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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 2  |  Page : 94-101

A randomized clinical trial of antimicrobial efficacy of photoactivated disinfection, conventional endodontic irrigation and their combination in primary endodontic infections


Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana, India

Date of Submission05-Jun-2020
Date of Acceptance27-Oct-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Dr. Varsha Jain
Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Sector 89, Kheri More, Faridabad - 121 002, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijohs.ijohs_20_20

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  Abstract 


Aim: The aim of this study is to evaluate and compare the antimicrobial efficacy of photoactivated disinfection (PAD), conventional irrigation (CEI), and combination of both against primary endodontic infections in vivo.
Methods: Active controlled and parallel design trial was registered with Clinical Trials Registry India under reference no. CTRI/2018/08/015478. Forty-eight qualified patients were randomly divided into three groups (n = 16 each) using the lottery method, Group-I: CEI, Group-II: PAD, Group-III: CEI and PAD. Access opening and working length were determined, and preinstrumentation sample (S1) was taken by inserting sterile paper point into the canal. Postinstrumentation sample (S2) was collected following biomechanical preparation and irrigation with respective interventions. Microbiological samples (S1, S2) were plated on brain heart infusion agar. Colony-forming units were counted using the classic bacterial counting method, and percentage bacterial reduction was determined.
Statistical Analysis: Inter- and intra-group comparison was made using repeated measures of ANOVA. Post hoc Tukey analysis was done for pairwise comparison with P < 0.05.
Results: In the intragroup comparison, statistically significant reduction was seen at S2 among all experimental groups. However, in intergroup comparison, mean bacterial reduction in Group III was significantly lower than the other groups. This difference was statistically significantly better between Group I and Group III; Group II, and Group III. However, no statistically significant difference was seen between Group I and Group II. Within the limitations of this study, it can be concluded that a combination of CEI and PAD showed significantly better antimicrobial activity against primary endodontic infections and can be recommended as an adjunct to facilitate maximum root canal disinfection.

Keywords: Irrigation, microbial reduction, sodium hypochlorite, toluidine blue


How to cite this article:
Mittal R, Tandan M, Jain V. A randomized clinical trial of antimicrobial efficacy of photoactivated disinfection, conventional endodontic irrigation and their combination in primary endodontic infections. Int J Oral Health Sci 2020;10:94-101

How to cite this URL:
Mittal R, Tandan M, Jain V. A randomized clinical trial of antimicrobial efficacy of photoactivated disinfection, conventional endodontic irrigation and their combination in primary endodontic infections. Int J Oral Health Sci [serial online] 2020 [cited 2021 Jun 15];10:94-101. Available from: https://www.ijohsjournal.org/text.asp?2020/10/2/94/309444




  Introduction Top


It is well known that diseases of the dental pulp and periradicular tissues are associated with microorganisms.[1] Microorganisms can exist within the root canals, dentinal tubules, accessory canals, canal ramifications, apical deltas, fins, and transverse anastomoses.[2] These microbes in the root canals grow not only as planktonic cells or in aggregates, co-aggregates, but they can also form biofilms consisting of a complex network of different microorganisms.[3] Thus, the success of endodontic treatment depends on how well the pathogenic microflora from the root canal system is eliminated, which allows periapical tissues to heal.[4]

The role of instrumentation during root canal therapy is to remove all of these bacteria along with vital tissue and nonvital tissue contributing to the infection to facilitate irrigation and expose the bacteria in the dentinal tubules to antibacterial agents. Thus, mechanical debridement along with chemical irrigation and intracanal medicaments aids in the removal of the bulk of microorganisms.[5]

Ideally, root canal irrigants should have a broad antimicrobial spectrum and high efficacy against anaerobic and facultative microorganisms organized in biofilms, dissolve necrotic pulp tissue remnants, inactivate endotoxin, prevent the formation of a smear layer during instrumentation or dissolve the latter once it has formed, be systemically nontoxic, be noncaustic to periodontal tissues, does not cause an anaphylactic reaction.[6] Various irrigants/medicaments are advised for disinfecting the root canal as well as for the removal of microorganisms from inaccessible areas.[7] Commonly used irrigants are sodium hypochlorite, ethylenediaminetetraacetic acid (EDTA), chlorhexidine gluconate, hydrogen peroxide, mixture of Tetracycline acid detergent, citric acid, maleic acid.[7],[8]

Sodium hypochlorite is the most common endodontic irrigant introduced by Walker in 1936.[9] It is used in concentration ranging from 0.2% to 5.25%.[10] It is a potent antimicrobial agent with a pH >11 and exhibits excellent tissue dissolving properties. It presents antimicrobial activity by irreversible inactivation of bacterial essential enzymes owing to hydroxyl ions and chloramination action.[11] However, it is unable to penetrate at greater depths in dentin. Long-standing exposure to increased concentrations of sodium hypochlorite might have a prejudicial result on dentin elasticity and flexural strength, thereby predisposing the tooth to vertical fracture.[12],[13] NaOCl irrigation leads to decreased bond strength between dentin and resin cements. Moreover, it has an unpleasant taste and does not remove the smear layer by itself. It also possess risk of emphysema on apical extrusion and discoloration of clothes.[13],[14] Thus, due to the potential side effects, safety concerns, and limitations in instrumentation methods, there is difficulty in complete cleaning of root canals, making it necessary to develop alternative procedures to optimize the disinfection process.[5]

Recently, divergent irrigation techniques and devices have been developed to upgrade the cleaning of the root canal system. Ultrasonic or sonic activation, negative pressure irrigation, sound wave-induced hydrodynamic forces, photon-induced photoacoustic streaming, lasers, and photoactivated disinfection (PAD) have all been promoted among the previous couple of years to enhance the penetration and efficiency of canal irrigation.[15] In dentistry, photodynamic therapy (PDT) is used for the treatment of gingivitis, periodontitis, periimplantitis, pericoronitis, oral-candidiasis, decontamination of decayed dentine, and disinfection of root canals.[4],[16]

PDT employs either ultraviolet or visible light, with a photosensitizer.[17] Different light sources can be used in PDT, such as light-emitting diode (LED) or lasers.[17],[18] Methylene blue and toluidine blue O (TBO) are the most common phenothiazine dyes used in dentistry. These dyes are activated in red light spectrum, i.e., 666 nm and 630 nm, respectively.[16],[18] The operating principle is that photosensitizer (PS) molecules attach to the membrane of bacteria. Irradiation with the light of a specific wavelength coordinated with the peak absorption of PS leads to the formation of singlet oxygen, which triggers the bacterial cell wall to rupture, killing the bacteria resulting in antibacterial effect.[10]

The antimicrobial effects of PDT on root canal microorganisms have been evaluated in several in vitro[16],[19],[20] and in vivo[10],[17],[21] studies, but no study has been reported that has been done in vivo that uses commercially available medical device – fotosan630 along with TBO photosensitizer for the specific purpose of PAD. The positive results of in vitro studies with Fotosan 630 became the inspiration to test this novel combination therapy in a clinical trial. Hence, this study was undertaken to evaluate the efficacy of PAD using TBO PS in primary endodontic infections and to compare it with that of conventional endodontic irrigation and its combination in patients.


  Methods Top


This study is a randomized, active controlled, parallel design, single-center and single-blinded clinical trial. It was designed and reported by adhering to consolidated standards of reporting trials statement. It was approved by ethical committee, and the study protocol was registered with Clinical Trials Registry of India before enrollment of patients for the study under reference no. CTRI/2018/08/015478. The written informed consent was obtained from all the participants.

Sample size and selection of patients

Patients referred for endodontic treatment were screened for enrollment at the Department of Conservative Dentistry and Endodontics. After comprehensive clinical and radiological examination, 66 patients of 15–55 years of age were enrolled. The sample size was estimated using Gpower software (version 3.0). The sample size of 48 (16 per group) was found to be sufficient for an alpha of 0.05, power 95%, 0.6 as effective size (difference in percentage reduction in bacterial colony counts from pre- to post-instrumentation). Patients with noncontributory medical history, single-rooted permanent teeth without previous restoration, with necrotic or infected pulp as diagnosed clinically and radiographically with the adequate coronal structure for proper isolation, temporization, and restoration were included. Patients with systemic conditions, acute periapical abscess, retreatment cases, patients on antibiotic therapy within 3 months, teeth with calcified canals, sinus opening, immature apex, internal and external resorption, or periodontal pockets >5 mm and pregnant women were excluded [Figure 1].
Figure 1: CONSORT 2010 flow diagram

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Sequence generation and intervention

The patients were divided using simple randomization method with an allocation ratio of 1:1:1. One of the investigators used the lottery method to implement the random allocation sequence, thereby assigning participants to intervention.

Clinical procedure

The oral cavity was disinfected with chlorhexidine solution. The tooth was anesthetized and isolated with rubber dam. Following caries removal, access cavity was prepared using sterile burs. Working length was determined with an electronic apex locator and it was confirmed radiographically. Moreover, to ensure surface decontamination and making certain that the sample is collected from the root canal system only, the tooth surface and surrounding operative field was swabbed with 5.25% NaOCl for 30 s and was inactivated with sterile 5% sodium thiosulfate.

Pretreatment sample (S1) was obtained under aseptic condition by injecting normal saline (5 ml) into the root canal and circumferentially pumping #10K file (1 mm short of working length). A sterile paper point was placed into the canal for 60 s and transferred to test tube containing freshly prepared peptone water as transport media. The obtained sample was sent to the laboratory within 30 min for processing. Biomechanical preparation was done using step-back technique up to master apical size #45 and step back till #60. After biomechanical preparation, the canal was dried, and the patients were randomly divided into three groups:

  • • Group I: Conventional endodontic irrigation (n = 16) Canals were irrigated with 10 mL of 2.5% sodium hypochlorite, 0.9% normal saline, 10 mL 17% EDTA solution and then final flush was done with 0.9% normal saline.
  • • Group II: PAD (n = 16) Canals were filled with 0.01% of the toluidine blue photosensitizer and stirred with #10K file to avoid bubbles. Endo light tip of Fotosan 630 was inserted into the canal approximately till the middle third or until resistance was met and irradiated for 30 s. Then, the final flush was done with 0.9% normal saline.
  • • Group III: Conventional Endodontic Irrigation with PAD (n = 16).


Canals were irrigated with 10 mL of 2.5% NaOCl, 0.9% normal saline, 10 mL of 17% EDTA solution and followed by a flush with 0.9% normal saline. Fotosan 630 was used for disinfection with toluidine blue as photosensitizer in the same manner as in Group II.

Postchemo-mechanical preparation, a sample (S2) was collected in the same manner as S1 after surface decontamination with 5.25% NaOCl for 30 s.

Homogenization, dilution, and seeding

Microbiological samples (S1, S2) were incubated for 30 min and shaken vigorously in a vortex mixture for 60 s. Decimal dilutions were performed in 4.5 ml of peptone water up to 10−6. Fifty microliter aliquots of these decimal solutions were seeded on to the plate surfaces containing brain heart infusion agar (HiMedia Laboratories Pvt. Ltd., India) with a micropipette using aseptic technique. Three paper point samples were taken for each tooth at both stages.

To check the quality of aseptic technique, the culture medium and peptone water were incubated at 37°C overnight without intentionally adding bacteria.

Cultivation conditions

Subsequently, the cultures were incubated for 24 h at 37°C in an incubator under aerobic conditions. Colony-forming units (CFUs) were counted after 24 h using the classic bacterial counting method.

Blinding

The microbiologist was blinded to the intervention performed in group of samples provided to him for culturing and counting of bacterial colony counts. Furthermore, to eliminate bias, the data analysts were kept blinded to the intervention. Because of the nature of interventions, the operator was not blinded to the interventions.

Patients were recalled after 7 days. The tooth was isolated using rubber dam, and disinfection was done by 0.2% chlorhexidine solution (Hexidine Mouthwash, ICPA Health Products Ltd.). The temporary restoration was removed using a round diamond bur and an air rotor handpiece. Teeth were obturated by lateral condensation technique using #45 gutta-percha (Metabiomed, India) as master cone (after radiographic verification) and AH Plus sealer (Dentsply, Germany). The access cavities were then restored with composite (Spectrum, Dentsply India Pvt. Ltd., India).

Statistical analysis

The normality of data was checked using Shapiro–Wilk test. Data reached normality. Thus, inferential statistics were performed using parametric tests of significance. Inter- and Intragroup comparison was made using repeated measures of ANOVA. Post hoc Tukey analysis was done for pairwise comparison. The level of significance was set at < 0.05.


  Results Top


The antibacterial efficacy was measured by counting CFUs from samples taken at different stages; that is, after access opening (S1), after biomechanical preparation and irrigation with respective irrigants (S2). Their mean bacterial reduction was calculated. When intragroup comparison of CFUs showed statistically significant reduction from S1 to S2 among all of the experimental groups [Figure 2].
Figure 2: Bar diagram of mean bacterial count at stages S1 and S2 for the three experimental groups

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When the intergroup comparison was made, mean bacterial counts in the postirrigation samples of Group III were found to be significantly lower than that among Group I and Group II [Figure 2]. The mean percentage reduction was statistically significant between Group I and Group III; Group II, and Group III. However, the difference was not statistically significant between Group I and Group II [Figure 3].
Figure 3: Mean percentage reduction of bacterial count ([S1 − S2]/S1 × 100) for conventional endodontic irrigation, photoactivated disinfection and their combination in primary endodontic infections

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  Discussion Top


PAD is a laser-induced photochemical disinfection, which is based on the activation of a nontoxic PS by low-level laser energy.[22] PAD combines a nontoxic photoactivatable dye or PS in combination with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state that will lead to the formation of cytotoxic reactive oxygen species (ROS).[23] Various combinations of light sources (diode laser at 630 nm, 660 nm, and 670 nm; Helium: Neon laser) and dyes (methylene blue, tolonium chloride) have been investigated and are commercially available.[22] The mechanism of action for ROS can be classified into those produced by Type I photochemical mechanism: free radicals such as hydroxyl radicals (HO.) and those produced by Type II photochemical mechanism: singlet oxygen (1O2) that directly destroy the microorganisms.[23] The ROS reacts strongly and destroys the microorganisms instantly and effectively.[24] The bactericidal effects of PDT have been observed to be due to the destruction of nucleic acid and/or the cytoplasmic membrane caused by these ROS.[25]

Based on the principles described above, a system has been developed for endodontic use consisting of a LED (Fotosan; CMS Dental, Copenhagen, Denmark). It has an endodontic tip with its apical size of 500 μm and a 0.03 taper in the apical part.[26] The energy output of the device is 2.000 mW/cm2–4.000 mW/cm2.[27] Rios et al. did an in vitro study to evaluate the antimicrobial effect of PDT using Fotosan 630 after the conventional disinfection protocol with 6% NaOCl. It was observed that PDT for 30 s alone exhibited a 2.9% survival rate of Enterococcus faecalis, whereas the combination of NaOCl followed by PDT, lowered the survival rate to 0.1%. Thus, Fotosan 630 device, along with TBO photosensitizer has the potential to be used as an adjunctive antimicrobial procedure in endodontic treatment.[28] These results were also in accordance with a study conducted by Poggio et al. who evaluated the antimicrobial effect of PAD using Fotosan 630 device and compared it with conventional 5.25% NaOCl irrigating solution and observed that PAD associated with 5% NaOCl showed the significantly higher antibacterial effects.[24]

Many in vitro[18],[19],[20] studies and few in vivo[10],[17],[21] studies have been done on the evaluation and comparison of antimicrobial efficacy of PAD, conventional endodontic irrigation, and their combination, but no study has been reported that has been done in vivo that uses commercially available medical device along with photosensitizer for the specific purpose of PAD. Therefore, this study was undertaken to comparatively evaluate the antimicrobial efficacy of PAD, conventional endodontic irrigation and their combination in primary endodontic infections by counting the colonies formed after culturing the samples taken from the patients at different stages, i.e., preinstrumentation (S1), and postinstrumentation (S2).

Teeth with acute clinical signs or with sinus tracts were excluded, as they have the greater and variable bacterial load.[29] Patients with previous restoration and retreatment cases were excluded as they form biomaterial-centered biofilms, which cause the bacteria to convert into a viable but noncultivable state.[30] Patients with the adequate coronal structure were included for proper isolation, temporization, and restoration. A negative control group with no irrigation/irrigation with the only saline was not included due to ethical concerns. In the present study, 2.5% sodium hypochlorite was used as a part of conventional irrigation (CEI) procedure owing to its potent antimicrobial action, its dissolution capacity of pulpal remnants, and collagen.

In the group undergoing PAD and combination of CEI and PAD, the canals were irradiated at 630 nm as recommended by the manufacturer for 30 s. Based on a study conducted by Schlafer et al. who evaluated PAD treatment of microorganisms in planktonic suspension using Fotosan 630 and recommended photosensitizer along with it and found that irradiation of 30 s achieved 100% bacterial reduction.[26] A 27G safe-ended/side irrigation tip was used as this prevents accidental extrusion of the solution apically.[31]

The results of this study demonstrated that percentage reduction in mean bacterial count from baseline to postirrigation was significantly higher in Group III as compared to that in Group I and Group II. The percentage reduction of bacterial count from baseline to postirrigation among Group II was higher than that of Group I, but this difference was not statistically significant.

In the present study, combination of sodium hypochlorite and PAD showed the highest reduction of the mean bacterial count. These results are in accordance with the results obtained from several in vitro studies.[10],[18],[20] The plausible reason for the better antimicrobial activity of the combination of conventional endodontic irrigation and PAD could be that the bacterial load was reduced initially by the CEI procedure and the remaining viable bacteria were disinfected by PAD.[32] Various in vitro studies have shown that PAD is active against a wide range of micro-organisms, such as Gram-positive and Gram-negative bacteria such as E. faecalis, Porphyromonas gingivalis, Fusobacterium nucleatum, P. gingivalis, Peptostreptococcus micros, Prevotella intermedia and decreases the intracanal microorganisms by accessing hard to reach areas.[17],[33] The ability of the Fotosan 630 device to access hard to reach areas can be attributed to its specially designed fiber tip, which distributes light homogeneously inside the root canal, thereby guaranteeing better photo-reaction and contributing to its high antimicrobial activity.[21] Rios et al. who evaluated the antimicrobial effect of PDT using a LED lamp (fotosan 630) with and without the conventional disinfection protocol of 6% NaOCl on extracted human teeth infected with E. faecalis and found that the bacterial survival rate of the NaOCl/TBO/light group (0.1%) was significantly lower (P < 0.005) than the NaOCl (0.66%) group.[28] Ng et al. conducted an ex vivo study to evaluate the antimicrobial effects of PDT on infected human teeth. The results showed that PDT significantly reduces residual bacteria within the root canal system and holds substantial promise as an adjunct to chemomechanical debridement.[34]

However, the percentage reduction observed with a combination of PAD and CEI (76.88%) was less as compared to that observed in several in vitro studies (90%–98%). This variation in results could be attributed to the different light parameters and wavelength, the concentration, type, and volume of photosensitizer used, and light delivery techniques.[20] It is evident in various in vitro studies that they are done for planktonic bacteria, whereas biofilms are more prevalent in clinical situations. One thousand-fold more resistant microbes are found in biofilms toward antimicrobial agents and host defense mechanisms than their planktonic counterparts. Furthermore, bacterial cells grow more slowly in biofilms than in their planktonic state and, therefore, take up antimicrobial agents more slowly.[35] Thus, initial biofilms in primary endodontic infections are more complex and difficult to disrupt and eradicate. In addition, lesser reduction than other studies can also be attributed to the anoxic root canal conditions.[17],[18] It is known that PAD utilizes photoactive compound (tolonium chloride) and a directly applied visible light (LED illumination at 635 nm), which form metachromatic complexes with lipopolysaccharides that can be photo-activated to cause oxygen ion release.[36] The oxygen ions are specifically toxic to a vital structural component of the target cells. However due to limited diffusion of the PS into the biofilm, results in restricted production of ROS that may interfere with the efficacy of a PDT for root canal disinfection.[37]

Furthermore, in the present study, it was found that PAD alone was as effective as sodium hypochlorite in reducing the number of bacteria. Although the difference was not statistically significant between the groups but the percentage of bacterial reduction was more in the PAD group. These results are in accordance with the results of Yildirim et al. and Xhevdet et al. who found that PDT was as efficacious as CEI with 5% NaOCl in reducing E. faecalis.[38],[39] This can be attributed to various factors that involve photosensitizer, photo-activating device, and intraoral conditions. There are two mechanisms of action that have been proposed for lethal damage caused to bacteria by PAD, (i) DNA damage (ii) damage to the cytoplasmic membrane, allowing cellular contents or inactivation of membrane transport systems and enzymes.[40] In this study, TBO photosensitizer was used, and it has been proved that it has a greater depth of penetration as compared to sodium hypochlorite. Williams et al. conducted a study on the photo-activated antibacterial action of TBO in a collagen matrix and in carious dentine and it is found that penetration depth of TBO dye after 30 s was about 300 μm and after 1 min was 400 μm where as in a study conducted by Giardino et al. penetration of sodium hypochlorite into human dentine ranged from 74 to 131 μm.[12],[41] The poorer action of NaOCl is probably caused by problems in penetration to the most peripheral parts of the root canal system such as fins, anastomoses, apical canal, lateral canals, and dentinal canals. Furthermore, the presence of inactivating substances for instance, pulp tissue, exudate from the peripheral area, and microbial biomass may counteract the effectiveness of NaOCl.[13]

Limitations and strengths

First, nonblinding of the operator may be a limitation, although a large experimental bias is unlikely. The selection bias was considered to be minimized because of the generation of a suitable randomized sequence. Even though PDT has significant advantages, potential adverse events as tooth discoloration have been reported in root canal treatment when TBO were used as PS. It is also important that future clinical studies clearly report adverse events associated with PDT so that an estimation of the benefit-to-risk ratio from the use of PDT is feasible. Nonetheless, there were no adverse effects mentioned in this study, quantification of total viable bacteria was used, instead of the quantification of species and genus separately. Therefore, future studies should be directed to assess the efficiency of promising molecular approaches by vital dye for the quantification of viable microbiota in the samples.


  Conclusion Top


Therefore, within the limitation of this study, it can be concluded that the combination of CEI with PAD has shown significant antimicrobial activity in primary endodontic infections when compared to conventional endodontic irrigation and PAD alone. Hence, PAD can be used as an adjunct to conventional endodontic irrigation in primary endodontic infections to ensure maximum disinfection.

Acknowledgment

I affirm that we have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultant ships, patent licensing arrangements, or honoraria), or involvement with any commercial organization with direct financial interest in the subject or materials discussed in the manuscript, nor have any such arrangements existed in the past years.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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