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ABSTRACTPhotoactivated disinfection is a new antimicrobial

method for root canal disinfection, based on photodynamictherapy.

Purpose: The goal of this study is to investigate theantimicrobial effect of photoactivated disinfection onexperimental biofilm from Enterococcus faecalis and Candidaalbicans, through scanning electron microscopy.

Material and Methods: Freshly extracted, one rootteeth were prepared with a sequence of rotary nickel-titaniumfiles (ProTaper ; Dentsply ; Mailefer ), irrigated, the externalroot canal surfaces isolated with nail polish and autoclaved.After the incubation with the experimental biofilm, the rootcanals were filled with photosensitizer – Toluidine Blue –0,01% and irradiated with Foto San(CMS Dental), 630 nm,2000mW/cm2 for 30 seconds.

SEM was performed on the coronal, middle and apicalthird of the root canal, for evaluation of the results.

Results and discussion: In the range of 600 to 8000,SEM showed significant reduction of microorganisms fromthe canal system. A large increase in microorganisms wasobserved, showing a disturbance in the cell membrane, aseffect from the activation of chromophore with the laser andthe penetration of the photosensitizer in dental tubules. In theapical third single microorganisms were observed. This maydue to decreased penetration of the photosensitizer,incomplete pervasion of MB in the biofilm or insufficientoxygenation.

Conclusion: FAD has the potential to be a goodalternative and addition to the conventional root canaldisinfection methods. SEM is a precise method for endodontictreatment result evaluation.

Key words: endodontic microorganisms; disinfection;scanning electron microscopy;

One of the reasons for unsuccessful endodontictreatment is the presence of persisting microbial flora andrecontamination of the root canal, triggered by micro leakage.

The infection of the endodontic space is alwayspolymicrobial. Even though during the initial endodontic

EFFICENCY OF PHOTOACTIVATED DISINFECTION

ON EXPERIMENTAL BIOFILM – SCANING

ELECTRON MICROSCOPY RESULTS

Ivan Filipov, Kremena Markova, Elena BoyadzhievaDepartment of Operative dentistry and Endodontics, Faculty of Dental Medicine,Medical University, Plovdiv, Bulgaria

Journal of IMAB - Annual Proceeding (Scientific Papers) 2013, vol. 19, issue 4ISSN: 1312-773X (Online)

treatment it is generally anaerobic and throughout retreatmentit is associated with Gr positive aerobes and facultativemicroorganisms, which are more influence resistant thenanaerobes. Most commonly associated with endodontic failuremicrobes are: Enterococcus faecalis, Streptococci,Staphylococci, Actinomyces, Pseudomonas, Candidaalbicans, Proprionibacterium, Porphyromonas, Prevotellaetc. [4, 8, 9, 20, 24].

Conventional root canal preparation and disinfectionmethods do not allow full elimination of the microbial flora.The presence of a smear layer decreases the effectiveness ofthe applied irrigants and their antimicrobial activity. Thebacterial biofilm is hard to eliminate and is resistant to a widenumber of antimicrobial agents.

The necessity to achieve maximum cleanness of theroot canal is the cause for the search of new alternativemethods for its disinfection. Such method is photoactivateddisinfection (FAD), based on photodynamic therapy (FDT).

The purpose of the following research is to find outthrough SEM the antibacterial effect of FAD on experimentalbiofilm from Enterococcus faecalis and Candida albicans.

MATERIALS AND METHODSThe research was performed on freshly extracted, one

root teeth. The teeth crowns were removed with a diamonddisk and the roots were shortened to the length of 13 mm.The root canals were prepared using a sequence of rotarynickel-titanium endodontic files (ProTaper; Dentsply;Maillefer) to final apical size of F3 irrigated with NaClO. Theexternal root surfaces were covered with two layers of nailpolish and the apical aperture was sealed with compositematerial (Filtek Z350, 3M). The root canals were irrigatedwith 17% EDTA for 2 minutes for smear layer removal andthen with 10ml distilled water. The teeth were then autoclavedat 121°C, 1.2 atm for 20 minutes.

Biofilm characterisationThe root canals were inoculated , through their whole

length, with planktonic suspension of Candida albicans andEnterococcus faecalis.

http://dx.doi.org/10.5272/jimab.2013194.383

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The teeth were then incubated in test tubes withnutrient, for 15 days, anaerobically at 37°C .

A photosensitizer was then applied – Toluidine Blue0.01% and photo activated disinfection was performed usingLED light source (FotoSan; CMS Dental; Denmark),producing red light with wavelength of 630 nm and intensity2000 mw/cm2 for 30 seconds. The root canals were cutlongitudinally at each 3 mm.

For evaluation of the results, scanning electronmicroscopy was performed in the range of 600 to 8000 onthe coronal, middle and apical third of the root canal.

RESULTSThe scanning electron microscopy shows open dentinal

tubules, after irrigation with sodium hypochlorite and EDTA.(fig. 1) The created biofilm is visible on SEM - graphy, aswell as the penetrated microorganisms in the dentinal tubules.(fig. 2)

The scanning electron microscopy showed significantreduction of microorganisms in coronal, middle and apicalthird of the root canal. (fig. 3, 4, 5) Some of themicroorganisms showed disturbance in the cell membrane, asan effect from the activation of chromophore with the redLED light of FotoSan and the penetration of photosensitizerin dentinal tubes.

Fig. 1. Clear root surface

Fig. 2. The experimental biofilm

Fig. 3. Coronal third of the root canal

Fig. 4. Middle third of the root canal

Regardless of the MO reduction, single microbes areobservable in the apical third of the root canal. (fig. 5)

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Fig. 5. Microbes in the apical third of the root canal

DiscussionPhotodynamic therapy is based on the idea that

nontoxic photosensitive agent, called photosensitizer,preferentially localizes in premalignant and malignant tissues.The PS is then activated by light with susceptible wavelengthand produces singlet oxygen and free radicals, which arecytotoxic for the target cells. [14]

Appliance of FDT for inactivation of microorganismswas first demonstrated more then 100 years ago. For the firsttime Andersen et al, 2007, [15], [17] used FDT in oral cavityin the mid 80s as supportive treatment for patients withchronic parodontitis.

Researches from the same year show thatphotodynamic therapy was used for elimination of infectionin the root canal in conjunction with the conventionalendodontic treatment.

In recent years it is assumed that FDT is able to “mark”microbes in the root canal in vitro [11, 25, 26] and in vivo[18, 27, 28] and can be useful as an additional method forits antimicrobial effect in routine endodontic practice.

FDT requires light with suitable wavelength andphotosensitizer in the presence of oxygen. The target cells areobject to a local photo damage, by the exuded during theirradiation oxygen species.

Phenothiazine dyes – Methylene blue (MB) andToluidine blue (TBO) are the most commonly usedphotosensitizers in dental practices.

Endodontic pathogens have different survival strategieswhen the conditions are unfavorable. The microbes penetratethe dentinal tubules [13] in 1000 ìm depth, creating a firmlybonded biofilm [7, 10].

Several publications have summarised incompletedestruction of the oral biofilm, when using MB – mediatedphoto activated disinfection. This is due to decreasedpenetration of the photosensitizer in the dentinal tubules,insufficient oxygenation, deficient pervasion of MB in thebiofilm.

There is evidence that phenothiazine PSs, includingMB and TBO, are substrates of multi-drug resistance, whichdecreases their efficiency [33].

In our research where we used TBO as PS, we alsoobserved single MO in the middle third, and the colonizationof MO on the apical third of the root canal.

Other possible explanation of the problem establishedin similar researches is that the high intensity of light maycause rapid consummation of molecular oxygen in the lowoxygenated dentinal tubules as a result the oxygen pressureis too low for O2 mediated reactions to take place. That’s whyit is so important for PS concentration, light intensity and timeof application to be precise.

Those obstacles can be avoided by optimization ofMB’s pharmaceutical characterization. In relation to this somestudies have been carried out on using polymer nanoparticlesas vehicle and resource of PS, chlorines and porphyrines [15],the so called antibody-pointed PS [19].

FAD requires a source of light that is absorbable bythe PS and activates its properties. Human body tissuesconduct red light, that’s why most of the PS are activated bylight in the range of 630 to 700 nm, which penetrate the tissueat a depth of 0.5 – 1.5 cm [15, 27]

Different light sources have been used for performingFAD – halogen, xenon, LED lamps, lasers [16, 17, 36, 37].

Phenothiazine dyes, used as red light activated PS,require longer light exposition to be triggered, which maycause excessive heat. High energetic red light lasers areexpensive and not every dental office can afford them. Diodelight sources are good alternative in PS activation, which isconfirmed by our study results.

In some cases redness and damage to healthy tissueshave been reported. That’s why the use of blue light in therange of 380 – 500 nm is also possible [39, 40]. Such lightsources are used daily in dental offices. The possibilities ofthe so-called “blue” PS have to be investigated. In vitrostudies find that when “blue” light is applied, normal gingivalfibroblastes proliferation is observed one week afterirradiation [39, 40].

CONCLUSIONFAD has the potential to be a good alternative to the

routine endondontic disinfection protocol. The interactionbetween PS and light with suitable wavelength provides awide effect spectrum. Resistance development is very unlikelywith FAD because of singlet oxygen and free radicals, andtheir interaction with some cell structures of MO.

The use of red light producing diode lamps is givinggood results in FAD of the root canals. SEM is a precisemethod for evaluating the cleanness of the root canal afterperforming FAD disinfection.

The results demonstrate the need for further researchon the optimal PS as well as the most effective parametersof the light source, associated with bacterial pathogens forachieving the best results possible.

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