Qiao et al.

Asian Pac J Dent 2015; 15: 33-40

Change of dentin permeability in different storage media after
resin coating
Hua Qiao, BS,a Rena Takahashi, DDS, PhD,a Toru Nikaido, DDS, PhD,a Syozi
Nakashima, DDSc,a Alireza Sadr, DDS, PhD,b Masaomi Ikeda, RDT, BSc, PhD,c andJunji Tagami, DDS, PhDaa

Department of Cariology and Operative Dentistry, Graduate School of Medical and Dental Sciences,bTokyo Medical and Dental University (TMDU), Tokyo, Japan, Department of Restorative Dentistry,c
University of Washington School of Dentistry, Seattle, WA, USA, and Oral Prosthetic Engineering,
Graduate School of Tokyo Medical and Dental University (TMDU), Tokyo, Japan
Purpose: The objective of this study was to evaluate the dentin permeability with/without resin coating indifferent storage media and periods.
Materials and Methods: Prepared bovine dentin disks were divided into three groups; 1) left uncoated as acontrol, 2) resin-coated with Clearfil SE Bond only, and 3) coated with the combination of SE and Clearfil
Protect Liner F. The hydraulic conductance of each specimen was measured after storage in either deionizedwater or artificial saliva. The hydraulic conductance value was expressed as the percentage of baseline
hydraulic conductance.
Results: For the control group, the mean percentage of hydraulic conductance to baseline hydraulic conductanceafter 1, 30, and 90 days were 103.7%, 126.0%, and 128.6% in deionized water, and 92.5%, 64.4%, and 62.2% in
artificial saliva, respectively. The mean percentage of hydraulic conductance to baseline hydraulic conductanceof coated with the combination of SE and Clearfil Protect Liner F after 1 day in artificial saliva yielded thelowest (2.5%) among all the groups.
Conclusion: Application of the resin coating to dentin surface demonstrated remarkable reduction of dentin
permeability over time.
(Asian Pac J Dent 2015; 15: 33-40.)
Key Words: artificial saliva, dentin permeability, hydraulic conductance, resin coating, storage media

Introduction
The post-operative sensitivity and pulpal irritation of the indirect restorations are major drawbacks inclinical dentistry.

The protection of the dentin and pulp from the mechanical, chemical, and biological stimuli

exerts clinical benefits for the patients.

According to the widely accepted Brannstrom’s hydrodynamic theory,

the exposed dentin provokes the movement of intertubular fluid, which activates pulpal nerves and causes pain.1
Based on the theory, reduction of dentin permeability leads the reduction of post-operative sensitivity and pulpal
irritation.

Various materials i.e. dentin desensitizers, varnishes, topical fluorides, and strontium chloride havebeen used to mitigate dentin sensitivity.2-5Some authors have used dentin bonding systems to reduce the dentin

permeability by sealing dentinal tubules.6,7
A resin-coating technique was introduced in the early 1990’s in order to overcome the problems of
post-operative sensitivity and pulpal irritation in preparations of the inlay/onlay and fullcrown restorations.8

In

this technique, after cavity preparation for indirect restoration and before taking an impression, the prepared
tooth surface is covered by the resin coating material, which is a dentin bonding system or a combination of adentin bonding system and a flowable resin composite.

It was demonstrated that the resin coating technique

improved the dentin bonding performance of resin cements,9-13 and prevented marginal leakage beneath therestorations.14,15

Nowadays thin-film coating materials have been developed for crown preparations.

resin coating technique is also effective to reduce coronal leakage of the endodontically treated teeth.

The

16

Several studies showed that the dentin adhesive materials are effective to seal the exposed dentin and33

Qiao et al.

Asian Pac J Dent 2015; 15: 33-40

reduce the dentin permeability.6,17
water,

18

On the other hand, it has been reported that adhesive materials absorb

which may affect dentin permeability after resin coating.

storage media for measurement of dentin permeability;

19,20

Deionized water has been often used as a

however, under clinical situation the coating

materials are likely to be exposed to saliva with contains a considerable amount of minerals and affect water
sorption and permeability in a different manner.

Thus, the effects of artificial saliva as a storage media tosimulate the oral cavity environment should be investigated.

However, there was little information in theliterature about the effects of different storage media and coating materials on dentin permeability with the lapseof time.
Thus, the objective of this study was to evaluate the dentin permeability with/without resin coating indifferent storage media and periods.

The null hypothesis tested was that coating material, storage media orstorage period did not affect dentin permeability.

Materials and Methods
Specimen preparation
Thirty-six bovine incisors, stored frozen, were used in this study.

The teeth were put in room temperaturefor 1 day before specimen preparation and were kept moist to prevent dehydration.
remove soft tissue remnants.

They were hand-scaled to

The labial surfaces of the teeth were ground to expose the dentin using a model

trimmer (Y-230, Yoshida, Tokyo, Japan).

Then dentin disks (approximately 10 mm in diameter and 0.75 mm

in thickness) were prepared using a slow-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) and adiamond point (F102R, GC, Tokyo, Japan) under running water.

Dentin disks were ground flat and ahomogeneous smear layer was created using 600-grit silicon carbide paper under running water for 30 s.

The

specimens were then cleaned in distilled water in an ultrasonic cleaner for 3 min to remove any debris and thesmear layer.

Subsequently, the permeability of each dentin specimen at baseline was measured according to theprocedures as described below.

The specimens were randomly allocated to three groups according to thefollowing dentin treatments: 1) dentin surface was left uncoated (control); 2) coated with a self-etching adhesive
system, Clearfil SE Bond (SE, Kuraray Noritake Dental, Tokyo, Japan)	
 (SE); and 3) dentin was coated with a
combination of SE and a micro-filled low-viscosity resin composite, Clearfil Protect Liner F (PLF, Kuraray
Noritake Dental) (SE+PLF).
Table 1.

Materials used in this study

Materials
Clearfil SE Bond

Code
SE

Lot No.
011580Compositions
Directions
Primer: 10-MDP, HEMA, hydrophilic
Apply for 20 s, blow air
dimethacrylate, photo-initiator, water
gently.
Bond: 10-MDP, Bis-GMA, HEMA, hydrophobic Apply, blow air mildly,
dimethacrylate, photo-initiator, silanatedand light cure for 10 s.
colloidal silica
Clearfil Protect
PLF
0073DA Bis-GMA, TEGDMA, fluoride-methyl
Light cure for 20 s.
Liner Fmethacrylate, camphorquinone, silanatedcolloidal silica, organic filler
All materials are manufactured by Kuraray Noritake Dental, Tokyo, Japan.
Abbreviations: 10-MDP, 10-methacryloyloxydecyl dihydrogen phosphate; HEMA, 2-hydroxyethyl methacrylate; Bis-GMA,
bis-phenol A diglycidylmethacrylate; TEGDMA, triethylene glycol dimethacrylate

Compositions and instructions of the materials used in this study were listed in Table 1.

In the SE andSE+PLF groups, the SE primer was first applied to the dentin surface for 20 s and dried with gentle air blow.
The SE bonding resin was then applied, dried with mild air blow and light-cured for 10 s using a halogen light34

Qiao et al.

Asian Pac J Dent 2015; 15: 33-40

curing unit (Optilux 501, Demetron Kerr, Danbury, CT, USA).

In the SE+PLF group, PLF was additionallyplaced on the cured SE adhesive with a micro-brush, and light cured for 20 s.

Furthermore, the specimens weredivided into two-subgroups according to the storage media (n=6). The specimens were stored at 37˚C in 50 mLof either deionized water or artificial saliva (1.0 mM CaCl2, 3.0 mM KH2PO4, 3.08 mM NaN3 and 100 mM NaCl,
pH 6.5) for 1, 30, and 90 days, respectively.

The storage media for each group was replenished every week.

Measurement of dentin permeability
Dentin permeability was measured using a fluid filtration system working under liquid pressure of 703
cmH2O.21

Each dentin disk was placed in a split-chamber device and between two rubber O-rings, which had

0.196 cm² inner surface area for infiltration of deionized water.

Each dentin disk was coated outside of the areaof O-ring with two-layers of nail varnish to maintain the exact position of the disk for repeated measurements.
The rate of fluid movement across dentin was measured by following the progress of a small bubble within 1.14
mm inner-diameter micro-capillary tube filled with deionized water lying on the top of a millimeter scale.
movement of the air bubble was metered every 5 min for successive 15 min.

TheThe average of the results ofevery 5 min was then calculated into hydraulic conductance (Lp) using the following equation:

Lp =

Q
P× A

where Lp is hydraulic conductance in µL min-¹ cm-² cmH2O-¹, Q is fluid flow in µL per min, P is hydrostaticpressure across dentin in cmH2O and A is the dentin surface area in cm².
Scanning electron microscopic (SEM) observations
The dentin disks of the coating groups (SE and SE+PLF) were prepared in the same manner as described in
the specimen preparations for the dentin permeability measurement.

Unpolymerized oxygen-inhibition layer ofthe coating layer on the dentin surface was removed with alcohol-soaked cotton pellets.
then embedded in an epoxy resin (Epoxicure, Buehler).

The dentin disks were

After curing the epoxy resin, the specimens weresectioned perpendicularly to the dentin surface using a low-speed diamond saw (Isomet) to obtain approximately
1 mm thick slices.

Each slice was subsequently polished with silicone carbide papers followed by diamondpastes (DP-Paste, Struers, Ballerup, Denmark) down to 0.25 µm particle size.

The specimens were cleaned

ultrasonically in distilled water for 5 min at the end of each step and dried at room temperature for 24 hours.
They were then subjected to argon-ion etching (EIS-1E, Elionix, Tokyo, Japan) for 6 min at 0.2 mA and 1 kV inorder to disclose the interfacial structure and finally gold sputter-coated (SC-701AT, Elionix) for SEM
observation.

Thickness of the coating layer for the SE and SE+PLF groups was measured by using an SEM(JSM-5310LV, JEOL, Tokyo, Japan) under ×500 magnification at three points of the coating layer, which wereat the center of the coating layer and at a distance of 1 mm from the center on either side.

The mean value ofthe thickness of the coating layers was calculated for each group (n=6).
Additionally, dentin disks without resin coating (control) were stored in either deionized water or theartificial saliva for 1, 30, and 90 days. Following the storage procedures, they were removed from the storage
media and dried in a desiccator for 1 day.

They were then fractured vertically with a wire cutter (YS-602,

YDM, Tokyo, Japan) at the center of the disk.

After gold-sputter coating, the dentin surface and inside of theirdentinal tubules at the fractured dentin disks were obliquely observed under SEM at ×2,000 magnification (n=3).
Statistical analysis
The results from dentin permeability measurement were statistically analyzed by the following methods.

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Asian Pac J Dent 2015; 15: 33-40

The Lp value obtained immediately after ultrasonic cleaning served as the baseline Lp for each specimen.

The

Lp values of each coating group after 1, 30, and 90 days were expressed in percentage of the baseline Lp (Lp%),
respectively.

The data were statistically analyzed with three-way ANOVA followed by multiple comparisonsusing t-test with Bonferroni corrections as post-hoc.
period.

The factors were coating, storage media and storage

All the statistical procedures were performed at a 95% confidence level with using Statistics package(ver. 16 for windows SPSS, Chicago, IL, USA).

Results
Measurement of dentin permeability
The mean Lp% and standard deviation for each group are summarized in Table 2.

ANOVA test revealed

that Lp% values were significantly affected by the three factors; coating material, storage media, and storage
period (p<0.001 for each).

The interaction among these three factors was also significant (p<0.001).

There

were significant differences between the control and SE groups, between the control and SE+PLF groups, andbetween the SE and SE+PLF groups, respectively (p<0.01).

The Lp% of the control group stored in deionizedwater significantly increased up to 30 days (p<0.05) and then plateaued (p>0.05), while the Lp% of the control
specimens stored in artificial saliva significantly decreased up to 30 days (p<0.05) and did not show any change(p>0.05) at 90 days when the measurement was repeated.

For the coating groups stored in deionized water,

there were significant differences between 1 day and 90 days (p<0.05), while for those stored in artificial saliva
there were no significant differences among 1, 30, and 90 days (p>0.05).

The lowest Lp% mean value wasobtained in the SE+PLF after 1 day storage in artificial saliva.
Table 2.

Hydraulic conductance, expressed as percentages (Lp%)

Coating
Control

Storage media
Deionized water
Artificial saliva

1 day
103.74 (1.69)
92.53 (4.67)

Storage time
30 days126.00 (12.77)a
64.35 (5.23)b

90 days128.62 (14.35)a64.24 (6.91)b

SE

Deionized water
Artificial saliva

12.01 (5.96)c
5.09 (2.53)e

18.87 (5.53)c,d7.52 (3.77)e

23.42 (5.82)d9.32 (4.51)eDeionized water
6.12 (1.84)f
10.31 (4.49)f,g
15.36 (6.70)ghhArtificial saliva
2.48 (1.25)
3.69 (0.92)
4.46 (1.51)h
All values are mean (SD).
In each row, the values marked by the same superscript lowercase letters are not significantly different (p>0.05).
SE+PLFSEM observations
The SEM images of the coating layers of the SE and SE+PLF groups were shown in Fig. 1.

Goodadaptation was found at the interface between the coating layer and dentin in both SE and SE+PLF groups.
The thickness of SE and SE+PLF was 22.1±3.7 µm and 126.0±19.5 µm, respectively.
The SEM images of specimens without resin coating (control) stored in either deionized water or theartificial saliva for 1, 30, and 90 days were shown in Fig. 2.

In the deionized water group, dentinal tubule

orifices were open and no precipitations were observed either on the dentin surface or inside of the dentinal
tubules at any storage period (Figs. 2a-c).

On the other hands, some precipitations were observed on the dentin

surface in the artificial saliva group, which gradually increased over the storage periods.
precipitations were not observed inside the dentinal tubules (Figs. 2d-f).

36

However, theQiao et al.

Asian Pac J Dent 2015; 15: 33-40

Fig. 1 SEM images of the dentin surface with SE (a) and SE+PLF (b): A, adhesive; D, dentin; R, resin composite

Fig. 2 SEM images of dentin surface and dentinal tubules of the dentin disks stored in deionized waterfor 1 day (a), 30 days (b), and 90 days (c), and in artificial saliva for 1 day (d), 30 days (e), and 90 days (f)While minimal changes are apparent in deionized water (a-c), deposition of mineral precipitants is evident in theartificial saliva with time (d vs. e and f).

Discussion
Both deionized water and artificial saliva were used as storage media in this vitro study.

The current

results indicated that there was a significant difference in dentin permeability between the two storage media
where the Lp% of control group increased in the deionized water, while in the artificial saliva it decreased.

Inline with this finding, SEM observation of the dentin surfaces in the control group (Fig. 2) revealed that dentinal
tubule orifices widened in deionized water over the storage periods (Figs. 2a-c).

On the other hand, in theartificial saliva group, a scattering of small precipitants, probably mineral crystals covering the dentinal tubule
orifices were observed (Fig. 2d).
(Figs. 2e and 2f).

Such precipitations were more obviously seen with longer storage periods

It should be noted that the precipitations were not observed inside the dentinal tubules.

It isspeculated that morphological changes inside the tubules and at peritubular dentin were too small to be detected.
In the deionized water group, mineral loss had likely taken place due to dissolution of peritubular dentin

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Asian Pac J Dent 2015; 15: 33-40

minerals, resulting in increased dentin permeability.

It was previously reported that storage of dentin resulted

in reduction of its mechanical properties due to demineralization in the unsaturated solution.22
In a solution saturated with regard to a mineral crystal, such as the artificial saliva used in this study, crystal
deposition from the solution may occur on the dentin surface or in the dentinal tubules and peritubular dentin
surfaces inside the tubules, resulting in constriction of the tubules and decreased dentin permeability.

These

hydrodynamic changes may be limited because the dentin permeability reached a plateau after 30 days in bothstorage media.
A dentin bonding agent has been used as a dentin desensitizer.

The adhesive systems contain hydrophilic

monomers and/or acidic monomers that in addition to the physical obstruction of tubule through surface
coverage and formation of resin tags, have a potential to coagulate with proteins in fluid proteins in the dentinal
tubules, resulting in reduction of the dentin permeability.23
exposed dentin is effective in the clinical situation.

Therefore, application of the resin coating to the24

Selection of dentin bonding system for the resin coating is an essential issue to obtain clinical success of theindirect restoration.

The two-step self-etch adhesive used in the current study was composed of a self-etchingprimer (SE primer) and a bonding resin (SE bond).

An acidic functional monomer, 10-methacryloxydecyl

dihydrogen phosphate (10-MDP) is present in both SE primer and SE bond.

The 10-MDP in the primer isconsidered to play multiple roles; to demineralize the smear layers and the underlying dentin, to promote
monomer penetration into dentin, and to react with calcium/hydroxyapatite intensively.25
al.,

26

reported that the hybrid layer formation is essential to obtain good dentin bonding.

Nakabayashi etRecently, theacid-base resistant zone (ABRZ) was found beneath the hybrid layer in the self-etch adhesive systems.27

It wasreported that acid resistance at the ABRZ forefront decreased when 10-MDP was excluded from the bonding
resin in an experimental two-step adhesive system.28

On the other hand, it has been reported that 10-MDP may

act as a hydrophilic component in the polymerized bonding layer and increase water sorption over time.28
The application of PLF on SE reduced dentin permeability.

PLF is a low-viscosity microfilled composite

mainly formulated using cross-linking dimethacrylates, such as bisphenol A glycidyl methacrylate (Bis-GMA).
Therefore, the PLF resin composite is more hydrophobic than the SE adhesive alone.

Application of PLF on

SE protects the underlying adhesive, and increases thickness of the coating layer and also increases
hydrophobicity of the coating layer.

The mean thickness of the coating layer of SE+PLF was 126.0 µm, which

was approximately five times thicker than that of the SE (22.1 µm).

Application of a flowable resin composite

on the cured adhesive can eliminate the oxygen inhibited layer of the adhesive, since the uncured resin in theoxygen inhibited layer is subsequently polymerized by diffusion of free radical from the flowable resin
composite.

It is believed that the adhesive itself is improved in quality (i.e. better cross linking, improved

conversion ratio and higher mechanical property), contributing to durable bonds at the interface.29-31
Nevertheless, it should be noted that none of the methods could completely eliminate dentin permeability
and reach zero Lp% in the present study.

It is inferred that dentin is inherently a permeable tissue, and acomplete elimination of its hydrolytic conductance may be structurally very difficult.

On the other hand, it has

been reported that dental composites absorb water and release unreacted monomers in aqueous environment.32
The water ingress into dental composites in the oral cavity, with time, leads to deterioration mainly due to ahydrolytic breakdown of the bond between silane and filler particles, filler–matrix debonding or even hydrolytic
degradation of the fillers.33

In addition to the degradation effects, water sorption may alter resins due to38

Qiao et al.

Asian Pac J Dent 2015; 15: 33-40

plasticizing effects through expansion, increasing the effective free volume and the ease of movement of chain
segments, thus reducing the elastic modulus of the material.
34

in both deionized water and artificial saliva.

These effects have been observed during storage

Similar to the control (uncoated) groups, significant differenceswere observed between artificial saliva and deionized water in both coated groups.

The higher permeability indeionized water was attributed to the effects of water on degradation of both the dentin and the resin-dentin
interface.35
Based on the present results, the null hypothesis that coating material, storage media or storage period did
not affect dentin permeability was rejected.

Further study should be carried out to evaluate the clinicalperformance of the resin coating for the indirect restorations.

Application of the resin coating to dentin surface

demonstrated significant reduction of dentin permeability over time.

However, reduction of dentin

permeability was strongly influenced by coating strategy and storage media.

Artificial saliva is more favorable

storage media than deionized water for in vitro permeability measurement.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Research (No. 25861792) from the Ministry of Education,
Science, Sports, Culture, and Technology, Japan.

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Correspondence to:
Dr. Rena Takahashi
Department of Cariology and Operative Dentistry, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University (TMDU)
1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Fax: +81-3-5803-0195 E-mail: renatakahashi@hotmail.com
Accepted December 26, 2015.
Copyright ©2015 by the Asian Pacific Journal of Dentistry.
Online ISSN 2185-3487, Print ISSN 2185-3479

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