ugmenting the Antibiofilm Efficacy of Advancedoninvasive Light Activated Disinfection with Emulsifiedxidizer and Oxygen Carrier
aji George, PhD, and Anil Kishen, MDS, PhD
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bstractn this study, we tested the hypothesis that the inclu-ion of an oxidizer and oxygen carrier in the photosen-itization formulation would facilitate comprehensiveisinfection of matured endodontic biofilm by light-ctivated disinfection (LAD). Photosensitizing formula-ions containing methylene blue (MB) and an oxygenarrier alone (perfluorodecahydronaphthalene) (PF1) orn combination with oxidizer (H2O2) (PF2) or their emul-ions formed with triton-X100 (Bio-Rad Laboratories,ercules, CA) in different proportions (PF3 and PF4)ere tested for photochemical properties and damage
o the biofilm structure using confocal laser scanningicroscopy. Conventional chemomechanical prepara-
ion, LAD using MB in water, and LAD using MB inmulsion (PF4) were also conducted on 10-week-oldnterococcus faecalis biofilm within root canals. MB inmulsion (PF4) was overall the most effective photo-ensitizer formulation for photooxidation, generation ofinglet oxygen (p � 0.001), and in disinfecting biofilmacteria. Advanced noninvasive LAD using a photosen-itizer formulation containing oxidizer and oxygenarrier disrupted the biofilm matrix and facilitatedomprehensive inactivation of biofilm bacteria. Thisodified photosensitizer formulation will have po-
ential advantages in endodontic disinfection. (Jndod 2008;34:1119 –1123)
ey Wordsiofilm, emulsion, endodontics, oxidizer, oxygen car-ier, photodynamic therapy
From the Department of Restorative Dentistry, Nationalniversity of Singapore, National University Hospital, Republicf Singapore.
Supported by National University of Singapore (NUS) ARFrant number R-224-000-024-112.
Address requests for reprints to Dr Anil Kishen, Depart-ent of Restorative Dentistry, Faculty of Dentistry, Nationalniversity of Singapore, National University Hospital, 5 Lowerent Ridge Road, Republic of Singapore 119074. E-mail ad-ress: [email protected]/$0 - see front matter
acterial biofilm in the root canal system that evokes inflammatory response can leadto apical periodontitis (1, 2). Conventionally, disinfection of the root canal is sought
y a “chemomechanical” approach that involves cleaning and shaping of the root canalystem by the application of a chemical disinfectant and mechanical instrumentation3). Nonetheless, this technique often fails to eradicate bacterial biofilms completely,ostly because of various microbiological and anatomical factors (2, 4 – 6). Endodon-
ic pathogens such as Enterococcus faecalis have been reported to form a biofilm evenn medicated root canals, which is regarded as one of the reasons for its persistence in
he posttreatment endodontic environment (7). The phenotypic and genotypic variationf biofilm bacteria when compared with their “free-floating” counterparts, comple-ented by the structure and composition of biofilm matrix, contribute to their high
ntimicrobial resistance (8 –10).Recently, many in vitro and in vivo studies have highlighted the potential of
ight-activated disinfection (LAD) that involves the use of a photosensitizer andow-level light for treating localized bacterial infections (11–13). During LAD, thehotoactivated photosensitizer molecule can either transfer an electron to theeighboring molecule (type-1 reaction) or its energy to molecular oxygen (type-2eaction) to generate highly reactive oxygen species, mostly singlet oxygen. Theilling of bacteria by LAD can be induced by either of these photoreactions; how-ver, a type 2 reaction, which generates highly reactive singlet oxygen (1 O 2 ), isegarded as the principle bactericidal agent. Unlike antibiotics, the emergence ofLAD-resistant” bacterial strains is highly unlikely because the oxygen-based freeadicals act on multiple targets in a bacterial cell (14, 15).
LAD conducted on endodontic biofilms has very often failed to achieve completeradication, prompting many workers to combine LAD with conventional antimicrobialtrategies for superior performances (16 –21). Nevertheless, many workers have over-ooked the necessity of designing “tissue-specific LAD conditions.” This is because thehysicochemical environment existing at the site of application may influence the “pho-
odynamic effect” during light activation of a photosensitizer. For example, the “hy-ooxygenic” nature of bacteria-infected anatomic sites such as a root canal may ad-ersely affect the outcome of LAD because molecular oxygen is a prerequisite for theeneration of singlet oxygen (1, 22). In addition, the cumulative increase in the thick-ess and calcification of biofilm matrix during maturation would create a photosensi-
izer and oxygen-concentration gradients across the thickness of biofilm (1, 9, 10, 22).Consequently, a combination of a photosensitizer and light may not be sufficient
or the effective eradication of endodontic biofilm. We had previously reported thedvantages of an advanced noninvasive light-activated disinfection (ANILAD) strategyor the eradication of endodontic biofilm (13, 23). In the first step of this dual-stagedpproach, sensitization was performed by using a photosensitizer dissolved in a formu-ation that enhanced not only the photochemical properties of photosensitizer but alsollowed better diffusion of a photosensitizer into the anatomic complexities of the rootanal system. In the second step, an oxygen-carrier solution was applied to enhance thexygen availability and to facilitate light propagation during the irradiation phase. Al-
hough ANILAD performed better than conventional LAD, the bactericidal effect wasignificantly less in matured biofilm models (24). The increased thickness and calcifi-
ation of matured biofilm matrix was thought to contribute to its resistance (9).
Augmenting Antibiofilm Efficacy of LAD with Emulsified Oxidizer and Oxygen Carrier 1119
An emulsified oxygen carrier and antibiotics have been reported tonhance wound healing and antimicrobial action against biofilm (25,6). However, a similar concept has not been tried in LAD. We hypoth-size that the inclusion of an oxidizer and an oxygen carrier in thehotosensitizing formulation could enhance the photooxidation poten-ial of LAD, and this could facilitate the disruption of the biofilm matrixnd more complete eradication of biofilm bacteria. Therefore, in thistudy, experiments were conducted with different photosensitizing for-ulations to examine (1) the ability to generate the photooxidation
roduct and singlet oxygen, (2) the efficacy to eradicate bacterial bio-ilm in an in vitro model, and (3) the efficacy to eradicate bacterialiofilm in an ex vivo model.
Materials and MethodsUnless otherwise stated, all chemicals and bacteriologic media
ere purchased from Sigma-Aldrich Inc (St Louis, MO). Methylenelue (MB), a phenothiazine dye, was used as the photosensitizer;erfluoro-decahydronaphthalene was used as the oxygen carrier;ydrogen peroxide (H2O2) (Cica Reagents, Japan) was used as thexidizer; and a nonionic detergent, triton-X100 (Bio-Rad Laborato-ies, Hercules, CA) was used as the surfactant. A diode laser ofavelength 664 nm (wavelength for MB excitation) with an outputnergy of 30 mW was used as the light source. The laser light waselivered by using an optical fiber of a 400-�m outer diameterPower Technology Inc., Little Rock, AR).
hemical AssaysFour different photosensitizing formulations were tested for model
ubstrate oxidation and singlet-oxygen generation: PF1, 50 �mol/L ofB in combination with perfluorodecahydronaphthalene; PF2- 50,mol/L of MB in combination with perfluorodecahydronaphthalene
nd H2O2 (66.6:33.3); PF3, 50 �mol/L of MB in an emulsion producedy mixing perfluorodecahydronaphthalene:H2O2:triton-X100 in the ra-
io 60:35:5; and PF4-50, �mol/L of MB in an emulsion produced byixing perfluoroecahydronaphthalene:H2O2:triton-X100 in the ratio
5:24.5:0.5.The photooxidizing activity of different photosensitizing formula-
ions was evaluated by fluorimetrically measuring the photooxidation ofodel substrate N-acetyl-L-tryptophanamide (NATA). MB, at a final
oncentration of 50 �mol/L in different formulations containing 10mol/L NATA, was taken in a fluorimetric cuvette. The test solution was
rradiated with 664 nm in order to induce the generation of oxidizinggents (23). The rate of decrease of NATA concentration with an in-reasing dose of irradiation was recorded by measuring the intensity of90 nm excited fluorescence emission spectrum at 300 to 400 nmypical of the tryptophanyl moiety of NATA.
Singlet-oxygen generation was assessed photometrically by using,3-diphenylisobenzofuran (DPBF), a singlet-oxygen scavenger. DPBFt a concentration of 100 �mol/L that corresponds to an absorbanceetween 1.5 and 2 at 420 nm was mixed with 10 �mol/L MB in different
est formulations (total volume 3 mL). The rate of singlet-oxygen pro-uction was related to the rate of decrease in DPBF concentration (cal-ulated from the absorbance at 420 nm using UV-VISIBLE Spectropho-ometer, Shimadzu, Kyoto, Japan) as a function of the irradiation dose.ll experiments were repeated in triplicate, and the statistical signifi-ances of mean value were analyzed by one-way analysis of variance,
nd p values less than 0.05 were considered significant.
120 George and Kishen
he Antibiofilm Efficacy of Augmented ANILADharacterization of Structural Damage to Biofilm by LAD:n Vitro
The structural damage to the biofilm caused by LAD was assessedn E. faecalis biofilms grown on a glass coverslip that was fixed cover-ng a grove (6-mm diameter) made at the bottom part of a petridish. E.aecalis cell suspension (100 �L containing �107cells/mL) preparedrom overnight grown culture in all-culture (AC) media was added ontohe saliva-coated glass-cover slip. Six petridishes containing the biofilmere tested in each group. After 7 days of incubation with periodical
eplacement of growth media, the biofilm was washed and photosensi-ized with either 100 �mol/L MB in water or PF4 (MB in an emulsionroduced by mixing perfluorodecahydronaphthalene:H2O2:triton-100 in the ratio 75:24.5:0.5). These biofilms were incubated in theark for 10 minutes, after which the excess of the photosensitizing
ormulation was partially replaced with perfluorodecahydronaphtha-ene and exposed to irradiation using a 664-nm diode laser with a totalluence of 31.84 J/cm2.
After LAD, the washed biofilms were stained using 20 �L PBSontaining 2 �L live/dead stain (Molecular Probes, Eugene, OR) andere observed using confocal laser scanning microscopy (CLSM) (9,7). Confocal illumination was provided by a Kr/Ar laser (488-nm ex-itation wavelength) fitted with a long-pass 514-nm emission filter. Nineindows from each sample were imaged using 60� water-immersion
enses. The optical sections of the biofilm structure were recorded andnalyzed by using FluoView software (Olympus Corporation, Tokyo,apan).
AD of Endodontic Bacterial Biofilm: Ex VivoThe institutional review board of the National University of Singa-
ore approved the collection and use of extracted human single-rootednterior teeth (from young adults ages 16 to 24 years) for this experi-ent.
Thirty tooth blocks were prepared by removing the crown at theevel of the cementoenamel junction and the apical third of the rootanal (�8 mm long). All specimens were instrumented using K-filesith sizes #20 to #40. The smear layer formed during mechanical shap-
ng was removed by rinsing the root canal with sodium hypochlorite1%) followed by EDTA (100 mmol/L). The prepared single-rootedooth specimens were incubated with 50 mL of AC media inoculated withsingle colony of E. faecalis as described elsewhere (23). Incubationas carried out for 10 weeks at 37°C with periodical replacement ofrowth media under constant shaking (120 rpm). After the incubationeriod, the tooth specimens were randomly divided into five equalroups and were treated as follows:
1. Control group: root canals were not subjected to any treatment.2. Root canal– treatment (RCT) group: root canals were subjected
to conventional cleaning and shaping procedures performed byusing a sequence of endodontic file sizes (#25 to #50 K-files;Maillefer Instruments SA, Ballaigues, Switzerland). The root ca-nals were repeatedly irrigated with 5 mL 5.2% sodium hypochlo-rite before and after instrumentation step using a 28-G needle anda syringe. EDTA 17% was also used at the end of instrumentationto remove the smear layer.
3. Conventional LAD group: root canals were subjected to minimalinstrumentation (using #25 K file). These root canals were filledwith 100 �mol/L MB in water and were irradiated with a 664-nmlaser light with a total energy of 31.84 J/cm2.
4. PF4 group: root canals were subjected to minimal instrumenta-tion (using #25 K file) followed by LAD using PF4. Here, the root
canals were filled with PF4 and were left in the dark for 10 min-
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utes. After the sensitization period, the excess of the photosensi-tizing formulation was partially replaced with perfluorodecahy-dronaphthalene, and the root canals were irradiated with a664-nm diode laser with a total fluence of 31.84 J/cm2.
5. RCT�PF4 group: root canals were subjected to the conventionalroot canal disinfection procedure similar to the conventional RCTgroup followed by LAD using PF4.
After the specified treatment, the tooth specimens were split openesiodistally, and dentine shavings were collected using a round bur of
-mm diameter held perpendicular to the root canal surface. The den-inal shavings were collected at the midpoint (coronoapically) from thewo halves (buccal and lingual) of each root (n � 12). These shavingsere inoculated into 1 mL of fresh AC medium and were incubated at7°C for 4 hours to enrich the number of bacteria. Serially dilutedamples (100 �L) were plated on AC agar as triplicate. Colony-formingnits were enumerated on the subsequent day, and the mean value ofog10 number of bacteria and the standard error were calculated. Be-ause the extreme low level of bacterial cells (�100 cells/mL) in thenitial inoculums may fail to show colony-forming units after 4 hours ofnrichment, the bacteriologic status of the dentine powders suspendedn growth media were assayed after 24 hours of incubation. The num-ers of tubes positive for bacterial cells were counted against the total
igure 1. (A) The oxidation of NATA caused by oxygen based-free radicals meaxidation of NATA that was not significantly different in PF3, PF4, and PF2. Hoompared with other sensitizing media. (B) The oxidation of DPBF indicatingations. Compared with any other formulation, PF4 showed a significant increroduction
umber of tubes. b
OE — Volume 34, Number 9, September 2008 Augmenti
Resultshemical Assays
The time-depended reduction in fluorescence intensity at 360 nmecause of the photoxidation of NATA is given in Figure 1A. LAD usingF1 showed significantly reduced NATA oxidation rate (1.21 �mol/L/in) compared with other photosensitizing formulations (p � 0.05).
he rate of DPBF bleaching indicative of singlet-oxygen generation wasnfluenced by the photosensitizing formulation used and was signifi-antly different for each test formulations (p � 0.001) (Fig. 1B). Thebility to produce singlet oxygen in different photosensitizing formula-ions was in the order PF4�PF1�PF3�PF2 that corresponded to aate of 1.95 �mol/L/min, 1.8 �mol/L/min, 1.39 �mol/L/min, and 0.55mol/L/min, respectively.
ntibiofilm Efficacy of Augmented ANILADharacterization of Structural Damage to Biofilm by LAD:n Vitro
CLSM analysis of biofilms before and after subjected to LAD arehown in Figure 2. Figure 2A shows the biofilm that was neither sub-ected to sensitization nor light irradiation. This biofilm was continuousithout marked variation in the thickness (17 �m). As evident fromigure 2B, the viability status of bacteria in the biofilm was not affected
as the reduction of NATA concentration. There was a dose-dependent (J/cm2)beyond 5.4J/cm2, PF1 showed a significantly reduced rate of NATA oxidationglet-oxygen generation during irradiation of different photosensitizing formu-the rate of oxidation of DPBF, showing the increased rate of singlet-oxygen
suredwever,the sinase in
y irradiation alone and the thickness of the biofilm was unchanged.
ng Antibiofilm Efficacy of LAD with Emulsified Oxidizer and Oxygen Carrier 1121
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iofilm subjected to LAD using MB dissolved in water increased theroportion of dead cells (Fig. 2D) and the biofilm thickness was 12 �m,hich was lower than the control (Fig. 2C). However, the treatment wasot sufficient to cause a major destruction of biofilm structure or inac-ivation of biofilm bacteria. Biofilms exposed to LAD using PF4 is shownn Figure 2F. The predominance of red color indicated that a largeroportion of the bacteria in the biofilm were killed under this condi-ion. Most importantly, the remnant of biofilm was thinner (6 �m) andiscontinuous, indicating marked disruption of the biofilm structure.
AD of Endodontic Bacterial Biofilm: Ex VivoThe results of different antibacterial treatment on endodontic bio-
ilm are presented in Table 1. Significant decreases in the viability of E.aecalis biofilms were observed under all the treatment conditionshen viable bacterial cells were enumerated after 4 hours of enrich-ent. When the biofilm was subjected to LAD using MB dissolved inater, there was a difference of 1.5log10 in the mean viable count thatorresponded to 96.89% reduction in viable bacteria compared withhe control group. Complete killing of bacteria was observed when theoot canals were subjected to RCT, LAD using PF4, and treatment com-rising RCT combined with LAD using PF4. However, bacteriologic eval-ation of dentine shaving after 24 hours of enrichment showed all theroups except PF4 and RCT�PF4 groups positive for bacterial growth.
A
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igure 2. The three-dimensional confocal laser scanning microscopy reconstruiofilm receiving no treatment, (B) the biofilm subjected to irradiation alone, (Co sensitization with MB followed by irradiation, (E) the biofilm subjected to P
lthough none of the tubes from conventional RCT group showed bac- t
122 George and Kishen
erial presence when plated after 4 hours of enrichment, 60% of thepecimens showed bacterial growth after 24 hours of enrichment. Theesults overall showed that unlike RCT that may reduce the number ofiable bacteria in an endodontic biofilm, LAD using PF4 can ensureore complete inactivation of endodontic bacterial biofilm.
f the biofilm subjected to LAD (inlet shows the sagittal section) (60�). (A) Theiofilm subjected to sensitization with 100 �mol/L MB, (D) the biofilm subjectedd (F) the biofilm subjected to PF4 and irradiation.
ABLE 1. The Efficacy of Different Endodontic Disinfection Techniques Testedn This Ex Vivo Study
TreatmentGroups
CFU (log10after 4 Hours of
Enrichment (�SD)
Percentage of ToothSpecimens Positive
for Bacteria after 24Hours of Enrichment
Control 7.147 (�0.601) 100Conventional LAD 5.639 (�0.066) 100Conventional RCT 0.0 60LAD using PF4 0.0 0Conventional 0.0 0RCT�LAD using
ooxidation and singlet-oxygen generation when oxygen carrier-oxi-izer emulsion was used in the photosensitizing formulation. Theseesults highlighted that during irradiation of oxygen carrier-oxidizermulsion, reactive oxygen species other than singlet oxygen were alsoenerated. Based on studies conducted by Gak et al. (28), the directnteraction of photoexcited MB molecules with H2O2 (type 1 mecha-ism) has been proposed to generate hydroxyl radicals and hydroper-xide radicals. This reaction, which may happen in the absence ofolecular oxygen, is important in eradicating bacterial growth under
educed oxygen tension as in the case of many in vivo environmentsncluding endodontic infection.
Earlier CLSM observations of matured biofilm have shown theormation of pockets of viable bacterial cells inside the mineralized
atrix of matured biofilm (8). Although studies by Wainwright et al.29) have indicated the efficiency of LAD in breaking down the matrixomponents of biofilm, Zanin et al. (30) suggested that bacterial killinguring LAD was mostly confined to the outer surface of the biofilm. Ourtudies showed that LAD using PF4 could breakdown the biofilm matrixnd inactivate bacteria as evident from the reduced thickness and theiscontinuity in biofilm structure and predominance of red fluores-ence, respectively (Fig. 2). The superior bactericidal action could beaused by the complementing function of oxygen carrier that ensuresdequate concentration of oxygen and oxidizer that degrade the biofilmatrix, thus facilitating the penetration of photosensitizer into the bio-
ilm. The increased photooxidation potential and singlet-oxygen gener-tion were thought to have collectively contributed toward the biofilmatrix disruption and bacterial inactivation during LAD using PF4.
It is evident from this study that “matured” endodontic bacterialiofilm can be effectively inactivated by LAD using PF4. Although con-entional endodontic disinfection procedure showed no viable bacteriafter 4 hours of the enrichment process, 60% of the root canal shavingsonfirmed bacterial growth after 24 hours of incubation. This observa-ion suggested the possibility of bacterial regrowth after disinfection.owever, LAD using PF4 alone or in combination with conventionalisinfection technique showed the absence of bacteria even after 24ours of incubation, suggesting complete bacterial inactivation. Thehemical assays used in this study showed no obvious difference in thehotooxidation potential of PF4 compared with other photosensitiza-ion formulations. However, compared with other formulations, PF4roduced significantly increased rate of singlet-oxygen generation. Thisncreased singlet-oxygen generation could be responsible for completenactivation and disruption of matured biofilm observed in this study.he findings from this investigation suggested that ANILAD using pho-osensitizer formulation containing an emulsion of oxidizer:oxygen car-ier will find significant application in the disinfection of endodonticiofilm.
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