Introduction as gingival crevicular fluid, hand piece oil,


concept of adhesion was introduced into the field of dentistry by Buonocore in
1955 (1). Adhesive dentistry rapidly expanded treatment possibilities and
revolutionized the way direct and indirect restorations were traditionally
performed. Paralleling the growing demand for adhesive restorations, dentine
bonding systems too have undergone an evolution to improve their bond strengths
as well as to reduce their technique sensitivity.

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Dentin bonding agents have evolved from the gold standard – etch and
rinse fifth generation adhesives to the present universal adhesives.
The different generations of dentin bonding agents have witnessed a change in
chemistry, mechanism of action, procedural steps and a varying degree of
clinical efficiency(2). A recent innovation in the one bottle adhesive systems
is their expansion to a more universal bond with 10-Methacryloyloxydecyl
dihydrogen phosphate (MDP) as the active ingredient. These universal bonding
agents can be used in all etch modes for both direct and indirect restorations.

Single Bond Universal(SBU),
marketed as Scotchbond Universal in USA, was the first commercial
universal adhesive and is popularly used by the clinicians worldwide (3,4,5,6). SBU
apart from MDP, also has methacrylate-modified
polyalkenoic acid copolymer (PAAC) in its composition.( Table/Figure 1)  Mitra and co-workers have reported that PAAC
bonds chemically to calcium in hydroxyapatite showing excellent long-term
clinical performance thereby further improving the bond strength (7).

Tetric N Bond Universal (TNBU)
is a relatively new Universal adhesive which has its matrix based on a combination of monomers of hydrophilic, hydrophobic
and intermediate nature allowing it to reliably bridge the gap between the
hydrophilic tooth substrate and the hydrophobic resin restorative.( Table/Figure 1) However studies using this bonding agent are scarce (8,9,10).

of the major problems associated with the use of adhesive systems is the
difficulty in obtaining a moisture-free clean tooth surface for adequate
bonding (11). Moisture control in the working field is particularly difficult
in situations such as equigingival or subgingival cavity margins, seating of
indirect restorations, newly erupted molars or when patients have limited mouth
opening (12). Contamination during the bonding process from sources such as
gingival crevicular fluid, hand piece oil, blood and saliva, can adversely
affect the quality of the bond predisposing it to microleakage at the
tooth-restoration interface. As a consequence, loss of the restoration,
recurrent caries, postoperative sensitivity and discoloration may occur (13).

Studies in the past have shown that salivary contamination has a
deleterious effect on bonding (14,15,16,17,18). But manufacturers are claiming that universal bonding agents are
resistant to salivary contamination. In accordance to this, study by
Santschi and colleagues concluded that saliva contamination did not affect the
bond strength of SBU (19).

the event of contamination, use of an appropriate decontaminating agent to
restore bond strengths has been advocated (20,21). Work
by Yoo et al. and Santschi et al. has shown that for all-in-one adhesives, washing, drying
and adhesive reapplication was the most effective decontamination protocol (12,19).

to date, studies which have investigated the effect of salivary contamination
on the universal bonding agents are scant and conflicting
(19, 22). Hence the aim of this study was to evaluate the influence of
salivary contamination and water rinsing as a decontamination method on the
shear bond strength of universal bonding agents.

Materials and Method

Type of study- Original
research (in- vitro study)

Name and place of the
institute- Y.M.T. Dental College and Hospital, Navi Mumbai, Maharashtra.

Time duration- April 2017-
September, 2017

Sample selection

freshly extracted intact, caries free human premolars were selected for the
study as per statistician’s recommendation (attached at
the end of the document). All the collected teeth were cleared of blood
and saliva and cleaned under tap water with a scaler and
stored in buffered isotonic saline solution. Teeth with cracks, restorations or
any anatomical deformities were excluded from the study.


Sample preparation and mounting of

Teeth were mounted in self-cure acrylic
resin (Dental Products India Ltd.). The occlusal surfaces of the teeth were sectioned off with a double face
diamond disc under water cooling to prepare flat dentin surfaces at a depth of
1.5 mm from the cuspal tip of the tooth. The dentin surface to be bonded
was ground with #600 SiC paper under running water to produce a standardized
smear layer.

Saliva collection

To achieve standardised salivary
contamination, unstimulated human saliva was collected from a single individual
at least one hour after any consumption of food or drink in a sterile beaker
and was used immediately.

of samples

were randomly divided into two groups of forty five samples each according to
the universal bonding agent used as follows;

Group I- Single
Bond Universal (3M ESPE)

Group II- Tetric®
N-Bond Universal (Ivoclar Vivadent)

forty five premolars in each adhesive group were further divided among three
experimental subgroups (n =15) as follows:

Subgroup 1- Control group:
The premolars in this group were not subjected to any contamination. The
adhesive was applied according to manufacturer’s instructions and light cured
for 10 seconds using Bluephase N® LED unit (Ivoclar Vivadent).

Subgroup 2- Contamination group: The
adhesive was applied according to manufacturer’s instructions. The specimens were covered with fresh
whole saliva for 20 seconds using a disposable brush. A gentle stream of air
was then applied for 2 seconds to dry the surface followed by light curing as
in subgroup 1.

Subgroup 3- Decontamination (Water rinse and reapplication): The
adhesive was applied according to manufacturer’s instructions. After saliva contamination
as in subgroup 2, the contaminated surface was rinsed for 60 seconds with a
stream of water from an air-water syringe. A gentle stream of air was then
applied for 2 seconds to dry the surface and adhesive was reapplied as a part of decontamination protocol and
light cured as in subgroup 1.

Composite placement

Teflon tube of 3 mm inner diameter and 4 mm length was placed on the surfaces.
The Teflon tube was filled with composite resin (Filtek TM Z350,
shade A2, 3M ESPE) in two horizontal increments wherein each increment was
tightly compressed and light cured for 20 seconds using Bluephase N® LED unit
(Ivoclar Vivadent). The Teflon tube
was removed and the resin cylinder additionally cured.

Preparation of samples for shear bond
strength analysis

prepared specimens were stored in distilled water at 37°C for 24 hours and
shear bond strength test was carried out using a Universal Testing Machine
(UNITEST 10, Acme Engineers, India) at a crosshead speed of 0.5 mm/min.

Two examiners evaluated the debonded surfaces at 10X magnification by using a stereomicroscope (Croma
Systems) to identify the mode of bond failure (adhesive, cohesive or mixed). Table/Figure 2


data obtained in the present study was subjected to statistical analysis using
One-way ANOVA test. The intra-group and inter-group comparison was subjected to
statistical analysis using Tukeys HSD test (p<0.05). Statistical Package for the Social Sciences (SPSS) software version 17 was used for statistical analysis. Results Shear bond strength (SBS) values obtained for different test groups with Single Bond Universal (SBU) and Tetric® N-Bond Universal (TNBU) are presented in Table/Figure 3. A drop in mean SBS was seen after salivary contamination for both the groups. As compared to the contamination group there was an increase in mean SBS in water rinsing group. The intergroup comparison showed that TNBU group showed significantly better results as compared to SBU group (p=0.000). The mode of failure in all groups was mainly adhesive Table/Figure 4. Discussion The present study was conducted to ascertain both the effect of salivary contamination and decontamination method on shear bond strength of two universal bonding agents- Single Bond Universal and Tetric N Bond Universal. The shear bond strength (SBS) of dentin was adversely affected by salivary contamination for both the adhesives. Further, statistical analysis revealed that the decontamination protocol had a significant increase in SBS of both the adhesives. In laboratory tests, the efficacy of dentine adhesion is often evaluated by its SBS. It is useful for a relative comparison of different adhesive systems and for screening new materials (23). The condition of the substrate that is the tooth structure and the chemical composition of the adhesive system influences the bond strength (24). As a result, enhancing the efficacy of adhesive restorative materials has been an area of active research. Pleffken and colleagues and Loguercio and colleagues suggested that active application of the adhesive on dentin improved the bonding performance as well as reduced the degradation rate of the adhesive systems (25,26). Hence in this study, adhesive was applied to the tooth surface in scrubbing action as instructed by the manufacturer to maximize the bond strength. During the study on bond strengths of universal bonding agents, Muñoz and coworkers observed that the self etch approach led to more stable bonds even after long-term water storage as against the etch and rinse approach which seemed to be ultra-structurally more susceptible to biodegradation over time(27). Hence in this study, the adhesive was used in self-etch mode. In the current study, natural human saliva was used as the contaminant. Using artificial saliva or saliva substitutes could have diminished the clinical significance of the study. Moreover, work from several researchers has deemed whole human saliva as an acceptable contaminant (16,19,28,29,37). Unstimulated saliva collected from single, healthy individual was used to reduce variability in pH of the saliva and electrolyte, enzyme, or protein content. In the present study, SBU has consistently shown lower bond strength values as compared to TNBU. This can be a result of PAAC in SBU competing with MDP by binding to the calcium present in hydroxyapatite. Another possibility could be the prevention of monomer infiltration during polymerization due to its high molecular weight (30). However, Mohamed Moustafa Awad on comparing the same adhesives concluded that, when applied in self-etch mode, both can infiltrate into dentin producing high quality interfacial morphology (8). Likewise, a study by Jayasheel and coworkers comparing shear bond strength of universal adhesives inferred that the bond strength values of the TNBU regardless of application mode were comparable to SBU making them reliable for working under different clinical conditions(9). Nevertheless, both the above studies were conducted under ideal conditions without taking salivary contamination into consideration. Saliva is composed mostly of water (99%) with immunoglobulins, polysaccharides, proteins, enzymes and a variety of electrolytes (31). Researchers have implicated proteins in saliva to be the main factors responsible for reduction in bond strength (14-18,32). It has been proposed that saliva macromolecules, especially glycoproteins, adsorbed on the enamel surface act as a barrier preventing complete wetting of resin, in turn, inhibiting the monomers from penetrating the collagen network of dentine (33). Moreover, salivary proteins compete with hydrophilic monomers during the hybridization process, preventing complete polymerization of the adhesive, thereby, further reducing bond strength (34,35). Furthermore, dilution of the adhesive by excess saliva produces a weak hybrid layer. Vitrebond™ copolymer, the patented product present in SBU is claimed to be moisture tolerant. But despite that we found that the SBS has reduced after salivary contamination. This may be due to adsorption of the biofilm and competition of the monomer during hybridization (36). Also degradation of Bis- GMA due to the hydrolytic enzymes of saliva has been reported which can further compromise bonding (37). Stage of saliva contamination is also critical towards its effect on bonding (34,38). In this study specimens were contaminated with saliva after application of bonding agent before light curing. Hence it evaluated the effect of salivary contamination on the uncured bonding agent as this would directly hamper the formation of hybrid layer. Salivary contamination before polymerization is particularly significant as Taneja and coworkers demonstrated greater decrease in bond strength by contamination at this stage (16). Moreover, Santschi has suggested that as the bonding agent is highly water soluble and most liable to dilution before polymerization (19).  Water rinsing is an easy choice to combat saliva contamination of a prepared tooth surface. In a study by Sattabanasuk and colleagues showed that simply rinsing saliva-contaminated enamel surfaces with water restores the bond strength (32).On the other hand, studies have demonstrated that conventional washing protocols do not completely remove the coating of salivary proteins on the enamel surface and a subsequent reapplication of the adhesive after water rinsing and air-drying restores bond-strength value (39). This could be attributed to increased rein- dentin interaction due to multiple coatings of adhesive (40,41,42). Erickson et al. and Cobanoglu et al. after evaluating several saliva decontamination procedures, proposed application of adhesive after rinsing and drying to be more reliable than just drying, rinsing (31,38). They suggested that washing and drying should remove the adhesive layer providing a demineralized surface non-infiltrated by monomers. Hence, water decontamination followed by reapplication of adhesive was the method of choice used for decontamination in this study. The type of dentin substrate used could alter also bond strength, as there could be inter-tooth discrepancy and dentinal tubule diameter variation with age and degree of mineralization (43,44). These variable factors were overcome by the use of teeth from patients whose ages ranged from 15 to 25 years and within six months of extraction. Stereoscopic microscopy helped us evaluate the nature of failure and further gave us an insight in the probable cause for failure. Cohesive mode of failure is persistent if bond strength is more than 20 Mpa (45). As most of the samples in the study had bond strength less than 20 Mpa, adhesive failure was common in this study.     There are limitations in simulating the oral environment in vitro indicating that the excellent physical properties of the adhesive resin that were obtained in vitro are not always attained in vivo. Lower bond strength and failure of adhesives in vivo can be attributed to exposure to oral environment including moisture contact, intraoral temperature, tooth flexure, higher C factor and bacterial enzymes (46). Further long term in vitro and in vivo studies are recommended to improve the understanding of the interaction effect of saliva with various bonding systems which have different chemistry and acidity. Bond durability and sealing ability of the samples decontaminated by the protocol as mentioned in the study after salivary contamination should be investigated. Ongoing research should be directed towards exploration for a novel dentin bonding agent that would be resistant to contamination. Conclusion Within the limitations of this in vitro study, it can be concluded that salivary contamination reduces the shear bond strength of universal adhesives to dentin. Reapplication of the adhesive after water rinsing and drying after salivary contamination improves the bond strength significantly. However, long-term in vivo studies are necessary to substantiate the clinical performance of this technique.