dental

materials
Dental Materials 19 (2003) 245±251
www.elsevier.com/locate/dental

Determination of residual double bonds in resin±dentin interface

by Raman spectroscopy
M. Miyazaki a,*, H. Onose a, N. Iida b, H. Kazama b
a
Department of Operative Dentistry, Nihon University School of Dentistry, 1-8-13, Kanda Surugadai, Chiyoda-Ku, Tokyo 101-8310, Japan
b
Research Laboratories, Tokuyama Corp., 40, Wadai, Tsukuba-city, Ibaraki 300-4247, Japan
Received 30 January 2001; revised 6 September 2001; accepted 4 December 2001

Abstract
Objectives: The quality of the hybrid layer is believed to be more important than the thickness of this layer. The purpose of this study was
to investigate a method to analyze the percentage of adhesive resin residual double bonds in the dentin±resin interface using laser Raman
spectroscopy.
Methods: Bovine dentin was treated with dentin adhesives and resin composite was bonded according to the manufacturers' instructions.
The specimens were sectioned parallel to dentinal tubules and the surfaces were then polished to 1 mm diamond pastes. Raman spectra were
recorded along a line perpendicular to the dentin±resin interface in steps of 0.2 mm. The measurement of residual CyC bond was made on a
relative basis by comparing the CyC unpolymerized methacrylate stretching vibration (1638 cm 21) against the CyO stretching mode of the
ester group (1719 cm 21). The percentage of residual double bonds including pendant and monomeric double bonds was calculated by
comparing the obtained ratio with that of uncured adhesive resin.
Results: The amount of residual double bonds in the hybrid layer varied from 10 to 25% compared to the uncured adhesives, a relatively
higher percentage was detected for Fluoro Bond (12.3±23.6%) and Single Bond (9.5±21.8%), and lower for Mac Bond II (10.6±18.0%) and
Mega Bond (10.7±16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin±
dentin interface.
Signi®cance: Laser Raman microscopy used was a useful tool for measuring the residual double bonds in the dentin±resin interface.
q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved.
Keywords: Laser Raman microscopy; Residual double bond; Dentin bonding; Hybrid layer

1. Introduction If the super®cial dentin is completely demineralized and

A strong and durable bond between resin restoration and
tooth is needed to prevent microleakage and formation of
marginal gap. Excellent adaptation of resin to dentin with
monomer penetration has been reported with scanning and
transmission electron microscopic studies [1±4]. The hybrid
layer is believed to be one of the most effective mechanisms
in dentin bonding [5]. Morphological examination of the
hybrid layer has revealed that the bond strength and sealing
ability of bonding systems are not related to the thickness of
this layer but seem to correlate with the quality of the dentin
substrate [6±8]. Although many advances have been made
in understanding the hybridization process, several impor-
tant questions remain with respect to physical and mechani-
cal properties of this layer.
* Corresponding author. Tel.: 181-3-3219-8141; fax: 181-3-3219-8347.
E-mail address: miyazaki-m@dent.nihon-u.ac.jp (M. Miyazaki).
0109-5641/03/$30.00 + 0.00 q 2003 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved.
PII: S 0109-564 1(02)00039-8
the resin monomer in®ltration is complete, the hybrid layer
consists of approximately 70 vol% of resin and 30 vol% of
collagen ®bers [9]. Mechanical properties of the adhesive
resin in the hybrid layer should play an important role in
creation of a durable bond [10]. Due to the solubility para-
meter theory that controls resin in®ltration [11,12], and
the presence of collagen ®bers, there is a probability that
incomplete resin penetration may occur [13,14]. The exis-
tence of a decalci®ed collagen layer not impregnated by
resin at the base of the hybrid layer might weaken the
dentin±resin bond [15]. Even if the full penetration into
the decalci®ed dentin occurs, the degree of double bond
conversion of adhesive resin in the hybrid layer is not
known. The presence of intrinsic water in dentin and
solvents such as ethanol and acetone in bonding agent
may affect polymerization of resinous components in the
hybrid layer [16]. Inadequate polymerization of the
adhesive monomers or small oligomers might attract
246 M. Miyazaki et al. / Dental Materials 19 (2003) 245±251

water, leading to their slow extraction and diminished bond-

Bonding systems used in this study (4-AET: 4-acryloxyethyltrimellitic acid, HEMA: 2-hydroxyethyl methacrylate, UDMA: urethane di-methacrylate, 5-MNSA: N-methacryloyl 5-aminosalicylic acid, Phenyl-P:
2-methacryloyloxyethyl phenyl hydrogen phosphate, MDP: 10-methacryloyloxydecyl dihydrogen phosphate; Bis-GMA: bisphenol-glycidyl methacrylate, MAC-10: 11-methacryloxy-11-undecarboxylic acid,

00687 A
Lot. no.

050113
ing properties. However, due to thin thickness of the hybrid

0ABJ
J225
layer, insuf®cient information with respect to the quality of
the resin±dentin interface is available. The presence of
oxygen is known to inhibit the polymerization of resin
composite. The bonding resin, which is placed in a very

Pal®que Estelite
Clear®l AP-X
thin layer, might be more susceptible to oxygen inhibition
Lite-Fil II A

Filtek Z250
of the polymerization. It has been reported that air thinning
the bonding resin prior to light irradiation signi®cantly
Resin

reduced dentin bond strength [17]. The thicker layer is

necessary to make the inner region of the bonding resin to
be less in¯uenced by oxygen inhibition. The effect of
oxygen was responsible for the formation of an inhibited
Lot no.

0797

0056

6AB
zone on the surface of the resin in contact with environmen-
004

tal air. The thickness of the oxygen inhibited layer is related

to monomer composition, mode of activation, and viscos-
ities of the resin [18].
Adhesive (main components)

It was the purpose of this study to investigate the degree

FB Bond (4-AET, HEMA,

Bonding Agent (MAC-10,

Bond (MDP, HEMA, Bis-

copolymer water, ethanol,

Single Bond (Bis-GMA,

of remaining uncured methacrylate carbon±carbon double

HEMA, polyalkenoic

bonds (RDB) in the resin±dentin interface including the

UDMA, Filler, CQ)

GMA, ®ller, CQ)

hybrid layer by use of layer Raman microscopy. The

Bis-GMA CQ)

hypothesis tested here was that the RDB in the resin±dentin

interface are different among the resin±dentin interface.
CQ)

2. Materials and methods

A: 079726 B:079732

2.1. Specimen preparation

A: 005 B: 003
A: 038 B:048

The combination of the bonding systems/resin compo-

sites used were three self-etching primer systems Mac
Lot no.

Bond II/Pal®que Estelite (Tokuyama Co., Tokyo, Japan),

7EC

Clear®l Mega Bond/Clear®l AP-X (Kuraray Co., Osaka,

Japan), and Imperva Fluoro Bond/Lite-Fil II A (Shofu
Inc., Kyoto, Japan), and a total-etch adhesive system Single
Conditioner (main components)

Bond/Z250 (3M Dental Products Division, St Paul, MN,

Etchant (35% phosphoric acid)
FB Primer (4-AET, HEMA,

USA) as listed in Table 1. The same adhesive resins without

Primer (5-MNSA, HEMA,

Primer (MAC-10, HEMA,

photoinitiators were provided by each manufacturer as

controls to calculate the percentage of RDB. A dental
photo-curing unit (Optilux 500, Demetron/Kerr, Danbury,
water, ethanol)

water, ethanol)

water, ethanol)

CT, USA) was connected to a variable transformer in order

to adjust the light intensity to 600 mW/cm 2 as measured
with a dental radiometer (Model 100, Demetron/Kerr).
Mandibular incisors extracted from 2 to 3 year old cattle
and stored frozen for up to 2 weeks were used as a substitute
for human teeth in this study [19±21]. It has been reported
that the adhesion to the super®cial layer of dentin showed no
Imperva Fluoro Bond

Clear®l Mega Bond

signi®cant differences between human and bovine dentin,

and the dentin bond strength decreased with the depth of
Mac Bond II

Single Bond

dentin because of the lower density of dentinal tubules [21].

CQ: camphorquinone)

System

Because the differences in tubule diameters and the number

of lateral branches may have some effect on dentin bond
strength [22], bovine super®cial dentin was used as a substi-
tute for human dentin.
Table 1

After removing the roots with a low-speed saw (Isomet,

Code

MB
LB
FB

SB

Buehler Ltd, Lake Bluff, IL, USA), the pulps were removed,
 M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 1. Typical Raman spectrum obtained from Si plate. Because the semiconductor such as Si is very simple consisting of only one Raman peak, this peak was
used for calibration the equipment before measurement.
and the labial surfaces were ground on wet 240-grit silicon
carbide paper to expose dentin. Final ®nish was accom-
plished with 600-grit silicon carbide paper followed by
ultrasonic cleaning with distilled water for 1 min. The
dentin surfaces were treated with primer/etchant and adhe-
sives were applied according to manufacturers' directions.
Resin composite as listed in Table 1 was condensed into a
Te¯on mold (4 mm diameter, 2 mm high) and exposed for
40 s. After 24 h in distilled water at 37 8C, the specimens
were embedded in self-curing epoxy resin (Epon 812,
Nisshin EM, Tokyo, Japan) and stored at 37 8C for 12 h.
After setting, the epoxy-embedded specimens were sec-
tioned parallel to dentinal tubules. The sectioned surfaces
of the cut halves were polished to high gloss (Ecomet 4/
Automet 2, Buehler Ltd) using silicon carbide papers of
600-, 1200- and 4000-grit, successively. Then, the surface
was mirror polished on a special soft cloth (Technofron,
Heraus Kulzer GmbH, Wehrheim, Germany) with diamond
paste (Hyprez Diamond Compounds, Engis Corp., Morton
Grove, IL, USA) to a particle size of 1 mm. Three specimens
originating from different bovine teeth were prepared for
each adhesive system.
2.2. Laser Raman microscopy
Raman spectra of the unpolymerized adhesive resins and
polymerized resins in the hybrid layer were obtained with a
computer-controlled laser Raman microscope (System
2000, Renishaw, UK), equipped to analyze the dentin±
resin interface. The sample was excited at a wavelength of
632.8 nm with the output level of 75 mW by a He±Ne laser
(GLG-5900, NEC Co., Tokyo, Japan) through an optical
microscope. The focus of the laser beam in conjunction
247
with the backthinned CCD camera provided a spatial reso-
lution of 0.6±0.8 mm determined as follows.
The spectral resolution of the laser Raman microscopy
was determined by use of a silicon solid plate, ion-coated
with thin Au. The plate was placed on a precision X±Y
stage and moved across from Au-coated to Au-uncoated
areas using 0.2 mm steps. Spectra of Si (520 cm 21) were
obtained at positions corresponding to each measuring
point and the relative intensities of the spectra were calcu-
lated (Fig. 1). Using curve ®tting data of this spectrum, the
spatial resolution was 1.6±1.9 mm, hence the spectral reso-
lution was determined to be spatial resolution of 0.6±
0.8 mm. Variations of Raman intensities of partially the
Au-coated silicon plate as a function of measuring points
are shown in Fig. 2. From the acquired data, a differential
rate was calculated, and the spatial resolution of the test
instrumentation was determined as 0.6±0.8 mm. This device
was also designed as a confocal microscope and sampling
depth of the Raman scattering from the 1 £ 1 mm 2 area was
calculated as 2 mm.
2.3. Residual double bonds calculation
The specimen was placed on the X±Y stage and the laser
beam was focused on the specimen surface through a 100 £
microscope objective. The specimen was moved relative to
the layer spot position by steps of 0.2 mm from the dentin
towards the adhesive resin, and the spectra were obtained at
positions corresponding to this interval across the dentin±
adhesive interface. When the measurement was performed
on dentin, the laser beam was focused on intertubular
dentin, avoiding dentinal tubules ®lled with adhesive
resin. Raman spectra of the uncured adhesive resins were
also recorded as the control. Measurements were repeated
248 M. Miyazaki et al. / Dental Materials 19 (2003) 245±251

Fig. 3. The Raman spectrum of cured MB in the region of 1560±

1800 cm 21. A few intense peaks were observed in this area.
Fig. 2. Obtained Raman intensities of Si as a function of each measuring
point from Au-coated to Au-uncoated areas by 0.2 mm steps. Differential
rates were calculated from the acquired data, and the spatial resolution of equations:
the test instrumentation was determined as 0.6±0.8 mm.
RDB %† ˆ Rcured =Runcured
three times with different specimens for each bonding where
system.
CyC absorbance area of hybrid layer
The acquired spectra in the region of interest (1500± Rcured ˆ
1800 cm 21) were analyzed using a curve ®tting program CyO absorbance area of hybrid layer
with the Raman microscope software (Renishaw Raman
CyC absorbance area of uncured resin adhesive
Software ver. 3.3.1., Renishaw, UK). Before curve ®tting, Runcured ˆ
a strong peak at 960 cm 21 (P±O stretching mode of hydro- CyO absorbance area of uncured resin adhesive
xyapatite) was eliminated from the obtained spectrum to
Care should be taken that the obtained percentages were
reduce the background effect, and then the spectrum of
based on the assumption that the linearity of the ratios
pure bovine collagen dentin was subtracted. The measure-
between uncured and cured states existed.
ment of RDB was made on a relative basis by comparing the
The area of dentin, hybrid layer, and adhesive resin were
CyC unpolymerized methacrylate stretching vibration
determined by observing changes in Raman intensities at
(1638 cm 21) to that of the CyO stretching mode of the
960 cm 21 (P±O stretching mode of hydroxyapatite) and
ester group (1719 cm 21).
1450 cm 21 (CH scissoring vibration of alkyl group). The
A band whose intensity does not change during polymer-
intensities of the peaks were plotted relatively from dentin
ization should be selected as a reference band. In this study,
to resin [26±32].
the CyO stretching mode of the ester group at 1719 cm 21
The gradual increase in intensity of the Raman peak at
was used as an internal standard, because some of the adhe-
960 cm 21 (hydroxyapatite) from resin to dentin side of
sive resins do not use aromatic ring containing monomers as
the resin±dentin interface was observed. On the other
base resins. It has been reported that the CyC stretching
hand, the intensity of the Raman peak at 1450 cm 21 (organic
mode at 1640 cm 21 can be in¯uenced by overlapping with
substrate) across the interface gradually decreased, indi-
the CyO stretching mode at 1719 cm 21, whose intensity has
cating gradual impregnation of bonding resin into the
tended to broaden during polymerization [23]. The
demineralized dentin. From the gradual changes of these
frequency of the CyO is affected by intra- and intermole-
Raman peaks, the width of each area was determined.
cular hydrogen bonding, so that the CyO vibration some-
One-way analysis of variance and Duncan multiple range
times is observed as a broadened doublet [24]. To eliminate
test was used to statistically evaluate the RDB among the
this effect, the area ratio after curve ®tting was used to
bonding systems used a ˆ 0:05†:
calculate the intensity of the bands [25].
The intensities for the individual bands (1638 and
1719 cm 21) were then obtained from the peak areas by the 3. Results
curve ®tting software, which was designed to calculate a
best-®t curve. In this way, the ratios of the two band areas The Raman spectrum of cured adhesive resin of Mac
were calculated at each measuring point, from dentin side to Bond II adhesive in the spectral region of 1560±
resin side of the dentin±resin interface. The percentage of 1800 cm 21 is shown in Fig. 3. Several intense peaks in the
RDB in the hybrid layer was calculated using the following Raman spectrum were observed. The band area at
 M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
Fig. 4. Representative intensity curves of the selected Raman bands
scanned across the resin±dentin interface. Since the C±H alkyl groups
also appeared in the collagen spectrum in dentinal substrate, the changes
in relative intensities of this peak were considered from the dentin to the
resin side.
1638 cm 21, which is assigned to CyC stretching vibration
of methacrylate was compared to the area at 1719 cm 21,
which is assigned to CyO stretching vibration of ester,
used as an internal standard. Intensity curves of the selected
Raman bands scanned across the resin±dentin interface
obtained with the P±O peak at 960 cm 21 and the C±H
peak at 1450 cm 21 (Fig. 4) allowed the estimated depth
of resin impregnation into dentin. Since the C±H band
obtained at 1450 cm 21 also appeared in the collagen spec-
trum, the changes in relative intensities of this peak were
considered from the dentin to the resin. These depths were
2.2±2.4 mm for Mac Bond II, 2.0±2.2 mm for Mega Bond
and Fluoro Bond, 6.4±6.8 mm for Single Bond.
Fig. 5 shows the changes in percentages of RDB in the
hybrid layer of each bonding system. The percent of residual
double bonds detected in the hybrid layer ranged from 9.5 to
23.6%, and was different among the bonding systems used
in this study. A relatively higher percentage of RDB was
detected for Fluoro Bond (12.3±23.6%) and Single bond
(9.5±21.8%), and lower for Mac Bond II (10.6±18.0%)
and Mega Bond (10.7±16.3%). No relationship was seen
between the percentage of remaining double bonds and
the location within the resin±dentin interface.
4. Discussion
The laser Raman spectroscopy is a useful analytical tech-
nique for study of the bonding structure of samples and
determining their composition. Using this technique, the
problems associated with morphological analysis of the
dentin±resin interface with infrared spectroscopy (IR) can
be avoided [33]. The specimens can be observed under
normal atmospheric conditions without ultra-thin sectioning
249
Fig. 5. Percentage of residual double bonds in the hybrid layer of the
bonding systems used in this study.
or other preparatory procedures that might damage the inter-
face. In contrast to conventional IR microscopy, water is a
weak Raman scatterer so that Raman spectra can be
acquired from moist dentin specimens. Despite these
advantages, the background from ¯uorescence dominates
the weaker Raman signal. The ¯uorescence is due to elec-
tronic excitation of the organic component when irradiated
by laser radiation. To help overcome the problem of high
background noise, a He±Ne laser was used as an excitation
source in this study. In addition, the light generated from a
He±Ne laser (632.8 nm) does not contribute to initiating the
polymerization reaction of visible-light cured resins that use
campohroquinone as a photoinitiator [34].
Several approaches are employed for measurement of
conversion in resin composites; the majority of analytical
studies have been done with the use of Fourier-Transmit IR
(FTIR) [35±38]. As a vibrational technique like FTIR,
Raman microscopy has been used to investigate conversion
pro®les of resin composite [23,24,39±41]. Since Raman
microscopy involves scattering rather than absorption, and
laser emission can be focused on the specimen surface,
specimens of any geometry and thickness can be analyzed
without destructive procedures. The molecular vibration
frequencies observed by FTIR and Raman microscopy are
nearly the same, and Raman scattering due to a symmetrical
vibration (such as CyC) is more sensitive than FTIR
scattering [33].
The micromechanical entrapment of resin in the
demineralized dentin substrate is of importance to create a
hybrid layer. The dentin±resin interface has been investi-
gated by use of micromorphological techniques and the
importance of resin penetration through the entire depth of
decalci®ed dentin has been emphasized. After in®ltration of
the resin monomers into the decalci®ed dentin, subsequent
polymerization of monomers is required to create a stable
bond. If the polymerization of these monomers is not
complete, hydrophilic monomers or small oligomers might
be extracted or hydrolyzed by the presence of nanoleakage
250 M. Miyazaki et al. / Dental Materials 19 (2003) 245±251
[13]. The quality of the hybrid layer and uniformity of resin
impregnation are important factors to understand contribu-
tions of the hybrid layer for dentin bonding mechanism.
The presence of water inside the dentin substrate might
interfere with the polymerization of resin adhesives [16].
Residual resin monomers may act as a plasticizer to alter
the mechanical properties of adhesive resin as well as hybrid
layer, leading to lower dentin bond strength. From the
results of this study, the amount of RDB of the adhesive
resin in the hybrid layer varied from 10 to 25%, which is
lower than reported for the restorative composite resins at
25±55% [42]. The amount of RDB in methacrylate groups
depends on the extent of polymerization reaction. For a
light-cured resin, the degree of conversion is affected by
light intensity, which provides the energy to excite the
photoinitiator. It is well known that the distance from
the light tip and the resin surface, and the thickness of the
material are the factors that would affect the conversion
[43]. Since the adhesive resin and the hybrid layer have
micron-sized thickness, light emission from the activator
light should be intense enough to polymerize the monomers
in this layer. Another reason for relatively lower amounts of
residual double bonds in the hybrid layer would be an
elution of monomers into water. The rate of elution of
components from composite resins has been reported to be
rapid during the initial period of immersion in solvent,
leading to substantial solution within hours [44]. Uncured
monomers in the hybrid layer may leach during the speci-
men preparation or storage in water thus reducing the
amount of uncured RDB found.
Relatively higher amounts of RDB in the hybrid layer
were observed with Fluoro Bond and Single Bond. The
degree of conversion appears to depend upon the monomer
composition of the resin and the concentration of initiator/
inhibitor. The conversion of a bis-GMA/TEGDMA mixture
is about 20% higher than that of UDMA/TGDMA mixture
[45]. The Fluoro Bond adhesive system utilizes UDMA as a
base resin. For Single bond, phosphoric acid was used as a
demineralizing treatment to remove the smear layer and to
expose collagen ®bers. The adhesive resin penetrates into
the demineralized dentin by displacing water inside the
collagen network. After in®ltration into the dentin, the
resin monomers should polymerize to create a strong
bond. It is well known that the presence of oxygen from
ambient air during placement of resin composite inhibits
the resin polymerization to some extent [46]. There might
be a possibility that oxygen present in the water around
collagen ®bers acts as an inhibitor of bonding resin poly-
merization for the bonding system, which utilizes a wet
bonding technique.
Due to the micron-size scale and compositional complex-
ity of the hybrid layer, it has been dif®cult earlier to analyze
the nature of the hybrid layer. With the use of the laser
Raman spectroscopy, the remaining double bonds and the
relative extent of conversion of commercial dentin bonding
agents in hybrid layer has been assessed. Further studies are
needed to determine the relationship between the amount of
remaining double bonds and bond strength durability.
Acknowledgements
The authors are indebted to Mr Kimura (Tokuyama Co.,
Tokyo, Japan) for his technical assistance. This work was
supported, in part, by grant in aid (C)(2) 12671861 from the
Ministry of Education, Science, Sports and Culture of Japan.
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