Keywords Contaminants - decontamination - dentin-bonding agents - microtensile - resin composite
Introduction
Current adhesive systems are applied using either an “etch-and-rinse,” “self-etch,”
or “selective etch” technique, which differs in how the adhesives are applied and
how they interact with tooth structures.[[1 ]] Etch-and-rinse systems comprise phosphoric acid to pretreat the dental hard tissues
before rinsing and subsequent application of an adhesive. In the etch-and-rinse approach,
adhesives are applied after phosphoric acid etching, whereas when using the self-etching
technique, the acid-etching step is eliminated, simplifying the procedure.[[2 ]] The evidence available today suggests that the choice between etch-and-rinse or
self-etch systems is often a matter of personal preference.[[3 ]] In general, the etch-and-rinse technique is frequently preferred for indirect restorations
and when large areas of enamel are still present.[[4 ]]
The effectiveness of dentin bonding systems after suitable clinical application protocols
is required to ensure the longevity of restorations. Clinically, many factors are
known to impair adhesion and retention of resin-containing restorative materials such
as the contamination of the operative field with oral fluids and microorganisms.[[5 ]],[[6 ]] To prevent this, the rubber dam is still the most important tool to use to guarantee
moisture control.[[7 ]],[[8 ]] However, moisture control is difficult in some clinical situations, such as caries
located at or near the gingival margin,[[9 ]] and contamination of the operative field with blood or saliva is likely to occur.[[6 ]]
Previous studies that have evaluated the effect of blood contamination during bonding
procedures have shown that this could lead to premature failure of the bond of light-cured
resin composites, even after several decontamination methods.[[10 ]],[[11 ]],[[12 ]] Contamination of operative field can occur at different critical times of the bonding
procedure: before or after acid etching, after application of adhesive without light
activation, after application of adhesive and light application, during insertion
of a resin composite in increments, and after restoration placement.[[10 ]],[[11 ]],[[13 ]],[[14 ]],[[15 ]] Furthermore, few studies[[16 ]],[[17 ]],[[18 ]] have evaluated the effect of blood contamination in conventional adhesive systems.
Thus, this study aimed to evaluate the bond strength of a two-step etch-and-rinse
adhesive system to dentin in the presence of blood contamination and to determine
which decontamination protocol is capable of recovering adhesion. The null hypotheses
to be tested were that (1) blood contamination will not impair the bond strength to
dentin and that (2) the decontamination protocols tested will be able to recover the
bond strength to dentin of a two-step etch-and-rinse adhesive.
Materials and Methods
Twenty freshly extracted bovine incisors were collected and stored in 0.5% Chloramine-T
solution for 7 days. Teeth were then kept in distilled water at 4°C until use.[[19 ]] The criteria for tooth selection included intact buccal enamel free of caries,
cracks, and damage due to extraction. Each tooth was examined under a stereomicroscope
to eliminate teeth with cracks or hypoplastic defects. All teeth were cleaned using
hand scalers and scalpels, and then, their roots were sectioned using a low-speed
diamond saw under water cooling, and their crowns were embedded in polyester resin
(Resina cristal, Comfibras, Porto Alegre – Brasil), allowing the buccal enamel surface
to be exposed. Then, the enamel was removed with an orthodontic grinder, and then,
the exposed dentin surface was wet-ground with 400- and 500-grit SiC abrasive papers
coupled to a universal polishing machine at a speed of 50 rpm, under constant water
irrigation.
Standardized cylindrical cavities were prepared in the flat dentin using a round-wheel
diamond bur (No. 3056, KG Sorensen, Alphaville, SP, Brazil) under water irrigation.
Diamond bur was replaced after every five preparations to ensure efficient cutting.
The cavity dimensions were 4.0 ± 0.1 mm in diameter and 1.0 ± 0.1 mm deep [[Figure 1 ]]. The teeth with the prepared cavities were divided into five experimental groups
of six teeth each, and these were randomly assigned to one of the five blood contamination
and decontamination protocols used [[Figure 2 ]]. Fresh human blood was collected from the fingertip of a volunteer (ethics committee
approval protocol No. 41/11 Dentistry/UFPel) at the same time that the restoration
processes were performed. In the blood contamination groups, the specimens were rinsed
with distilled water and were dried with sterile paper towels.[[20 ]],[[21 ]]
Figure 1: Schematic representation of the microtensile bond strength process used. (a) Root
sectioning. (b). Crown embedded in polyester resin. (c) Dentin surface exposed. (d)
Cylindrical cavity preparation. (e) Bonding procedures. (f) Increments of micro-hybrid
resin composite. (g) Specimens sectioning. (h) Microtensile bond strength beams
Figure 2: Experimental groups and decontamination protocols
The restorative procedures were performed using a two-step etch-and-rinse adhesive
system (Single Bond; 3M ESPE, St Paul, MN, USA) applied in accordance with the manufacturer’s
recommendations. Phosphoric acid gel (Scotchbond etchant; 3M ESPE, St Paul, MN, USA)
was applied during 15 s and then rinsed for 10 s. Excess water was removed using sterile
paper towels, leaving dentin moist. Then, two consecutive coats of adhesive were applied
to the etched dentin using a microbrush. A stream of air was applied for 5 s between
each coat. The adhesive was light cured using a light-emitting diode (LED) photopolymerization
unit (Radii Cal, SDI Limited, Victoria, Australia) with a light irradiance of 900
mW/cm2. After light curing the adhesive, a microhybrid resin composite (Filtek Z-250;
3M ESPE, St. Paul, MN, USA) was applied in four increments of approximately 1.0 mm
thick. Each increment was light cured for 20 s with a light polymerizing unit equipped
with a LED visible light source (Radii Cal; SDI, Bayswater, Victoria, Australia).
After storage in distilled water at 37°C for 24 h, the specimens were sectioned perpendicular
to the bond interfaces in the mesiodistal and buccolingual directions, using a slow-speed
diamond saw (Isomet Saw 1000 Precision, Buehler Ltd., Lake Bluff, IL, USA) to obtain
resin-dentin beams with a cross-sectional area of approximately 0.5 mm2. Six beams
from each tooth were obtained, providing 30 sticks per group for the microtensile
bond strength (μTBS) test. Half of the beams were tested after 24 h and the other
half, after 6 months of storage in distilled water at 37°C (n = 15). The beams were attached to a microtensile testing device with cyanoacrylate
glue (Super Bonder Gel, Loctite® Corp., Henkel Technologies, Diadema, SP, Brazil),
and the μTBS was tested in a universal mechanical testing machine (DL 500, EMIC®,
Pinhais, PR, Brazil), at a crosshead speed of 0.5 mm/min and a load cell of 100 N.
The μTBS values were expressed in MPa by dividing the load (N) applied at the time
of the fracture by the cross-sectional area of the bonded interface (μTBS = F/A).
The fracture modes were evaluated by a single observer, using a light microscope (Mobiloskop;
Renfert, Hilzingen, Germany) at ×100 and ×500. Failure modes were classified as adhesive,
cohesive within dentin, and cohesive within resin composite or mixed failure.
The data were analyzed to check normality (Shapiro–Wilk test) and homoscedasticity
(Levene’s test). Two-way analysis of variance (ANOVA) was used to evaluate the effect
of contamination step and storage time on the μTBS. Multiple comparison procedures
were performed using Tukey’s test (α = 5%). Data were analyzed and plotted with SigmaPlot
12 software (Systat Software Inc., San Jose, CA, USA).
Results
The results of the μTBS test are summarized in [[Figure 3 ]]a. The two-way ANOVA test revealed that μTBS was influenced by both the decontamination
protocol and storage time (P < 0.05); however, the interaction between these two variables was not significant
(P = 0.529).
Figure 3: Microtensile bond strength (a) and distribution of failure modes (b). Columns under
the same horizontal line indicate no differences between aging times for each group.
Different capital or lowercase letters indicate differences between groups within
24 h and 6 months, respectively
In the μTBS test at 24 h, the highest bond strength values were observed for the Control
Group, followed by Group 1; the lowest μTBS values were observed for Group 3. After
6 months of storage in distilled water, the μTBS values decreased in all groups; the
Control Group showed the highest values, while the Group 2 showed the lowest μTBS
values; however, these differences were not statistically significant. The intergroup
analysis revealed that μTBS values for Groups 3 and 4 remained stable.
The numbers and percentages of failure modes in each group are shown in [[Figure 3 ]]b. The results of failure mode analysis after 24 h demonstrated that adhesive type
failure mode was predominant in all groups, followed by mixed-type failure. After
6 months of aging, the results of failure mode analysis demonstrated that the adhesive
type failure mode remained predominant in all groups, followed by the cohesive in
dentin failure type.
Discussion
In this study, the influence of blood contamination on the bond strength of a two-step
etch-and-rinse adhesive to dentin was investigated. Besides, the decontamination protocol
that would be capable of recovering the bond strength of this adhesive system was
determined. Since the statistical analysis revealed that blood contamination impaired
the bond strength of a resin composite to dentin and that none of the decontamination
protocols tested were able to recover the bond strength, the null hypotheses tested
in this study were rejected.
In this study, freshly drawn blood, collected at the same time that the experiment
was being performed, was used to contaminate the dentin surfaces. No anticoagulants
were used since studies in the literature have shown that the addition of an anticoagulant
may reduce the bond strength.[[10 ]],[[22 ]] Considering this variable, and other factors such as the adhesive system used,
the step when contamination occurs, and substrate type, it was difficult to make comparisons
with previous studies that investigated the blood contamination of adhesive restorations.
The results obtained in this study proved that when compared with the control group,
any blood contamination at any of the stages of adhesive system application decreased
the μTBS, both in 24 h and after 6 months. The literature has shown that the blood
is capable of interacting with the dentin surface, and the content of proteins, macromolecules
of fibrinogen, and platelets may form a thin film on the dentin surface, which may
make it difficult for the adhesive to infiltrate into the treated dentin, thereby
weakening the bond strength.[[23 ]] Furthermore, residual blood proteins could remain on the polymerized bond surface
and eliminate an oxygen-inhibited layer, which has the potential of preventing copolymerization
between the successive increments of resin composite material.[[24 ]]
Among the decontamination protocols, when the contamination occurred after the application
of acid and before the application of the adhesive system (Group 1), the bond strength
values were higher than those in the other contaminated groups, both in 24 h and after
6 months. These results could be explained by the cleaning processes performed after
blood contamination, which were able to eliminate a large part of the blood proteins
deposited on the dentin surfaces. In addition, it could be hypothesized the application
of primer cleaned or hydrolyzed blood on the dental surface.[[25 ]] However, as no values equal to those of the control group were obtained, rinsing
with water was shown to be insufficient to achieve complete decontamination of the
dentin surface.
When the contamination occurred after the application of the adhesive system (Groups
2 and 3), the decrease in bond strength could be attributed to the degradation of
the adhesive components of the contaminated adhesive layer, rather than to its removal.
Furthermore, the presence of excessive humidity trapped in the degraded components
in the dentinal tubules may have impaired bonding between the subsequent resin composite
layers (increments).[[26 ]]
Moreover, in this study, it could be demonstrated that after the dentin surface had
been contaminated, it was not recommendable to re-etch the contaminated surface. In
Groups 3 and 4, in which a re-etching procedure was performed after blood contamination,
lower μTBS values were also observed. A possible explanation for this could be that
re-etching the dentin surface could produce an excessive layer of demineralized dentin,
which could be not totally penetrated by the adhesive system, allowing the formation
of a fragile adhesive area.[[27 ]]
This study showed the negative effect that blood contamination has on the bond to
dentin when using a two-step etch-and-rinse adhesive system. Besides, it could be
established that recovering the bond to blood contaminated dentin surfaces did not
depend only on careful cleaning with distilled water, and other cleaning agents should
be tested in further studies.
Conclusions
The findings of this study proved that blood contamination significantly impaired
the bond strength of two-step etch-and-rinse adhesives to dentin. In addition, none
of the decontamination protocols tested were capable of recovering the bond strength.
Therefore, when the dentin surface has been contaminated with blood during the restoration
bonding procedures with the use of two-step etch-and-rinse adhesives, the dentin surface
should be re-prepared with a rotary cutter to prevent impairment of the bond efficiency
of the adhesive system.
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