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Pit and fissure sealant assessment using

Quantitative light-induced fluorescence (QLF)

technology

Sang-Mi Nam

The Graduate School

Yonsei University

Department of Dentistry

Pit and fissure sealant assessment using

Quantitative light-induced fluorescence (QLF)

technology

Directed by Professor Baek Il Kim

A Dissertation

Submitted to the Department of Dentistry

And the Graduate School of Yonsei University

in partial fulfillment of the requirements

for the degree of Doctoral of Philosophy in Dental Science

Sang-Mi Nam

June 2020

ACKNOWLEDGEMENTS

Dr. Elbert

i

CONTENTS

LIST OF FIGURES v

LIST OF TABLES vii

ABSTRACT viii

I. INTRODUCTION 1

II. MATERIALS AND METHODS 9

2.1. Study design 9

2.2. In vitro study 11

2.2.1. Tooth selection 11

2.2.2. Preparation of the specimen 11

2.2.3. Pit and fissure sealant application and microleakage formation 12

2.2.4. QLF-D image taking 13

2.2.5. Fluorescence assessment and analysis using QLF-D 15

2.2.6. Histological analysis 17

2.3. Clinical image study 20

2.3.1. Study subject 20

ii

2.3.2. Image assessments of Q-ray pen 22

2.3.3. Fluorescence image assessment and analysis using Q-ray pen 22

2.4. Clinical trial 25

2.4.1. Study subject 25

2.4.2. Visual and tactile assessments 26

2.4.3. Q-ray view combined assessment 28

2.5. Statistical analysis 30

2.5.1. In vitro study (Study 1) 30

2.5.2. Clinical image study (Study 2) 30

2.5.3. Clinical trial (Study 3) 31

III. RESULTS 32

3.1. In vitro study results (Study 1) 32

3.1.1. Comparison of histological distribution by microleakage formation 32

3.1.2. Distribution of fluorescence variables according to the histological score

33

3.1.3. QLF-D images and magnification microscope images of microleakage in

pit and fissure sealant according to the histological score 35

3.1.4. Correlation of variable between histology and fluorescence values

36

iii

3.1.5. Validity of QLF-D 37

3.1.5.1. AUC analysis for diagnostic accuracy of fluorescence variables 37

3.1.5.2. Sensitivity, specificity, and AUC curves of fluorescence variables

at outer half microleakage of the pit and fissure sealant 38

3.1.5.3. Sensitivity, specificity, and AUC curves of fluorescence variables

at inner half microleakage of the pit and fissure sealant 39

3.1.5.4. Sensitivity, specificity, and AUC curves of fluorescence variables

at underlying fissure microleakage of the pit and fissure sealant 40

3.2. Clinical image study results (Study 2) 41

3.2.1. Study population 41

3.2.2. Distribution of fluorescence variables according to pit and fissure sealant

status 41

3.2.3. Interrater reliability between White-light image and Fluorescence with

white-light image 44

3.2.4. Distribution of marginal plaque between White-light image and

Fluorescence with white-light image 46

3.3. Clinical trial results (Study 3) 47

3.3.1. Study population 47

3.3.2. Interrater reliability between VT and Q-ray view with VT 48

3.3.3. Intrarater reliability between VT and Q-ray view with VT 51

iv

3.3.3.1. Intrarater reliability of marginal plaque assessment of pit and fissure

sealant 53

3.3.3.2. Intrarater reliability of marginal discoloration assessment of pit and

fissure sealant 54

3.3.3.3. Intrarater reliability of marginal integrity assessment of pit and

fissure sealant 55

3.3.3.4. Intrarater reliability of retention assessment of pit and fissure sealant

56

3.3.3.5. Intrarater reliability of caries assessment of pit and fissure sealant

57

IV. DISCUSSIONS 58

V. CONCLUSIONS 68

REFERENCES 70

ABSTRACT (IN KOREAN) 76

v

LIST OF FIGURES

Figure 1. A schematic diagram of the study design 10

Figure 2. QLF-D camera (QLF-D Biluminator TM) 14

Figure 3. (A) A representative occlusal fluorescence image. (B) A magnified image for analysis. (C) A designed patch area around margin of sealant. (D) A reconstructed image based on the fluorescence of the sound area. The blue line indicates the sound reference area, whereas the red line indicates the deactivated area. (E) The fluorescence difference between the original and reconstructed images. (F) Analysis results from the margin of sealant. (G) A total of 4 analyzed patches in single tooth sample. 16

Figure 4. Sample preparation for observing the cross-sectional view of mesial and distal pit

18

Figure 5. Schematic diagram of the cross-sectioned specimen for measuring microleakage and dye penetration 19

Figure 6. Q-ray pen TM 21

Figure 7. (A) A representative occlusal white light image. (B) A representative occlusal fluorescence image. (C) A reconstructed image based on the fluorescence of the sound area. The blue line indicates the sound reference area, whereas the red line indicates the deactivated area. (D) Analysis results from the margin of sealant. (E) Analyzed patches in single tooth sample. 24

Figure 8. Q-ray view TM 29

Figure 9. Box plot showing ΔF, ΔFmax values related to histological score 34

Figure 10. Representative QLF-D images (white-light and fluorescence images) and a magnification microscope images(×50) of specimens according to histological score 35

Figure 11. Sensitivity, specificity, and AUC curves of fluorescence variables at outer half microleakage of the pit and fissure sealant 38

vi

Figure 12. Sensitivity, specificity, and AUC curves of fluorescence variables at inner half microleakage of the pit and fissure sealant 39

Figure 13. Sensitivity, specificity, and AUC curves of fluorescence variables at underlying fissure microleakage of the pit and fissure sealant 40

Figure 14. Comparison of kappa values for interrater reliability between white-light image and fluorescence with white-light image 44

Figure 15. Comparison of mean kappa values for interrater reliability between VT and Q-ray view 49

Figure 16. Representative images of marginal plaque (A, B), marginal discoloration (C, D), and caries (E, F) at pit and fissure sealant margin. White-light image (A, C, E), Q-ray pen image (B, D, F) 50

Figure 17. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view 52

Figure 18. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view of marginal plaque assessment 53

Figure 19. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view of marginal discoloration assessment 54

Figure 20. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view of marginal integrity assessment 55

Figure 21. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view of retention assessment 56

Figure 22. Comparison of mean kappa values for intrarater reliability between VT and Q-ray view of caries assessment 57

vii

LIST OF TABLES

Table 1. Photographing condition of QLF-D in this study 14

Table 2. Criteria of image evaluation for pit and fissure sealant status 23

Table 3. Criteria of evaluation for pit and fissure sealant status 27

Table 4. Histological distribution of specimens by microleakage formation 32

Table 5. Distribution of Fluorescence variables according to the histological score after microleakage formation 33

Table 6. Correlation coefficients between histology and fluorescence variables of microleakage 36

Table 7. Area under the ROC curve of fluorescence variables at each histology criteria 37

Table 8. Distribution of fluorescence variables according to pit and fissure sealant status 42

Table 9. Intrarater reliability of two images examination methods for evaluating pit and fissure sealant status 43

Table 10. Interrater reliability of two images examination methods for evaluating pit and fissure sealant status 45

Table 11. Interater reliability of two images examination methods for evaluating pit and fissure sealant status 45

Table 12. Distribution of marginal plaque in white-light image and fluorescence with white-light image 46

Table 13. Demographic data 47

Table 14. Interrater reliability of two examination methods for evaluating pit and fissure sealant status 49

Table 15. Intrarater reliability of two examination methods for evaluating pit and fissure sealant status 51

viii

Abstract

Pit and fissure sealant assessment using Quantitative light-

induced fluorescence (QLF) technology

Sang-Mi Nam

Department of Dentistry

The Graduate School, Yonsei University

(Directed by professor Baek-Il Kim)

Currently, the evaluation of the retention status of the pit and fissure sealant is subjective,

so limitations on validity and reliability have been reported. Attempts have been made to

evaluate the pit and fissure sealant using a variety of optical equipment, but it has been

difficult to utilize in clinical practice due to the limited objectivity and reproducibility. The

purpose of this study was to evaluate the validity and reliability of an optical technique

ix

(Quantitative light-induced fluorescence, QLF) that quantifies fluorescence reactions to

detect microleakage of pit and fissure sealants.

The first study used 160 specimens from a total of 40 human tooth with pit and fissure

sealants. Fluorescence images of tooth specimens, which captured by QLF technology,

were analyzed using a specific analysis program. The mesial and distal pit margin were

analyzed, and this was the same site as histological evaluation.

The fluorescence values (ΔF, ΔFmax) of QLF-D according to the depth of microleakage

were measured, and the correlation between depth and each fluorescence value was

analyzed. In addition, sensitivity, specificity and AUC were calculated according to

histological evaluation results. As a result, all fluorescence values of QLF-D showed a

strong correlation with the depth of microleakage (Spearman's correlation coefficients, rho;

-0.72; p <0.001). The diagnostic accuracy of good or better was shown at the threshold of

all microleakage depths (AUC = 0.83 – 0.93, p <0.001) in the AUC analysis result using

the ROC curve.

For the second study, 29 occlusal surfaces were captured from human tooth with pit

and fissure sealant using a Q-ray pen. Total 129 sites of interest from these images were

selected, and one trained examiner evaluated it according to each clinical evaluation

criterion. An examiner also analyzed fluorescence values (ΔF, ΔFmax, ΔR) to obtain

quantitative values. Another three clinicians trained four clinical evaluation criteria

(marginal plaque, marginal discoloration, retention, and caries), and then performed image

x

analysis. White light and fluorescent image captured by Q-ray pen was used for analysis,

and interrater agreement was calculated after each image evaluation. Fluorescence values

(ΔF, ΔFmax, ΔR) were calculated from fluorescent image, and these values (ΔF, ΔFmax)

except for ΔR showed significant differences according to clinical criteria in the retention

category (p <0.05). For evaluation retention and caries of pit and fissure sealant, interrater

agreement showed fair agreement - substantial agreement (0.26-0.41, 0.33-0.67) in white

light image, but it showed higher value in fluorescent image (0.43-0.57, 0.40-0.72), which

means moderate agreement- substantial agreement.

For study 3, total 58 teeth were examined from 15 volunteers among 3rd grade dental

hygiene departments who had at least one tooth that had been treated with pit and fissure sealant

six months ago. In order to compare conventional method (visual and tactile inspection) and

combination method with Q-ray view for tooth with pit and fissure sealant, interrater and

intrarater agreements were calculated from three trained examiners. For marginal plaque,

marginal discoloration, caries assessment, interrater agreement showed fair agreement -

moderate agreement (0.22-0.57, 0.36-0.57, 0.43-0.61) in conventional method, while it showed

statistically high value in combination method with Q-ray view (0.81-0.89, 0.69-0.88, 0.8-0.9),

which means substantial agreement - almost perfect agreement. Intrarater agreement also

showed similar result that combination method with Q-ray view showed higher value (0.81-

0.83, 0.57-0.89, and 0.69-0.91, respectively) than conventional method.

xi

Therefore, by identifying the excellent diagnostic ability of QLF technology for pit and

fissure sealant evaluation, we confirmed that it can be used not only for microleakage

detection but also retention evaluation in clinical practice.

Keywords: caries, discoloration, microleakage, pit and fissure sealant, plaque, quantitative

light-induced fluorescence(QLF) technology, retention

1

Pit and Fissure Sealant Evaluation

using Quantitative light-induced fluorescence (QLF) technology

Sang-Mi Nam

Department of Dentistry

The Graduate School, Yonsei University

(Directed by professor Baek-Il Kim)

I. INTRODUCTION

Dental care is now shifting from treatment to prevention-oriented as the public's interest

in oral health increases. Therefore, prevention-oriented treatment plans are being

2

emphasized in the field of dentistry (National Center for Health 2010). In Korea, the first

molar pit and fissure sealant insurance for children aged 6 to 14 was applied from

December 1, 2009, and the insurance coverage of the first and second molar was expanded

from October 1, 2012 to those under the age of six. In addition, from October 1, 2017,

preventive care benefits were expanded to cover the first and second molars under the age

of 18. The dental field has recently made significant scientific advances in dental caries

with dental restoration materials and innovative preventive technologies, but dental caries

is still considered a high incidence pathology. Overall, half of dental caries is found in pit

and fissure of permanent teeth (Ekstrand et al. 1995), and the occlusal caries in children

and adolescents ranged from 67% to 90% (Brown, Wall, and Lazar 2000; Kaste et al. 1996).

This is due to a direct relationship between the internal form of the occlusion groove-fossa

system and the caries pressure (Ekstrand et al. 1995). It has been reported that occlusal

surfaces are more likely to be bacterial accumulation, there is a difference between pit-and-

fissure plaque and smooth-surface plaque, and it is difficult to remove the plaque from the

occlusal surfaces (Kidd and Fejerskov 2004; Loesche 1986).

Pit and fissure sealant was introduced in 1967 by Cueto and Buonocore to prevent

occlusal caries (Cueto and Buonocore 1967). Pit and fissure sealants are

micromechanically bonded by applying a liquid material to the occlusal surfaces (pits and

fissures), mainly forming barrier against acids and preventing mineral loss in the teeth

(Schwendicke et al. 2015). Regarding the application criteria of the pit and fissure sealant,

Beauchamp et al. claimed that it is possible to apply a sealant if the cavity is invisible due

3

to initial caries confined to the enamel based on the results of a comprehensive analysis of

several studies (Beauchamp et al. 2009). Pit and fissure sealants are also reported to be

effective and safe in preventing or inhibiting the progression of non-cavitated carious

lesions (Ahovuo-Saloranta et al. 2013; Wright et al. 2016). It was reported that dental caries

prevention effect for pit and fissure sealant was 99% in permanent teeth and 87% in

deciduous teeth (Cueto and Buonocore 1967), and it was similar (Chestnutt et al. 2017) or

superior to fluoride varnish (Wright et al. 2016).

However, pit and fissure sealant has weak physical properties, it fractures well at the

marginal area, causing partial microleakage, which causes dental caries and fall off

(Hatibovic-Kofman, Wright, and Braverman 1998). Kidd. defined microleakage as the

passage of bacteria, oral fluid, molecules, or ions between teeth and restorations (Kidd

1976). This may cause restoration discoloration, facilitation of fractures, recurrent caries,

irritable teeth and pulp necrosis (Going 1972). In addition, Jensen et al. reported that the

preventive effect of pit and fissure sealant appears when microleakage is minimized (Jensen

and Handelman 1980). Therefore, microleakage is an important factor affecting the success

and failure of the pit and fissure sealant.

Futatsuki et al. reported that the dropout rate after 3 months of application of the pit and

fissure sealant was 14.4%, and a partial or complete dropout rate of 7.0% between 3 and 6

months (Futatsuki et al. 1995). In addition, it is reported that the pit and fissure sealant

shows a drop rate of 5–10% in the occlusal surface 1 year after application (Feigal 1998;

Futatsuki et al. 1995). The causes of early dropout of representative pit and fissure sealant

4

are as follows; (1) When a sealant is applied to a tooth with caries on the occlusal surface,

not on the healthy tooth surface due to the selection error of the target tooth, (2) When

plaque remains in the pit and fissure of the occlusal surface, (3) In the case of sealant

treatment, water or saliva remains after acid corrosion and washing. For these reasons, the

sealant cannot penetrate into the teeth and the holding power is reduced (Lee 2011). Taylor

and Gwinnet. reported that the factors influencing retention were the pit and fissure

geometry, the presence of organic residues in the fissure, and the physical and chemical

properties of the sealant (Taylor and Gwinnett 1973). Gwinnet. suggested to fully utilize

the acid corrosion effect through proper cleaning in the fissure and prevention of

contamination by saliva during the procedure to obtain proper retention of the pit and

fissure sealant (Gwinnett 1967). Therefore, one of the factors for the success of the pit and

fissure sealant is the holding force between the tooth and the sealant. Failure to apply the

sealant correctly can result in premature dropout and secondary caries.

The main cause of pit and fissure sealant drop out is when the sealant is applied without

performing a complete dry state after etching (Disney and Bohannan 1984; Ripa 1985). The

microcavity of the etched enamel surface can be closed under the influence of moisture from

saliva contamination. Therefore, the penetration of the sealant into the microcavity is prevented,

and an insufficient resin tag is formed, thereby reducing the holding power of the sealant

(Hormati, Fuller, and Denehy 1980). In addition, since the etched enamel surface absorbs

saliva as soon as it is contaminated with saliva, the surface activity is reduced, which affects the

reduction of surface bonding force (Buonocore 1971). The retention problem of how long a

5

pit and fissure sealant can be maintained is constantly being raised (Beauchamp et al. 2009;

Kim, Lee, and Lee 2007). Therefore, it is necessary to evaluate the retention of sealant

periodically after application of sealant. Traditionally, the retention force of pit and fissure

sealant has been evaluated by visual examination and tactile examination (Antonson et al. 2012;

Guler and Yilmaz 2013; Ünal et al. 2015). To examine whether the occlusal sealant and the

marginal part of the tooth surface are sealed, the clinician's visual and tactile examination is

widely used as a traditional evaluation method (Lee 2006). However, these methods can be

detected when lesions have progressed to a depth of at least 300 to 500 μm outside the enamel

(Stookey 2005). In addition, it is a method of evaluating whether the boundary between sealant

and tooth is smooth subjectively using the sense of the inspector. Therefore, there is a limitation

that it is not objective due to low validity and reliability (Lee 2006).

Some studies have been reported to objectively detect the maintenance of the sealant

using various optical device due to the limitations of the traditional pit and fissure sealant

evaluation method. Deery et al. confirmed the validity and reproducibility of caries

detection of occlusal surfaces before and after application of clear sealant by using laser

fluorescence equipment, DIAGNOdent. As a result of the study, DIAGNOdent was less

feasible and reproducible than the clinical visual evaluation (Deery et al. 2006). Other study

using DIAGNOdent showed false-positive results due to restorative materials or foreign

substances (Hitij and Fidler 2008). In a study evaluating the pit and fissure sealant margin

using micro-CT, it was possible to detect the preservation status of the sealant margin (Chen

et al. 2010), but it is considered to be difficult to apply to the evaluation of the retention of

6

the sealant clinically due to the high radiation exposure hazard. Recently, studies have been

conducted to identify defects in the tooth-restoration interface using SS-OCT (Swept-

source optical coherence tomography) (Bakhsh et al. 2011; Oancea 2015). However, OCT

is observed in a tomographic image with a depth of the teeth enlarged by 1.63 times due to

the influence of the optical distance, and it is impossible to acquire an image of a deep

region of a biological tissue due to a limitation in the depth of transmission because of the

characteristics of the light source (Hariri et al. 2012). This has the limitation that the

subjective criteria of the inspector can influence the evaluation of the sealant's marginal

preservation status and holding power, and does not reflect the exact depth.

Quantitative light-induced fluorescence (QLF) is an optical device that quantifies the

difference in loss of fluorescence from sound enamel and lesions by irradiating 405nm blue

visible light to the teeth (Pretty 2006). Ozyoney et al. was comparatively evaluate QLF and

clinical examination in marginal preservation status of pit and fissure sealant. The

difference in fluorescence loss (ΔF) of QLF showed higher agreement rate than clinical

examination in evaluating caries and dropout of sealant materials (Ozyoney et al. 2005).

Recently developed as a digital camera type QLF-D (Quantitative light-induced

fluorescence-digital), it is possible to detect red fluorescence expressed in the bacterial

metabolite porphyrin (Kim 2013). Plaques that have penetrated the microleakage site of the

pit and fissure sealant margin can be confirmed by a red autofluorescence reaction from

QLF-D. Also, dental caries showed relatively dark compared to sound enamel due to loss

of fluorescence, so early caries can be easily detected (Kim 2011). In addition, it is easy to

7

check whether secondary caries occurs (Kim 2013), and a useful equipment to monitor the

progress of the lesion because it can quantify minute mineral changes in the initial caries

(Ellwood, Gomez, and Pretty 2012). A Q-ray pen (Aiobio Inc., Seoul, Korea) that is more

portable than QLF-D has been developed, but there has been no report using it in the field

of pit and fissure sealant. Furthermore, a handy portable Q-ray view (Aiobio Inc., Seoul,

Korea) was developed to be useful in clinical trials, and attempts were made to evaluate

the restoration using it, and Oh et al. reported its feasibility (Oh et al. 2014). However,

previous studies only reported the difference between the fluorescence loss (ΔF) and the

caries and loss of sealant materials of the pit and fissure sealant using QLF technology.

There have been no reports of studies evaluating the difference from the gold standard test

method through histological evaluation according to the degree of microleakage. The visual

examination with Q-ray view simply confirmed the presence or absence of a pit and fissure

sealant, so clinical evaluation of factors affecting the maintenance status of the sealant is

insufficient.

Based on the necessity of this research, this study intends to evaluate the validity and

reliability of QLF technology as a tool for detecting pit and fissure sealants. In the first in vitro

study, we tried to evaluate the feasibility by comparing microleakage around the pit and fissure

sealant with histological evaluation of gold standard using QLF-D.

The second clinical images study was quantified according to the evaluation criteria of

clinical conditions (marginal plaque, marginal discoloration, retention, caries) using Q-ray pen

8

images obtained from subjects with teeth coated with pit and fissure sealant in clinical practice

and evaluated reliability by comparing the white light and white– and fluorescent images.

The objective of the third clinical trial study was to compare the reliability of the traditional

method (visual and tactile inspection) with the new method (combination of traditional method

and Q-ray view) in assessing clinical maintenance status (marginal plaque, marginal

discoloration, marginal integrity, retention, and caries) of pit and fissure sealant.

9

II. MATERIALS AND METHODS

2.1 Study design

This in vitro study was intended to evaluate the fluorescence expression pattern

according to the depth of microleakage around the pit and fissure sealant using QLF-D and

to confirm the diagnostic accuracy by comparing it with histology criteria, which is a gold

standard. The QLF images study was intended to quantitatively analyze the pit and fissure

sealant state in the fluorescence image of the Q-ray pen, and to evaluate the reliability by

comparing the test using a white light image and a fluorescence image. The clinical trial

attempted to evaluate reliability by comparing the retention status of pit and fissure sealant

with the conventional visual and tactile assessment and Q-ray view (Figure 1).

10

Figure 1. A schematic diagram of the study design

11

2.2. In vitro study

2.2.1. Tooth selection

This in vitro study was approved by the Institution Review Board for Clinical Research

of the Yonsei dental hospital to use the extracted human teeth (IRB No. 2-2017-0044). Prior

to the study, all participants were informed of the purpose and method of the study, and

informed consent was obtained. The extracted teeth were immediately frozen at -20°C. The

research teeth were assigned a unique number after removing the dental plaque. For this

study, 44 permanent molars of ICDAS code 0-1 were used. Two examiners classified the

occlusal surface of the teeth as naked eyes, and if there were disagreements, they were

checked and classified together.

2.2.2. Preparation of the specimen

The remaining tissue and deposits attached to the extracted tooth surface were removed

with a manual scaler, and a brush was mounted on a low-speed handpiece to remove plaque

on the occlusal surface with a fluorine-free pumice. Each tooth is assigned a unique number,

and the root is cut using a low-speed handpiece (Lasungmedics, Korea) and a cutting tool

(Diamond discs, NTI-Kahla GmbH, Germany) and then molded using orthodontic resin on

an acrylic block (15x12x6 mm) (Ortho-jet, Lang Dental Mfg. Co., Inc., USA).

12

2.2.3. Pit and fissure sealant application and microleakage formation

In order to induce microleakage around the pit and fissure sealant, previous study (Hevinga

et al. 2007) have contaminated moisture or saliva prior to application of the pit and fissure

sealant so that the sealant penetrates incompletely to form a microleakage. Similar to the

previous method, 22 of the molded teeth were treated with sealant according to the

manufacturer's instructions, and the remaining 22 teeth formed microleakage by contaminating

saliva before applying the sealant. The pit and fissure of all teeth was etched for 15 seconds

using 35% phosphoric acid etchant (3M-ESPE, USA), washed with a water sprayer for 10

seconds, and dried sufficiently. ClinproTM (3M-ESPE, USA) was used as the pit and fissure

sealant material, and Dr's LightTM (Good Doctors Co., Ltd, Korea) was used for light curing.

For formation of no microleakage group, the sealant was applied according to the

manufacturer's instructions and then light cured for 40 seconds. For microleakage

formation, 0.1 ml of saliva (human saliva) was placed on the pit and fissure of the occlusal

surface of the tooth for 10 seconds, the excess saliva was absorbed with a Kimtech science

wiper, and a sealant was applied and light cured for 40 seconds. Then, referring to the

previous study (Jeong et al. 2014), all specimens were immersed in distilled water for 48

hours for the purpose of reproducing the intraoral environment by inducing hydration

expansion of the sealant.

13

2.2.4. QLF-D image taking

To evaluate the microleakage of the pit and fissure sealant, each tooth specimen was captured

by using a Quantitative Light-induced Fluorescence-Digital system (QLF-D biluminator TM,

Inspektor Research systems BV, Amsterdam, The Netherlands) in a dark room under the same

conditions. The QLF-D is a single lens reflex (SLR) camera with two light-emitting diodes

(LEDs) lamps mounted on the front of a 60mm macro lens. In the visible light region, 12 LEDs

of blue light (405 nm) and 4 LEDs of white light are attached, and different light sources can

be irradiated (Figure 2). The QLF-D camera was perpendicular to the occlusal surface of the

specimen and maintained a distance of 10 cm, and captured white light images and fluorescent

images continuously under the same conditions (Table 1).

14

Figure 2. QLF-D camera (QLF-D BiluminatorTM)

Table 1. Photographing condition of QLF-D in this study

White light Blue light

Shutter speed 1/13s 1/50

Aperture value 14.0 10.0

ISO speed 250 1600

Pixel size 2592 × 1728 2592 × 1728

15

2.2.5. Fluorescence assessment and analysis using QLF-D

All images were analyzed using specific program (QA2 v1.26, Inspektor Research Systems

BV, Amsterdam, The Netherlands) to quantify the fluorescence of the microleakage detected in

the pit and fissure sealant margins.

To quantify the fluorescence of the microleakage detected in the pit and fissure sealant

margins, images taken using a dedicated analysis program (QA2 v1.26, Inspektor Research

Systems BV, Amsterdam, The Netherlands) were analyzed. The cross section of each pit was

marked with a water-based pen on an acrylic block molded with a crown to analyze the mesial

and distal pit of each specimen and cut the same site in histological evaluation. When the sound

part was selected as the patch in the mesial and distal pit margin of the sealant, the microleakage

site showed relatively fluorescence loss. The average fluorescence loss, which represents the

amount of fluorescence lost compared to the sound portion of the region of interest (ROI), was

calculated as ΔF [%]. The maximum fluorescence loss, which represents the largest amount of

fluorescence loss, was calculated as ΔFmax [%] (Figure 3).

16

Figu

re 3

. (A

) A re

pres

enta

tive

occl

usal

fluo

resc

ence

imag

e. (B

) A m

agni

fied

imag

e fo

r ana

lysi

s. (C

) A d

esig

ned

patc

h ar

ea

arou

nd m

argi

n of

seal

ant.

(D) A

reco

nstru

cted

imag

e ba

sed

on th

e flu

ores

cenc

e of

the

soun

d ar

ea. T

he b

lue

line

indi

cate

s the

soun

d re

fere

nce

area

, whe

reas

the

red

lin

e in

dica

tes

the

deac

tivat

ed a

rea.

(E)

The

flu

ores

cenc

e di

ffere

nce

betw

een

the

orig

inal

and

reco

nstru

cted

imag

es. (

F) A

naly

sis r

esul

ts fr

om th

e mar

gin

of se

alan

t. (G

) A to

tal o

f 4 an

alyz

ed p

atch

es in

sing

le

toot

h sa

mpl

e.

17

2.2.6. Histological analysis

Nail varnish was applied twice on the tooth surface except for 1mm of the margin of the

sealant, and dried to prevent unnecessary dye penetration from the specimens. Then, it was

immersed in a 1% methylene blue solution for 24 hours to allow dye to penetrate between the

sealant and the tooth surface (Hevinga et al. 2007). After each tooth is washed and dried under

running water, the mesial pit and distal pit sectioned longitudinally in the bucco-lingual

direction using an automatic cutter (sectioned longitudinally) and 4 cut sections per tooth, i.e.

two opposite tooth sections were obtained for each pit (mesial, distal). The mesial and distal

pit of each tooth were recorded by selecting the higher score of dye penetration among the

same two tooth surfaces when viewed from the bucco-lingual direction, and 4 histological

scores per tooth were obtained (Figure 4).

In order to evaluate the microleakage of the exposed cut surface and the degree of

penetration of the dye, it was observed with an optical microscope (Carl Zeiss Axio imager

A1m, Oberkochen, Germany) at a magnification of 50, and classified and evaluated

according to prior criteria (Ovrebö and Raadal 1990) (Figure 5).

18

Figure 4. Sample preparation for observing the cross-sectional view of mesial and distal pit

19

The buccal and lingual parts of each tooth section were recorded by observing the degree

of dye penetration, respectively, and the histological score was selected for the more dye

penetration in each mesial and distal pit. Microleakage score is as follows; Score o, no dye

penetration; Score 1, dye penetration restricted to the outer half of the pit and fissure sealant;

Score 2, dye penetration to the inner half of the pit and fissure sealant; Score 3: dye

penetration into the underlying fissure.

Figure 5. Schematic diagram of the cross-sectioned specimen for measuring microleakage

and dye penetration

20

2.3. Clinical image study

2.3.1. Study subject

In this study, data was collected based on subjects who voluntarily participated in dental

clinics, dental hospitals, and dental university hospitals among three medical institutions

located in Seoul from January 2017 to March 2020. Total 28 teeth with pit and fissure sealant

were captured on the occlusal surface using a Q-ray pen. Twenty-nine occlusal images were

used for analysis, and 12 difficult-to-read photos were excluded.

In order to evaluate the validity of the evaluation method using a fluorescent image from

Q-ray pen (Aiobio Inc., Seoul, Korea, Figure 6), one skilled examiner evaluated the method

using the white light and the fluorescent image in combination.

The examination was conducted twice to identify the difference between image

evaluation methods depending on the presence or absence of fluorescent image. Three

skilled clinicians performed a 1st examination using only white light images, and white

light and fluorescence images were used for evaluation in the 2nd examination. The three

examiners were trained in the evaluation criteria using white light and fluorescence image

before evaluation. In order to minimize the effect between the first and second

examinations, each test was divided, and all images were presented as random. The

evaluation was conducted with a washout period of about 2 hours between the first test and

the second test.

21

Figure 6. Q-ray penTM

22

2.3.2. Image assessments of Q-ray pen

The evaluation items of white light images were classified into four categories: marginal

plaque, marginal discoloration, retention, and caries (Ünal et al. 2015). In the fluorescence

image, evaluation criteria considering the fluorescence expression pattern were set and

scored by reflecting the principles and characteristics of QLF for three items (marginal

plaque, marginal discoloration, caries) that can be evaluated (Table 2).

2.3.3. Fluorescence image assessment and analysis using Q-ray pen

To compare the image evaluation method and the quantitative analysis value, one trained

examiner performed white light and fluorescence image inspection on the occlusal surface

of the sealant according to the evaluation criteria. All images were re-evaluated a week

later to evaluate reproducibility. To obtain quantified values from fluorescence images, all

images were analyzed using a specific analysis program. All images were analyzed using

a specific analysis program (QA2 v1.26, Inspektor Research Systems BV, Amsterdam, The

Netherlands) to obtain quantified values from fluorescent images. A patch was designated

to calculate the fluorescence value of each area of interest since the pit and fissure sealant

is widely distributed on the occlusal surface. Fluorescence parameter was calculated as the

average fluorescence loss (∆F), maximum fluorescence loss (∆Fmax), and the difference in

red fluorescence (∆R) due to metabolism of microorganisms (Figure 7).

23

Tabl

e 2.

Crit

eria

of i

mag

e ev

alua

tion

for p

it an

d fis

sure

seal

ant s

tatu

s

24

Figure 7. (A) A representative occlusal white light image. (B) A representative occlusal

fluorescence image. (C) A reconstructed image based on the fluorescence of the sound area.

The blue line indicates the sound reference area, whereas the red line indicates the

deactivated area. (D) Analysis results from the margin of sealant. (E) Analyzed patches in

single tooth sample.

25

2.4. Clinical trial

2.4.1. Study subject

This study was conducted from May to June 2017 at a dental hygiene department of a

university located in Dongdaemun-gu, Seoul. The subjects who participated in the study were

15 volunteers among 3rd grade dental hygiene departments. Subjects had at least one tooth that

had been treated with pit and fissure sealant six months ago, and total 58 teeth were examined.

The first examination of the pit and fissure sealant was evaluated using visual and tactile

inspection method by 3 experienced examiners, and 2nd examination was performed by

using the Q-ray view together with the first inspection method. All examiners were trained

and calibrated on evaluation criteria using white light and fluorescent image prior to the

examination. To ensure that the 1st and 2nd examinations were not affected by each other,

evaluation sheets were prepared for each test. In addition, the evaluation was conducted

with a washout period of about 2 hours between the examinations.

26

2.4.2. Visual and tactile assessments

All subjects were brushed to remove the plaque around the pit and fissure sealant prior to

the examination, and the dental chair was examined under the same illumination. After the

occlusal surface of the specimen was sufficiently dried, the examiner evaluated it with a

naked eye. The evaluation items are classified into five categories according to previous

study (Ünal et al. 2015); marginal plaque microbial membrane, marginal discoloration,

marginal integrity, retention, and caries (Table 3).

27

Table 3. Criteria of evaluation for pit and fissure sealant status

Surface characteristic Score Clinical Criteria QLF Criteria

Marginal plaque

0 No plaque No red fluorescence changes

1 Presence of plaque accumulation limited to margin

Red fluorescence emission limited to margin

2 Presence of plaque accumulation around sealant including margin

Red fluorescence emission around sealant including margin

Marginal discoloration

0 No discoloration No fluorescence changes

1 Presence of marginal discoloration less than 50% Fluorescence loss less than 50%

2 Presence of marginal discoloration more than 50% Fluorescence loss more than 50%

Marginal integrity

0 Not detectable with an explorer Not detectable with an explorer

1 Margin detectable with an explorer

Margin detectable with an explorer

2 Crevice detectable with an explorer

Crevice detectable with an explorer along the margin of visible width and depth

Retention

0 Completely retained material Completely retained material

1 Partially lost material Partially lost material

2 Totally lost material Totally lost material

Caries

0 No caries No fluorescence changes

1 Initial caries (no cavity) Slight fluorescence loss and/or red fluorescence emission

2 Enamel or dentin caries (cavity formation)

Distinct fluorescence loss and/or red fluorescence emission

28

2.4.3. Q-ray view combined assessment

The same sealant tooth was evaluated using visual, palpation, and Q-ray views (Aiobio

Inc., Seoul, Korea, Figure 8), and all evaluation items were identical. Of the five evaluation

items, three items (marginal plaque, marginal discoloration, and caries) were modified for

fluorescence image evaluation.

First, the marginal plaque evaluation was evaluated with or without red fluorescence. This is

based on the rationale that it is possible to detect dental plaque with red fluorescence using QLF

technology (Kim 2011). The score was classified as follows; Score 0, no red fluorescence

observed; Score 1, red fluorescence was observed only at the margin of the sealant due to the

presence of dental plaque; Score 2, red fluorescence was observed around the teeth and sealant.

Next, the marginal discoloration item were modified based on the results of previous

studies (Lee et al. 2018) that can distinguish the discoloration characteristics using

fluorescence images. This previous study identified by using QLF technology that non-

cariogenic and cariogenic discoloration can be distinguished, and as the intensity of

discoloration increases, fluorescence disappears and dark discoloration is observed.

Accordingly, marginal discoloration was classified as follows; Score 0, no fluorescence

change; Score 1, the fluorescence loss less than 50%; Score 2, florescence loss more than

50%.

Evaluation criteria for caries were scored by setting criteria reflecting both fluorescence loss

and red fluorescence expression. QLF technology can quantify the mineral change of the initial

29

carious lesion with fluorescence loss (Ellwood, Gomez, and Pretty 2012), and detect the

increased bacterial activity in caries lesions with red fluorescence (Kim and Kim 2017). Based

on this rationale, caries evaluation items were classified as follow; Score 0, no fluorescence

change; Score 1, slight fluorescence loss and/or red fluorescence emission; Score 2, distinct

fluorescence loss and/or red fluorescence emission.

Figure 8. Q-ray view TM

30

2.5. Statistical analysis

All analyzes of this study were at a significance level of 0.05 using statistical programs

(R core team (2018). R: A language and environment for statistical computing. R

Foundation for Statistical computing, Vienna, Austria. URL https: //www.R-project.org/.).

2.5.1. In vitro study (Study 1)

The correlation between histological evaluation results (gold standard) and fluorescence

parameter values was calculated using Spearman's rank correlation. The sensitivity,

specificity, and area under the ROC (AUC) of each fluorescence variable were analyzed to

evaluate the validity of the fluorescence variables ΔF and ΔFmax that detect each score in

the histology classification.

2.5.2. Clinical image study (Study 2)

Independent sample t-test was performed to quantitatively analyze the pit and fissure

sealant using a fluorescent image. Cohen's kappa value was used to calculate the

consistency within the examiner. Fleiss kappa and Cohen's kappa values were used to

calculate the agreement between the three examiners on the results of white-light and

fluorescence image evaluation.

31

2.5.3. Clinical trial (Study 3)

Fleiss kappa and Cohen's kappa values were used to calculate the inter- and intra-

examiner agreement between conventional method (visual and tactile inspection) and

combination method with Q-ray view. In order to test whether the difference between the

mean kappa values of conventional and combination method was significant, it was

analyzed by paired t-test.

32

III. RESULTS

3.1. In vitro study results (Study 1)

3.1.1. Comparison of histological distribution by microleakage formation

Four of the 44 extracted teeth were damaged after histological evaluation and distilled

water deposition. In total, 160 tooth surfaces (40 specimens) were analyzed and it includes

22 sealants formed according to the manufacturer's instructions and 18 teeth forming

microleakage (Table 4).

Table 4. Histological distribution of specimens by microleakage formation

Histology Microleakage (N=160)

0 35 (21.88%)

1 39 (24.38%)

2 41 (25.63%)

3 45 (28.13%)

33

3.1.2. Distribution of fluorescence variables according to the histological score

In order to quantitatively evaluate the microleakage of the pit and fissure sealant,

variables of average fluorescence loss (ΔF) and maximum fluorescence loss (ΔFmax) were

used. In each histological evaluation, the absolute values of ΔF and ΔFmax increased as the

depth of microleakage by methylene blue dye penetration increased (Figure 9). The

fluorescence parameter from QLF-D increased with increasing histological classification

criteria at all stages (p<0.05, Table 5).

Table 5. Distribution of fluorescence variables according to the histological score after

microleakage formation

Histology N Fluorescence variables

ΔF ΔFmax

0 35 -6.1 ± 0.8a -7.3 ± 2.5 a

1 39 -7.2 ± 1.6b -11.2 ± 4.4 b

2 41 -9.5 ± 2.5c -15.9 ± 4.6 c

3 45 -11.0 ± 2.8d -19.4 ± 7.5 d

All values are expressed as mean ± standard deviations. Different letters within the same column indicate significant differences between groups by Games-Howell post hoc analysis at a=0.05.

34

Figu

re 9

. Box

plo

t sho

win

g ΔF

, ΔF m

ax v

alue

s rel

ated

to h

isto

logi

cal s

core

35

3.1.3. QLF-D images and magnification microscope images of microleakage in pit and

fissure sealant according to the histological score

Figure 10 shows the result of confirming the ΔFmax value and dye penetration pattern

according to the histological score stage. The absolute value of ΔFmax and dye penetration

increased with each step of the histological score.

Figure 10. Representative QLF-D images (white-light and fluorescence images) and a

magnification microscope images (×50) of specimens according to histological score

36

3.1.4. Correlation of variable between histology and fluorescence values

Fluorescence parameters (ΔF and ΔFmax) and histological score of the microleakage site

evaluated by QLF-D showed a statistically significant correlation (p<0.001, Table 6). The

correlation coefficient of histological classification and each variable showed high value

with both ΔF and ΔFmax of -0.72 (Table 6).

Table 6. Correlation coefficients between histology and fluorescence variables of microleakage

Histology Fluorescence variables

ΔF ΔFmax

Histology 1

ΔF -0.72*** 1

ΔFmax -0.72*** 0.95*** 1

*** Spearman’s rank correlation analysis, P<0.001

37

3.1.5. Validity of QLF-D

3.1.5.1. AUC analysis for diagnostic accuracy of fluorescence variables

The AUC of each fluorescence variables (ΔF, ΔFmax) was presented at optimal cutoff

points calculated from three diagnostic thresholds to evaluate the feasibility of diagnostic

ability according to the depth of microleakage of pit and fissure sealant using QLF-D (Table

7). In all diagnostic criteria, the AUC of the fluorescence parameters ΔF and ΔFmax values

showed high value. When the outer half microleakage of the pit and fissure sealant

(histological score 0 vs 1-3) was cutoff, the AUC of ΔFmax value was 0.91, which showed

very good diagnostic ability.

Table 7. Area under the ROC curve of fluorescence variables at each histology criteria

Histology criteria Fluorescence variables ROC

AUC 95% CI

Histo 0/1-3 ΔF 0.88 0.82-0.93

ΔFmax 0.91 0.87-0.96

Histo 0-1/2,3 ΔF 0.90 0.86-0.95

ΔFmax 0.89 0.84-0.94

Histo 0-2/3 ΔF 0.83 0.77-0.90

ΔFmax 0.83 0.76-0.89

AUC, Area under the curve; CI, confidence interval Histo; Histological score

38

3.1.5.2. Sensitivity, specificity, and AUC curves of fluorescence variables at outer half

microleakage of the pit and fissure sealant

Sensitivity, specificity and AUC were calculated respectively to assess the diagnostic

validity of distinguishing between no microleakage and outer half microleakage

(histological score 0 vs 1-3) in pit and fissure sealant. As a result of histological

examination, when the outer half microleakage was used as a diagnostic threshold, the

AUC value of ΔFmax was 0.91, which showed the highest validity. In ΔF value, the

sensitivity was 79.2, the specificity was 91.4, and in ΔFmax value, the sensitivity was 82.4,

and the specificity was 91.4 (Figure 11).

Figure 11. Sensitivity, specificity, and AUC curves of fluorescence variables at outer half

microleakage of the pit and fissure sealant

39

3.1.5.3. Sensitivity, specificity, and AUC curves of fluorescence variables at inner half

microleakage of the pit and fissure sealant

Sensitivity, specificity and AUC were calculated respectively to assess the diagnostic

validity of distinguishing between outer half microleakage and inner half microleakage

(histological score 0-1 vs 2-3) in pit and fissure sealant. Histological examination showed

that the AUC value of ΔF was 0.90 when the inner half microleakage was used as the

diagnostic threshold, which showed the highest validity. In ΔF value, sensitivity 84.9,

specificity was 82.4, in ΔFmax value, sensitivity was 95.3, and specificity was 68.9 (Figure

12).

Figure 12. Sensitivity, specificity, and AUC curves of fluorescence variables at inner half

microleakage of the pit and fissure sealant

40

3.1.5.4. Sensitivity, specificity, and AUC curves of fluorescence variables at underlying

fissure microleakage of the pit and fissure sealant

Sensitivity, specificity and AUC were calculated respectively to assess the diagnostic

validity of distinguishing between inner half microleakage and underlying fissure

microleakage (histological score 0-2 vs 3) in pit and fissure sealant. When the underlying

fissure microleakage was the diagnostic threshold, the AUC value of ΔF was the highest

validity (0.83) in histological examination. In ΔF value, sensitivity 80.0, specificity was

78.3, in ΔFmax value, sensitivity was 82.2, and specificity was 73.0 (Figure 13).

Figure 13. Sensitivity, specificity, and AUC curves of fluorescence variables at underlying

fissure microleakage of the pit and fissure sealant

41

3.2. Clinical image study results (Study 2)

3.2.1. Study population

In this study, images of occlusal surfaces of 41 pit and fissure sealant teeth from 28

volunteers were obtained. A total of 129 sites were evaluate except for 12 teeth that are difficult

to judge.

3.2.2. Distribution of fluorescence variables according to pit and fissure sealant status

Fluorescence images of pit and fissure sealant captured with a Q-ray pen were used for

analysis obtaining quantitative values such as the average fluorescence loss (ΔF), the maximum

fluorescence loss (ΔFmax), and the average red fluorescence change (Average change of red

fluorescence; ΔR). One examiner evaluated the presence or absence of sealant microleakage

for each fluorescence value. As a result, the absolute values of ΔF, ΔFmax, and ΔR increased

when microleakage was present excluding ΔR in the retention (p<0.05, Table 8). When

retesting to evaluate the reliability of results, the intrarater agreement showed a very good

reliability of 0.89 or more (Table 9).

42

Tabl

e 8.

Dis

tribu

tion

of fl

uore

scen

ce v

aria

bles

acc

ordi

ng to

pit

and

fissu

re se

alan

t sta

tus

Fluo

resc

ence

var

iabl

es

N

ΔF

ΔF

max

ΔR

M

ean±

SD

P-va

lue*

Mea

n±SD

P-

valu

e* M

ean±

SD

P-va

lue*

Mar

gina

l pl

aque

Non

e 27

-4

.62±

4.22

<

0.00

1 -7

.59±

10.6

6 <

0.00

1 1.

46±7

.60

< 0.

001

Pres

ence

10

2 -9

.08±

3.61

-18.

75±1

3.27

13.5

9±13

.73

Mar

gina

l di

scol

orat

ion

Non

e 10

9 -7

.74±

4.25

0.

009

-14.

57±1

2.09

<

0.00

1 10

.01±

13.1

9 0.

042

Pres

ence

20

-1

0.37

±2.6

7

-26.

43±1

6.59

16.7

2±14

.75

Ret

entio

n

Non

e 12

3 -7

.91±

3.89

0.

003

-15.

76±1

2.99

0.

013

10.6

0±13

.33

0.09

1

Pres

ence

6

-13.

04±6

.45

-2

9.66

±18.

41

20

.23±

17.1

6

Car

ies

Non

e 10

2 -7

.16±

3.56

<

0.00

1 -1

1.94

±7.9

0 <

0.00

1 7.

33±1

1.67

<

0.00

1

Pres

ence

27

-1

1.94

±4.1

3

-33.

28±1

6.83

25.1

3±11

.01

All

valu

es a

re e

xpre

ssed

as m

ean

± st

anda

rd d

evia

tions

. * P-

valu

es w

ere

obta

ined

by

inde

pend

ent s

ampl

e t-t

est.

43

Table 9. Intrarater reliability of two images examination methods for evaluating pit and

fissure sealant status

WL: white-light image assessment F: white-light with fluorescence image assessment All values represent Cohen’s kappa values.

Measurement parameters

Marginal

plaque

Marginal

discoloration Retention Caries

WL F WL F WL F WL F

Intrarater - 1.000 0.965 0.970 0.885 1.000 0.974 1.000

44

3.2.3. Interrater reliability between White-light image and Fluorescence with white-

light image

The maintenance status of pit and fissure sealant was evaluated by classifying it into

four categories. First, the marginal plaque was not able to be analyzed because all values

were evaluated as score 0 in the white light image by two examiners. Interrater agreement

was higher when both white and fluorescence images were used (Table 10, 11, Figure 14).

Figure 14. Comparison of kappa values for interrater reliability between white-light image

and fluorescence with white-light image

45

Table 10. Interrater reliability of two images examination methods for evaluating pit and

fissure sealant status

WL: white-light image assessment F: white-light with fluorescence image assessment All values represent Cohen’s kappa values.

Table 11. Interrater reliability of two images examination methods for evaluating pit and

fissure sealant status

WL: white-light image assessment F: white-light with fluorescence image assessment All values represent Fleiss’ kappa values.

Inter

rater

Measurement parameters

Marginal

plaque

Marginal

discoloration Retention Caries

WL F WL F WL F WL F

A and B - 0.66 0.33 0.24 0.41 0.51 0.33 0.40

A and C - 0.59 0.40 0.31 0.26 0.57 0.29 0.67

B and C - 0.53 0.09 0.24 0.40 0.43 0.67 0.72

Interrater

Measurement parameters

Marginal

Plaque

Marginal

discoloration Retention Caries

WL F WL F WL F WL F

A and B and C - 0.59 0.28 0.25 0.33 0.50 0.46 0.60

46

3.2.4. Distribution of marginal plaque between White-light image and Fluorescence

with white-light image

When examining the presence or absence of plaque, the agreement rate between

examiners was 0.0 ̶ 33.3% in the white-light image, whereas the examination using both

white and fluorescence showed a higher agreement of 53.5 ̶ 72.1% (Table 12).

Table 12. Distribution of marginal plaque in white-light image and fluorescence with

white-light image

WL: white-light image assessment F: white-light with fluorescence image assessment All values represent n (%).

Rater

Marginal Plaque

WL F

None Plaque None Plaque

A 129 (100%) 0 (0%) 36 (27.9%) 93 (72.1%)

B 86 (66.7%) 43 (33.3%) 52 (40.3%) 77 (59.7%)

C 129 (100%) 0 (0%) 60 (46.5%) 69 (53.5%)

47

3.3. Clinical trial results (Study 3)

3.3.1. Study population

In this study, 58 teeth were evaluated in a total of 15 subjects. The average age of the study

subjects was 21.5 years, and the mandibular premolar ratio was the highest with 41.4% (Table

13).

Table 13. Demographic data

Variable Total (N=58)

Age (years)

Mean 21.5

Range 20-27

Sex

Female 15 (100%)

Male 0 (0%)

Dental arch

Maxilla 20 (34.5%)

Mandible 38 (65.5%)

Tooth

Premolar 37 (63.8%)

Molar 21 (36.2%)

48

3.3.2. Interrater reliability between VT and Q-ray view with VT

The evaluation of the maintenance status of the pit and fissure sealant was classified into

five categories. In all three categories, marginal plaque, marginal discoloration, and caries,

assessment with Q-ray view was statistically significantly higher than visual and tactile

assessments in interrater agreement (Figure 15, Table 14). Among them, the agreement rate

of marginal plaque and caries items was the same, and the difference in agreement rate

between visual and tactile assessment (0.22-0.57) and assessment with Q-ray view (0.81-

0.89) was the highest in the marginal plaque assessment items (Table 14).

The presence of dental plaques in the pit and fissure sealant margin is difficult to detect

with the naked eye, but red fluorescence from plaque was observed clearly at the margin

of the sealant when using Q-ray view (Figure 16-A, B). The marginal discoloration of the

sealant was detected as darker than the sound area due to loss of fluorescence in the

discolored area when observing the Q-ray view (Figure 16-C, D). In the case of initial caries,

it was difficult to confirm the initial caries by visual and tactile assessment, but the

fluorescence disappeared when observed with a Q-ray view. When using the Q-ray view,

the caries lesion showed relatively dark fluorescence or red fluorescence due to the activity

of the bacteria compared to the sound tooth (Figure 16-E, F).

49

Table 14. Interrater reliability of two examination methods for evaluating pit and fissure

sealant status

VT: visual and tactile assessment All values represent Cohen’s kappa values.

Figure 15. Comparison of mean kappa values for interrater reliability between VT and

Q-ray view VT: visual and tactile assessment *Exhibited significant differences in mean kappa value between two examination methods (paired t-test, p<0.05)

Inter rater

Measurement parameters

Marginal Plaque

Marginal discoloration

Marginal integrity

Retention Caries

VT Q-ray view

VT Q-ray view

VT Q-ray view

VT Q-ray view

VT Q-ray view

A and B 0.47 0.89 0.55 0.78 0.40 0.30 0.70 0.57 0.61 0.90

A and C 0.57 0.81 0.57 0.69 0.32 0.32 0.57 0.71 0.43 0.84

B and C 0.22 0.84 0.36 0.88 0.63 0.69 0.63 0.62 0.56 0.80

50

Figure 16. Representative images of marginal plaque (A, B), marginal discoloration (C, D),

and caries (E, F) at pit and fissure sealant margin. White-light image (A, C, E), Q-ray pen

image (B, D, F).

51

3.3.3. Intrarater reliability between VT and Q-ray view with VT

In intrarater agreement, Q-ray view combined assessment showed higher agreement than

visual and tactile assessment. in marginal plaque (0.81-0.83), marginal discoloration (0.57-

0.89), caries (0.69-0.91). In particular, marginal plaque showed statistically significant

differences. In the Q-ray view combined assessment, marginal integrity and retention

evaluation items showed similar agreement with visual and tactile assessment (Table 15,

Figure 17).

Table 15. Intrarater reliability of two examination methods for evaluating pit and fissure

sealant status

VT: visual and tactile assessment All values represent kappa values.

Intrarater

Measurement parameters

Marginal Plaque

Marginal discoloration

Marginal integrity Retention Caries

VT Q-ray view VT Q-ray

view VT Q-ray view VT Q-ray

view VT Q-ray view

A 0.54 0.83 0.55 0.77 0.45 0.30 0.78 0.70 0.65 0.78

B 0.49 0.81 0.48 0.57 0.33 0.41 0.73 0.78 0.42 0.69

C 0.50 0.83 0.48 0.89 0.68 0.79 0.79 0.79 0.60 0.91

52

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53

3.3.3.1. Intrarater reliability of marginal plaque assessment of pit and fissure sealant

As a result of calculating the degree of agreement within the examiner in the marginal

plaque assessment, visual and tactile assessment (0.49-0.54) showed a moderate agreement,

while Q-ray view assessment (0.81-0.83) showed an almost perfect agreement. In all three

examiners, the Q-ray view combined assessment showed higher agreement than the visual

and tactile assessment (Figure 18).

Figure 18. Comparison of mean kappa values for intrarater reliability between VT and

Q-ray view of marginal plaque assessment VT: visual and tactile assessment

54

3.3.3.2. Intrarater reliability of marginal discoloration assessment of pit and fissure sealant

As a result of calculating the consistency within the examiner in the marginal

discoloration assessment, visual and tactile assessment (0.48-0.55) showed moderate

agreement, but Q-ray view assessment (0.57-0.89) was excellent (substantial agreement)

or very good (almost perfect agreement). In all three examiners, the Q-ray view combined

assessment showed higher agreement than the visual and tactile assessment (Figure 19).

Figure 19. Comparison of mean kappa values for intrarater reliability between VT and

Q-ray view of marginal discoloration assessment VT: visual and tactile assessment

55

3.3.3.3. Intrarater reliability of marginal intergrity assessment of pit and fissure sealant

The intrarater agreement for marginal integrity showed that visual and tactile assessment

was 0.33-0.68, which means from fair to substantial agreement, and assessment with Q-ray

view was 0.30-0.79, which means from fair agreement to excellent agreement. All three

examiners showed similar agreement between assessment with Q-ray view and visual and

tactile assessment (Figure 20).

Figure 20. Comparison of mean kappa values for intrarater reliability between VT and

Q-ray view of marginal integrity assessment

VT: visual and tactile assessment

56

3.3.3.4. Intrarater reliability of retention assessment of pit and fissure sealant

The retention assessment showed that visual and tactile assessment (0.73-0.79) had

excellent (substantial agreement) agreement, and Q-ray view combined assessment (0.70-

0.79) showed excellent (substantial agreement) agreement. All three examiners showed

similar agreement between Q-ray view combined assessment and visual and tactile

assessment (Figure 21).

Figure 21. Comparison of mean kappa values for intrarater reliability between VT and

Q-ray view of retention assessment VT: visual and tactile assessment

57

3.3.3.5. Intrarater reliability of caries assessment of pit and fissure sealant

Intrarater reliability in caries assessment showed that visual and tactile assessment was

0.42-0.65, which means from moderate to substantial agreement, and Q-ray view combined

assessment was 0.69-0.91, which means from substantial to very perfect agreement. In all

three examiners, the Q-ray view combined assessment showed higher agreement than the

visual and tactile assessment (Figure 22).

Figure 22. Comparison of mean kappa values for intrarater reliability between VT and

Q-ray view of caries assessment VT: visual and tactile assessment

58

IV. DISCUSSION

The dental caries prevention function of the pit and fissure sealant is possible only when this

material remains on the restored tooth and is well combined with the enamel to prevent

microleakage (Hatibovic-Kofman, Butler, and Sadek 2001; Pope et al. 1996; Symons, Chu,

and Meyers 1996). There is always a risk that microleakage caused by insufficient sealing of

the pit and fissure sealant will progress to macroleakage (Kidd, Joyston-Bechal, and Beighton

1995; Mjör 2005). The most important factor for the retention of the pit and fissure sealant is

the penetration of the material into the fissure. It is important to penetrate deep into the fissure

in the process of applying the pit and fissure sealant. Because air bubbles or occlusal force can

weaken the sealant and form partial loss or microleakage (Carlos et al. 1984; Symons, Chu,

and Meyers 1996; Taylor and Gwinnett 1973). Pit and fissure sealant products generally have

less filler content than composite resins, so they have low mechanical properties such as

abrasion resistance or marginal toughness. Materials with weak physical properties may cause

clinical problems such as fractures and abrasions where the occlusal force is strongly applied

or in thin marginal areas (Futatsuki et al. 1995). Therefore, an objective and accurate diagnosis

of the current state after applying the pit and fissure sealant is essential in clinical practice.

The purpose of this study was to evaluate the validity and reliability of QLF technology

as a tool to detect pit and fissure sealant.

In the Study 1, microleakage generated at the marginal areas of the pit and fissure sealant

was confirmed using QLF-D, which is a fluorescence detection device, and the

59

fluorescence pattern according to the depth of the microleakage was evaluated. QLF-D can

monitor not only the current state but also the detailed change by presenting the

fluorescence change as a quantitative value (Ando et al. 2004; Pretty, Edgar, and Higham

2002). Fluorescence parameters of QLF-D showed a high level of diagnostic accuracy

compared to the gold standard, and confirmed the possibility of clinical use of QLF

technology for evaluation of pit and fissure sealant.

In the Study 1 results, fluorescence expression appeared in two patterns according to the

depth of the microleakage, and it could be subdivided in 4 steps. The first fluorescence

expression pattern is Delta F (ΔF, %), which represents the average amount of fluorescence

loss, which indicates that the margins of the pit and fissure sealant show darker lines. The

ΔF value increased significantly as histological score increased, and a relatively strong

correlation could be confirmed (Table 4, 5, Figure 9). According to previous studies, it was

found that the ΔF value indicating the degree of mineral loss was similar to an index

reflecting the histological lesion depth (Gmür et al. 2006). In addition, the ΔF value is used

as a clinical guideline that provides suitable treatment methods according to the severity of

the lesion (Alammari et al. 2013). The darker line between the pit and fissure sealant and

the enamel is thought to be due to the increased porosity due to the loss of minerals in the

microleakage area and the lowest reflectance of light in the deepest region.

The second fluorescence expression pattern is ΔFmax (ΔFmax, %), which represents the

maximum amount of fluorescence loss, and refers to the deepest portion. Previous studies

reported that ΔFmax (ΔFmax, %) means the darkest region among fluorescence variables and

60

reflects the deepest point of the region (Jun et al. 2016). Similar to the previous studies, the

results of this study also significantly increased the ΔFmax value as the histological score

increased. In addition, there was a high correlation between ΔFmax and the degree of

penetration of dyes evaluated for microleakage (Table 5, Figure 9). The ΔF value can be

measured differently depending on the ROI measurement (Pretty et al. 2002). However,

ΔFmax can detect the fluorescence loss of deep lesions excellently below the pit and fissure

of the occlusal surface (Angmar-Månsson and ten Bosch 2001; van der Veen and de

Josselin de Jong 2000). In addition, since the ΔFmax value represents the deepest region, the

ROI of the tooth crack does not change significantly (Jun et al. 2016). Therefore, it is

considered to be appropriate to use the ΔFmax fluorescence parameter when analyzing

microleakage of pit and fissure sealant quantitatively.

In this study, the tooth with pit and fissure sealant was captured using QLF-D and the

fluorescence parameters were compared with histological scores for each methylene blue

dye penetration. As a result, the dye penetration increased as the fluorescence parameters

increased (Figure 10). Conventional methods for evaluating microleakage were radiation

isotopes, bacteria, air pressure, neutron activity analysis, scanning electron microscopy,

interface penetration, and use of dyeing solutions (Going 1972). Among them, the dyeing

solution is most useful because it is simple, and methylene blue has a small molecular

weight and high penetration (Hatibovic-Kofman, Wright, and Braverman 1998). Therefore,

in this study, methylene blue was used for histological evaluation of microleakage

penetration according to the method of Overbo and Raddal (Ovrebö and Raadal 1990).

61

As a result of evaluating the diagnostic accuracy according to each threshold, the

fluorescence parameter was appropriate to classify the level of microleakage. ΔFmax showed

high sensitivity, specificity, and AUC (0.91) value when the outer half microleakage

(histological score 0/1) was cutoff. When the inner half microleakage (histological score

1/2) was cutoff, ΔF showed high sensitivity, specificity, and AUC (0.90) value confirming

a very good diagnostic ability. All fluorescence values showed an AUC of 0.83 or higher

and excellent diagnostic ability. This result showed that QLF-D can detect changes in pit

and fissure sealant and accurately quantify microleakage. Therefore, it is considered to be

very useful for evaluating the maintenance state after the pit and fissure sealant.

In Study 2, the maintenance status of the pit and fissure sealant was identified in a

fluorescence image captured by Q-ray pen and evaluated according to the fluorescence

value. In addition, the reliability of the test using both white light and fluorescence images

of the Q-ray pen was evaluated to confirm the possibility of clinical use of QLF technology.

Park et al. confirmed that they can be used interchangeably because there is no significant

difference in fluorescence loss (ΔF) value when comparing diagnostic accuracy for

detection of initial caries lesions by QLF device (Park et al. 2018).

It was confirmed that the presence or absence of marginal plaque showed a significant

difference in fluorescence variables (Table 8). The agreement for plaque detection was 0-

33% in the white light image test, whereas it was 53.5-72.1% in both white and

fluorescence image (Table 12). This was consistent with the results of Ozyoney et al

(Ozyoney et al. 2005), which compared the conventional test (3.9%) and QLF test (52.7%)

62

in plaque detection. This can be explained that plaque accumulated due to microleakage

between the sealant and the enamel was observed in red in the fluorescence image, making

detection easy.

As a result of the marginal discoloration evaluation, there was a significant difference in

the fluorescence variable, but the agreement between examiners using both white light and

fluorescent images was similar (Table 8, 10, 11, Figure 14). This is thought to have

limitations in the detection of fluorescence images because the discoloration state of the pit

and fissure sealant teeth included in this study is not variously distributed. Retention

evaluation items showed significant differences in the fluorescence variables ΔF and ΔFmax,

and the agreement between examiners was improved when both white- and fluorescent

images were used compared to when only white light images were used (Table 8, 10, 11,

Figure 14). It was consistent with previous studies that agreement was higher when QLF

technology was used in detecting oral restorations (Oh et al. 2014). This is because the

fluorescence expression of QLF was able to confirm the partial loss of material easily.

In caries assessment and interrater agreement were higher when both white and

fluorescent images were used than only used white-light image (Table 8, 10, 11, Figure 14).

This was consistent with the results of previous studies that interrater agreement for caries

assessment showed higher when QLF technology was used (Oh et al. 2014). It is difficult

to identify caries in white light images, but it is considered that it can be evaluated more

objectively using fluorescent images.

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To evaluate the maintenance of pit and fissure sealant, Study 3 compared the reliability

of conventional (visual and tactile inspection) and combination method using Q-ray view.

As a result, it was confirmed the possibility of using QLF technology to objectively

examine the retention status of the restoration. By using the fluorescence reaction detected

from QLF technology in the clinical field, it is possible to more objectively and easily detect

various factors related to the retention state of the pit and fissure sealant.

As a result of Study 3, marginal plaque, marginal discoloration, and caries, which

showed high reliability when using a Q-ray view, are clinically important factors. The

criteria for evaluating the success of pit and fissure sealant in the clinical field is that the

sealant teeth have no marginal discoloration and caries, and margin cannot be felt when

using explorer (Cho et al. 2003).

This study compared interrater agreement between conventional (visual and tactile

inspection) and combination method using Q-ray view for evaluating the retention status

of pit and fissure sealant. The result showed that the agreement of combination method was

higher than conventional method (Figure 15, Table 14). These results were the same in the

intrarater agreement (Figure 17, Table 15). In the results of Ozyoney et al., retention status

of pit and fissure sealant could be easily detected when using QLF technology. This

previous study evaluated the sealant after 24 months by two examiners about retention

(partially lost), marginal plaque, marginal discoloration, and caries, and it can’t be found

in conventional method (Ozyoney et al. 2005). The adhesion of pit and fissure sealant must

be ensured to prevent caries (Dennison, Straffon, and Smith 2000). The microleakage

64

between the enamel and the sealant causes marginal discoloration (Kidd 1976), and

facilitates the accumulation of bacterial membranes at the restoration interface (Dunkin and

Chambers 1983), allowing the bacteria to penetrate into the restoration margins (Fraser

1929; Harrison 1964; Krauss and Krauss 1959; Rose et al. 1955; Seltzer 1955). This, in turn,

causes dropout of the sealant (Futatsuki et al. 1995) or secondary caries in the margin of

the restoration (Hatibovic-Kofman, Wright, and Braverman 1998). Therefore, when

clinically evaluating teeth with pit and fissure sealant, it is essential to check for marginal

plaque, marginal discoloration, and caries. In this case, it will be more accurate and

objective evaluation if the QLF system that proved the feasibility in this study is used.

In particular, when the dental plaque of the pit and fissure sealant margin was examined

using the Q-ray view, the reliability was highest with a level of 'very good' and the deviation

of interrater and intrarater was the lowest. When bacteria are infiltrated into the

microleakage of the pit and fissure sealant, plaque is deposited and secondary caries is

generated (Going 1972). According to previous studies, QLF technology can detect

metabolites of specific bacteria in the oral cavity with red fluorescence, so it is possible to

quantify mature pathogenic plaques with red fluorescence distribution and intensity

(Coulthwaite et al. 2006; Lee et al. 2013). QLF technology showed higher reliability and

validity compared to visual inspection in plaque detection (Han et al. 2015). Red

fluorescence appears in an increased condition of pathogenic bacteria, so it is possible to

evaluate the prognosis of future lesion progression in the pre- disease stage (Kim and Kim

2017). The previous studies evaluated the pit and fissure sealant retention using QLF

65

technology under various criteria (retention, plaque, marginal discoloration, and caries),

and plaque detection can be easily detected with QLF among them. It is consistent with the

results of this study (Ozyoney et al. 2005). This previous study also reported that the

deposited plaque identified only 2.5% of the total teeth in the naked eye, but 18.5% plaque

can be detected using QLF technology. It is possible to clearly detect plaques with red

fluorescence by using QLF technology, which are early stage pathological factors affecting

the retention status of pit and fissure sealant. When evaluated with QLF technology, plaque

is considered to be the most reliable and easy factor for sealant retention.

When the Q-ray view was used to evaluate the marginal discoloration at the enamel and

sealant interface, the discolored areas were observed with dark fluorescence, so both the

interrater and intrarater agreement were higher than the visual and tactile assessment. Lee

et al. reported that cariogenic discoloration is distinguished using QLF technology. The

discoloration portion of the fluorescence image was clearly observed compared to visual

inspection, and the discoloration in the fluorescence image was darker as the intensity of

discoloration increased (Lee et al. 2018). This study also showed a dark margin of pit and

fissure sealant when observed using the Q-ray view. This may be due to the formation of

porosity, where was the loss of minerals, by the plaque accumulation in the margin, and

this part was affected by the chromogen of the external discolored material. In visual and

tactile assessment, the distinction of marginal discoloration was subjective and ambiguous,

but the Q-ray view confirmed that it was possible to objectively detect detailed marginal

discoloration.

66

In caries evaluation of sealant retention, compared to visual and tactile assessment, the

intrarater agreement was improved when the Q-ray view was used, and the interrater

agreement also showed a high level of 0.84 (p<0.05). Diniz et al. reported the results of

detecting secondary caries around restoration using QLF, and the inter-examiner agreement

was 0.9, which was higher than the agreement of visual inspection (0.85) (Diniz et al. 2016).

In this previous study, the interrater agreement was higher than this study, probably because

of the difference in the selection of teeth with secondary caries. Our study selected only

teeth with initial caries that occurred around the sealant, while previous studies included

teeth with moderate caries that occurred around the resin inlay. Therefore, most examiners

could clearly evaluate the cavitated tooth with naked eyes in the previous study. In contrast,

our study used initial caries, which are ICDAS code 0-1 caries, making it difficult to

identify caries by visual and tactile inspection. In this case, however, QLF can be used to

objectively evaluate the presence or absence of secondary caries, because the lesion with

the loss of minerals can be clearly detected by fluorescence loss. Therefore, QLF will be

more beneficial in preventing secondary caries on pit and fissure sealant.

Study 3 was an in vivo study, and it was not possible to evaluate the feasibility through

histological analysis because it targeted the teeth in the oral cavity. In particular, there were

limitations in the presence of dental caries around the pit and fissure sealant, accurate

diagnosis of depth and extent, and classification of marginal discoloration and caries. In

addition, various factors can affect the pit and fissure sealant dropout, and there is a

limitation that retention evaluation is impossible when the sealant is dropped. Nevertheless,

67

it was confirmed that the Q-ray view was very useful in evaluating the retention of pit and

fissure sealant. This suggests that the Q-ray view is likely to be used not only for evaluation

tools but also for improving the durability of preventive treatment. Since the microleakage

or fracture is frequently generated after the treatment of the pit and fissure sealant, it is

necessary to find factors affecting retention or to check the condition of the teeth before

treatment by using the Q-ray view. In future studies, it is necessary to evaluate the retention

status of pit and fissure sealant according to various factors such as the tooth condition

before application of the sealant and retention period after application. In addition, it is

considered that there is a need to find a way to use it to improve the durability of preventive

measures by using it in clinical practice.

68

V. CONCLUSIONS

This study evaluated the validity of the fluorescence pattern according to the depth of

microleakage in pit and fissure sealant with extracted initial caries (ICDAS code 0-1). In

addition, each fluorescence characteristic was compared with the clinical maintenance

status, and reliability was evaluated. Therefore, we confirmed the possibility of clinical use

of QLF technology as a detection tool for pit and fissure sealant.

The specific conclusions of this study are as follows.

1. Fluorescence values of the sealant microleakage showed significant differences

according to the depth of microleakage, and a correlation with histological score (r

= -0.72 and r = -0.72 in ΔF and ΔFmax, respectively, p<0.001).

2. Fluorescent value showed excellent validity in the diagnosis of microleakage of

sealant by confirming high sensitivity, specificity and AUC at all thresholds (AUC

= 0.83 – 0.91, p <0.001).

3. As a result of evaluating the sealant microleakage using a Q-ray pen, the

fluorescence values (ΔF and ΔFmax) showed significant differences except ΔR in the

retention (p < 0.05).

4. In the image evaluation of the sealant microleakage captured from the Q-ray pen,

the interrater agreement was higher in the fluorescent image (53.5-72.1%) than white

light image (33.3%).

69

5. In the interrater agreement for microleakage of pit and fissure sealant captured by

Q-ray pen, retention and caries in the fluorescence image were 0.43-0.57 and 0.40-

0.72, respectively, higher than white light images (0.26-0.41, 0.33-0.67).

6. For evaluation of microleakage of pit and fissure sealant, assessment with Q-ray

view showed high interrater agreement in marginal plaque, marginal discoloration,

and caries (0.81-0.89, 0.69-0.88, and 0.80-0.90, respectively), while visual and

tactile assessments showed lower agreement (0.22-0.57, 0.36-0.57, and 0.43-0.61,

respectively).

7. Intrarater agreement with Q-ray view for microleakage of pit and fissure sealant in

marginal plaque, marginal discoloration, caries showed significantly higher

agreement (0.81-0.83, 0.57-0.89, and 0.69-0.91, respectively) than visual and tactile

assessment (0.49-0.54, 0.48-0.55, and 0.42-0.65, respectively).

From these research results, it was confirmed that QLF technology is a valid

technology for quantitatively classifying microleakage of pit and fissure sealant using

fluorescence properties. In addition, the Q-ray pen image and Q-ray view test showed

that it can be used clinically through high interrater agreement. Therefore, it is expected

to be useful for more accurate and objective evaluation and monitoring of the condition

of the pit and fissure sealant using QLF technology in the clinical field.

70

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ABSTRACT (IN KOREAN)

QLF technology

< >

.

.

(Quantitative light-

induced fluorescence, QLF) .

40 160

. QLF-D

77

. QLF-D

(ΔF, ΔFmax)

. , AUC

. QLF-D

(Spearman’s correlation coefficients, rho; -0.72; p <0.001).

ROC curve AUC threshold good

(AUC = 0.83 – 0.93, p <0.001).

Q-ray pen

29 . 129

1

(ΔF, ΔFmax, ΔR) . 3 4

( , , , ) ,

Q-ray pen 2 ( , )

.

, ΔR

(ΔF, ΔFmax, ΔR)

(p < 0.05).

fair agreement - substantial agreement(0.26-0.41, 0.33-0.67)

78

moderate agreement- substantial

agreement(0.43-0.57, 0.40-0.72) .

6 3 15 58

. 3

Q-ray view ,

. , ,

fair agreement

- moderate agreement(0.22-0.57, 0.36-0.57, 0.43-0.61) Q-ray view

substantial agreement - almost perfect agreement(0.81-0.89, 0.69-0.88, 0.8-

0.9) .

, , Q-ray view (0.81-0.83,

0.57-0.89, 0.69-0.91) .

QLF technology

.

.

: , , , , ,

quantitative light-induced fluorescence(QLF) ,