EFFICENCY OF PHOTOACTIVATED DISINFECTION ON EXPERIMENTAL BIOFILM – SCANING ELECTRON MICROSCOPY RESULTS

383 ABSTRACT Photoactivated disinfection is a new antimicrobial method for root canal disinfection, based on photodynamic therapy. Purpose: The goal of this study is to investigate the antimicrobial effect of photoactivated disinfection on experimental biofilm from Enterococcus faecalis and Candida albicans, through scanning electron microscopy. Material and Methods: Freshly extracted, one root teeth were prepared with a sequence of rotary nickel-titanium files (ProTaper ; Dentsply ; Mailefer ), irrigated, the external root canal surfaces isolated with nail polish and autoclaved. After the incubation with the experimental biofilm, the root canals 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 apical third 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 from the canal system. A large increase in microorganisms was observed, showing a disturbance in the cell membrane, as effect from the activation of chromophore with the laser and the penetration of the photosensitizer in dental tubules. In the apical third single microorganisms were observed. This may due to decreased penetration of the photosensitizer, incomplete pervasion of MB in the biofilm or insufficient oxygenation. Conclusion: FAD has the potential to be a good alternative and addition to the conventional root canal disinfection methods. SEM is a precise method for endodontic treatment result evaluation.

One of the reasons for unsuccessful endodontic treatment is the presence of persisting microbial flora and recontamination of the root canal, triggered by micro leakage.
Conventional root canal preparation and disinfection methods do not allow full elimination of the microbial flora.The presence of a smear layer decreases the effectiveness of the applied irrigants and their antimicrobial activity.The bacterial biofilm is hard to eliminate and is resistant to a wide number of antimicrobial agents.
The necessity to achieve maximum cleanness of the root canal is the cause for the search of new alternative methods for its disinfection.Such method is photoactivated disinfection (FAD), based on photodynamic therapy (FDT).
The purpose of the following research is to find out through SEM the antibacterial effect of FAD on experimental biofilm from Enterococcus faecalis and Candida albicans.

MATERIALS AND METHODS
The research was performed on freshly extracted, one root teeth.The teeth crowns were removed with a diamond disk and the roots were shortened to the length of 13 mm.The root canals were prepared using a sequence of rotary nickel-titanium endodontic files (ProTaper; Dentsply; Maillefer) to final apical size of F3 irrigated with NaClO.The external root surfaces were covered with two layers of nail polish and the apical aperture was sealed with composite material (Filtek Z350, 3M).The root canals were irrigated with 17% EDTA for 2 minutes for smear layer removal and then with 10ml distilled water.The teeth were then autoclaved at 121°C, 1.2 atm for 20 minutes.

Biofilm characterisation
The root canals were inoculated , through their whole length, with planktonic suspension of Candida albicans and Enterococcus faecalis. http://dx.doi.org/10.5272/jimab.2013194.383 The teeth were then incubated in test tubes with nutrient, for 15 days, anaerobically at 37°C .
A photosensitizer was then applied -Toluidine Blue 0.01% and photo activated disinfection was performed using LED light source (FotoSan; CMS Dental; Denmark), producing red light with wavelength of 630 nm and intensity 2000 mw/cm 2 for 30 seconds.The root canals were cut longitudinally at each 3 mm.
For evaluation of the results, scanning electron microscopy was performed in the range of 600 to 8000 on the coronal, middle and apical third of the root canal.

RESULTS
The scanning electron microscopy shows open dentinal tubules, after irrigation with sodium hypochlorite and EDTA.(fig. 1) The created biofilm is visible on SEM -graphy, as well as the penetrated microorganisms in the dentinal tubules.(fig.2) The scanning electron microscopy showed significant reduction of microorganisms in coronal, middle and apical third of the root canal.(fig.3, 4, 5) Some of the microorganisms showed disturbance in the cell membrane, as an effect from the activation of chromophore with the red LED light of FotoSan and the penetration of photosensitizer in dentinal tubes.

Discussion
Photodynamic 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 wavelength and produces singlet oxygen and free radicals, which are cytotoxic for the target cells.[14] Appliance of FDT for inactivation of microorganisms was first demonstrated more then 100 years ago.For the first time Andersen et al, 2007, [15], [17] used FDT in oral cavity in the mid 80s as supportive treatment for patients with chronic parodontitis.
Researches from the same year show that photodynamic therapy was used for elimination of infection in the root canal in conjunction with the conventional endodontic 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 for its antimicrobial effect in routine endodontic practice.
FDT requires light with suitable wavelength and photosensitizer in the presence of oxygen.The target cells are object to a local photo damage, by the exuded during the irradiation oxygen species.
Phenothiazine dyes -Methylene blue (MB) and Toluidine blue (TBO) are the most commonly used photosensitizers in dental practices.
Endodontic pathogens have different survival strategies when the conditions are unfavorable.The microbes penetrate the dentinal tubules [13] in 1000 ìm depth, creating a firmly bonded biofilm [7,10].
Several publications have summarised incomplete destruction of the oral biofilm, when using MB -mediated photo activated disinfection.This is due to decreased penetration of the photosensitizer in the dentinal tubules, insufficient oxygenation, deficient pervasion of MB in the biofilm.
There is evidence that phenothiazine PSs, including MB and TBO, are substrates of multi-drug resistance, which decreases their efficiency [33].
In our research where we used TBO as PS, we also observed single MO in the middle third, and the colonization of MO on the apical third of the root canal.
Other possible explanation of the problem established in similar researches is that the high intensity of light may cause rapid consummation of molecular oxygen in the low oxygenated dentinal tubules as a result the oxygen pressure is too low for O2 mediated reactions to take place.That's why it is so important for PS concentration, light intensity and time of application to be precise.
Those obstacles can be avoided by optimization of MB's pharmaceutical characterization.In relation to this some studies have been carried out on using polymer nanoparticles as 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 by the PS and activates its properties.Human body tissues conduct red light, that's why most of the PS are activated by light in the range of 630 to 700 nm, which penetrate the tissue at a depth of 0.5 -1.5 cm [15,27] Different light sources have been used for performing FAD -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 may cause excessive heat.High energetic red light lasers are expensive and not every dental office can afford them.Diode light sources are good alternative in PS activation, which is confirmed by our study results.
In some cases redness and damage to healthy tissues have been reported.That's why the use of blue light in the range of 380 -500 nm is also possible [39,40].Such light sources are used daily in dental offices.The possibilities of the so-called "blue" PS have to be investigated.In vitro studies find that when "blue" light is applied, normal gingival fibroblastes proliferation is observed one week after irradiation [39,40].

CONCLUSION
FAD has the potential to be a good alternative to the routine endondontic disinfection protocol.The interaction between PS and light with suitable wavelength provides a wide effect spectrum.Resistance development is very unlikely with FAD because of singlet oxygen and free radicals, and their interaction with some cell structures of MO.
The use of red light producing diode lamps is giving good results in FAD of the root canals.SEM is a precise method for evaluating the cleanness of the root canal after performing FAD disinfection.
The results demonstrate the need for further research on the optimal PS as well as the most effective parameters of the light source, associated with bacterial pathogens for achieving the best results possible.