Optical properties of bovine dentin when irradiated by Nd:YAG and a black dentifrice aimed at treating dentin erosion Daísa L. Pereira 1, Matheus Del Valle 2, Gabriela V. Gomes 1, Denise M. Zezell 1, Patricia A. Ana 2 1 Center for Lasers and Applications, IPEN-CNEN/SP, Av Prof. Lineu Prestes, 2242, Sao Paulo, Brazil 2 Center for Engineering, Modeling and Applied Social Sciences, Federal University of ABC (UFABC), Av. da Universidade s/n, Sao Bernardo do Campo, Brazil patricia.ana@ufabc.edu.br Abstract. Dental erosion has been extensively studied as a risk factor for tooth loss or injure, and the early diagnosis of lesions is essential for avoiding greater damages. Optical Coherence Tomography (OCT) is a potential tool for early diagnosis of demineralization. In this study, this technique was used to analyze the optical changes of dentin samples irradiated with Nd:YAG laser using a black dentifrice as photoabsorber, then submitted to an erosive cycling. 75 slabs of bovine root dentin were randomized into 5 groups: G1 untreated; G2 treated with acidulated phosphate fluoride gel (APF-gel, [F - ]=1.23%, ph=3.3 to 3.9); G3- irradiated with Nd:YAG laser (100µs, 1064nm, 0.6W, 10Hz) without photoabsorber; G4 irradiated with Nd:YAG laser using a coal paste as photoabsorber; G5 irradiated with Nd:YAG laser using a black dentifrice as photoabsorber. All samples were submitted to a 3-day erosive demineralization (Citric acid 1%, ph=3.6, 5min, 2x/day) under agitation, and remineralization (artificial saliva, ph=7, 120min) cycling. The samples were evaluated by OCT before treatments (baseline), after treatments and after erosive cycling. Optical attenuation coefficient (µ) was calculated using a Matlab routine, and the statistical analysis was performed ( =0.05). It was observed a significant decrease on µ values after all treatments. Also, the µ values decreased after erosive cycling, except for the groups G3 and G5. It was concluded that OCT technique is capable to distinguish among sound, treated and demineralized dentin. As well, the black paste was efficient to act as a photoabsorber, helping the Nd:YAG laser to decrease dentin erosion. Keywords: Optical coherence tomography, infrared laser, tooth erosion.
2 1 Introduction Dental erosion is defined as an irreversible loss of tooth enamel and/or dentin caused by extrinsic or intrinsic acids, in the absence of biofilm. It is an oral demineralization process whose prevalence has increased significantly in the last few decades. The etiology of the erosive process is multifactorial, and the follow-up of the patient is fundamental to prevent the wear development and progression. Infrared pulsed lasers are described as a successful method for caries and erosion prevention [1] due to the chemical, morphological and crystallographic changes resulting from heating. However, substances that act as photoabsorber are necessary when Nd:YAG laser is used in dental hard tissues due to the low absorption of 1064 nm photons by these tissues, aiming to decrease the risk of heat injury in deeper tissues [2]. Nowadays, the coal paste is the most used photoabsorber during Nd:YAG laser irradiation; however, it has the disadvantage of staining the tissues. In this way, the search for new biocompatible substances that act as a photoabsorber is still necessary. The Optical Coherence Tomography (OCT) is a non-destructive and non-invasive technique which provides transversal images of biological structures, called B-scans, contactless with the sample, with no use of ionizing radiation, and allows its clinical use without any side effects to the patient. A near-infrared (NIR) light source is used to shine the sample, providing a penetration of units of millimeters. The light is scattered through the sample and the backscattered light is detected by the OCT equipment; in this way, OCT imaging provides optical information of tissues that can be related with their composition and microstructure. The optical attenuation coefficient ( ) of the light is obtained by OCT and is related to the characteristics of the tissues [3], such as the loss of water and demineralization. In this way, this study aims to analyze the optical attenuation coefficient from the OCT signal in bovine dentin to distinguish sound, treated and demineralized tissue, as well as to verify the efficacy of a new photoabsorber on the effects of Nd:YAG laser on dentin demineralization. 2 Materials and Methods After the approval from the Animal Ethics Committee of the Instituto de Pesquisas Energeticas e Nucleares (CEUA-IPEN 149/2014), 75 bovine root dentin slabs (5 x 5 x 2 mm) were obtained, polished and cleaned with deionized water in ultrasonic bath for 10 min. The samples were randomized in five experimental groups (n=15): Group 1- Untreated (control); Group 2 Treated with acidulated phosphate fluoride gel (APF-gel); Group 3 irradiated with Nd:YAG laser (1064nm, 100µs, 0.6W, 10Hz, 60mJ, 84.9J/cm 2 ) without photoabsorber; Group 4 irradiated with Nd:YAG laser using a coal paste as photoabsorber; Group 5 irradiated with Nd:YAG laser using a black dentifrice (Black is White, Curaprox ) as photoabsorber. The APF-gel ([F - ]=1.23%, ph=3.3 to 3.9 -Flutop Gel, SSWhite, Rio de Janeiro, Brazil) was applied to the dentin slabs with a cotton swab for 4 min, followed by washing with distilled and deionized water during 1 min and dried with absorbent paper. The laser irradiation was performed using a Nd:YAG laser device (Power Laser TM ST6, Lares Research, Chico, CA, USA), which operates at wavelength of 1064 nm and temporal width of
3 100 μs. The energy is delivered through a fiber optic system with 300 μm of spot size. Nd:YAG laser was used to irradiate all slabs surfaces in contact mode using a highprecision motorized translator (ESP300, Newport Corporation). Before irradiation, the energy per pulse was calibrated by an energy/power meter (FieldMaster, Coherent, Santa Clara, CA, USA). After treatments, all samples were submitted to a 3-day erosive demineralization (Citric acid 1%, ph=3.6, 5min, 2x/day) under agitation, and remineralization (artificial saliva, ph=7, 120min) cycling, in order to create an initial erosion lesion. Before treatments (baseline), after treatments and after erosive challenge, the measurement of the optical attenuation coefficient was performed using an optical coherence tomography system (Callisto, Thorlabs Inc., Newton, NJ, USA), central wavelength of 930 nm, pixel resolution pixel resolution of 1.52 µm x 3.34 µm, lateral resolution of 8 µm and axial resolution of 7 µm. The tomographic images were obtained from 3 previously demarcated scanning lines (3 B-scans) located in the center of each sample. To calculate the optical attenuation coefficient ( ), it was developed a routine in MATLAB software (MathWorks, EUA), in which a region of interest (ROI) was isolated. Then, each depth line (A-scan) from the B-scan was analyzed, where the intensity of the decay of the signal was modeled using an exponential fitting, according to the Beer-Lambert like equation presented in Equation 1 [4]. ( ) ( ) ( ) ( ) is the intensity at the depth and is the initial intensity. The number two multiplying the exponential parameters differs from the original Beer-Lambert equation due to the incident (light source) and returning (backscattered) signal path. The of the image was obtained by the mean of all calculated in each A-scan. The statistical analysis was performed, considering the level of significance of 5%. For untreated group, t-paired test was performed and for the other groups ANOVA+Tukey were performed. 3 Results Figure 1 shows the average of optical attenuation coefficient obtained for all experimental groups of this study. In untreated, treated with APF-gel, treated with laser and treated with laser and coal paste groups, it was observed that optical attenuation coefficients after erosion cycling were significantly lower (p<0.05) than the baseline values. Optical attenuation coefficients after cycling in Group laser with dentifrice showed no statistically significant difference when compared to the untreated samples. However, only in the group treated with laser with dentifrice, the values obtained after erosion lesions were significantly higher (p<0.05) than the immediately treated samples.
4 Fig. 1. Average of optical coefficient attenuation values of all experimental groups. Bars denote standard errors. Distinct letters indicate statistically significant differences (p<0.05) according to the appropriate comparison test. 4 Discussion Although the Nd:YAG laser is not absorbed by the main chromophores of dental hard tissues (water and hydroxyapatite) [5], it is a laser with wide application to prevent the onset of caries and erosion lesions [6]. The coal paste is the main photoabsorber applied before Nd:YAG laser irradiation, but another substances such as Indian ink were tested [7]. However, the search for a photoabsorber that is efficient, biocompatible, easy to apply and do not stain the tissues is still necessary. The black dentifrice used in this study seems to have all these characteristics; in addition, it has 1450 ppm fluoride in its composition, which would be a major benefit for patients at high caries risk. Previous studies performed by our group suggest that this black dentifrice can be used as a photoabsorber to increase the absorption of Nd:YAG photons by enamel and dentin surface, since dentin treated with black dentifrice + Nd:YAG laser presented higher microhardness values after erosive challenge when compared to untreated dentin, as well as decreased the proportion of calcium to phosphate and promoted melting and recrystallization of dentin surface, in a similar way than the promoted by coal paste + Nd:YAG laser irradiation [8]. The parameter of optical attenuation coefficient (µ) is an optical measurement that can be calculated trough the OCT images and it can be related to the identification of sound and demineralized tissues. Decrease in optical attenuation coefficient values can indicate that the demineralization promoted by acids, such as the citric acid used in this study, creates empty spaces in the structure of dentin that increases the number of interfaces and, as a consequence, increases the scattering of light and decreases the values of optical attenuation coefficients [4]. However, there is a discrepancy on liter-
5 ature concerning the interpretation of optical attenuation coefficient values related to demineralization, since other authors relate that the optical attenuation rises with the increase of intercrystalline spaces in the dental hard tissue structure due to the demineralization [3]. In the present study, it was evidenced that the optical attenuation coefficient values significantly decreased after erosive cycling, which agrees with the reported by Maia et al. (2015) [4] and Sowa et al. (2011) [9]. In this way, the optical coefficient attenuation can distinguish healthy from the eroded dentin, as it was reported by Pereira et al. (2018) [10]. It was also evidenced that the APF-gel application and Nd:YAG laser irradiation, with or without any photoabsorber, significantly decreased the optical attenuation coefficient values, which indicates that the treatments alter the optical characteristics of dentin. However, these changes are not related to tissue demineralization, but to some biochemical change, such as water content. After erosive cycling, the APF-gel and laser with coal paste treated groups did not prevent dentin demineralization, since it was observed the decrease in the optical attenuation coefficient after erosive cycling. However, the group treated with laser irradiation without photoabsorber presented similar optical attenuation coefficient values after cycling when compared to values before cycling. This finding suggests that there was no progress of demineralization in this group, probably due to the thermal effect of laser irradiation [2]. The group treated with black dentifrice and laser irradiation showed significantly higher values of optical attenuation coefficient, similar to those presented by healthy dentin, which suggests a greater protective activity. Probably, this phenomenon refers to the thermal action of the laser irradiation [2], potentiated by the photoabsorber itself, in addition to the presence of fluoride in the dentifrice, whose synergistic action protected the dentine from the erosive challenge. It is important to notice that the erosive challenge employed in this study lasted for a short period of time, and that studies with longer erosive challenges are necessary to evaluate the durability of the treatment. Also, it was evidenced the potential of OCT technique to identify the optical changes on dentin due to APF-gel application and Nd:YAG laser irradiation, as well as the ability to monitor the lesions and to quantify the effect of the proposed treatments in a non-invasive, high resolution and real time manner. 5 Conclusion According to the results obtained in this study, it was possible to conclude that the OCT technique is able to quantify dentin erosion lesions, as well as to identify the treatments performed on dentin. As well, it was evidenced the potential use a dentifrice to potentialize the effect of Nd:YAG laser for preventing dentin erosion.
6 6 Acknowledgements The authors would like to thank to PROCAD-CAPES (88881.068505/2014-01), National Institute of Photonics (CNPq/INCT 465763/2014-6), CAPES for the scholarship and Multiuser Central Facilities (UFABC) for the experimental support. References 1. Lussi, A. et al.: Dental erosion An overview with emphasis on chemical and histopathological aspects. Caries Res. 45, S2 S12 (2011). 2. Boari, H.G.D. et al.: Absorption and thermal study of dental enamel when irradiated with Nd:YAG laser with the aim of caries prevention. Laser Phys. 19, 1 7 (2009). 3. Cara A.C. et al.: Evaluation of two quantitative analysis methods of optical coherence tomography for detection of enamel demineralization and comparison with microhardness. Lasers Surg Med. 46(9), 666-71 (2014). 4. Maia, A.M.A. et al: Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography. J. Biophotonics 9(6), 1 7 (2015). 5. Seka, W. et al.: Light deposition in dental hard tissue and simulated thermal response. J. Dent. Res. 74(4), 1086 1092 (1995). 6. Zezell, D.M. et al.: Nd:YAG laser in caries prevention: a clinical trial. Lasers Surg Med. 41(1), 31-5 (2009). 7. E. Jennett, M. et al.: Dye-enhanced ablation of enamel by pulsed lasers. Dent. Res. 73, 1841 (1994). 8. Pereira, D.L. et al.: New Photoabsorber for Nd:YAG Laser Irradiation of Dental Hard Tissues to Prevent Erosion. In: Proceeding of the 6 th European Division Congress of the World Federation for Lasers in Dentistry, Thessaloniki, Greece (2017). 9. Sowa, M.G, et al.: A comparison of methods using optical coherence tomography to detect demineralized regions in teeth. J Biophotonics 4, 814 823 (2011). 10. Pereira, D.L. et al.: Variation on Molecular Structure, Crystallinity, and Optical Properties of Dentin Due to Nd:YAG Laser and Fluoride Aimed at Tooth Erosion Prevention. Int. J. Mol. Sci. 19(2), (2018).