TITLE: 
Effect of ultrasonic activation of photosensitizer dye temoporfin (Foscan) on antimicrobial photodynamic therapy: An ex vivo study



ABSTRACT.AIM:
The aim of this study is to evaluate the effectiveness of ultrasonic activation of photosensitizer (PS) drug Foscan in the elimination of endodontic pathogen from root canal system.

ABSTRACT.MATERIALS AND METHODS:
The minimal bactericidal concentration of “Foscan” was determined using disc diffusion and broth dilution method. Forty-eight extracted single-rooted premolars with periapical pathology were used for the study. After access opening, the first microbial sample was taken. The teeth were then randomly divided into four groups: Group 1: Chemomechanical debridement (CMD), Group 2: CMD and passive ultrasonic irrigation (CMD + PUI), Group 3: CMD and antimicrobial photodynamic therapy (aPDT), and Group 4: CMD and aPDT with activation by ultrasonics. After antimicrobial protocols, the second microbiological sample was collected. The samples were analyzed by multiplex polymerase chain reaction for the effectiveness of four antimicrobial protocols on Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, and Treponema denticola.

ABSTRACT.STATISTICAL ANALYSIS USED:
Wilcoxon signed-rank test and Mann–Whitney U-test with the significant level P < 0.05, using IBM SPSS statistics 20 version software.

ABSTRACT.RESULTS:
Foscan at concentration of 3.125 μg/ml was chosen for antimicrobial analysis. The prevalence of T. denticola, F. nucleatum, P. gingivalis, and P. intermedia was 79.17%, 70.84%, 70.83%, and 58.33%, respectively. Group 4 showed highest bacterial reduction of 99.51%, followed by Group 1 which showed bacterial reduction of 97.35%.

ABSTRACT.CONCLUSIONS:
Foscan can be used as an effective PS dye at low concentration with minimal tendency for tooth discoloration. Ultrasonic activation of PS dye facilitated better diffusion into dentinal tubules and biofilm obtaining greater bacterial reduction.

BODY.INTRODUCTION:
Endodontic failures are usually related to inadequate cleaning and disinfection of the root canal system. This is due to the presence of plethora of microorganisms residing within the canal lumen, dentinal tubules, and anatomic complexities which cannot be accessed by the instruments or chemical agents alone.[1]
The effective delivery of irrigants into the anatomic complexities can be enhanced using sonic and ultrasonic devices as well as apical negative-pressure irrigation.[23] Ultrasonic activation of irrigant enhances its mechanical cleansing of canal due to cavitation and acoustic microstreaming. Ultrasonics also effectively removes smear layer, dislodges the biofilm, and leads to bacterial destruction.[34]
Antimicrobial photodynamic therapy (aPDT) is one of the novel methods employed to supplement the disinfection of root canal. It generally employs a photosensitizer (PS) which on light irradiation of appropriate wavelength produces reactive oxygen species and singlet oxygen which are very lethal to microorganisms.[56] Methylene blue and toluidine blue are the most commonly used PSs in endodontics.[789] There is a surge for newer and effective PS that has no dark toxicity and that can be used at low concentration.
5,10,15,20-Tetra(m-hydroxyphenyl) chlorin (mTHPC) with the generic name ''temoporfin'' and the proprietary name ''Foscan'' has been introduced since 2000. Foscan is a second-generation PS derived from chlorophyll-a. The red light activation at 650 nm allows for greater photoactivation and deeper tissue penetration. It has also been shown to yield higher1O2 at lower drug concentration and requires light doses as low as 10 J/cm2.[1011]
The aim of this study was to evaluate the effectiveness of photodynamic therapy in elimination of root canal flora when activated with both ultrasonics and light.

BODY.MATERIALS AND METHODS:
BODY.DETERMINATION OF ANTIBACTERIAL CONCENTRATION OF PHOTOSENSITIZER DYE:
Foscan (CAT NO: 17333, Cayman Chemical Company, USA) is available as powder was mixed with solvent dimethyl sulfoxide (Finar Limited, India) to prepare stock solution of 100 μg/ml of drug concentration. The bacteria Enterobacter aerogenes was procured from the National Collection of Industrial Microorganisms (NCIM 5139, Pune) and cultured in Muller-Hinton agar. Different concentrations of PS dye were prepared from the stock solution, and the microbial activity was evaluated using disk method and broth dilution method. The minimal bactericidal concentration of the drug was found to be of 3.125 μg/ml.

BODY.LIGHT SOURCE:
The PS dye Foscan was activated with red light of wavelength of 652 ± 4 nm operated with light source (LIT 600, Apoza Enterprise CO; LTD, Taiwan) at intensity of 400 mW/cm2 and Fluence energy of 240 J/cm2 for activation. The light was irradiated through disposable fiber optic tip of 200 μm diameter into the apical portion of root canal.

BODY.TOOTH SPECIMENS:
Forty-eight single-rooted mandibular premolar teeth with radiographic evidence of periapical pathology were selected. Before extraction, patient consent was taken and the extracted teeth were immediately stored in normal saline.
BODY.THE FIRST MICROBIOLOGICAL SPECIMEN:
After access opening, the working length was determined with size 15 K-file (Mani Files, Brussels, Belgium). The apical closure was done with glass-ionomer cement (GC Corporation, Japan). The pulp chamber and root canal were flooded with normal saline (Parenteral Drugs India Limited, Navlakha, Indore, Madhya Pradesh, India). The 15 K-file was used in a forward and backward motion to collect the necrotic debris. The handle of the file was separated and the remaining part of the file was placed in microcentrifuge tube containing 1 ml of Tris-EDTA (TE) transport medium. Three sterile paper points were left inside the root canal for 1 min each and collected in microcentrifuge tube. These samples were stored in a container at 4°C until bacterial analysis.


BODY.TREATMENT GROUPS:
Forty-eight teeth were randomly divided into four main groups with 12 teeth per group as follows:

Group 1: Chemomechanical debridement (CMD)
Group 2: CMD with passive ultrasonic irrigation (CMD + PUI)
Group 3: CMD with photodynamic therapy (CMD + PDT)
Group 4: CMD with PDT activated by ultrasonics (CMD + PDT + PUI).



BODY.INSTRUMENTATION, IRRIGATION, AND ANTIMICROBIAL PROTOCOLS:
All the root canals were instrumented in crown-down approach. The coronal flaring was done using Gates-Glidden Drills no. 3, 2 and 1. The canal was enlarged to obtain apical size equivalent to file size 35, and the irrigation was performed as per Mozo et al.[12] and Miranda et al.[13] A volume of 10 ml of 3% sodium hypochlorite (NaOCl) (Neelkanth Healthcare Ltd., Jodhpur, India) was used between each of the instruments and delivered into the canal using 30-gauge NaviTip needle (Ultradent Products, Inc, South Jordan) attached to disposable plastic syringe. At the end of the instrumentation, final irrigation was performed with 1 ml of 17% EDTA (Neelkanth Healthcare Ltd., Jodhpur, India) left in situ for 3 min, followed by 1 ml of 3% NaOCl for additional 3 min for smear layer removal.
PUI was performed as per Tennert et al.[14] (Group 2). The canal was flooded with 3% NaOCl and ultrasonically activated using U-file size 20 tip (Mani Files, Brussels, Belgium). The U-file was used 2 mm short of the working length at frequency of 24–32 KHz for 60 s.
aPDT was performed as per De Paz LC[15] (Group 3), and the canal was filled to the level of access cavity with Foscan dye using 30-gauge NaviTip needle. The dye was left in the root canal for 60 s as incubation time. The canal was then irradiated with fiber-optic tip of diameter 200 μ for 1 min. The fiber-optic tip was moved spirally from apical to cervical during the irradiation time for uniform diffusion of light throughout the entire canal lumen. The canal was then flushed with 10 ml of normal saline solution to remove the PS dye.
For Group 4, the samples were treated same as Group 3 except that the PS dye was activated with U-file for 1 minute allowing the dye to penetrate into the dentinal tubules and to reach the target bacterial cells within the biofilm.

BODY.POSTTREATMENT SAMPLE COLLECTION:
Following antimicrobial treatment of all groups, the final sample was taken after filling the canal with sterile saline solution. Three paper points were sequentially inserted to the working length for 1 min each and placed in sterile microcentrifuge tube. To determine the survivability of bacteria within the dentinal tubule, 40 K-file was used up to the working length to collect the dentinal shavings.[16] The file was placed in microcentrifuge tube along with paper points.

BODY.POLYMERASE CHAIN REACTION ANALYSIS:
BODY.DNA EXTRACTION:
The first and final samples were sent to a microbiology laboratory for identification of bacteria and for further analysis. Briefly, the samples were vortex-mixed and centrifuged to collect the cells. The pellet was suspended in 300 μl of lysis buffer plus lysozyme (5 mg/ml) and incubated at 37°C for 1 h. Then, proteinase-K was added, and after 1-h incubation at 65°C, the DNA was extracted with phenol and chloroform-isoamyl alcohol treatment. The DNA extracted from each sample was assayed by multiplex polymerase chain reaction (PCR), for the detection of Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, and Treponema denticola.[17]


BODY.POLYMERASE CHAIN REACTION DETECTION:
The multiplex PCR was performed using specific primers for the 16S rRNA gene of each bacterium. PCR amplification reactions were carried out in a reaction mixture in a final volume of 100 μl consisting of 10 μl of DNA sample, and 90 μl of reaction mixture containing 30 pmol of each primer, 200 μM of a mixture of deoxynucleoside triphosphates, 1.5 mM MgCl2 buffer, 50 mM KCl, and 2.5 U Hot Start Taq™ DNA polymerase. The PCR protocol was as follows: 98°C for 15 min followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min, and a final step of 72°C for 10 min. PCR amplification was performed in an iCycler system. Amplicons were detected by electrophoresis of 20 μl of samples from each PCR tube in a 2% agarose gel in Tris-acetate-EDTA buffer for 2 h at 80 V [Figure 1]. The amplification products were visualized and photographed under an ultraviolet light transilluminator after 30 min of ethidium bromide (1 μg/ml) staining. The frequency of sites positive for each microbiota was reported.[1819]
Figure 1Electrophoretic image showing the identification of four bacteria at different base pairs

BODY.STATISTICAL ANALYSIS:
The prevalence of F. nucleatum, P. intermedia, P. gingivalis, and T. denticola and mean bacterial reduction were calculated in percentages. Intergroup comparison of effectiveness in bacterial reduction was calculated using Wilcoxon signed-rank test and Mann–Whitney U-test. All analyses were performed with the significant level P < 0.05, using IBM Statistical Package for the Social Sciences (SPSS) statistics 20 version software (IBM).


BODY.RESULTS:
The prevalence of F. nucleatum, P. intermedia, P. gingivalis, and T. denticola was calculated from first microbiological samples of all groups. T. denticola was the most commonly isolated bacterial species accounting for 79.17%. The other bacterial species F. nucleatum, P. gingivalis, and P. intermedia accounted for 70.84%, 70.83% and 58.33%, respectively.
T. denticola was more resistant to antimicrobial protocol employed in this study with percentage mean reduction of 90.14% when all groups were compared. F. nucleatum showed percentage mean reduction of 96.98%. The bacterial species P. gingivalis and P. intermedia were sensitive to the antimicrobial protocols employed with percentage mean reduction of 99.15% and 99.16%, respectively [Table 1].
Table 1Percentage of mean bacterial reduction from preoperative to postoperative samples
When total bacterial reduction was taken into consideration, Group 4 (CMD + PDT + PUI) showed better elimination of bacteria when compared to other groups [Figure 2]. This was followed by Group 1 (CMD), Group 2 (CMD + PUI), and Group 3 (CMD + PDT). The differences in bacterial reduction between the groups were not statistically significant, and all the protocols were almost equally effective in elimination of bacteria [Table 2].
Figure 2Intergroup comparision of effectiveness in bacterial reduction
Table 2Bacterial count mean reduction comparison between the groups

BODY.DISCUSSION:
Foscan at concentration of 3.125 μg/ml was used in this study which showed bactericidal effect on E. aerogenes. Kranz et al.[10] used mTHPC enriched in liposomes against Enterococcus faecalis at concentration of 10, 30, and 50 μM and showed that 50 μM of mTHPC at 100 J/cm2 light fluence irradiated for 33 s was effective against E. faecalis. The concentration used in this study was effective against the four obligatory anaerobes analyzed in this study.
Cameron[20] has shown that activation of NaOCl with PUI for a period of 3–5 min is sufficient to completely remove the smear layer in instrumented root canals. In our study, 20 size ultrasonic file activated for 1 min at 2 mm short of working length achieved bacterial reduction better than PDT but was not statistically significant.
The nonsignificant difference between the groups in our study may be because of employing NaOCl in all the groups. Nevertheless, when PDT was activated with ultrasonics (Group 4), 99.51% of bacterial reduction was seen compared to Group 1 (97.36%), Group 2 (96.70%), and Group 3 (91.86%).
PDT group showed less bacterial reduction compared to CMD in the present study. This may be due to the difference in the bacterial load of the initial samples, inability of PS to penetrate the biofilm, and presence of anatomic complexities.[21] Since there is a low concentration of available oxygen within the root canal, the production of cytotoxic oxygen derivatives may also be reduced. Furthermore, the penetration of light necessary for activation of PS dye may be limited. All these factors could have caused reduced efficiency in bacterial reduction compared to other groups. Because of the inability of PS to reach anatomic complexity and enter into biofilm,[22] ultrasonics was used in this study to activate the PS and increase its efficiency.
Tennert et al.[22] increased the efficiency of toluidine blue by combining the dye with chelating agents and also by activating with ultrasound energy. EDTA-based or citric acid-based toluidine blue and ultrasonic activation of PS dye led to significant reduction of E. faecalis compared to conventional PDT. The result of this present study also showed near complete elimination of bacteria when the PS was activated with ultrasonics and is in agreement with Tennert et al. and Ghinzelli et al.[23]

BODY.CONCLUSIONS:
Foscan dye at concentration of 3.125 μg/ml and energy density of 240 J/cm2 activated at 650 nm was able to achieve near complete elimination of the bacteria tested in this study. The use of ultrasonics to activate PS dye allowed better penetration of dye into anatomic complexity, dentinal tubules, and into biofilm resulting in total bacterial reduction of 99.51%.
BODY.FINANCIAL SUPPORT AND SPONSORSHIP:
Nil.

BODY.CONFLICTS OF INTEREST:
There are no conflicts of interest.

Question: Compared to CMD and aPDT with activation by ultrasonics. what was the result of Chemomechanical debridement (CMD) or CMD and passive ultrasonic irrigation (CMD + PUI) or  CMD and antimicrobial photodynamic therapy (aPDT) on bacterial reduction?
Evidence: The differences in bacterial reduction between the groups were not statistically significant, and all the protocols were almost equally effective in elimination of bacteria
Answer: no significant difference