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Clinical Performance of Self-etching


Saliva Contamination

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Adhesives with lication
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Moustafa N. Abousheliba

Purpose: The aim of this clinical study was to investigate the influence of salivary exposure on microtensile
bond strength of one self-etching adhesive resin.
Materials and Methods: Thirty carious premolars were divided into two groups: a rubber-dam was either inserted before or after cavity preparation and the cavities were restored using direct composite resin. After extraction, the teeth were sectioned into microbars and the microtensile bond strength (MTBS) test was conducted
(_ = 0.05). Scanning electron microscopy (SEM) and silver nitrate staining to detect nanoleakage were conducted to evaluate the sealing quality of the bonded interface.
Results: Statistical analysis revealed that placement of rubber-dam before cavity preparation resulted in significantly higher MTBS values (24 2 MPa) and predominantly cohesive failures (85%). SEM of specimens exposed
to saliva revealed the presence of structural defects at the tooth/resin interface and pull-out of shorter resin
tags. Silver nitrate nanoleakage confirmed these findings as well.
Conclusion: It is recommended to insert a rubber-dam before commencing with cavity preparation in order to ensure strong and durable bond strength.
Keywords: salivary contamination, microtensile bond strength, SEM.
J Adhes Dent 2011; 13: 489493
doi: 10.3290/j.jad.a19648

Submitted for publication: 09.01.10; accepted for publication: 31.05.10.

stablishment of strong and durable bond strength


between the tooth structure and the restoration has
always been the ultimate goal for any adhesive system.
Ease of handling and reduction of application steps
became the driving force for the introduction of the latest generation of adhesive systems, known as single
step self-etching adhesives, which rely basically on
self-etching primers for achieving good bond strength to
both enamel and dentin.9 Nevertheless, some of these
systems require more than one step for achieving adequate bonding to the tooth structure.
During routine cavity preparation, the tooth surface
is contaminated with a smear layer consisting of a mixture of debris, saliva, freshly cut tooth structure, food

a Lecturer,

Dental Biomaterials Department, Faculty of Dentistry, Alexandria


University, Egypt.

Correspondence: Dr. M. Aboushelib, Dental Biomaterials Department, Faculty


of Dentistry, Alexandria University, Egypt. Tel: +2-300-002-012-300-0853, Fax:
+2-300-002-03-560-0466. e-mail: Info@aboushelib.org

Vol 13, No 5, 2011

remnants, bacteria, and blood. Before application of any


adhesive system, the tooth is isolated from the oral cavity
using different types of rubber-dams and suction systems
to prevent re-contamination of the tooth surface with saliva during application of the adhesive system and the
composite resin restoration.8
It has been previously reported that saliva contains
several hydrolytic enzymes capable of reacting with the
tooth structure through different biochemical mechanisms which could alter the external surface of the tooth
structure and thus compromise bond strength.7 There
are studies confirming that contamination of the bonded
area with saliva significantly reduced bond strength of
self-etching adhesive resins.1,21 Re-conditioning of the
contaminated surface restored the original bond strength
values,7 as re-priming of the contaminated surface was
successful in restoring initial bond strength values.15
Depending on the adhesive system used, self-etching
adhesive systems were less influenced by salivary contamination, as these systems were designed to partially
remove or modify the existing smear layer.16 Moreover,
the time of contamination also influenced bond strength
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Preparation of the Specimens


Fifteen patients visiting a private orthodontic clinic were
selected based on the previously mentioned criteria.
On the left side, a simple occlusal cavity (2 to 3 mm
deep) was prepared in the carious premolar using a
diamond point and air turbine. Patients were instructed
to touch the prepared cavity walls with the tip of their
tongue for a period of 1 min to insure complete wetting with saliva (saliva-contaminated specimens). A
rubber-dam was inserted (Optra dam, Ivoclar Vivadent;
Schaan, Liechtenstein), and the premolar on the right
side was prepared accordingly (isolated specimens).
Both prepared cavities were gently dried with com-

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Patients and Selection Criteria


In this study, simple occlusal box-shaped cavities (2.5
mm deep, 3 mm long, 3 mm wide) were prepared under
local anesthesia in mandibular premolars intended for
extraction for orthodontic reasons. Patients and teeth
were selected based on the following criteria: presence of superficial caries not deeper than 2 mm, two
premolars involved (one on each side), sound coronal
structure, good oral hygiene, age between 12 and 19
years, and at least 3 years between insertion of the restoration and time of extraction. Patients were informed
that their extracted premolars were to be used as research specimens and that the restoration technique
was testing one variable in the study. The procedure
was approved by the ethics committee and necessary
consent forms were signed by the parent or guardian of
each patient.

MATERIALS AND METHODS

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values.8 This influence was not observed for glass-ionomer cement, which was less sensitive to salivary contamination; however, it increased probability of failure at
low stresses.10 Etch-and-rinse adhesive systems require
adequate cleaning of the surface and proper removal
of the smear layer in order to expose the orifices of the
dentinal tubules.14,17 The presence of a thick smear layer
or excessive surface contamination could interfere with
the formation of adequate resin tags. In contrast, several studies reported no reduction in bond strength as
a result of surface contamination before acid etching,
which is related to the powerful potential of phosphoric
acid in removing surface contaminants.3,19 It must also
be mentioned that contamination of the surface would
have a more negative effect on the established bond if it
occurred after acid etching.
Currently, there is insufficient clinical data on the influence of salivary contamination on the performance of
self-etching adhesive resins and on the mechanism of
interaction of salivary enzymes on freshly cut tooth surfaces. The aim of this study was to clinically investigate
the influence of exposure of freshly cut dentin to saliva
prior to application of one self-etching adhesive resin. The
proposed hypothesis was that exposure to saliva would
significantly compromise long-term bond strength.

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pressed air (1 short jet at a distance of f4orcm for 3 s)
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to remove excess water and saliva, then a two-step
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self-etching adhesive resin (Clearfil SE Bond, Kuraray;
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Osaka, Japan) was applied following manufacturers
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structions. After digital shade selection (Spectroshade,

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Aboushelib

Spectro Shade Micro; Verona, Italy), the cavities were


incrementally restored using a restorative composite
resin (Filtek Z250, 3M ESPE; Seefeld, Germany). Patients (n = 15) were followed up every 3 months during
their orthodontic visits. All patients were treated by
one qualified dentist.
Microtensile Bond Strength Test
After at least three years of orthodontic treatment, the
restored teeth were extracted, cleaned of soft tissue,
and disinfected. The root was horizontally cut 2 mm
below the cervical line using a diamond wheel, then
the coronal sections were cut into microbars (5 mm
long and 1 mm cross section) using a precision cutting
instrument (Isomet 1000, Buehler; Lake Bluff, IL, USA).
At least 2 sound microbars were retrieved from each
tooth: one was tested and the other stored in water
for a period of 4 years for a later study. The microbars
were glued to the attachment unit using a light-curing
adhesive resin (Clearfil SE) and taking care to center
the dentin/composite resin interface at the free space
of the specimen holder. Tensile load was axially applied on the bonded interface at a crosshead speed of
0.5 mm/min using a 100 N load cell (Instron 6022;
High Wycombe, UK). Stress/strain diagrams were obtained from the universal testing machine, and load at
failure was extracted from computer generated files.
Pre-test failures were assigned a value of 0 MPa and
included in the statistical analysis.
Examination of the Fractured Surfaces
Both the dentin and composite resin sides of the fractured microbars were ultrasonically cleaned, dried, gold
sputter coated, and examined with a scanning electron
microscope (XL 30, Philips; Eindhoven, the Netherlands). The chemical composition of the fractured surfaces was examined using spot x-ray diffraction analysis
(EDAX; Mahwah, NJ, USA).
Nanoleakage of the Bonded Interface
After cutting the specimens for the microtensile bond
strength test, the remaining tooth segments were used
to assess nanoleakage of the bonded interface. The
entire surface of every segment was isolated with red
nail varnish, leaving only 1 mm of the bonded interface
exposed. The coated segments were then immersed
in a buffered silver nitrate solution for 6 h, after which
they were washed under running water for 20 min. The
segments were then immersed in x-ray developing liquid
under fluorescent light for 24 h to fix the silver nitrate
particles. Each segment was then sectioned in the
bucco-lingual direction and the cut surface was polished
using silicon carbide paper (1000 and 1200 grit). The
specimens were then dried for 4 days at 36C using an
electrical induction incubator, carbon coated, and finally
The Journal of Adhesive Dentistry

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Fig 1 SEM image, the tooth side of a saliva-contaminated


specimen demonstrating interfacial failure of a fractured specimen. The dentin side demonstrated partially opened dentinal
tubules (1000X).

Fig 2 SEM image of the composite resin side of the previous


specimen demonstrating resin tag pull-out, leading to partially
opened dentinal tubules in the previous specimen. Observe
that most tubules demonstrated short resin tags. Magnification: 1000X.

examined under SEM for the presence of nanoparticles


of silver across the bonded interface.20 The presence of
silver particles was confirmed using spot EDAX analysis,
which detected silver peaks in the examined spot (1 m
x 1 m).
The thickness of the salivary coat on the prepared
surface of all teeth (isolated and contaminated) was determined immediately before application of the self-etching adhesive using a digital ultrasonic industrial coating
thickness meter (PosiTector 200 B/Adv, DeFelsko; NY,
NY, USA) following ASTM standard D6132-04 for organic
coatings. The pH of the applied self-etching primer 15 s
after the coating was applied was also measured using a
digital pH probe.

specimens (100% cohesive failure in resin cement)


(Fig 1). None of the tested specimens demonstrated
a true mixed failure. Nevertheless, there were 7 saliva
contaminated specimens which demonstrated minor
scattered traces of the adhesive resin on the examined
dentinal surface, representing less than 15% of the entire bonding surface.
SEM examination revealed that exposure to saliva resulted in the formation of short resin tags which were
easily pulled out of the dentinal tubules during load application, resulting in interfacial failure (Fig 2). Additionally,
these specimens revealed the presence of microbubbles
of air located immediately above the resin impregnated
smear layer which were associated with the fractured surface. Isolated specimens demonstrated a predominantly
cohesive fracture and longer resin tags (Fig 3). Using a
digital coating thickness gauge, the average thickness of
the salivary coat on contaminated teeth was found to be
16 m, while isolated specimens demonstrated a 3- to
5-m wet film on the surface. The pH of the self-etching
primer increased from 2.2 to 5.5 for the saliva contaminated surface, and to 3.2 for the isolated specimens
15 s after application.
EDAX analysis of the dentinal surface before and after exposure to saliva did not reveal any change in the
chemical composition of dentin, with the exception of a
slight increase in the percentage of nitrogen for the saliva
contaminated specimens (quantitative analysis was not
straightforward). Nanoleakage assessment indicated the
presence of silver particles above the hybrid layer and at
the orifice of dentinal tubules for all saliva contaminated
specimens (Fig 4). Nanoleakage was observed between
the adhesive resin and the composite restorative material
for only 3 isolated specimens.

Statistical Analysis
One-way analysis of variance (ANOVA) was selected for
analyzing the data. According to the level of significance
(_ = 0.05), sample size (n = 30), and medium effect
size (F = 0.4), the test of choice had adequate power
to detect significant differences between test groups,
which could validate clinical relevance.

RESULTS
Statistical analysis revealed that exposure to saliva
prior to application of the rubber-dam resulted in significant reduction in the MTBS values (18 3 MPa),
compared to the isolated specimens (24 2 MPa).
Additionally, exposure to saliva resulted in an increase
in the percentage of interfacial failure (21 specimens,
ie, 70% of the tested specimens) compared to entirely
cohesive failure in the resin cement for the isolated
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Fig 3 SEM image demonstrating cohesive fracture in the resin


cement, which was the most common failure pattern in the
isolated group. Magnification: 500X.

Fig 4 SEM image in back scatter mode (electron dense materials such as silver nitrate appears white) demonstrating silver
nitrate nanoleakage between dentin (right side) and resin cement. Magnification: 2500X.

DISCUSSION

of reacting with dentin and resulting in structural changes.


EDAX analysis of dentin after exposure to saliva indicated
a slight increase in the weight percent of nitrogen, which
could be related to the chemical interaction between dentin and other chemicals containing amino groups.
In a previous study, it was observed that an increase
in the thickness of resin adhesive (as a result of multiple
coating) resulted in a reduction of MTBS bond values
compared to a single coat, but simultaneously resulted
in a reduction of silver nitrate nanoleakage,6 which is related to the ability of the thicker layer to compensate for
polymerization shrinkage. In the present study, contamination with saliva resulted both in the reduction of bond
strength and an increase in nanoleakage, which were
directly related to interference with the resin tag formation
mechanism and with the presence of structural defects
observed at the hybrid layer.11
After three years of clinical service (loading and thermal cycling), the self-etching adhesive was capable of
maintaining strong and durable bond strength values
(24 MPa), as well as resisting biochemical degradation
and hydrolysis during water storage.12,13 When isolated
from saliva, self-etching adhesive resin demonstrated an
adequate seal, as shown by its resistance to silver nitrate
nanoleakage. However, when the tooth surface was intentionally exposed to saliva, a reduction in bond strength,
structural defects, and nanoleakage were observed.16,18
The reduction in the acidity of primers upon contact
with saliva is related to the buffering capacity of saliva;
in turn, the self-etching capacity on the saliva-contaminated specimens was reduced, leading to the observed
structural defects observed at dentin/resin interface.4 In
presence of a 16-m layer of saliva on the surface, the
performance of the self-etching primer was disturbed,
leading to a deterioration of the established bond after 3
years of clinical service.

Different tests have been designed to measure the


bond strength between adhesive resins and the tooth
structure. The microtensile bond strength test has
become a popular method in the dental literature, as
it offers several advantages. Obtaining few microbars
from one small-sized specimen is a direct advantage
of this technique, which is of great benefit considering
the dimensions of the tested specimens in this study.
Moreover, the direction of the applied tensile load perpendicular to the bonded interface results in a direct
evaluation of the established bond, and reduces misleading cohesive fractures as observed with the shear
test as a result of stress concentration at the loading
point.2 Nevertheless, the microtensile bond strength
test requires investing considerable time and effort to
avoid damaging the fragile specimens.
The findings of this study revealed that exposure of
freshly cut dentin to saliva prior to application of rubberdam resulted both in the reduction of the bond strength
values and an increase in the percentage of interfacial
fracture (debonding failure). Structural defects at the dentin/hybrid layer interface weakened the established bond
and increased nanoleakage in the form of penetration of
nanoparticles of silver nitrate.5 The proposed hypothesis
was thus accepted.
Several theories have been proposed to explain the reaction between saliva and dentin. Saliva contains several
hydrolytic enzymes, such as amylase, collagenase, esterase, and ptyaline, all of which are capable of degrading
the proteins and collagen fibers, which could compromise
the structural stability of the hybrid layer as a result of
depletion of collagen fibers or due to the presence of
a thick smear layer.7,17,21 Besides proteases and collagenase, saliva is full of immune mediators also capable
492

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REFERENCES

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Part of the materials used in this study was provided by the research enhancement program (Alex-rep HTL-023-09) supported by
Alexandria University.

ACKNOWLEDGMENTS

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Placement of rubber-dam before cavity preparation will


significantly improve the clinical performance of selfetching bonding resins and reduce the chance of delamination failure.

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CONCLUSION

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Clinical relevance: It is recommended that rubberdam be placed to prevent saliva contamination before starting cavity preparations, in order to improve
the long-term performance of self-etching adhesives.

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