In Vitro Evaluation of Er:YAG and Nd:YAG Laser Irradiation on Root Canal Walls A Preliminary Study

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1 In Vitro Evaluation of Er:YAG and Nd:YAG Laser Irradiation on Root Canal Walls A Preliminary Study Publication Claudia Goya a, Bruno Lopes da Silveira b, Ana Cecília Corrêa Aranha b, Denise Maria Zezell c, Koukichi Matsumoto d, Carlos de Paula Eduardo e a Graduate Student, Lasers in Dentistry, Special Laboratory of Lasers in Dentistry (LELO FOUSP), University of São Paulo, São Paulo, SP, Brazil. b PhD Student, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, São Paulo, SP, Brazil. c Associate Professor - Lasers and Applications Center Energy and Nuclear Research Institute IPEN, São Paulo, SP, Brazil. d Professor and Head, Department of Endodontics, Showa University School of Dentistry, Kitasensoku, Ohta-Ku, Tokyo, Japan. e Full Professor, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, São Paulo, SP, Brazil. Purpose: This study aimed to compare the in vitro effects of Nd:YAG and Er:YAG laser irradiation on root canal walls using scanning electron microscopy (SEM), and evaluate apical leakage and temperature changes in external root surfaces. Materials and Methods: Seventy-four recently extracted human teeth with single, straight roots were used. The teeth were prepared to 1 mm short of the apical amen a conventional technique using K-files and assigned to different groups: G1) no laser irradiation/control; G2) Nd:YAG laser/helicoidal technique; G3) Nd:YAG laser/vertical technique; G4) Er:YAG laser/helicoidal technique; G5) Er:YAG laser/vertical technique; G6) Er:YAG and Nd:YAG laser/helicoidal technique; G7) Er:YAG and Nd:YAG laser/vertical technique. The Nd:YAG laser parameters were 100 mj, 15 Hz, 0.5 J/cm 2, and those of the Er:YAG laser were 160 mj, 10 Hz, 0.9 J/cm 2, four times at 2 mm/s, with 20-s intervals. Temperature changes were recorded thermographically. All teeth were examined light microscopy or scanning electron microscopy (SEM). Results: No apical leakage was observed in the teeth irradiated with Nd:YAG laser alone or in association with Er:YAG laser. However, in the teeth irradiated with Er:YAG laser, more pronounced leakage was observed. SEM observation showed that Nd:YAG laser irradiation caused melting and crystallization on dentin surfaces. Er:YAG laser samples showed a clean, debris-free surface with opened dentinal tubules. Specimens irradiated the combination of the two lasers showed a melted layer covering the dentinal tubules. The temperature increase did not exceed 6 C. Conclusions: This study demonstrated that Nd:YAG laser irradiation with or without adjunct Er:YAG laser irradiation is a potentially suitable means of root canal treatment, since morphological alterations are desirable and it did not cause thermal damage to adjacent tissues. Keywords: apical leakage, dentin, dentinal tubules, endodontics, Er:YAG, Nd:YAG, scanning electron microscopy, thermographic evaluation. J Oral Laser Applications: 2007; 7: Submitted publication: ; accepted publication: In endodontics, effective cleaning of the root canal system is essential ensuring a successful, long-lasting outcome. During endodontic instrumentation, various morphological changes occur on the root canal walls, including organic and mineral debris and smear layer mation. Theree, conventional cleaning and removal of debris and smear layer are important steps in the endodontic procedure. According to Takeda et Vol 7, No 1,

2 al 1 and Ramalho et al, 2 the success of endodontic treatment depends on the comprehensive cleaning and decontamination of the root canal system; however, the removal of the smear layer, remaining pulp tissue, and inorganic debris produced the endodontic treatment can be responsible the leakage between canal walls and filling material after obturation, which may harbor microorganisms that ultimately result in periapical pathosis. Until now, endodontic treatment has been based on the use of chemical irrigating solutions, such as sodium hypochlorite (NaOCl) and ethylene diamine tetraacetate (EDTA), associated with mechanical instrumentation to remove smear layer. Many studies have been developed focusing on materials and techniques to clean the dentin root canal walls completely. Laser irradiation has been widely introduced in endodontic treatment as an aid to disinfection and the removal of debris and smear layer from instrumented root canal walls. 3 Laser irradiation produces different effects on the same tissue, and the same laser can produce various effects in different tissues. Due to its effects and development of optical fibers that allow the laser light to enter into the root canal, its clinical use as a coadjuvant in endodontic treatment is promising. 3-7 Since the development of the ru laser Maiman, scientific interest in the use of lasers in dentistry has increased. 8,9 The CO 2 laser was the first to be used apical sealing in vitro. Although the aims were not achieved, the data were sufficiently convincing to encourage other investigations. 10 Since then, a number of studies concerning laser application in endodontics have been published. 11 In various laser systems used in dentistry, the emitted energy can be delivered into the root canal system a thin optical fiber (Nd:YAG, Er,Cr:YSGG, argon, diode, Er:YAG) or a hollow tube (CO 2 and Er:YAG). 3 The Nd:YAG laser irradiation causes morphological changes in the root canal walls melting and solidifying the dentin layer, including the smear layer. The irradiation can induce the mation of droplets that cover the dentinal tubules, thus representing the etching factor canal sealing with occlusive material. 12 On the other hand, morphological changes caused Er:YAG laser irradiation on the root canal wall are determined residual material vaporization and dentin tubule opening, there increasing dentin permeability after canal sealing. It also causes reduction of bacteria due to the high temperatures achieved. 13 Despite the beneficial potential of lasers in endodontics, thermal injury is the most important problem as- Publication sociated with it. The temperature limit avoiding thermal injury to the external surface of the root and periodontal tissues on application of the laser was found to be 47 C. 3,14 The aims of this study were to verify the morphological alterations in root canal walls after Nd:YAG and Er:YAG laser irradiation, assess apical marginal leakage after obturation of irradiated teeth, and to determine thermal variations during intracanal irradiation. MATERIALS AND METHODS Sample Preparation Seventy-four extracted single-rooted human teeth were obtained after approval the Ethics Committee and stored in 0.1% aqueous thymol solution. The teeth were cleaned with Gracey curettes to remove visible calculus. The working length was determined radiographs, 1 mm distant from the apical amen. Root canals were biomechanically prepared to their full lengths up to k-file 45. Cervical and middle thirds of all canals were debrided with Gates-Glidden drills providing standardized access to the middle and apical thirds of the canals. Irrigation with 2.5% NaOCl solution was alternately permed between each file and Gates- Glidden drills. The root canals were then dried with paper points. Then the teeth were divided into 7 groups (n = 10): G1: control group, no laser irradiation G2: Nd:YAG laser irradiation helicoidal technique with dry root canal G3: Nd:YAG laser irradiation vertical technique with dry root canal G4: Er:YAG laser irradiation helicoidal technique with sodium hypochlorite irrigation G5: Er:YAG laser irradiation vertical technique with sodium hypochlorite irrigation G6: Er:YAG laser irradiation with wet root canal; then Nd:YAG laser irradiation helicoidal technique with dry root canal G7: Er:YAG laser irradiation with wet root canal; then Nd:YAG laser irradiation, vertical technique with dry root canal Laser Systems Nd:YAG laser (American Dental Laser; Birmingham, MI, USA), which has a wavelength of 1064 nm, was 46 The Journal of Oral Laser Applications

3 used according the following parameters: 0.32 mm diameter quartz fiber in contact with the surface, 100 mj, 15 Hz, 1.5 W, 0.5 J/cm 2, and 100 μ pulse width. Nankin ink (black ink) was used as an intracanal initiating agent because of low Nd:YAG laser wavelength absorption hard tissue. According to the literature, satisfactory results were presented when the canal walls were painted with nankin ink. 15,16 Er:YAG laser (Kavo Key Laser 1242, Kavo Dental; Jena, Germany), which has a wavelength of 2940 nm, was used according to the following parameters: 0.47 mm diameter tip in contact with the surface, 160 mj and 10 Hz (panel settings), 0.54 transmission factor, 0.9 J/cm 2 of energy density, 250 and 500 μs pulse duration, 2 mm/s fiber movement speed with 50/10 fiber. Irradiation Techniques For both techniques, to ensure that the flexible glass fiber reached the physiological apex, the determined length of the root canal was transferred exactly to the fiberoptic waveguide. The fiber is first positioned in place at the apex without activating the laser and only then is the fiber moved over the root canal walls from the apex to the crown. In the helicoidal technique, the fiber tip was introduced into the full length of the root canal irradiating from the apical to the cervical area with gentle spiralling movements at 2 mm/s ascendant speed. This procedure was repeated 4 times, with 20-s intervals between applications (see Gutknecht et al 17 ). In the vertical technique, the fiber tip was introduced into the full length of the root canal irradiating from the apical to the cervical area with gentle vertical movements at 2 mm/s ascendant speed. This procedure was repeated 4 times, with 20-s intervals between applications (see Anic et al 18 ). SEM Morphological Analysis Five teeth from each group were sectioned longitudinally into two symmetrical sections. The sections were first fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer 48 h at room temperature. The specimens were dehydrated in graduated alcoholic solutions and submitted to chemical drying in hexamethyl disilazane (HMDS) until its complete evaporation, and then covered with a 15-μm platinum layer analysis in a scanning electron microscope (JSM 1220, JEOL; Tokyo, Publication Japan). Electron micrographs were taken from the apical area of each tooth at 500X magnification. Apical Leakage After laser irradiation, specimens were irrigated with physiological saline solution and then the canals were dried with absorbent paper tips. A gutta-percha point (no. 45) was adapted and endodontic cement (Canals N, Showa Yakuhin; Tokyo, Japan) placed. Material excesses were removed and all roots were sealed with 2 layers of finishing gloss (synthetic resin) except in apical area. Then, specimens were immersed in 0.5% methylene blue aqueous solution, ph 7.2, 48 h at 37 C. The specimens were washed in distilled water and the teeth were longitudinally cut with a diamond disk (Isomet-Buehler; Lake Bluff, IL, USA) dividing them into a mesial and a distal part. Analysis of leakage was permed three standardized and independent, blinded examiners all the groups and specimens under stereomicroscopy (SMZ-10, Nikon; Tokyo, Japan) at 35X magnification, concentrating on the apical part of the root. Linear infiltration of the dye solution was measured with a digital caliper. Statistical analysis of this step was done using the Kappa test to determine the interexaminer agreement, and the Kruskal-Wallis test at a p < 0.01 level of significance. Thermal Evaluation SCIENCE Temperature changes on root surfaces during irradiation were measured during and immediately after laser irradiation using a thermovision device (AGEMA, Infrared System AD; Danderyd, Sweden) linked to a personal computer at room temperature (24 C). To standardize the experimental conditions during measurements, the samples were fixed using clamps, and an ice block was placed at a distance of 10 cm from the samples. Laser irradiation was permed according to the technique described above. The image of the thermal change on the external surface while undergoing laser irradiation was captured the thermograph camera and registered on a computer screen (Fig 1). The gradual color scale indicated the thermal changes on external root surface. The thermal changes were not registered in groups 6 and 7 because the technique used in these groups was similar to that used in groups 2 to 5. Vol 7, No 1,

4 Fig 1 Thermograph camera (AGEMA, Infrared System AB, Danderyd, Sweden) connected to a computer temperature analysis. RESULTS Apical Leakage SEM Analysis Publication Scanning electron micrographs of group 1 specimens, without irradiation, showed a thick smear-layer deposit over the dentin surfaces. In Nd:YAG laser irradiated specimens (G2 and G3), the dentin surface presented a melted and re-solidified layer, characteristic of Nd:YAG laser irradiation. In some cases, the smear layer was melted into the resolidified material. The final appearance was a vitrified surface with droplets totally or partially covering dentin tubules (Fig 5). In Er:YAG laser irradiated specimens (G4 and G5), photomicrographs showed changes in the dentin surface due to tissue ablation creating irregularities and craters. These specimens presented a clean surface with opened dentin tubules; debris and smear layer over tubules were lacking (Fig 6). In Er:YAG laser irradiated specimens followed Nd:YAG laser irradiation (G6 and G7), dentin walls presented a clean surface with droplets due to melting and resolidification resulting from the Nd:YAG laser irradiation, covering the dentin tubules (Fig 7). In the apical area, it was possible to observe that the entire dentinal surface was reached the Nd:YAG laser fiber. Irrespective of the root canal treatments, no morphological differences were perceived between the helicoidal and vertical techniques. Differences were noted in relation to the laser type used and their combination. Because the mean apical leakage values did not possess normality, a nonparametric Kruskal-Wallis analysis was permed. Mean leakage values and standard deviations are illustrated in Table 1. Specimens without any treatment showed the significantly greatest stain infiltration (p 0.01), presenting a mean value of 1.16 mm. The results showed that Nd:YAG laser irradiation was capable of preventing leakage after analysis on the spectroscopy as observed in a representative image (Fig 2). In contrast, the Er: YAG laser groups (G4 and G5) presented higher leakage values, 0.28 and 0.44 mm, respectively. ice that in Fig 3, apical leakage is more pronounced than in the Nd:YAG specimens. No leakage was observed when Er:YAG and Nd:YAG laser were used in succession (Fig 4). This could be due to the cleaning and shaping properties of the Er:YAG laser and Nd:YAG laser irradiation. The dentinal tubules were occluded. Thermal Changes The distribution in temperature in the dentin surface was registered bee and after irradiation. Figure 8 shows the initial temperature as seen on the screen. Thermal changes in root surfaces from each group are described in Table 2. Although the Er:YAG irradiation in both vertical and helicoidal techniques presented a reduction in time to return to the initial temperature, it was found that there was no significant difference between the initial and final temperature. No differences were found between the two types of laser used. In addition, the thermographic study showed that the temperature increase did not exceed 6 C (Figs 9 and 10). DISCUSSION Successful endodontic treatment depends on many factors, such as appropriate chemomechanical preparation, type of irrigant, filling material, and technique. 48 The Journal of Oral Laser Applications

5 Publication Table 1 Apical leakage mean values and standard deviations (SD) G1 G2 G3 G4 G5 G6 G7 Description control Nd:YAG Nd:YAG Er:YAG Er:YAG Er: YAG + Er: YAG + helicoidal vertical helicoidal vertical Nd:YAG Nd:YAG helicoidal vertical Mean Values (mm) and SD 1.16± ± ± ± ± ± ±0.0 a b b c C b b *Similar letters indicate no statistically significant differences. Fig 2 Longitudinal cut of a Nd:YAG laser irradiated surface. Observe that there is no infiltration in the apical area. Fig 3 Longitudinal cut of an Er:YAG laser irradiated surface. Observe that more leakage is visable after irradiation. Fig 4 Longitudinal cut of an Er:YAG associated with Nd:YAG laser irradiation. Due to Nd:YAG laser irradiation, tubules were sealed and apical leakage was not observed. The final obturation achieved hermetic sealing avoids fluid, microorganism and metabolic product exchanges between the root canal and periapical tissues. 3 The presence of a smear-layer deposition on root canal walls is a contentious topic, since its presence can modify marginal sealing, creating an empty space between the dentin wall and the obturation. 1,2,19 Mc- Comb and Smith 20 verified SEM that smear-layer Vol 7, No 1,

6 Publication Fig 5 Representative scanning electron micrograph of dentinal walls irradiated Nd:YAG laser. Original magnification 500X. Fig 6 Representative scanning electron micrograph of dentinal walls irradiated Er:YAG laser. Original magnification 500X. Fig 7 Representative scanning electron micrograph of dentinal walls irradiated Er:YAG laser associated with the Nd:YAG laser. Original magnification 500X. deposition can penetrate into the dentin tubules to an extent of 40 μm, and its removal would allow the dentin tubule entrances to be unobstructed. Furthermore, this procedure has been recommended because it prevents microorganisms in dentin tubules from producing gaps at the cement/wall interface which could lead to leakage and consequently colonization bacteria One of the recent methods removing smear layer is laser application. 19,25 It is already well established in the literature that the Er:YAG laser is effective in removing debris and smear layer from root canal walls. In the present study, samples treated with Er:YAG laser mostly presented clean areas in the apical region, whereas the control root canal walls predominantly exhibited areas covered smear layer. The results can be explained the absorption of the Er:YAG laser wavelength in water and hydroxyapatite. Further, after irrigation, Er:YAG laser specimens presented dentin walls of irregular shape and granular appearance, without the presence of debris and thus widely opened dentinal tubules. This result is in agreement with Paghdiwala et al, 26 who showed a clean and polished surface under moist conditions after Er:YAG laser irradiation with 70 mj and 6 Hz. Both studies indicate that the Er:YAG laser has a potential application in endodontic periapical procedures. In addition, researchers have shown that the zone of thermal damage and carbonization following Er:YAG laser application on soft tissues and bone is appreciably less compared with other lasers, and theree its use may result in improved healing and diminished postoperative discomt. Another potential benefit of Er:YAG laser irradiation is the elimination of microorganisms in the root canal system. One of the reasons we chose Er:YAG and Nd: YAG lasers the present study was that they have an antibacterial effect in vitro against several types of bacteria Bacterial elimination is possible under safe 50 The Journal of Oral Laser Applications

7 Publication Table 2 Thermal changes in irradiated teeth Group Technique Laser Irradiation Apical area Medial area Return to the initial temperature G2 helicoidal Nd:YAG 1st 3 C 1 C 2 min 2nd 6 C 1 C G3 vertical Nd:YAG 1st 2 C 4 C 2 min 2nd 5 C 4 C G4 helicoidal Er:YAG 1st 3 C 2 C 1 min 15 s 2nd 5 C 2 C G5 vertical Er:YAG 1st 2.6 C 2 C 1 min 19 s 2nd 4 C 2 C Fig 8 Thermographic image of tooth bee irradiation. ice that the initial temperature is 24 C. Fig 9 Thermographic image of irradiated tooth Nd:YAG laser, helicoidal technique. ice the initial temperature was 24 C and there was an increase of 1 C in the apical area after irradiation. parameters. Pashley 21 considered that a smear layer containing bacteria or bacterial products might provide a reservoir of irritants. Thus, complete removal of the smear layer would eliminate bacterial irritants from the root canal system. Laser irradiation of one specific wavelength produces different effects on the same tissue at different parameters, and the same laser wavelength can produce varying effects in different tissues. In the present study in the Nd:YAG groups, nankin ink was used in order to increase the surface energy absorption, resulting in the melting of dentin, yielding a morphological appearance of lava. Zhang et al 15 and Goya et al 16 used Nankin ink as a dye solution in a Nd:YAG laser technique in root canals and concluded that it is an effective means to seal walls, as it promoted melting and re-solidification. However, its application in clinical situations is restricted to posterior teeth because of the risk of dentin and enamel pigmentation. Some authors found a decrease in dentin permeabil- Fig 10 Thermographic image of irradiated tooth Nd:YAG laser, helicoidal technique. Observe that the initial temperature was 24 C and there was an increase of 6 C in the apical area. This observation is related to the higher temperature increase showed in the study. Temperature returned to baseline 2 min after the second irradiation. Vol 7, No 1,

8 ity in Nd:YAG laser irradiated root canals after obturation Comparative studies between Nd:YAG and other lasers showed that Nd:YAG laser irradiation was more effective in reducing apical leakage with black ink as an initiator with parameters of 2 W, 20 Hz, 4 s. In the present study, Nd:YAG laser irradiation after root canal preparation promoted a significant reduction in dentin permeability after blue methylene staining. This reduction can be explained the sealing of dentin tubules laser irradiation through the process of melting and re-crystallization. This was also observed in previous studies. 16,30 Nd:YAG laser irradiation is useful decreasing dentin hypersensitivity due to its ability to seal dentinal tubules. 31,32 When the Er:YAG laser was associated with the Nd:YAG laser, the objective was to clean and open the dentin tubules the ablation process and then close them the melting and re-solidification procedure. In the present study, photomicrographs showed that the dentin surfaces irradiated Er:YAG laser were clean, debris free, and without smear-layer mation. Thus, after Nd:YAG irradiation, all obturated teeth, irrespective of the association with Er:YAG laser, showed surfaces that were hermetically sealed, preventing stain penetration into the root canal. It could be observed that the surface presents an aspect of melting and recrystallization, characteristic of the Nd:YAG laser irradiation (Fig 4). Correlating it to the apical leakage, no infiltration the dye solution was observed. Clinically, Nd:YAG laser irradiation shows potential as an aid to apical healing in infected teeth and in reducing postoperative pain. Inamoto et al 33 found satisfactory results with regard to tissue healing in apical lesions irradiated Nd:YAG laser (150 mj, 10 Hz, 5 s) in rats. With regard to thermal changes on dental surfaces, it was found that the temperature increase on the external surface did not exceed 10 C. Furthermore, a higher thermal increase occurred when Nd:YAG laser was applied, with an increase of 5 C in the vertical technique and 6 C in the helicoidal technique. Machida et al 38 related a temperature increase of less than 2 C when Er:YAG laser was associated with a water spray root canal preparation. Kimura et al 39 found that 1 min of laser irradiation increased the temperature in the apical area less than 6 C and 3 C in the middle area. Bachmann et al 40 in the same year established that the helicoidal irradiation technique presented the lowest temperature increases (1 C to 3 C) compared to the stationary and mixed techniques. An increase of 6 C the Nd:YAG laser and 5 C the Er:YAG laser is not considered to be harmful to Publication the adjacent tissues. Bone tissue has a 10 C temperature increase survival limit over a period of 1 min of irradiation. 14 In a finite element study, Gutknecht et al 41 showed that thermal damage of the surrounding tissue can be avoided if treatment instructions including power settings and time are followed. It should be considered that there is a cumulative temperature increase resulting in a higher temperature with each irradiation. In the present study, a 20-s interval was enough specimens to return to their initial temperature. The key to success with the clinical use of laser in endodontics depends on energy delivery, irradiation time, pulse duration, focus area and type of the irradiated tissue. Considering that the results of this study did not show thermal increases above the maximum temperature supported periodontal tissue, it is possible to suggest in vivo research under the same irradiation conditions to assure that this technique is suitable clinical use. CONCLUSIONS Under the conditions of this study, it was possible to conclude that: a) Nd:YAG laser irradiation with or without adjunct Er:YAG laser was capable of reducing apical leakage; b) the use of helicoidal vs vertical technique did not influence the results; c) thermal increase with two types of laser irradiation was not significant; d) scanning electron microscopy confirmed the findings of the previous studies; e) both lasers and techniques, if used under correct protocols, are effective and do not cause injuries to adjacent tissues. ACKNOWLEDGMENTS The authors wish to express their gratitude to the Special Laboratory of Lasers in Dentistry (LELO) at the University of São Paulo (Brazil), to FAPESP the financial support, and Kavo the interchange accomplished with LELO. REFERENCES 1. Takeda FH, Harashima T, Kimura Y, Matsumoto K. Efficacy of Er:YAG laser irradiation in removing debris and smear layer on root canal walls. J Endod 1998;24: Ramalho KM, Marques MM, Apel C, Meneguzzo DT, Eduardo CP, Gutknecht N. Morphological analysis of root canal walls after Er:YAG and Nd:YAG laser irradiation: a preliminary SEM investigation. J Oral Laser Appl 2005;5: Stabholz A, Sahar-Helft S, Moshonov J. Laser in Endodontics. Dent Clin North Am 2004;48: The Journal of Oral Laser Applications

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Lineu Prestes, 2227 Cidade Universitária, Cep São Paulo SP, Brazil. Tel: , Fax: cpeduard@usp.br Vol 7, No 1,