PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Endod. Author manuscript; available in PMC 2012 February 1.
Published in final edited form as:
PMCID: PMC3034089
NIHMSID: NIHMS260716

Endodontic photodynamic therapy ex vivo

Abstract

Objective

To evaluate the anti-microbial effects of photodynamic therapy (PDT) on infected human teeth ex vivo.

Materials and Methods

Fifty-two freshly extracted teeth with pulpal necrosis and associated periradicular radiolucencies were obtained from 34 subjects. Twenty-six teeth with 49 canals received chemomechanical debridement (CMD) with 6% NaOCl and twenty-six teeth with 52 canals received CMD plus PDT. For PDT, root canal systems were incubated with methylene blue (MB) at concentration of 50 µg/ml for 5 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm2. The contents of root canals were sampled by flushing the canals at baseline and following CMD alone or CMD+PDT and were serially diluted and cultured on blood agar. Survival fractions were calculated by counting colony-forming units (CFU). Partial characterization of root canal species at baseline and following CMD alone or CMD+PDT was performed using DNA probes to a panel of 39 endodontic species in the checkerboard assay.

Results

The Mantel-Haenszel chi-square test for treatment effects demonstrated the better performance of CMD+PDT over CMD (P=0.026). CMD+PDT significantly reduced the frequency of positive canals relative to CMD alone (P=0.0003). Following CMD+PDT, 45 of 52 canals (86.5%) had no CFU as compared to 24 of 49 canals (49%) treated with CMD (canal flush samples). The CFU reductions were similar when teeth or canals were treated as independent entities. Post-treatment detection levels for all species were markedly lower for canals treated by CMD+PDT than were for those treated by CMD alone. Bacterial species within dentinal tubules were detected in 17/22 (77.3%) and 15/29 (51.7%) of canals in the CMD and CMD+PDT group, respectively (P= 0.034).

Conclusion

Data indicate that PDT significantly reduces residual bacteria within the root canal system, and that PDT, if further enhanced by technical improvements, holds substantial promise as an adjunct to CMD.

Keywords: Photodynamic therapy, methylene blue, endodontic disinfection, ex vivo

INTRODUCTION

Endodontic treatment is the clinical management of a microbiological problem (1) and the main target of treatment is the microorganisms residing within the root canal system (2). However, the complexity of the root canal system makes complete debridement and removal of bacteria with instrumentation, irrigation and intracanal medicaments virtually impossible (3). In addition, current endodontic procedures require very good technical skills, and use medicaments whose effectiveness has never been definitively proven in human clinical trials. Three systematic reviews (46) on the outcome of primary non-surgical root canal treatment summarized findings from longitudinal clinical studies published up to 2006, in which treatments were carried out by undergraduate students, graduate students, general dental practitioners or specialists. The estimated success reported in these studies was 75% (6) and 78% (4, 5). In a recent systematic review by Ng et al. (2010) that included fourteen studies published between 1993 and 2007, the pooled proportion of teeth surviving over 2–10 years following root canal treatment was found to range between 86% and 93% (7). However, Wu et al. (2009) reported several factors that contribute to the overestimation of successful outcomes after primary root canal treatment: A high percentage of cases confirmed healthy by periapical radiography reveal apical periodontitis on cone beam computed tomography and by histology; extractions and retreatments were rarely recorded as failures; and the recall rate was often < 50% in longitudinal clinical studies (8). General dentists perform about 75% of root canal procedures (9), and thus it might be anticipated that failure rates are even greater in general practice (6). When strict radiographic criteria were used, the success rates were approximately 66%, 75%, 77% and 85% for treatments carried out by general dental practitioners, undergraduate students, graduate students and specialists, respectively (6). Given that more than 20 million root canals are performed yearly in the U.S. (10), approximately 2 million endodontic failures could be avoided by better disinfection procedures. The development of adjunctive antibacterial therapeutic strategies to CMD therefore becomes important in the evolution of methods to target residual microorganisms in the root canal system.

Photodynamic therapy (PDT) was developed as a therapy for cancer and is based on the concept that a non-toxic photosensitizing agent, known as photosensitizer, can be preferentially localized in premalignant and malignant tissues and subsequently activated by light of the appropriate wavelength to generate singlet oxygen and free radicals that are cytotoxic to cells of the target tissue (11). In recent years, PDT has been employed to target microorganisms in root canals in vitro (1228) and in vivo (2932) suggesting its usefulness as an adjunct to current endodontic disinfection techniques. Methylene blue (MB) is a well-established photosensitizer that has been used in PDT for targeting various gram-positive and gram-negative oral bacteria (33) and was previously employed to study the effect of PDT on endodontic disinfection (14, 19, 20, 22, 25, 26). MB has been used as a photosensitizing agent for almost nine decades (34). It has been used for the detection of mucosal premalignant lesions (35) and as a marker dye in surgery (36). The hydrophilicity of MB (37), along with its low molecular weight and positive charge allows passage across the porin-protein channels in the outer membrane of gram-negative bacteria (38). MB, whose intravenous administration is FDA approved for methemoglobinemia, predominantly interacts with the anionic macromolecule lipopolysaccharide and results in the generation of MB dimers (38), which participate in the photosensitization process (38).

The objective of the present study was to evaluate the antimicrobial effects of MB-mediated PDT in a stringent and clinically relevant evaluation using naturally human infected teeth ex vivo treated immediately upon their extraction. Teeth with radiographic evidence of periradicular lesions were chosen because they were guaranteed to be grossly infected, which mimics the clinical situation that leads to higher failure rates (2). The use of naturally-infected teeth, which contain a much broader range of pathogens and deeper penetration into tubules than any in vitro model system provides an excellent test of the potential of PDT in achieving root canal disinfection.

MATERIALS AND METHODS

Collection of teeth and groups

Fifty-two freshly extracted teeth with pulpal necrosis and radiographic evidence of periradicular lesions were obtained from 34 subjects in the Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Boston. Permission to collect extracted teeth was authorized by Institutional Review Board-approved informed consent. Patients had no systemic disease and had not taken any antibiotics in the previous 3 months. Following extraction, teeth were placed into individual sterile vials and transferred within 30 minutes to the Applied Molecular Photomedicine Laboratory at The Forsyth Institute for preparation and experimentation. The external surface of each tooth was cleaned with 10% povidone-iodine. After 5 min, the disinfectant was removed from the surface with isopropyl alcohol and the tooth was decoronated apical to the roof of the pulp chamber with a sterile rotating diamond saw (#911H, Brasseler USA, Savannah, GA) set at 20,000 rpm. Teeth were assigned to two groups. The first group comprised 26 teeth (5 incisors, 3 canines, 6 premolars, 12 molars) with 49 canals that received only chemomechanical debridement (CMD group) (Fig. 1). The second group comprised 26 teeth (4 incisors, 2 canines, 6 premolars, 14 molars) with 52 canals that received CMD followed by PDT (CMD+PDT group) (Fig. 1). In eight subjects, more than one tooth was obtained (22 teeth). In these cases, teeth were randomly allocated to one of the above groups.

Figure 1
Fifty-two teeth were assigned to two experimental groups (CMD and CMD+PDT). The number of canals from each tooth that received treatment is provided. Numbers highlighted in bold indicate incompletely disinfected teeth. The number of positive canals following ...

Baseline bacterial samplings

In both groups, a baseline microbial sample of the root canal was taken. The canal was completely filled with pre-reduced anaerobically sterilized (PRAS) Ringer’s solution using a sterile Monoject tuberculin syringe with 27-G X 1/2-in. detachable needle (Sherwood Medical, St. Louis, MO). A sample was collected by introducing an ISO size 10 K-type file to a working length of 0.5 mm short of the apical foramen and then agitated in the canal solution in the canal for 60 seconds. The file was then removed and the file handle was cut off under aseptic conditions and put in a 1.5 ml microcentrifuge tube containing 1 ml PRAS Ringer’s solution. The canal contents were aspirated using the same syringe as above and added in the tube containing the file.

Root canal treatment

Chemomechanical debridement was performed in teeth of both groups using standard K files and 0.04 mm/mm taper number 7 series 29 Ni-Ti rotary Profiles™ (Dentsply Maillefer, Tulsa, OK) to achieve a master apical file size of .465 (ISO equivalent) for distal canals of mandibular molars, palatal canals of maxillary molars, and all single rooted teeth. Number 6 0.04 mm/mm taper Ni-Ti rotary Profiles™ were used to achieve a master apical file size of .360 (ISO equivalent) for buccal canals of maxillary molars and mesial canals of mandibular molars. RC Prep® (Premium Products, Plymouth Meeting, PA) was used as a lubricant during instrumentation and canals were irrigated with 10 cc of 6% sodium hypochlorite (NaOCl) throughout the instrumentation sequence. All irrigants used were dispensed using a 30 gauge Max-I-Probe (Dentsply Maillefer). After canal preparation an aliquot of 1 ml of 17% ethylenediaminetetraacetic acid (EDTA) solution was left in situ for 3 min for smear layer removal, and was replaced by 1 ml of 6% NaOCl for 3 minutes.

Post-SET bacterial sampling

Immediately after chemomechanical disinfection, each specimen in the CMD group was aseptically mounted on a rubber dam attached to a rack. The contents of root canals were sampled by flushing the root canals with a coronal application of 1-ml of sterile phosphate buffered saline (PBS) with a Pro Rinse® 30 gauge irrigation needle (Dentsply Maillefer) (Fig. 2). The bacterial suspension was collected in a 1.5 ml microcentrifuge tube positioned below the apical foramen and bacterial yielding was measured spectrophotometrically for each sample. After vortexing for 20 seconds, serial dilutions were prepared and 100 µl aliquots were inoculated onto blood agar and incubated anaerobically for 7 days.

Figure 2
Post-flushing of tooth specimen under rubber dam.

Photodynamic therapy

Following CMD, specimens in the CMD+PDT group were treated by MB-mediated PDT. Methylene blue (Sigma, St Louis, MO) was dissolved in sterile PBS and filter-sterilized immediately prior to use. The final concentration used was 50 µg/ml (134 µM). The ultraviolet-visible absorption spectra of MB in PBS were recorded from 200 to 800 nm using quartz cuvettes with 1 cm path length on a diode-array spectrophotometer and were characterized by a long-wavelength maximum at 665 nm as shown previously (14).

All individual specimens were aseptically mounted on a rubber dam, with the rubber dam frame attached to a rack. Then the canals were filled to the level of the access cavity with MB solution using a Pro Rinse® 30 gauge irrigation needle (Dentsply Maillefer) for 5 minutes. Following incubation, the canal was dried with a paper cone. Light was then applied in the root canal system of the specimens in appropriate groups for 2.5 minutes followed by a break of 2.5 minutes and a second light exposure for 2.5 minutes. The irradiation source was a diode laser (BWTEK Inc., Newark, DE) with an output power of 1 Watt and a central wavelength of 665 nm. The system was coupled to a 250-µm diameter optical fiber (22) that was mechanically notched over a one-centimeter length at approximately one-millimeter intervals (Schoelly Imaging Inc., Worcester, MA). The fiber was able to uniformly distribute light at 36° within the root canal. The power density was 100 mW/cm2 and the total energy fluence dose was 30 J/cm2. The fiber optic was wiped with ethanol after the completion of each light exposure.

Post-PDT bacterial sampling

The contents of root canals were sampled by flushing the root canals as described above. Serial dilutions were prepared and 100 µl aliquots were inoculated onto blood agar and incubated anaerobically for 7 days.

Dentinal Shavings

Following flushing of tooth specimens, intracanal dentinal shavings were removed from the CMD group (9 teeth with 22 canals) and CMD+PDT group (12 teeth with 29 canals) and gathered in an microcentrifuge tube containing 1.5 ml of BHI. Briefly, a 21 mm length nickel-titanium rotary file (#25, Sequence, Brasseler, Savannah, Georgia) with a tip diameter of 0.25 mm and a taper of 0.06 mm/mm was inserted to length in each 12 and 14 mm tooth specimen (i.e. at length, the 21 mm Sequence file protruded 9 mm and 7 mm beyond each 12 mm and 14 mm tooth specimen root tip respectively). This generated a circumferential dentinal tubule penetration of 205 to 455µ or 205 to 485µ for each 12 mm or 14 mm tooth specimen respectively measured from each root tip to coronal level.

Microbial analysis

The microbial composition of root canals before and after treatment was assayed using a whole genomic probe assay as described previously (22). Tris-EDTA buffer (1.5 ml) was added to the plates and the bacterial colonies were harvested using glass rods. The cell suspensions were placed into individual Eppendorf tubes and sonicated for 10 sec to break up clumps. The optical density (OD) of each suspension was adjusted to a final OD of 1.0, which corresponded to approximately 109 cells. Ten µl of the suspension (107 cells) was removed and placed in another Eppendorf tube with 140 µl of TE buffer and 150 µl of 0.5M NaOH. The samples were lysed and the DNA was placed in lanes on a positively charged nylon membrane using a Minislot device (Immunetics, Cambridge, MA, USA). After fixation of the DNA to the membrane, the membrane was placed in Miniblotter 45 (Immunetics) with the lanes of DNA perpendicular to the lanes of the device. Digoxigenin-labeled whole genomic DNA probes against 39 species found in endodontic infections (39) were hybridized in individual lanes of the Miniblotter. After hybridization, the membranes were washed at high stringency and the DNA probes were detected using antibody to digoxigenin conjugated with alkaline phosphatase for chemifluorescence detection. Signals were detected using AttoPhos substrate (Amersham Life Science, Arlington Heights, IL, USA) and were scanned using a Storm Fluorimager (Molecular Dynamics, Sunnyvale, CA, USA). Computer-generated images were analyzed to determine the fluorescence intensity associated with each sample and probe. Two lanes in each membrane contained DNA standards with 1 ng (105 bacteria) and 10 ng (106 bacteria) of each species. The sensitivity of the assay was adjusted to permit detection of 104 cells of a given species by adjusting the concentration of each DNA probe. The measured fluorescence intensities were converted to absolute counts by comparison with the standards on the same membrane. Failure to detect a signal was recorded as zero.

Statistical Analysis

The principal endpoint calculated for each canal was the residual level of colony-forming units (CFUs) following treatment relative to the pretreatment CFU level (residual %CFUs). For multi-rooted teeth the value for each tooth was the average of the canal values. Treatment effects were evaluated in a logistic model using generalized estimating equations (GEE) to account for correlations between canals from the same tooth. An indicator (0/1) variable was included to estimate and adjust for single-rooted/multi-rooted teeth. Similar analyses were done for teeth stratified as single/multi-rooted using Mantel-Haenszel analysis.

RESULTS

Figure 1 shows the distribution of 52 teeth in the CMD (26 teeth) and CMD+PDT (26 teeth) groups. The number of canals from each tooth that received either treatment is also provided. These numbers are highlighted in bold when teeth were incompletely disinfected following treatment and the number of positive canals is given in parentheses. The results clearly demonstrated the better performance of CMD+PDT over CMD. The summary Mantel-Haenszel chi-square test for treatment effects was significant (P= 0.026). Overall, 13 of 26 teeth (50%) were positive following CMD, whereas 6 of 20 teeth (30%) were positive following CMD+PDT. Among single-rooted teeth, 5 of 14 teeth (35.7%) were positive following CMD, whereas only 1 of 12 teeth (8.3%) was positive following CMD+PDT. Among multi-rooted teeth, 8 of 12 teeth (66.7%) were positive following CMD and 5 of 14 teeth (35.7%) were positive following CMD +PDT.

CMD+PDT significantly reduced the frequency of positive canals relative to CMD alone (P= 0.0003) (Table 1). Canals from single-rooted teeth were less likely to be positive post-treatment than canals from multi-rooted teeth (P= 0.10). Following CMD+PDT, 45 of 52 canals (86.5%) had no CFU as compared to 24 of 49 canals (49%) treated with CMD (Table 1) (canal flush samples). Post-treatment microbial levels were low as a percent of pretreatment levels (%CFU). For CMD+PDT only 7 of 52 canals (13.5%) were positive and all had %CFU less than 0.1% of pretreatment CFU levels. However, following CMD 25 of 49 canals were positive (51%) and 22.4% of canals had post-treatment values greater than 0.1% of pretreatment levels (Table 1). The CFU reductions were similar when teeth or canals were treated as independent entities. Analysis stratified by tooth type indicated that post treatment %CFU values were more often positive and also at higher levels of infection in canals that received CMD relative to canals that have received CMD+PDT (P<0.0001).

TABLE 1
Frequency of Root Canal Infection after CMD or CMD+PDT Treatment

The microbial composition of canal biofilms (canal flush samples) was studied by checkerboard DNA-DNA hybridization. Pre- and post-treatment frequencies (+/−) were obtained for 39 species found in endodontic infections with whole genomic probes for 45 canals that received CMD+PDT and 44 that received CMD alone (Fig. 3). The number of canals positive for each species pretreatment was quite high and the pattern was similar for both treatments. Post-treatment detection levels for all species were systematically and markedly lower for canals treated by CMD+PDT than for those treated by CMD alone. Key endodontic pathogens resisting intracanal disinfection procedures (40) were dramatically reduced (Fig. 3, highlighted in grey).

Figure 3
Pre- and post-treatment detection frequencies for 39 species found in endodontic infections by checkerboard DNA-DNA hybridization with whole genomic probes for 45 canals treated by CMD+PDT and 44 treated by CMD alone.

The frequency of dentinal infection (after debridement up to 485 µm) was also evaluated (Table 2). In the CMD group, tubules from 17/22 canals (77.3%) were positive after treatment, while in the CMD+PDT group tubules from 15/29 (51.7%) canals were positive (P= 0.034).

TABLE 2
Frequency of Dentinal Infection (after debridement up to 485 µm) after CMD or CMD+PDT Treatment

DISCUSSION

The present study was built on the interdependent foundations of: 1) developing an in vitro model for testing PDT (14, 20, 22); 2) the utilization of an FDA approved drug – methylene blue (MB) – as the photosensitizer (14, 20, 22); 3) the development of a novel light delivery system that maximizes the distribution of light within the entire anatomy of the root canal system (22); 4) the ongoing refinement of light and drug dosimetry (14, 20, 22, 41); and 5) the assessment of PDT safety (41). The hypothesis of this study was that near complete elimination of residual root canal bacteria could be achieved using PDT as an adjunctive procedure to SET in chronically-infected extracted human teeth ex vivo. Our findings show that MB-mediated PDT significantly enhanced the effect of CMD. Four in vivo studies have also suggested the potential of PDT as an adjunctive technique to eliminate residual root canal bacteria after CMD (2932). Toluidine blue-mediated PDT offered a means of destroying microorganisms remaining after using sodium hypochlorite alone (29) or citric acid and sodium hypochlorite as co-irrigants (30). PDT significantly enhanced the effect of CMD in teeth with necrotic pulps using a conjugate between polyethyleneimine chlorine e6 conjugate (31) and toluidine blue (32).

The incomplete bacterial killing in dentinal tubules following PDT may be due to: a) Incomplete MB penetration in the tubules that may be related to binding interactions with dentin components; b) Failure of MB to penetrate canal biofilms; and c) Insufficient oxygenation. We have proposed the encapsulation of MB within poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles that may offer a novel nano-platform for enhanced drug delivery and photodestruction of canal biofilms (28). These nanoparticles have a hydrophobic core part made up of PLGA (hydrophobic) and PEO-PPO (surfactant) molecules (polyethylene oxide-polypropylene oxide). Surfactant chains project outwards from the surface of the core part because of their hydrophilic end groups (hydroxyl, quarternary ammonium). This creates a gradient from the hydrophilic end groups outside to increased hydrophobicity towards the core of nanoparticles. Hydrophilic end groups provide an anchoring effect for retention of nanoparticles to negatively-charged membranes. Due to the hydrophobic-hydrophilic orientation of the surfactant molecules, they provide good wettability to enhance interaction on/within bacterial membranes. Infiltration of dentinal tubules by MB-loaded nanoparticles has recently been demonstrated (28). George and Kishen (19) dissolved MB in a mixture composed of glycerol, ethanol and water (30:20:50) and showed greater penetration of MB into dentinal tubules. Our future studies will explore the use of ultrasonic waves for enhancement of the transdentinal movement and penetration of MB in canal biofilms. It has been demonstrated that an irrigant in conjunction with ultrasonic vibration, which generates acoustic streaming and continuous movement of the irrigant, increases the effectiveness of the cleaning of root canal (42). Regarding insufficient oxygenation, the application of perfluoro-decahydro-napthalene in the root canal system was proposed as a carrier of oxygen for enhancement of the PDT effect (26). The basic properties of perfluorocarbons and perfluorocarbon emulsions relevant to their use as oxygen delivery systems were briefly reviewed (43). A Phase III clinical trial in cardiopulmonary bypass surgery, with a protocol that included both augmented-acute normovolemic hemodilution and intraoperative autologous donation, was interrupted following the observation of adverse events. At this time point, there is not enough information concerning the toxicity of these compounds to utilize them.

The results obtained from this study are very promising. The use of naturally infected teeth, which contain a broader range of pathogens than in vitro model systems, provide an excellent test of the potential of PDT in achieving root canal disinfection. However, since some living bacteria were still present in dentinal tubules following PDT, further refinement and enhancement of the PDT procedure may be necessary. The effect of biophysical means and surface tension-reducing agents on the transdentinal penetration of MB as well as the effect of supplemental hyper-oxygenation should be evaluated.

ACKNOWLEDGEMENTS

This work was supported by NIDCR grant RO1-DE-16922.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The authors deny any conflicts of interest related to this study.

REFERENCES

1. Figdor D. Apical Periodontitis: A very prevalent problem. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002;94:651–652. [PubMed]
2. Nair PN. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med. 2004;15:348–381. [PubMed]
3. Siqueira JF, Jr, Rôças IN, Paiva SS, Magalhães KM, Guimarães-Pinto T. Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing. Oral Microbiol Immunol. 2007;22:266–271. [PubMed]
4. Lewsey JD, Gilthorpe MS, Gulabivala K. An Introduction to meta-analysis within the framework of multilevel modelling using the probability of success of root canal treatment as an illustration. Community Dent Health. 2001;18:131–137. [PubMed]
5. Basmadjian-Charles CL, Farge P, Bourgeois DM, Lebrun T. Factors influencing the long-term results of endodontic treatment: a review of the literature. Int Dent J. 2002;52:81–86. [PubMed]
6. Ng Y-L, Mann V, Rahbaran S, Lewsey J, Gulabivala K. Outcome of primary root canal treatment: systematic review of the literature - Part 1. Effects of study characteristics on probability of success. Int Endod J. 2007;40:921–939. [PubMed]
7. Ng Y-L, Mann V, Gulabivala K. Tooth survival following non-surgical root canal treatment: a systematic review in the literature. Int Endod J. 2010;43:171–189. [PubMed]
8. Wu M-K, Shemesh H, Wesselink PR. Limitations of previously published systematic reviews evaluating the outcome of endodontic treatment. Int Endod J. 2009;42:656–666. [PubMed]
9. Nash KD, Brown LJ, Hicks ML. Private practicing endodontists: Production of endodontic services and implications for workforce policy. J Endod. 2002;28:699–705. [PubMed]
10. Brown LJ, Nash KD, Johns BA, Warren M. ADA Health policy Resources Center Dental Health Policy Analysis Series. Chicago, IL: 2003. The Economics of Endodontics.
11. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q. Photodynamic therapy. J Natl Cancer Inst. 1998;90:889–905. [PubMed]
12. Silbert T, Bird PS, Milburn GJ, Walsh L. Disinfection of root canals by laser dye photosensitization. J Dent Res. 2000;79:569.
13. Seal GJ, Ng YL, Spratt D, Bhatti M, Gulabivala K. An in vitro comparison of the bactericidal efficacy of lethal photosensitization or sodium hyphochlorite irrigation on Streptococcus intermedius biofilms in root canals. Int Endod J. 2002;35:268–274. [PubMed]
14. Soukos NS, Chen PS, Morris JT, Ruggiero K, Abernethy AD, Som S, Foschi F, Doucette S, Bammann LL, Fontana CR, Doukas AG, Stashenko PP. Photodynamic therapy for endodontic disinfection. J Endod. 2006;32:979–984. [PubMed]
15. Garcez AS, Núñez SC, Lage-Marques JL, Jorge AO, Ribeiro MS. Efficiency of NaOCl and laser-assisted photosensitization on the reduction of Enterococcus faecalis in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102:e93–e98. [PubMed]
16. Williams JA, Pearson GJ, Colles MJ. Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals. J Dent. 2006;34:363–371. [PubMed]
17. Garcez AS, Ribeiro MS, Tegos GP, Núñez SC, Jorge AO, Hamblin MR. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med. 2007;39:59–66. [PMC free article] [PubMed]
18. George S, Kishen A. Advanced noninvasive light-activated disinfection: assessment of cytotoxicity on fibroblast versus antimicrobial activity against Enterococcus faecalis. J Endod. 2007;33:599–602. [PubMed]
19. George S, Kishen A. Photophysical, photochemical, and photobiological characterization of methylene blue formulations for light-activated root canal disinfection. J Biomed Opt. 2007;12:034029. [PubMed]
20. Foschi F, Fontana CR, Ruggiero K, Riahi R, Vera A, Doukas AG, Pagonis TC, Kent R, Stashenko PP, Soukos NS. Photodynamic inactivation of Enterococcus faecalis in dental root canals in vitro. Lasers Surg Med. 2007;39:782–787. [PubMed]
21. Bergmans L, Moisiadis P, Huybrechts B, Van Meerbeek B, Quirynen M, Lambrechts P. Effect of photo-activated disinfection on endodontic pathogens ex vivo. Int Endod J. 2008;41:227–239. [PubMed]
22. Fimple JL, Fontana CR, Foschi F, Ruggiero K, Song X, Pagonis TC, Tanner AC, Kent R, Doukas AG, Stashenko PP, Soukos NS. Photodynamic treatment of endodontic polymicrobial infection in vitro. J Endod. 2008;34:728–734. [PMC free article] [PubMed]
23. Fonseca MB, Júnior PO, Pallota RC, Filho HF, Denardin OV, Rapoport A, Dedivitis RA, Veronezi JF, Genovese WJ, Ricardo AL. Photodynamic therapy for root canals infected with Enterococcus faecalis. Photomed Laser Surg. 2008;26:209–213. [PubMed]
24. George S, Kishen A. Augmenting the antibiofilm efficacy of advanced noninvasive light activated disinfection with emulsified oxidizer and oxygen carrier. J Endod. 2008;34:1119–1123. [PubMed]
25. George S, Kishen A. Influence of photosensitizer solvent on the mechanisms of photoactivated killing of Enterococcus faecalis. Photochem Photobiol. 2008;84:734–740. [PubMed]
26. Lim Z, Cheng JL, Lim TW, Teo EG, Wong J, George S, Kishen A. Light activated disinfection: an alternative endodontic disinfection strategy. Aust Dent J. 2009;54:108–114. [PubMed]
27. Souza LC, Brito PRR, Machado de Oliveira JC, Alves FRF, Moreira EJL, Sampaio-Filbo HR, Rôças IN, Siqueira JF., Jr Photodynamic therapy with two different photosensitizers as a supplement to instrumentation/ irrigation procedures in promoting intracanal reduction of Enterococcus Faecalis. J Endod. 2010;36:292–296. [PubMed]
28. Pagonis TC, Chen J, Fontana CR, Devalapally H, Ruggiero K, Song X, Foschi F, Dunham J, Skobe Z, Yamazaki H, Kent R, Tanner AC, Amiji MM, Soukos NS. Nanoparticle-based endodontic antimicrobial photodynamic therapy. J Endod. 2010;36:322–328. [PMC free article] [PubMed]
29. Bonsor SJ, Nichol R, Reid TM, Pearson GJ. An alternative regimen for root canal disinfection. Brit Dent J. 2006;22:101–105. [PubMed]
30. Bonsor SJ, Nichol R, Reid TM, Pearson GJ. Microbiological evaluation of photo-activated disinfection in endodontics (an in vivo study) Brit Dent J. 2006;25:337–341. [PubMed]
31. Garcez AS, Núñez SC, Hamblin MR, Ribeiro MS. Antimicrobial effects of photodynamic therapy on patients with necrotic pulps and periapical lesion. J Endod. 2008;34:138–142. [PMC free article] [PubMed]
32. Pinheiro SL, Schenka AA, Neto AA, de Souza CP, Rodriguez HM, Ribeiro MC. Photodynamic therapy in endodontic treatment of deciduous teeth. Lasers Med Sci. 2009;24:521–526. [PubMed]
33. Harris F, Chatfield LK, Phoenix DA. Phenothiazinium based photosensitisers-photodynamic agents with a multiplicity of cellular targets and clinical applications. Curr Drug Targets. 2005;6:615–627. [PubMed]
34. Wainwright M, Mohr H, Walker WH. Phenothiazinium derivatives for pathogen inactivation in blood products. J Photochem Photobiol B. 2007;86:45–58. [PubMed]
35. Ojetti V, Persiani R, Nista EC, Rausei S, Lecca G, Migneco A, Cananzi FC, Cammarota G, D’Ugo D, Gasbarrini G, Gasbarrini A. A case-control study comparing methylene blue directed biopsies and random biopsies for detecting pre-cancerous lesions in the follow-up of gastric cancer patients. Eur Rev Med Pharmacol Sci. 2007;11:291–296. [PubMed]
36. Creagh TA, Gleeson M, Travis D, Grainger R, McDermott TE, Butler MR. Is there a role for in vivo methylene blue staining in the prediction of bladder tumour recurrence? Br J Urol. 1995;75:477–479. [PubMed]
37. Wainwright M, Phoenix DA, Marland J, Wareing DR, Bolton FJ. A study of photobactericidal activity in the phenothiazinium series. FEMS Immunol Med Microbiol. 1997;19:75–80. [PubMed]
38. Usacheva MN, Teichert MC, Biel MA. The interaction of lipopolysaccharides with phenothiazine dyes. Lasers Surg Med. 2003;33:311–319. [PubMed]
39. Brito LC, Teles FR, Teles RP, França EC, Ribeiro-Sobrinho AP, Haffajee AD, Socransky SS. Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. J Clin Microb. 2007;45:3039–3049. [PMC free article] [PubMed]
40. Siqueira JF, Jr, Rôças I. Clinical Implications and Microbiology of Bacterial Persistence after Treatment Procedures. J Endod. 2008;34:1291–1301. [PubMed]
41. Xu Y, Young MJ, Battaglino RA, Morse LR, Fontana CR, Pagonis TC, Kent R, Soukos NS. Endodontic antimicrobial photodynamic therapy: safety assessment in mammalian cell cultures. J Endod. 2009;35:1567–1572. [PMC free article] [PubMed]
42. Gutarts R, Nusstein J, Reader A, Beck M. In vivo debridement efficacy of ultrasonic irrigation following hand-rotary instrumentation in human mandibular molars. J Endod. 2005;31:166–170. [PubMed]
43. Riess JG. Perfluorocarbon-based oxygen delivery. Artif Cells Blood Substit Immobil Biotechnol. 2006;34:567–580. [PubMed]