International Journal of
Molecular Sciences Article
New Irradiation Method with Indocyanine Green-Loaded Nanospheres for Inactivating Periodontal Pathogens Yasuyuki Sasaki 1,2 , Jun-ichiro Hayashi 1, *, Takeki Fujimura 1,2 , Yuki Iwamura 1,2 , Genta Yamamoto 1 , Eisaku Nishida 1 , Tasuku Ohno 1 , Kosuke Okada 1 , Hiromitsu Yamamoto 3 , Takeshi Kikuchi 1 , Akio Mitani 1 and Mitsuo Fukuda 1,2 1
2 3
*
Department of Periodontology, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651, Japan; [email protected] (Y.S.); [email protected] (T.F.); [email protected] (Y.I.); [email protected] (G.Y.); [email protected] (E.N.); [email protected] (T.O.); [email protected] (K.O.); [email protected] (T.K.); [email protected] (A.M.); [email protected] (M.F.) Division of Periodontal Health Promotion, Dental Hospital, Aichi Gakuin University, Nagoya, Aichi 464-8651, Japan Department of Pharmaceutical Engineering, School of Pharmacology, Aichi Gakuin University, Nagoya, Aichi 464-8650, Japan; [email protected] Correspondence: [email protected]; Tel.: +81-52-759-2150
Academic Editor: Michael R. Hamblin Received: 16 November 2016; Accepted: 10 January 2017; Published: 13 January 2017
Abstract: Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive strategy for periodontitis treatments. However, use of aPDT for periodontal treatment is complicated by the difficulty in accessing morphologically complex lesions such as furcation involvement, which the irradiation beam (which is targeted parallel to the tooth axis into the periodontal pocket) cannot access directly. The aim of this study was to validate a modified aPDT method that photosensitizes indocyanine green-loaded nanospheres through the gingivae from outside the pocket using a diode laser. To establish this trans-gingival irradiation method, we built an in vitro aPDT model using a substitution for gingivae. Irradiation conditions and the cooling method were optimized before the bactericidal effects on Porphyromonas gingivalis were investigated. The permeable energy through the gingival model at irradiation conditions of 2 W output power in a 50% duty cycle was comparable with the transmitted energy of conventional irradiation. Intermittent irradiation with air cooling limited the temperature increase in the gingival model to 2.75 ◦ C. The aPDT group showed significant bactericidal effects, with reductions in colony-forming units of 99.99% after 5 min of irradiation. This effect of aPDT against a periodontal pathogen demonstrates the validity of trans-gingival irradiation for periodontal treatment. Keywords: photodynamic therapy; indocyanine green; diode laser
1. Introduction Periodontitis is caused by anaerobic bacteria, which form a biofilm on tooth surfaces or in the periodontal pocket. These actions provoke an excessive and aggressive immune reaction in the host, and cause collateral damage to periodontal tissues [1]. Removal of the biofilm and elimination of periodontal pathogens from the periodontal pocket is the main purpose of treatment for this disease [2]. Scaling and root-planing (SRP) is a non-surgical method of mechanical debridement to eliminate calculus, plaque, and contaminated root cementum/dentine from the periodontal pocket and usually results in significant clinical improvement. However, complete eradication of pathogenic bacteria from
Int. J. Mol. Sci. 2017, 18, 154; doi:10.3390/ijms18010154
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 154
2 of 11
Int. J. Mol. Sci. 2017, 18, 154
2 of 11
results in significant clinical improvement. However, complete eradication of pathogenic bacteria from areas that are inaccessible to periodontal instruments is difficult. Matia et al. reported that even areas are inaccessible to periodontal instruments is difficult. Matia etinal. reported that even after afterthat non-surgical mechanical debridement, residual calculus was present 37.7% of furcation areas [3]. non-surgical mechanical debridement, residual calculus was present in 37.7% of furcation areas [3]. As an adjunct to SRP in inaccessible areas, various other treatment strategies have been As an [4–7], adjunct to SRPthe in use inaccessible areas, variousand other treatment evaluated including of lasers. CO 2, Nd:YAG, Er:YAG lasers strategies have been have shownbeen to be evaluated [4–7], including the use of lasers. CO , Nd:YAG, and Er:YAG lasers have been shown to be 2 therapy, with clinical effects including the removal effective modalities in non-surgical periodontal effective modalities in non-surgical periodontal therapy, with clinical effects including the removal of of calculus, detoxification of the root surface, and bacterial lysis [8,9]. However, these high-level calculus, detoxification of the root thermal surface, damage and bacterial [8,9]. However, these high-level lasers lasers can produce unacceptable to thelysis dental pulp and periodontal tissues [10]. can produce unacceptable thermal damage to the dental pulp and periodontal tissues [10]. Recently, antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative Recently, antimicrobialadjunctive photodynamic therapy (aPDT) has beenThe proposed as mechanism an alternative strategy to conventional treatment for SRP [11–14]. general ofstrategy aPDT is tochemical conventional adjunctive treatment for SRP [11–14]. The general mechanism of aPDT is chemical sterilization by the highly reactive oxygen species (ROS) formed by irradiation of a sterilization by the highly reactive laser oxygen (ROS)wavelength. formed by irradiation of a photosensitizer photosensitizer with a low-level of species appropriate This ROS sterilization has been with a low-level laser ofeven appropriate This ROS sterilization hasa been shown towe be effective shown to be effective againstwavelength. resistant bacterial species [15,16]. In recent study, validated even resistant bacterial [15,16]. In a recentWe study, we validated the principle of using the against principle of using aPDT species for periodontal therapy. employed an 810-nm diode laser with aPDT for periodontal therapy. We employed an 810-nm diode laser with indocyanine green-loaded indocyanine green-loaded nanospheres coated with chitosan (ICG-Nano/c) to demonstrate nanospheres coated with chitosan (ICG-Nano/c) to demonstrate positive nano-spherization, positive charging of the chitosan coating nano-spherization, of the photosensitizer, andcharging resultant ofimproved the chitosan coating of the photosensitizer, and resultant improved antibacterial effects of aPDT [17]. antibacterial effects of aPDT [17]. Furcation is is a formidable challenge in the treatment of periodontitis, because of theof Furcationinvolvement involvement a formidable challenge in the treatment of periodontitis, because morphological complexity and irregular structure of the bone defects in these areas [18]. Conventional the morphological complexity and irregular structure of the bone defects in these areas [18]. irradiation methods for aPDTmethods can produce effects in furcations (Scheme 1A).in Conventional irradiation for insufficient aPDT can sterilization produce insufficient sterilization effects This is because the direction of insertion of the fiberofinto the periodontal pocket—From furcations (Scheme 1A). This is because the optical direction insertion of the optical fiber intothe the gingival margin and parallel to the tooth axis—Limits the amount of light penetrating into the furcation, periodontal pocket—From the gingival margin and parallel to the tooth axis—Limits the amount of and thus the photosensitizer is excitedand onlythus partially in these areas. is A excited novel irradiation method, in light penetrating into the furcation, the photosensitizer only partially in these which directional limitation is overcome, sufficient light toisthe furcationmay to resolve areas.this A novel irradiation method, in whichmay thisdeliver directional limitation overcome, deliver this problem (Scheme sufficient light to the1B). furcation to resolve this problem (Scheme 1B).
(A)
(B)
Scheme1. 1.Advantages Advantagesofofa anovel novelirradiation irradiationmethod methodfor forextending extendingaPDT aPDTinto intoinaccessible inaccessibleareas areas Scheme (schematic). (A) Conventional irradiation method via the periodontal pocket (intra-pocket (schematic). (A) Conventional irradiation method via the periodontal pocket (intra-pocket irradiation). irradiation). light cannot be delivered to (e.g., inaccessible areas to the Sufficient light Sufficient cannot be delivered to inaccessible areas furcation) due (e.g., to thefurcation) irradiationdue direction irradiation direction being parallel to the tooth axis; (B) Novel trans-gingival irradiation from outside being parallel to the tooth axis; (B) Novel trans-gingival irradiation from outside the periodontal pocket the periodontal pocket (external light passing throughdeep the gingiva can penetrate (external irradiation): light passing irradiation): through the gingiva can penetrate into inaccessible areas.deep into inaccessible areas.
Most biological soft tissues have low light absorption properties in the near infrared (NIR) Most biological soft tissues have low light absorption properties in the near infrared (NIR) spectral regions, especially between 600–1300 nm [19]. This spectral range is called the “tissue optical spectral regions, especially between 600–1300 nm [19]. This spectral range is called the “tissue optical window” [20], within which light can be transmitted into deep layers of soft tissue without significant window” [20], within which light can be transmitted into deep layers of soft tissue without absorption [21]. The penetration depth into soft tissue for wavelengths 800 nm) and its absorption in plasma protein [23]. Recently, ICG and its derivatives have also been applied as NIR fluorescent peak at 805 nm in plasma protein [23]. Recently, ICG and its derivatives have also been applied as NIR
Int. J. Mol. Sci. 2017, 18, 154
3 of 11
Int. J. Mol.probes Sci. 2017, 18, 3 of 11 fluorescent for154optical imaging because of their optical properties. That is, their excitation and fluorescence wavelengths fall within the tissue optical window [24]. Thus, for weoptical hypothesized that of trans-gingival irradiation diode laser could deliver probes imaging because their optical properties. Thatusing is, theiraexcitation and fluorescence sufficient energyfalltowithin excitetheICG in optical sub-gingival wavelengths tissue window areas [24]. such as the furcation, where conventional methods Thus, for intra-pocket irradiation fail to penetrate. of the present was to we hypothesized that trans-gingival irradiationThe usingpurpose a diode laser could deliverstudy sufficient energyintoanexcite ICGmodel, in sub-gingival areas such as the furcation,methods where conventional methods ascertain, in vitro if aPDT using external irradiation can produce efficacious for intra-pocket fail to penetrate. The purpose theICG-Nano/c present study was to ascertain, bactericidal effects. irradiation We investigated the antibacterial effectsof of activated by this new in an in vitro model, if aPDT using external irradiation methods can produce efficacious bactericidal irradiation method, and describe the optimization of conditions for its future clinical application.
effects. We investigated the antibacterial effects of ICG-Nano/c activated by this new irradiation method, and describe the optimization of conditions for its future clinical application. 2. Results 2. Results
2.1. Selection of Gingival Model 2.1. Selection of Gingival Model
To select a suitable tissue material for a model mimicking human gingiva, three types of tissue To select a suitable tissue material for a modelthrough mimicking human threeduring types ofdiode tissuelaser slices were prepared and the permeable energies these weregingiva, measured slices were prepared and the permeable energies through these were measured during diode irradiation. The permeable energies through each type of tissue were increased proportionallaser to peak irradiation. The permeable energies through each type of tissue were increased proportional to peak power output (Figure 1). The energy through the fresh beef slice was the lowest in all settings of power output (Figure 1). The energy through the fresh beef slice was the lowest in all settings of power power output. Low permeability of energy was the most important property for the gingival model, output. Low permeability of energy was the most important property for the gingival model, so beef so beef was selected as the most suitable material for use in subsequent experiments. was selected as the most suitable material for use in subsequent experiments.
Figure 1. Selection a gingivalmodel modelaccording according to of of thethe laser. BarsBars denote the the Figure 1. Selection of aofgingival to the thepermeability permeability laser. denote permeable energies transmitted through three types of raw meat with different contents of myoglobin. permeable energies transmitted through three types of raw meat with different contents of The tissue with the lowest permeability was selected as the best gingival model. Data are the myoglobin. The tissue with the lowest permeability was selected as the best gingival model. Data are mean ± standard deviation. * p < 0.05 (n = 3). the mean ± standard deviation. * p < 0.05 (n = 3).
2.2. Optimization of Power Output for External Irradiation
2.2. Optimization of Power Output for External Irradiation For trans-gingival aPDT to produce a sufficient inhibitory effect against bacteria, the permeable
For trans-gingival to produce a sufficient effect bacteria, the energy through the aPDT gingival model must be equalinhibitory to or higher thanagainst that transmitted by permeable direct energy through Direct the gingival model must belaser equal to or higher that transmitted by direct irradiation. irradiation by the diode apparatus used inthan the present study produced irradiation. irradiation diode apparatus used in W the(Figure present adequateDirect bactericidal effectsby forthe aPDT at a laser peak power output of 0.7 2A,study Figureproduced A1). adequate effects for aPDT at a peak output of 0.7 W (Figure 2A,inFigure A1). This 2 This 2bactericidal log10 reduction in bacterial viability waspower the same efficacy as that described our previous [17]. The transmitted energywas of this condition detected in by our the power meter log10report reduction in bacterial viability thedirect sameirradiation efficacy as that described previous report 0.4 W (Figureenergy 2B). An equivalent levelirradiation of permeablecondition energy was achievedby by the indirect irradiation [17]. was The≈transmitted of this direct detected power meter was through the gingival model at a peak power output of 2 W (Figure 2B). ≈0.4 W (Figure 2B). An equivalent level of permeable energy was achieved by indirect irradiation through the gingival model at a peak power output of 2 W (Figure 2B).
Int. J. Mol. Sci. 2017, 18, 154 Int. Int.J.J.Mol. Mol.Sci. Sci.2017, 2017,18, 18,154 154
4 of 11 44of of11 11
(A) (A)
(B) (B)
Figure 2. Optimization of power output for external irradiation. (A) effect of with Figure output forfor external irradiation. (A)Bactericidal Bactericidal effecteffect ofaPDT aPDT with Figure 2. 2. Optimization Optimizationofofpower power output external irradiation. (A) Bactericidal of aPDT ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus used in ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus used in with ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by direct this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by direct used in this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by irradiation in with (B) of for external irradiation incombination combination withICG-Nano/c; ICG-Nano/c; (B)Determination Determination ofoptimal optimal poweroutput output for external direct irradiation in combination with ICG-Nano/c; (B) Determination of power optimal power output for irradiation. Permeable energies transmitted through the gingival model at different power outputs irradiation. Permeable energies energies transmitted through the gingival model atmodel different power outputs external irradiation. Permeable transmitted through the gingival at different power (black were with energy of irradiation with (black column) were compared compared with the the transmitted energy of direct direct irradiation with power outputscolumn) (black column) were compared withtransmitted the transmitted energy of direct irradiation withaaapower power output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are the output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are the the mean ± standard deviation. * p < 0.05 (n = 3). mean ± standard deviation. * p < 0.05 (n = 3). mean ± standard deviation. * p < 0.05 (n = 3).
2.3. 2.3.Absorption Absorption of Permeable Energy byPhotosensitizers Photosensitizers 2.3. Absorptionof ofPermeable PermeableEnergy Energy by by Photosensitizers To To ascertainifif if the thelight lighttransmitted transmittedthrough throughthe thegingival gingivalmodel modelretained retainedthe theability abilityto toexcite excitethe the To ascertain ascertain the light transmitted through the gingival model retained the ability to excite the photosensitizer, the absorption permeable energy by 33 photosensitizer,the theabsorption absorption of permeable energy by ICG-Nano/c ICG-Nano/c was investigated. investigated. Figure photosensitizer, ofof permeable energy by ICG-Nano/c was was investigated. FigureFigure 3 shows shows that—After passing through the gingival model—Significantly less energy penetrates shows that—After passing through the gingival model—Significantly less energy penetrates that—After passing through the gingival model—Significantly less energy penetrates through the through solution layer medium. that through the the ICG-Nano/c ICG-Nano/c solution layerthan than through the control controlThis medium. This result result suggested suggested that ICG-Nano/c solution layer than through thethrough control the medium. result This suggested that the energy the energy penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, the energy penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, thus, could be thus, be to thus,could could beused used toexcite excitethe thephotosensitizer. photosensitizer. used to excite the photosensitizer.
Figure 3. Absorption Absorptionby the photosensitizer photosensitizer of of permeable permeable energy energy transmitted transmitted through through the the gingival gingival Figure 3. by the model. chart shows the penetration in experimental scenarios. Control: The chart shows the energy penetration in different experimental scenarios. Control: model.The The chart shows theenergy energy penetration indifferent different experimental scenarios. Control:gingival gingival model and control ICG-Nano/c: gingival ICG-Nano/c. Data the ±± gingival model andmedium; control medium; ICG-Nano/c: gingival model and ICG-Nano/c. are the model and control medium; ICG-Nano/c: gingival model model and and ICG-Nano/c. Data are are Data the mean mean mean ± standard deviation. * p < 0.05 (n = 3). standard deviation. * p < 0.05 (n = 3). standard deviation. * p < 0.05 (n = 3).
2.4. 2.4. Comparison ComparisonofofCooling CoolingEffects Effects Avoidance of hyperthermic hyperthermic damage damage to to gingival gingival tissue tissue is is of of paramount paramount importance to to external external Avoidance of paramount importance irradiation irradiationmethods require higher higher irradiation irradiation energy energy than than conventional conventional intra-pocket intra-pocket methods because because these these require irradiation. irradiation.Figure Figure44shows showsaa comparison comparison of of four four cooling cooling methods methods for for the the trans-gingival irradiation Figure the trans-gingival trans-gingival irradiation model. model. The Thehighest highestrise risein temperatureon the gingival gingival model model was was observed observed during during The highest rise in temperature temperature on the the surface surface of of the continuous continuous irradiation irradiation(15.27 (15.27 °C °Cincrease increase after after55 min minof of irradiation), irradiation), whereas whereasintermittent intermittent irradiation irradiation
Int. J. Mol. Sci. 2017, 18, 154
5 of 11
Int. J. Mol. Sci. 2017, 18, 154
5 of 11
continuous irradiation (15.27 ◦ C increase after 5 min of irradiation), whereas intermittent irradiation with with air cooling limited the just2.72 2.72°C◦ at C 5atmin 5 min (Figure 4A). Temperature air cooling limited theincrease increase to just (Figure 4A). Temperature changeschanges in the in ◦ ICG-Nano/c solution were also compared, with the smallest increase (4.94 °C at 5 min) recorded the ICG-Nano/c solution were also compared, with the smallest increase (4.94 C at 5 min) recorded during intermittent irradiation withair aircooling cooling (Figure (Figure 4B). during intermittent irradiation with 4B).
(A)
(B) Figure 4. Temperature changes during external irradiation with different cooling paradigms.
Figure 4. Temperature changes during external irradiation with different cooling paradigms. (A) Temperature changes at the surface of the gingival model; (B) Temperature changes in the (A) Temperature changes at the surface of the gingival model; (B) Temperature changes in the ICG-Nano/c solution beneath the gingival model. The mean value of the highest temperatures within a ICG-Nano/c solution beneath the gingival is model. The mean value heat distribution captured by thermography indicated in polygonal linesof (n the = 3).highest Cooling temperatures strategies within a heat distribution captured by thermography is indicated in polygonal (n = 3). Cooling were air cooling (air blowing at 2 L/min) and/or intermittent irradiation (10 s break lines in radiation every strategies were air cooling (air blowing at 2 L/min) and/or intermittent irradiation (10 s break in 60 s). radiation every 60 s). 2.5. Bactericidal Effect of aPDT with Laser Transmitted through the Gingival Model
2.5. Bactericidal Effect of aPDT Laser Transmitted throughifthe Gingival Model irradiation showed The ultimate aim of thewith present study was to ascertain aPDT with external a sufficient bactericidal effect against periodontal pathogenic bacteria to be considered for
The ultimate aim of the present study was to ascertain if aPDT with external irradiation trans-gingival periodontal therapy in vivo. Using the conditions optimized above (2 W; 50% duty showed a sufficient bactericidal effect against periodontal pathogenic bacteria to be considered for cycle with a pulse of 100 ms; intermittent irradiation with air cooling), we tested the bacterial trans-gingival periodontal vivo. post-irradiation Using the conditions optimized (2 W; 50% viability after irradiation.therapy Figure 5inshows colony-forming unit above (CFU) counts on a duty cyclelogarithmic with a pulse of 100 ms; intermittent irradiation with air cooling), we tested the scale. The viability of Porphyromonas gingivalis was decreased significantly in all ofbacterial the viability irradiation. 5 shows post-irradiation colony-forming (CFU) counts on aPDTafter groups (1, 3, and 5 Figure min) compared with the control group, and the efficacyunit of aPDT increased proportional to theThe duration of irradiation (Figure 5A).gingivalis The most was significant reduction in bacterialin all a logarithmic scale. viability of Porphyromonas decreased significantly viability observed was on the order of 4 log 10 (99.99% reduction) when the mixture was irradiated of the aPDT groups (1, 3, and 5 min) compared with the control group, and the efficacy offoraPDT 5 min.proportional After irradiation for duration 3 min, bacterial viability (Figure was reduced ordersignificant of 2 log10 (96.71%), increased to the of irradiation 5A). by Theanmost reduction in equivalent to the aPDT effect of direct irradiation for 1 min reported previously [17]. External bacterial viability observed was on the order of 4 log10 (99.99% reduction) when the mixture was irradiation for 1 min produced a reduction of 78.12%. To confirm that this bactericidal effect was irradiated for 5 min. After irradiation for 3 min, bacterial viability was reduced by an order of 2 log10 caused by aPDT, a culture of P. gingivalis mixed with or without ICG-Nano/c was irradiated by a (96.71%), the aPDT effect direct irradiation for 15B). minBacterial reportedviability previously External diodeequivalent laser for 3tomin through the of gingival model (Figure was [17]. reduced irradiation for 1 min produced a reduction of 78.12%. To confirm that this bactericidal effect was significantly in the aPDT group, but not in the control, ICG-Nano/c-only, or laser-only groups. caused by aPDT, a culture of P. gingivalis mixed with or without ICG-Nano/c was irradiated by a diode laser for 3 min through the gingival model (Figure 5B). Bacterial viability was reduced significantly in the aPDT group, but not in the control, ICG-Nano/c-only, or laser-only groups.
Int. J. Mol. Sci. 2017, 18, 154
6 of 11
Int. J. Mol. Sci. 2017, 18, 154
6 of 11
(A)
(B)
Figure 5. Bactericidal effect of aPDT with ICG-Nano/c in the external irradiation model.
Figure 5. Bactericidal effect of aPDT with ICG-Nano/c in the external irradiation model. (A) Bactericidal effects with irradiation of different durations (1, 3, and 5 min) were shown by (A) Bactericidal effects with irradiation of different durations (1, 3, and 5 min) were shown by quantification of viable cell counts; (B) Bactericidal effects with or without ICG-Nano/c and laser quantification of cell counts; (B)quantification Bactericidalofeffects or without ICG-Nano/c and laser irradiation forviable 3 min were shown by viable with cell counts. P. gingivalis (108 CFU/mL) 8 CFU/mL) irradiation for 3ICG-Nano/c min were shown by quantification of (10 viable cell every counts. P. by gingivalis (10 mixed with were irradiated intermittently s pause 60 s) the diode laser at a mixed with ICG-Nano/c irradiated intermittently (10cooling. s pauseData every s) mean by the± diode laser at peak power output of were 2 W in a 50% duty cycle with air are60 the standard deviation. p < 0.05of (n 2 = 3). a peak power *output W in a 50% duty cycle with air cooling. Data are the mean ± standard deviation. * p < 0.05 (n = 3). 3. Discussion
3. Discussion This report describes, for the first time, the bactericidal effectiveness of a new trans-gingival method of photoactivation that permits aPDT by laser irradiation from outside the periodontal pocket.
This report describes, for the first time, the bactericidal effectiveness of a new trans-gingival Studies of laser irradiation of the gingival surface have focused on acceleration of healing in wounds or method of photoactivation that permits aPDT bytolaser irradiation outside the periodontal lesions. Here, we developed an irradiation method transmit energy, infrom a trans-gingival manner, to pocket. Studies of laser irradiation of theThe gingival surface focused onmethod acceleration of healing in activate a photosensitizer for aPDT. advantages of have this irradiation for aPDT over wounds or lesions. Here, we developed an irradiation method to transmit energy, in a trans-gingival conventional intra-pocket irradiation are: an operative procedure is facilitated; reduced invasiveness (and to thus, less discomfort) due to no requirement insertion of a laser into the periodontal manner, activate a photosensitizer for aPDT. for The advantages of probe this irradiation method for and effective delivery of appropriate light energy intooperative areas that would otherwise be inaccessible. aPDTpocket; over conventional intra-pocket irradiation are: an procedure is facilitated; reduced Blood-borne pigments such as hemoglobin, and bilirubinof are majorprobe causesinto of the invasiveness (and thus, less discomfort) due to nooxyhemoglobin, requirement for insertion a laser absorbance of visible radiation in the dermis [22]. However, maintenance of a physiologic level of periodontal pocket; and effective delivery of appropriate light energy into areas that would otherwise blood-borne chromophores in a gingival sample ex vivo is not possible because its blood supply is be inaccessible. terminated upon removal from the living body. Myoglobin is an iron-containing porphyrin pigment, Blood-borne pigments such as hemoglobin, oxyhemoglobin, and bilirubin are major causes of as are hemoglobin and oxyhemoglobin, and its absorption spectrum resembles that of hemoglobin. absorbance of visible in the dermis [22].myoglobin) However, as maintenance of atophysiologic level of Therefore, we usedradiation muscle tissues (which contain gingival models mimic the wall blood-borne chromophores in a gingival sample ex vivo is not possible because its blood supply is of the periodontal pocket. Beef proved to be more effective than pork or chicken for the reduction of terminated upon removal from the living body. Myoglobin an iron-containing pigment, the permeable energy. Hence, laser light penetrating throughisthese beef slices shouldporphyrin be able to pass actual and gingiva. At a laser peakand power output of 2 spectrum W, the power transmitted the as arethrough hemoglobin oxyhemoglobin, its absorption resembles that though of hemoglobin. gingival ≈0.4 tissues W. Anders et al.contain reportedmyoglobin) that the percentage transmission of 810 Therefore, wemodel used was muscle (which as gingival models of to light mimic the wall wavelengthpocket. throughBeef the gastrocnemius of rats was 7.42%, through the of thenm periodontal proved to be muscle more effective than porkand or chicken for overlying the reduction skin was 24.63% [25], data that are consistent with our results. of the permeable energy. Hence, laser light penetrating through these beef slices should be able to We calculated that ≥2 W of power output was required to transmit sufficient energy for pass through actual gingiva. At a laser peak power output of 2 W, the power transmitted though trans-gingival aPDT. This output power is higher than that used in conventional aPDT (200–500 mW) the gingival model was ≈0.4 W. Anders et al. reported that the percentage transmission of light of [12,26], and increased the risk of causing histologic damage by hyperthermia. However, we 810 nm wavelength through theincrease gastrocnemius ratsawas 7.42%, throughirradiation the overlying suppressed this temperature in tissue muscle to 20 °C,to andtransmit that transient reversible changes are caused by temperature increases of >5 °C [27]. We conclude that the influence of for trans-gingival aPDT. This output power is higher than that used in conventional aPDT temperature by thisand irradiation method is minimal andhistologic non-injurious, but that periodic application (200–500 mW) [12,26], increased the risk of causing damage by hyperthermia. However, of a laser and continuous air cooling during irradiation are fundamental to hyperthermia ◦ we suppressed this temperature increase in tissue to 15 °C if they are not employed). with air cooling. Reports have suggested that irreversible reactions such as hyperthermal denaturation of tissue proteins occur at temperature increases of >20 ◦ C, and that transient reversible changes are caused by temperature increases of >5 ◦ C [27]. We conclude that the influence of temperature by this irradiation method is minimal and non-injurious, but that periodic application of a laser and
Int. J. Mol. Sci. 2017, 18, 154
7 of 11
continuous air cooling during irradiation are fundamental to hyperthermia suppression (because the temperature rise is >15 ◦ C if they are not employed). We confirmed that laser light penetrating through the gingival model retains its ability to excite the photosensitizer. Our data are in accordance with studies showing that a laser transmitted through tissue maintains its coherence [28]. To confirm this finding, we measured the absorbance of permeable energy by the ICG-Nano/c solution instead of detecting ROS generation directly because measuring ROS is technically very difficult due to its short lifetime (1 × 10−6 s). Bacterial viability was reduced by an order of 2 log10 and 4 log10 after external irradiation for 3 and 5 min, respectively. The bactericidal effect of this irradiation method was equivalent to that described in our report on aPDT with ICG-Nano/c [17] and in other studies of aPDT reporting ICG-mediated inhibition of the planktonic Staphylococcus aureus and Pseudomonas aeruginosa cells with a maximum reduction of 99% [15]. Conversely, aPDT using other photosensitizing dyes produced more substantial effects. For instance, methylene blue excited with a 670 nm laser reduced the viability of P. gingivalis by an order of 7 log10 [29]. However, the ability of 670 nm-light to penetrate through the mucosa is lower than that of light at 810 nm and, thus, is not suitable for trans-gingival irradiation. ICG has been used as a photosensitizer for photothermal therapy because it has higher calorific properties compared with other photosensitizers following laser irradiation [30,31], and the associated temperature increase may enhance its bactericidal effect. Kranz et al. reported that NIR irradiation of high concentration ICG heated a bacterial solution to >60 ◦ C and caused bacterial inactivation due to this hyperthermic effect. They suggested that the generation of heat was thereby influenced by the concentration of the applied ICG and the total exposure value [26]. In our study, the temperature in the ICG-Nano/c solution was
Molecular Sciences Article
New Irradiation Method with Indocyanine Green-Loaded Nanospheres for Inactivating Periodontal Pathogens Yasuyuki Sasaki 1,2 , Jun-ichiro Hayashi 1, *, Takeki Fujimura 1,2 , Yuki Iwamura 1,2 , Genta Yamamoto 1 , Eisaku Nishida 1 , Tasuku Ohno 1 , Kosuke Okada 1 , Hiromitsu Yamamoto 3 , Takeshi Kikuchi 1 , Akio Mitani 1 and Mitsuo Fukuda 1,2 1
2 3
*
Department of Periodontology, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651, Japan; [email protected] (Y.S.); [email protected] (T.F.); [email protected] (Y.I.); [email protected] (G.Y.); [email protected] (E.N.); [email protected] (T.O.); [email protected] (K.O.); [email protected] (T.K.); [email protected] (A.M.); [email protected] (M.F.) Division of Periodontal Health Promotion, Dental Hospital, Aichi Gakuin University, Nagoya, Aichi 464-8651, Japan Department of Pharmaceutical Engineering, School of Pharmacology, Aichi Gakuin University, Nagoya, Aichi 464-8650, Japan; [email protected] Correspondence: [email protected]; Tel.: +81-52-759-2150
Academic Editor: Michael R. Hamblin Received: 16 November 2016; Accepted: 10 January 2017; Published: 13 January 2017
Abstract: Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjunctive strategy for periodontitis treatments. However, use of aPDT for periodontal treatment is complicated by the difficulty in accessing morphologically complex lesions such as furcation involvement, which the irradiation beam (which is targeted parallel to the tooth axis into the periodontal pocket) cannot access directly. The aim of this study was to validate a modified aPDT method that photosensitizes indocyanine green-loaded nanospheres through the gingivae from outside the pocket using a diode laser. To establish this trans-gingival irradiation method, we built an in vitro aPDT model using a substitution for gingivae. Irradiation conditions and the cooling method were optimized before the bactericidal effects on Porphyromonas gingivalis were investigated. The permeable energy through the gingival model at irradiation conditions of 2 W output power in a 50% duty cycle was comparable with the transmitted energy of conventional irradiation. Intermittent irradiation with air cooling limited the temperature increase in the gingival model to 2.75 ◦ C. The aPDT group showed significant bactericidal effects, with reductions in colony-forming units of 99.99% after 5 min of irradiation. This effect of aPDT against a periodontal pathogen demonstrates the validity of trans-gingival irradiation for periodontal treatment. Keywords: photodynamic therapy; indocyanine green; diode laser
1. Introduction Periodontitis is caused by anaerobic bacteria, which form a biofilm on tooth surfaces or in the periodontal pocket. These actions provoke an excessive and aggressive immune reaction in the host, and cause collateral damage to periodontal tissues [1]. Removal of the biofilm and elimination of periodontal pathogens from the periodontal pocket is the main purpose of treatment for this disease [2]. Scaling and root-planing (SRP) is a non-surgical method of mechanical debridement to eliminate calculus, plaque, and contaminated root cementum/dentine from the periodontal pocket and usually results in significant clinical improvement. However, complete eradication of pathogenic bacteria from
Int. J. Mol. Sci. 2017, 18, 154; doi:10.3390/ijms18010154
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2017, 18, 154
2 of 11
Int. J. Mol. Sci. 2017, 18, 154
2 of 11
results in significant clinical improvement. However, complete eradication of pathogenic bacteria from areas that are inaccessible to periodontal instruments is difficult. Matia et al. reported that even areas are inaccessible to periodontal instruments is difficult. Matia etinal. reported that even after afterthat non-surgical mechanical debridement, residual calculus was present 37.7% of furcation areas [3]. non-surgical mechanical debridement, residual calculus was present in 37.7% of furcation areas [3]. As an adjunct to SRP in inaccessible areas, various other treatment strategies have been As an [4–7], adjunct to SRPthe in use inaccessible areas, variousand other treatment evaluated including of lasers. CO 2, Nd:YAG, Er:YAG lasers strategies have been have shownbeen to be evaluated [4–7], including the use of lasers. CO , Nd:YAG, and Er:YAG lasers have been shown to be 2 therapy, with clinical effects including the removal effective modalities in non-surgical periodontal effective modalities in non-surgical periodontal therapy, with clinical effects including the removal of of calculus, detoxification of the root surface, and bacterial lysis [8,9]. However, these high-level calculus, detoxification of the root thermal surface, damage and bacterial [8,9]. However, these high-level lasers lasers can produce unacceptable to thelysis dental pulp and periodontal tissues [10]. can produce unacceptable thermal damage to the dental pulp and periodontal tissues [10]. Recently, antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative Recently, antimicrobialadjunctive photodynamic therapy (aPDT) has beenThe proposed as mechanism an alternative strategy to conventional treatment for SRP [11–14]. general ofstrategy aPDT is tochemical conventional adjunctive treatment for SRP [11–14]. The general mechanism of aPDT is chemical sterilization by the highly reactive oxygen species (ROS) formed by irradiation of a sterilization by the highly reactive laser oxygen (ROS)wavelength. formed by irradiation of a photosensitizer photosensitizer with a low-level of species appropriate This ROS sterilization has been with a low-level laser ofeven appropriate This ROS sterilization hasa been shown towe be effective shown to be effective againstwavelength. resistant bacterial species [15,16]. In recent study, validated even resistant bacterial [15,16]. In a recentWe study, we validated the principle of using the against principle of using aPDT species for periodontal therapy. employed an 810-nm diode laser with aPDT for periodontal therapy. We employed an 810-nm diode laser with indocyanine green-loaded indocyanine green-loaded nanospheres coated with chitosan (ICG-Nano/c) to demonstrate nanospheres coated with chitosan (ICG-Nano/c) to demonstrate positive nano-spherization, positive charging of the chitosan coating nano-spherization, of the photosensitizer, andcharging resultant ofimproved the chitosan coating of the photosensitizer, and resultant improved antibacterial effects of aPDT [17]. antibacterial effects of aPDT [17]. Furcation is is a formidable challenge in the treatment of periodontitis, because of theof Furcationinvolvement involvement a formidable challenge in the treatment of periodontitis, because morphological complexity and irregular structure of the bone defects in these areas [18]. Conventional the morphological complexity and irregular structure of the bone defects in these areas [18]. irradiation methods for aPDTmethods can produce effects in furcations (Scheme 1A).in Conventional irradiation for insufficient aPDT can sterilization produce insufficient sterilization effects This is because the direction of insertion of the fiberofinto the periodontal pocket—From furcations (Scheme 1A). This is because the optical direction insertion of the optical fiber intothe the gingival margin and parallel to the tooth axis—Limits the amount of light penetrating into the furcation, periodontal pocket—From the gingival margin and parallel to the tooth axis—Limits the amount of and thus the photosensitizer is excitedand onlythus partially in these areas. is A excited novel irradiation method, in light penetrating into the furcation, the photosensitizer only partially in these which directional limitation is overcome, sufficient light toisthe furcationmay to resolve areas.this A novel irradiation method, in whichmay thisdeliver directional limitation overcome, deliver this problem (Scheme sufficient light to the1B). furcation to resolve this problem (Scheme 1B).
(A)
(B)
Scheme1. 1.Advantages Advantagesofofa anovel novelirradiation irradiationmethod methodfor forextending extendingaPDT aPDTinto intoinaccessible inaccessibleareas areas Scheme (schematic). (A) Conventional irradiation method via the periodontal pocket (intra-pocket (schematic). (A) Conventional irradiation method via the periodontal pocket (intra-pocket irradiation). irradiation). light cannot be delivered to (e.g., inaccessible areas to the Sufficient light Sufficient cannot be delivered to inaccessible areas furcation) due (e.g., to thefurcation) irradiationdue direction irradiation direction being parallel to the tooth axis; (B) Novel trans-gingival irradiation from outside being parallel to the tooth axis; (B) Novel trans-gingival irradiation from outside the periodontal pocket the periodontal pocket (external light passing throughdeep the gingiva can penetrate (external irradiation): light passing irradiation): through the gingiva can penetrate into inaccessible areas.deep into inaccessible areas.
Most biological soft tissues have low light absorption properties in the near infrared (NIR) Most biological soft tissues have low light absorption properties in the near infrared (NIR) spectral regions, especially between 600–1300 nm [19]. This spectral range is called the “tissue optical spectral regions, especially between 600–1300 nm [19]. This spectral range is called the “tissue optical window” [20], within which light can be transmitted into deep layers of soft tissue without significant window” [20], within which light can be transmitted into deep layers of soft tissue without absorption [21]. The penetration depth into soft tissue for wavelengths 800 nm) and its absorption in plasma protein [23]. Recently, ICG and its derivatives have also been applied as NIR fluorescent peak at 805 nm in plasma protein [23]. Recently, ICG and its derivatives have also been applied as NIR
Int. J. Mol. Sci. 2017, 18, 154
3 of 11
Int. J. Mol.probes Sci. 2017, 18, 3 of 11 fluorescent for154optical imaging because of their optical properties. That is, their excitation and fluorescence wavelengths fall within the tissue optical window [24]. Thus, for weoptical hypothesized that of trans-gingival irradiation diode laser could deliver probes imaging because their optical properties. Thatusing is, theiraexcitation and fluorescence sufficient energyfalltowithin excitetheICG in optical sub-gingival wavelengths tissue window areas [24]. such as the furcation, where conventional methods Thus, for intra-pocket irradiation fail to penetrate. of the present was to we hypothesized that trans-gingival irradiationThe usingpurpose a diode laser could deliverstudy sufficient energyintoanexcite ICGmodel, in sub-gingival areas such as the furcation,methods where conventional methods ascertain, in vitro if aPDT using external irradiation can produce efficacious for intra-pocket fail to penetrate. The purpose theICG-Nano/c present study was to ascertain, bactericidal effects. irradiation We investigated the antibacterial effectsof of activated by this new in an in vitro model, if aPDT using external irradiation methods can produce efficacious bactericidal irradiation method, and describe the optimization of conditions for its future clinical application.
effects. We investigated the antibacterial effects of ICG-Nano/c activated by this new irradiation method, and describe the optimization of conditions for its future clinical application. 2. Results 2. Results
2.1. Selection of Gingival Model 2.1. Selection of Gingival Model
To select a suitable tissue material for a model mimicking human gingiva, three types of tissue To select a suitable tissue material for a modelthrough mimicking human threeduring types ofdiode tissuelaser slices were prepared and the permeable energies these weregingiva, measured slices were prepared and the permeable energies through these were measured during diode irradiation. The permeable energies through each type of tissue were increased proportionallaser to peak irradiation. The permeable energies through each type of tissue were increased proportional to peak power output (Figure 1). The energy through the fresh beef slice was the lowest in all settings of power output (Figure 1). The energy through the fresh beef slice was the lowest in all settings of power power output. Low permeability of energy was the most important property for the gingival model, output. Low permeability of energy was the most important property for the gingival model, so beef so beef was selected as the most suitable material for use in subsequent experiments. was selected as the most suitable material for use in subsequent experiments.
Figure 1. Selection a gingivalmodel modelaccording according to of of thethe laser. BarsBars denote the the Figure 1. Selection of aofgingival to the thepermeability permeability laser. denote permeable energies transmitted through three types of raw meat with different contents of myoglobin. permeable energies transmitted through three types of raw meat with different contents of The tissue with the lowest permeability was selected as the best gingival model. Data are the myoglobin. The tissue with the lowest permeability was selected as the best gingival model. Data are mean ± standard deviation. * p < 0.05 (n = 3). the mean ± standard deviation. * p < 0.05 (n = 3).
2.2. Optimization of Power Output for External Irradiation
2.2. Optimization of Power Output for External Irradiation For trans-gingival aPDT to produce a sufficient inhibitory effect against bacteria, the permeable
For trans-gingival to produce a sufficient effect bacteria, the energy through the aPDT gingival model must be equalinhibitory to or higher thanagainst that transmitted by permeable direct energy through Direct the gingival model must belaser equal to or higher that transmitted by direct irradiation. irradiation by the diode apparatus used inthan the present study produced irradiation. irradiation diode apparatus used in W the(Figure present adequateDirect bactericidal effectsby forthe aPDT at a laser peak power output of 0.7 2A,study Figureproduced A1). adequate effects for aPDT at a peak output of 0.7 W (Figure 2A,inFigure A1). This 2 This 2bactericidal log10 reduction in bacterial viability waspower the same efficacy as that described our previous [17]. The transmitted energywas of this condition detected in by our the power meter log10report reduction in bacterial viability thedirect sameirradiation efficacy as that described previous report 0.4 W (Figureenergy 2B). An equivalent levelirradiation of permeablecondition energy was achievedby by the indirect irradiation [17]. was The≈transmitted of this direct detected power meter was through the gingival model at a peak power output of 2 W (Figure 2B). ≈0.4 W (Figure 2B). An equivalent level of permeable energy was achieved by indirect irradiation through the gingival model at a peak power output of 2 W (Figure 2B).
Int. J. Mol. Sci. 2017, 18, 154 Int. Int.J.J.Mol. Mol.Sci. Sci.2017, 2017,18, 18,154 154
4 of 11 44of of11 11
(A) (A)
(B) (B)
Figure 2. Optimization of power output for external irradiation. (A) effect of with Figure output forfor external irradiation. (A)Bactericidal Bactericidal effecteffect ofaPDT aPDT with Figure 2. 2. Optimization Optimizationofofpower power output external irradiation. (A) Bactericidal of aPDT ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus used in ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus used in with ICG-Nano/c by direct irradiation with a diode laser. The diode laser emitted by the apparatus this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by direct this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by direct used in this study had a peak power output of 0.7 W and showed an appreciable aPDT effect by irradiation in with (B) of for external irradiation incombination combination withICG-Nano/c; ICG-Nano/c; (B)Determination Determination ofoptimal optimal poweroutput output for external direct irradiation in combination with ICG-Nano/c; (B) Determination of power optimal power output for irradiation. Permeable energies transmitted through the gingival model at different power outputs irradiation. Permeable energies energies transmitted through the gingival model atmodel different power outputs external irradiation. Permeable transmitted through the gingival at different power (black were with energy of irradiation with (black column) were compared compared with the the transmitted energy of direct direct irradiation with power outputscolumn) (black column) were compared withtransmitted the transmitted energy of direct irradiation withaaapower power output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are the output of 0.7 W (white column), shown in B to have the required bactericidal efficacy. Data are the the mean ± standard deviation. * p < 0.05 (n = 3). mean ± standard deviation. * p < 0.05 (n = 3). mean ± standard deviation. * p < 0.05 (n = 3).
2.3. 2.3.Absorption Absorption of Permeable Energy byPhotosensitizers Photosensitizers 2.3. Absorptionof ofPermeable PermeableEnergy Energy by by Photosensitizers To To ascertainifif if the thelight lighttransmitted transmittedthrough throughthe thegingival gingivalmodel modelretained retainedthe theability abilityto toexcite excitethe the To ascertain ascertain the light transmitted through the gingival model retained the ability to excite the photosensitizer, the absorption permeable energy by 33 photosensitizer,the theabsorption absorption of permeable energy by ICG-Nano/c ICG-Nano/c was investigated. investigated. Figure photosensitizer, ofof permeable energy by ICG-Nano/c was was investigated. FigureFigure 3 shows shows that—After passing through the gingival model—Significantly less energy penetrates shows that—After passing through the gingival model—Significantly less energy penetrates that—After passing through the gingival model—Significantly less energy penetrates through the through solution layer medium. that through the the ICG-Nano/c ICG-Nano/c solution layerthan than through the control controlThis medium. This result result suggested suggested that ICG-Nano/c solution layer than through thethrough control the medium. result This suggested that the energy the energy penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, the energy penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, penetrating through the gingival model was absorbed by the ICG-Nano/c liquid and, thus, could be thus, be to thus,could could beused used toexcite excitethe thephotosensitizer. photosensitizer. used to excite the photosensitizer.
Figure 3. Absorption Absorptionby the photosensitizer photosensitizer of of permeable permeable energy energy transmitted transmitted through through the the gingival gingival Figure 3. by the model. chart shows the penetration in experimental scenarios. Control: The chart shows the energy penetration in different experimental scenarios. Control: model.The The chart shows theenergy energy penetration indifferent different experimental scenarios. Control:gingival gingival model and control ICG-Nano/c: gingival ICG-Nano/c. Data the ±± gingival model andmedium; control medium; ICG-Nano/c: gingival model and ICG-Nano/c. are the model and control medium; ICG-Nano/c: gingival model model and and ICG-Nano/c. Data are are Data the mean mean mean ± standard deviation. * p < 0.05 (n = 3). standard deviation. * p < 0.05 (n = 3). standard deviation. * p < 0.05 (n = 3).
2.4. 2.4. Comparison ComparisonofofCooling CoolingEffects Effects Avoidance of hyperthermic hyperthermic damage damage to to gingival gingival tissue tissue is is of of paramount paramount importance to to external external Avoidance of paramount importance irradiation irradiationmethods require higher higher irradiation irradiation energy energy than than conventional conventional intra-pocket intra-pocket methods because because these these require irradiation. irradiation.Figure Figure44shows showsaa comparison comparison of of four four cooling cooling methods methods for for the the trans-gingival irradiation Figure the trans-gingival trans-gingival irradiation model. model. The Thehighest highestrise risein temperatureon the gingival gingival model model was was observed observed during during The highest rise in temperature temperature on the the surface surface of of the continuous continuous irradiation irradiation(15.27 (15.27 °C °Cincrease increase after after55 min minof of irradiation), irradiation), whereas whereasintermittent intermittent irradiation irradiation
Int. J. Mol. Sci. 2017, 18, 154
5 of 11
Int. J. Mol. Sci. 2017, 18, 154
5 of 11
continuous irradiation (15.27 ◦ C increase after 5 min of irradiation), whereas intermittent irradiation with with air cooling limited the just2.72 2.72°C◦ at C 5atmin 5 min (Figure 4A). Temperature air cooling limited theincrease increase to just (Figure 4A). Temperature changeschanges in the in ◦ ICG-Nano/c solution were also compared, with the smallest increase (4.94 °C at 5 min) recorded the ICG-Nano/c solution were also compared, with the smallest increase (4.94 C at 5 min) recorded during intermittent irradiation withair aircooling cooling (Figure (Figure 4B). during intermittent irradiation with 4B).
(A)
(B) Figure 4. Temperature changes during external irradiation with different cooling paradigms.
Figure 4. Temperature changes during external irradiation with different cooling paradigms. (A) Temperature changes at the surface of the gingival model; (B) Temperature changes in the (A) Temperature changes at the surface of the gingival model; (B) Temperature changes in the ICG-Nano/c solution beneath the gingival model. The mean value of the highest temperatures within a ICG-Nano/c solution beneath the gingival is model. The mean value heat distribution captured by thermography indicated in polygonal linesof (n the = 3).highest Cooling temperatures strategies within a heat distribution captured by thermography is indicated in polygonal (n = 3). Cooling were air cooling (air blowing at 2 L/min) and/or intermittent irradiation (10 s break lines in radiation every strategies were air cooling (air blowing at 2 L/min) and/or intermittent irradiation (10 s break in 60 s). radiation every 60 s). 2.5. Bactericidal Effect of aPDT with Laser Transmitted through the Gingival Model
2.5. Bactericidal Effect of aPDT Laser Transmitted throughifthe Gingival Model irradiation showed The ultimate aim of thewith present study was to ascertain aPDT with external a sufficient bactericidal effect against periodontal pathogenic bacteria to be considered for
The ultimate aim of the present study was to ascertain if aPDT with external irradiation trans-gingival periodontal therapy in vivo. Using the conditions optimized above (2 W; 50% duty showed a sufficient bactericidal effect against periodontal pathogenic bacteria to be considered for cycle with a pulse of 100 ms; intermittent irradiation with air cooling), we tested the bacterial trans-gingival periodontal vivo. post-irradiation Using the conditions optimized (2 W; 50% viability after irradiation.therapy Figure 5inshows colony-forming unit above (CFU) counts on a duty cyclelogarithmic with a pulse of 100 ms; intermittent irradiation with air cooling), we tested the scale. The viability of Porphyromonas gingivalis was decreased significantly in all ofbacterial the viability irradiation. 5 shows post-irradiation colony-forming (CFU) counts on aPDTafter groups (1, 3, and 5 Figure min) compared with the control group, and the efficacyunit of aPDT increased proportional to theThe duration of irradiation (Figure 5A).gingivalis The most was significant reduction in bacterialin all a logarithmic scale. viability of Porphyromonas decreased significantly viability observed was on the order of 4 log 10 (99.99% reduction) when the mixture was irradiated of the aPDT groups (1, 3, and 5 min) compared with the control group, and the efficacy offoraPDT 5 min.proportional After irradiation for duration 3 min, bacterial viability (Figure was reduced ordersignificant of 2 log10 (96.71%), increased to the of irradiation 5A). by Theanmost reduction in equivalent to the aPDT effect of direct irradiation for 1 min reported previously [17]. External bacterial viability observed was on the order of 4 log10 (99.99% reduction) when the mixture was irradiation for 1 min produced a reduction of 78.12%. To confirm that this bactericidal effect was irradiated for 5 min. After irradiation for 3 min, bacterial viability was reduced by an order of 2 log10 caused by aPDT, a culture of P. gingivalis mixed with or without ICG-Nano/c was irradiated by a (96.71%), the aPDT effect direct irradiation for 15B). minBacterial reportedviability previously External diodeequivalent laser for 3tomin through the of gingival model (Figure was [17]. reduced irradiation for 1 min produced a reduction of 78.12%. To confirm that this bactericidal effect was significantly in the aPDT group, but not in the control, ICG-Nano/c-only, or laser-only groups. caused by aPDT, a culture of P. gingivalis mixed with or without ICG-Nano/c was irradiated by a diode laser for 3 min through the gingival model (Figure 5B). Bacterial viability was reduced significantly in the aPDT group, but not in the control, ICG-Nano/c-only, or laser-only groups.
Int. J. Mol. Sci. 2017, 18, 154
6 of 11
Int. J. Mol. Sci. 2017, 18, 154
6 of 11
(A)
(B)
Figure 5. Bactericidal effect of aPDT with ICG-Nano/c in the external irradiation model.
Figure 5. Bactericidal effect of aPDT with ICG-Nano/c in the external irradiation model. (A) Bactericidal effects with irradiation of different durations (1, 3, and 5 min) were shown by (A) Bactericidal effects with irradiation of different durations (1, 3, and 5 min) were shown by quantification of viable cell counts; (B) Bactericidal effects with or without ICG-Nano/c and laser quantification of cell counts; (B)quantification Bactericidalofeffects or without ICG-Nano/c and laser irradiation forviable 3 min were shown by viable with cell counts. P. gingivalis (108 CFU/mL) 8 CFU/mL) irradiation for 3ICG-Nano/c min were shown by quantification of (10 viable cell every counts. P. by gingivalis (10 mixed with were irradiated intermittently s pause 60 s) the diode laser at a mixed with ICG-Nano/c irradiated intermittently (10cooling. s pauseData every s) mean by the± diode laser at peak power output of were 2 W in a 50% duty cycle with air are60 the standard deviation. p < 0.05of (n 2 = 3). a peak power *output W in a 50% duty cycle with air cooling. Data are the mean ± standard deviation. * p < 0.05 (n = 3). 3. Discussion
3. Discussion This report describes, for the first time, the bactericidal effectiveness of a new trans-gingival method of photoactivation that permits aPDT by laser irradiation from outside the periodontal pocket.
This report describes, for the first time, the bactericidal effectiveness of a new trans-gingival Studies of laser irradiation of the gingival surface have focused on acceleration of healing in wounds or method of photoactivation that permits aPDT bytolaser irradiation outside the periodontal lesions. Here, we developed an irradiation method transmit energy, infrom a trans-gingival manner, to pocket. Studies of laser irradiation of theThe gingival surface focused onmethod acceleration of healing in activate a photosensitizer for aPDT. advantages of have this irradiation for aPDT over wounds or lesions. Here, we developed an irradiation method to transmit energy, in a trans-gingival conventional intra-pocket irradiation are: an operative procedure is facilitated; reduced invasiveness (and to thus, less discomfort) due to no requirement insertion of a laser into the periodontal manner, activate a photosensitizer for aPDT. for The advantages of probe this irradiation method for and effective delivery of appropriate light energy intooperative areas that would otherwise be inaccessible. aPDTpocket; over conventional intra-pocket irradiation are: an procedure is facilitated; reduced Blood-borne pigments such as hemoglobin, and bilirubinof are majorprobe causesinto of the invasiveness (and thus, less discomfort) due to nooxyhemoglobin, requirement for insertion a laser absorbance of visible radiation in the dermis [22]. However, maintenance of a physiologic level of periodontal pocket; and effective delivery of appropriate light energy into areas that would otherwise blood-borne chromophores in a gingival sample ex vivo is not possible because its blood supply is be inaccessible. terminated upon removal from the living body. Myoglobin is an iron-containing porphyrin pigment, Blood-borne pigments such as hemoglobin, oxyhemoglobin, and bilirubin are major causes of as are hemoglobin and oxyhemoglobin, and its absorption spectrum resembles that of hemoglobin. absorbance of visible in the dermis [22].myoglobin) However, as maintenance of atophysiologic level of Therefore, we usedradiation muscle tissues (which contain gingival models mimic the wall blood-borne chromophores in a gingival sample ex vivo is not possible because its blood supply is of the periodontal pocket. Beef proved to be more effective than pork or chicken for the reduction of terminated upon removal from the living body. Myoglobin an iron-containing pigment, the permeable energy. Hence, laser light penetrating throughisthese beef slices shouldporphyrin be able to pass actual and gingiva. At a laser peakand power output of 2 spectrum W, the power transmitted the as arethrough hemoglobin oxyhemoglobin, its absorption resembles that though of hemoglobin. gingival ≈0.4 tissues W. Anders et al.contain reportedmyoglobin) that the percentage transmission of 810 Therefore, wemodel used was muscle (which as gingival models of to light mimic the wall wavelengthpocket. throughBeef the gastrocnemius of rats was 7.42%, through the of thenm periodontal proved to be muscle more effective than porkand or chicken for overlying the reduction skin was 24.63% [25], data that are consistent with our results. of the permeable energy. Hence, laser light penetrating through these beef slices should be able to We calculated that ≥2 W of power output was required to transmit sufficient energy for pass through actual gingiva. At a laser peak power output of 2 W, the power transmitted though trans-gingival aPDT. This output power is higher than that used in conventional aPDT (200–500 mW) the gingival model was ≈0.4 W. Anders et al. reported that the percentage transmission of light of [12,26], and increased the risk of causing histologic damage by hyperthermia. However, we 810 nm wavelength through theincrease gastrocnemius ratsawas 7.42%, throughirradiation the overlying suppressed this temperature in tissue muscle to 20 °C,to andtransmit that transient reversible changes are caused by temperature increases of >5 °C [27]. We conclude that the influence of for trans-gingival aPDT. This output power is higher than that used in conventional aPDT temperature by thisand irradiation method is minimal andhistologic non-injurious, but that periodic application (200–500 mW) [12,26], increased the risk of causing damage by hyperthermia. However, of a laser and continuous air cooling during irradiation are fundamental to hyperthermia ◦ we suppressed this temperature increase in tissue to 15 °C if they are not employed). with air cooling. Reports have suggested that irreversible reactions such as hyperthermal denaturation of tissue proteins occur at temperature increases of >20 ◦ C, and that transient reversible changes are caused by temperature increases of >5 ◦ C [27]. We conclude that the influence of temperature by this irradiation method is minimal and non-injurious, but that periodic application of a laser and
Int. J. Mol. Sci. 2017, 18, 154
7 of 11
continuous air cooling during irradiation are fundamental to hyperthermia suppression (because the temperature rise is >15 ◦ C if they are not employed). We confirmed that laser light penetrating through the gingival model retains its ability to excite the photosensitizer. Our data are in accordance with studies showing that a laser transmitted through tissue maintains its coherence [28]. To confirm this finding, we measured the absorbance of permeable energy by the ICG-Nano/c solution instead of detecting ROS generation directly because measuring ROS is technically very difficult due to its short lifetime (1 × 10−6 s). Bacterial viability was reduced by an order of 2 log10 and 4 log10 after external irradiation for 3 and 5 min, respectively. The bactericidal effect of this irradiation method was equivalent to that described in our report on aPDT with ICG-Nano/c [17] and in other studies of aPDT reporting ICG-mediated inhibition of the planktonic Staphylococcus aureus and Pseudomonas aeruginosa cells with a maximum reduction of 99% [15]. Conversely, aPDT using other photosensitizing dyes produced more substantial effects. For instance, methylene blue excited with a 670 nm laser reduced the viability of P. gingivalis by an order of 7 log10 [29]. However, the ability of 670 nm-light to penetrate through the mucosa is lower than that of light at 810 nm and, thus, is not suitable for trans-gingival irradiation. ICG has been used as a photosensitizer for photothermal therapy because it has higher calorific properties compared with other photosensitizers following laser irradiation [30,31], and the associated temperature increase may enhance its bactericidal effect. Kranz et al. reported that NIR irradiation of high concentration ICG heated a bacterial solution to >60 ◦ C and caused bacterial inactivation due to this hyperthermic effect. They suggested that the generation of heat was thereby influenced by the concentration of the applied ICG and the total exposure value [26]. In our study, the temperature in the ICG-Nano/c solution was
