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Photodiagnosis and Photodynamic Therapy 13 (2016) 114–119 Contents lists available at ScienceDirect Photodiagnosis and Photodynamic Therapy journal homepage: www.elsevier.com/locate/pdpdt Differences in the intensity of light-induced fluorescence emitted by resin composites Bo-Ra Kim, Si-Mook Kang, Gyung-Min Kim, Baek-Il Kim ∗ Department of Preventive Dentistry & Public Oral Health, BK21 PLUS Project, Oral Science Research Institute, Yonsei University College of Dentistry, 50-1 Yonsei-ro, Seodaemun-Gu, Seoul 03722, Republic of Korea a r t i c l e i n f o Article history: Received 24 September 2015 Received in revised form 11 December 2015 Accepted 7 January 2016 Available online 12 January 2016 Keywords: Detection Fluorescence Resin composites Quantitative light-induced fluorescence technology a b s t r a c t Background: The aims of this study were to compare the intensities of fluorescence emitted by different resin composites as detected using quantitative light-induced fluorescence (QLF) technology, and to compare the fluorescence intensity contrast with the color contrast between a restored composite and the adjacent region of the tooth. Methods: Six brands of light-cured resin composites (shade A2) were investigated. The composites were used to prepare composite discs, and fill holes that had been prepared in extracted human teeth. Whitelight and fluorescence images of all specimens were obtained using a fluorescence camera based on QLF technology (QLF-D) and converted into 8-bit grayscale images. The fluorescence intensity of the discs as well as the fluorescence intensity contrast and the color contrast between the composite restoration and adjacent tooth region were calculated as grayscale levels. Results: The grayscale levels for the composite discs differed significantly with the brand (p < 0.001): DenFil (10.84 ± 0.35, mean ± SD), Filtek Z350 (58.28 ± 1.37), Premisa (156.94 ± 1.58), Grandio (177.20 ± 0.81), Charisma (207.05 ± 0.77), and Gradia direct posterior (211.52 ± 1.66). The difference in grayscale levels between a resin restoration and the adjacent tooth was significantly greater in fluorescence images for each brand than in white-light images, except for the Filtek Z350 (p < 0.05). However, the Filtek Z350 restoration was distinguishable from the adjacent tooth in a fluorescence image. Conclusions: The intensities of fluorescence detected from the resin composites varied. The differences between the composite and adjacent tooth were greater for the fluorescence intensity contrast than for the colors observed in the white-light images. © 2016 Elsevier B.V. All rights reserved. 1. Introduction The commercially available resin composite material used in dentistry now have color and translucency properties that are close to those of human teeth. The use of a layering technique using enamel and dentin shading, and the development in dental adhesives technology have also contributed to improvements in the esthetics of resin restorations [1]. These technical developments mean that well-placed tooth-colored resin restorations can now fulfill the esthetic demands of patients. However, this also means that detecting resin restorations can be a challenging task for dental professionals during oral examinations. ∗ Corresponding author at: Department of Preventive Dentistry & Public Oral Health, Yonsei University College of Dentistry, 50-1 Yonsei-Ro, Seodaemun-Gu, Seoul 03722, Republic of Korea. Fax: +82 2 392 2926. E-mail addresses: [email protected] (B.-R. Kim), [email protected] (S.-M. Kang), [email protected] (G.-M. Kim), [email protected] (B.-I. Kim). http://dx.doi.org/10.1016/j.pdpdt.2016.01.005 1572-1000/© 2016 Elsevier B.V. All rights reserved. The ability to accurately detect a tooth-colored resin restoration can affect the repair procedure applied to a damaged restoration and the removal of excessive materials in a clinical situation, as well as the results of mass dental examinations such as in forensic analysis and epidemiological studies [2–5]. It is well established that the presence of a resin restoration can been confirmed by visual and tactile inspections with air-drying during a general oral examination [6]. However, since this method is influenced by subjective decisions made by the examiner, the diagnosis accuracy can vary with the experience of the examiner. Extra radiography investigations can be used to compensate for this limitation by supporting the detection of radiopaque resin materials and pathologic alteration of the tooth around the restoration [7]. However, it is still difficult to discriminate some resin materials and teeth due to recommendations to use resin composite with similar or higher radiopacity than the tooth tissue when evaluating small defects or caries [8]. In addition, radiography investigations expose the patient to the potentially detrimental effects of ionizing radiation, B.-R. Kim et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 114–119 given that it has been reported that the risk of meningioma may be elevated among those with frequent exposure to dental X-rays and younger age groups [9]. Therefore, the development of an alternative method that is noninvasive and intuitive would be valuable to clinicians for use in dental inspections. Differences in the intensity and wavelength of the fluorescence emitted from teeth and resin restorations illuminated by certain wavelengths of light have recently been reported [3,5,10–12]. It is known that such differences are attributable to the different chemical compositions of teeth and resin composites [4,13–15]. Teeth exhibit bluish autofluorescence when illuminated by ultraviolet (UV), and its intensity is believed to be most affected by fluorophores in organic components of dentin, whose fluorescence is greater than that of enamel [13]. The visible fluorescence intensity of a tooth may be affected by the layering of dentin and enamel [14], while the visible fluorescence of a resin composite is induced by luminescent elements, such as europium, ytterbium, or other rare-earth elements. These additives are included in glass fillers to improve the appearance of the resin [4,15], and their presence results in the excitation and emissions appearing within different wavelength ranges. A method that utilizes this property could improve visual inspections involving evaluations of the esthetic appearance and the detection of a tooth-colored resin restoration. Several previous studies have shown that a UV light source is useful for discriminating teeth and resin restorations based on fluorescence [11,12,15,16]. However, the use of UV light requires protective gear such as a dental dam due to this radiation being harmful to the oral tissues [3]. In addition, these studies have been limited by the difficulty of interpreting the results since most such studies have involved spectral analyses. The usefulness of visible light that is safer than UV light has also been investigated, and it was found that the absolute fluorescence intensities detected from teeth and composites decreased when the excitation intensity increased within the wavelength range of 400–500 nm, whereas the relative difference in intensities between the teeth and composites increased [10]. In addition, a device based on quantitative light induced-fluorescence (QLF) technology, consisting of a visible-light lamp (about 405 nm) and a long-pass filter (>520 nm), has been shown to increase the validity and intraexaminer agreement over that obtained with standard white light images when detecting resin restorations of shade B3 in a restored tooth [17]. However, the potential of visible-light sources for identifying various resin composite materials from tooth structures using specific fluorescence properties—and usefulness of these lights sources in clinical situations—remain to be established. Based on the above-mentioned considerations, this study had two objectives: (1) to compare the intensities of the fluorescence emitted from different resin composites as detected using a combination of a 405-nm light source and a specific filter, and 115 (2) to compare the fluorescence intensity contrast and the color differences detected from a resin restoration and the adjacent region of the human tooth. 2. Materials and methods 2.1. Specimen preparation The fluorescence intensities of the following six brands of light-cured resin composites of shade A2 were investigated: Charisma (Heraeus Kulzer, Hanau, Hesse, Germany), DenFil (Vericom, Anyang, Republic of Korea), Filtek Z350 (3 M ESPE, St. Paul, MN, USA), Gradia direct posterior (GC, Tokyo, Japan), Grandio (VOCO, Cuxhaven, Niedersachsen, Germany), and Premisa (Kerr, Orange, CA, USA). The details of the following six products are listed in Table 1. Two types of specimens were prepared: (1) composite discs made of each composite product and (2) human teeth restored with each product. 2.1.1. Resin discs In order to prepare composite discs that were 3 mm wide and 2 mm thick, a portion from each composite product was packed into a rubber mold in increments of 1 mm and polymerized for 40 s with a light-emitting diode (LED) curing unit that had a luminous power density of 1200 mW/cm2 (EliperTM S10, 3 M ESPE). To form a flat and smooth surface, the disc in the mold was covered with a Mylar strip and a slide glass on each side, pressed down by a 1-kg weight for 3 min, and polymerized by light exposure. Eleven discs of each product were constructed, one of which was used as a baseline to take photographs of the specimens. All discs were immersed in distilled water at 37 ◦ C for 24 h until taking photographs for use in image analysis. 2.1.2. Human teeth specimens Extracted human premolars and molars were collected according to the protocol approved by Institutional Review Board (Approval No. 2-2014-0023) of Yonsei University Dental Hospital in Republic of Korea. Calculus and debris were removed from the teeth surfaces using a hand scaler. Seven teeth with a shade similar to that of a ready-made resin disc of each composite product were selected by one examiner when the teeth had been wetted with distilled water. A standardized hole (1.5 mm deep and 2.0 mm in diameter) was made on the smooth surface with a round bur attached to a low-speed handpiece (32,000 rpm). The inside of the hole was etched with 37% phosphoric acid gel (B&E Etch-37, B&E Korea, Gwangmyeong, Republic of Korea) for 15 s and then washed thoroughly with water. After applying a bonding solution (ONE-STEP® , Bisco, Schaumburg, IL, USA) in accordance with the manufacturer’s instructions, the hole was restored by filling with a Table 1 List of analyzed dental resin composites of shade A2. Brand name Composition Filler type Manufacturer Charisma Bis-GMA, barium aluminium fluoride glass, silicium dioxide Bis-GMA, TEGDMA, UDMA, barium aluminosilicate, fumed silica Bis-GMA, Bis-EMA, UDMA, TEGDMA, PEGDMA, non-agglomerated/non-aggregated silica filler, non-agglomerated/non-aggregated zirconia filler, aggregated zirconia/silica cluster filler UDMA, prepolymerized filler, silica, fluoro-alumino-silicate glass Bis-GMA, TEGMDA, nanohybrid filler Microhybrid Microhybrid Heraeus Kulzer, Hanau, Hesse, Germany Vericom, Anyang, Republic of Korea Nanofiller 3 M ESPE, St. Paul, MN, USA Microhybrid GC Corporation, Tokyo, Japan Nanohybrid Voco, Cuxhaven, Niedersachsen, Germany Kerr, Orange, CA, USA DenFil Filtek Z350 Gradia direct posterior Grandio Premisa Bis-GMA, TEGDMA, prepolymerized filler, barium glass, silica filler Nanohybrid 116 B.-R. Kim et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 114–119 portion from each resin composite product in increments of 1 mm followed by light curing for 40 s using the LED curing unit. Finally, the restoration surface was polished using water and a white stone bur attached to a contra-angle handpiece. All restored human teeth were immersed in distilled water at 37 ◦ C for 24 h until taking photographs for use in image analysis. 2.2. Assessments of fluorescence intensity Each of the 10 dried composite disc constructed from each product was placed on top of the baseline disc, and fluorescence images were obtained to investigate the fluorescence of the composite product. For the restored human tooth, photographs were obtained in a wet condition with excess moisture removed using a dried cotton roll. All of the photographs of discs and restored teeth were obtained using a fluorescence digital camera based on the QLF technology (QLF-D BiluminatorTM 2+, Inspektor Research Systems, Amsterdam, The Netherlands) to obtain both white-light and fluorescence images with the following parameters: ISO speed of 250, shutter speed of 1/13 s, and aperture value of 14.0 for the whitelight images; and ISO speed of 1600, shutter speed of 1/15 s, and an aperture value of 13.0 for the fluorescence images. The QLF-D device had a customized digital single-lens reflex (DSLR) camera equipped with 4 white LEDs and 12 blue LEDs that emit light at 405 nm, and a filter set, which means that it can obtain white-light and fluorescence photographs in a single shot. The intensities of the fluorescence detected in the resin composites and teeth images were calculated as grayscale levels. For the first purpose of the study, only fluorescence images of resin discs were used for the analysis. The fluorescence images of resin discs of each brand were converted from RGB (red, green, and blue) images into 8-bit grayscale images using the weighted conversion [12] function of standard image analysis software (Image J version 1.47, National Institutes of Health, USA). The grayscale level was calculated in a region of interest in the center of the resin disc after conversion to a monochrome image. The grayscale level was stored as an 8-bit value, which meant that the intensity scale ranged from 0 (black) to 255 (white) according to the brightness. The mean grayscale level of ten discs for each group was calculated, and then the mean values were compared between the products. The fluorescence image of the restored tooth was converted into a monochrome image, and the mean grayscale level was obtained from the adjacent region of the tooth as well as region of the restored resin [12]. The difference between these two grayscale levels represented the fluorescence intensity contrast between the two substances. The white-light image of the restored tooth was also converted into a monochrome image and the difference in the grayscale levels was calculated in the same way to allow the second purpose of the present study to be achieved. 2.3. Statistical analysis All analyzes were performed using standard statistical software (IBM® SPSS® Statistics version 20, IBM, Armonk, NY, USA), with a probability cutoff for statistical significance of 0.05. Differences in fluorescence intensities measured as grayscale levels among the resin composite products were compared using one-way ANOVA, and statistical significance was determined by the Tukey post-hoc test. The paired t-test was used to compare differences in grayscale levels between the restored resin and adjacent region of the tooth obtained from the converted fluorescence and white-light images for each product. Fig. 1. Mean grayscale levels detected from converted fluorescence images of composite discs. Different letters above the standard deviation bars showed significant differences in the mean grayscale levels (p < 0.001). 3. Results Fig. 1 shows a graph of the mean grayscale levels calculated from grayscale images of composite discs constructed from each product. The fluorescence intensities based on the grayscale levels in ascending order were as follows: DenFil (10.84 ± 0.35, mean ± SD), Filtek Z350 (58.28 ± 1.37), Premisa (156.94 ± 1.58), Grandio (177.20 ± 0.81), Charisma (207.05 ± 0.77), and Gradia direct posterior (211.52 ± 1.66). The value for DenFil indicates that there was hardly any fluorescence, while the other values indicate that there was also a wide diversity of fluorescence intensity values for the other five products. All of the differences in the fluorescence intensities among the six products were statistically significant (p < 0.001). Fig. 2 shows representative white-light, fluorescence, and the corresponding converted grayscale images of restored teeth with each resin composite product. Comparing the restored resin region with the adjacent region of the tooth in a fluorescence image revealed that the fluorescence levels of two of the resin composites (DenFil and Filtek Z350) were lower that for the teeth, while the other four products were brighter than the teeth. Overall the fluorescence of the teeth and restored resin composites appeared bluish and black/bluish, respectively. Table 2 lists the mean differences in grayscale levels between the restored resin region and adjacent tooth region as obtained from the converted grayscale white-light and fluorescent images. For the six tested resin composites, the grayscale level contrast between the two regions obtained from the converted white-light images ranged from 7 to 12 depending on the resin product, while the contrasts obtained from the converted fluorescence images ranged from 15 to 61. This indicates that the difference in grayscale levels based on the difference in fluorescence between the restored resin and adjacent tooth was significantly greater than those values based on the color difference between the two regions, with the exception of the teeth specimen restored with Filtek Z350 (p < 0.05). However, since that restored resin composite exhibited only weak fluorescence, which meant that it could be clearly separated from tooth material in the fluorescence images (Fig. 2). 4. Discussion Most studies that have investigated the different fluorescence properties of teeth and resin composites have been based on spectral analyses [5,14,18,19]. Although these various studies have involved slight differences in absorption and excitation spectra, the various resin materials showed difference maxima in fluorescence B.-R. Kim et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 114–119 117 Fig. 2. Original images (A–F) and the corresponding converted grayscale images (A’–F’), and fluorescence images (a–f) and the corresponding converted grayscale images (a’–f’) of restored teeth obtained by QLF-D. The brand names of the restored resin composites are as follows: DenFil (A, A’, a, a’), Filtek Z350 (B, B’, b, b’), Premisa (C, C’, c, c’), Grandio (D, D’, d, d’), Charisma (E, E’, e, e’), and Gradia direct posterior (F, F’, f, f’). intensity. This property has been found for different brands with the same shade, for the same brand with different shades, and for resin restorations layered by two types of the materials [5,14,18,19]. Nevertheless, this spectral information could be of limited use in clinical situations. In general, a process is required to prepare the specimen in order to obtain detailed and objective data, and it is difficult to apply the analysis apparatus directly to the oral cavity. The present study aimed to overcome these limitations by using a custom camera that can be used to obtain photographs of the fluorescence emitted from resin materials and tooth tissue when the materials are excited by a visible-light source. This study found that the fluorescence intensities varied in the fluorescence images of the tested materials. Moreover, the fluorescence could be determined as being darker or brighter than that of tooth as well as the fluorescence intensities could be calculated as numerical values using image analysis. It is difficult to compare directly the results obtained in this study with those from previous studies due to the use of light sources of different wavelengths and different resin materials. Although the present study did not perform spectral analysis, some of the findings are comparable with those of previous studies. The fluorescence emitted by the resin composites and teeth in the present study was bluish in color, and the brightness of the individual resin composites varied. These results are consistent with those of previous studies that have used UV light and visible light of different wavelengths [5,10,11]. The fluorescence of resin materials is caused by the presence of fluorescent additives in fillers, and the type and content of various components and the ratio of filler and resin may vary between manufacturers [15], which could result in variations in the intensity of the fluorescence emitted from various resin materials varying under the same fluorescence-inducing condition. The condition under which QLF technology is used for inducing and detecting fluorescence may also lead to differences in the fluorescence intensity of resin products. The excitation wavelength that induces the largest emission intensity varied between different resin composites. An early study found that natural teeth emitted a maximum fluorescence intensity at about 450 nm for excitation by UV light at 365 nm [16]. A recent study that made direct measurements with a spectrometry demonstrated that teeth—which are composed of enamel and dentin—absorbed wavelengths in the range of 250–300 nm and emitted fluorescence whose intensity was highest at around 490 nm and gradually reduced as the wavelength increased to around 700 nm [14]. The shape of the absorption and emission spectra of resin materials were found to be similar to those of tooth, but various resin composite products revealed absorption peaks at 250–450 nm and emission peaks at 450–485 nm [3,5,14]. In addition, some resin restorative products could be detected in teeth using a light source of 380 nm but not one of 365 nm in a previous study [11]. It is therefore possible that the blue light source (with an emission peak at 405 nm) used in the present study is not the most suitable for inducing the high- Table 2 Differences in grayscale levels between each resin restoration and the adjacent region of the tooth obtained from the fluorescence images, and the difference in color obtained from the white-light images. Brand name of resin composite DenFil Filtek Z350 Premisa Grandio Charisma Gradia direct posterior Difference in grayscale levels between a resin restoration and the adjacent region of the tooth In white-light images In fluorescence images 9.31 (6.87) 9.97 (4.79) 11.62 (4.86) 10.41 (4.96) 8.80 (3.69) 7.44 (4.53) 22.68 (5.14) 14.68 (5.02) 30.36 (2.06) 35.96 (6.07) 61.26 (5.05) 57.77 (7.93) p-value* 0.016 0.153 <0.001 <0.001 <0.001 <0.001 The difference in grayscale levels were obtained from converted 8-bit grayscale images of the white-light and fluorescence images, and they were presented in mean (standard deviation). * p-values were obtained by paired t-test at ˛ = 0.05. 118 B.-R. Kim et al. / Photodiagnosis and Photodynamic Therapy 13 (2016) 114–119 est fluorescence intensity from some of the tested resin products. Another possibility is that the filter set mounted on the QLF-D filtered out certain wavelengths at which some resin materials might emit greater fluorescence. These backgrounds provide a reason for the reduced fluorescence that was observed from DenFil and Filtek Z350 on the fluorescence images taken by QLF-D. Nevertheless, the results obtained in the current study indicate that the use of a fixed QLF-D condition to induce fluorescence from resin restorations and teeth made it possible to differentiate the fluorescence emissions from different materials and between the two substances. The fluorescence intensity contrast between the region restored with Filtek Z350 and the adjacent region of the tooth, as measured in converted fluorescence images, did not differ significantly from the color contrast between these two regions as detected in a white-light image (Table 2). However, the restored Filtek Z350 was distinguishable as darker fluorescence relative to the adjacent tooth in the original fluorescence image (Fig. 2). This reduced fluorescence tendency of Filtek Z350 is consistent with the findings of a previous study that evaluated the relative fluorescence intensity contrast between the tooth material and resin composites of the same brand (with trademarked names of Filtek Z250 and Z100) [12]. That study also found that the fluorescence contrast between the tooth and resin composite material was more pronounced for the Filtek materials than for Charisma, with the fluorescence being darker for the former. However, the Charisma materials exhibited a higher fluorescence intensity than enamel in the present study, which contrast with the result found in the previous study due to a UV light source with a wavelength range of 340–400 nm and a filter with a cutoff wavelength of 405 nm being used. In addition, in accordance with the present study, another previous study demonstrated that the intensity of the blue–green light with a wavelength range of 450–600 nm emitted by Filtek Z350 of shade A2 was slightly lower than that emitted by an enamel/dentin combination specimen [14]. These results indicate that the fluorescence-based device used in the present study that combined a light source and a filter set can provide reliable evaluations of the fluorescence intensity of resin materials and teeth. The intensity of the fluorescence of various composite products varied in grayscale level under a constant illumination condition. In the present study, the hue, saturation, and intensity values of the QLF-D fluorescence images of the resin discs were evaluated using image analysis software before producing the monochrome images; this confirmed that the colors of the fluorescence emitted were similar for the various brands of resin composites under investigation. The hue values of five of the six tested resin composites were similar (from 116 to 124, out of a possible range from 0 to 255). However, while the fluorescence emitted by DenFil had a different hue, its intensity was very low, appearing almost black with a mean intensity value of 10, and so it can be considered that there was actually little difference in the color of the fluorescence detected for all six resin brands [20]. It was therefore determined that grayscale levels could be used to differentiate the intensities of fluorescence between resin composites in this study. The present study was subject to some limitations. The first stemmed from the device used to obtain the white-light and fluorescence photographs. This study utilized QLF technology with a DSLR camera, which can result in different absolute fluorescence intensities being measured depending on the camera settings. Moreover, obtaining fluorescence images that are either too bright or too dark will make it difficult to differentiate the color and fluorescence of the materials. Therefore, future studies should attempt to identify the best camera settings for capturing optimal QLF-D images. The second limitation was that resin composite products of only one shade (A2) were used. Further research is needed to evaluate the fluorescence properties of sealant [21], combinations of different composites [22], and changes in the fluorescence intensity according to the thickness of the resin materials [3], while considering the clinical relevance of the QLF technology used in this study. The technology was originally developed for the detection of early dental caries and dental plaque by representing the loss of fluorescence and red fluorescence using a visible light source rather than UV light [23–25]. In order to use this technique to detect toothcolored composite restoration in clinical fields, it was necessary to determine whether the fluorescence images obtained by the technology can be used to discriminate different composite products and improve the ability of composite restoration, ensuring that it is more sensitive than the naked eye. The results obtained in this study demonstrate that variations in the intensities of the fluorescence emitted by resin composites can be detected in images of light-induced fluorescence. The composite restoration exhibits fluorescence that is greater or less than that of a tooth, and the differences in the fluorescence intensity between the composite and the adjacent tooth is greater than that in the colors evident in white-light images. These results indicate that the lightinduced fluorescence technology provides superior capabilities for detecting restored composites relative to simply using the naked eye. 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