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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx

Available online at www.sciencedirect.com

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Photoactivation of curcumin and sodiumhypochlorite to enhance antibiofilmefficacy in root canal dentin

Prasanna Neelakantana,∗, Cheng Qing Chenga,Vinoddhine Ravichandrana, Teresa Maoa, Priyanka Sriramana,Swetha Sridharana, Chandana Subbaraoa, Subash Sharmaa,Anil Kishenb

a Biofilm Research Cluster and Department of Conservative Dentistry and Endodontics,Saveetha Dental College and Hospitals, Saveetha University, Chennai, Indiab Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Canada

KEYWORDSBiofilm;Confocal microscopy;Curcumin;Enterococcusfaecalis;Photosensitizer;Sodium hypochlorite

SummaryBackground: To test the effect of ultrasonic or light activated curcumin and sodium hypochloriteagainst Enterococcus faecalis biofilms in vitro.Methods: E. faecalis biofilms were grown within root canals (n = 175) and divided into 7 groups(n = 25). Group 1, sterile saline; group 2, 3% sodium hypochlorite; group 3, 3% sodium hypochlo-rite activated with ultrasonic files (30 s cycles for 4 min); group 4, 3% sodium hypochloriteirradiated with blue light (1200 mw/cm2 for 4 min); group 5, curcumin (2.5 mg/mL); group 6,curcumin (2.5 mg/mL) activated with ultrasonic files (30 s cycles for 4 min); group 7, curcumin(2.5 mg/mL) irradiated with blue light. The biofilms’ ultrastructure was examined using scan-ning electron microscopy. Bacterial viability was assessed by confocal microscopy. Data wereanalyzed by one-way ANOVA and Student—Newman—Keuls test (P = 0.05). The quantitative anal-ysis of the colony-forming units was carried out from dentinal shaving and analyzed by One-wayANOVA and Tukey multiple comparison test (P = 0.05).Results: All treatment groups showed a significantly higher percentage of dead bacteria thanthe saline control (P < 0.05). The percentage of dead bacteria was significantly higher when lightactivated curcumin was used (P < 0.05). At both depths (200 and 400 microns), light activatedcurcumin showed no growth of bacteria.

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Conclusions: Light activation produced significantly higher antibacterial efficacy than ultrasonicagitation, with light activated cuwithin the root canal lumen and© 2014 Elsevier B.V. All rights re

∗ Corresponding author at: Plot 1500, 16th Main Road, Anna Nagar Wesfax: +91 044 2616 3639.

E-mail address: prasanna neelakantan@yahoo.com (P. Neelakantan).

http://dx.doi.org/10.1016/j.pdpdt.2014.10.0111572-1000/© 2014 Elsevier B.V. All rights reserved.

l. Photoactivation of curcumin and sodium hypochlo-n. Photodiagnosis and Photodynamic Therapy (2014),

rcumin producing the maximum elimination of biofilm bacteria dentinal tubules.served.

t, Chennai, Tamil Nadu, India. Tel.: +91 98847 54914;

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hotodynamic therapy has shown great potential in thereatment of localized bacterial infections [1]. It involveshe photoactivation of a photosensitizer with low-energyoherent or non-coherent light, in the presence of oxygeno produce reactive oxygen species, such as hydroxyl radi-als, superoxides and singlet oxygen. These reactive oxygenpecies act on multiple targets in a bacterial cell, resultingn instantaneous killing. Since photodynamic therapy resultsn the instantaneous killing of bacteria, generation of pho-odynamic therapy-resistant bacteria is highly unlikely [2].hough this method has been shown to be effective againstoth gram-positive and gram-negative bacteria, they areore effective against the gram-positive species [2].Chemomechanical preparation of the root canal sys-

em aims at complete eradication of intracanal microbialiofilms, which is unachievable with the current materialsnd techniques [3,4]. The root canal anatomy poses sev-ral challenges in disinfection owing to the complexities thatrevent irrigant penetration into the anatomical complex-ties within the root canal. Therefore, the role of irrigantctivation becomes important to potentiate the antimicro-ial efficacy of an endodontic irrigant within the root canals.f late, research has been focused on the use of sonics,ltrasonics and laser/light energy for the intracanal acti-ation of irrigants [5—7]. The primary goal of combining auitable endodontic irrigant with activation methods is notnly to achieve marked reduction of bacterial biofilms fromhe main canal but also from the anatomical complexitiesnd dentinal tubules [5]. Photodynamic therapy is known toe less toxic to mammalian cells and less damaging to dentinltrastructure [8].

Medicinal plants represent a rich source of antimicro-ial agents. They are an important source of many potentnd powerful drugs [9]. Wide ranges of medicinal plantxtracts are established to possess antioxidant and immune-odulatory properties with minimum toxic/side effects

10]. These agents can find significant application as topicalntimicrobials where host cells are located in the vicinity.urrently, there has been a growing trend to seek naturalgents for dental treatment. Curcumin is such an antimi-robial agent, which can be used as a photosensitizer [11].hus, curcumin may not only exhibit antimicrobial prop-rties but also produce photodynamic effects to furtherotentiate its antimicrobial efficacy.

Enterococcus faecalis, a facultative anaerobic gram-ositive coccus, is implicated in the reinfection of theoot-filled teeth, owing primarily to their ability to adhereo the root dentin, resist some of the antimicrobials usedithin root canals, survive hard environmental conditions,evelop antibiotic resistant strains, and form communi-ies organized in biofilm, thereby enabling it to becomeighly resistant to phagocytosis, antibodies, and antimi-robials than non—biofilm-producing organisms [12,13]. Inddition, they show an exceptional ability to form robustiofilms on root dentin with homogeneous deposition of

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xtracellular polymeric matrix. Therefore this microorgan-sm has been utilized as a model organism to compare thentibiofilm efficacy of different antimicrobials in endodon-ics [6—8,10,11,13].

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The current study was undertaken to examine thentibiofilm efficacy of ultrasonically agitated and lightctivated curcumin and sodium hypochlorite irrigation onatured monospecies E. faecalis biofilms grown on rootentin in vitro. The null hypothesis of the study was thathe ultrasonic agitation or light activation did not improvehe antibiofilm efficacy of curcumin and sodium hypochloriterrigation in root dentin.

aterials and methods

ample preparation

ingle-rooted mandibular premolars with a closed apexn = 175] were used in the study based on a protocolpproved by the Institutional Review Board and Ethics Com-ittee of the University. The teeth were collected in 0.01%

odium hypochlorite solution and maintained hydrated untilse. The crowns were sectioned and the root lengths weretandardized to 15 mm. The working length was defined as

mm short of the apical foramen. The root canals were pre-ared with Mtwo rotary nickel titanium instruments [VDWmBH, Munich, Germany] to an apical size of 35/0.04 tapersing 3% sodium hypochlorite as the irrigant.

The biofilm tooth model employed in this study wasdapted from Lin et al. [14]. Buccal and lingual grooves wereade to split the tooth longitudinally. The split halves were

eapproximated using utility wax placed over the root tip toimulate an in vivo closed apical system that provided resis-ance to irrigant flow by creating an apical vapor lock effect14,15]. Smear layer was removed by placing the sectionsn an ultrasonic bath of 5.25% sodium hypochlorite and 17%thylene diamine tetraacetic acid for 4 min each, rinsed interile water for 1 min and autoclaved (20 min at 121 ◦C).

acterial inoculation and biofilm generation

. faecalis [ATCC 29212] was plated on brain heart infusionroth supplemented with 1.5% [wt/vol] agar and incu-ated anaerobically at 37 ◦C for 24 h. A single colony of. faecalis was collected from the agar plate and sus-ended in sterile brain heart infusion broth. Root specimensere placed in sterile centrifuge tubes containing 3 mL. faecalis suspension [1 × 108 mL−1] and incubated undernaerobic conditions at 37 ◦C for 4 weeks. Fresh broth waseplaced every second day to remove dead cells and tonsure bacterial viability. After incubation, the specimensere removed from the tubes aseptically and rinsed with

terile phosphate-buffered saline to remove the cultureedium and nonadherent bacteria. Dentin sections [n = 4]ere observed by a field emission scanning electron micro-

cope [FE-SEM; JSM-7500F, JEOL Ltd., Tokyo, Japan] to verifyhe presence of E. faecalis biofilms on the dentin surfacesFigure 1].

reatment of biofilms

l. Photoactivation of curcumin and sodium hypochlo-n. Photodiagnosis and Photodynamic Therapy (2014),

he sectioned teeth were reassembled and placed in a stoneasing [14] after which they were randomly divided into

treatment groups [n = 25]: group 1, sterile saline; group

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Photoactivation of curcumin

Fig. 1 Scanning electron microscopic examination of spec-imens after generation of 4-week-old biofilms of E. faecalis

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(1000×). Note the dense aggregates of the microorganismswithin and over the dentinal tubules.

2, 3% sodium hypochlorite [Parcan, Septodont, Saint-Maur-des-fossés Cedex, France]; group 3, 3% sodium hypochloriteactivated with ultrasonic files [IrriSafe, Satelec Acteon,Merignac, France]; group 4, 3% sodium hypochlorite acti-vated with blue light [Bluephase LED, Ivoclar Vivadent,Liechtenstein]; group 5, curcumin [2.5 mg/mL]; group6, curcumin [2.5 mg/mL] activated with ultrasonic files[IrriSafe]; group 7, curcumin [2.5 mg/mL] activated withblue light [Bluephase LED].

For groups 3 and 6, the irrigants were activated for 30 sfollowing that fresh irrigant was placed into the canal andthe process repeated until a total irrigation time of 4 minwas reached. Groups 4 and 7 were activated with the bluelight (wavelength of 380—515 nm, intensity of 1200 mW/cm2

for 4 min), with the light guide tip in contact with the canalorifice. All irrigation procedures were performed at roomtemperature under aseptic conditions by the same opera-tor. After irrigation, the specimens were neutralized with 5%sodium thiosulfate solution for groups 2—4 and neutralizingbroth [Hi Media Labs] for groups 5—7.

Confocal laser scanning microscopic examination

Root sections from each group [n = 30 sections] were stainedwith fluorescent LIVE/DEAD BacLight Bacterial Viability stain[Molecular Probes, Eugene, OR, USA] and viewed using aconfocal laser scanning microscope [LSM 510, Carl Zeiss,Jena, Germany]. Four random areas of the biofilm on eachdentin section were scanned with a 2 �m step size. Simul-taneous dual-channel imaging was used to display the greenand red fluorescence. Three-dimensional reconstruction ofthe confocal laser scanning microscopic images was per-formed [OsiriX Imaging Software, Geneva, Switzerland] and

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quantified using the BioimageL software [16]. The ratio ofdead cells [red fluorescence to total fluoresence ratio] wascalculated. Furthermore, the amount of dead bacteria wasquantified at two regions of interest [200 and 400 microns]

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rom the pulp—dentin junction toward the cementum toetermine how effectively the irrigant activation methodsisinfected the dentinal tubules of the root canal system.tatistical analysis of the data was done using one-way anal-sis of variance and Student—Newman—Keuls. The alpharror was set at P = 0.05.

entin powder analysis

entin debris from the root samples [n = 20 sections] wasarvested at 2 depths (200 and 400 microns) using Gateslidden drills nos. 4 and 5 [Mani Inc., Tochigi-Ken, Japan],

espectively [17], and collected in 1 mL of sterile braineart infusion broth and incubated in an anaerobic environ-ent at 37 ◦C for 24 h. The content of each microcentrifuge

ube was serially diluted, 100 �L of broth in 100 �L of nor-al saline for 5 times. Five microliters of this sample waslated on brain heart infusion agar plates and incubatedor 24 h. The microbial colony-forming units count [CFU/mL]as counted and the data were statistically analyzed withne-way ANOVA followed by Tukey multiple comparisonP = 0.05].

esults

onfocal laser scanning microscopic [CLSM] analysis of bio-olume and viable/dead cells in biofilm structure:

The data obtained from the confocal microsopic analysisf biofilms is tabulated [Table 1]. The mean biofilm heightn the control group was 428.32 ± 72.16 �m. Figure 2A—Fhows the three-dimensional reconstruction of biofilm struc-ures obtained from different groups. The biofilms in theoot canal lumen were completely destroyed in all thexperimental groups [groups 2—7]. Consequently, only theercentage of live and dead bacteria within the dentinalubules was calculated. The percentage of dead bacte-ia was significantly higher when light activated curcuminas used [group 7, Figure 2F], while the least percent-ge of dead bacteria was found in the saline treated groupP < 0.05]. The superficial layer [200 microns] demonstratedhe least amount of live bacteria in all the test irrigationroups, which was significantly lesser than the control groupP < 0.05]. At 400 microns, there was no significant differ-nce in the proportion of dead cells present in the groups—6 [P > 0.05].

entin powder analysis for the quantitativessessment of the viable biofilm bacteria

ata from this analysis showed a significant reduction ofiable bacteria in all groups compared to the controlP < 0.05]. At 200 microns depth, all experimental groupsxcept group 5 and the control saline group showed a

l. Photoactivation of curcumin and sodium hypochlo-n. Photodiagnosis and Photodynamic Therapy (2014),

log reduction of bacteria [no growth]. Light activated cur-umin showed no growth [7 log reduction] of bacteria at 400icrons and the colony-forming units/mL was significantly

ess than the other groups [P < 0.05].

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4 P. Neelakantan et al.

Table 1 Percent of apparently dead bacterial cells in the overall biofilm biomass and bacterial colony-forming units (CFU/mL)within the dentinal tubules at 200 and 400 microns depth, assessed by confocal laser microscopy and microbial culture analysisafter different treatment regimes.

Group Biofilm mass Dead cells %at200 micronsdepth

Dead cells%at400 micronsdepth

CFU/mL at200 microns

CFU/mL at400 microns

Saline (Group 1) 1.975 ± 0.94a 2.86 ± 0.87a 0.94 ± 0.07a 1.5 × 109 ± 0.72 × 109a 1.9 × 109 ± 0.36 × 109a

Sodium hypochorite (Group2)

58.57 ± 5.62b 64.79 ± 3.18b 43.27 ± 3.88b No growthb 3.2 × 104b

Sodium hypochlorite —ultrasonic (Group 3)

62.34 ± 4.17b 73.65 ± 2.31b 55.91 ± 3.74b No growthb 1.5 × 104b

Sodium hypochlorite — light(Group 4)

74.87 ± 5.87b 81.86 ± 3.29b 62.65 ± 2.86b No growthb 1 × 103b

Curcumin (Group 5) 31.6 ± 3.12c 37.16 ± 2.97c 20.15 ± 3.17c 4.2 × 104 ± 0.81 × 104c 4.8 × 104

±0.43 × 104b

Curcumin — ultrasonic(Group 6)

38.96 ± 4.64c 42.81 ± 2.48c 27.32 ± 1.87c No growthb 3.0 × 104b

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iscussion

ulp and periapical pathoses are recognized as hostesponses to biofilm mediated infection [18]. Therefore,otent antibiofilm strategies that can eliminate bacterialiofilm are an important requisite in endodontic therapy.he core objective of an antibiofilm strategy in endodontics

s to bring about destruction of biofilm structure within theoot canal system [18,19]. Sodium hypochlorite is the mostommonly recommended root canal irrigant. It is a potentntimicrobial agent with an ability to bring about destruc-ion of root canal biofilms [20]. However, its proteolyticature has a detrimental effect on dentin microhardness,ltrastructural integrity, modulus of elasticity and flexuraltrength [21]. It is cytotoxic to the periapical tissues in thevent of inadvertent extrusion into the periradicular region22]. Irrigant activation/agitation is an approach to enhancehe penetration of the antibacterial irrigant so as to increasehe degree of bacterial killing within these locations [19].he inability of the current root canal irrigation strategies tofficiently eliminate bacterial biofilms from the anatomicalomplexities and dentinal tubules still remains to be a majorhallenge [18]. This signifies the need for new irrigants thatould have the positive attributes of sodium hypochloritend potential for enhancement.

The effectiveness of antimicrobial photodynamic therapyn root canal disinfection has been demonstrated by sev-ral in vitro and in vivo studies [11,12,23,24]. The highlyeactive oxygen species produced during photoactivationnteract with the amino acids molecules in dentin colla-en to promote cross-linking [25,26]. This increase in theumber of intermolecular collagen bonds has been foundo enhance the resistance of dentin collagen to bacteria-ediated enzymatic degradation and improve the fracture

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oughness of dentin [25—27]. Low-energy level red and nearnfrared light is known to penetrate dentin tissue/dentinalubules without inducing untoward effects on dentin matrix27]. Therefore, the current study examined the efficacy of

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hotodynamically activated curcumin to eliminate biofilmnd disinfect dentin tissue in vitro. Few studies have focusedn the antibiofilm activity of photodynamic activation ofurcumin [27], but no study till date has investigated theffect of photodynamic activation on antibiofilm activity ofurcumin used as a root canal irrigant. The findings fromhis study showed that photodynamic activation enhancedhe antibiofilm activity of curcumin on the root canal lumennd the dentinal tubules when compared to the controls.ence, the null hypothesis needs to be rejected. The supe-ior antibiofilm efficacy and significant bacterial killing fromhe dentinal tubules was exemplified by the colony-formingnit analysis of the dentin powder.

This study compared the antibiofilm activity of curcuminnd sodium hypochlorite subjected to photoactivation orltrasonic activation, using two methods: Confocal lasercanning microscopy and bacterial culture based technique.onfocal microscopy provides valuable information on thebility of a material to kill microorganisms. Its ability to ren-er data in a three-dimensional fashion helps in obtainingnformation about the biofilm structure following treatment28]. Colony-forming units analysis of the dentin powderided in determining quantitatively, the degree of bacterialeduction within the dentinal tubules. Confocal microscopyas used to determine the percentage of dead and liveacteria, and is a more valid method to study the efficacy ofntimicrobial agents on bacterial biofilm structure [20,28].

The resistance of biofilm bacteria to antimicrobialgents could be several times more than their planktonicounterparts due to the relative impenetrability of thentimicrobials into biofilms [29], emphasizing the needor activation methods. Ultrasonically activated irrigationnvolves the transmission of acoustic energy from the oscil-ating file to the irrigant to allow movement of irrigants

l. Photoactivation of curcumin and sodium hypochlo-n. Photodiagnosis and Photodynamic Therapy (2014),

aterally into the anatomical eccentricities of the rootanal system [30]. This study showed that ultrasonic acti-ation of curcumin and sodium hypochlorite resulted inigher percentage of dead bacteria than the control groups.

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Photoactivation of curcumin 5

Fig. 2 Three-dimensional reconstructions of confocal laser scanning microscopic images of E. faecalis biofilms. Images (A—F)show the biofilm after treatment with the experimental groups: (A) [sodium hypochlorite], (B) [sodium hypochlorite with ultrasonicactivation], (C) [sodium hypochlorite with light activation], (D) [curcumin], (E) [curcumin with ultrasonic activation], (F) [curcumin

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with light activation]. Note the high proportion of red channel

(F).

This finding could be attributed to the higher rate of cat-alytic decomposition or degassing of hypochlorite [31].Consequently, application of a fresh solution of sodiumhypochlorite is important to exploit this property [32].Ultrasonically activated curcumin showed significantly lowerpercentage of dead bacteria than ultrasonically activatedhypochlorite. This finding highlighted that unlike in the case

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of sodium hypochlorite, ultrasonic activation was ineffec-tive in enhancing the antibacterial properties of curcumin.Previous reports on the antibiofilm efficacy of ultrasoni-cally activated hypochlorite are not conclusive [33,34]. This

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d bacteria] in the group treated with light activated curcumin

ifference could be attributed to the methodologicalariables used in the studies such as: biofilm model, con-entration of sodium hypochlorite used and the method ofvaluation.

Conventional photoactivated disinfection in root canalreatment employs one of the two photosensitizers (methy-ene blue or toluidene blue) after the use of sodium

l. Photoactivation of curcumin and sodium hypochlo-n. Photodiagnosis and Photodynamic Therapy (2014),

ypochlorite. These photosensitizers need light within theavelength range of 620—660 nm to elicit their effect, whicheeded dedicated light sources [35]. A recent study demon-trated that use of conventional photosensitizers after

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hemomechanical preparation of root canals with sodiumypochlorite did not offer significant effect in the reductionf E.faecalis [35]. Hence, novel photosensitizers need to bevaluated for better clinical performance of photoactivatedisinfection.

The present study demonstrated that photodynamic acti-ation of curcumin was able to bring about the highestercentage of bacterial killing [97.32 ± 3.29%]. Photokillingy curcumin has been demonstrated previously [13]. How-ver, this appears to be the first report on the applicationf curcumin as a photosensitizer for root canal disinfection.he basic structure of curcumin with the presence of twoighly conjugated electron systems renders it as an effec-ive photosensitizer. On irradiation with light, active oxygenpecies are produced. Curcumin exhibits strong absorptionf light within the wavelength of 420—430 nm. Furthermore,t forms inter and intramolecular hydrogen bonds whichnfluence the photophysical properties both in the normaltate and on excitation by light [36,37] It has been proposedhat on illumination, curcumin produces hydrogen perox-de as an intermediate, which is toxic to bacterial cells13]. Another important moiety in curcumin is the keto-nol form, which is considered primarily responsible forhe phototherapeutic effects [38,39]. An important advan-age of curcumin is its ability to exhibit its lethal effectsithout binding or being in close proximity to bacteria

13]. Nevertheless, binding of the PS to the cell wall ofacteria potentially increases the sensitivity of the microbeo light [38]. The usefulness of curcumin as a potentialnter-appointment intracanal medicament is being investi-ated. Future research should also be directed to elucidatehe exact mechanisms of photokilling by curcumin in thendodontic environment. Furthermore, the efficacy of pho-oactivated curcumin after chemomechanical root canalreparation with sodium hypochlorite needs to be assessed.

In conclusion, light activated disinfection of rootanal systems using curcumin as photosensitizer showeduperior antibiofilm activity than ultrasonic activation.hotoactivated curcumin demonstrated significantly higherntibacterial activity than sodium hypochlorite.

onflict of interest

he authors declare no conflicts of interest.

eferences

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