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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 2  |  Page : 108-114

Antimicrobial efficacy of photosensitized nanoparticles, diode laser, conventional, and sonic irrigation systems against enterococcus faecalis in root canals – An in vitro study


Departments of Conservative Dentistry and Endodontics, ITS Dental College, Greater Noida, Uttar Pradesh, India

Date of Submission31-Jan-2021
Date of Decision06-Aug-2021
Date of Acceptance01-Feb-2022
Date of Web Publication01-Jul-2022

Correspondence Address:
Dr. Ananya Sharma
Department of Conservative Dentistry and Endodontics, ITS Dental College, Greater Noida, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_38_21

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  Abstract 


Aim: This study aims to evaluate and compare the antimicrobial effect of Photosensitized Nanoparticles, Diode Laser (DL), and Conventional and Sonic Irrigation Systems against Enterococcus faecalis in root canals – An in vitro Study.
Materials and Methods: The root canals of 104 extracted human single-rooted teeth were prepared, and E. faecalis was incubated in the root canals for 1 week. The teeth were randomly divided into four experimental groups and one control group (n = 20): No treatment was done, sodium hypochloride (NaOCl) group (n = 20): Canals irrigated with5 ml of 2.5% NaOCl for 60 s, NaOCl and Endoactivator group (n = 20): Canals irrigated with 5 ml of 2.5% NaOCl for 60 s followed by activation of NaOCl with Endoactivator for 30 s, NaOCl and 910 nm DL group (n = 20): (1 W 5 times for 5 s) canals irrigation with5 ml of 2.5% NaOCl for 60 s followed by Photodynamic Therapy (PDT) with 910 nm DL, silver nanoparticles (AgNPs)/Indocyanine green (ICG)/DL group (n = 20): DL modified PDT with 5 min of irrigation with 5 mL AgNPs (100 ppm)/ICG/910 nm DL (200 mW, 30 s). A reduction in colony count was assessed by counting colony-forming units by Digital Colony Counter.
Results: Significant reductions were noted in E. faecalis colony counts in all groups (P < 0.05). The greatest reduction in colony count (99.93%) was noted in the NaOCl and DL group followed by NaOCl and Endoactivator group; however, the differences in this respect between the AN/ICG/DL group and NaOCl group were not significant (P > 0.05).
Conclusions: NaOCl and 910 nm DL were more successful in reducing the number of E. faecalis colony counts followed by NaOCl and Endoactivator group but it can also be concluded that PDT with ICG, an 910 nm DL, and AgNPs has the potential to be used as an adjunct for disinfection of the root canal system.

Keywords: Diode laser, Enterococcus faecalis, indocyanine green dye, nanoparticles, silver nanoparticles, sodium hypochloride, sonic irrigation


How to cite this article:
Sharma A, Kochhar R, Kumari M. Antimicrobial efficacy of photosensitized nanoparticles, diode laser, conventional, and sonic irrigation systems against enterococcus faecalis in root canals – An in vitro study. Endodontology 2022;34:108-14

How to cite this URL:
Sharma A, Kochhar R, Kumari M. Antimicrobial efficacy of photosensitized nanoparticles, diode laser, conventional, and sonic irrigation systems against enterococcus faecalis in root canals – An in vitro study. Endodontology [serial online] 2022 [cited 2022 Aug 8];34:108-14. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/2/108/349564




  Introduction Top


The long-cherished goal of endodontic treatment has been to eliminate infectious agents or substantially reduce the microbial load from the root canal.[1] During root canal infection, the microenvironment of root canal favors the selection of few bacterial species like Enterococcus faecalis, Streptococcus anginosus, and Fusobacterium Nucleatum.[2] E. faecalis is a nonfastidious, therapy resistant, Gram-positive facultative anaerobe that can proficiently invade dentinal tubules, survive chemomechanical instrumentation and intracanal medication, adapt to altered nutrient supply and remain viable within the dentinal tubules.[3] It can penetrate into the dentinal tubules and is capable of biofilm formation.[4] Hence, E. faecalis has gained interest of endodontists over the last few decades due to its complex nature. This calls for the need to eradicate this microorganism from the root canals.

Bacterial biofilm is highly resistant to conventional irrigants because of formation of the extracellular polymeric matrix. Hence, efficient disinfection of the root canal system is important for the prevention of reinfection.[5] It is achieved by a combination of mechanical instrumentation, along with the use of disinfecting solutions and placement of intracanal medicaments between subsequent appointments but large areas of the root canal system remain untouched, and therefore a lot has been expected from using various combinations of disinfecting solutions and irrigation devices.[6]

Irrigants have been traditionally delivered using a syringe and needle. The problem with this irrigation technique is inadequate replacement of the irrigant throughout the root canal system and the high surface tension of sodium hypochlorite (NaOCl) prevents direct contact of the irrigant with the dentinal walls of the anatomical complexities.[7]

Several mechanical devices have been developed to improve the penetration and effectiveness of irrigation in peripheral areas of the root canal space. The efficiency of sonic devices is based on hydrodynamic phenomenon to facilitate the disruption of biofilms and make the cell membrane of bacteria more permeable to NaOCl.

Apart from conventional irrigation, the use of high-power Diode lasers (DLs) is an innovative approach for disinfection, providing access to formerly unreachable parts of the tubular network, due to their ability to penetrate dental tissues better than irrigant solutions. Photodynamic therapy (PDT) is an adjunct method for the inactivation of bacteria. In PDT, a reaction occurs between light and a photosensitizer in the presence of oxygen. This reaction generates reactive oxygen species and causes oxidative damage to the respective. Indocyanine green (ICG) has gained special attention as a new photosensitizer. It is generally believed that ICG affects the target cell mainly through a photothermal effect rather than a photochemical effect and performs its bactericidal effect through oxidative stress cells.[2] Silver nanoparticles (AgNPs) have gained interest to be used as irrigants due to their antimicrobial properties and polyanionic/polycationic nature.[4],[8]

Therefore, this study was done using the method where the root canals were contaminated with E. faecalis at a particular concentration and then the reduction in colony-forming units (CFUs) was evaluated after using various irrigants.

The null hypothesis was that there is no difference in antimicrobial efficacy of Photosensitized Nanoparticles, DL, Conventional and Sonic Irrigation Systems against E. faecalis in root canals?


  Materials and Methods Top


Sample selection

For the present study, 104 single-rooted, single canal teeth extracted due to periodontal disease or carious lesion were taken as study samples. Approval was obtained from Ethical Committee of the ITS Dental Studies and Research (Reference No. Director PG Studies/ITSCDSR/L/2018/114). Sample size estimation was done by using GPower software (version 3.0). The sample size was estimated for F test and ANOVA: Omnibus fixed one way, for five groups with equal sample size was chosen. A minimum total sample size of 100 (20 per group) was found to be sufficient for an alpha of 0.05, power of 80%, 0.36 as effect size (assessed for the difference in S mutans colony counts among different study groups). Teeth were stored in 10% formalin and roots were cleaned with curette. The following exclusion criteria were established: Teeth with immature root apices, Root canal curvatures, Root Caries, Previous endodontic treatment, Fractured root, Cracks.[9]

Preparation of specimen

One hundred and four human single canal teeth were decoronated to a standardized root length of 12 mm using diamond discs. The teeth were radiographically confirmed to have a single canal.

A working length of 11 mm was established by introducing #10 or #15 K file into the canal until the apical foramen and then subtracting 1 mm from the length and were prepared using ProTaper Rotary System up to size F3 as master apical file.[10]

Each canal was irrigated with 1 ml of 2.5% NaOCl between each instrument and final irrigation was done with 1 ml of 17% EDTA for 2 min followed by final rinse with 1 ml of 2.5% NaOCl and 1 ml of saline solution. Canals were dried with the help of sterile paper points.

The apical foramen of each tooth was sealed with composite resin and root surfaces were covered with dental varnish and embedded in acrylic resin.[10]

Sterilization of specimen

Specimens were collected in Eppendorf tube and autoclaved at 121° C at 15 atm pressure for 15 min.[10]

Cultivation of Enterococcus faecalis

The suspension was prepared by mixing a pure culture of E. faecalis ATCC29212 grown in BHI Agar for 24 h at a concentration of 1.5 × 108 CFU/ml (0.5 McFarland) in BHI Broth.[10]

Bacterial inoculation of root canals

Prepared root canals were filled with 15 μL of suspension using insulin syringe, carried to entire length by #15 K file and incubated at 37°C for 7 days at 100% humidity.

Canals were reinoculated at 4th and 6th days after initial inoculation and after the incubation period, any residual medium was removed with sterile paper points.[9]

SEM analysis

Four samples were stored in 10% buffered formalin and subjected to Scanning Electron Microscopy to visualize the pattern of colonization. They were longitudinally split sectioned using a diamond fissure bur and a chisel. The samples were dehydrated in aqueous ethanol solutions in ascending order (25%, 50%, 75%, 95%, and absolute alcohol twice), for 20 min , mounted on aluminum scanning electron microscopic stubs and sputter coated with a gold–palladium alloy under a vacuum. The samples were examined using a scanning electron microscope (JSM6510 LV JEOL, Japan) [Figure 1].[9]
Figure 1: Pictures of sem analysis of 4 samples at 2000X (a), 5000X (b) AND 10,000(c) showing pattern of colonisation of Enterococcus faecalis

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Sampling

Remaining 100 samples (n = 100) were randomly allocated in four experimental groups (n = 20) each and control group (n = 20).

Experimental procedures

Group 1: Control group (n = 20) Negative control: No irrigation was performed.[9]

Group 2: Conventional irrigation (n = 20) Irrigation was done with 5 ml of 2.5% NaOCl for 60 s using 5 ml 30 gauge side vented needles which were kept 2 mm short of working length.[9]

Group 3: 2.5% NaOCl using endoactivator irrigation system (n = 20) Irrigation was done with 5 ml of 2.5% NaOCl for 30 s followed by NaOCl activation for 30 s using Endoactivator. The red tip instrument size 25, with 0.04 taper was placed 2 mm short of the working length and was moved up and down in short vertical strokes.[9]

Group 4: 2.5% NaOCl plus DL (n = 20) Irrigation was done with 2.5% NaOCl and left in canal for 5 min, dried with paper point then irradiation with 940 nm DL (1W, CW) was done 5 times for 5 s each time, with a 15 s interval between irradiation. It was delivered 1 mm short of working length in circling movements from apical part toward coronal part.[9]

Group 5: silver nanoparticles (AgNPs)/ICG/DL (n = 20) Suspension of 100 ppm AgNPs and ICG in 1:40 ratios was injected in root canal following canal irradiation with 940 nm DL (200 mW, CW) after 5 min for 30 s.[10]

Microbiological procedure

Following experimental procedures canals were filled with 10 μL 0.9% Physiologic Saline solution and 3 aspiration delivery cycles were performed following which the canal content was finally aspirated and transferred to Eppendorf Tube containing 90 μL 0.9% Physiologic Saline to achieve a 10 × 1 dilution rate.

After dilution and agitation in vortex for 1 min, aliquots of 10 μL were plated on BHI agar plate and incubated for 48 h at 37°C in 100% humidity.

Following incubation, the growth of E. faecalis was counted using digital colony counter and were compared [Flowchart 1].[9]



Statistical analysis

Intergroup comparison of Colony Counts was done using one-way Analysis of Variance test [Table 1]. Post hoc pairwise comparison was done using Tukey's test [Table 1]. The Statistical analysis was performed by using IBM SPSS Statistics for Windows, version 17.0, Armonk, NY, USA: IBM Corp. The significance level was set at 5%.
Table 1: Descriptives of mean colony counts , standard deviation and significance among different study groups

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  Results Top


The mean Colony Count of all the five groups was found to be significantly different from each other (P < 0.05). It was found that the mean Colony Counts of Group 1 (control group) was found be significantly higher than that among all other study groups. The Mean Colony Counts among Group 2 and Group 5 specimens were found to be significantly higher than that among Group 3 and Group 4. No statistically significant differences could be found among Group 2 and Group 5. Furthermore, no statistically significant differences could be found among Group 3 and Group 4.


  Discussion Top


The null hypothesis was rejected as a difference was found in the antimicrobial efficacy of Photosensitized Nanoparticles, DL, and Conventional Irrigation Systems against E. faecalis in root canals. The antimicrobial efficacy of irrigants in microbiology is evaluated by two assays, Disc Diffusion Assay and Minimum Inhibitory Concentration Assay. However, they do not simulate the oral environment.

Therefore, this study used the method where the root canals were contaminated with E. faecalis at a particular concentration and then the reduction in CFUs was evaluated. That gave the exact efficacy of the various methods of irrigation being used in the study.

The result showed that minimum colony counts were seen in group in which irrigation was done with NaOCl followed by activation with DL indicating that it has more antibacterial efficacy when compared to other irrigants such as NaOCl alone or AgNPs with ICG dye and DL. These findings could be attributed to the fact that DL has a high penetration of around 500 microns in the irregularities of root canal system and also this improvement of the antibacterial activity of NaOCl solution has been attributed to the cavitational effect of laser and irrigation solution.

The strong absorption of DL energy in NaOCl causes vaporization and the formation of large elliptical vapor bubbles. The vapor bubbles cause a volume expansion of up to 1600 times the original volume of an irrigant, with a high intracanal sheer pressure that drives the fluid into the deep dentinal tubules and can remove the smear layer, and potentially destroy the bacterial cell. These results justified the literature published by Wang et al.[11] in 2018, Tokuc et al.[12] in 2019, which stated that the use of laser activated irrigation (LAI) with NaOCl promoted the antibacterial efficacy of sodium hypochlorite against E. faecalis. Other studies done in the following years by Souza et al.[13] in 2010, Nunes et al.[14] in 2011, Cheng et al.[15] in 2012, Yao et al.[16] in 2012, Bago et al.[9] in 2012, Balić et al.[17] in 2015 and Liu et al.[18] in 2018 stated that the use of LAI with NaOCl promoted the antibacterial efficacy of sodium hypochlorite against E. faecalis. The literature published that does not support our study was by Meire et al.[19] in 2009 where the laser systems were less effective than NaOCl in reducing E. faecalis, both in aqueous suspension and in the infected tooth model which can be attributed to the fact that in this study the time of exposure to 2.5% NaOCl contact with E. faecalis was 15 min whereas for KTP and Nd: YAG laser it was 25 s; PAD for 150s.

Reduction in colony count was more in the group in which NaOCl was agitated with Endoactivator than NaOCl alone. This could be due to the fact that disinfection agents such as NaOCl require direct contact with the bacteria which is often impossible in peripheral areas of the root canal such as anastomoses, fins, and the most apical part of the main root canal. The EndoActivator system has been reported to provide deeper penetration of an irrigant to all areas of the endodontic space, and to effectively clean debris from lateral canals, remove the smear layer and dislodge clumps of simulated biofilm. This is in accordance with the study done by Bago et al.[15] in 2012. The other studies which showed a similar result were done by Pasqualini et al.[20] in 2010, Jonathan et al.[27] in 2013, Srivastava et al.[10] in 2017, Pedullà et al.[21] in 2019. The literature published that does not support our study was by Abdel Latif et al.[22] in 2016, which stated that there is no difference in reduction in mean colony count when irrigation was done with either NaOCl alone or NaOCl agitated with Endoactivator. These differences could be due to wide apical preparation to size 40 which facilitates insertion of irrigation devices short of working length as well as distribution and exchange of irrigants and by Rodig et al.[23] in 2017 which used NaOCl and Qmix 2in1 irrigants agitated by Endoactivator and alone, stated that difference in antimicrobial efficacy depends on type of irrigant used.

Reduction in colony count was almost the same when irrigation was done with NaOCl photodynamically activated by DL) and NaOCl agitated with endoactivator with the former group being more effective. This is accordance with the study done by Bago et al.[9] in 2013. The reason attributed to this could be the different but effective mechanism of action of both DL and endoactivator as discussed in the earlier section. This also supported by other studies done by Jonathan et al.[12] in 2013.

The reduction in colony count was more in the group in which irrigation was done with AgNPs, ICG Dye followed by photodynamic activation with DL than the control group. The literature published that supports this is done by Wu et al.[24] in 2014.This can be due to the properties of AgNPs. They interact with multiple targets in the microbial cell, such as cell membrane, enzymes, and plasmids, simultaneously providing the bacteria least capacity to gain resistance.

The reduction in colony count was more in the group in which irrigation was done with AgNPs, ICG Dye followed by photodynamic activation with DL than NaOCl alone but not stastically significant. This is in accordance with the study done by Beltes et al.[25] in 2017, Afkhami F et al.[26] in 2017. The reason could be that PDT can effectively decrease the number of bacteria present in the biofilm as near-IR (infrared) wavelengths are more capable of penetrating into tissues compared with other light spectra. ICG is a photosensitizer used for near-IR wavelengths with an optical density (absorbance) at around 800 nm.

The reduction in colony count was found to be more in the group in which irrigation was done with NaOCl and DL and NaOCl followed by agitation with Endoactivator than the group in which AgNP, indocynine green dye was used followed by photodynamic activation with DL for irrigation. There has been no previously published literature that favors or contradicts these results obtained from this study, so this can serve as a scientific evidence for further researches to be carried out in this context.


  Conclusions Top


Within the limitation of this study, it can be concluded that NaOCl and 940 nm DL was most successful in reducing the number of E. faecalis colony counts followed by NaOCl agitation with Endoactivator. It can also be concluded that PDT with ICG, and 940-nm DL, and AgNPs has the potential to be used as an adjunct for disinfection of the root canal system. However, further research and clinical trials are required to assess antimicrobial efficacy of NaOCl with DL and AgNPs with ICG Dye and DL against E. faecalis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Srivastava S, Kukreja M, Kharbanda S, Grover R, Paliwal A. Effect of different methods of agitation on penetration of an endodontic irrigant. Endodontology 2017;29:156-9.  Back to cited text no. 10
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Souza LC, Brito PR, de Oliveira JC, Alves FR, Moreira EJ, Sampaio-Filho HR, et al. Photodynamic therapy with two different photosensitizers as a supplement to instrumentation/irrigation procedures in promoting intracanal reduction of Enterococcus faecalis. J Endod 2010;36:292-6.  Back to cited text no. 13
    
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