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ORIGINAL ARTICLE
Year : 2020  |  Volume : 23  |  Issue : 9  |  Page : 1188-1193

Antimicrobial effects of sodium hypochlorite and Er,Cr:YSGG laser against Enterococcus faecalis biofilm


1 Department of Infectious, Near East University, Faculty of Medicine, Diseases and Clinical Microbiology, Nicosia, Cyprus
2 DentX Dental Hospital, Near East University, Vocational School of Health Services, Nicosia, Cyprus
3 Department of Microbiology, Near East University, Vocational School of Health Services, Nicosia, Cyprus

Date of Submission10-Dec-2018
Date of Acceptance06-Aug-2020
Date of Web Publication10-Sep-2020

Correspondence Address:
Dr. M Guvenir
Department of MIcrobiology, Near East University, Vocational of Health Services, Nicosia
Cyprus
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_632_18

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   Abstract 


Aims: The aim of this study was to investigate the antimicrobial effect of Erbium, chromium-doped yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser with and without NaOCl solution. A total of 81 extracted human mandibular premolar teeth were used. Materials and Methods: After root canal preparation and sterilization, the samples were inoculated with E. faecalis for 24 hours. The specimens were divided into 4 experimental groups. Group 1 (n = 25) was irradiated with 2 W laser, group 2 (n = 25) was irradiated with 0.75 W laser in combination with 2.5% NaOCl, group 3 (n = 25) was irrigated with 5% NaOCl and group 4 (n = 6) was not treated. Statistical analysis was performed by using Wilcoxon Signed Ranks, Mann-Whitney U and Kruskal-Wallis H tests. Results: The combination of 0.75 W laser with 2.5% NaOCl regime was found to be just as effective at inhibiting the growth of E. faecalis and sterilization of all root canals as 5% NaOCl irrigation (P > 0.001). The 2 W laser had significant bactericidal effect in infected root canals however it did not eradicate all bacteria. The SEM observations were in accordance with the microbiologic findings. Conclusions: Within the limitations of this laboratory study, NaOCl irrigation improved the antimicrobial effect of Er,Cr:YSGG laser irradiation. When the toxic effects of high percentage of NaOCl was considered, the combination of low-powered laser and low concentration of NaOCl can be used as an effective disinfection method in root canal treatment.

Keywords: Disinfection, Enterococcus faecalis, laser, NaOCI, root canal treatment


How to cite this article:
Suer K, Ozkan L, Guvenir M. Antimicrobial effects of sodium hypochlorite and Er,Cr:YSGG laser against Enterococcus faecalis biofilm. Niger J Clin Pract 2020;23:1188-93

How to cite this URL:
Suer K, Ozkan L, Guvenir M. Antimicrobial effects of sodium hypochlorite and Er,Cr:YSGG laser against Enterococcus faecalis biofilm. Niger J Clin Pract [serial online] 2020 [cited 2020 Oct 20];23:1188-93. Available from: https://www.njcponline.com/text.asp?2020/23/9/1188/294685




   Introduction Top


Instrumentation and irrigation in endodontic treatment is an essential stage for the elimination or the reduction of microorganism numbers during root canal preparation.[1] Although microorganisms can be eliminated from the root canal system with the use of mechanical instrumentation, some microorganisms may continue to persist with in the isthmic, lateral canals, fins, ramifications, and anatomical structures.[2]

Enterococcus faecalis (E. faecalis), is a Gram-positive aerobic cocci, and is a primary microorganism responsible for endodontic post-treatment disease.[3],[4]E. faecalis is able to adapt to the adverse conditions found within the canal, due to its ability to form a biofilm. The biological properties of the biofilm allow it to adhere and colonize the root canal, while allowing it to persist under nutrient deprived and alkaline conditions.[5] The adaptive behavior of E. faecalis however, makes the eradication of this bacterium from infected root canals difficult.[6] Its prevalence in such infections ranges from 22% to 77%[7] and has been widely used as a valuable microbiological pathogen for in vitro studies due to its ability to successfully colonize the root canal in a biofilm-like style, invade dentinal tubules, and resist endodontic treatment.[8]

In an irrigation regime, sodium hypochlorite (NaOCl) is used in concentrations from 0.5% to 5.25% and is the most commonly used antimicrobial irrigate,[9] mainly because of its potent antimicrobial action and ability to dissolve organic matter and necrotic tissue.[9],[10] At low concentrations, it is however ineffective against some specific microorganisms.[11] The biocompatibility of NaOCl is inversely proportional to its concentration,[12] that is, NaOCl reduces the mechanical properties of dentine.[13] Furthermore, in dentistry the application of NaOCl can have associated complications, including injury to the skin surface, oral mucosa and eyes, damage to clothing, air emphysema, and allergic reactions.[14] One of the new antimicrobial approaches includes the use of lasers. Laser radiation was introduced into endodontic treatment as an adjunctive disinfection procedure after the chemo-mechanical root canal preparation.[15] Unlike, conventional methods, laser lights are able to reach areas which may not be inaccessible with conventional techniques (e.g., bacteria deeply located in the fins, lateral canals isthmuses, and dentinal tubules).[5] It is also a procedure which improves the removal of intracanal debris and the smear layer.[16] The Erbium, chromium-doped yttrium, scandium, gallium, and garnet (Er, Cr:YSGG) laser system uses hydrokinetic energy—the laser energy heats the air and water directly in front of the laser beam to deliver energy onto the rear surface of atomized water molecules with the aim of accelerating them to a higher speed.[17]. Some studies recommend the use of lasers in root canal disinfection[5],[18] where others claim that the results of antimicrobial activity of lasers are not adequate.[19],[20]

The aim of this study was to evaluate whether chemical modification with the addition of NaOCl was able to improve the antimicrobial effects of the Er, Cr:YSGG laser system on E. faecalis, in artificially infected human root canals. This study was based on the hypothesis that the combination of Er, Cr:YSGG laser with NaOCl may enhance the antimicrobial effect compared to conventional disinfection methods.


   Materials and Methods Top


Sample preparation

Eighty-nine freshly extracted human mature permanent mandibular premolar teeth were obtained from orthodontic patients. Patients teeth which were having single root canal and a closed apex were selected, whereas patients teeth having curved roots, more than one canal or any abnormal shape were discarded leaving a total of 81 human teeth which were used in the study. Patients were informed about the investigation and written informed consent was given by all patients to allow the use of their teeth in the current study.

The teeth were stored in saline solution following extraction. Bone, calculus, and soft tissues on the root surface were removed with curettes. The teeth were sectioned at 12 mm from the apex to separate the crowns from the roots using a diamond disk (Isomet 1000; Buehler, Lake Bluff, IL, USA). The working length was set at 1 mm short of apical foramen. Root canals were prepared using the step-back technique with Ni-Ti K-files (K Files NITI FLEX, Dentsply, Germany) up to # 50 and Gates Glidden burs (#2, 3, and 4) (JS Dental, Ridgefield, USA) which allowed entrance of the laser fiber to the apical area. The root canals were irrigated with 0.9% saline solution only, no EDTA (ethylenediaminetetraacetic acid) was applied during preparation. One investigator was assigned to perform all of the root canal preparations (LO). After the preparation process, the canals were closed by using flowable composite resin (Clearfil Majesty Flow, Kuraray Medical Inc) in their apical region by means of bonding to prevent bacterial leakage, whereas cotton pellets were used to seal the canal orifices followed by temporary filling material (3M ESPE, Cavit G). The roots were mounted vertically into Eppendorf tubes partially filled with acrylic resin. Once the acrylic resin set, the temporary filling material and cotton pellet were removed.

In order to standardize the incubation and sterilization procedure, 8 specimens were randomly selected and were sterilized by autoclaving at 121°C for 15 min.

Microbial inoculation

Specimens were assigned into groups in a double-blind manner. Initially four specimens were selected to standardize the inoculation process. A standard bacterial culture of E. faecalis (ATCC 29212) was obtained from the Culture Collection. E. faecalis was incubated for 24 hours on 5% sheep blood agar (Merck Millopore) at 37°C. 0.5 McFarland microbial suspension of E. faecalis colonies in Brain Heart Infusion Broth was used (Oxoid Microbiology Products-UK). Then 10 μl of the microbial suspension was transferred into the canal lumen using sterile needles and incubated at 37°C for 24 hours. Following incubation, the microbial suspension was removed from the canal lumen and cultured on Sheep blood agar to obtain the number of colony-forming units. Microbiological procedures were performed in a blind manner.

Treatment protocol

After standardization of the procedure, the specimens were sterilized and inoculated according to the procedure above. Subsequently 81 specimens were randomly divided into 4 groups and treated according to the following protocols:

Group 1 (n = 25): The root canals received 2 W Er, Cr:YSGG laser treatment, output power of 0.45, 20 Hz, 25% water, and 35% air.

Group 2 (n = 25): The root canals received 0.75 W Er, Cr:YSGG laser treatment, output power of 0.45, 20 Hz, 0% water, and 0% air, combined with 2.5% NaOCl.

Group 3 (n = 25): 5% NaOCI was used as a root canal irrigation solution. The samples were irrigated with 2 ml of NaOCl for 1 minute.

Group 4 (n = 6): The root canals did not receive any treatment and served as the control group.

During laser irradiation, each sample was treated with one lasing cycle, which consisted of four irradiations of ten seconds each with 5-second intervals in between, and the fiber tip was inserted all the way down into the apex. The laser was then activated and the root canal was irradiated from the apical to cervical region with helicoidal movements. One investigator was assigned to perform all of the irradiations (LM).

Microbiological evaluation

Following laser treatment, the root canals were rinsed with 1 ml of saline solution (0.9%). From each root canal 10 μl of remnant saline solution was removed using a sterile needle and cultured on sheep blood agar at 37°C for 24 hours in order to obtain the number of bacterial colonies (cfu/ml). The colonies were then counted and the total number of bacteria (colony forming units per ml of the extraction fluid) was assessed.

Scanning electron microscopy (SEM)

Twelve samples were prepared as described before without the bacterial contamination and randomly subdivided into 3 groups. Three groups were compared (2 W, 0.75 W + 2.5% NaOCl, 5% NaOCl) to evaluate the morphologic changes induced by laser irradiation and to observe the remnants of smear layer on the root canal walls by using scanning electron microscopy (JSM 6400, JEOL, Tokyo, Japan). The preparation of the samples involved making longitudinally grooves on the buccal and lingual surfaces of the root with a diamond bur. These grooves were used as guidance to split the roots into two pieces. The samples were gold sputtered and mounted on metal stubs for examination. Apical, middle, and coronal thirds of the samples were evaluated separately with the aid of scanning electron microscopy.

Statistical analysis

Descriptive statistics for the whole data were calculated and provided within the text. In each experimental group, the change in the number of E. faecalis were compared with Wilcoxon Signed Ranks test. The difference in number of E. faecalis was compared between the groups with Kruskal Wallis and in case of statistical significance, pairwise group comparisons were performed by Mann Whitney U test with Bonferroni correction. The significance level was accepted as P = 0.05 level. Statistical analysis was carried out using the SPSS 12.0.1 (SPSS, Chicago, ILL) software program.


   Results Top


Comparison of laser and NaOCI treatment methods

The data obtained for Er, Cr:YSGG laser and NaOCl groups are presented in [Table 1]. At 2 W laser power setting 50% elimination of E. faecalis was achieved whereas 100% elimination of E. faecalis was obtained using 5% NaOCl only and also with the combination of 2.5% NaOCl and 0.75 W laser irradiation. The number of E. faecalis was significantly reduced in each experimental group (P < 0.001).
Table 1: Mean values of bacterial counts (E. faecalis) before/after laser irradiation and NaOCI irrigation

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Scanning electron microscopy analysis

One image was taken for each third (apical, middle, and cervical) of the specimens from all groups. Due to the helicoidal movement of laser tip, 2 W laser (group 1) irradiated dentin was characterized by partial removal of smear layer. The irradiated surfaces showed irregular areas, which looked like cracks and fissures but there was no sign of either carbonization or melting [Figure 1]. Further down the apical, less dentinal tubules were observed [Figure 2]. As the laser output energy decreased in group 2 (0.75 W + NaOCl), the crack and fissure appearance also diminished [Figure 3]. However, some areas also showed inefficient cleanliness, presenting occlusion of dentinal tubules by a smear layer due to irregular laser irradiation [Figure 4].
Figure 1: SEM photo showing the samples treated with 2 W Er,Cr:YSGG laser

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Figure 2: SEM photo showing the samples treated with 2 W Er,Cr:YSGG laser

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Figure 3: SEM photo showing the samples treated with 0.75 W Er,Cr:YSGG laser and NaOCl

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Figure 4: SEM photo showing the samples irrigated with NaOCl

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Examination of the specimen with SEM after NaOCI treatment (group 3) revealed that the entire dentinal wall was covered by a dense, homogeneous smear layer [Figure 5]. The smear layer was getting thicker from the coronal to apical third of the teeth. Moreover, the smear layer in NaOCI irrigated teeth was excessive compared to those that were laser irradiated.
Figure 5: SEM photo showing the samples irrigated with NaOCl

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   Discussion Top


For endodontic treatment to be successful bacteria and their byproducts need to be eliminated from infected root canals. Current treatment plans involving rotary instruments and chemical irrigation fail to completely eliminate microorganisms from inside the root canal, increasing the risk of the development of treatment-resistant bacterial infections.[5] A final disinfection irrigation protocol that is often implemented includes the use of sodium hypochlorite (NaOCl) followed by ethylenediaminetetraacetic acid (EDTA) and chlorhexidine (CHX) or NaOCl (9). NaOCl ionizes in water into sodium (Na+) and hypochlorite ion (OCl-), and establishes an equilibrium with hypochlorous acid (HOCl), which affects the vital functions of the microbial cell,[21],[22] making it an effective antimicrobial substance in root canal irrigation.[9],[23],[24] The teeth were stored in saline solution following extraction. One of the disadvantages of using of NaOCl is its toxicity, especially in the case of overflowing to the apical foramen.[9],[25] NaOCl is unselective and can damage human cells, dentine, and periodontal tissues with clinical consequences.

Although a number of studies have investigated ways in which to eradicate the E. faecalis biofilm with the use of various NaOCl concentrations, the recommended concentration to eradicate the biofilm remains conflicted. Furthermore, there is no consensus on the irrigation period required to eliminate the bacteria from the canal system. The use of different methodologies between the studies may have contributed to the conflicting results. Lower concentrations are often used as is the case in the current study in order to overcome NaOCl's toxicity in the case where it overflows into the apical foramen.[20],[26] Therefore, a potentially safer alternative to 5% NaOCl irrigation could be implemented when combined with Er, Cr:YSGG laser, where concentration levels of NaOCl could potentially be lowered to 2.5%.

The results from the current study demonstrated that laser treatment alone showed a significant reduction in bacterial numbers by approximately 50% when compared with the control group (P < 0.001) [Table 1]. It is, however, a treatment which should not be applied as it is insufficient at reducing the bacterial population inin vivo endodontic infections. Full (100%) elimination was obtained with the combined procedure of laser irradiation and chemical irrigation (group 2). This group proved to be an effective treatment method for the disintegration of root canal bacteria which is encased within a biofilm.

The efficacy of laser systems in endodontic disinfection is dependent on the specific wavelength and the interaction between the laser beam and the target structures.[27] Therefore, the use of laser systems in endodontic decontamination can be considered as a safe procedure as long as the correct parameters are used, furthermore it is a procedure which does not cause adverse effects on dental and periodontal tissues. Recently Yavari et al.[26] stated that 2 W and 3 W powers of Er, Cr:YSGG had antibacterial effects on E. faecalis in root canals. Arnabat et al.[18] indicated that 2 W laser for 60 seconds was as effective as 5% NaOCI when it came to reducing bacterial numbers. Yamazaki et al.[28] also revealed that carbonization and crack formation occurred all over the root canal surface when irradiation above 2 W was used, in the current study power settings of 0.75 and 2 W were applied.

This study also compared the effects of NaOCl and Er, Cr:YSGG laser on smear layer and the morphological alterations on dentinal surfaces. EDTA was not used since it has the potential to remove the smear layer after root canal preparation.[29],[30] The removal potential of Er, Cr:YSGG laser on smear layer was evaluated in the absence of EDTA. It has previously been demonstrated that mechanical instrumentation creates a smear layer of calcified detritus that adheres to the dentinal surface. In order to decrease the amount of smear layer on root canal surfaces, irrigants, and antiseptic agents are used, enhancing the effect of hand instrumentation and improving the efficiency of the sodium hypochlorite. However, studies have shown that a combination of NaOCl and EDTA removes the smear layer only partially.[29],[30],[31]

A correlation between the duration of Er, Cr:YSGG laser irradiation and its effectiveness in removing a smear layer and debriding the root canal walls was also noted by DiVito et al.[32] It was demonstrated that root canals irradiated for 40 s were significantly cleaner than those irradiated for 20 s. This result is in line with that of the current study, which demonstrated that standardized instrumentation, followed by a final Er, Cr:YSGG laser irradiation resulted in moderate smear layer removal when compared with traditional irrigation methods.

SEM studies demonstrated that the Er, Cr:YSGG laser at power settings of 0.75 and 2 W, removes the smear layer and debris from the root canal walls and opens up the dentinal tubule orifices, which would allow practitioners to seal the root canal tightly. Findings from previous studies have indicated that after irradiation without water spray, carbonization is seen in the enamel and dentin and is associated with an irregular structure and with many microholes.[28],[33],[34] As a result, throughout the current study laser irradiation with a water-air delivery system was applied which showed no melting or crystallization as was the case with the Schoop et al.' study,[33] however only cracks and fissures was observed in this investigation. It can be concluded that using Er, Cr:YSGG laser with a water-air delivery system decreases the possibility of melting and carbonization.


   Conclusion Top


Considering all the variables that were evaluated in the current study it can be concluded that the lower wavelengths can provide an additional aid in endodontic disinfection and can allow the operator to improve the predictability and the prognosis of endodontic treatments. In fact, even if the reduction of bacterial load through conventional chemo-mechanical treatments of root canals is sufficient, in most cases, to obtain a favorable prognosis, the possibility of increasing the bactericidal effect through the use of laser systems must be given serious consideration. Further research however is required to evaluate the clinical applications of the laser treatment system.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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