Effect of Saliva Contamination on the Bond Strength of Dentin Adhesives to Central and Peripheral Primary Dentin in vitro
Ayca T. Ulusoy
The bond strength of adhesives to dentin have been shown to be affected by
a number of different factors, including intrinsic properties of the prepared
dentin, various types of contamination and the chemical composition of the adhesive
agent. The present study investigates the microtensile bond strength (μTBS)
of two different bonding systems at different dentinal areas of primary dentin
after saliva contamination. Caries-free primary molars were randomly divided
into four groups (n = 10) for μTBS. Prime and Bond NT (etch-and-rinse)
and Clearfil Protect Bond (two step self-etch) adhesives were tested under the
following conditions: (a) control, (b) contamination with saliva prior to adhesive
application. Following adhesive and composite superstructure application μTBS
was measured. Maximum load at failure (N) was recorded and converted to MPa.
Statistical analysis was carried out using one-way ANOVA with Tukeys
test. No statistically significant difference was found between the μTBS
of the contaminated and control groups in the central region of primary dentin
for either adhesive system tested (p>0.05). However, saliva contamination
resulted in significant reductions in bond strength in the peripheral region
(p<0.05) for both adhesive systems tested. In the saliva contaminated groups,
μTBS was higher in the central region than in the peripheral region. The
etch and rinse adhesive performed better than the two-step self-etching adhesive
under saliva contamination in both the peripheral and central regions of primary
dentin. Results indicate that saliva contamination should be avoided when restoring
primary teeth with proximal cavities using both Prime and Bond NT (etch and
rinse) and Clearfil Protect Bond (two step self-etch) adhesives. However, confirmatory
studies are needed before conclusive recommendations can be made for clinical
Received: February 27, 2012;
Accepted: June 13, 2012;
Published: June 30, 2012
A rise in aesthetic expectations and improvements in bonding systems have led
to the widespread use of resin-based bonding systems for the restoration of
primary teeth (Pashley et al., 1982; Powers
et al., 2003). The bond strength of adhesives to dentin have been
shown to be affected by a number of different factors, including intrinsic properties
of the prepared dentin (e.g., depth, tubule diameter, morphology, calcium concentrations),
various types of contamination (gingival fluid, blood, saliva, hand-piece oil)
and the chemical composition of the adhesive agent (Fritz
et al., 1998; Prati and Pashley, 1992; Van
Meerbeek et al., 2003). The two main alternatives currently used
for dentin bonding are etch and rinse and self-etch adhesives (Hitmi
et al., 1999). The latter are particularly attractive in pediatric
dentistry, since, they require fewer steps and less time, which helps to avoid
contamination of the operative field (Sattabanasuk et
al., 2006). However, many carious lesions in primary teeth are located
in areas that are difficult to isolate, especially near or at the gingival margin,
where saliva contamination is more likely to occur (Tagami
et al., 1990). The effect of saliva contamination on the bond strength
of adhesive systems to dentin is controversial. Several studies (Jacobsen
and Soderholm, 1995; Van Meerbeek et al., 2003,
2010) have shown saliva contamination to significantly
reduce the bond strength of dentin adhesives, while others have reported no
such reductions (Gwinnett, 1992; Humphrey
and Williamson, 2001). Moreover, there is no consensus about the relationship
between dentin region and bond strength (Fritz et al.,
1998). The clinical performance of adhesive restorations is affected by
the strength of the bond between the adhesive agent and dentin, making it important
to determine the effects of saliva contamination on bond strength, however,
there is no study to date exploring the effects of saliva contamination on adhesive
bond strength to different sites of primary dentin. Therefore, this study aimed
to evaluate the μTBS of two different bonding agents at different dentinal
areas after saliva contamination.
MATERIALS AND METHODS
Tooth preparation: Details of the materials and application procedures
tested are given in Table 1. The study was conducted using
40 caries-free human primary second molars that were exfoliated or extracted
for orthodontic reasons. Teeth were stored in distilled water at 4°C for
a maximum period of three months before use (Kitasako et
al., 2000). Teeth were cleaned of debris and embedded in an acrylic
mold to 2 mm below the cervical line for adaptation to a microcut device. The
occlusal surfaces were sectioned perpendicular to the long axis of the tooth
using a low-speed diamond saw under water cooling in order to expose superficial
dentin within 1-2 mm of the Dentino-enamel Junction (DEJ). Exposed dentin surfaces
were inspected with a light microscope (Olympus SZ61, Tokyo, Japan) to ensure
that no enamel remained. A uniform, flat dentin surface and smear layer were
created by abrading each specimen with 600-grit carbide paper under water (Hosoya,
|| Composition and application mode of adhesive systems used
In this study
|PENTA: Dipentaerythritol penta acrylate monophosphate, MDP:
10-methacryloyloxydecyl dihydrogen phosphate, 4META: 4-methacryl olyl oxyethyl
trimellitate anhydride, MDPB: 12-methacryloyloxy-dodecylpyridinium bromide,
UDMA: Urethane dimethacrylate, Bi-GMA: Bisphenol glycidyl methacrylate,
HEMA: 2-hydroxyethyl methacrylate
Experimental design: Teeth were then randomly distributed into four
groups of 10 teeth each and prepared as follows:
||Group 1: Prime and Bond NT (Dentsply/DeTrey Konstanz,
Almanya) (saliva contaminated group). Dentin surfaces were acid-etched with
36% phosphoric acid gel (Scotchbond Etching Gel, 3M ESPE, St. Paul, MN,
USA) for 15 sec, thoroughly washed and gently air dried for 2 sec and contaminated
with 0.01 mL of fresh human saliva 30 collected from a single donor and
applied with a micropipette. Saliva was left undisturbed for 10 sec (Sattabanasuk
et al., 2006) and the contaminated dentin was then gently dried
for 10 sec from a distance of 1 cm. (Jacobsen and Soderholm,
1995) Prime and Bond NT dentin adhesive was applied according to the
manufacturers instructions (Table 1). A hybrid resin
composite material (TPH, De Trey/Dentsply, Konstanz, Germany) was used to
prepare a superstructure of approximately 5 mm in height to provide sufficient
bulk for microtensile bond-strength testing. Resin composite was applied
in 2-3 layers and each layer was cured for 40 sec
||Group 2: Prime and Bond NT (control group). Dentin surfaces and
superstructures were prepared as in group 1, but without saliva contamination
||Group 3: Clearfil Protect Bond (Kuraray America, New York, USA)
(saliva contaminated group). The primer of this two-step dentin adhesive
system was applied to the dentin surfaces according to the manufacturers
instructions. Dentin surfaces were contaminated as in group 1. Following
contamination, the dentin bonding agent was applied according to the manufacturers
instructions (Table 1) and superstructures were created
using a resin composite (Clearfil AP-X, Kuraray Medical Inc., Japan), as
in group 1
||Group 4: Clearfil Protect Bond (control group). Dentin surfaces
and superstructures were prepared as in group 3, but without saliva contamination
Measurement of microtensile bond strength: Teeth were visually assessed
and marked with indelible ink to identify the central region (between the pulp
horns) and the peripheral region (between the pulp horns and the DEJ) and were
then stored in distilled water at 37°C for 24 h.
Teeth were then sectioned using a water-cooled low-speed Isomet 1,000 diamond
micro-slicing saw (Buehler, Lake Bluff, IL, USA) across the adhesive interface
along the x and y axis to obtain stick-shaped specimens with a bonding area
of 1±0.2 mm2. No failures occurred during specimen production.
The beam-shaped specimens were fixed by their ends to a microtensile bond-strength
testing device (Force Gauge 200 X 0.2 N, Scales Galore, A Division of Itin Scale
Co., Inc. 431 Avenue Brooklyn, USA) using a cyanoacrylate adhesive (502, Eva
Bond group, Japan) and tested in tension at a crosshead speed of 1.0 mm min-1.
Bond strength (MPa) for each specimen was calculated as the failure load (N)
divided by the cross-sectional area of the bonded interface.
Statistical analysis: Shapiro-Wilk test was applied and showed a normal
distribution of data. Mean μTBS of the groups were calculated using one
way analysis of variance (ANOVA) and Tukeys
test was used to identify significant differences between group, with the level
of significance set at p<0.05 (Table 2). Statistical analysis
was performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL.
Means and standard deviations of microtensile bond strength values (MPa) for
superficial central dentin and superficial peripheral dentin are given in Table
||Mean micro tensile bond strengths (MTBS) and standard deviation
for a two-step etch and rinse adhesive (Prime and Bond NT), a two-step self
etch adhesive (Clearfil Protect Bond) and all in one dentin adhesive (I
bond) to superficial peripheral and superficial central dentin
|Differences in superscript letters indicate statistically
significant differences within columns and differences in superscript numbers
indicate significant differences within rows at p<0.05
In the central region, no statistically significant differences were found
between the μTBS to superficial primary dentin of the saliva-contaminated
and control groups for either adhesive system tested (p>0.05). However, in
the peripheral regions, saliva contamination significantly reduced the μTBS
of both tested adhesives to peripheral superficial primary dentin (p<0.05).
Whereas the non-contaminated Prime and Bond NT etch and rinse adhesive specimens
(group 2) had a mean μTBS of 38.4±17.4 MPa to peripheral dentine,
the contaminated Prime and Bond NT specimens (group 1) had a mean μTBS
of only 24.2±11.1 MPa. Similarly, the non-contaminated Clearfil Protect
Bond two-step adhesive specimens (group 4) had a mean μTBS of 26.2±12.4
MPa, compared to only 15.9±10.9 MPa for the contaminated specimens (group
Prime and Bond NT had significantly higher mean μTBS than Clearfil Protect
Bond in saliva contamination groups of both region.
Stereomicroscope evaluation of samples showed mainly adhesive fractures in
The present study examined the effect of saliva contamination on the μTBS
of two adhesive systems to peripheral and central superficial primary dentin.
Saliva contamination was found to decrease the μTBS of both adhesive systems
to peripheral dentin, but did not affect the μTBS of either system to central
The search for restorative materials with improved adhesive capacity has been
the object of considerable research in recent years. Evaluating bond strength
to dental hard tissue is an important element in developing a better understanding
of the clinical performance of bonding systems (Phrukkanon
et al., 2003). However, there is insufficient information currently
available on the effects of saliva contamination on adhesive bond strength to
different regions of primary tooth dentin. Contamination by saliva, blood and
gingival crevicular fluid is a major clinical problem encountered during restorative
dental treatment, especially when the cavity margins are near or at the gingival
margins. While the problem of contamination can be exacerbated by a lack of
cooperation in small children, which precludes the use of a rubber dam for isolation,
resin-based materials are considered innately susceptible to dentinal moisture
contamination, which has been shown to adversely affect bonding properties (Taskonak
and Sertgoz, 2002; Van Meerbeek et al., 2003).
The complex biological nature of dentin may affect the μTBS of adhesives
in different ways (Marshall et al., 1997). For
example, the number of dentin tubules and location of the bonding area may alter
the μTBS of adhesives (Finer and Santerre, 2004).
Pashley (1989) reported that while tubule density and
peritubuler dentin area decreases with distance from the pulp, intertubuler
dentin area increases with distance from the pulp. Previous studies (Hansen
and Munksgaard, 1989; Hosoya, 1994; Staehle,
1999) have shown that dentin bond strength gradually decreases from the
superficial to the deeper layers of dentin due to the decrease in intertubular
dentin ratio. Standardization of dentin is difficult in primary molars due to
the wide coronal pulpal chamber relative to the outer diameter of the tooth.
In this study, standardization was achieved by removing occlusal enamel to a
depth of 2 mm apical to the occlusal pit and using the superficial dentin only
(Nikaido et al., 1998).
Variations in dentin structure and composition occur not only with differences
in depth, but from region to region as well. When compared to central dentin,
peripheral dentin has fewer dentin tubules and they are oriented oblique or
perpendicular to the long axis of the tooth. In theory, resin tags are unable
to form in areas where the dentin tubules are located perpendicular to the long
axis of the tooth (Pashley et al., 1998). Gwinnett
(1992) has shown that μTBS is negatively affected by resin infiltration
into both the dentin tubules and the intertubular matrix in areas where the
dentin tubules are exposed perpendicular to their long axis. A study by Cabrera
and Macora (2007) that looked at the μTBS of resin-based composites
to gingival, central and incisal enamel showed that μTBS decreased significantly
with increases in the distance from the centre of the curing mass. In the present
study, μTBS in the central region was higher than in the peripheral region
for both adhesive systems in the saliva contaminated groups. In theory, proper
bonding requires the shrinkage vectors of resin-based restorations to be oriented
towards the bonded interfaces (Pashley, 1989) so that
the resin composite is pulled away from the peripheral zones during polimerization
(Van Meerbeek et al., 2000). In the present study,
reductions in bond strength in the peripheral region can be attributed to reduced
dentinal tubule density as well as the orientation of shrinkage vectors from
the peripheral to the central zones. Saliva is a very dilute solution composed
of more than 99% water as well as immunoglobulin, glycoprotein, enzymes, mucins,
nitrogenous products and a variety of electrolytes. Excess water from saliva
had been reported to cause over wetting of dentin surfaces and reduce the bond
strength of dentin adhesives. Salivary glycoprotein may also be absorbed and
accumulate on the bonded surface, thus, interfering with proper adhesion and
high-molecular-weight macromolecules in saliva may diffuse into the dentin tubules
(Cabrera and Macora, 2007; El-Kalla
and Garcia-Godoy, 1997; Hashimato et al., 2006)
and complete with hydrophilic monomers during the hybridization process, causing
a reduction in bond strength. Finally, enzymes in human saliva have been shown
to degrade the Bis-GMA in composite and this hydrolytic activity may also contribute
to the breakdown of the bonded interface (Hiraishi et
al., 2003; Park and Lee, 2004).
Studies examining the effects of saliva contamination on bond strength have
had conflicting results. Fritz et al. (1998)
claimed that saliva contamination decreased the μTBS of one-bottle, self-etch
dentin adhesives to dentin by an average of 50%, whereas, Hansen
and Munksgaard (1989) found that saliva contamination did not effect the
shear bond strength of one-bottle, self-etch adhesives. Similarly, a study by
El-Kalla and Garcia-Godoy (1997) reported no differences
in the bond strength of one-bottle adhesives to either contaminated or non-contaminated
dentin surfaces. Park and Lee found that saliva contamination reduced the bond
strength of both two-steps, self-etch and etch and rinse adhesive systems to
dentin. In the present study, saliva contamination was also found to significantly
reduce the μTBS of both a two-step, self-etch adhesive and an etch and
rinse adhesive to superficial primary dentin in the peripheral region; however,
saliva contamination resulted in only a slight, insignificant reduction in μTBS
to superficial primary dentin in the central region. Prior to this study, no
clear differences have been demonstrated in regional bond strength values under
saliva contamination, especially for primary tooth dentin. In the present study,
saliva contamination was blot-dried after conditioning in group 1 and after
primer application in group 3. This means that the water filled collagen layer
will collapse and that a dried protein film will be adsorbed to the dentin surface.
The protein adsorbing properties of dentin have been reported previously (Cabrera
and Macora, 2007). When blot-dried, the protein components of saliva will
form a film on the dentin surface and these proteins will be adsorbed by the
collapsed collagen (Cabrera and Macora, 2007). Both
the collapse of the collagen layer and the protein film will prevent adhesive
from penetrating the exposed collagen network and forming a sound hybrid layer.
The findings of the present study showing lower adhesive bond strengths in the
peripheral region when compared to the central region of the saliva-contaminated
groups indicate that this mechanism would have a greater effect in the peripheral
region of primary tooth dentin than in the central region. This result may be
due to the regional differences of bonding strength would change with the property
of adhesive materials.
The present study found that the etch and rinse adhesive system tested (Prime
and Bond NT) performed better than the two-step, self-etching adhesive system
tested (Clearfil Protect Bond) in both the central and peripheral regions when
saliva contamination was present. It is possible that an increase in dentin
wetness due to the presence of saliva inhibits the ability of water-based adhesives
(such as Clearfil Protect Bond) to evaporate as easily and completely as ethanol
and acetone-based adhesives (such as Prime and Bond NT) and poor evaporation
and thus retention of water may result in a mechanical weakening of the adhesive
layer and hence lower bond strengths. Previous studies have also found etch
and rinse adhesive systems to exhibit higher bond strengths than self-etch adhesive
systems (Can Say et al., 2006; Senawongse
et al., 2004).
In conclusion, this study found saliva contamination to have a significant
negative affect on the bond strength of adhesives to peripheral primary dentin,
but not to central primary dentin. When saliva contamination was present, the
etch and rinse adhesive system tested performed better than the two-step self-etching
adhesive system tested in both regions. Results indicate that saliva contamination
should be avoided when restoring primary teeth with proximal cavities using
both Prime and Bond NT (etch and rinse) and Clearfil Protect Bond (two step
self- etch) adhesives. However, confirmatory studies are needed before conclusive
recommendations can be made for clinical practice.
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