The efficacy of dentine adhesive to sclerotic dentine

The efficacy of dentine adhesive to sclerotic dentine
Mizuho Kusunoki*, Kazuo Itoh, Hisashi Hisamitsu, Sadao Wakumoto
Department of Operative Dentistry, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ohta-Ward, Tokyo 145-8515, Japan
Received 13 March 2000; revised 12 January 2001; accepted 12 May 2001
Abstract
Objective. To evaluate the effect of a dentine bonding system to sclerotic dentine in comparison with normal dentine.
Methods. The efficacy of the dentine bonding system to sclerotic dentine was examined by measuring wall-to-wall polymerization
contraction gap width. The dentine cavity wall was pretreated with an experimental dentine bonding system with and without a dentine
primer. The dentine primer was glyceryl mono-methacrylate (Blemmer GLM, NOF Corp., Tokyo, Japan) (GM), which contained esterified
methacrylate with a polyvalent alcohol, which is similar to 2-HEMA. The structure of sclerotic dentine and the changes to that structure
caused by etching were observed using a scanning electron microscope (SEM).
Results. With GM priming, complete marginal integrity was obtained regardless of the type of dentine. Without GM priming, complete
marginal integrity was obtained in half of the specimens of the sclerotic dentine, and was not obtained in any of the specimens of normal
dentine. In the SEM study, the structure of sclerotic dentine was considered to be viable for adhesion. However, this was not the case when
etched with phosphoric acid.
Conclusion. It was concluded that sclerotic dentine had a clear advantage over normal dentine with regard to the adaptation of resin
composites. Therefore the structure of sclerotic dentine possesses a naturally derived structure to which a primer may attach. Sclerotic
dentine is part of the body’s natural defenses and should be preserved. It should not be exposed to acid etching which would damage its
structure. q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Sclerotic dentine; Dentine primer; Contraction gap; SEM observation
1. Introduction
In 1982, the bonding mechanism of dental adhesive was
defined as the hybrid layer formation in the superficial
substrate dentine, after dentine conditioning with citric acid
containing ferric chloride, in which 4-methacryloxyethyl
trimellitate anhydride (4-META) was diluted in methyl
methacrylate (MMA) [1]. In addition, improvement of the
priming effect of the dentine bonding system was suggested
by the chemical reaction of the amino group with
glutaraldehyde [2]. However, the priming mechanism of
2-hydroxyethyl methacrylate (2-HEMA) without glutaraldehyde
was reported to be based on the expansion of the
micro space within the collagen network in which the
monomer infiltrated easily to form the hybrid layer [3]. In
both of these dentine bonding systems, the dentine was
etched with citric acid or phosphoric acid, which significantly
decalcified the dentine cavity wall. In previous
* Corresponding author. Tel.: þ81-3-3787-1151x291; fax: þ81-3-3787-
1229.
E-mail address: mizuho@senzoku.showa-u.ac.jp (M. Kusunoki).
Journal of Dentistry 30 (2002) 91–97
0300-5712/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0300-5712(02)00003-9
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reports, it was claimed that decalcification on the dentine
cavity wall by dentine conditioner or self-etching dentine
primer deteriorated the marginal integrity of the resin
composite in the dentine cavity [4,5]. This was due to the
functional monomers in the dentine bonding agent, which
were designed to attack inorganic components in the tooth
substance [6,7]. Furthermore, the experimental dentine
bonding agent from which the functional monomer was
completely omitted, was unable to prevent the contraction
gap formation [8]. Therefore it was speculated that the
marginal adaptation was established by polymerization of
the resin composite to the dentine with an intermediary
monomer, which binds specifically to calcium.
However, Yukitani et al. noted that multiple application
of the dentine bonding agent forms a thick bonding layer,
like a dental cement film, on the acid etched dentine
adhesive surface. This is needed to obtain the marginal
integrity of the total-etch wet dentine bonding system [9].In
addition, Sano et al. discussed the nanoleakage that occurs
within the hybrid layer [10,11]. While using the total-etch
wet dentine bonding system, the nanoleakage resulted from

92
Table 1
Dentine sclerosis scale
Category Criteria
1 No sclerosis present
Dentine is light yellow or whitish color with little
discoloration
Dentine is opaque, with little translucency or transparency
2 More than category 1 but ,50% of way between categories
1 and 4
3 Less than category 4 but .50% of way between categories
1 and 4
4 Significant sclerosis present
Dentine is dark yellow or even discolored (brownish)
Glassy appearance of dentine, with significant translucency
or transparency evident
Based on scale developed by Dr Steven E. Duke of the University of
Texas Health Science Center at San Antonio, and modified by the
Department of Operative Dentistry at the University of North Carolina
School of Dentistry.
inefficient infiltration and polymerization of the adhesive in
the hybrid layer.
Most of the mentioned reports, however, were performed
using caries-free teeth as a substrate, even though normal
dentine does not require any treatment. However, often the
clinical target is sclerotic dentine which varies from normal
dentine and is usually observed adjacent to caries, cervical
defects and exposed root surfaces. In sclerotic dentine, the
dentine tubules are closed by deposits of an inorganic
component and the dentine permeability is significantly
reduced [12,13]. Sano et al. reported that the micro tensile
strength of the dentine adhesive to the cervical sclerotic
dentine was significantly decreased compared to that to the
normal dentine [14–16]. Such a finding suggested that the
impregnation of the resin conditioner into the dentine
substrate was limited in the case of the sclerotic dentine
which might cause an unreliable hybrid layer formation.
The purpose of the present study was to examine the
efficacy of a dentine bonding system for sclerotic dentine,
and to observe the structure of sclerotic dentine and the
changes to that structure after etching with phosphoric acid.
2. Materials and methods
2.1. Contraction gap measurement
The extracted human teeth used were physiologically
discolored, highly transparent and caries free. Sclerotic
dentine was identified according to the North Carolina dentine
sclerosis scale [17] (Table 1). The teeth used for sclerotic
dentine were classified as being in category 3 or 4 and the teeth
used for normal dentine were classified as being in category 1.
An example of each tooth is given in Fig. 1.
The proximal enamel of a extracted human tooth was
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97
Fig. 1. Normal teeth (left) and physiologically discolored, highly
transparent teeth (right). It was concluded that the sclerotic dentine of
teeth, such as those of the right, is effective for adhesion.
eliminated to produce a flat surface and then a cylindrical
cavity, approximately 3 mm in diameter and 1 mm in depth
was prepared in the exposed dentine. The cavity wall was
cleaned with 0.5 mol/l neutralized ethylene diamine tetraacetic
acid (EDTA, Dojin, Wako Pure Chemical Industries
Ltd, Osaka, Japan) (pH 7.4) for 60 s followed by rinsing and
drying. The cavity was then primed with 35 vol% of
glyceryl mono-methacrylate (GM) solution for 60 s followed
by thorough drying. A commercial dual-cured
dentine bonding agent containing 10-methacryloxydecyl
dihydrogen phosphate (10-MDP) (Clearfil Photo Bond,
Kuraray, Okayama, Japan) was then applied to the cavity
followed by 10 s irradiation. Finally, the commercial light
cured resin composite (Silux Plus, 3M, MN, USA) was
inserted into the cavity. The composite surface was pressed
flat on a glass plate mediated with a plastic matrix and
irradiated for 40 s using a visible light source (White Light,
Takarablmont Co., Osaka, Japan). The specimens were
stored for 10 min in tap water at room temperature
(24 ^ 1 8C). The slightly over-filled resin composite was
eliminated with a wet carborundum paper and the exposed
cavity margin was polished with a linen cloth mediated with
an alumina slurry with a grain size of 0.03 mm. The
marginal integrity was inspected under a light microscope
(Orthoplane, Leitz, Wetzlar, West Germany). A screw
micrometer (Eyepiece Digital, Leitz, Wetzlar, West
Germany) mounted on an ocular lens was used to measure
the contraction gap width at eight points located every 458
along the cavity margin at a magnification 1024 £ . The
contraction gap value was presented by the sum of the
diametrically opposing gap width in percentage to the cavity
diameter and the maximum of the four values was given as
the maximum contraction gap value of the specimen (Fig. 2).
In the experimental groups, the glyceryl mono-methacrylate
priming was omitted and the dentine bonding agent was
applied in the cavity after the dentine conditioning with
EDTA. Forty specimens in total were made consisting of ten

specimens each from the primer-treated and nonprimertreated
groups from the normal and sclerotic dentine groups.
The data was analyzed using a nonparametric Mann–
Whitney U-test because the data was not a normal
distribution. The dentine types (normal and sclerotic) and
two treatments (with and without GM) were the two
independent factors.
2.2. SEM observation of the dentine surfaces
The structure of sclerotic dentine and the change of the
dentine surfaces after either EDTA conditioning for 60 s or
phosphoric acid (Etchant, 3M Co., St Paul, MN, USA) etching
for 30 s was observed by using a scanning electron microscope
(S-4700, Hitachi, Tokyo, Japan). A 0.5 mol/l neutralized
EDTA solution (pH 7.4) was used for 60 s to remove only the
smear layer on all specimens. The specimens were dehydrated
in a gradual alcohol solution in which the concentration was
increased from 70 to 95% (70, 80, 90, 95%) for 30 min and
99% for two 15 min periods, critical point dried and sputtercoated
with palladium and platinum.
3. Results
3.1. Contraction gap measurement
The contraction gap measurements are presented in
Table 2
Contraction gap width in cylindrical dentine cavity
GM priming Without GM priming
Sclerotic dentine 0 (10) 0.016 ^ 0.016 (5)
Normal dentine 0 (10) 0.078 ^ 0.029 (0)
N ¼ 10; mean ^ SD of the marginal gap width in percentage to the
cavity diameter. The number of gap free specimens out of 10 is in
parentheses. GM: 35 vol% of glyceryl mono-methacrylate. Values joined
by a line are not significantly different statistically (Mann–Whitney U-test
p . 0.05).
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97 93
Fig. 2. Contraction gap measurement.
Table 2. Complete marginal integrity was obtained with the
experimental dentine bonding system regardless of the type
of dentine. In the nonprimer-treated groups for normal
dentine, a gap was observed in all the specimens. However,
in the nonprimer-treated groups of sclerotic dentine, a gap
was observed in half of the specimens. The difference
among these nonprimer-treated groups was significant
(Mann–Whitney U-test, p , 0.05).
3.2. SEM observation of the dentine surfaces
The SEM images of sclerotic dentine were varied
compared to normal dentine. In one specimen of sclerotic
dentine, almost all the tubules were completely closed with
rod-like structures containing spherical objects, which were
considered to be deposits of an inorganic component (Fig.
3). In another specimen, the lumina of the tubules contained
mineralized cubic crystals (Fig. 4). In normal dentine, the
intertubular dentine and the peritubular dentine were clearly
observed. The lumina tubules were wide and the inside
walls was smooth. At each part of the surface, the opening
of the branches were clearly recognizable.
The change of the dentine surfaces after either EDTA
conditioning or phosphoric acid was observed in the same
part of the teeth, because the structure of sclerotic dentine
was different in each specimens. The images in Figs. 5 and 7
were of the same normal dentine specimen. Similarly, the
images in Figs. 6 and 8 were also of the same sclerotic
dentine specimen. When conditioning with EDTA, only the
smear layer was completely removed (Figs. 5 and 6). When
etching with phosphoric acid, not only was the smear layer
removed but also inorganic components were lost. Hence
SEM images of sclerotic dentine and normal dentine were
similar (Figs. 7 and 8). After etching silica (a component of
the etchant) remained on the surface and the openings of the
lumina were extended due to the loss of the peri-tubular
dentine forming a funnel-shaped configuration. This part of
the peri-tubular dentine is shown with an arrow in Fig. 9.

94
Fig. 3. The fractured surface of sclerotic dentine. Rod-like structures
containing spherical objects are observable on the walls of tubules
( £ 2000). The bar represents 5 mm.
4. Discussion
The primary requirement for the dentine bonding system
is to prevent the separation of the unpolymerized resin
composite paste from the dentine cavity wall throughout the
polymerization contraction of the resin composite. As
demonstrated by Asmussen, the interaction between the
efficacy of the dentine bonding system and the polymerization
contraction stress could be evaluated by the contrac-
Fig. 4. The fractured surface of sclerotic dentine. Mineralized cubic crystals
are observable on the walls of tubules ( £ 10,000). The bar represents 1 mm.
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97
Fig. 5. The normal dentine surfaces conditioned with EDTA for 60 s. Only
the smear layer is removed ( £ 2000). The bar represents 5 mm.
tion gap width measurement of the resin composite restored
in the cylindrical dentine cavity [18]. Hasegawa et al. noted
that marginal adaptation of the resin composites could not
be predicted by the tensile bond strength measurement. In
the cavity, the polymerization contraction of resin composite
produces a marginal gap when the marginal integrity is
not obtained. The contraction gap is impossible to detect in
the tensile bond strength measurement, because the resin
composite paste flows toward the flat dentine surface only.
Fig. 6. The sclerotic dentine surfaces conditioned with EDTA for 60 s.
Almost all the tubules are closed. The structure of sclerotic dentine is
different from that of normal dentine ( £ 2000). The bar represents 5 mm.

Fig. 7. The same specimen as Fig. 5. Normal dentine surfaces etched with
phosphoric acid for 30 s. The opening of the tubules has been widened
( £ 2000). The bar represents 5 mm.
Therefore only the wall-to-wall polymerization contraction
gap width measurement can judge whether the dentine
bonding system meets the primary requirement [19].
In the previous papers, it was reported that when using
the experimental dentine bonding system the contraction
gap formation of the light activated resin composite was
completely prevented [4,20]. The first step is to condition
with 0.5 mol/l neutralized EDTA solution (pH 7.4) for 60 s
to remove only the smear layer and to cause as little
decalcification as possible to the dentine. This is considered
important because the functional monomer in the bonding
agent combines with calcium in the dentine. The most
valuable advantage of this EDTA conditioning was that the
possibility of the nanoleakage which was observed at the
profound layer of the hybrid was prevented. The second step
is to prime with 35 vol% glyceryl mono-methacrylate (GM)
solution to prevent both the monomer infiltration into the
dentine and the water from coming up through the dentine
tubules. In addition, the contraction gap formation was
impossible to prevent completely with 2-HEMA priming
because the functional monomer infiltrated into the 2-
HEMA primed dentine. Consequently the monomer concentration
at the adhesive interface was also reduced. The
GM solution was completely effective in obtaining the
marginal integrity of the resin composite because it
prevented the monomer diffusion into the dentine and kept
the monomer concentration just beneath the cavity wall
high, which was observed as the high density zone by
transmission electron microscope [21]. In this bonding
procedure, the bonding layer was prepared in the extreme
superficial dentine at the adhesive interface. The third step is
to bond with a commercial dual cured bonding agent
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97 95
Fig. 8. The same specimen as Fig. 6. Sclerotic dentine surfaces etched with
phosphoric acid for 30 s. It is similar to the image in Fig. 7. The structure of
the sclerotic dentine is considered to be effective for adhesion was collapsed
by phosphoric acid. ( £ 2000) The bar represents 5 mm.
containing a functional monomer (10-MDP). Thus the
concentration of both the calcium and the functional
monomer at the adhesive interface was decreased and
unpolymerized resin composite easily separated from the
dentine cavity wall [6,7].
If the dentine cavity wall is decalcified by phosphoric
acid, the dentine adhesive should be repeatedly applied to
Fig. 9. The fractured surface of the normal dentine etched with phosphoric
acid. The top of the dentine is decalcified forming a funnel-shaped
configuration shown with an arrow ( £ 2000). The bar represents 5 mm.

96
the surface of the hybrid layer. This is because a single
application of the dentine adhesive is ineffective in
preventing contraction gap formation. In the total-etch wet
bonding system, the resin composite may not have bonded
to the top surface of the hybrid layer but to the surface of the
thick bonding layer. The dentine adhesive applied primarily
might have penetrated into the decalcified dentine and
formed the hybrid layer. Furthermore, Gwinnett et al.
proposed the total-etch wet bonding system, and noted that
the first application was needed to remove water in the
decalcified layer using the alcohol in the adhesives. The
second application was necessary to form a thick bonding
layer on the top of the hybrid layer, which was easily
identified as the luster surface with the naked eye [22–24].
In addition, Pachuta and Meiers reported that the microleakage
of the resin-modified glass ionomer cement (Fuji II
LC, GC, Tokyo, Japan) was not affected by etching with
phosphoric acid [25]. It was noted that the velocity of
polymerization of the resin-modified glass ionomer cement
was small, similar to a chemically cured resin composites.
The resin-modified glass ionomer cement (Fuji II LC)
required a longer period of time to completely polymerize
than resin composite and the velocity of the chemically
cured resin composites was smaller than light activated
ones, therefore the contraction gap decreased [26,27].
The experimental dentine bonding system could be
reproduced in vivo. Despite the fact that a rubber dam was
not employed in the mouth, similar results were found in
vivo and in vitro [28,29]. Therefore the amount of moisture
in the mouth appears to have had only a negligible effect on
the adaptation of the resin composite.
As demonstrated in this study, the contraction gap
formation was completely prevented in half of the specimens
prepared even when the sclerotic dentine cavity wall
was not primed with GM solution. Such an effect similar to
GM priming was possibly caused by the limited monomer
diffusion into the sclerotic dentine. The functional monomer
could not infiltrate into the sclerotic dentine and the
monomer concentration at the adhesive interface was kept
near the surface therefore creating the potential for the
marginal adaptation of the resin composite. Due to the GM
priming, the monomer infiltration into the sclerotic dentine
was further decreased and the reduction of the monomer
concentration at the adhesive interface was prevented
completely. It was speculated that the effect was caused
by differences of structure between sclerotic and normal
dentine. However, the structure of sclerotic dentine which
was observed after EDTA conditioning and was considered
to be advantageous for adhesion, was lost because of
decalcification by phosphoric acid. After phosphoric acid
etching, the structure of sclerotic dentine and normal
dentine were alike. Chiba et al. claimed that the surface of
dentine should not be etched with a strong acid because the
contraction gap width was well correlated with the degree of
softening of dentine by a dentine conditioner [4]. Thus the
resin composite bonded to the surface of the extremely thin
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97
bonding layer formed on the EDTA-conditioned dentine
surface.
From a clinical point of view, sclerotic dentine, which is
frequently observed adjacent to a carious region, cervical
defect or on an exposed root surface should be preserved as
the substrate because it is suitable for bonding. In addition, a
dentine conditioner such as phosphoric acid or citric acid
should not be applied on the sclerotic dentine because the
acid etched dentine may promote monomer diffusion into
dentine and cause the monomer content on the adhesive
interface to be decreased.
References
[1] Nakabayashi N, Kojima K, Masuhara E. The promotion of adhesion
by the infiltration of monomers into tooth substrates. J Biomed Mater
Res 1982;16:265–73.
[2] Munksgaard EC, Asmussen E. Bond strength between dentine and
restorative resins mediated by mixture of HEMA and glutaraldehyde.
J Dent Res 1984;63:1087–9.
[3] Sugizaki J. The effect of the various primers on the dentin adhesion of
resin composites—SEM and TEM observation of the resin impregnated
layer and adhesion promoting effect of the primers. Jpn J
Conserv Dent 1991;34(1):228–65. in Japanese.
[4] Chiba H, Itoh K, Wakumoto S. Effect of dentin cleansers on the
bonding efficacy of dentin adhesive. Dent Mater J 1989;8(1):76–85.
[5] Chigira H, Manabe A, Hasegawa T, et al. Efficacy of various
commercial dentin bonding systems. Dent Mater 1994;10:363–8.
[6] Wu J, Itoh K, Yamashita T, et al. Effect of 10% phosphoric acid
conditioning on the efficacy of a dentin bonding system. Dent Mater J
1998;17(1):21–30.
[7] Manabe A, Tani C, Yamashita T, et al. Bonding is obtained by high
Ca-content and functional monomer. J Dent Res (Special issue B)
1998;77:656.
[8] Manabe A, Itoh K, Tani C, et al. Effect of the functional monomer in
commercial dentin bonding agents use of an experimental dentin
bonding system. Dent Mater J 1999;18(1):116–23.
[9] Yukitani W, Hasegawa T, Yamashita T, et al. Bonding efficacy of
single bond dental adhesive system to dentin. Jpn J Conserv Dent
1998;42(Autumn):101. in Japanese.
[10] Sano H, Takatsu T, Ciucchi B, et al. Nanoleakage; leakage within the
hybrid layer. Oper Dent 1995;20(1):18–25.
[11] Sano H, Yoshiyama M, Ebisu S, et al. Comparative SEM and TEM
observations of nanoleakage within the hybrid layer. Oper Dent 1995;
20(4):160–7.
[12] Vasiliadis L, Darlin AI, Levers BGH. The histology of sclerotic
human root dentin. Arch Oral Biol 1983;28(8):693–700.
[13] Aoyama I, Katagiri M, Suga S. Ultrastructure of sound and sclerosed
dentinal tubules viewed by scanning electron microscopy. Proceedings
of the VI International Conference on X-ray Optics and
Microanalysis; 1973. p. 881–5.
[14] Yoshiyama M, Sano H, Ebisu S, et al. Regional strength of bonding
agents to cervical sclerotic dentin. J Dent Res 1996;75(6):1404–13.
[15] Nakajima M, Sano H, Burrow MF, et al. Tensile bond strength and
SEM evaluation of caries-affected dentin using dentin adhesives.
J Dent Res 1995;74(10):1679–88.
[16] Yoshiyama M, Carvalho RM, Sano H, Horner JA, Brewer PD,
Pashley DH. Regional bond strengths of resins to human root dentin.
J Dent 1996;24(6):435–42.
[17] Heymann HO, Bayne SC. Current concepts in dentin bonding:
focusing on dentinal adhesion factors. JADA 1993;124:27–35.
[18] Asmussen E. Composite restorative resins. Composition versus

wall-to-wall polymerization contraction. Acta Odontol Scand 1975;
33:337–44.
[19] Hasegawa T, Itoh K, Koike T, et al. Effect of mechanical properties of
resin composites on the efficacy of the dentin bonding system. Oper
Dent 1999;24:323–30.
[20] Chigira H, Manabe A, Itoh K, et al. Efficacy of glyceryl methacrylate
as a dentin primer. Dent Mater J 1989;8(2):194–9.
[21] Chigira H, Itoh K, Tachikawa T, et al. Bonding efficacy and interfacial
microstructure between resin and dentin primed with glyceryl
methacrylate. J Dent 1998;26(2):157–63.
[22] Gwinnett AJ. Moist versus dry dentin. Its effect on shear bond
strength. Am J Dent 1992;5(3):127–9.
[23] Gwinnett AJ, Kanca III J. Micromorphological relationship between
resin and dentin in vivo and in vitro. Am J Dent 1992;5:19–23.
M. Kusunoki et al. / Journal of Dentistry 30 (2002) 91–97 97
[24] Gwinnett AJ, Kanca III J. Interfacial morphology of resin composite
and shiny erosion lesions. Am J Dent 1992;5:315–7.
[25] Pachuta SM, Meiers JC. Dentin surface treatment and glass ionomer
microleakage. Am J Dent 1995;8(4):187–90.
[26] Kusunoki M, Itoh K, Hisamitsu H, Wakumoto S. Marginal adaptation
of commercial compomers in dentin cavity. Dent Mater J 1998;17(4):
321–7.
[27] Kato H, Itoh K, Wakumoto S. The bonding efficacy of chemically and
visible light cured composite systems. Dent Mater J 1988;7(1):13–8.
[28] Yanagawa T, Chigira H, Manabe A, et al. Adaptation of a resin
composite in vivo. J Dent 1996;24:71–5.
[29] Tani C, Itoh K, Ohba M, et al. Cavity adaptation of resin composite in
canine cavity in vivo. Dent Mater J 1998;17(3):195–204.