While the total number Colony Forming Unit (CFU) of the first samples was 551170 CFU/ml, after instrumentation, irrigation and applying Ca(OH)2 the CFU count had dropped significantly to total 391300 CFU/ml. In culture results, 44% of the microbial flora of root canals was found to be aerobe while 46% of the flora was anaerobe. Most observed aerobe bacteria species was Alpha hemolitic streptococcus with a ratio of 28.8%. Most observed anaerobe bacteria species was Veillonella spp with a ratio of 22.8%. In all experiment groups, when the final measurements of difference in the decrease of both aerobe and anaerobe bacteria were evaluated (Table 2). Statistically significant difference was only seen in Group III in which Ca(OH)2 was applied after cleaning and shaping procedures with hand instrument (p<0.05). There is a significant decrease in bacteria elimination in Group III compared to other groups. When initial and final changes in cultures were evaluated, Group IV registered the highest level of bacteria compared to other groups in which cleaning and shaping procedures were carried out with hand instrument without using Ca(OH)2 (p=0.0036). Groups in which Ca(OH)2 was applied proved to be significantly better in eliminating bacteria in root canals compared to groups in which Ca(OH)2 was not applied (Figure 1). Cleaning and shaping procedures with hand instrument were significantly better than rotary instrument groups in eliminating bacteria from root canals (p=0.03) (Figure 2). DISCUSSION An effective endodontic treatment requires the eradication of bacteria or at least the reduction in bacterial counts in the root canal space.14 Chemo-mechanical preparation of root canal using different types and concentrations of irrigation 17 CLINICAL DENTISTRY AND RESEARCH Table 1. The total distribution of bacterial types in the first visit and second visit Aerobe Bacteria Patient Prevalance n CFU % Alfa Hem. Strep. 34,5 253500 28,81664 Coagulase-Stap 29,14 420800 47,83449 Neisseria 14,54 10400 1,182221 S. Viridans 5,46 17100 1,943844 Difteroid 5,46 24100 2,73957 Other 10,9 153800 17,48323 Total 100% 879700 100% Anaerobe Bacteria Patient Prevalance n CFU % Veillonella ssp 34,70 16870 22,86837 Actinomyces 8,69 6000 8,133388 Fusobacterium 13,17 8200 11,11563 Eurobacterium lim 4,34 10000 13,55565 Peptostreptococus 8,69 11500 15,58899 Bacteriodes cap 4,34 5000 6,777823 Prevotella 4,34 5300 7,184492 Other 21,73 10900 14,77565 Total 100% 73770 100% CFU: Colony Forming Unit Table 2. Result of one week treatment according to groups Groups Result of one week treatment Rotary with Ca(OH)2 Rotary w/o Ca(OH)2 Manual with Ca(OH)2 Manual w/o Ca(OH)2 n % n % n % n % Elimination 7 53,8 0 0 14 100 2 12,5 Repeated-growth 2 15,4 5 41,7 0 0 2 12,5 Stagnation 0 0 2 16,7 0 0 0 0 Reduction 2 15,4 2 16,7 0 0 2 12,5 New 2 15,4 4 25 0 0 10 62,5 Total 13 100 13 100 14 100 16 100 n: The number of species of microorganism solution has been shown to be critical step for reduction of bacterial populations in the root canal but about 40% to 60% of the canals still yield positive cultures after instrumentation and irrigation.9,15 Bacteria in the infected root canal are mostly anaerobic bacteria.16 Our study shows that 44% of the microbial flora of root canals was aerobe 18 while 46% was anaerobe. According to the results reported by Sundquist et al.17, Byström & Sundqvist18, Baumgartner & Falker,19 Siqueira & Lopes,20 and Sassone et al.21, the most microbial flora in infected root canals is anaerobic bacteria. These authors concluded that the infection in infected root canal is mainly polymicrobial, with anaerobic flora being less Infected Root Canals Treated with Calcium Hydroxide 90 77,8 80 70 60 46,4 50 % 40 25 30 20 10 7,4 7,1 7,1 0 0 Elimination Repeated stagnation Repeated growth With Ca(OH)2 4,3 7,4 7,4 Repeated reduction New Without Ca(OH)2 Figure 1. The effect of Ca(OH)2 on bacterial flora 100 80 % 60 Group 1 Group 2 Group 3 Group 4 40 20 0 Elimination Repeated growth Repeated stagnation Repeated reduction New Figure 2. Results of one week treatment according to groups frequent. The results reached in our study conflict with those reported by the mentioned authors. This may be explained by the method we applied for the identification of microbes in root canal. Although cleaning, shaping and irrigating the canal greatly reduce the number of bacteria,16 it is generally believed that the number of bacteria can also be controlled by placing an inter-appointment dressing such as Ca(OH)2 within the prepared root canal.9 Estrela et al.22 analyzed the antimicrobial properties of Ca(OH)2 and concluded that the latter affects the cell membrane of both aerobe and anaerobe bacteria. Sjögren et al.10 clinically evaluated the antibacterial effect of Ca(OH)2 as a short-term intra-canal dressing by applying the medicament for 7 days in root canals of teeth with periapical lesions. These authors showed that the 7 day dressing effectively eliminated bacteria which survived biomechanical instrumentation of the canal. The present study also shows that groups in which Ca(OH)2 was applied were significantly better in eliminating bacteria in root canals compared with groups in which Ca(OH)2 was not applied. After the final measurement, a statistically significant difference was observed only in Group III (treated with Ca(OH)2 and hand instruments). Our results parallel those of Sakamoto et al.23 showing the success of hand instrumentation and treatment with Ca(OH)2 in 97% of cases. De Rossi et al.24 investigated the effect of Ca(OH)2 in infected root canals using hand and rotary instruments and showed that, regardless of the instrumentation technique, the use of intra-canal dressing is important in the endodontic treatment for the elimination of bacteria in the root canal. The present study demonstrates that instrumentation, irrigation and applying Ca(OH)2 significantly reduces the total number of microorganisms, namely approximately to 16 % of the original number. Peters et al.16 found that the total number of microorganisms was reduced by 0.18 % after instrumentation, irrigation and dressing with Ca(OH)2 on infection in pulpless teeth with periapical lesions. They also concluded that such reduction was not related to the use of Ca(OH)2 for 4 weeks. The difference in our findings might relate to the duration of the use of Ca(OH)2 and the transport media used. In our study, Ca(OH)2 was applied for one week as opposed to four weeks. In this study, we analyzed the effectiveness of hand and rotary instrumentation in the elimination of bacteria from root canal. Our results show that hand instruments are far better than rotary instruments in cleaning and shaping (p=0.03). Even if the crown–down technique was the basis for the preparation with rotary files, taper and apical diameter caused changes in the preparation. Similarly, in the preparation with hand files, the taper and apical diameter was different. In all Groups, master apical file was determined according to each tooth’s number and anatomy. This might have had an impact on dentin elimination and bacterial reduction. ProTaper instruments have active cutting surface which allows such instruments to progress easily in the root canal and remove more infected dentin from the root canal compared with hand instruments. However, although rotary instruments appear to be better than hand instruments in reducing bacteria counts, in this study, the Groups prepared with hand files turned out to be better than rotary files on bacterial reduction. The results of this study conflict with those of Chuste-Guillot et al.25, Limongi et al.26 and Beun et al.27 who concluded that hand and rotary instruments were not significantly different in eliminating bacterial flora from root canal. Evans et al.28 also showed no difference between the hand-file step-back and NiTi Quantec systems regarding pulp and pre-dentin elimination. 19 CLINICAL DENTISTRY AND RESEARCH CONCLUSION Our findings show that regardless of the technique and the instrument, a similar quantity of bacterial reduction may be achieved. A good root canal disinfection may be obtained by mechanical preparation coupled with the application of Ca(OH)2 for one week. REFERENCES 1.Sjögren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J 1997; 30: 297-306. 2.Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root filled teeth with apical periodontitis. Int Endod J 1998; 31: 1-7. 3.Ingle JI, Himel VT, Hawrich CE. Endodontic Cavity Preparation. In: Ingle JI, Backland LK, editors. Endodontics. Hamilton: BC Decker Inc; 2002. p. 405-570. 4.Peters OA, Peters CI. Cleaning and shaping of the root canal system. In: Cohen S, Burns RC, editors. Pathways of the pulp. St. Louis: Mosby 2006. p. 339-342. 5.Siqueira JF, Lima KC, Magalhaes FAC, Lopes HP, Uzeda M. Mechanical reduction of the bacterial cell number inside the root canal by three instrumentations techniques. 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