Effect of saliva and blood contamination on the bond strength of self-etching adhesive system- An in vitro study.

AIM: The aims of this study were to determine the effect of saliva and blood contamination on the shear bond strength of self-etching adhesive to enamel and dentin; and, to compare the difference in bond strength due to contamination beforeand after application of the self-etch adhesive.
MATERIALS AND METHODS: 40 human mandibular molars were wet ground on both buccal and lingual surfaces to prepare flat superficial enamel and dentin surfaces. They were randomly divided into two groups (n = 40) based on the substrate (enamel and dentin). Each group was further divided into five subgroups (n = 8) based on the type of contamination it was subjected to, and the step in the bonding sequence when the contamination occurred (before or after adhesive application). Fresh saliva and fresh human blood were applied either before or after the application of Xeno III(®) self-etching adhesive system (SES). Composite resin was applied as inverted, truncated cured cones that were subjected to shear bond strength test. STATISTICAL ANALYSIS: One-way analysis of variance (ANOVA) and Tukey's Honestly Significant Difference (HSD) test were used.
RESULTS: Statistically significant reduction in the bond strength was shown after both saliva and blood contamination before and after Xeno III(®) application (P< 0.05). Bond strength is significantly reduced after contamination with blood as compared to saliva.
CONCLUSIONS: When self-etching adhesive systems are used, saliva and blood contamination significantly decrease the bond strength of the adhesive to enamel and dentin of the tooth.

INTRODUCTION

The increasing demand for esthetic restorations has generated intensive research of adhesive materials. Successful adhesion to dental hard tissues is a fundamental requirement prior to the insertion of resin based composites.[1-3] Any contamination of the prepared surface by saliva, blood or gingival crevicular fluid should be avoided in order to achieve a successful and durable bond between the resin composite and the tooth.[45] Achieving good moisture control is a common problem encountered in restorative dentistry, especially when rubber dam isolation is not feasible.[26] Many carious lesions are found in areas where it is difficult to obtain appropriate isolation for the use of dentin bonding agents, especially when the site is near or at the gingival margin.[2] Contemporary adhesive systems can be classified into two groups: total-etching and self-etching systems.[5]

Self-etching adhesives are commonly employed for bonding procedures, as they are user friendly and yield good clinical results.[47] Several recent studies suggested that self-etch adhesives may be more resistant to salivary contamination because hydrophilic adhesive solutions, specifically products with acetone or ethanol based, may displace or diffuse through the salivary film to reach the underlying hydroxyapatite or collagen.[248] However, the effect of blood contamination on the adhesive properties of 1-step self-etching adhesives has not been fully understood.[24910] The aims of this present in vitro study were to determine the effect of saliva and blood contamination on shear bond strength of self-etching adhesive to enamel and dentin, and to compare the difference in bond strengths due to contamination before and after application of self-etch adhesive.

MATERIALS AND METHODS

40 freshly extracted non- carious human mandibular molars were stored in distilled water containing 0.2% thymol solution and used for the study. The teeth were cleaned using an ultrasonic cleaner and mounted in self curing acrylic resin. Samples were wet ground on both buccal and lingual surfaces using a series of silicon carbide discs to prepare flat superficial enamel and dentin surfaces. They were randomly divided into two groups (n = 40) based on the substrate as follows: Group I – enamel and Group II – dentin. Each group was further randomly divided into five subgroups (n = 8) based on the type of contamination subjected to, and the step in the bonding sequence when contamination occurred, that is, before or after adhesive application.

A commercially available self-etching adhesive Xeno III® (De Trey Dentsply, Germany) was used for the study [Table 1]. Fresh unstimulated saliva from the oral cavity and fresh human blood from the fingertip of one of the investigators was collected at the same time that the specimens were prepared for the study.

Table 1

Manufacturers, composition and application procedure of Xeno III® in the study

Control group (E and D: n = 8): In this group, the specimens were not subjected to either saliva or blood contamination. Self-etching adhesive was applied to the enamel and dentin of each specimen according to the manufacturer's instructions.

Contamination group 1 (E1 and D1: n = 8): Enamel and dentin surfaces were subjected to contamination with saliva for 15 seconds using a micro brush. After the contamination, the surfaces were rinsed for 10 seconds with water steam from an air-water syringe, followed by a gentle blast of air for 10 seconds to dry the surface. The self-etching adhesive was applied in the same manner as the control group.

Contamination group 2 (E2 and D2: n = 8): The same procedure as in group 1 was followed here, and the specimens were subjected to contamination with blood.

Contamination group 3 (E3 and D3: n = 8): The self-etching adhesive was applied to both enamel and dentin as in the control group. The surfaces were then subjected to contamination with saliva for 15 seconds, and then rinsed and dried as in group 1.

Contamination group 4 (E4 and D4: n = 8): The same procedure as in group 3 was followed except that the specimens were subjected to contamination with blood.

Table 2 and Figure 1 represent the groups and a summary of the experimental protocol, respectively.

Table 2

Experimental groups and protocol of the study

Figure 1

Schematic representation of the experimental protocol

A 3 mm diameter polytetrafluoroethylene mold was used to create an inverted, and truncated cone of composite (Filtek Z-250®, shade A1 - 3M ESPE, U.S.A.). The composite was inserted in two sections, and each one was photocured for 20 seconds (Bluephase C5®, Ivoclar-Vivadent AG, Liechtenstein). All specimens were stored at 37 °C in water for 24 hours before testing, to simulate oral environment. After storage, the specimens were transferred to the universal testing machine individually, and then subjected to shear bond strength analysis at a crosshead speed of 1.0 mm/minute at the Bangalore Integrated System Solutions (P) Ltd (BiSS), Bengaluru, Karnataka. Bond strengths were expressed in MPa.

RESULTS

The data obtained in the present study was subjected to statistical analysis using One-way analysis of variance (ANOVA), followed by Tukey's honestly significant difference (HSD) test. Regarding the enamel groups, the one-way ANOVA was performed to compare all experimental groups with the control, and a statistically sig-nificant difference between the tested groups was detected (F = 105.785 and P< 0.05). Tukey's multiple comparisons test between subgroups showed that E4 showed a statistically signifi-cant difference in bond strength when compared to the other experimental groups [Table 3]. Regarding the dentin groups, one-way ANOVA also detected a statistically significant difference be-tween the tested groups (F = 58.239 and P< 0.05). Tukey's multiple comparisons test between subgroups showed that D4 showed a statistically signifi-cant difference in bond strength when compared to the other experimental groups. Groups D1 and D3 showed no significant difference due to contamination on dentin either with saliva or blood before application of adhesive [Table 3]. It was observed that contaminated groups which were subjected to rinse and dry treatment before adhesive application showed higher shear bond strength values than after adhesive application.

Table 3

Shear bond strengths for tested groups (Mean ± Standard Deviation, MPa) in the study

DISCUSSION

Saliva and blood are two common contaminants that can affect the prepared tooth surface during placement of adhesives in restorative dental treat-ment. This study was designed to investigate their effect during different stages in the application of a self-etch adhesive. Self-etching adhesive systems have become very popular with clinicians due to the “no-rinse” concept and reduced post-operative sensitivity. Acid etching is not required; therefore, the problem of over-drying or over wetting the dentin is eliminated. This reduces the potential for post-operative sensitivity and bonding problems associated with movement of dentinal fluid through the patent dentinal tubules. The technique-sensitivity associated with bonding to a dehydrated collagen matrix is also eliminated. Self-etching adhesive systems provide a faster application due to a reduced number of components and application steps.[2410-12] Self-etching adhesives systems may be classified into mild, moderate and strong, based on their pH, and, consequently, their ability to dissolve the smear layer and demineralize the underlying dentin.[1314] Xeno III®, an intermediary strong single step self-etch adhesive with an acidic pH of 1.4, has shown better micromechanical interlocking to enamel and dentin when compared to mild self-etch adhesives.[21516] In this study, to simulate gingival bleeding in a laboratory setting, freshly drawn blood was used as the contaminant. Also, studies in literature search have shown that[2910] the addition of an anticoagulant may decrease the bond strength.

Enamel results of this study reveal that saliva and blood contamination prior to the application of an adhesive showcomparatively higher bond strength values than contamination after adhesive application. The single bottle adhesives contain solvents such as ethanol and acetone that seem to denature the glycoprotein sugars, and remove the salivary contamination.[6] Xeno III® containing ethanol might have exerted this effect. Another explanation for this result is that the application of self-etch primer cleans or hydrolyzes blood on the enamel surface.[17] The present study showed a slight reduction in the shear bond strength values when contaminated with saliva after light curing of the adhesive, which was statistically significant. This could be due to the adsorption of glycoproteins onto the poorly polymerized adhesive surface, which results in oxygen inhibition.[1518] The result of the present study is in agreement with the result of an earlier study done by Fritzet al. using a one-step adhesive, which concluded that in a one bottle adhesive system, any contamination of the already cured adhesive layer seriously compromises the bond strength.[815] Hitmi and others also reported that salivary contamination of the cured adhesive layer decreased shear bond strength dramatically.[19]

Blood contamination after light curing of Xeno III® resulted in the most significant decrease of shear bond strength compared to saliva contamination, which is in accordance with the findings of other authors.[4610] Remnants of blood proteins which were not completely removed, could have impaired adhesion between the layers of adhesive and composite. In addition, rinsing of contaminant after adhesive application could have disrupted the oxygen-inhibited and unpolymerized layer.[21020] Our dentin results also showed significantly reduced bond strengths when contamination was done after light curing of self-etching adhesive. Dentin contamination prior to application of bonding agent by both saliva and blood showed almost similar bond strength values. The influence of blood contamination on bond strength to dentin can be attributed to its macromolecules such as fibrinogen and platelets, which can form a film on the dentin surface which was also confirmed by Barakat and Powers.[21]

When contamination occurred following light curing of the adhesive, there is statistically significant decrease in shear bond strength. This was in agreement with the results of the study done by Fritz et al.[8] using one bottle adhesive systems, which concluded that any contamination of the already cured adhesive layer seriously compromised the bond strength. They put forward mainly three different hypotheses for the reduction in shear bond strength values:

Adsorption of glycoproteins to the poorly polymerized adhesive surface, thus preventing adequate co-polymerization;

compromise of the co-polymerization with the subsequent resin layer, by removal of the oxygen inhibited un-polymerized surface layer, during rinsing and drying; insufficient filling of the collagen mesh with resin.[815]

Adhesive application after rinsing and drying of contaminants showed better bond strength. This is because cleaning procedure was able to remove the contaminants from tooth surface andalso render a better adhesion, which is in agreement with the results obtained by many studies.[246101518]

The current study has several limitations, one being that the bond strength after reapplication of an adhesive after rinsing and drying of contaminant and before the composite restoration, was not studied. Other limitation is that the bond failure by microscopic means was not studied. The need for further studies with a better experimental design and using different types of adhesives is felt.

CONCLUSIONS

Within the scope of the current study, the following conclusions were drawn:

Both blood and saliva contamination significantly decreased the shear bond strength, before and after application of Xeno III® on both the enamel and dentin.

Blood contamination decreased the bond strength more than saliva.

Both contaminants showed decrease in the bond strength more after adhesive application, than before.

Prevention of contamination is critical for good adhesion even in self-etching adhesive systems which have been designed to be less technique sensitive.