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Effect of different diode laser wavelengths on root dentin decontamination infected with Enterococcus faecalis
Journal of Photochemistry & Photobiology, B: Biology 176 (2017) 1–8
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Effect of different diode laser wavelengths on root dentin decontamination infected with Enterococcus faecalis☆
MARK
Caroline Cristina Borgesa, Carlos Estrelab, Fabiane Carneiro Lopesa, Regina Guenka Palma-Dibba, Jesus Djalma Pecoraa, Cyntia Rodrigues De Araújo Estrelab, Manoel Damião de Sousa-Netoa,⁎ a b
Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil Faculty of Dentistry, Federal University of Goiás, Goiânia, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Enterococcus faecalis Diode laser Chlorhexidine Sodium hypochlorite
The objective of this study was to evaluate the antibacterial effect and the ultrastructural alterations of diode laser with different wavelengths (808 nm and 970 nm) and its association with irrigating solutions (2.5% sodium hypochlorite and 2% chlorhexidine) in root dentin contaminated by a five days biofilm. Thirteen uniradicular teeth were sectioned into 100 dentin intraradicular blocks. Initially, the blocks were immersed for 5 min in 17% EDTA and washed with distilled water for 5 min, then samples were sterilized for 30 min at 120 °C. The dentin samples were inoculated with 0.1 mL of E. faecalis suspension in 5 mL BHI (Brain Heart Infusion) and incubated at 37 °C for 5 days. After contamination, the specimens were distributed into ten groups (n = 10) according to surface treatment: GI - 5 mL NaOCl 2.5%, GII - 5 mL NaOCl 2.5% + 808 nm diode (0.1 W for 20 s), GIII - 5 mL NaOCl 2.5% + 970 nm diode (0.5 W for 4 s), GIV - 808 nm diode (0.1 W for 20 s), GV - 970 nm diode (0.5 W for 4 s), GVI - CHX 2%, GVII - CHX 2% + 808 nm diode (0.1 W for 20 s), GVIII - CHX 2% + 970 nm diode (0.5 W for 4 s), GIX - positive control and GX - negative control. Bacterial growth was analyzed by turbidity and optical density of the growth medium by spectrophotometry (nm). Then, the specimens were processed for analysis ultrastructural changes of the dentin surface by SEM. The data was subject to the One-way ANOVA test. GI (77.5 ± 12.1), GII (72.5 ± 12.2), GIII (68.7 ± 8.7), GV (68.3 ± 8.7), GVI (62.0 ± 5.5) and GVII (67.5 ± 3.3) were statistically similar and statistically different from GIV (58.8 ± 25.0), GVIII (59.2 ± 4.0) and control groups (p < 0.05). SEM analysis showed a modified amorphous organic matrix layer with melted intertubular dentin when dentin samples were irradiated with 970 nm diode laser; erosion of the intertubular dentin in blocks submitted to 808 nm diode laser irradiation; and an increased erosion of the intertubular dentin when 2.5% NaOCl was associated to the different wavelengths lasers. All the therapeutic protocols were able to reduce the bacterial contingent in dentin blocks, and the association of diode laser and solutions did not significantly improve the reduction of the bacterial contingent.
1. Introduction
techniques and favoring the persistence of tissue and bacteria remnants [4]. In addition, the dentin has a tubular characteristic with a conical conformation, wherein, the larger diameter of the tubules is located near the lumen of the canal, which allows the penetration of bacteria in deeper areas of the dentin [5]. The depth of bacterial penetration in the dentin tubules is on average 500 μm [6]. The bacterial species Enterococcus faecalis, which has a high prevalence in cases of persistent apical periodontitis [7], is known to extend to a still greater depth, penetrating 800–1000 μm in the dentinal tubules after three weeks of incubation [8].
The disinfection of the root canal system aims to eliminate irritants such as bacteria, their products and pulp tissue remains, providing a favorable environment for the repair of the periapical tissues [1] through the action of endodontic instruments aided by irrigation solutions and intracanal medication [2,3]. However, variations in the internal anatomy of root canals such as flattening, presence of isthmuses, recesses and ramifications can interfere on the success of the disinfection, hindering the instrumentation
☆ Acknowledgement "The authors deny any conflicts of interest. We affirm that we have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements or honoraria), or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past three years." ⁎ Corresponding author at: Rua Célia de Oliveira Meirelles 350, 14024-070, Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (M.D.d. Sousa-Neto).
http://dx.doi.org/10.1016/j.jphotobiol.2017.09.009 Received 27 April 2017; Received in revised form 25 August 2017; Accepted 11 September 2017 Available online 13 September 2017 1011-1344/ © 2017 Elsevier B.V. All rights reserved.
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were left in sterile saline until use.
Therefore, diode laser at different wavelengths (970 nm and 808 nm) was developed for endodontic application, and has thin optical fibers, with a diameter of 200–320 μm, which allow adaptation to the reduced dimensions and curvatures of the root canals, enabling the decontamination along the root canal [9]. Due to the near-infrared wavelength, the diode laser is able to reach deeper layers of the dentin [10], acting where the irrigating solutions are incapable of acting. In addition, the diode laser is able to increase dentin permeability and remove the smear layer in the intraradicular dentin [11,12] without altering its chemical structure [13]. The persistence of bacteria in the root canals is a challenge for Endodontics once the control and elimination of microorganisms are necessary to reach the success of endodontic treatment [14]. Since irrigation solutions have limitations on penetration depth in the dentin tubules [15,16] and that the eradication of root canal bacterial species has not yet been achieved, further studies using auxiliary tools are necessary to ensure a successful endodontic treatment. The purpose of the present study was to evaluate the antibacterial effect and the ultrastructural alterations of diode laser with different wavelengths (808 nm and 970 nm) and its association with irrigating solutions (2.5% sodium hypochlorite and 2% chlorhexidine) in root dentin contaminated by a five days biofilm.
2.1. Standardization of the Bacterial Indicator The bacterial strain used in this study was Enterococcus faecalis (ATCC 29212) inoculated in 7 mL brain heart infusion (BHI, Difco Laboratories, Detroit, USA) and incubated at 37 °C for 24 h. Twentyfour hours prior to specimen contamination, the bacteria were again cultured on the surface of the BHI agar following the same incubation conditions. The bacterial inoculum was obtained by resuspending the cells in saline at a final concentration of approximately 3 × 108 cells mL-1, adjusted for the McFarland turbidity standard 1 standardized by UV spectrophotometer (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) [17]. 2.2. Formation of the Biofilm on the Dentin Blocks Surface After the standardization of the bacterial inoculum, 0.1 mL of Enterococcus faecalis suspension was dispensed into 5 mL of BHI contained in test tubes (15 × 150mm). Hereafter, the blocks of dentin that were so far in sterile saline were transferred to this new solution containing Enterococcus faecalis and stored at 37 °C for five days to allow bacterial fixation. Ten blocks were kept contaminated throughout the experiment as a positive control to verify bacterial viability, while 10 uncontaminated blocks were maintained in 5 mL of sterile BHI as a negative control to ensure sterility of the sample. After bacterial fixation in the dentin blocks, UV spectrophotometer analyses (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) was performed, comparing with the negative control group, which remained sterile throughout the experiment. The UV spectrophotometer allows the BHI medium without contamination to be set to zero. After the spectrophotometer analysis of all the samples, ensuring the contamination of the samples, the specimens were randomly distributed in ten groups, according to the surface treatment to which they were submitted.
2. Material and Methods The experimental protocol was approved by the local ethical committee (19,811,113.0.0000.5083). Maxilary anterior teeth and mandibular pre-molars extracted by orthodontic or periodontal reasons were collected and stored in 0.1% thymol solution at 9 °C for 48 h. The teeth were washed in running water for 24 h and had its external surface ultrasonically cleaned (Profi II Ceramic, Dabi Atlante Ltda., Ribeirão Preto, SP, Brazil). Then, teeth were examined macroscopically with a magnifying glass (10 × magnification), and radiographed in the ortho and mesio-radial directions and later evaluated with the aid of a negatoscope. Thus, thirteen teeth were selected according to the following inclusion criteria: uniradicular teeth, with fully formed roots and single root canal, absence of calcification, curvatures and resorptions, without flattening and absence of previous endodontic treatment. The samples were fixed in acrylic plates with low melt godiva (DFL, Porto Alegre, RS, Brazil). Then, teeth were attached to a fastening device in the cutting machine (Labcut 1010, Erios, São Paulo, SP, Brazil), and sectioned perpendicularly with a diamond saw to the long axis of the root in order to obtain two slices of 2 mm thickness of the cervical third of each teeth, thus obtaining 26 slices. The middle and apical root thirds and the crowns were discarded, and only the cervical slice was used in this study. Each cervical dentin slice was sectioned twice. The first section was performed in the sagittal plane of the slice dividing it in half, and the second section in the frontal plane. The blocks were adjusted with a fine polishing disc (3 M ESPE Dental Products, St. Paul, MN, USA) and their measurements were verified using a digital caliper (Mitutoyo, Suzano, SP, Brazil) in order to obtain the standardized dimensions of 4 mm height, 4 mm width and 2 mm thickness, totalizing 104 blocks of dentin. One hundred blocks were randomly selected for this study. The blocks were placed in a 150 mL becker with 17% ethylenediaminetetraacetic acid (EDTA) (Formula and Action, São Paulo, SP, Brazil) and kept under stirring on a tube shaker (Vortex, Model AP 56, Presidente Prudente, SP, Brazil) for 5 min. Subsequently, the same process was performed, however, using distilled water. The blocks were kept hydrated in a 150 mL becker and were incubated for 24 h. Afterward, the becker distilled water was renewed so that the blocks were kept hydrated during sterilization in an analogue vertical autoclave (Idealclave, Stermax, Barueri, São Paulo, Brazil) for 30 min at 120 °C. After sterilization, the blocks were incubated in BHI at 37 °C for 48 h to confirm the absence of bacteria by visual analysis. The blocks
2.3. Disinfection Protocols The groups treated with sodium hypochlorite (groups I, II and III) were immersed in 5 mL of 2.5% sodium hypochlorite (Fitofarma, Lt. 20,442, Goiânia, GO, Brazil) contained in the Petri dish (K13-0035, KASVI, Goiânia, Brazil) for 5 min. The blocks were then pinched and transferred to a gauze pad over another Petri dish to remove excess sodium hypochlorite. Group II, after immersion in 2.5% sodium hypochlorite, the intraradicular dentin was irradiated with 808 nm laser (DMC Whitening Lase II, São Carlos, SP, Brazil), with an optical fiber tip with 200 μm diameter, perpendicularly to the surface, on all faces of the dentin blocks. In each face, irradiation with light in continuous mode (100 mW), power of 0.1 W, for 20 s, totalizing 2 W, with energy density 31,847 J/cm2 (Table 1) was performed. Group III, after the same immersion process as Group II, was irradiated with 970 nm diode laser (Sirona Dental, Benshein, HE, Germany) Table 1 Parameters established for diode laser use.
2
Parameters
808 ± 10 nm diode laser
970 ± 15 nm diode laser
Diameter of the fiber optic tip Mode of application Duration Power Energy density
200 μm
200 μm
Continuous wave 20 s 2W 31,847 J/cm2
Continuous wave 4s 2W 159,23 J/cm2
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Table 2 Mean and standard deviation of microbial activity (nm) before and after dentinal surface treatment and visual analysis of bacterial contamination before treatment (CB1) and after treatment (CB2). Dentinal surface treatments
CB1 n = 10
Mean and standard deviation of optical density (nm)
CB2 n = 10
Mean and standard deviation of optical density (nm)
NaOCl 2.5% NaOCl 2.5% + 808 nm diode laser NaOCl 2.5% + 970 nm diode laser 808 nm diode laser 970 nm diode laser CHX 2% CHX 2% + 808 nm diode laser CHX 2% + 970 nm diode laser Positive control Negative control
+ + + + + + + + + −
0.8175 0.8081 0.7718 0.8473 0.8711 0.8229 0.8326 0.7902 0.7974 0 ± 0
+ + + + + + + + + −
0.1754 0.2220 0.2427 0.3474 0.2745 0.3090 0.2696 0.3224 0.7918 0 ± 0
± ± ± ± ± ± ± ± ±
0.082 0.061 0.081 0.034 0.077 0.073 0.032 0.025 0.045
± ± ± ± ± ± ± ± ±
0.102 0.100 0.072 0.205 0.072 0.029 0.020 0.031 0.144
*The positive and negative symbols corresponds to visual analysis: (+) represents turbidity (−) absence of turbidity. Table 3 Mean and standard deviation values of the percentage of reduction of the number of bacteria. Dentine surface treatment
Optical density mean values (nm)
NaOCl 2.5% NaOCl 2.5% + 808 nm diode laser NaOCl 2.5% + 970 nm diode laser 808 nm diode laser 970 nm diode laser CHX 2% CHX 2% + 808 nm diode laser CHX 2% + 970 nm diode laser
77.5 72.5 68.7 58.8 68.3 62.0 67.5 59.2
± ± ± ± ± ± ± ±
12.1 (68.8–86.1) A 12.2 (63.6–81.5) AB 8.7 (62.3–74.8) AB 25.0 (41.0–76.7) B 8.7 (62.1–74.5) AB 5.5 (58.2–66.0) AB 3.3 (55.1–69.9) AB 4.0 (56.4–62.0) B
*Different upper case letters indicate statistical difference of the dentinal surface treatments in the bacterial reduction (Tukey's p < 0.05).
with an optical fiber tip of 200 μm diameter perpendicularly to the surface on all faces of the dentin blocks. On each face, irradiation was performed with light in continuous mode, power of 0.5 W, for 4 s, totaling 2 W, with energy density 159.23 J/cm2 (Table 1). Group IV was irradiated with diode laser 808 nm and group V with diode laser 970 nm, according to parameters previously described, without previous immersion. The tips of both lasers were analyzed between the applications, with the aid of a magnifying glass, in order to observe the presence of damages in its surface. When the surface was irregular or burned, the tip was sectioned perpendicular to the long axis of the tip, so as not to damage the next irradiation. The samples of groups VI, VII and VIII were immersed in 2% chlorhexidine according to the same procedure used for the treatment with NaOCl. Group VII was irradiated with 808 nm diode laser and group VIII with 970 nm diode laser according to the previously described parameters. Ten blocks of group IX were kept contaminated throughout the experiment at 37 °C to verify bacterial viability and maintained (positive control). Meanwhile, 10 blocks of group X were stored in 5 mL sterile BHI at 37 °C to ensure the sterility of the sample (negative control). After the dentin surface treatments, the blocks were stored in 7 mL BHI at 37 °C for 48 h. Then each block was transferred to a tube containing 7 mL of BHI added with Tween 80 and sodium thiosulphate neutralizers (P.A., Lab Art, Campinas, SP, Brazil) and incubated at 37 °C for 48 h.
Fig. 1. Percentage of the decrease of antimicrobial activity after treatment.
observation. In fact, the amount of absorbed and dispersed light is proportional to the mass of cells in the luminous path. Thus, for the turbidimetric measurements of the cell mass, a UV Spectrophotometer was used. In the microbiological analysis, a 0.1 mL inoculum obtained from the BHI was transferred to 5 mL of Letheen Broth under identical incubation conditions. Gram staining was used in BHI cultures to verify contamination and growth and was microscopically examined. The microbial concentration was analyzed using a spectrophotometer (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) adjusted for reading at wavelength λ = 600 nm, adopting the McFarland scale 1, which corresponds to the absorbance of 0.137 nm after zero reading of the sterilized culture medium and at room temperature.
2.5. Ultrastructural Evaluation of Dentin by Scanning Electron Microscopy (SEM) Five samples from each group were randomly selected and submitted to scanning electron microscopy (SEM) for analysis of the dentin surface. Samples were fixed in an ascending scale of ethanol concentrations (25°, 50°, 75° for 20 min in each, 95° for 30 min and 100° for 1 h). The specimens were then fixed in metal cylinders and then metallized by a ~ 300 Å gold layer in metallizer (Jeol, JSM-6610, equipped with EDS, Thermo scientific NSS Spectral Imaging, Tokyo, Japan). In order to perform the qualitative analysis, 1500 × and 5000 × magnification photomicrographs of the intraradicular dentin were performed for each sample, totalizing 10 images for each group using the scanning electron microscope (JSM-IT300, Joel, Tokyo, Japan). The
2.4. Microbiological Analysis Microbial growth was analyzed by the turbidity of the culture medium. This method is based on light scattering as it passes through a sample. Suspended cells absorb the light and the dispersion of light that pass through them makes a culture appear blurred by visual 3
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Fig. 2. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000× magnification. I - negative control; II - positive control.
(68.3 ± 8.7), VI (CHX 2%) (62.0 ± 5.5), VII (CHX 2% + laser diode 808 nm) (67.5 ± 3, 3) (p > 0.05); and statistically different from group IV (laser diode 808 nm) (58.8 ± 25.0), VIII (CHX 2% + diode 970 nm) (59.2 ± 4.0) and control groups (p < 0.05) (Table 3 and Fig. 1). The qualitative analysis of the images obtained by scanning electron microscope revealed dentinal tubules with open and regular contour in the negative control group (Fig. 2IAB). The intertubular dentine appeared regular, free of debris and bacteria. The group that maintained contamination throughout the treatment (positive control) (Fig. 2IIAB) revealed dense biofilm covering the dentin surface. The openings of the dentinal tubules were obliterated. Dentin treated with 2.5% sodium hypochlorite (Fig. 3IAB) showed regions with intertubular dentin erosion with an irregular dentin surface and a dispersed biofilm. Surface treatment with 2% chlorhexidine solution (Fig. 3IIAB) revealed dense biofilm covering the surface, making it impossible to observe the possible changes that the solution could cause to the dentin surface. After the dentin was irradiated with 808 nm diode laser, regions with erosion of the intertubular dentin were observed, making the dentin surface irregular (Fig. 4IAB), and the biofilm appeared dispersed. The association of 2.5% sodium hypochlorite with the 808 nm laser (Fig. 4IIAB), the effect of the 808 nm diode laser was increased, showing an irregular surface with melted dentin. Dentin submitted to 2% chlorhexidine and 808 nm diode laser treatment (Fig. 4IIIAB) presented a surface similar to the group treated only with 2% chlorhexidine. When irradiated with 970 nm diode laser, the dentin surface showed an amorphous organic matrix and melted intertubular dentin with partial dentinal tubule opening, characterizing an irregular surface (Fig. 5IAB) with a dispersed biofilm on its surface. The images obtained from dentin blocks submitted to 2.5% sodium hypochlorite and 970 nm
following aspects of the dentin surface were observed: opening or obliteration of dentinal tubules, regular or irregular surface, presence or absence of exposed collagen fibers and presence or absence of biofilm. 2.6. Statistical Analysis Once the data presented a normal distribution (Shapiro-Wilk, p > 0.05) and homogeneity of variance (Levene test, p > 0.05), parametric tests were used. The ANOVA one factor was used to evaluate the influence of disinfection protocols on the percentage decrease of Enterococcus faecalis contingent. Tukey test was used for comparisons between the groups. The probability level was 95% and all analyzes were performed in SPSS software. 3. Results In the visual and spectrophotometry analysis of all the blocks before treatments, contamination was demonstrated with the exception of the blocks that remained sterilized (negative control group), showing the effectiveness of the bacterial contamination procedure (Table 2). Through the microbial activity analysis before and after the dentin surface treatments, a significant statistical difference (p < 0.05) was observed for all treatments, except for the blocks that remained contaminated (group IX - positive control) and blocks that remained sterilized (negative X-control group). Therefore, the treatments performed significantly decreased the number of bacteria. When analyzing the percentage of antimicrobial activity (nm) before and after surface treatments, it was observed that the treatment with 2.5% NaOCl (group I) had the highest percentage of antimicrobial activity decrease (77.5 ± 12, 1) statistically similar to groups II (NaOCl 2.5% + laser diode 808 nm) (68.7 ± 8.7), III (NaOCl 2.5% + laser diode 970 nm) (68.7 ± 8.7), Group V (laser diode 970 nm) 4
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Fig. 3. Scanning electron microscopy images at different magnifications. A - 1500× magnification; B - 5000× magnification. I - treatment with 2.5% NaOCl; II - treatment with 2% CHX.
is similar to the study by Moritz et al. [22], irradiation was performed only once, which may justify the lower percentage of bacterial reduction when compared to the study by Moritz et al. [22] which achieved almost complete elimination of bacteria on the second irradiation. Therefore, neither the laser nor the solutions and their associations evaluated in the present study were able to reach the deeper layers of dentin, not allowing the complete elimination of the bacteria. Regarding the application of diode laser (808 nm and 970 nm) and after irrigation with 2.5% sodium hypochlorite and 2% chlorhexidine solutions, it was not possible to observe a significant improvement in the reduction of the bacterial contingent. These results corroborate with Mehrvarzfar et al. [23] and Castelo-Baz et al. [24], that when analyzing the effect of laser diode alone and in combination with solutions on infected root canals, did not observe statistical differences between groups. These results show that even after the application of irrigation solutions, 970 nm and 808 nm diode laser diodes were not able to reach sufficient depth to eliminate the entire bacterial contingent. Beer et al. [9] verified higher reduction values of bacterial contingent when compared to the present study, in which treatment with 808 nm diode laser showed the lowest reduction in the number of bacteria. Romeo et al. [25] evaluated the effectiveness of 980 nm diode laser to kill Enterococcus faecalis in experimentally infected root canals and found higher reduction value of the bacterial contingent to the laser treatment combined with sodium hypochlorite when compared to irrigation treatment without irradiation, different from the present study that did not observe improvement in the decontamination of the root dentine when hypochlorite was associated to laser irradiation. However, the differences in those results may be related to the methodology, once the contamination period in the present study was 5 days, when compared to Beer et al. [9], that used a contamination period of only 2 h, and Romeo et al. [25] that evaluated the decontamination of 72 h biofilms. Thus, the bacteria would be in less mature stages, which
diode laser treatment (Fig. 5IIAB) showed irregular surface with intertubular dentine erosion and biofilm on its surface. The dentin submitted to the 2% chlorhexidine and 970 nm diode laser treatment (Fig. 5IIIAB) showed an amorphous organic matrix with partial dentinal tubule exposure. 4. Discussion The development phase of the biofilm in the antimicrobial evaluation is very important and may be a determining factor for the greater resistance to different types of antimicrobial strategies [18]. Currently, most studies evaluate the antimicrobial properties of irrigation solutions, involving both bacterial stages of growth, planktonic and biofilm [19], which makes it difficult to compare the results obtained by different types of laser, as well as to verify the effective action of the lasers in situations where the bacteria present in the root canal system are not in their planktonic stage. In this sense, in the present study 2-mm-thick (2000 μm) intraradicular dentine blocks contaminated with a five-day biofilm were used to evaluate different diode laser wavelengths (808 nm and 970 nm). The antibacterial action of the 970 nm diode laser, verified in the present study, is directly related to the heating of the dentin substrate to which the bacteria are bound [20], which resulted in the bacterial contingent reduction (68.3 ± 8.7) statistically similar to the group irradiated with 808 nm diode laser, corroborating to previous studies [9,21]. The surface irradiation with 808 nm diode laser resulted in 58.8% reduction of the bacterial contingent. According to Moritz et al. [22], gram-negative bacteria are reached immediately after the first irradiation, whereas gram-positive bacteria such as E. Faecalis, evaluated in the present study, need to be irradiated repeatedly for bacterial cell wall disruption. Although the energy density used in the present study 5
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Fig. 4. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000 × magnification. I - 808 nm diode laser; II - 808 nm diode laser + 2.5% NaOCl; III - 808 nm diode laser + 2% CHX.
blocks by SEM, irradiation with 808 nm diode laser promoted an irregular surface with erosion regions in the intertubular dentine, which differs from other studies in which there was fusion of dentinal tubules after applying the laser diode [9,10,34]. The different results can be justified due to the position of application of the laser to the substrate surface [35]. In the present study, the application of the laser was perpendicular to the surface of the dentin blocks, whereas in other studies the application was performed along the root canal by helical motions [9,13,35]. On the other hand, when analyzing the surface of the dentin blocks irradiated with 970 nm diode laser an amorphous organic matrix and melted intertubular dentine was observed. According to Jhingan et al. [36], after irradiation with 980 nm diode laser (2 W), the analysis of the intraradicular dentinal surface by means of scanning electron microscopy, evidenced the melting of the dentin layer making it irregular as
allowed an easier removal and consequent bacterial death. Regarding the irrigation solutions used alone, treatment with 2.5% sodium hypochlorite showed similar results regarding the bacterial decrease (77.5 ± 12.1) when compared to the 2% chlorhexidine group (62.0 ± 5.5), in agreement with previous studies comparing these solutions at different concentrations [26–28]. Both solutions have antimicrobial properties, and sodium hypochlorite has the capacity to promote the disinfection and solvency of organic and inorganic tissues [29,30] and chlorhexidine, normally used as an alternative solution, has properties of substantivity, prolonged antimicrobial effect and low cytotoxicity [31–33]. From the microbiological point of view, any solution can be used during the treatment of infected teeth, however, the different properties of irrigating solutions should influence the choice of the auxiliary solution. When evaluating the surface morphological changes of the dentin 6
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Fig. 5. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000 × magnification. I - 970 nm diode laser; II - 970 nm diode laser + 2.5% NaOCl; III - 970 nm diode laser + 2% CHX.
present study, when evaluating the effect of different irrigation protocols on root dentin structure through SEM and transmission electron microscopy. Intraradicular dentin presented structural alterations caused by 2.5% sodium hypochlorite, whereas treatment with chlorhexidine, distilled water and saline solution preserved the collagen structure, and were unable to dissolve the organic content. Thus, in the evaluation of the influence of the diode lasers with different wavelengths on the dentin surface, the present study concluded that the application of laser diode 970 nm and 2.5% sodium hypochlorite had similarity in the decontamination of the root dentin blocks surface, although they presented morphological changes in the dentin surface. However, further studies should be carried out to find the effectiveness of the diode laser in root canals contaminated by mature biofilms, in conditions closer to the clinical reality, as well as its influence on the dentin morphology in the prognosis of endodontic treatment.
also observed by Alfredo et al. [10] and Faria et al. [37]. These observed morphological changes can be attributed to the thermal effect caused by superheating and subsequent surface cooling when the laser interacts with the dentinal tissue [10,11]. This phenomenon explains the melted intertubular dentine and irregular surface promoted by the 970 nm diode laser. Regarding the association of 808 nm diode laser with 2.5% sodium hypochlorite solution, there was a potentiation of the morphological alterations of the dentin compared to the laser treatment without immersion. When the laser diode 970 nm was associated with 2.5% sodium hypochlorite, it was possible to observe a greater erosion of the intertubular dentine compared to the treatment with 970 nm diode laser without immersion. However, when chlorhexidine was associated with the lasers, there was a minor alteration of the dentin structure. Wagner et al. [38] obtained similar results to those obtained in the 7
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[18] Z. Lim, J.L. Cheng, T.W. Lim, E.G. Teo, J. Wong, S. George, et al., Light activated disinfection: an alternative endodontic disinfection strategy, Aust. Dent. J. 54 (2009) 108–114. [19] K. Jhajharia, A. Parolia, K.V. Shetty, L.K. Mehta, Biofilm in endodontics: a review, J. Int. Soc. Prev. Community Dent. 5 (2015) 1–12. [20] N. Gutknecht, T.S. Al-Karadaghi, M.A. Al-Maliky, G. Conrads, R. Franzen, The bactericidal effect of 2780 and 940 nm laser irradiation on Enterococcus faecalis in bovine root dentin slices of different thicknesses, Photomed. Laser Surg. 34 (2016) 11–16. [21] N.R. Kanumuru, R. Subbaiah, Bacterial efficacy of Ca(oH)2 against E.faecalis compared with three dental lasers on root canal dentin- an invitro study, J. Clin. Diagn. Res. 8 (2014) ZC135–137. [22] A. Moritz, N. Gutknecht, U. Schoop, K. Goharkhay, O. Doertbudak, W. Sperr, Irradiation of infected root canals with a diode laser in vivo: results of microbiological examinations, Lasers Surg. Med. 21 (1997) 221–226. [23] P. Mehrvarzfar, M.A. Saghiri, A. Asatourian, R. Fekrazad, K. Karamifar, G. Eslami, et al., Additive effect of a diode laser on the antibacterial activity of 2.5% NaOCl, 2% CHX and MTAD against Enterococcus faecalis contaminating root canals: an in vitro study, J. Oral Sci. 53 (2011) 355–360. [24] B. P.M.B. Castelo–Baz, M. Ruiz–Pinon, Combined sodium hypochlorite and 940 NM diode laser treatment against mature E. facials boxfuls in-vitro, Lasers Med. Sci. 3 (2012) 116–121. [25] U. Romeo, G. Palaia, A. Nardo, G. Tenore, V. Telesca, R. Kornblit, A. Del Vecchio, A. Frioni, P. Valenti, F. Berlutti, Effectiveness of KTP laser versus 980 nm diode laser to kill Enterococcus faecalis in biofilms developed in experimentally infected root canals, Aust. Endod. J. 41 (2015) 17–23. [26] N. Flach, D.E. Bottcher, C.C.F. Parolo, L.B. Firmino, M. Malt, M.L. Lammers, et al., Confocal microscopy evaluation of the effect of irrigants on Enterococcus faecalis biofilm: an in vitro study, Scanning 38 (2016) 57–62. [27] I.N. Rocas, J.C. Provenzano, M.A. Neves, J.F. Siqueira Jr., Disinfecting effects of rotary instrumentation with either 2.5% sodium hypochlorite or 2% chlorhexidine as the main irrigant: a randomized clinical study, J. Endod. 42 (2016) 943–947. [28] J.F. Siqueira Jr., I.N. Rocas, S.S. Paiva, T. Guimaraes-Pinto, K.M. Magalhaes, K.C. Lima, Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 104 (2007) 122–130. [29] F. Baratto-Filho, J.R. De Carvalho Jr., L.F. Fariniuk, M.D. Sousa-Neto, J.D. Pecora, A.M. Da Cruz-Filho, Morphometric analysis of the effectiveness of different concentrations of sodium hypochlorite associated with rotary instrumentation for root canal cleaning, Braz. Dent. J. 15 (2004) 36–40. [30] C. Estrela, C.R. Estrela, E.L. Barbin, J.C. Spano, M.A. Marchesan, J.D. Pecora, Mechanism of action of sodium hypochlorite, Braz. Dent. J. 13 (2002) 113–117. [31] L. Bazvand, M.G. Aminozarbian, A. Farhad, H. Noormohammadi, S.M. Hasheminia, S. Mobasherizadeh, Antibacterial effect of triantibiotic mixture, chlorhexidine gel, and two natural materials Propolis and Aloe Vera against Enterococcus faecalis: an ex vivo study, Dent. Res. J. (Isfahan) 11 (2014) 469–474. [32] B.P. Gomes, M.E. Vianna, A.A. Zaia, J.F. Almeida, F.J. Souza-Filho, C.C. Ferraz, Chlorhexidine in endodontics, Braz. Dent. J. 24 (2013) 89–102. [33] Z. Mohammadi, L. Giardino, F. Palazzi, S. Asgary, Agonistic and antagonistic interactions between Chlorhexidine and other endodontic agents: a critical review, Iran Endod J. 10 (2015) 1–5. [34] M. Esteves-Oliveira, C.A. De Guglielmi, K.M. Ramalho, V.E. Arana-Chavez, C.P. De Eduardo, Comparison of dentin root canal permeability and morphology after irradiation with Nd:YAG, Er:YAG, and diode lasers, Lasers Med. Sci. 25 (2010) 755–760. [35] P.R. Alves, N. Aranha, E. Alfredo, M.A. Marchesan, A. Brugnera Junior, M.D. SousaNeto, Evaluation of hollow fiberoptic tips for the conduction of Er:YAG laser, Photomed. Laser Surg. 23 (2005) 410–415. [36] P. Jhingan, M. Sandhu, G. Jindal, D. Goel, V. Sachdev, An in-vitro evaluation of the effect of 980 nm diode laser irradiation on intra-canal dentin surface and dentinal tubule openings after biomechanical preparation: scanning electron microscopic study, Indian J. Dent. 6 (2015) 85–90. [37] M.I. Faria, M.D. Sousa-Neto, A.E. Souza-Gabriel, E. Alfredo, U. Romeo, Y.T. SilvaSousa, Effects of 980-nm diode laser on the ultrastructure and fracture resistance of dentine, Lasers Med. Sci. 28 (2013) 275–280. [38] M.H. Wagner, R.A. Da Rosa, J.A. De Figueiredo, M.A. Duarte, J.R. Pereira, M.V. So, Final irrigation protocols may affect intraradicular dentin ultrastructure, Clin Oral Investig (2016).
5. Conclusions According to the results obtained in this study, it can be concluded that all the therapeutic protocols were able to reduce the bacterial contingent, and that the 970 nm diode laser showed similar antibacterial potential to the sodium hypochlorite solution, and when associating the diode lasers and the studied solutions there was no significant improvement in reducing the bacterial contingent. References [1] S. Saha, R. Nair, H. Asrani, Comparative evaluation of propolis, metronidazole with chlorhexidine, calcium hydroxide and Curcuma longa extract as intracanal medicament against E.faecalis- an invitro study, J. Clin. Diagn. Res. 9 (2015) ZC19–21. [2] F. Afkhami, S.J. Pourhashemi, M. Sadegh, Y. Salehi, M.J. Fard, Antibiofilm efficacy of silver nanoparticles as a vehicle for calcium hydroxide medicament against Enterococcus faecalis, J. Dent. 43 (2015) 1573–1579. [3] S. Kiran, S. Prakash, P.R. Siddharth, S. Saha, N.E. Geojan, M. Ramachandran, Comparative evaluation of smear layer and debris on the canal walls prepared with a combination of hand and rotary ProTaper technique using scanning electron microscope, J. Contemp. Dent. Pract. 17 (2016) 574–581. [4] M.A. Versiani, R. Ordinola-Zapata, A. Keles, H. Alcin, C.M. Bramante, J.D. Pecora, et al., Middle mesial canals in mandibular first molars: a micro-CT study in different populations, Arch. Oral Biol. 61 (2016) 130–137. [5] R.G. Ribeiro, M.A. Marchesan, R.G. Silva, M.D. Sousa-Neto, J.D. Pecora, Dentin permeability of the apical third in different groups of teeth, Braz. Dent. J. 21 (2010) 216–219. [6] J.L. Brittan, S.V. Sprague, E.L. Macdonald, R.M. Love, H.F. Jenkinson, N.X. West, In vivo model for microbial invasion of tooth root dentinal tubules, J. Appl. Oral Sci. 24 (2016) 126–135. [7] C.H. Stuart, S.A. Schwartz, T.J. Beeson, C.B. Owatz, Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment, J. Endod. 32 (2006) 93–98. [8] S. Ran, S. Gu, J. Wang, C. Zhu, J. Liang, Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo, Eur. J. Oral Sci. (2015 Aug 21), http://dx. doi.org/10.1111/eos.12202 [Epub ahead of print] PubMed PMID: 26296719. [9] F. Beer, A. Buchmair, J. Wernisch, A. Georgopoulos, A. Moritz, Comparison of two diode lasers on bactericidity in root canals—an in vitro study, Lasers Med. Sci. 27 (2012) 361–364. [10] E. Alfredo, A.E. Souza-Gabriel, S.R. Silva, M.D. Sousa-Neto, A. Brugnera-Junior, Y.T. Silva-Sousa, Morphological alterations of radicular dentine pretreated with different irrigating solutions and irradiated with 980-nm diode laser, Microsc. Res. Tech. 72 (2009) 22–27. [11] M.A. Marchesan, A. Brugnera-Junior, A.E. Souza-Gabriel, S.R. Correa-Silva, M.D. Sousa-Neto, Ultrastructural analysis of root canal dentine irradiated with 980nm diode laser energy at different parameters, Photomed. Laser Surg. 26 (2008) 235–240. [12] T.S. Al-Karadaghi, R. Franzen, H.A. Jawad, N. Gutknecht, Investigations of radicular dentin permeability and ultrastructural changes after irradiation with Er,Cr:YSGG laser and dual wavelength (2780 and 940 nm) laser, Lasers Med. Sci. 30 (2015) 2115–2121. [13] F.C. Lopes, R. Roperto, A. Akkus, O. Akkus, A.E. Souza-Gabriel, M.D. Sousa-Neto, Effects of different lasers on organic/inorganic ratio of radicular dentin, Lasers Med. Sci. 31 (2016) 415–420. [14] M. Fernandes, I. De Ataide, Nonsurgical management of periapical lesions, J. Conserv. Dent. 13 (2010) 240–245. [15] M.C. Valera, S.A. Oliveira, L.E. Maekawa, F.G. Cardoso, A. Chung, S.F. Silva, et al., Action of Chlorhexidine, Zingiber Officinale, and calcium hydroxide on Candida Albicans, Enterococcus faecalis, Escherichia Coli, and endotoxin in the root canals, J. Contemp. Dent. Pract. 17 (2016) 114–118. [16] P. Pladisai, R.S. Ampornaramveth, P. Chivatxaranukul, Effectiveness of different disinfection protocols on the reduction of bacteria in Enterococcus faecalis biofilm in teeth with large root canals, J. Endod. 42 (2016) 460–464. [17] C. Estrela, M.D. Sousa-Neto, D.R. Alves, A.H. Alencar, T.O. Santos, J.D. Pécora, A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis, Braz. Dent. J. 23 (2012) 645–653.
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Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Effect of different diode laser wavelengths on root dentin decontamination infected with Enterococcus faecalis☆
MARK
Caroline Cristina Borgesa, Carlos Estrelab, Fabiane Carneiro Lopesa, Regina Guenka Palma-Dibba, Jesus Djalma Pecoraa, Cyntia Rodrigues De Araújo Estrelab, Manoel Damião de Sousa-Netoa,⁎ a b
Department of Restorative Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil Faculty of Dentistry, Federal University of Goiás, Goiânia, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Enterococcus faecalis Diode laser Chlorhexidine Sodium hypochlorite
The objective of this study was to evaluate the antibacterial effect and the ultrastructural alterations of diode laser with different wavelengths (808 nm and 970 nm) and its association with irrigating solutions (2.5% sodium hypochlorite and 2% chlorhexidine) in root dentin contaminated by a five days biofilm. Thirteen uniradicular teeth were sectioned into 100 dentin intraradicular blocks. Initially, the blocks were immersed for 5 min in 17% EDTA and washed with distilled water for 5 min, then samples were sterilized for 30 min at 120 °C. The dentin samples were inoculated with 0.1 mL of E. faecalis suspension in 5 mL BHI (Brain Heart Infusion) and incubated at 37 °C for 5 days. After contamination, the specimens were distributed into ten groups (n = 10) according to surface treatment: GI - 5 mL NaOCl 2.5%, GII - 5 mL NaOCl 2.5% + 808 nm diode (0.1 W for 20 s), GIII - 5 mL NaOCl 2.5% + 970 nm diode (0.5 W for 4 s), GIV - 808 nm diode (0.1 W for 20 s), GV - 970 nm diode (0.5 W for 4 s), GVI - CHX 2%, GVII - CHX 2% + 808 nm diode (0.1 W for 20 s), GVIII - CHX 2% + 970 nm diode (0.5 W for 4 s), GIX - positive control and GX - negative control. Bacterial growth was analyzed by turbidity and optical density of the growth medium by spectrophotometry (nm). Then, the specimens were processed for analysis ultrastructural changes of the dentin surface by SEM. The data was subject to the One-way ANOVA test. GI (77.5 ± 12.1), GII (72.5 ± 12.2), GIII (68.7 ± 8.7), GV (68.3 ± 8.7), GVI (62.0 ± 5.5) and GVII (67.5 ± 3.3) were statistically similar and statistically different from GIV (58.8 ± 25.0), GVIII (59.2 ± 4.0) and control groups (p < 0.05). SEM analysis showed a modified amorphous organic matrix layer with melted intertubular dentin when dentin samples were irradiated with 970 nm diode laser; erosion of the intertubular dentin in blocks submitted to 808 nm diode laser irradiation; and an increased erosion of the intertubular dentin when 2.5% NaOCl was associated to the different wavelengths lasers. All the therapeutic protocols were able to reduce the bacterial contingent in dentin blocks, and the association of diode laser and solutions did not significantly improve the reduction of the bacterial contingent.
1. Introduction
techniques and favoring the persistence of tissue and bacteria remnants [4]. In addition, the dentin has a tubular characteristic with a conical conformation, wherein, the larger diameter of the tubules is located near the lumen of the canal, which allows the penetration of bacteria in deeper areas of the dentin [5]. The depth of bacterial penetration in the dentin tubules is on average 500 μm [6]. The bacterial species Enterococcus faecalis, which has a high prevalence in cases of persistent apical periodontitis [7], is known to extend to a still greater depth, penetrating 800–1000 μm in the dentinal tubules after three weeks of incubation [8].
The disinfection of the root canal system aims to eliminate irritants such as bacteria, their products and pulp tissue remains, providing a favorable environment for the repair of the periapical tissues [1] through the action of endodontic instruments aided by irrigation solutions and intracanal medication [2,3]. However, variations in the internal anatomy of root canals such as flattening, presence of isthmuses, recesses and ramifications can interfere on the success of the disinfection, hindering the instrumentation
☆ Acknowledgement "The authors deny any conflicts of interest. We affirm that we have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements or honoraria), or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past three years." ⁎ Corresponding author at: Rua Célia de Oliveira Meirelles 350, 14024-070, Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (M.D.d. Sousa-Neto).
http://dx.doi.org/10.1016/j.jphotobiol.2017.09.009 Received 27 April 2017; Received in revised form 25 August 2017; Accepted 11 September 2017 Available online 13 September 2017 1011-1344/ © 2017 Elsevier B.V. All rights reserved.
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were left in sterile saline until use.
Therefore, diode laser at different wavelengths (970 nm and 808 nm) was developed for endodontic application, and has thin optical fibers, with a diameter of 200–320 μm, which allow adaptation to the reduced dimensions and curvatures of the root canals, enabling the decontamination along the root canal [9]. Due to the near-infrared wavelength, the diode laser is able to reach deeper layers of the dentin [10], acting where the irrigating solutions are incapable of acting. In addition, the diode laser is able to increase dentin permeability and remove the smear layer in the intraradicular dentin [11,12] without altering its chemical structure [13]. The persistence of bacteria in the root canals is a challenge for Endodontics once the control and elimination of microorganisms are necessary to reach the success of endodontic treatment [14]. Since irrigation solutions have limitations on penetration depth in the dentin tubules [15,16] and that the eradication of root canal bacterial species has not yet been achieved, further studies using auxiliary tools are necessary to ensure a successful endodontic treatment. The purpose of the present study was to evaluate the antibacterial effect and the ultrastructural alterations of diode laser with different wavelengths (808 nm and 970 nm) and its association with irrigating solutions (2.5% sodium hypochlorite and 2% chlorhexidine) in root dentin contaminated by a five days biofilm.
2.1. Standardization of the Bacterial Indicator The bacterial strain used in this study was Enterococcus faecalis (ATCC 29212) inoculated in 7 mL brain heart infusion (BHI, Difco Laboratories, Detroit, USA) and incubated at 37 °C for 24 h. Twentyfour hours prior to specimen contamination, the bacteria were again cultured on the surface of the BHI agar following the same incubation conditions. The bacterial inoculum was obtained by resuspending the cells in saline at a final concentration of approximately 3 × 108 cells mL-1, adjusted for the McFarland turbidity standard 1 standardized by UV spectrophotometer (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) [17]. 2.2. Formation of the Biofilm on the Dentin Blocks Surface After the standardization of the bacterial inoculum, 0.1 mL of Enterococcus faecalis suspension was dispensed into 5 mL of BHI contained in test tubes (15 × 150mm). Hereafter, the blocks of dentin that were so far in sterile saline were transferred to this new solution containing Enterococcus faecalis and stored at 37 °C for five days to allow bacterial fixation. Ten blocks were kept contaminated throughout the experiment as a positive control to verify bacterial viability, while 10 uncontaminated blocks were maintained in 5 mL of sterile BHI as a negative control to ensure sterility of the sample. After bacterial fixation in the dentin blocks, UV spectrophotometer analyses (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) was performed, comparing with the negative control group, which remained sterile throughout the experiment. The UV spectrophotometer allows the BHI medium without contamination to be set to zero. After the spectrophotometer analysis of all the samples, ensuring the contamination of the samples, the specimens were randomly distributed in ten groups, according to the surface treatment to which they were submitted.
2. Material and Methods The experimental protocol was approved by the local ethical committee (19,811,113.0.0000.5083). Maxilary anterior teeth and mandibular pre-molars extracted by orthodontic or periodontal reasons were collected and stored in 0.1% thymol solution at 9 °C for 48 h. The teeth were washed in running water for 24 h and had its external surface ultrasonically cleaned (Profi II Ceramic, Dabi Atlante Ltda., Ribeirão Preto, SP, Brazil). Then, teeth were examined macroscopically with a magnifying glass (10 × magnification), and radiographed in the ortho and mesio-radial directions and later evaluated with the aid of a negatoscope. Thus, thirteen teeth were selected according to the following inclusion criteria: uniradicular teeth, with fully formed roots and single root canal, absence of calcification, curvatures and resorptions, without flattening and absence of previous endodontic treatment. The samples were fixed in acrylic plates with low melt godiva (DFL, Porto Alegre, RS, Brazil). Then, teeth were attached to a fastening device in the cutting machine (Labcut 1010, Erios, São Paulo, SP, Brazil), and sectioned perpendicularly with a diamond saw to the long axis of the root in order to obtain two slices of 2 mm thickness of the cervical third of each teeth, thus obtaining 26 slices. The middle and apical root thirds and the crowns were discarded, and only the cervical slice was used in this study. Each cervical dentin slice was sectioned twice. The first section was performed in the sagittal plane of the slice dividing it in half, and the second section in the frontal plane. The blocks were adjusted with a fine polishing disc (3 M ESPE Dental Products, St. Paul, MN, USA) and their measurements were verified using a digital caliper (Mitutoyo, Suzano, SP, Brazil) in order to obtain the standardized dimensions of 4 mm height, 4 mm width and 2 mm thickness, totalizing 104 blocks of dentin. One hundred blocks were randomly selected for this study. The blocks were placed in a 150 mL becker with 17% ethylenediaminetetraacetic acid (EDTA) (Formula and Action, São Paulo, SP, Brazil) and kept under stirring on a tube shaker (Vortex, Model AP 56, Presidente Prudente, SP, Brazil) for 5 min. Subsequently, the same process was performed, however, using distilled water. The blocks were kept hydrated in a 150 mL becker and were incubated for 24 h. Afterward, the becker distilled water was renewed so that the blocks were kept hydrated during sterilization in an analogue vertical autoclave (Idealclave, Stermax, Barueri, São Paulo, Brazil) for 30 min at 120 °C. After sterilization, the blocks were incubated in BHI at 37 °C for 48 h to confirm the absence of bacteria by visual analysis. The blocks
2.3. Disinfection Protocols The groups treated with sodium hypochlorite (groups I, II and III) were immersed in 5 mL of 2.5% sodium hypochlorite (Fitofarma, Lt. 20,442, Goiânia, GO, Brazil) contained in the Petri dish (K13-0035, KASVI, Goiânia, Brazil) for 5 min. The blocks were then pinched and transferred to a gauze pad over another Petri dish to remove excess sodium hypochlorite. Group II, after immersion in 2.5% sodium hypochlorite, the intraradicular dentin was irradiated with 808 nm laser (DMC Whitening Lase II, São Carlos, SP, Brazil), with an optical fiber tip with 200 μm diameter, perpendicularly to the surface, on all faces of the dentin blocks. In each face, irradiation with light in continuous mode (100 mW), power of 0.1 W, for 20 s, totalizing 2 W, with energy density 31,847 J/cm2 (Table 1) was performed. Group III, after the same immersion process as Group II, was irradiated with 970 nm diode laser (Sirona Dental, Benshein, HE, Germany) Table 1 Parameters established for diode laser use.
2
Parameters
808 ± 10 nm diode laser
970 ± 15 nm diode laser
Diameter of the fiber optic tip Mode of application Duration Power Energy density
200 μm
200 μm
Continuous wave 20 s 2W 31,847 J/cm2
Continuous wave 4s 2W 159,23 J/cm2
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Table 2 Mean and standard deviation of microbial activity (nm) before and after dentinal surface treatment and visual analysis of bacterial contamination before treatment (CB1) and after treatment (CB2). Dentinal surface treatments
CB1 n = 10
Mean and standard deviation of optical density (nm)
CB2 n = 10
Mean and standard deviation of optical density (nm)
NaOCl 2.5% NaOCl 2.5% + 808 nm diode laser NaOCl 2.5% + 970 nm diode laser 808 nm diode laser 970 nm diode laser CHX 2% CHX 2% + 808 nm diode laser CHX 2% + 970 nm diode laser Positive control Negative control
+ + + + + + + + + −
0.8175 0.8081 0.7718 0.8473 0.8711 0.8229 0.8326 0.7902 0.7974 0 ± 0
+ + + + + + + + + −
0.1754 0.2220 0.2427 0.3474 0.2745 0.3090 0.2696 0.3224 0.7918 0 ± 0
± ± ± ± ± ± ± ± ±
0.082 0.061 0.081 0.034 0.077 0.073 0.032 0.025 0.045
± ± ± ± ± ± ± ± ±
0.102 0.100 0.072 0.205 0.072 0.029 0.020 0.031 0.144
*The positive and negative symbols corresponds to visual analysis: (+) represents turbidity (−) absence of turbidity. Table 3 Mean and standard deviation values of the percentage of reduction of the number of bacteria. Dentine surface treatment
Optical density mean values (nm)
NaOCl 2.5% NaOCl 2.5% + 808 nm diode laser NaOCl 2.5% + 970 nm diode laser 808 nm diode laser 970 nm diode laser CHX 2% CHX 2% + 808 nm diode laser CHX 2% + 970 nm diode laser
77.5 72.5 68.7 58.8 68.3 62.0 67.5 59.2
± ± ± ± ± ± ± ±
12.1 (68.8–86.1) A 12.2 (63.6–81.5) AB 8.7 (62.3–74.8) AB 25.0 (41.0–76.7) B 8.7 (62.1–74.5) AB 5.5 (58.2–66.0) AB 3.3 (55.1–69.9) AB 4.0 (56.4–62.0) B
*Different upper case letters indicate statistical difference of the dentinal surface treatments in the bacterial reduction (Tukey's p < 0.05).
with an optical fiber tip of 200 μm diameter perpendicularly to the surface on all faces of the dentin blocks. On each face, irradiation was performed with light in continuous mode, power of 0.5 W, for 4 s, totaling 2 W, with energy density 159.23 J/cm2 (Table 1). Group IV was irradiated with diode laser 808 nm and group V with diode laser 970 nm, according to parameters previously described, without previous immersion. The tips of both lasers were analyzed between the applications, with the aid of a magnifying glass, in order to observe the presence of damages in its surface. When the surface was irregular or burned, the tip was sectioned perpendicular to the long axis of the tip, so as not to damage the next irradiation. The samples of groups VI, VII and VIII were immersed in 2% chlorhexidine according to the same procedure used for the treatment with NaOCl. Group VII was irradiated with 808 nm diode laser and group VIII with 970 nm diode laser according to the previously described parameters. Ten blocks of group IX were kept contaminated throughout the experiment at 37 °C to verify bacterial viability and maintained (positive control). Meanwhile, 10 blocks of group X were stored in 5 mL sterile BHI at 37 °C to ensure the sterility of the sample (negative control). After the dentin surface treatments, the blocks were stored in 7 mL BHI at 37 °C for 48 h. Then each block was transferred to a tube containing 7 mL of BHI added with Tween 80 and sodium thiosulphate neutralizers (P.A., Lab Art, Campinas, SP, Brazil) and incubated at 37 °C for 48 h.
Fig. 1. Percentage of the decrease of antimicrobial activity after treatment.
observation. In fact, the amount of absorbed and dispersed light is proportional to the mass of cells in the luminous path. Thus, for the turbidimetric measurements of the cell mass, a UV Spectrophotometer was used. In the microbiological analysis, a 0.1 mL inoculum obtained from the BHI was transferred to 5 mL of Letheen Broth under identical incubation conditions. Gram staining was used in BHI cultures to verify contamination and growth and was microscopically examined. The microbial concentration was analyzed using a spectrophotometer (Spectrophotometer Model Nova 1600 UV, Piracicaba, SP, Brazil) adjusted for reading at wavelength λ = 600 nm, adopting the McFarland scale 1, which corresponds to the absorbance of 0.137 nm after zero reading of the sterilized culture medium and at room temperature.
2.5. Ultrastructural Evaluation of Dentin by Scanning Electron Microscopy (SEM) Five samples from each group were randomly selected and submitted to scanning electron microscopy (SEM) for analysis of the dentin surface. Samples were fixed in an ascending scale of ethanol concentrations (25°, 50°, 75° for 20 min in each, 95° for 30 min and 100° for 1 h). The specimens were then fixed in metal cylinders and then metallized by a ~ 300 Å gold layer in metallizer (Jeol, JSM-6610, equipped with EDS, Thermo scientific NSS Spectral Imaging, Tokyo, Japan). In order to perform the qualitative analysis, 1500 × and 5000 × magnification photomicrographs of the intraradicular dentin were performed for each sample, totalizing 10 images for each group using the scanning electron microscope (JSM-IT300, Joel, Tokyo, Japan). The
2.4. Microbiological Analysis Microbial growth was analyzed by the turbidity of the culture medium. This method is based on light scattering as it passes through a sample. Suspended cells absorb the light and the dispersion of light that pass through them makes a culture appear blurred by visual 3
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Fig. 2. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000× magnification. I - negative control; II - positive control.
(68.3 ± 8.7), VI (CHX 2%) (62.0 ± 5.5), VII (CHX 2% + laser diode 808 nm) (67.5 ± 3, 3) (p > 0.05); and statistically different from group IV (laser diode 808 nm) (58.8 ± 25.0), VIII (CHX 2% + diode 970 nm) (59.2 ± 4.0) and control groups (p < 0.05) (Table 3 and Fig. 1). The qualitative analysis of the images obtained by scanning electron microscope revealed dentinal tubules with open and regular contour in the negative control group (Fig. 2IAB). The intertubular dentine appeared regular, free of debris and bacteria. The group that maintained contamination throughout the treatment (positive control) (Fig. 2IIAB) revealed dense biofilm covering the dentin surface. The openings of the dentinal tubules were obliterated. Dentin treated with 2.5% sodium hypochlorite (Fig. 3IAB) showed regions with intertubular dentin erosion with an irregular dentin surface and a dispersed biofilm. Surface treatment with 2% chlorhexidine solution (Fig. 3IIAB) revealed dense biofilm covering the surface, making it impossible to observe the possible changes that the solution could cause to the dentin surface. After the dentin was irradiated with 808 nm diode laser, regions with erosion of the intertubular dentin were observed, making the dentin surface irregular (Fig. 4IAB), and the biofilm appeared dispersed. The association of 2.5% sodium hypochlorite with the 808 nm laser (Fig. 4IIAB), the effect of the 808 nm diode laser was increased, showing an irregular surface with melted dentin. Dentin submitted to 2% chlorhexidine and 808 nm diode laser treatment (Fig. 4IIIAB) presented a surface similar to the group treated only with 2% chlorhexidine. When irradiated with 970 nm diode laser, the dentin surface showed an amorphous organic matrix and melted intertubular dentin with partial dentinal tubule opening, characterizing an irregular surface (Fig. 5IAB) with a dispersed biofilm on its surface. The images obtained from dentin blocks submitted to 2.5% sodium hypochlorite and 970 nm
following aspects of the dentin surface were observed: opening or obliteration of dentinal tubules, regular or irregular surface, presence or absence of exposed collagen fibers and presence or absence of biofilm. 2.6. Statistical Analysis Once the data presented a normal distribution (Shapiro-Wilk, p > 0.05) and homogeneity of variance (Levene test, p > 0.05), parametric tests were used. The ANOVA one factor was used to evaluate the influence of disinfection protocols on the percentage decrease of Enterococcus faecalis contingent. Tukey test was used for comparisons between the groups. The probability level was 95% and all analyzes were performed in SPSS software. 3. Results In the visual and spectrophotometry analysis of all the blocks before treatments, contamination was demonstrated with the exception of the blocks that remained sterilized (negative control group), showing the effectiveness of the bacterial contamination procedure (Table 2). Through the microbial activity analysis before and after the dentin surface treatments, a significant statistical difference (p < 0.05) was observed for all treatments, except for the blocks that remained contaminated (group IX - positive control) and blocks that remained sterilized (negative X-control group). Therefore, the treatments performed significantly decreased the number of bacteria. When analyzing the percentage of antimicrobial activity (nm) before and after surface treatments, it was observed that the treatment with 2.5% NaOCl (group I) had the highest percentage of antimicrobial activity decrease (77.5 ± 12, 1) statistically similar to groups II (NaOCl 2.5% + laser diode 808 nm) (68.7 ± 8.7), III (NaOCl 2.5% + laser diode 970 nm) (68.7 ± 8.7), Group V (laser diode 970 nm) 4
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Fig. 3. Scanning electron microscopy images at different magnifications. A - 1500× magnification; B - 5000× magnification. I - treatment with 2.5% NaOCl; II - treatment with 2% CHX.
is similar to the study by Moritz et al. [22], irradiation was performed only once, which may justify the lower percentage of bacterial reduction when compared to the study by Moritz et al. [22] which achieved almost complete elimination of bacteria on the second irradiation. Therefore, neither the laser nor the solutions and their associations evaluated in the present study were able to reach the deeper layers of dentin, not allowing the complete elimination of the bacteria. Regarding the application of diode laser (808 nm and 970 nm) and after irrigation with 2.5% sodium hypochlorite and 2% chlorhexidine solutions, it was not possible to observe a significant improvement in the reduction of the bacterial contingent. These results corroborate with Mehrvarzfar et al. [23] and Castelo-Baz et al. [24], that when analyzing the effect of laser diode alone and in combination with solutions on infected root canals, did not observe statistical differences between groups. These results show that even after the application of irrigation solutions, 970 nm and 808 nm diode laser diodes were not able to reach sufficient depth to eliminate the entire bacterial contingent. Beer et al. [9] verified higher reduction values of bacterial contingent when compared to the present study, in which treatment with 808 nm diode laser showed the lowest reduction in the number of bacteria. Romeo et al. [25] evaluated the effectiveness of 980 nm diode laser to kill Enterococcus faecalis in experimentally infected root canals and found higher reduction value of the bacterial contingent to the laser treatment combined with sodium hypochlorite when compared to irrigation treatment without irradiation, different from the present study that did not observe improvement in the decontamination of the root dentine when hypochlorite was associated to laser irradiation. However, the differences in those results may be related to the methodology, once the contamination period in the present study was 5 days, when compared to Beer et al. [9], that used a contamination period of only 2 h, and Romeo et al. [25] that evaluated the decontamination of 72 h biofilms. Thus, the bacteria would be in less mature stages, which
diode laser treatment (Fig. 5IIAB) showed irregular surface with intertubular dentine erosion and biofilm on its surface. The dentin submitted to the 2% chlorhexidine and 970 nm diode laser treatment (Fig. 5IIIAB) showed an amorphous organic matrix with partial dentinal tubule exposure. 4. Discussion The development phase of the biofilm in the antimicrobial evaluation is very important and may be a determining factor for the greater resistance to different types of antimicrobial strategies [18]. Currently, most studies evaluate the antimicrobial properties of irrigation solutions, involving both bacterial stages of growth, planktonic and biofilm [19], which makes it difficult to compare the results obtained by different types of laser, as well as to verify the effective action of the lasers in situations where the bacteria present in the root canal system are not in their planktonic stage. In this sense, in the present study 2-mm-thick (2000 μm) intraradicular dentine blocks contaminated with a five-day biofilm were used to evaluate different diode laser wavelengths (808 nm and 970 nm). The antibacterial action of the 970 nm diode laser, verified in the present study, is directly related to the heating of the dentin substrate to which the bacteria are bound [20], which resulted in the bacterial contingent reduction (68.3 ± 8.7) statistically similar to the group irradiated with 808 nm diode laser, corroborating to previous studies [9,21]. The surface irradiation with 808 nm diode laser resulted in 58.8% reduction of the bacterial contingent. According to Moritz et al. [22], gram-negative bacteria are reached immediately after the first irradiation, whereas gram-positive bacteria such as E. Faecalis, evaluated in the present study, need to be irradiated repeatedly for bacterial cell wall disruption. Although the energy density used in the present study 5
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Fig. 4. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000 × magnification. I - 808 nm diode laser; II - 808 nm diode laser + 2.5% NaOCl; III - 808 nm diode laser + 2% CHX.
blocks by SEM, irradiation with 808 nm diode laser promoted an irregular surface with erosion regions in the intertubular dentine, which differs from other studies in which there was fusion of dentinal tubules after applying the laser diode [9,10,34]. The different results can be justified due to the position of application of the laser to the substrate surface [35]. In the present study, the application of the laser was perpendicular to the surface of the dentin blocks, whereas in other studies the application was performed along the root canal by helical motions [9,13,35]. On the other hand, when analyzing the surface of the dentin blocks irradiated with 970 nm diode laser an amorphous organic matrix and melted intertubular dentine was observed. According to Jhingan et al. [36], after irradiation with 980 nm diode laser (2 W), the analysis of the intraradicular dentinal surface by means of scanning electron microscopy, evidenced the melting of the dentin layer making it irregular as
allowed an easier removal and consequent bacterial death. Regarding the irrigation solutions used alone, treatment with 2.5% sodium hypochlorite showed similar results regarding the bacterial decrease (77.5 ± 12.1) when compared to the 2% chlorhexidine group (62.0 ± 5.5), in agreement with previous studies comparing these solutions at different concentrations [26–28]. Both solutions have antimicrobial properties, and sodium hypochlorite has the capacity to promote the disinfection and solvency of organic and inorganic tissues [29,30] and chlorhexidine, normally used as an alternative solution, has properties of substantivity, prolonged antimicrobial effect and low cytotoxicity [31–33]. From the microbiological point of view, any solution can be used during the treatment of infected teeth, however, the different properties of irrigating solutions should influence the choice of the auxiliary solution. When evaluating the surface morphological changes of the dentin 6
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Fig. 5. Scanning electron microscopy images at different magnifications. A - 1500 × magnification; B - 5000 × magnification. I - 970 nm diode laser; II - 970 nm diode laser + 2.5% NaOCl; III - 970 nm diode laser + 2% CHX.
present study, when evaluating the effect of different irrigation protocols on root dentin structure through SEM and transmission electron microscopy. Intraradicular dentin presented structural alterations caused by 2.5% sodium hypochlorite, whereas treatment with chlorhexidine, distilled water and saline solution preserved the collagen structure, and were unable to dissolve the organic content. Thus, in the evaluation of the influence of the diode lasers with different wavelengths on the dentin surface, the present study concluded that the application of laser diode 970 nm and 2.5% sodium hypochlorite had similarity in the decontamination of the root dentin blocks surface, although they presented morphological changes in the dentin surface. However, further studies should be carried out to find the effectiveness of the diode laser in root canals contaminated by mature biofilms, in conditions closer to the clinical reality, as well as its influence on the dentin morphology in the prognosis of endodontic treatment.
also observed by Alfredo et al. [10] and Faria et al. [37]. These observed morphological changes can be attributed to the thermal effect caused by superheating and subsequent surface cooling when the laser interacts with the dentinal tissue [10,11]. This phenomenon explains the melted intertubular dentine and irregular surface promoted by the 970 nm diode laser. Regarding the association of 808 nm diode laser with 2.5% sodium hypochlorite solution, there was a potentiation of the morphological alterations of the dentin compared to the laser treatment without immersion. When the laser diode 970 nm was associated with 2.5% sodium hypochlorite, it was possible to observe a greater erosion of the intertubular dentine compared to the treatment with 970 nm diode laser without immersion. However, when chlorhexidine was associated with the lasers, there was a minor alteration of the dentin structure. Wagner et al. [38] obtained similar results to those obtained in the 7
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[18] Z. Lim, J.L. Cheng, T.W. Lim, E.G. Teo, J. Wong, S. George, et al., Light activated disinfection: an alternative endodontic disinfection strategy, Aust. Dent. J. 54 (2009) 108–114. [19] K. Jhajharia, A. Parolia, K.V. Shetty, L.K. Mehta, Biofilm in endodontics: a review, J. Int. Soc. Prev. Community Dent. 5 (2015) 1–12. [20] N. Gutknecht, T.S. Al-Karadaghi, M.A. Al-Maliky, G. Conrads, R. Franzen, The bactericidal effect of 2780 and 940 nm laser irradiation on Enterococcus faecalis in bovine root dentin slices of different thicknesses, Photomed. Laser Surg. 34 (2016) 11–16. [21] N.R. Kanumuru, R. Subbaiah, Bacterial efficacy of Ca(oH)2 against E.faecalis compared with three dental lasers on root canal dentin- an invitro study, J. Clin. Diagn. Res. 8 (2014) ZC135–137. [22] A. Moritz, N. Gutknecht, U. Schoop, K. Goharkhay, O. Doertbudak, W. Sperr, Irradiation of infected root canals with a diode laser in vivo: results of microbiological examinations, Lasers Surg. Med. 21 (1997) 221–226. [23] P. Mehrvarzfar, M.A. Saghiri, A. Asatourian, R. Fekrazad, K. Karamifar, G. Eslami, et al., Additive effect of a diode laser on the antibacterial activity of 2.5% NaOCl, 2% CHX and MTAD against Enterococcus faecalis contaminating root canals: an in vitro study, J. Oral Sci. 53 (2011) 355–360. [24] B. P.M.B. Castelo–Baz, M. Ruiz–Pinon, Combined sodium hypochlorite and 940 NM diode laser treatment against mature E. facials boxfuls in-vitro, Lasers Med. Sci. 3 (2012) 116–121. [25] U. Romeo, G. Palaia, A. Nardo, G. Tenore, V. Telesca, R. Kornblit, A. Del Vecchio, A. Frioni, P. Valenti, F. Berlutti, Effectiveness of KTP laser versus 980 nm diode laser to kill Enterococcus faecalis in biofilms developed in experimentally infected root canals, Aust. Endod. J. 41 (2015) 17–23. [26] N. Flach, D.E. Bottcher, C.C.F. Parolo, L.B. Firmino, M. Malt, M.L. Lammers, et al., Confocal microscopy evaluation of the effect of irrigants on Enterococcus faecalis biofilm: an in vitro study, Scanning 38 (2016) 57–62. [27] I.N. Rocas, J.C. Provenzano, M.A. Neves, J.F. Siqueira Jr., Disinfecting effects of rotary instrumentation with either 2.5% sodium hypochlorite or 2% chlorhexidine as the main irrigant: a randomized clinical study, J. Endod. 42 (2016) 943–947. [28] J.F. Siqueira Jr., I.N. Rocas, S.S. Paiva, T. Guimaraes-Pinto, K.M. Magalhaes, K.C. Lima, Bacteriologic investigation of the effects of sodium hypochlorite and chlorhexidine during the endodontic treatment of teeth with apical periodontitis, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 104 (2007) 122–130. [29] F. Baratto-Filho, J.R. De Carvalho Jr., L.F. Fariniuk, M.D. Sousa-Neto, J.D. Pecora, A.M. Da Cruz-Filho, Morphometric analysis of the effectiveness of different concentrations of sodium hypochlorite associated with rotary instrumentation for root canal cleaning, Braz. Dent. J. 15 (2004) 36–40. [30] C. Estrela, C.R. Estrela, E.L. Barbin, J.C. Spano, M.A. Marchesan, J.D. Pecora, Mechanism of action of sodium hypochlorite, Braz. Dent. J. 13 (2002) 113–117. [31] L. Bazvand, M.G. Aminozarbian, A. Farhad, H. Noormohammadi, S.M. Hasheminia, S. Mobasherizadeh, Antibacterial effect of triantibiotic mixture, chlorhexidine gel, and two natural materials Propolis and Aloe Vera against Enterococcus faecalis: an ex vivo study, Dent. Res. J. (Isfahan) 11 (2014) 469–474. [32] B.P. Gomes, M.E. Vianna, A.A. Zaia, J.F. Almeida, F.J. Souza-Filho, C.C. Ferraz, Chlorhexidine in endodontics, Braz. Dent. J. 24 (2013) 89–102. [33] Z. Mohammadi, L. Giardino, F. Palazzi, S. Asgary, Agonistic and antagonistic interactions between Chlorhexidine and other endodontic agents: a critical review, Iran Endod J. 10 (2015) 1–5. [34] M. Esteves-Oliveira, C.A. De Guglielmi, K.M. Ramalho, V.E. Arana-Chavez, C.P. De Eduardo, Comparison of dentin root canal permeability and morphology after irradiation with Nd:YAG, Er:YAG, and diode lasers, Lasers Med. Sci. 25 (2010) 755–760. [35] P.R. Alves, N. Aranha, E. Alfredo, M.A. Marchesan, A. Brugnera Junior, M.D. SousaNeto, Evaluation of hollow fiberoptic tips for the conduction of Er:YAG laser, Photomed. Laser Surg. 23 (2005) 410–415. [36] P. Jhingan, M. Sandhu, G. Jindal, D. Goel, V. Sachdev, An in-vitro evaluation of the effect of 980 nm diode laser irradiation on intra-canal dentin surface and dentinal tubule openings after biomechanical preparation: scanning electron microscopic study, Indian J. Dent. 6 (2015) 85–90. [37] M.I. Faria, M.D. Sousa-Neto, A.E. Souza-Gabriel, E. Alfredo, U. Romeo, Y.T. SilvaSousa, Effects of 980-nm diode laser on the ultrastructure and fracture resistance of dentine, Lasers Med. Sci. 28 (2013) 275–280. [38] M.H. Wagner, R.A. Da Rosa, J.A. De Figueiredo, M.A. Duarte, J.R. Pereira, M.V. So, Final irrigation protocols may affect intraradicular dentin ultrastructure, Clin Oral Investig (2016).
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