Elemental Distributions and Microtensile Bond Strength of the
Adhesive Interface to Normal and Caries-Affected Dentin
Masatoshi Nakajima,1 Yuichi Kitasako,1 Mamiko Okuda,1 Richard M. Foxton,2 Junji Tagami1,3
1
Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical
and Dental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113-8549, Japan
2
Department of Conservative Dentistry, Guy’s, King’s and St. Thomas’ Dental Institute, Kings College London,
London SE1-9RT, United Kingdom
3
Center of Excellence Program for Frontier Research on Molecular, Destruction and Reconstruction of Tooth and Bone,
Tokyo Medical and Dental University, Tokyo, Japan
Received 31 March 2004; revised 6 July 2004; accepted 8 July 2004
Published online 20 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30149
Abstract: The aim of this study was to evaluate the microtensile bond strength (␮TBS) and
the elemental contents of the adhesive interface created to normal versus caries-affected
dentin. Extracted human molars with coronal carious lesions were used in this study. A
self-etching primer/adhesive system (Clearfil Protect Bond) was applied to flat dentin surfaces
with normal and caries-affected dentin according to the manufacturer’s instructions. After
24 h water storage, the bonded specimens were cross-sectioned and subjected to a ␮TBS test
and electron probe microanalysis for the elemental distributions [calcium (Ca), phosphorus
(P), magnesium (Mg), and nitrogen (N)] of the resin– dentin interface after gold sputtercoating. The ␮TBS to caries-affected dentin was lower than that of normal dentin. The
demineralized zone of the caries-affected dentin–resin interface was thicker than that of
normal dentin (approximately 3 ␮m thick in normal dentin; 8 ␮m thick in caries-affected
dentin), and Ca and P in both types of dentin gradually increased from the interface to the
underlying dentin. The caries-affected dentin had lost most of its Mg content. The distributions of the minerals, Ca, P, and Mg, at the adhesive interface to caries-affected dentin were
different from normal dentin. Moreover, a N peak, which was considered to be the collagenrich zone resulting from incomplete resin infiltration of exposed collagen, was observed to be
thicker within the demineralized zone of caries-affected dentin compared with normal dentin.
© 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 72B: 268 –275, 2005
Keywords:
resin– dentin interface; elemental content; demineralized dentin; hybrid layer
INTRODUCTION
Carious dentin consists of two distinct layers: an outer layer
of bacterially infected dentin, and an inner layer of affected
dentin.1 The caries-affected dentin is uninfected and remineralizable, and should be preserved during clinical treatment.
Therefore, during cavity preparation for an adhesive restoration, after caries removal, large areas of the cavity floor are
composed of caries-affected dentin. However, previous studies have reported that the bond strength to caries-affected
dentin was lower than that of normal dentin and a thicker
hybrid layer was created in caries-affected dentin.2– 4
Correspondence to: M. Nakajima (e-mail: nakajima.ope@tmd.ac.jp)
Contract grant sponsor: Center of Excellence Program for Frontier Research on
Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and
Dental University
© 2004 Wiley Periodicals, Inc.
268
The dentinal caries process consists of a dynamic process
of cyclic episodes of demineralization and remineralization.
The physical and chemical characteristics of caries-affected
dentin are very different from those of normal dentin. Cariesaffected dentin is softer than normal dentin because it is
partially demineralized.2,5–7 Crystals in intertubular cariesaffected dentin are scattered and randomly distributed, with
larger apatite crystallites and wider intercrystalline spaces
compared with normal dentin.8 Moreover, most tubules of
caries-affected dentin are occluded with mineral deposits.6,9
The increased porosity of intertubular dentin in caries-affected dentin permits deeper etching of the intertubular dentin.2 However, the presence of mineral crystals inside dentinal tubules of caries-affected dentin prevents resin tag formation during bonding.4 Electron probe microanalysis
(EPMA) has been used for semiquantitative analysis of mineral elements in tooth tissues.10 Much of the magnesium (Mg)
in dentin is located in the hypermineralized peritubular dentin
269
EPMA STUDY OF RESIN–DENTIN INTERFACE
matrix lining each tubule. A reduction in Mg content of
dentin starts before the beginning of a decrease in calcium
(Ca) or phosphorus (P) content occurs in dentinal caries.11,12
Changes in Mg content could be the first sign of carious
demineralization and may indicate a loss of peritubular dentin
matrix.13 These results may indicate that the elemental contents are different between normal and caries-affected dentin
surfaces as bonding substrates.
EPMA has been used for studies on mineralization and
fluoride uptake in tooth substrates adjacent to restorative
materials.14 –19 However, few studies have used EPMA for
evaluation of the hybrid layer at the resin– dentin interface.20,21 Han et al.20 analyzed Ca and nitrogen (N) distributions at the adhesive interface of total-etch systems, in which
a N-rich layer was detected. The bulk chemical composition
of dentin is mineral, organic matrix, and water. When dentin
is completely demineralized, 90 wt % of the dry dentin matrix
is composed of type I collagen made up of N-containing
amino acids. Using light microscopy, exposed protein was
identified at the resin– dentin interface with the total-etch
technique, which would be due to inadequate adhesive penetration into demineralized dentin.22 In that study, the density
of protein-staining material had a peak at the bottom of the
demineralized zone.
Self-etch adhesive systems partially demineralize the dentin surface and simultaneously infiltrate resin monomer into
the dentin matrix, resulting in the creation of a hybrid layer
containing scattered apatite crystals. The acidic functional
monomer present in the self-etching primer may interact with
Ca ions to form insoluble Ca salts.23 The crystalline characteristics of the dentin matrix may affect the bond strength of
self-etch adhesive systems. The presence of solubilized Ca
within the partially demineralized zone may promote chemical interactions with acidic functional monomers. Moreover,
the bond strength of a self-etching primer/adhesive system to
demineralized dentin was reported to be dependent on the
application time of a calcium phosphate precipitating solution.24 Therefore, an examination of elemental distributions
in a hybrid layer created by a self-etch system seems to be
necessary in order to compare the quality of the hybrid layers
in normal and caries-affected resin-bonded dentin.
The aim of this study was to examine the microtensile
bond strength (␮TBS) and the elemental distributions (Ca, P,
Mg, and N) of the resin– dentin interface created in normal
versus caries-affected dentin by a self-etching primer/adhesive system. The hypotheses tested were that: 1. the ␮TBS to
caries-affected dentin is lower than to normal dentin; and 2.
there is a difference between the elemental distributions of
the adhesive interface in normal versus caries-affected dentin.
TABLE I. Composition of Self-Etching Primer/Adhesive System
(Protect Bond; Kuraray Medical) Used in This Study
Antibacterial primer: MDP, HEMA, MDPB, dimethacrylates,
photoinitiator, water
Fluoride-releasing bonding agent: MDP, HEMA, dimethacrylates,
photoinitiator, surface-treated sodium fluoride crystals, silanated
colloidal silica
MDP, 10-methacryloyloxydecyl dihydrogen phosphate; MDPB, 12-methacryloyloxydodecylpyridinum bromide; HEMA, 2-hydroxyethyl methacrylate.
saline containing 0.2% sodium azide to inhibit microbial
growth for no longer than 1 week until preparation. After
removal of the occlusal enamel, grinding was performed with
600-grit SiC paper under running water according to the
combined criteria of visual examination and Caries Detector
staining (Kuraray Medical Inc., Tokyo, Japan) to obtain flat
caries-affected dentin surfaces, with the surrounding, yellow
dentin being classified as normal dentin as previously described.2,3
␮TBS Test
MATERIALS AND METHODS
A self-etching primer/adhesive system (Clearfil Protect Bond,
PB; Kuraray Medical) was applied to the flat dentin surface
including both normal and caries-affected dentin according to
the manufacturer’s instructions. The self-etching primer contains an antibacterial monomer, 12-methacryloyloxydodecylpyridinium bromide (MDPB), and the adhesive (Table I)
contains surface-treated NaF (sodium fluoride crystals) as a
fluoride-releasing material.4,25 Seven teeth were treated with
the PB primer for 20 s using a sponge and gently air-dried.
The PB adhesive was then applied and light-cured for 10 s
using a halogen light-curing unit (Optilux 500; Demetron,
Danbury, CT). Composite buildups were performed using 1to 1.5-mm-thick increments of Clearfil AP-X (Kuraray Medical) to a height of 4 –5 mm and each layer was light cured for
20 s. After 1 day of storage in tap water at 37°C, the
resin-bonded teeth were vertically sectioned into 4 or 5 0.7mm-thick slabs using a low-speed diamond saw (Isomet;
Buehler Ltd., Lake Bluff, IL) under water lubrication. The 26
slabs were hand-trimmed into an hourglass shape with approximately 1-mm2 cross-sectional areas isolated by normal
or caries-affected dentin using a fine diamond bur according
to the technique for the microtensile bond test as previously
described.26,27 The specimens were attached to a table-top
material tester (EZ-test; Shimadzu Co., Kyoto, Japan) with a
cyanoacrylate adhesive (Zapit; DVA, Anaheim, CA) and
subjected to microtensile testing at a crosshead speed of 1
mm/min (Figure 1). After testing, their failure modes were
determined under optical microscopic observation (⫻20).
Bond strength data were statistically analyzed by Student t
test (p ⬍ 0.05).
Specimen Preparation
EPMA
Eleven extracted human molars with coronal carious lesions
were used in this study. They were stored at 4°C in isotonic
For EPMA, the remaining six slabs of resin-bonded dentin
were polished with diamond pastes down to a particle size of
270
NAKAJIMA ET AL.
Figure 1. Schematic illustration of the microtensile bond testing, how the bonded tooth was vertically
sectioned into multiple slabs, each of which was trimmed to an hourglass shape for measurement of
tensile bond strength to normal or caries-affected dentin.
0.25 ␮m. After gold sputter-coating, the samples were analyzed for the elements of Ca, P, Mg, and N at the resin– dentin
interface using an electron probe microanalyzer of WDS type
with the ZAF correction method (EPMA 1610; Shimadzu) at
an accelerating voltage of 15 kV and a probe current of 100
nA and a probe diameter of 1 ␮m. This EPMA is also
equipped with a Layered-Synthetic-Microstructure of a specialized analyzing crystal to increase sensitivity to N. Line
analysis and color mapping, which depended on the X-ray
intensity of each element was then undertaken by EPMA.
Additionally, elemental distributions of the dentin surface
after treatment with the PB primer were examined. Dentin
disks including caries-affected dentin from four teeth were
prepared and polished with diamond pastes down to a particle
size of 0.25 ␮m. After application of the PB primer, the
primer components were removed with 50% acetone in water, and then the dentin disks were dehydrated in ascending
grades of acetone followed by the hexamethyldisilazane
(HMDS)-drying method.28 The elements (Ca, P, Mg) on the
treated dentin surface were detected by EPMA
RESULTS
mens failed cohesively in dentin. The specimens of cariesaffected dentin showed more cohesive failure in dentin than
those of normal dentin.
EPMA of the resin– dentin interfaces showed that the
demineralized zone (lower contents of Ca and P) was approximately 3 ␮m thick in normal dentin, but in caries-affected
dentin it was much thicker (approximately 8 ␮m thick, Figures 2 and 3). For both types of resin– dentin interfaces, Ca
and P contents gradually increased through the demineralized
zone to a level similar to that of the underlying dentin (Figure
3). N peaks were observed in the demineralized zones in both
interfaces, and an increase in N content occurred from the
interface, peaked, then decreased to a level similar to that of
the underlying dentin (Figure 3). Small amounts of Mg were
detected in the underlying normal dentin, but were less detectable in caries-affected dentin (Figure 2).
Mapping images (Ca, P, Mg) of normal and caries-affected dentin surfaces treated with the PB primer are shown
in Figure 4. The dentinal tubules were open in normal dentin.
TABLE II. ␮TBSs and Demineralized Dentin Thicknesses
Normal Dentin
The ␮TBS results are shown in the Table II. The ␮TBS to
caries-affected dentin was lower than that to normal dentin
( p ⬍ 0.05). In both types of dentin, the major mode of failure
showed adhesive failure between the bottom of the adhesive
layer and the top of the dentin, whereas some of the speci-
Bond strength
DD thickness
Caries-Affected Dentin
43.5 ⫾ 11.1 (14)
3.2 ⫾ 0.2 (6)c
a
29.4 ⫾ 7.5 (12)b
8.4 ⫾ 1.3 (6)d
Bond strength values are mean ⫾ SD (number of specimens) in megapascals. DD,
demineralized dentin thicknesses in micrometers. Values identified with different superscript letters are significantly different ( p ⬍ 0.05) by Student t test.
EPMA STUDY OF RESIN–DENTIN INTERFACE
Figure 2. Mapping images of elemental distributions (Ca, P, Mg, N) and backscatter electronic image
(BEI) of the adhesive interface to normal and caries-affected dentin by EPMA. D, dentin; A, adhesive
resin; T, tubules. N-rich zones were seen in both adhesive interfaces. Although some Mg was detected
in normal dentin, it was less detectable in caries-affected dentin.
Figure 3. Line analysis of Ca, P, Mg, and N at the adhesive interface by EPMA. (a) Normal dentin; (b)
caries-affected dentin. N peaks in both types of dentin were seen. Demineralized zones in normal
dentin and caries-affected dentin were 3.5 and 7 ␮m thick, respectively. D, dentin; A, adhesive resin;
T, tubules. The line analysis was performed along the center portion of two white lines.
271
272
NAKAJIMA ET AL.
Figure 4. Mapping images of elemental distributions (Ca, P, Mg) in the treated surface of normal and
caries-affected dentin. Mineral deposits, in which Mg was detected, were seen in dentinal tubules of
caries-affected dentin. Intertubular dentin of caries-affected dentin had lost most of its Mg content.
Figure 5. Light microscopic photographs and EPMA mapping images of carious tooth. (A) Crosssectioned specimen of carious tooth; (B) specimen A stained by caries detector solution; (C) mapping
images of elemental distributions (Ca, P, Mg) and backscatter electronic image of specimen A by
EPMA. Mg-depleted area corresponds well to the morphological changes seen in light microscope
images (A,B).
EPMA STUDY OF RESIN–DENTIN INTERFACE
In caries-affected dentin, the dentinal tubules were occluded
with mineral deposits, in which Mg was identified; in the
intertubular dentin, the amount of Mg detected was less.
DISCUSSION
A self-etching primer simultaneously demineralizes intact
dentin covered with a smear layer and infiltrates the dentin
matrix with resin monomer. Transmission electron microscopy has shown that the hybrid layer of a self-etching primer/
adhesive system in normal dentin is formed by partial demineralization of dentin, leaving residual apatite crystallites
scattered within the hybrid layer.4 In the present study, the
Ca, P, and Mg contents gradually increased from the demineralized resin-bonded dentin. In addition, because the selfetching systems do not use a rinsing step, the solubilized Ca
and phosphate ions remain dispersed in the primer resin that
infiltrate the hybrid layer interface to a level similar to that of
the underlying dentin in normal dentin. This result indicates
that dentin demineralization by the self-etching primer gradually decreased depending on dentin depth. Therefore, the
hybrid layer created by a self-etching primer/adhesive system
would contain minerals, and their amounts may be different
between the top and bottom of the hybrid layer. Moreover, a
N peak was present in the demineralized zone. Han et al.20
reported that N-rich layers were detected at the resin– dentin
interface with total-etch systems using EPMA, in which the
wet-bonding technique exhibited a thinner N layer than the
dry-bonding technique. They speculated that this N-rich layer
was collagen-rich, because 90 wt % of the N in the dry
demineralized dentin matrix is due to the presence of insoluble type I collagen although N is also present in the remaining 10 wt % of the noncollagenous protein. The dry-bonding
technique causes collagen fibrils in demineralized dentin to
collapse upon themselves during bonding procedures, resulting in an interference in resin monomer penetration. However, the wet-bonding technique prevents collapse of the
collagen fibril meshwork thus facilitating resin monomer
penetration into demineralized dentin. Therefore, there is less
resin within the created hybrid layer using the dry-bonding
technique, so that collagen fibrils would be more dense within
the hybrid layer of the dry-bonding technique compared with
the wet-bonding technique. A self-etching primer/adhesive
system has been reported to prevent shrinkage of collagen
fibrils during bonding procedures because this system simultaneously demineralizes and penetrates resin monomer into
dentin and avoids the rinsing and drying step.29 Therefore,
the density of collagen fibrils should not be different between
demineralized and underlying intact dentin. Our results indicate that a N-rich layer was identified within the dentin
components. We speculate that the N peak in the line scan
indicates more exposure of collagen fibrils resulting from
incomplete infiltration of resin monomer into demineralized
dentin compared with the underlying intact dentin packed by
minerals.
273
Incomplete resin infiltration and polymerization within
demineralized dentin produce leakage pathways through
nanometer-sized channels, which has been termed “nanoleakage.”30 These nanoleakage pathways would permit fluid penetration within hybrid layers, which might affect the durability of resin– dentin interfaces with respect to resin hydrolysis.31 The naked collagen fibrils seem to be the result of an
elution of resinous materials within the hybrid layer, that
were found to be depleted after long-term water storage.31
However, the nanoleakage patterns of caries-affected dentin
were reported to be different from those of normal dentin.32
The ␮TBS of the PB system to caries-affected dentin was
significantly lower than that of normal dentin. This result is in
agreement with a previous study using the PB system.4 The
demineralized zone in caries-affected dentin was thicker than
that in normal dentin in the present study. In previous studies,
hybrid layers in caries-affected dentin were thicker than those
in normal dentin because of an easier diffusion of the acidic
conditioner and resin monomers into the increased porosities
of caries-affected intertubular dentin.2 Previous transmission
electron microscopy (TEM) research has shown that the
hybrid layer created by the PB system to normal dentin was
0.5–1 ␮m thick, whereas that to caries-affected dentin was
3– 8 ␮m thick.4 These hybrid layer values are thinner than our
current results (approximately 3 ␮m thick in normal dentin; 8
␮m thick in caries-affected dentin) for the thickness of the
demineralized zone in normal and caries-affected dentin.
However, the thickness between the resin-bonded interface
and the maximum N content was 1 ␮m in normal dentin and
4 –5 ␮m in caries-affected dentin (Figure 3), which are in
agreement with the previous TEM results.4 These results may
indicate that the dentin beneath the hybrid layers in both types
of dentin was partially demineralized by the self-etching
primer. Moreover, the N peaks of all images in normal dentin
were sharp and narrow, whereas those in caries-affected
dentin were broader (Figure 3). This indicates that the exposed collagen and/or collagen-rich zone in the bottom of or
beneath the hybrid layer of caries-affected dentin appeared to
be much thicker than that of normal dentin. Most of the
dentinal tubules in caries-affected dentin are occluded by
acid-resistant mineral crystals,6,9 which interfere with monomer infiltration and resin tag formation.4 The thicker collagen-rich zone (i.e., N-rich zone) in caries-affected dentin
might have been caused by less lateral infiltration of resin
monomer from the dentinal tubules and deeper dentin demineralization by the self-etching primer. Furthermore, this
zone might be weaker and more porous. TEM observation
showed a porous zone in caries-affected dentin beneath the
hybrid layer but none in normal dentin.4 Additionally, the
debonded specimens of caries-affected dentin exhibited more
cohesive failures in dentin than those of normal dentin.4,33
The presence of a thicker collagen-rich porous zone at the
adhesive interface may give rise to reduced tensile bond
strength to caries-affected dentin and compromise the integrity of bonded restorations with dentinal margins exposed to
the oral environment.
274
NAKAJIMA ET AL.
The dentinal caries process consists of cyclical phases of
demineralization and remineralization. The reduction in Mg
content was found to start before that of Ca and P in carious
dentin.12 For our pilot EPMA evaluation, caries-affected dentin, as well as caries-infected dentin showed much lower Mg
content compared with intact dentin (Figure 5), although the
densities of Ca and P in caries-affected dentin were relatively
similar to intact dentin (Figure 5). However, larger apatite
crystals were reported in remineralized dentin after carious
demineralization, compared with apatite crystals in intact
dentin.8,12 This may be due to the dissolution of CO3- and
Mg-rich apatite and the re-precipitation of CO3- and Mg-poor
apatite.34,35 Indeed, Mg was barely present in the adhesive
interface of caries-affected dentin in the present study. However, Mg was identified in the mineral deposits occluding the
tubules of caries-affected dentin. Intratubular minerals in
carious dentin consist of apatite and large rhombohedral
crystals of Mg-substituted ␤-tricalcium phosphate (whitlokite).36 Differences in the mineral phases of normal and
caries-affected dentin and the presence of intratubular crystals might influence the formation and durability of the hybrid
layer. Further research is necessary to evaluate the quality of
hybrid layer and/or chemical interaction of resin-apatite
within the hybrid layer created in caries-affected dentin as
well as normal dentin.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
CONCLUSIONS
The ␮TBS to caries-affected dentin was lower than that of
normal dentin. The distributions of the minerals, Ca, P, and
Mg, at the adhesive interface to caries-affected dentin were
different from normal dentin, and the demineralized zone of
caries-affected dentin was thicker than that of normal dentin.
The N peak, which was considered to be the collagen-rich
zone resulting from incomplete resin infiltration of exposed
collagen, was observed to be thicker within the demineralized
zone of caries-affected dentin compared with normal dentin.
The thicker collagen-rich zone might affect bond strength
durability as well as the initial bond strength to caries-affected dentin. Further research is required to evaluate bonding durability to caries-affected dentin.
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