Amit Arvind Agrawal1*, Swapnil Kolhe2, Amit Sope3 and Dinesh Erlewad4
1Department of Periodontics and Implantology, MGV’s KBH Dental College and Hospital, Nasik, Maharashtra, India
2Department of Conservative Dentistry and Endodontics, MGV’s KBH Dental College and Hospital, Nasik, Maharashtra, India
3Department of Cell Immunology and Biology, University of North Texas Health Science Center, Texas, USA
4Principle Dentist, 208, Sterling Center, M.G road, Camp, Pune 411001, Maharashtra, India
Received: 03 May, 2016; Accepted: 11 May, 2016; Published: 13 May, 2016
Dr. Amit Arvind Agrawal, BDS, MDS, MPhil, Professor, Department of Periodontics and Implantology, MGV’s KBH Dental College and Hospital, Mumbai-Agra road, Panchvati, Nashik-422003, Maharashtra, India, Tel: +919822107562; E-mail:
Agrawal AA, Kolhe S, Sope A, Erlewad D (2016) Root Canal Disinfection Potential of 5.25% Sodium Hypochlorite, 2% Chlorhexidine and 810nm Diode Laser-A Comparative In vitro Antimicrobial Study. Int J Oral Craniofac Sci 2(1): 035-038. 10.17352/2455-4634.000016
© 2016 Agrawal AA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Root canal disinfection; Sodium hypochlorite; Chlorhexidine; Diode laser; Enterococcus fecalis
E.faecalis: Enterococcus faecalis; Nacl: Sodium Chloride; DNA: Deoxyribonucleic Acid; RNA: Ribonucleic Acid; NaOCl: Sodium Hypochlorite; DTA: Ethylene Diamino Tetra Acetic Acid; CHX: Chlorhexidine; DL: Diode Laser; CB group: Control-Baseline Group; C BMP group: ‘Control-Biomechanical Preperation’ Group; NCIM: National Collection of Industrial Micro-organisms; ATCC: American Type Culture Collection; BMP: Bio-Mechanical-Preperation; LASER: Light Amplification of Stimulated Emission of Radiation; Nd: YAG: Neodymium-doped Yttrium Aluminum Garnet; W: Watts
Background: The eradication of persisting bacteria, such as Enterococcus faecalis, is crucial for the long-term preservation of the endodontically treated tooth.
Context and Purpose of the study: The aim of this research was to evaluate and compare the root canal disinfection potential of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate and 810nm diode laser against control.The aim of this research was to evaluate and compare the root canal disinfection potential of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate and 810nm diode laser against control.
Results: Adjunctive use of chemical disinfection by either 5.25% sodium hypochlorite or 2% chlorhexidine led to 100% microbial eradication as against diode laser which achieved 97.7% reduction as compared to baseline microbial count and 68.42% reduction after mechanical cleaning at the same dilutions.
Main findings: Chemicals used in the study achieved greater disinfection than diode laser irradiation.
Conclusions: 5.25% sodium hypochlorite or 2% chlorhexidine can be efficiently used as an adjuvant to mechanical root canal cleaning.
Brief summary: A total of 20 extracted teeth, sectioned at cement-enamel junction, were divided into four groups of five teeth each. Control group: mechanical cleaning only; three test groups: mechanical cleaning followed by disinfection with 5.25% sodium hypochlorite or 2% chlorhexidine or 810nm diode laser. Pre and Post treatment microbial samples were collected and cultured.
Potential Implications: A thorough mechanical instrumentation is crucial for success of any endodontic therapy and chemical or laser irradiation will only be helpful as an adjuvant.
The major cause of endodontic failure is the survival of microorganisms in the apical portion of root filled teeth, of which, E.faecalis is considered one of the primary organisms in patients with post treatment endodontic infection . Enterococci were first placed under genus streptococcus, however studies demonstrated a more distant relationship with streptococci . In 1984, enterococci were given a formal genus status after DNA-DNA and DNA-RNA hybridization. They are gram positive facultative anaerobic coccoid bacteria which can occur singly, in pairs or as short chains. Enterococci grow at temperatures ranging from 10-450C, at pH 9.6 and in 6.5% (NaCl) sodium chloride and can survive at 600C for 30 minutes. E.faecalis in particular, possesses certain virulence factors including lytic enzymes, aggregation substance, pheromones and lipoteichoic acid . E.faecalis has the ability to establish monoinfections in medicated root canals. The organism has the ability to acquire, accumulate and share extra-chromosomal elements, encoding virulence traits, which help to colonize, compete with other bacteria, resist host defense mechanisms and produce pathological changes directly through the production of toxins or indirectly through the induction of inflammation. E.faecalis has the advantage of forming biofilms, hence it has a certain degree of protection and homeostasis. Biofilms grow in a nutrient-deprived ecosystem as it concentrates trace elements and nutrients by physical trapping and electrostatic interaction. The bacterial cells residing in a biofilm communicate, exchange genetic materials and acquire new traits. This communication takes place by quorum sensing. E.faecalis is also known to resist intra canal medicaments like calcium hydroxide by maintaining pH homeostasis .
An in-vitro study by Hohscheidt et al5 to evaluate the effect of different endodontic auxillary chemical substance such as (NaOCl) sodium hypochlorite, EDTA (ethylene diamino tetra acetic acid), 2%CHX (chlorhexidine) gel, 2% CHX liquid in different combinations, concluded that none of the tested substances could completely eliminate the E.fecalis from the root canal space. In addition, few in-vitro studies [6-8], have evaluated the disinfection potential of diode laser following chemo-mechanical procedures against E.fecalis, and concluded that 980nm diode laser can even eliminate bacteria that has immigrated into dentin, thus being able to increase the success rate in endodontic therapy. To our knowledge there is no study which has evaluated the disinfection potential of sodium hypochlorite, chlorhexidine and 810nm diode laser together.
The aim of this in-vitro microbial research was to evaluate and compare the root canal disinfection potential of 5.25% NaOCl, 2% CHX and 810nm diode laser (DL) against control (C). Our baseline null hypothesis was that 5.25%NaOCl, 2% CHX and 810nm diode laser are equally effective in eradication of E.fecalis from root canal space in vitro.
Materials and Methods
Twenty extracted single rooted teeth were sectioned at the cemento-enamel junction and the roots were prepared by step back technique to #30 K- file (Maillefer, Dentsply) at the apical end. All the teeth were then sterilized by autoclaving. The root canal spaces were then filled with liquid MRS medium containing pure culture strains of E.fecalis* (NCIM no. 5024) (ATCC no. 14506) and inoculated for 24 hrs in an incubator. E.fecalis culture was obtained from National Collection of Industrial Micro-organisms (NCIM), National Chemical Laboratory (NCL), Pune-411008, India.
Twenty samples were divided into 4 groups of 5 teeth each, the groups were as follows:
a. Control group:
i. Control Baseline (CB group) – 2 teeth.
ii. Control BMP, (C BMP group) – 3 teeth.
b. 2% Chlorhexidine group (CHX)
c. 5.25% sodium hypochlorite group (NaOCl)
d. Diode LASER group (Laser)
All the sample teeth along with the experimental materials were kept in the laminar air flow, and following techniques were performed depending on the study group.
After 24 hrs of inoculation, verification of count of bacteria inoculated in root canal was done with 2 samples (CB group), out of total 5 samples in control group, which displayed innumerable/uncountable colonies in MRS medium. The inner wall of remaining 3 teeth (C BMP group), were cleaned mechanically (for 1 min) using K files (Maillefer, Dentsply), followed by sterile saline irrigation (10ml) using 30 guage Max I probe needle.
2% Chlorhexidine (CHX) group
Mechanical cleaning with K files (Maillefer, Dentsply), was done for 1 min by brushing technique, followed by 5ml irrigation of 2% chlorhexidine (Dentochlor, Ammdent, Amrit Chemical, Mohali, India) for 30 seconds using 30 gauge Max I probe needle; followed by sterile saline irrigation (10ml).
5.25% sodium hypochlorite (NaOCl) group
Mechanical cleaning with K files (Maillefer, Dentsply), was done for 1 min by brushing technique, followed by 5ml irrigation of 5.25% sodium hypochlorite solution (Prime Dental Products, Mumbai, India) for 30 seconds using 30 guage Max I probe needle, followed by sterile saline irrigation (10ml).
Diode LASER (Laser) group
Mechanical cleaning with K files (Maillefer, Dentsply) was done for 1 min, followed by irradiation with a diode LASER (Picasso, Dentsply) in non-contact mode, continuous wave, 3W setting. A single cycle consisted of an exposure for 5sec and a rest of 15 sec, total 5 such cycles were completed for each of the 5 samples in this group. The fiber from the laser hand piece (tip diameter 30 microns) was introduced into the root canal up to the apex and then the laser is activated. The fiber was guided in an apical to coronal direction with circular movements. Finally irrigation was done using 10ml of sterile saline.
After these disinfection stages, three paper points (#20, Maillefer, Dentsply) were inserted, one by one, in the canal of each of 20 sample teeth and then transferred to a test tube containing 1ml of peptone water. The test tube was vortexed to dislodge any microbial colonies attached to the paper point. 1 ml of this peptone water is diluted with 9ml of sterile saline; 1ml from the resultant 1:10 dilution of bacteria is then mixed with 15ml of MRS medium using pour plating method and inoculated for 48 hrs.
Furthermore, 1ml of the 1:10 dilution solution is again diluted with 9 ml of sterile saline; 1ml from the resultant 1:100 dilution of bacteria is then mixed with 15ml of MRS medium using pour plating method and inoculated for 48 hrs.
Still further, 1ml of the 1:100 dilution is diluted with 9 ml of sterile saline; 1ml from the resultant 1:1000 dilution of bacteria is then mixed with 15ml of MRS medium using pour plating method and inoculated for 48 hrs. However, this 1:1000 dilution was required only in control group samples, where in the initial dilution, innumerable colonies were obtained.
After 48 hrs, all petri-dishes were recovered from the inoculation chamber and the colonies were physically counted. Only 2 plates had such innumerable count that it could have been impossible to get an exact count. In these samples a higher dilution (1:1000) count was considered.
Table 1 shows the number of colonies in each group and at various dilutions. At 1:10 dilution, the baseline microbial count in untreated samples was uncountable but after biomechanical preparation (BMP) the count reduced to an average of 30.5 colonies. At 1:100 dilution, this ratio was 66.33 to 4.75, (Figure 1a,b) that means there was about 92.83% reduction in the microbial count simply by thorough mechanical cleaning. This finding further strengthens the fact that all disinfecting agents can be an adjuvant to mechanical cleaning and never a replacement. In two test groups, that using chlorhexidine and sodium hypochlorite irrigation, the colony counts were zero at 1:10 or 1:100 dilutions (Figure 1c,d). However in the diode laser group, an average of 1.5 colonies were noted at 1:100 dilution (Figure 1e), which is 97.73% reduction as compared to baseline microbial count and 68.42% reduction after mechanical cleaning at the same dilutions.
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