Absorption and Thermal Study of Dental Enamel when Irradiated with Nd:YAG Laser with the Aim of Caries Prevention 1

ISSN 1054-660X, Laser Physics, 2009, Vol. 19, No. 7, pp. 1463 1469. Pleiades Publishing, Ltd., 2009. Original Russian Text Astro, Ltd., 2009. LASER METHODS IN CHEMISTRY, BIOLOGY, AND MEDICINE Absorption and Thermal Study of Dental Enamel when Irradiated with Nd:YAG Laser with the Aim of Caries Prevention 1 H. G. D. Boari a, P. A. Ana a, C. P. Eduardo b, G. L. Powell c, and D. M. Zezell a, * a Centro de Lasers e Aplicações IPEN/CNEN-SP, Av. Prof. Lineu Prestes, 2242, ZIP 05508-900, São Paulo, Brazil b Departamento de Dentística, FOUSP, Av. Prof. Lineu Prestes, 2229, ZIP 05508-000, São Paulo, Brazil c School of Medicine, University of Utah Health Sciences Center, 15 North Medical Drive East, ZIP 84132, Salt Lake City, Utah, USA *e-mail: zezell@usp.br Received February 9, 2009 Abstract It is widely recognized that Nd:YAG can increase enamel resistance to demineralization; however, the safe parameters and conditions that enable the application of Nd:YAG laser irradiation in vivo are still unknown. The aim of this study was to determine a dye as a photoabsorber for Nd:YAG laser and to verify in vitro a safe condition of Nd:YAG irradiation for caries prevention. Fifty-eight human teeth were selected. In a first morphological study, four dyes (waterproof India ink., iron oxide, caries indicator and coal paste) were tested before Nd:YAG laser irradiation, under two different irradiation conditions: 60 mj/pulse and 10 Hz (84.9 J/cm 2 ); 80 mj/pulse and 10 Hz (113.1 J/cm 2 ). In a second study, the enamel surface and pulp chamber temperatures were evaluated during laser irradiations. All dyes produced enamel surface melting, with the exception of the caries indicator, and coal paste was the only dye that could be completely removed. All irradiation conditions produced temperature increases of up to 615.08 C on the enamel surface. Nd:YAG laser irradiation at 60 mj/pulse, 10 Hz and 84.9 J/cm 2 promoted no harmful temperature increase in the pulp chamber (ANOVA, p < 0.05). Among all dyes tested, the coal paste was an efficient photoabsorber for Nd:YAG irradiation, considered feasible for clinical practice. Nd:YAG laser at 84.9 J/cm 2 can be indicated as a safe parameter for use in caries prevention. PACS numbers: 81.40.-z, 87.15.-v, 87.63.Hg, 87.64.Ee DOI: 10.1134/S1054660X09070160 1 INTRODUCTION Laser irradiation is a technology that is widely used in dentistry. Since the development of the first Ruby laser by Maiman in 1962, lasers were severely tested to improve the traditional procedures used in dentistry, mainly with respect to caries removal [1] and detection [2], cavity preparation [3, 4], adhesion [3], root canal treatment [5], periodontics [6], caries prevention [7 14], surgeries [15] and other applications. Nd:YAG lasers have been suggested as a potential tool for caries prevention because of the heating effects on enamel surface [12, 13, 16 18], speculated as being the main factor in inducing crystallographic and compositional changes that favor an increase in enamel resistance to subsurface demineralization in vitro [19, 20]. However, as opposed to what happens with pigmented tissues, the wavelength of Nd:YAG laser (λ = 1064 nm) is not effectively absorbed by dental enamel (absorption coefficient is less than 4 10 2 cm 1 ) [20, 21]; therefore, the use of photoabsorbers before Nd:YAG laser irradiation on dental hard tissues is reported [22,23]. The aim of applying a dark dye before laser irradiation is to increase the absorption of the laser beam at 1 The article is published in the original. the surface of the enamel. In this case, the heat produced due to laser absorption by the coating material is transmitted into the adjacent enamel. This technique certifies the deposit of a short laser pulse energy to a small volume of tissue, avoiding excessive penetration of the laser beam into deeper dental structures, and consequently, less risk of damage to dental pulp. The waterproof India ink is the dye most used as a photoabsorber for Nd:YAG pulsed laser [22 26]. However, the esthetics of treated teeth can be harmed because of it is difficult to remove, particularly from pits and fissures. A dye for application before Nd:YAG laser irradiation should have the following characteristics: biocompatibility, low surface tension, be easy to remove and help to promote enamel surface melting. Enamel surface melting requires heating up to 1200 C [17, 19, 20]. When this temperature range is reached, it is speculated that several chemical changes have occurred [19, 20] in the enamel microstructure, which can favor the clinical use of Nd:YAG lasers for preventive purposes. Conversely, with the purpose of choosing an irradiation condition for the purpose of clinical applications, the fluences used must be safe for ensuring the vitality of the pulp and periodontal tissues [27]. Increases of over 5.6 C can be considered potentially aggressive to the vitality of the pulp and temper- 1463

1464 BOARI et al. Cumulative value, arb. units 16 14 12 10 8 6 4 2 5 10 25 50 75 90 95 99 99.9 Particle diameter, mm Fig. 1. Distribution of triturated coal particles (%) in the coal paste. atures higher than 16 C can result in complete pulp necrosis [28]. Although there are several in vitro [3, 5 7, 12, 22, 23, 29 31] and in vivo [1, 13, 15, 24 26] studies that have applied Nd:YAG laser on enamel surfaces, there are few that determine the temperature rises during laser irradiation on the enamel surface and in pulp. Therefore, this in vitro study had two objectives: to choose a suitable dye for clinical application before Nd:YAG laser irradiation on enamel, and to determine the irradiation condition that lead to increase in pulp temperature that may be considered safe during Nd:YAG irradiation in vivo for caries prevention. 1. MATERIAL AND METHODS The Nd:YAG laser (Pulse Master 1000 ADT USA) used in this work emits pulses at 1.064 µm, with a 300 µm quartz fiber optic delivery system operating in contact mode (spot size of 300 µm), temporal width of 100 µs and operates at 10 100 repetition rate. Irradiation was performed perpendicularly to the sample with the fiber end in contact with the surface. The irradiation conditions and groups varied according to the experiments of this study. Fifty-eight human teeth were obtained from the Human Teeth Bank of the University of São Paulo after approval from the Human Ethics Committee of the Nuclear and Energy Research Institute. 1.1. First Experiment: Dye Selection Twenty-four human molar and twenty-four maxillary incisor teeth were selected for this experiment. After extraction, the teeth were cleaned and stored in physiological solution at room temperature for no longer than 1 month. Teeth with caries lesions or enamel structure cracks were excluded. The teeth were randomly divided into four groups, according to the biocompatible dyes selected for this experiment: Group 1DS: waterproof India ink. (control group); Group 2DS: black ink used as make up eyes, as eye liner (iron oxide); Group 3DS: caries indicator (Seek, Ultradent, USA); Group 4DS: coal paste. The coal paste was composed of coal triturated in a porcelain mortar for 10 min, resulting in particles 10 µm in diameter, diluted in equal parts of deionized water and 99% ethanol. This mix formed a paste of a fluid consistency that allowed it to be applied with a thin brush. Figure 1 shows the distribution of triturated coal particles. All dyes used in this experiment were applied with a brush in a layer thickness of approximately 100 µm on both smooth enamel of incisor teeth and occlusal (pits and fissures) molar surfaces. After dye application, the teeth were irradiated with Nd:YAG laser under two different conditions: 60 mj per pulse and 10 Hz repetition rate (84.9 J/cm 2 ); 80 mj per pulse and 10 Hz repetition rate (113.1 J/cm 2 ). Laser irradiation was performed by a single operator, scanning the entire enamel area three times, and dyes were reapplied before each laser scanning. After laser irradiation, the smooth enamel samples were cleaned by ultrasonic bath and were fixed in a 2% glutaraldehyde solution for 2 h. The samples were immediately perfused with a phosphate buffered solution 0.1 M at room temperature, rinsed with distilled water and then dehydrated in a graded series of alcohol solutions (50, 70, 80, 90, 95, and 100%) for 10 min at each concentration. These samples were sputtered with a 15 µm thick gold layer and submitted to scanning electron microscopy analysis (Phillips XI, Eindhover, Holland). The molar samples were analyzed by visual inspection to observe the difficulty of removing the dark pigment. 1.2. Second Experiment: Temperature Evaluation During laser irradiation, the surface temperatures of enamel were recorded using a thermographic camera and the pulp temperature was measured using thermocouples. For this experiment, ten human third molar teeth were selected, cleaned and stored in physiological solution at room temperature. The residual pulp tissue was removed from these teeth using endodontic type K-files introduced through apical access.

ABSORPTION AND THERMAL STUDY OF DENTAL ENAMEL 1465 Results of macroscopic and microscopic analysis for dye selection Dyes Enamel India Ink. Make up Caries indicator Coal paste Melting and resolidification Well defined Well defined Without characteristics Well defined of enamel at SEM analysis Presence of traces at visual inspection Present Present Present Absent After the analysis of the dye selection experiment, the coal paste was applied before laser irradiation in this experiment. The coal paste was prepared as before. The teeth selected for the temperature evaluation experiment were randomly divided into two groups, according to the laser irradiation conditions: Group 1TE: laser irradiation at 60 mj per pulse and 10 Hz repetition rate (84.9 J/cm 2 ); Group 2TE: laser irradiation at 80 mj per pulse and 10 Hz repetition rate (113.1 J/cm 2 ). At the time of the experiment, the root canals of all specimens were filled with a thermally-conductive paste (thermal conductivity of 1.67 W/m K) and a calibrated K-type thermocouple (chrome-alumel Omega Engineering, Stamford, CT), with a 0.05 mm diameter probe and sensitive to temperature variations between 0.1 C and 100.0 C assembly, was introduced into the pulp chamber. The thermal conductivity of soft tissue is comparable to that of water (0.58 W/m K). Thermal paste with higher thermal conductivity than that of water was used to assure more efficient heat transmission to the thermocouple. The temperature sensitive end of the probe was placed at the closest distance to the area to be irradiated, and its location was controlled radiographically. The thermocouple apparatus was connected via an analogue-to-digital converter linked to a computer, and time and temperature data were recorded at a sampling rate of 20 Hz, with a temperature resolution of 0.1 C. Samples were fixed and immersed in a waterfilled heating circulator at a standardized temperature of 37 C, with only the coronal part of the tooth not being submerged. The temperature changes on the enamel surface during and immediately after laser irradiation were also measured using a thermographic camera (ThermaCam FLIR SC3000 Systems, Sweden), which stores infrared images and data at rates of up to 900 Hz. This experiment was performed at a room temperature of 25 C, 47% relative humidity of the air and considering teeth emissivity of 0.91. The thermographic camera was positioned at a distance of 0.1 m from the samples, and the laser handpiece was positioned in contact mode with the enamel surface. The area of interest was isolated at a focal length of 0.1 m using an internal macro lens. The infrared images obtained were recorded at rates of 60 and 900 Hz for subsequent analysis. After the application of coal paste on the occlusal surface of samples, as performed in experiment 1, laser irradiation was performed by a single operator, scanning the entire enamel area three times, and the coal was reapplied before each laser scanning. Laser irradiation and thermal recordings were synchronized and thermal recording was terminated thirty seconds after the end of the laser irradiation. The measurements recorded by thermographic camera and thermocouples were plotted against time for each group; data were analyzed by ANOVA, and when p < 0.05, the difference among the treatments was assessed by Tukey s test. 2. RESULTS 2.1. Dye Selection During visual inspection, it was observed that the caries indicator was the dye that was the most difficult to remove on both smooth and occlusal surfaces. The waterproof India ink was also difficult to remove from occlusal surfaces. However, the coal paste was the dye that was easily removed either from smooth or occlusal surfaces. The result of the dye selection study is summarized in table. When waterproof India ink., dark make-up eyeliner ink and coal paste was applied to the groups, the scanning electron microscopy analysis showed irregularities and depressions, characteristics of melting of the enamel, with zones of fusion and resolidification in a mosaic pattern. In samples that were covered with caries indicator, a small degree of melting and zones with smoother surfaces than those produced with the other dyes, similar to the appearance of sound enamel were observed. These results can be seen in Fig. 2. 2.2. Temperature Evaluation Figure 3 shows the difference in temperature in the pulp chamber when samples were irradiated at different energies. At the energy of 60 mj per pulse (84.9 J/cm 2 ), a minimal effect on the pulp temperature was noted and the temperature increase was approximately 1.0 C. The highest rise was noted when samples were irradiated with 80 mj energy per pulse

1466 BOARI et al. (a) 10 µm 10 µm (b) (c) 10 µm (d) 10 µm (e) 10 µm Fig. 2. Scanning electron microscopy of enamel after irradiation with Nd:YAG laser at 60 mj per pulse (84.9 J/cm 2 ) without any dye (a), caries indicator (b), coal paste (c), dark ink for makeup (d), and coated with waterproof India ink, and (e) original magnification is 3500. (113.1 J/cm 2 ), when an increase of 5.0 C was recorded. A maximum surface temperature peak of 615.08 C was recorded during Nd:YAG laser irradiation at 60 mj and 10 Hz (84.9 mj/cm 2 ). The average rise recorded by the thermographic camera was 313.90 C at all parameters tested in this experiment. The temperature peaks were coincident with the laser interaction with the coal particles, which caused some microexplosions that corresponded to ejection of the coal paste. The remaining coal paste was removed after laser irradiation. When analyzing the graphs of surface and pulp temperatures, a delay of approximately 1 second between the increase in surface temperature and any detectable increase in pulp temperature can be seen. This can be attributed to the thermal diffusibility of the teeth and the dissipation of the surface heat. When irradiation stopped, the surface temperature decreased rapidly and returned to baseline temperatures. This did not happen with the pulp temperature, which needed more than 30 s to return to its baseline. 3. DISCUSSION It is known in the literature that Nd:YAG laser has a large potential to prevent dental caries, since this wavelength can alter the permeability, crystallinity and acid-solubility of enamel, promoting an increase in its resistance to demineralization [7, 31]. Because Nd:YAG laser has high absorption in black pigment and poor absorption in both water and hydroxyapatite, the application of a photoabsorber is used in order to enhance tissue absorption in the nearinfrared range for ablation and prevention actions in dental tissues. Without a photoabsorber, Nd:YAG laser would not be completely absorbed on the enamel surface, and an amount of energy would be reflected,

ABSORPTION AND THERMAL STUDY OF DENTAL ENAMEL 1467 Temperature, C 6 5 4 3 2 1 0 84.9 J/cm 2 113.1 J/cm 2 Energy density, J/cm 2 Fig. 3. Maximum temperature rises in pulp chamber during Nd:YAG laser irradiation at 60 mj per pulse (84.9 J/cm 2 ) and 80 mj per pulse (84.9 J/cm 2 ). scattered or transmitted to dentin, which could compromise pulp vitality [7, 22]. Although a large number of dyes have been tested as photoabsorber for Nd:YAG laser action in enamel, waterproof India ink was shown to be the appropriated dye for application to enamel surfaces before Nd:YAG pulsed laser irradiation [22 26]. However, this ink can compromise the appearance of teeth because of leaving traces, which makes it unsuitable esthetically when laser is used with for the purpose of caries prevention. Bahar et al. [32] suggested removal of the residual ink with 99% ethanol. Our experience showed that even with this procedure, detritus of the ink can stay on the occlusal surfaces for weeks, mainly in pits and fissures. The wettability of this ink favors its penetration into dental pits, in which the base is bigger than the entrance, favoring its permanence in this area. Considering the results obtained in the first experiment of the present study, a suitable result was obtained applying the coal paste followed by laser energy of 60 mj per pulse and 10 Hz (84.9 J/cm 2 ), repeating the procedure of applying dye followed by laser irradiation three times. This technique guaranteed melting and recrystallization of enamel surfaces, and at the same time, the coal was quickly and easily removed from occlusal surfaces [27, 29]. According to the Hess [23] study, the application of Nd:YAG laser at an energy output of 30 or 70 mj per pulse on enamel without a dye was unable to produce morphological changes; however, when a black initiator was applied, the same fluence promoted a conditioning of the surface. In the present study, the laser energy of 60 and 80 mj per pulse after the application of India ink produced melting and recrystallization of the enamel and these morphological findings indicate a high absorption of laser irradiation on the surface. The morphological findings of coal paste are similar to those obtained when waterproof India ink was applied, which guarantees the efficiency and feasibility of this new dye. For preventing caries, it has been suggested that laser irradiation should produce alterations in tooth hard tissues, which increase the resistance to demineralization [7, 11, 14, 31, 33]. Indeed, under specific conditions, physical, morphological and chemical changes that occur in irradiated enamel improve the superficial hardness and produce more resistant surfaces. Increased resistance to demineralization has been reported to be due to the decreased solubility of enamel resulting from an alteration in the composition of the mineral phase, such as the loss of organic matter and carbonate [11]. Besides that, changes on Ca/P ration are reported on irradiated surfaces [14]. A variety of mechanisms have been suggested for the acid-resistance induced in enamel treated with laser irradiation: (1) the melting, fusion and recrystallization fusion of enamel surface could decrease the enamel permeability [34]; (2) changes in ultra-structure of enamel such as, the reduction of water and carbonate contents, the increase in the hydroxyl ion contents, formation of pyrophosphates and the decomposition of proteins, promote a decrease in solubility of enamel [11, 16, 19, 20]. These crystallographic changes are produced by heating the enamel with laser energy at temperatures ranging from 100 to 650 C. In temperature ranges from 650 to 1100 C, products that favor a decrease in solubility are formed in the enamel, which was evidenced in previous studies [35, 36]. At 1100 C, all carbonate is eliminated and there is formation of new crystalline phases (α-tcp and β-tcp phases), which are less resistant to demineralization [36, 37]. In fact, these temperature increments can influence procedures such as adhesion of resins [3] and ablation [4]. Hydroxyapatite crystals melt at a temperature close to 1200 C [37], temperature that can be reached during irradiations with infrared or femtosecond lasers [4]. Therefore, when Nd:YAG laser promotes enamel melting and recrystallization, it was supposed that this temperature was reached during laser irradiation, which confirms the second mechanism of inducing resistance to demineralization. In experiment 2 of this present study, however, maximum temperature increases of 615.1 C were recorded at the enamel surface, which is in disagreement with the above discussion. Considering that the pulse length of Nd:YAG laser is 100 µs, the 900 Hz recording rate of thermographic camera seems to be unable to detect the highest temperature peaks. One of the main concerns regarding the use of lasers for caries prevention is heat generation on the pulp. The observations of experiment 2 indicate that the critical threshold of 5.6 C, reported as the temperature that starts irreversible pulp inflammation [28], can be exceeded when Nd:YAG is used at 80 mj

1468 BOARI et al. energy per pulse (113.1 J/cm 2 ). On the other hand, Nd:YAG laser irradiation at 60 mj per pulse (84.9 J/cm 2 ) promotes an increase of 1 C in pulp temperature, being considered safe to pulp vitality. These results agree with the literature information that most of the lasers systems promote an increase in pulp temperature dependent on the power setting [3, 5, 6, 27, 38]. It is important to point out that dentin thickness has a considerable effect on heat generation in the pulp. As reported by White et al. [39], Nd:YAG laser irradiated with a power output of 0.7 W (approximately 87 J/cm 2 ) induces an increase of 43.2 C in a remaining dentin thickness of 0.2 mm and induces an increment of 5.8 C in a dentin thickness of 2.0 mm. In the present study, laser irradiation with a power output of 0.6 W (84.9 J/cm 2 ) promoted an increase of 1 C on the occlusal surface of molar teeth, where the enamel presented a thickness of 1.5 mm and the dentin had an approximate thickness of 3.0 mm. Another important point to consider is that in the present study, non-carious teeth were used, and because of the large amount of water in carious lesions, there could be more excessive heat transfer to the pulp in decayed teeth. Taking into account the poor thermal conductivity of dentin and considering that human teeth present a large variation in volume and weight, the clinician must evaluate the physical conditions of dental hard tissue in order to suit the exposition time to avoid an unsafe thermal effect on soft tissues. The laser parameters used in the present study were based on other in vivo studies [1, 24 26] in the literature, and the results support the histological evidences [40, 41]. 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