Keywords
confocal laser scanning microscope - eroded dentin - resin tag length - total-etch
adhesive - universal adhesive
Introduction
Qualities of adhesive restorations are determined by three main crucial factors that
include condition of tooth, adhesive material properties, and the clinical procedure.
In a joint effort to promote the bonding between adhesive system and tooth structure,
clinical adhesive system has made a giant leap over the years. Despite having various
generations of adhesive system, the most preferred adhesive system are the fifth generation
(total-etch) and the seventh generation (self-etching system).
The principle of total-etch technique is to use acid etching for removal of smear
layer or smear plug and also expose the collagen matrix, which is followed by a subsequent
application of a single bottle self-priming bonding agent that fuses the primer and
adhesive component. Presence of any incomplete collagen expansion leads to impaired
penetration of the resin and compromise bonding capacity of these systems. The self-etch
system may consist of two bottle system, where the first bottle is the combination
of acid and primer and the second bottle is the bonding agent. Another variation of
the self-etch adhesive system is the all-in-one bottle system.[1]
Self-etch adhesive systems are superior to total-etch systems due to the reduction
of possibilities of iatrogenic induced clinical mishaps throughout acid conditioning,
rinsing, and drying, which are more common in total-etch systems.[2] On the contrary, self-etch system may fail to etch the enamel surface as efficiently
as phosphoric acid, in achieving depth of penetration into the tubules.[3]
[4]
The modern dentistry has observed development of various generations of bonding agents
in the evolution of adhesive dentistry over the decades. Invention of universal adhesive
can be considered as a great milestone in adhesive dentistry. It is also known as
“multipurpose” or “multimode” adhesive due to its flexible nature, which can be used
as either total-etch or self-etch technique.[5]
[6]
However, it is challenging to use the same bottle of adhesive for all types of tooth
structure such as sound, carious, attrited, eroded, or any other noncarious lesions
as the nature of the tooth substrate varies according to the tooth condition.[3] Although the focus is more on difference between sound or carious lesions, the noncarious
lesions especially erosive tooth wear is also a common clinical finding. Increased
incidence of teeth erosion is reported in adults and children due to current diet,
lifestyle, oral hygiene habits, eating disorders, or gastrointestinal diseases.[7]
[8]
[9]
[10] The common findings are erosion involving the maxillary anterior palatal surfaces
that affects patient’s aesthetics, functional occlusal efficiency, and also incisal
guidance.[8]
The depth of tooth erosion depends on the amount of dentin exposure. Moderate-to-severe
erosive tooth wear gives a significant clinical challenge for dental practitioners
in terms of complexity of treatment and longevity of the restoration. The treatment
modality for tooth erosion initially was focused more on invasive procedures such
as indirect all-ceramic restorations, resin-bonded palatal metal alloy veneers, but
currently the focus has shifted to a minimally invasive dentistry like direct composite
restorations.[11]
[12] Of late, self-adhering flowable resin composites were introduced eliminating the
prerequisite of application of etch and bond.[13] However, various authors have reported more adhesive failures compared with cohesive
failures in eroded teeth.[14]
[15]
The adhesive techniques are critical, and the success of direct restorations is impeded
by inadequate adhesion and hybrid layer degradation. Nowadays, researchers are focusing
on the development of surface treatments to enhance adhesion or a decrease in hybrid
layer degradation. Gabriela et al reported that arginine dentine pretreatment had
positive effect on adhesion especially with self-etch adhesive system.[16]
From the reported studies, it was observed that eroded dentin exhibited lower bond
strength compared with sound dentin, but this might also be affected by the age factor.
A clinical based study conducted by Federlin et al has showed that class V restorations
bonded to the unprepared surface of eroded dentin for a period of 12 months, displayed
good retention regardless of the type of restoration.[17] However, another similar study reported high rates of restoration failures up to
44 to 50%.[18] The effect of mode of adhesion and chlorhexidine on the microtensile strength of
a universal bonding agent to sound and caries-affected dentins reported by Lima et
al showed that the microtensile bond strength had no significant difference between
sound dentin and caries-affected dentin.[19]
According to Yabuki et al, bonding in eroded enamel was observed to be lower than
in sound enamel. Bond strength of the restoration of eroded dentin are being questioned
in day-to-day practice, and only few reports explained the relationship between the
bond strength and the restoration especially using universal adhesive system.[20]
Thus, there is a paucity of evidence that explains the relationship between total-etch
and self-etch technique, especially on eroded dentin using universal adhesive system.
Additionally, interfacial microscopic examination of resin dentin interface is conventionally
done by using optical microscope and SEM.[21] However, CLSM has emerged as a better nondestructive reliable tool to assess the
subsurface characteristics of resin dentin interface.[22]
[23]
Hence, the aim of this study was to compare the resin-dentin interface of normal and
eroded dentin by using universal adhesive system and total-etch adhesive system with
the aid of confocal laser scanning microscope (CLSM). The alternate research hypothesis
was the resin-dentin interface of sound dentin is better than eroded dentin by using
universal adhesive system, and formation of the resin tag and hybrid layer in total-etch
system is longer and thicker compared with self-etch system in eroded dentin.
Materials and Methods
Experimental Design
A randomized in vitro controlled study was conducted. Study protocol was submitted
to Research management Centre, MAHSA University, and the ethical approval was obtained.[24]
Forty caries-free human premolars extracted for the purpose of orthodontic treatment
were collected and sterilized according to guidelines published in Occupational Safety
and Health Administration and the Centre for Disease Control and Prevention.[23]
Sample Size Estimation
Sample size was estimated based on two means formula. The sampling was performed by
G Power Software. Estimated sample size is 10 per group and 40 samples in total.[23]
Sample Preparation
All the teeth samples were mounted in wax block, and the occlusal surfaces of the
teeth were ground by using slow speed diamond disc with copious water supply until
all the occlusal enamel layer was removed and a flat section of superficial dentin
was exposed. The teeth were then randomly divided into two groups of 20 samples each.
Control group A representing sound dentin (20 teeth) and test group-B representing
eroded dentin (20 teeth), respectively. Ten teeth from each group were further randomly
subdivided into subgroups A1 and B1 representing total-etch system, and A2 and B2
representing self-etch system, respectively ([Fig. 1]). After the sectioning is completed, all the samples were stored in S artificial
saliva in respective labeled containers till further use at 37°C. The composition
of the artificial saliva used in this study were KCl (16.1 mM), NaCl (14.4 mM), K2HPO4 (2.0-mM), MgCl2•6H2O (0.3 mM), CaCl2•2H2O (1.0 mM), and sodium carboxymethyl cellulose (0.10 g% ; CMC-Na) at pH of 7.0.[20]
Fig. 1 Flow chart of the methodology.
Selection of teeth and division of teeth was done by another investigator to avoid
the selection bias by the operator.
Erosive Protocol for the Test Group Samples (B1 and B2)
The teeth in test group underwent erosive protocol that is similar to the study conducted
by Yabuki et al, which involves immersion of test group teeth (eroded dentin) in 1.23%
citric acid solution (pH = 2.1, 6.4 × 102 mol/L) for 1 minute every 12 hours followed
by incubating in artificial saliva at 37°C. This cycle was repeated for five times
in 3 days.[20]
In the control group, the sound dentin sample teeth were simply incubated in artificial
saliva at 37°C for 3 days without exposure to erosive cycle.
Fluorescent Labeling of Bonding Agent
Rhodamine B dye with 0.01%wt mixed with 5 mL of universal adhesive agent (G-Premio
Bond, GC Corporation, Tokyo, Japan) as suggested by Júnior et al was used to obtain
an intense fluorescence to aid in the visualization of the bonding agent distribution
on the resin-dentin interface.[25] The materials used in this study are specified in [Table 1].
Table 1
Materials used for the study
|
Trade name
|
Material details
|
Manufacturer
|
Lot no
|
|
FineEtch
|
37% Phosphoric acid
|
Spident Co., Ltd
|
FE18240
|
|
G-Premio Bond
|
Universal adhesive system
|
GC Corporation, Tokyo, Japan
|
1712092
|
|
G-ænial Universal Flo
|
Flowable composite restorative material
|
GC Corporation, Tokyo, Japan
|
1806182
|
Restorative Procedure
Following manufacturer’s instructions, the restorative procedure in groups A1 and
B1 was performed with total-etch adhesive technique, while A2 and B2 restorative procedures
were performed by using self-etch adhesive technique ([Table 2]). The flow chart of the restorative procedure is shown in [Fig. 2].
Table 2
Group description
|
Group
|
Description
|
|
A1
|
Sound dentin, total-etch
|
|
A2
|
Sound dentin, self-etch
|
|
B1
|
Eroded dentin, total-etch
|
|
B2
|
Eroded dentin, self-etch
|
Fig. 2 Flow chart of the restorative procedure.
Total-Etch Adhesive Technique
In total-etch adhesive technique, A1 and B1 exposed dentin surface of each tooth was
treated with 37% phosphoric acid gel for 20 seconds, then rinsed with water for 5
seconds, and gently dried using three-way syringe with gentle airstream for 5 seconds.
The universal adhesive agent (G-Premio Bond, GC Corporation, Tokyo, Japan) was then
applied uniformly on the tooth occlusal surface for 10 seconds with a disposable applicator
tip, dried with gentle airstream for 5 seconds at a 3-cm distance to evaporate the
solvent, and light cured for 10 seconds using a LED device with a power output of
600 Mw/cm2. Composite build up was done by using a flowable nanocomposite (G-ænial Universal
Flo, GC Corporation) until a 2-mm occlusal height was achieved. Siqveland wide matrix
band and retainer was placed around the tooth followed by occlusal restoration. Once
the top surface of the restoration was light cured for 20 seconds, the matrix band
was removed and again light cured from all four sides for 20 seconds to ensure adequate
curing of the restoration. The tooth samples were placed in their respective labeled
containers and incubated in artificial saliva at 37°C.
Self-Etch Adhesive Technique.
In self-etch adhesive group, the A2 and B2 exposed dentin surface of each tooth were
dried by gentle airstream using three-way syringe, and then the universal adhesive
agent was applied on the tooth occlusal surface for 10 seconds with a disposable applicator
tip, dried with gentle airstream for 5 seconds at a 3-cm distance to evaporate the
solvent, and light cured for 10 seconds using a LED device with a power output of
600 Mw/cm2. This was followed by composite restoration as explained in 2.6.1.
Assessment of Resin-Dentin Interface
After completion of the restorative procedures, two longitudinal sections were made
along the long axis of the teeth in the middle portion of the tooth to obtain a tooth
section measuring approximately 0.5 mm thickness using a slow speed diamond disc under
copious water supply ([Fig. 3]). The sectioned samples were further grinded on a carborundum stone to achieve a
smooth uniform, even surface. The samples were pat dried with a paper towel and mounted
on the glass slide with a coverslip.
Fig. 3 Longitudinal sectioning of the tooth. The thickness of the longitudinal sectioning
of the tooth should be 0.5 mm thickness.
The resin-dentin interface of the tooth samples in both test and control groups was
examined to observe resin tag length and hybrid layer thickness by using CLSM (LSM
5 Pascal Exciter, Zeiss, Germany) under ×10 magnification and Argon laser illumination
at 50% intensity with 514 nm excitation wavelength. Confocal slits were set at 25
μm with a 536 nm long-pass filter ([Fig. 4]).
Fig. 4 Confocal laser scanning microscope image of resin-dentin interface. Confocal images
using a ×10 objective, Argon laser illumination at 50% intensity using a 514 nm excitation
wavelength. Confocal slits were set at 25 µm with a 536 nm long-pass filter.
The images were analyzed and the average of three readings was recorded for each parameter.
The resin tag length and hybrid layer thickness were measured by using Image Browser
Software (Zeiss, Germany) in micrometer by a single trained examiner. Prior to the
experimental study, intraexaminer calibration was done and the reliability was verified
at two different time intervals in two weeks gap. Intraclass correlation coefficient
(ICC) reliability index value was estimated to be greater than 0.9.
Statistical Analysis
The data obtained was tabulated and analyzed by using independent t-test.
Results
Resin dentin interface as observed under confocal microscopy was evaluated in this
in vitro study. Resin tag length and hybrid layer thickness were studied to assess
the difference between sound and eroded dentin by using total-etch and self-etch adhesive
systems. The mean values of the resin tag length and thickness of hybrid layer for
all the groups were tabulated in [Tables 3] and [4], respectively. Both the normal and eroded dentin exhibited formation of resin tags
and hybrid layer. The confocal images of sound and eroded dentin using total-etch
and self-etch system are depicted in [Figs. 5] and [6], respectively.
Table 3
Mean ± standard deviation of resin tag length in sound and eroded dentin
|
Sound dentin
|
Eroded dentin
|
p-Value
|
|
Total-etch system
|
104.94 ± 6.27
|
82.52 ± 3.9
|
p ≤ 0.001**
|
|
Self-etch system
|
19.6 ± 2.11
|
27.01 ± 3.28
|
p ≤ 0.001**
|
|
p-Value
|
p ≤ 0.001**
|
p ≤ 0.001**
|
|
Table 4
Mean ± standard deviation of hybrid layer thickness in sound and eroded dentin
|
Sound dentin
|
Eroded dentin
|
p-Value
|
|
Total-etch system
|
6.71 ± 0.41
|
5.45 ± 0.25
|
p ≤ 0.001**
|
|
Self-etch system
|
2.36 ± 0.22
|
3.61 ± 0.48
|
p ≤ 0.001**
|
|
p-Value
|
p ≤ 0.001**
|
p ≤ 0.001**
|
|
Fig. 5 Confocal laser scanning microscope images of (A) total-etch versus (B) self-etch in sound dentin. Total-etch system in sound dentin displays longer resin
tags and thicker hybrid layer compared self-etch system in sound dentin.
Fig. 6 Confocal laser scanning microscope images of (A) total-etch versus (B) self-etch in eroded dentin. Total-etch system in eroded dentin displays longer resin
tags and thicker hybrid layer compared with self-etch system in eroded dentin.
Resin Tag Length
With total-etch adhesive system, the sound dentin was superior to eroded dentin with
longer resin tag length which has a mean length of 104.94 ± 6.27 µm while the eroded
dentin has a mean length of 82.52 ± 3.9 µm, and the difference was statistically significant
(p ≤ 0.001). In contrast to the above, with self-etch system, the mean resin tag length
was significantly greater in eroded dentin (27.01 ± 3.28 µm) than that of the sound
dentin (19.6 ± 2.11 µm) at p ≤0.001. When the total-etch and self-etch adhesive systems were compared, the resin
tag length was longer in total-etch adhesive system compared with self-etch adhesive
system irrespective of sound or eroded dentin and was statistically significant (p ≤ 0.001).
Hybrid Layer Thickness
Using total-etch adhesive system, the hybrid layer thickness observed was better in
sound dentin with a mean thickness of 6.71 ± 0.41 µm than in eroded dentin with mean
thickness of 5.45 ± 0.25 µm. This difference was statistically significant (p ≤ 0.001). In contrast, the hybrid layer showed a significantly greater thickness
in eroded dentin than in sound dentin (3.61 ± 0.48 vs. 2.36 ± 0.22 µm, respectively)
with self-etch adhesive (p ≤ 0.001). The hybrid layer thickness produced by total-etch adhesive system were
thicker compared with self-etch adhesive system. The difference of hybrid layer thickness
produced in sound and eroded dentin using total-etch adhesive and self-etch adhesive
system were statistically significant.
Discussion
In this in vitro confocal study, the resin tag length and hybrid layer thickness were
analyzed at the resin dentin interface. Extracted human teeth were used as the samples
in this study to mimic in vivo situation closely to evaluate bond strength. All 40
extracted tooth samples were stored in 10% formalin for 14 days as per Center for
Disease Control and Prevention recommendation. This is to prevent the risk of cross
infection as extracted teeth are considered as a potential source of blood borne pathogens(OSHA).
The extracted teeth were ground superficially on a carborundum stone with water as
lubricant until the complete section of occlusal dentin was exposed. Selection of
dentin layer is an important factor to be considered in the study design, as it was
reported that different layers of dentin display different level of bond strength.
Dentin layer can be classified as superficial, middle, or deep dentin. Bond strength
of dentin bonding agent decreases as the depth of the dentin increases. Pegado et
al also evaluated the microtensile bond strength to deep and superficial permanent
dentin using different bonding agents, and concluded that the bond strength obtained
in superficial dentin was significantly higher than in deep dentin for all adhesive
systems tested.[26]
The usage of superficial dentin for maximum bond strength in this study was validated
by McCabe et al.[27] The test group samples underwent erosive cycle or pH cycling as described by Yabuki
et al and Zimmerli et al to depict an erosive condition.[7]
[20] The erosive cycle protocol was done by using 1.23% citric acid due to its low pH
with low calcium and fluoride concentration.[28] Citric acid also simulates acidic drinks, which is a prime cause for dental erosion.
Although in vitro erosive protocol may not depict the exact oral clinical environment,
alternate exposures to short cycles of acid, followed by immediate immersion into
artificial saliva can simulate demineralization and remineralization cycle as seen
in oral cavity.[29] The pH of the citric acid used in this study was 2.1 to mimic the pH of the regular
acidic beverages consumed and available in the market.[7]
Resin dentin interface is usually evaluated in vitro by using dye penetration studies
to detect bond failure at the enamel-resin interface. Confocal laser scanning microscopy
(CLSM) is a valuable technique for the visualization of bonding structures such as
a hybrid layer in dentin.[30]
[31] CLSM has gained reputation over the years in evaluation of interfacial microscopic
examination of adhesive agents due to its laser light characteristics and minimal
aberration and diffraction artifacts as compared light microscopy.[13] CLSM technique requires very low concentration of fluorochromes, which are soluble
in the adhesive liquids without altering their properties.[13]
[23]
[32]
CLSM was chosen in this study as it can generate noninvasive serial optical sectioning
of intact specimens eliminating the artifacts arising with manual sectioning as seen
in scanning electron microscopy (SEM) and transmission electron microscopy techniques
(TEM).[23] CLSM was also reported to render a detailed information than the SEM because of
its nondestructive nature and elimination of other artifacts such as shrinking, swelling,
or detachments that can happen with SEM. Moreover, CLSM gives the possibility to distinguish
the components of bonding agents that can be visualized up to 100 µm below the surface.[22]
[32]
[33]
To distinguish the resin-dentin interface under confocal microscopy, the universal
adhesive agent was incorporated with a concentration of 0.1 mg/mL of Rhodamine B dye
before application on the dentin surface, as it easily penetrates into the dentin
and act as a contrast agent.[25]
[34]
Depending on the type of group, the restorative procedures were done on all the samples.
Resin-dentin interface, has a thin layer of resin-reinforced dentin that binds the
two different materials together on a molecular level which is often called as hybrid
layer. It prevents leakage and develops high resistance to acidic medium. The penetration
of adhesive agent into the dentin tubules forms the resin tag. In conventional total-etch
system, acidic pretreatment removes the smear layer covering the dentin thus exposing
the dentin tubules.
Upon application of bonding agent, it spreads on the dentinal layer and penetrates
into the dentin tubules, thus forming the resin tags. In self-etch system, acid is
already incorporated (all-in-one bottle system) to remove the smear layer. However,
the removal of the smear layer is inadequate and is much lesser compared with total-etch
system.
The restored teeth were ground to 0.5 mm thickness, and the middle portion of the
tooth were taken and fixed on a glass slide and coverslip to assess the parameters
under the CLSM. CLSM aids in distinguishing and measurement of the hybrid layer and
resin tags.[23] The average of three readings per sample were recorded to minimize the human errors.
In the present study, it was observed that the total-etch system had better permeability
compared with self-etch system in both sound and eroded dentin. This observation can
be explained based on the following reasons:
-
The lower pH of phosphoric acid in total-etch adhesive system serves as a strong etchant
which could have led to demineralization and increased permeability of the dentin.[23]
-
The total-etch technique is a moist bonding technique. It increases the resin tag
formation by preventing the collapse of the collagen fibrils by maintaining the collagen
fibril separation.[18]
In self-etch adhesive system, there is no rinsing involved. Thus, the lower pH of
self-etch adhesive leads to formation of dentin by-products, which limits the level
of demineralization and penetration to the superficial layer of the dentin. Additionally,
the smear layer contains mineral components that may neutralize the acidic component
of the self-etch adhesive, thereby affecting the demineralization of the tooth surface.[35]
Present study results were in agreement with other studies done on sound dentin, where
in total-etch system was shown to have a better bonding capacity compared with self-etch
adhesives by using various analytic techniques.[1]
[13]
[36]
[37] However, bonding in eroded teeth is a great challenge. Augusto et al and Milosevic
et al have reported more adhesive failure than cohesive failure in eroded dentin which
clearly indicates failure in bonding agent. In erosive teeth, due to structural dissolution,
the erosion leads to mineral loss and exposure of the collagen fibrils and also dentin
tubules.[14]
[15]
[36] Emergence of universal adhesive system may possibly offer a solution for bonding
in eroded teeth.
In the present study, the difference in bonding ability of eroded and sound dentin
was assessed with two different adhesive systems. It was observed that total-etch
system had greater length of resin tags and hybrid layer thickness in sound dentin
compared with eroded dentin. This could be due to the difference in the mineral content
of the tooth. The erosive cycle subjects the tooth to acidic medium which demineralizes
the tooth surface leading to mineral loss and collapse of the collagen meshwork. Thus,
it limits the penetration of the bonding agent into the dentin tubules despite having
acid etching using 37% phosphoric acid, which removes the smear layer.[20]
However, with self-etch adhesive system, the resin tag length and thickness of hybrid
layer were more pronounced in the eroded dentin compared with sound dentin. This could
be due to incomplete removal of smear layer using self-etch adhesive system in sound
dentin. Erosive cycle using acidic medium causes as the tooth surface to undergo some
amount of acidic pretreatment leading to demineralization of dentin and removal of
smear layer. This finding corresponds to the scanning electron microscope observation
by Augusto et al wherein eroded dentin had etched pattern of tooth surface.[15]
Laser scanning microscope study by Yabuki et al also confirmed that eroded teeth demonstrated
irregular surface patterns which depicts erosion and etching pattern on the dentin.[20] This, explains the cause of greater penetration of resin tags into the dentin tubules
of eroded dentin compared with sound dentin, where the demineralization and acid exposure
was limited.
Limitations of the Study
Oral environment related studies always aim to simulate the oral condition as much
as possible as it is considered as the ultimate testing environment to predict the
restoration behavior. However, oral environment is complex and biodiverse in nature.
Thus, in vitro models hold a high significance in providing a consistent environment
of oral cavity and aids in studying product degradation in intraoral condition.[38]
In this in vitro study, the teeth collected were stored in artificial saliva to simulate
the oral cavity fluid and placed in incubator at 37°C to simulate body temperature.
The scope of this research was limited to superficial dentin where erosion may involve
deeper layers of dentin depending on the severity.
Future Recommendations
Further studies involving the bond strength to middle and deep dentin layers may be
beneficial to understand bonding in these dentin layers. Additionally, long-term observations
from in vivo study in a controlled environment will be more ideal to predict the longevity
of composite restoration on eroded teeth. This study can also be extended to compare
resin-dentin interface of various commercially available universal adhesive agents
on eroded dentin.
Conclusion
The findings of the present research can be summarized as follows:
-
Both sound and eroded dentin displayed greater resin tag length and thickness of hybrid
layer in total-etch system than self-etch system.
-
In total-etch system, sound dentin displayed greater resin tag length and thickness
of hybrid layer compared with eroded dentin.
-
In self-etch system, eroded dentin displayed increased resin tag length and thickness
of hybrid layer compared with sound dentin.
Within the limitations of the present study, the resin-dentin interface of sound dentin
was superior to eroded dentin by using total-etch system as it presented longer resin
tags and thick hybrid layer. Thus, we failed to reject the alternate hypothesis. Contrary
to the above, the resin tag length and hybrid layer thickness observed in self-etch
system was longer and thicker in eroded dentin than with the use of total-etch system.
Thus, we accept reject the second alternate hypothesis.
