Original Research ABSTRACT. Anuj Bhardwaj, Natanasabapathy Velmurugan 1, Sumitha 1, Suma Ballal 1

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1 Original Research Efficacy of passive ultrasonic irrigation with natural irrigants (Morinda citrifolia juice, Aloe Vera and Propolis) in comparison with 1% sodium hypochlorite for removal of E. faecalis biofilm: An in vitro study Anuj Bhardwaj, Natanasabapathy Velmurugan 1, Sumitha 1, Suma Ballal 1 Departments of Conservative Dentistry and Endodontics, Modern Dental College and Research Centre, Gandhi Nagar, Indore, Madhya Pradesh, 1 Meenakshi Ammal Dental College and Hospital, MAHER University, Maduravoyal, Chennai, Tamil Nadu, India Submission : Review completed : Accepted : ABSTRACT Aim: Present study evaluated the efficacy of natural derivative irrigants, Morinda citrifolia juice (MCJ), Aloe Vera and Propolis in comparison to 1% sodium hypochlorite with passive ultrasonic irrigation for removal of the intraradicular E. faecalis biofilms in extracted single rooted human permanent teeth. Materials and Methods: Biofilms of E. faecalis were grown on the prepared root canal walls of 60 standardized root halves which were longitudinally sectioned. These root halves were re approximated and the samples were divided into five groups of twelve each. The groups were, Group A (1% NaOCl), Group B (MCJ), Group C (Aloe vera), Group D (Propolis) and Group E (Saline). These groups were treated with passive ultrasonic irrigation (PUI) along with the respective irrigants. The root halves were processed for scanning electron microscopy. Three images (X2.5), coronal, middle and apical, were taken for the twelve root halves in each of the five groups. The images were randomized and biofilm coverage assessed independently by three calibrated examiners, using a four point scoring system. Results: 1% NaOCl with passive ultrasonic irrigation (PUI) was effective in completely removing E. faecalis biofilm and was superior to the natural irrigants like MCJ, Aloe vera and Propolis tested in this study. Conclusion: 1% NaOCl used along with passive ultrasonic irrigation was effective in completely removing E. faecalis biofilm when compared to natural irrigants (MCJ, Aloe Vera and Propolis). Key words: Aloe vera, E. faecalis, morinda citrifolia, propolis, passive ultrasonic irrigation Enterococci are part of the normal human flora, and are usually found in relatively small concentrations in the mouth. E. faecalis of the enterococci group is the most frequently isolated species in tooth with persistent infection after root canal treatment (RCT). [1] E. faecalis have the ability to survive endodontic treatment procedures by resisting high concentrations of intracanal Address for correspondence: Dr. Anuj Bhardwaj E mail: dranuj_84@yahoo.co.in 35 Quick Response Code: Access this article online Website: PMID: *** DOI: / medicaments and wide variations in ph. [2] Adaptive process of microbial growth as biofilm enables the microorganisms to survive the harsh growth conditions. In a biofilm, the microbes get adsorbed onto a solid non shedding surface, and are embedded in a common self produced extra cellular matrix. [3] The altered microbial genetic and metabolic processes, along with the extra cellular matrix, are thought to resist the actions of antimicrobials. [4] These bacterial biofilms are still present in untreated and treated root canals of teeth evincing apical periodontitis. [5] So, recognizing the potential role of E. faecalis is important to develop strategies to control infections by this organism. Reduction of bacteria within the root canal system is achieved by mechanical cleaning and shaping with the use of hand and/or rotary instruments. [6,7] A significant portion of the root canal walls may not be encompassed during instrumentation [8,9] leaving out large areas of biofilm.

2 Irrigation to disperse the biofilm is therefore essential as an adjunct to mechanical debridement of the root canal. The irrigant primarily acts as a vehicle for the removal of debris and infected material. [6] In addition, depending on the irrigant used, it may have additional bactericidal [10] and tissue dissolving properties. [11] Synthetic irrigant like 2% Chlorhexidine (CHX) [12] and 1% Sodium hypochloride (NaOCl) have been used successfully for removing E. faecalis biofilm. [13] But apical extrusion of the synthetic irrigants has adverse periapical reactions. [14] Along with the adverse periapical reactions, the constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for natural alternatives. Recently, Murray et al. [15] evaluated Morinda citrifolia juice in conjunction with Ethylenediaminetetraacetic acid (EDTA) as a possible alternative irrigant to NaOCl. Aloe Vera (synonym: Aloe barbadensis Miller) belongs to the Liliaceal family. Cosmetic and some medicinal products are made from the mucilaginous tissue in the centre of the Aloe Vera leaf and are called as Aloe Vera gel. Total leaf extracts contain anthraquinones. [16] Morinda citrifolia is considered as an important folk medicine. Its juice has a broad range of therapeutic effects including antibacterial, antifungal, antiviral, antitumor, antihelminthic, analgesic, hypotensive, anti inflammatory, and immune enhancing effects when this juice (MCJ) was used as an endodontic irrigant. [15] Propolis, also known as bee glue and bee propolis, is a brownish resinous substance collected by bees, mainly from plants. It is a potent antimicrobial, antioxidant, and anti inflammatory agent. [17] Morinda citrifolia and Propolis have proven effectiveness against E. faecalis in dentine of extracted teeth. [18] According to Bhuva et al. [13] when 1% NaOCl and saline were used with passive ultrasonic irrigation (PUI), their effect was better and was statistically significant in comparison to conventional irrigation. Hence, the usage of PUI might increase the efficacy of the irrigants. The use of PUI with these natural alternatives as root canal irrigants might help in increasing their efficacy in removal of E. faecalis biofilm. Hence, the purpose of this study is to compare the efficacy of passive ultrasonic irrigation with natural irrigants (MCJ, Aloe Vera and Propolis) in comparison with 1% sodium hypochlorite for removal of E. faecalis intraradicular biofilm in human extracted teeth. MATERIALS AND METHODS Sixty freshly extracted single rooted teeth were obtained and autoclaved individually at 121 C for 15 min. To prepare the root canals, the crowns of the teeth were removed, and the root lengths standardized to 15 mm. Patency of each canal was established first with a size 10 K files (Dentsply Maillefer, Ballaigues, Switzerland) followed by use of size 5 Gates Glidden bur (Dentsply Maillefer) to flare the coronal aspect of each canal. This was followed by the use of size 4 and size 3 Gates Glidden burs to further enlarge the canal coronally. The root canals were prepared with the F3 ProTaper rotary file (Dentsply Maillefer) to the full working length. Each canal was then instrumented to size 50 with K File, 1 mm short of the full working length. Irrigation with 1% sodium hypochlorite solution was performed throughout the instrumentation and canals were rinsed copiously with sterile saline solution to flush away any residual irrigants. Each root was placed into a block of freshly mixed additional silicone impression material, within a plastic specimen jar, ensuring that the coronal end of the prepared root was in flush with the surface of the silicone block. Once the impression material had set, each root and corresponding silicone block were numbered so that they could subsequently be matched up. Each root half was then sectioned longitudinally through the root canal using a 0.3 mm thickness diamond disc with low speed saw. One half from each root was selected for biofilm growth. The root halves were placed back into their corresponding silicone index, to ensure good re approximation. The silicone block and the chosen root half were marked, to ensure the correct orientation of each root in its block. The root halves were then removed from the silicone indices and immersed in a 17% solution of EDTA for 1 min to remove the smear layer, after which they were washed thoroughly with water and autoclaved. The root halves not being used for biofilm growth were left hydrated in labelled sterile flasks which were later used for re approximation to their respective half in order to carry out the irrigation protocol. Biofilm culture An E. faecalis biofilm (ATCC 29212) was grown on each selected root half using a standardized biofilm growth protocol. The strain was cultured anaerobically at 37 C on Fastidious Anaerobe Agar. Starter cultures were set up in filter sterilized modified fluid universal medium (mfum), which were incubated anaerobically at 37 C for 3 hrs, until the growth appeared moderately turbid. Each root was incubated with 3 ml of the E. faecalis culture in 24 well trays. The medium was 36

3 replaced after a period of 24, 48 and 72 hours after which the tray was removed from the anaerobic cabinet and stored at 4 C. The 60 root half pairs were re approximated and divided into five groups of 12 each. The five groups were: Group A (1%NaOCl), Group B (MCJ), Group C (Aloe Vera), Group D (Propolis) and Group E (Saline). Each sample in the group was treated with 4 ml of respective solution energized with a size 15 ultrasonic file for two min. The irrigation time in these groups was divided as follows: 30 sec syringe irrigation, 20 sec passive ultrasonic irrigation, 20 sec syringe irrigation, 20 sec passive ultrasonic irrigation followed by 30 sec of syringe irrigation. Irrigation protocol A Single operator performed all irrigation procedures. A 27 gauge irrigating needle was used for irrigation in groups A, B, C, D and E. The syringes were pre measured to ensure that the correct amount of irrigant was used on each occasion. A rubber stop was placed on each irrigating needle at a length of 14 mm from the tip to prevent its insertion beyond this depth. Conventional syringe irrigation was carried out using digital pressure with the forefinger only. The needle was moved back and forth in the canal gently whilst the irrigation was being performed, ensuring that the needle did not bind in the canal. The irrigation was performed at a rate ensuring that only half of the irrigant volume was used after 1 min of irrigation. 1.5 ml of irrigant was used in the first 30 sec of irrigation, 1 ml in between the two periods of passive ultrasonic irrigation and then 1.5 ml of irrigant in the final stage of irrigation. A total of 4 ml of irrigant was used for the protocol in various groups used in the study. The PUI was standardized by performing two 20 sec cycles of activated irrigation during the 2 min irrigation period. The passive ultrasonic irrigation was carried out with a size 15 ultrasonic file (Satelec, Merignac Cedex, France), which was used dry in a root canal. The power setting on the ultrasonic unit (Satelec, Merignac Cedex, and France) was kept at one quarter of maximum for all of the roots. The same ultrasonic unit was used throughout the study. Every effort was made to minimize contact of the file with the root canal walls during passive ultrasonic irrigation. Preparation of specimens After each root had been subjected to its allocated irrigation protocol, it was immediately immersed in 37 phosphate buffered glutaraldehyde fixative for a minimum of 4 h, followed by glutaraldehyde wash solution. The samples were immersed in ascending grades of ethanol for 10 min at successive concentrations of 10%, 50%, 70%, 90% and then two cycles of 100%. The specimens were immersed for 15 min in one part of hexamethyldisilazane (HMDS) (Alpha Lab chem, India) to two parts of absolute ethanol, then 15 min in one part HMDS to one part 100% ethanol. This was followed by 15 min in one part 100% ethanol to two parts HMDS, and finally two cycles of twenty min in 100% HMDS. The specimens were dried on clean lint free tissue. The root halves were then mounted on stub plates and gold sputter coated. The scanning electron microscope (Hitachi, S 3400N, High Technologies Co., Japan) was used for observations. Three scanning electron microscope (SEM) images were taken of each root half at the following positions along the midline of the canal: Three millimetres from the coronal end Coronal area. Six millimetres from the coronal end Middle area. Nine millimetres from the coronal end Apical area. These specific points were identified using a standardized protocol. To assess approximately the same area of each specimen, the three defined reference points were measured and marked on each root half with a scalpel blade prior to microscopy. High magnification images of the root specimens were also taken, to observe the samples in more detail. Three images were acquired for each of the 60 root halves, with a total of 180 images being obtained. Three examiners (specialist Endodontists n = 2, Endodontic postgraduate n = 1) that were unaware of the aim of the study assessed the SEM images in the following sequence: Session 1 Calibration session. Session 2 Randomized SEM images 1. Session 3 Randomized SEM images 2 (different sequence to session 1). A four point scoring system was devised to allow the E. faecalis biofilms to be assessed semi quantitatively as per Bhuva et al. [13] The scoring system formulated for the SEM images was as follows: Less than 5% biofilm coverage of the root canal wall. Biofilm coverage between 5-33% of the root canal wall. Biofilm coverage between 34-66% of the root canal wall. Biofilm coverage between % of the root canal wall. Three examiners were trained using a number of calibration images in the study. A number of different images

4 representing each score were used to ensure that the examiners were exposed to slight variations in the appearance of the biofilm. A calibration test was conducted by the examiners to confirm a high level of agreement. Two random sequences were obtained from 1 to 180 using a random number sequence generator. Two different Power Point presentations were created of all 180 images using the sequences. The observation sessions were conducted on two separate occasions, one week apart, to assess examiners agreement. Statistical analysis The data were analysed [Tables 1-3] using SPSS 15.0 software. Differences between the five groups at each of the three levels were assessed using Pearson Chi squared analysis. RESULTS At the three levels evaluated, 1% NaOCl with PUI was the most effective in removing biofilm compared to the natural irrigants (Propolis, Aloe Vera and MCJ) used. 1% NaOCl could completely remove the biofilm at the three levels as shown in [Figure 1]. There was no statically significant difference between Propolis, Aloe Vera and MCJ groups at the three levels. There was statistically significant difference between 1% NaOCl group and the other three groups (irrigants (Propolis, Aloe Vera and MCJ) at the three levels (coronal, middle and apical) (P < 0.001). Table 1: Scores of biofilm removal in coronal third Score NaOCl Saline Aloe vera Propolis MCJ Total MCJ=Morinda citrifolia juice Table 2: Scores of biofilm removal in middle third Score NaOCl Saline Aloe vera Propolis MCJ Total MCJ=Morinda citrifolia juice Table 3: Scores of biofilm removal in apical third Score NaOCl Saline Aloe vera Propolis MCJ Total MCJ=Morinda citrifolia juice DISCUSSION The model in this study utilized a mono species of E. faecalis for biofilm formation on the root canal walls of extracted single rooted teeth. Scanning electron microscopy (SEM) was used to evaluate the E. faecalis biofilms and the effects of the tested irrigation protocols with PUI on these microbial communities as it has been used frequently employed to observe intraradicular biofilms. In this study, the in vitro model used, provided a well standardized anatomical setup which allowed for comparison of different irrigants. Various techniques have been employed to develop and eradicate biofilm. The method used in this study was developing and removing of the biofilm within the root canal system. Hence, the intricacies of tubules and the inert buffering properties of dentin may play a major role in the action of the irrigants used. Various irrigants and medicaments like Triphala, [19] calcium hydroxide, [18] 2% povidone iodine, [18] MTAD, [20] citric acid, [21] chlorhexidine, [18] tetraclean, [20] green tea polyphenols, [19] EDTA [21] and phosphoric acid [18] have been tried for removal of E. faecalis biofilm, but have not been very efficient. Whereas, in this study we carried out irrigation in the root canal with ultrasonics and irrigants to see their efficacy in removing the biofilm which was developed on the tooth structure (intraradicular) so as to closely simulate the oral environmental conditions. Irrigation time on the efficacy of passive ultrasonic irrigation (PUI) is not clear. One study claimed an increased removal of the smear layer after 5 min of PUI as opposed to 3 min. [22] Whereas, in the study of Sabins et al., [23] no significant difference was found between 30 and 60 s of PUI in dentine debris removal from the root canal. In their study NaOCl was injected into the canal using syringe followed by PUI and same was followed in this study. The effect of each irrigation protocol was evaluated at three levels of each root canal. The reference points chosen were at 3 mm as coronal, 6 mm as middle and 9 mm as apical from the coronal end of each canal. There were no observed differences between the three levels in any of the experimental or control groups. It may have been anticipated that there would have been less effective biofilm removal apically. Another probable reason is the use of single rooted teeth were used which were instrumented to size 50, 1 mm short of the full working length, with K File which permitted unimpeded needle penetration to the full length of each canal. Ram Z [24] demonstrated that the depth of needle insertion was the single most important variable affecting the efficacy of root canal irrigation. The width of 38

5 [Downloaded free from on Tuesday, August 06, 2013, IP: ] Click here to download free Android application for this journal Bhardwaj, et al. Figure 1: SEM pictures of various irrigants removing the Enterococcus faecalis bio film from the root canal walls ( 2.5) the root canal, the ratio of root canal width to the irrigating needle, has also been an important variable.[25] Bhuva et al.[13] have shown that 1% NaOCl with PUI was effective in completely removing intraradicular E. faecalis biofilm. However, contradictory finding by Harrison 39 et al.[26] showed that when 1% NaOCl used with PUI was effective but could not completely remove the E. faecalis in experimentally infected root canals. However, in this study 1% NaOCl was effective in completely removing the E. faecalis biofilm at all the three levels (coronal, middle and apical).

6 None of the natural irrigants (Propolis, Aloe Vera and MCJ) were effective in complete removal of E. Faecalis biofilm. The probable reasons for superior results shown by 1% NaOCl can be attributed to: Sodium hypochlorite has bactericidal action. [27,28] The physical properties of NaOCl have an effect on the transmission of ultrasound energy to the irrigant. For example, bubbles formed in salt water tend to be more numerous, particularly the smallest bubbles, and are less prone to coalesce than bubbles in fresh water. [29] Vapor (chloride when NaOCl is used) could diffuse into the bubble during bubble expansion and the bubble dynamics depend on the concentration of the gas dissolved in the liquid, the temperature of the liquid and amounts of surface active impurities. [30] The probable reasons for the results shown by Aloe Vera, MCJ and propolis can be attributed to: Aloe Vera extracts contain anthraquinones. [16] MCJ has a broad range of therapeutic effects including antibacterial, antifungal, antiviral, antitumor, antihelminthic, analgesic, hypotensive, antiinflammatory. [15] Propolis is a potent antimicrobial, antioxidant, and anti inflammatory agent. [17] Morinda citrifolia and Propolis have proven effectiveness against E. faecalis in dentine of extracted teeth. [18] CONCLUSION Within the limitations of the study it was concluded that: 1% NaOCl used along with passive ultrasonic irrigation was effective in completely removing E. faecalis biofilm when compared to natural irrigants (MCJ, Aloe Vera and Propolis). ACKNOWLEDGEMENT The authors deny any conflicts of interest related to this study. REFERENCES 1. Sedgley CM, Lennan SL, Clewell DB. Prevalence, phenotype and genotype of oral enterococci. Oral Microbiol Immunol 2004;19: Love RM. Enterococcus faecalis A mechanism for its role in endodontic failure. Int Endod J 2001;34: Portenier I, Waltimo TM, Haapasalo M. 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7 canal system: An in vitro study. J Endod 1983;9: Leighton TG. The Acoustic Bubble, Chapter 1 and 2. New York: Academic Press; Brenner MP, Hilgenfeldt S, Lohse D. Single bubble sonoluminescence. Rev Mod Phys 2002;74: How to cite this article: Bhardwaj A, Velmurugan N, S, Ballal S. Efficacy of passive ultrasonic irrigation with natural irrigants (Morinda citrifolia juice, Aloe Vera and Propolis) in comparison with 1% sodium hypochlorite for removal of E. faecalis biofilm: An in vitro study. Indian J Dent Res 2013;24: Source of Support: Nil, Conflict of Interest: None declared. Staying in touch with the journal 1) Table of Contents (TOC) alert Receive an alert containing the TOC when a new complete issue of the journal is made available online. To register for TOC alerts go to 2) RSS feeds Really Simple Syndication (RSS) helps you to get alerts on new publication right on your desktop without going to the journal s website. You need a software (e.g. RSSReader, Feed Demon, FeedReader, My Yahoo!, NewsGator and NewzCrawler) to get advantage of this tool. RSS feeds can also be read through FireFox or Microsoft Outlook Once any of these small (and mostly free) software is installed, add as one of the feeds. 41