We aimed to determine whether dye-ehanced quantitative light-induced fluorescence (DEQLF), wherein the porous structure of carious lesions is filled in with a fluorescent dye, can be used to quantitatively and intuitively distinguish between active and inactive carious lesions. To simulate active and inactive caries, lesions were artificially formed on 126 bovine specimens. They were demineralized with 1% carbopol solution for 3 (A3), 5 (A5), and 10 days (A10). Half the specimens in each group were remineralized with 2% NaF (I3, I5, and I10). The specimens were dehydrated for 10 s and dyed with 100-µM sodium fluorescein for 10 s. Images were captured with a QLF-digital 2+ Biluminator. Fluorescence intensity of the lesions (ΔG) between the groups and absolute changes in ΔG (|ΔΔG|) between dehydration and dye penetration were compared using the independent t-test. ΔG in A3, A5, and A10 were higher than those in I3, I5, and I10 (P<0.001). In |ΔΔG| comparisons, dye penetration was 3.1-3.7 times higher than dehydration in the active group (P<0.001), and was 1.7-2.2 times lower than dehydration in I3 and I5 (P<0.05), with no significant difference in I10. DEQLF may be used to clinically evaluate early caries activity, and longitudnally monitor changes in lesion activity.
Research Article
Lesion Activity Assessment of Early Caries Using Dye-enhanced Quantitative Light- Induced Fluorescence
https://doi.org/10.21203/rs.3.rs-422166/v1
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Caries activity
Caries lesion acitivity assessment
Dye-enhanced quantitative light-induced fluorescence
Early caries
Quantitative light-induced fluorescence
Surface porosity
Imbalances in the dynamic process of tooth demineralization and remineralization lead to the development of early caries lesions with an intact surface1,2,3,4. In this dynamic process, the dominance of demineralization increases the surface porosity of caries lesions, while the dominance of remineralization decreases surface porosity5,6. Caries lesions that continue to demineralize are defined as active, whereas those in which the demineralization has stopped and remineralization has begun are defined as inactive7,8. Active caries lesions continue to demineralize and progress due to a continuous increase in surface porosity. In contrast, in inactive caries lesions with predominant remineralization, the surface porosity reduces, which either stops the progression of the lesion or promotes recovery to sound tissue5,9. Therefore, surface porosity may be an important factor in determining lesion activity in early caries.
Quantitative methods for measuring the surface porosity of early caries lesions have been used to objectively determine lesion activity. Micro-computed tomography (micro-CT) and transverse microradiography (TMR) may be used to evaluate the lesion porosity by measuring the mineral content via X-ray absorption profiles. Previous studies have reported that the surface layer of an active lesion is less porous than that of an inactive lesion10,11. However, micro-CT and TMR require extracted or sliced teeth as specimens; therefore, the two methods cannot be used clinically. Additionally, the radiation emitted by micro-CT and TMR may be harmful to the human body. Therefore, it is necessary to develop a non-invasive method to evaluate surface porosity in order to determine lesion activity in early caries, which can be easily used in the clinic.
The visual-tactile method has been widely used for lesion activity assessment. It depends on the roughness and reflection of the lesion surface, which are properties related to surface porosity7,9,12. For example, when the surface porosity increases, the surface layer of the caries lesion becomes irregular, resulting in rough texture, dull-whitish color, and loss of luster13,14,15. In previous studies that have quantitatively evaluated roughness and reflection, no difference was observed in roughness between active and inactive lesions. However, qualitative studies have shown that the reflection of active lesions is significantly higher than that of inactive lesions13. Therefore, reflection may be used to determine lesion activity. However, when measuring reflection in the clinic, the teeth are first dried with compressed air. This becomes an unreliable subjective observation as variations can occur according to the skill and ability of the examiner while observing the dull-whitish-colored lesion.
In dentistry, there have been efforts to objectively and quantitatively evaluate caries lesions using optical technologies. Among them, quantitative light-induced fluorescence (QLF) technology uses blue visible light of 405 nm to detect oral diseases (e.g., dental caries, dental abrasion, tooth fissure, and pathogenic dental plaque)16,17,18,19,20,21,22,23. When an early caries lesion is irradiated with blue visible light, it emits a relatively dark autofluorescence compared to sound enamel due to light scattering by the porous structure of the surface layer24,25,26. When early caries lesions are dried with compressed air (the “dehydration method”), the active lesions show significantly darker autofluorescence than the inactive lesions14. However, it is difficult to intuitively use the dehydration method for lesion activity assessment due to the subtle differences in fluorescence intensity between active and inactive lesions occurring from dehydration in the clinic27.
A fluorescent dye may be used by clinicians to detect and monitor invisible early caries lesions, as it can penetrate into the porous structure of caries lesions by capillary action28. Dye-enhanced QLF (DEQLF) is a method of imaging and quantifying fluorescence by penetrating a fluorescent dye into a caries lesion29,30,31. A previous study that evaluated artificial root caries lesions using DEQLF has reported that as the lesions were demineralized, there was better penetration of the fluorescent dye into the lesion, resulting in a brighter autofluorescence32. Therefore, the aim of this study was to determine whether DEQLF could be used to distinguish between active and inactive lesions. We hypothesized that active and inactive caries lesions can be quantitatively and intuitively distinguished using DEQLF.
In Fig. 2, the histological and fluorescence images of the representative specimens are presented side-by-side to allow for intuitive comparison. Lesion depth (Ld) (mean ± standard deviation; unit: µm) showed no significant differences between the active and inactive groups in A3 (38.7 ± 8.2) and I3 (43.7 ± 10.6), and in A5 (56.6 ± 13.2) and I5 (45.7 ± 16.5). However, A10 (116.6 ± 11.4) was significantly larger compared to I10 (76.2 ± 14.1, P < 0.001). Yellow arrows in the histological image indicate the remineralized surface layer of the caries lesions. While the caries lesions in I3 and I5 had a completely remineralized surface layer, those in I10 had an incompletely remineralized surface layer. In the fluorescence images of the specimens in all active groups, the caries lesions emitted the yellow-green fluorescence of the fluorescent dye. Among the inactive groups, yellow-green fluorescence was not observed in the caries lesions in I3 and I5, whereas the caries lesion in I10 emitted the yellow-green fluorescence of the fluorescent dye, point-by-point.
The ΔG of the specimens between the active and inactive groups were compared (Table 1). After dehydration, ΔG in all active groups was significantly lower than those in all inactive groups (P < 0.001). In contrast, after dye penetration, ΔG in all active groups was significantly higher than that in all inactive groups (P < 0.001).
|
Demineralization period (day) |
Dehydration |
Dye penetration |
||||
|---|---|---|---|---|---|---|
|
AX |
IX |
P-value |
AX |
IX |
P-value |
|
|
3 |
-21.1 (6.6) |
-9.9 (5.0) |
< 0.001 |
24.0 (12.6) |
-9.7 (4.8) |
< 0.001 |
|
5 |
-27.4 (5.3) |
-9.0 (6.8) |
< 0.001 |
38.9 (11.3) |
-10.2 (5.5) |
< 0.001 |
|
10 |
-38.9 (7.3) |
-18.7 (8.7) |
< 0.001 |
46.4 (16.2) |
-12.6 (9.8) |
< 0.001 |
| Mean (standard deviation). | ||||||
| ΔG means the brightness of caries lesion in respect with that of sound enamel (unit: %). | ||||||
| AX indicates active lesions that demineralize for X days; IX indicates inactive lesions that re-mineralize for 10 days after demineralization for X days. | ||||||
On comparing the change in fluorescence (|ΔΔG|) of the caries lesions due to dehydration and dye penetration among all active groups (AX), dye penetration was 3.1-times (A3 and A5) and 3.7-times (A10) significantly higher than dehydration (P < 0.001, Fig. 3). In the inactive groups, dye penetration was 2.2-times (I3) and 1.7-times (I5) significantly lower than dehydration (P < 0.05). There was no significant difference in |ΔΔG| of the specimens in I10 between the two methods.
In this study, we investigated whether DEQLF could be used to differentiate between active and inactive lesions, thereby acting as a valid tool for lesion activity assessment of early caries. On visual observation of the fluorescence images obtained with DEQLF, the fluorescent dye penetrated the caries lesions and thus, emitted brighter autofluorescence in case of the active lesions, whereas in the inactive lesions, it was confirmed that the fluorescent dye did not penetrate the caries. DEQLF was used to easily distinguish whether the early caries lesions were active or inactive by observing autofluorescence with the naked eye in caries lesions. By calculating the G value, the lesion activity of early caries could be quantitatively evaluated.
The fluorescent dye penetrated the active lesions but not the inactive lesions (Fig. 2). The emitted autofluorescence of the active caries lesions was brighter than that of the sound enamel and showed positive G values, whereas the emitted autofluorescence of the inactive caries lesions was darker than that of the sound enamel and showed negative G values (Table 1). This is similar to the results of previous studies in which active caries lesions showed brighter fluorescence than inactive lesions when DEQLF was performed on artificial root caries lesions32. These results can be explained based on the fact that the porosity of a caries lesion’s surface increases proportionally as the number and diameter of microchannels in the surface layer increase33,34. Likewise, in the histological evaluation in this study, it was confirmed that the active caries lesions have no remineralized surface layer, which also means that active caries lesions have a more porous surface than inactive lesions (Fig. 2), due to which, a larger amount of fluorescent dye could penetrate the caries lesion simultaneously.
In the DEQLF image, the inactive caries lesions of I10 emitted partially bright autofluorescence due to penetration of the fluorescent dye into the lesions, while the inactive caries lesions of I3 and I5 emitted dark autofluorescence as the fluorescent dye did not penetrate the lesions (Fig. 2). In contrast, in the histological images, the inactive caries lesions of I3 and I5 had a “completely remineralized” surface layer with mineral content similar to that of normal enamel. However, the inactive caries lesions of I10 had a mineral content similar to that of demineralized lesions and an “incompletely remineralized” surface layer. When the caries lesions were treated with a high concentration of fluoride (such as 2% sodium fluoride used in this study), the remineralization of the surface layer differed according to the lesion severity35. Therefore, it is considered that the caries lesions of I10 had an “incompletely remineralized surface layer” due to the low remineralization of a high concentration of fluoride at a relatively deep lesion depth. Based on this, DEQLF may be used to detect inactive lesions in which the lesion surface is incompletely remineralized by observing the autofluorescence of a fluorescent dye that is partially emitted within the lesion. DEQLF may also be used to evaluate the extent to which the lesion has remineralized.
DEQLF, the method of dye penetration after dehydration, can help clinicians intuitively assess lesion activity. In this study, when only dehydration was performed, both the active and inactive groups emitted darker fluorescence than the sound enamel (Table 1). In previous studies that used QLF to observe early caries lesions during dehydration, both active and inactive lesions emitted dark fluorescence resulting from the evaporation of water from the porous structure of the lesion, as the refractive index changed from water (1.33) to air (1.00)14,27,33. However, it is difficult for clinicians to facilitate evaporation of such a small amount of water as that observed in the early caries lesions, and to assess lesion activity by visually observing or quantitatively measuring the result of evaporation (change in the refractive index of the lesion). In contrast, in this study, it was possible to clearly differentiate between active and inactive lesions using DEQLF, as the dye penetration highlighted the active lesions as bright fluorescence and the inactive lesions as dark fluorescence (Fig. 2). The change in fluorescence (|ΔΔG|) by DEQLF in the active groups was 3.1−3.7 times higher than that by dehydration, and the |ΔG| by DEQLF in the inactive groups was 1.7−2.2 times lower than the |ΔΔG| by dehydration (Fig. 3). Therefore, these results show that DEQLF may be used to clearly and intuitively assess lesion activity rather than using the dehydration method to visualize and quantify the porous structure of early caries lesions.
Fluorescein sodium was used as the fluorescent dye in this study. Previous studies have reported that porphyrin derivatives are mainly used as fluorescent dyes for caries lesion assessment with laser fluorescence (LF)36,37,38,39,40. However, the porphyrin derivatives are not suitable as a fluorescent dye for DEQLF, since they already exist in natural caries lesions as bacterial metabolites20,41,42,43,44. Consequently, when performing DEQLF on natural caries lesions with porphyrin derivatives, it is impossible to distinguish the red fluorescence between the fluorescent dye and the bacterial metabolite. In contrast, fluorescein sodium is produced only outside the body and does not form an ionic bond with enamel28,45. Moreover, since fluorescein sodium emits yellow-green autofluorescence, it can be easily distinguished from the red autofluorescence of porphyrin derivatives on the caries lesions. However, since this study used artificial lesions to simulate active and inactive lesions in the laboratory, it was not possible to reflect all features (dental plaque, dental calculus, and anatomical morphology) of natural caries lesions. Therefore, it is useful as a fluorescent dye for DEQLF to evaluate the lesion activity of natural caries.
This is the first study to distinguish between active and inactive lesions in artificial caries using DEQLF. We confirmed that there was a difference in the autofluorescence emitted from the active and inactive lesions of artificial caries using DEQLF. Active lesions emitted brighter yellow-green autofluorescence than sound enamel, whereas inactive caries lesions emitted darker autofluorescence than sound enamel after performing DEQLF. It may be considered a supportive modality for clinicians in the intuitive and quantitative assessment of lesion activity in early caries, based on different fluorescence patterns and surface porosity.
The DEQLF method can intuitively and quantitatively distinguish between active and inactive lesions based on differences in autofluorescence. Therefore, DEQLF might be useful as an objective assessment of lesion activity. The method has ample scope to become a useful tool for assessing lesion activity with a single observation and for longitudinal monitoring of lesion activity in clinical practice. Additionally, DEQLF consists of a simple procedure; therefore, even dentists with little experience in evaluating lesion activity of early caries can easily use this in clinical settings and for large groups.
Specimen preparation
Bovine maxillary jaws were purchased in the slaughterhouse for preparing bovine tooth specimen. 63 teeth with no dental cracks, dental caries, dental fluorosis, or enamel hypoplasia on the labial surfaces were obtained from the jaws using a low-speed diamond disk (NTI-KAHLA GmbH, Kahla, Germany). And then, the teeth were cut to make 126 slabs (4 × 3 × 3 mm) using the low-speed diamond disk (NTI-KAHLA GmbH). The slab with a labial surface facing outward was embedded in acrylic molds (20 × 12 × 7 mm) with 9-mm diameter holes using self-cured resin (Ortho-Jet, Lang Dental Manufacturing, Wheeling, Illinois, USA; Fig. 1A). The surface of the specimen was polished using a water-cooled polishing machine (RB 209 Minipol, R&B Inc., Daejeon) with 400-, 800-, and 1200-grit abrasive papers (SiC sandpaper, R&B Inc., Daejeon, South Korea). Half of the surface was covered with an acid-resistant and non-fluorescent transparent varnish to protect the sound enamel.
Caries lesion formation
In this study, caries lesions were artificially formed to simulate active and inactive lesions. Demineralized and remineralized lesions were used to reproduce the active and inactive lesions, respectively.
The specimens were equally distributed among three active groups (AX, where X indicates the demineralization period; Fig. 1A). The demineralizing gel contained 0.1 M lactic acid gel (pH 4.8) with 1% carbopol (Carbopol® ETD 2050 polymer, Noveon Inc., Wickliffe, Ohio, USA), and was 50% saturated with hydroxyapatite (calcium phosphate tribasic Sigma, St. Louis, MO, USA)46. The specimens were immersed in 40 mL of the solution at 37°C during the demineralization period of each group (3, 5, and 10 days, respectively). After the specified durations, the specimens were removed from the solution and were washed with distilled water.
Half of the specimens from each group (21 per group) were reassigned to three inactive groups (IX, where X indicates the demineralization period; Fig. 1A). The remineralization procedure consisted of four steps35. First, the specimens were immersed in 3 mL of 2% sodium fluoride solution for 4 min. Second, the solution remaining on the surface of the specimen was washed with distilled water. Third, the specimens were immersed in artificial saliva (0.22% mucin, 30 mM KCl, 10 mM KH2PO4, 13 mM NaCl, and 3 mM CaCl2, final pH 6.8; refilled daily) for the rest of the day. Finally, the solution remaining on the surface of the specimen was rinsed with distilled water. The entire remineralization procedure was repeated for 10 days. After the remineralization process was complete, the acid-resistant varnish covering the surface was removed using acetone. Except for one specimen, in which the nail varnish was removed in the I10 group during the demineralization process, 125 specimens were used until the end of this study. The specimens were placed in a dark container and stored in a refrigerator at 4°C until performing DEQLF.
Dye-enhanced QLF (DEQLF)
DEQLF is as a process comprising dehydration and subsequent dye penetration (Fig. 1B). First, cotton pellets saturated with distilled water were placed on the surface to hydrate the specimens for 60 s. The specimens were then dehydrated for 10 s using compressed air14,33. Cotton pellets saturated with the fluorescent dye, 100 µM fluorescein sodium salt (Sigma-Aldrich, St. Louis, MO, USA) in 50% ethanol solution, were placed on the surface of the dried specimens for 10 s47. White light and fluorescent images of the specimens were captured after hydration, dehydration, and dye penetration using QLF-digital 2 + Biluminator (QLF-D, Inspector Research Systems BV, Amsterdam, The Netherlands). Imaging conditions were as follows: white light image (shutter speed 1/90s, aperture value 13.0, ISO speed 1600) and fluorescent image (shutter speed 1/30s, aperture value 13.0, ISO speed 1600).
Image analysis
All images were analyzed using an image analysis software (Image-Pro PLUS 6.0, Media Cybernetics, Rockville, MD, USA). The fluorescence images of all specimens were converted into 8-bit grayscale images using the “convert to” function (Fig. 1C). The area of interest (AOI) of sound enamel and caries lesions was drawn along the margins of the sound enamel and caries lesions, respectively. The mean gray levels in the AOIs of sound enamel (Gs) and caries lesions (Gc) measured using an 8-bit grayscale image. The fluorescence intensity (ΔG) of the caries lesion with respect to sound enamel was calculated using the following formula:
. A positive ΔG indicates that the caries lesion emits brighter fluorescence compared to the sound enamel. In contrast, a negative ΔG means that the caries lesion emits darker fluorescence compared to the sound enamel. To measure the fluorescence change in the caries lesion according to the assessments (dehydration and dye penetration), ΔΔG (ΔG after assessment – ΔG before assessment) was calculated before and after the assessments, respectively. To be able to compare the difference in G, the absolute ΔΔG (lΔΔGl) was used as the fluorescence parameter.
Histological analysis
The straight line containing the point with the highest gray level parallel to the major axis of the specimen was set as the AOI. The specimen was cut vertically twice using a microtome (TechCut 4™, Allied High Tech Products, Compton, CA, USA) for the acquisition of the sectioned tooth, including the AOI. It was ground using 800-grit abrasive papers until the thickness of the sectioned tooth was 150 µm. Histological images (×400 magnification) of the sectioned teeth were obtained using a polarizing microscope. In the histological image, the Ld of early caries was calculated using an image analysis program (Image Pro PLUS 6.0, Media Cybernetics).
Statistical analysis
All statistical analyses were performed using SPSS version 21.0 (IBM Corp., Armonk, NY, USA). The difference in ΔG and Ld between the active (AX) and inactive (IX) groups, and the difference in |ΔΔG| between the specimens that underwent dehydration and dye penetration was evaluated using an independent t-test (α = 0.05).
Acknowledgments
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C0129) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI20C2114).
Author contributions statement
S.-W. Park designed the work, acquired and analysed the data, and have drafted the work
S.-M. Kang acquired and analysed the data
H.-S. Lee and S.-K. Kim, and E. de Josselin de Jong interpreted the data
E.-S. Lee substantively revised the draft
B.-R. Kim designed of the work
B.-I. Kim built the conception and substantively revised the draft
Competing Interest
Inspektor Research Systems BV provided the salary for author EdJdJ, but it did not have any role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. EdJdJ’s involvement in this research was under the auspices of his status as adjunct professor at Yonsei University College of Dentistry supported by BK21 FOUR Project. The specific role of EdJdJ was to provide his expertise regarding the fluorescence technology. This does not alter the author’s adherence to the policies of Scientific Reports on sharing data and materials. EdJdJ holds several patents with respect to QLF technology. The remaining authors declared no conflicts of interest.
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Competing interest reported. Inspektor Research Systems BV provided the salary for author EdJdJ, but it did not have any role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. EdJdJ’s involvement in this research was under the auspices of his status as adjunct professor at Yonsei University College of Dentistry supported by BK21 FOUR Project. The specific role of EdJdJ was to provide his expertise regarding the fluorescence technology. This does not alter the author’s adherence to the policies of Scientific Reports on sharing data and materials. EdJdJ holds several patents with respect to QLF technology. The remaining authors declared no conflicts of interest.