| Abstract|| |
Background & Objectives
Formation of hybrid layer is thought to be the main mode of adhesion between dentin and resin. But, the longetivity of this hybrid layer is questionable as it is subjected to hydrolytic degradation over time. Hence, the concept of reverse hybrid layer on application of NaOCI is contemplated to bring about better bonding. Studies till date, have focused on the effects of NaOCI on sound tooth structure. Hence, this comparative study evaluated the effect of NaOCI on shear bond strength of two fifth generation single bottle adhesive agents on demineralized and remineralized dentin surfaces.
88 humans premolars were taken and divided into two main groups (group 1 and 2) and bonded with two fifth generation bonding agents Adper Single Bond 2 and Excite respectively.. Each of these groups were further divided into three subgroups Control, Demineralizing and reminerlizing group. The specimens in each subgroups were subjected to either acid etching alone or acid etching and NaOCI pretreatment. The teeth were then stored in distilled water for I month and subjected to shear bond testing. Stereomicroscopic evaluation of the fracture mode at debonded interfaces was done. SEM was used to evaluate the resin-dentin interface.
No statistically significant difference existed between the two bonding agents (group I and 2) when the three subgroups were compared either without or with NaOCI application. A statistical significant difference existed for each of the bonding agents between the demineralized and remineralized subgroups. The subgroups subjected to NaOCI pretreatment showed better bond strength values, though statistically not significant.
Interpretation and conclusion
Bond strength values to demineralized dentin surface was higher than remineralized dentin surface both without and with NaOCI application.
Sodium hypochlorite is an effective deproteinizing agent and its application results in the formation of a Reverse Hybrid Layer. This formed a more stable interface and would delay the process of hydrolytic degradation overtime.
Keywords: Bonding agent; Remineralizing solution; Demineralized solution: Hybrid layer. Sodium hypochlorite.
|How to cite this article:|
Vinutha M, Manjunath M K. Invitro evaluation of the effect of sodium hypochlorite on shear bond strength of dentin bonding agent on demineralized and remineralized dentin. J Conserv Dent 2007;10:64-73
|How to cite this URL:|
Vinutha M, Manjunath M K. Invitro evaluation of the effect of sodium hypochlorite on shear bond strength of dentin bonding agent on demineralized and remineralized dentin. J Conserv Dent [serial online] 2007 [cited 2021 May 11];10:64-73. Available from: https://www.jcd.org.in/text.asp?2007/10/2/64/42296
| Introduction|| |
One of the goals in adhesive dentistry is to develop materials that can replace dental tissue in form, function and esthetics. It is clinically important to enhance the adhesion between dentin and restorative resin, leading to better retention of restoration, preventing marginal leakage and consequently, reducing the incidence of secondary caries. The stability of the bond between composite resin and dentin is of critical importance for longetivity of restoration. 
The acid etching technique put forth by Buonocore in the year 1955, relies on micromechanical retention created by enamel acid etching and subsequent penetration of a blend of polymerizable monomer into the interprismatic spaces to form resin tags. 
In contrast, bonding to dentin is more complicated and achieving predictable bonding has long been a goal and challenge in restorative dentistry. The high organic content of dentin, heterogeneous nature, along with its tubular structure and outward fluid flow makes dentin bonding difficult to attain. Several current dentin adhesives rely on the permeation of hydrophilic monomers into acid-etched moist dentin; subsequent polymerization creates a transitional zone of resin reinforced dentin called "hybrid layer" or "resin-dentin interdiffusion zone" which was first described by Nakabayashi in the year 1982. 
The main limitations imposed by the total etch, wet bonding technique is derived from the observation that, the degree of wetness might influence the dentin bond strength. Additionally the acid exposed noninfiltrated collagen fibres undergo hydrolytic degradation when exposed to fluid for long periods. Due to these limitations of the total etch technique, alternative dentin bonding strategies have been proposed, like self etch adhesives and deproteinization. 
Sodium hypochlorite is a non-specific proteolytic agent that effectively removes organic compounds at room temperature  . A "reverse hybrid layer" has been shown to form when this technique is applied to sound dentin, and bond strength assays using this technique have shown system dependent high-bond strength values. ,
Although, most bond strength testing is done on normal dentin for convenience, clinically most bonding substrates are not normal dentin, but rather caries affected and infected dentin. In a carious lesion the de and remineralized sites exists concomitantly in vivo situation. However, it is difficult if not impossible to mimic the same in vitro. Hence, studies demonstrating the effect of Sodium hypochlorite (NaOCI) on demineralized and remineralized dentin is to be carried out for better understanding of the bonding mechanism and bond strength analysis. Studies till date, have focused on the effect of NaOCI on sound tooth structure, the results of which are controversial.
Variations have been reported regarding the bond strength values obtained using the various single bottle bonding agents available at present.  This study is aimed at determining the bond strength values to demineralized and remineralized dentin using the 5 th generation adhesives after subjecting to NaOCI pretreatment, hence attempting to simulate in vivo condition and better understanding of dentin bonding.
| Methodology|| |
88 recently extracted, caries free human premolars were obtained for the present study. The teeth were thoroughly hand scaled, stored in water and used within one month of extraction.
| Materials|| |
Details of the materials used in the present study are given in [Table 1].
a. Artificial saliva - pH 6.7
b. Demineralization solution - pH 4.4
c. Remineralization solution - pH 9
Equipment and accessories used in the study
- A Teflon (polytetrafluoroethylene) mold to create composite resin buttons of 3mm diameter and 2mm thickness.
- A stabilizing device to stabilize the specimens during its curing stages.
- A rectangular block of 1 cm x 1 cm x 2 cm made of aluminium was used to embed the experimental tooth specimens.
- A visible light curing unit Translux (Heraeus Kulzer) 600 mw/cm 2
- Knife edge chisel of4mmwidth
- Acustomizedjig to hold the aluminium block.
- Instron testing machine model 1011
- Stereomicroscope LEICAWILDM3Z
- Scanning electron microscope JEOL 100 CX-II
Preparation of the specimen
The teeth were taken out of distilled water and crowns ground on the occlusal surface just below the DEJ, in an orthodontic model trimmer using water as a coolant. This obtained a flat ground occlusal surface perpendicular to the long axis of the tooth, exposing dentin of not less than 3 mm with a thin peripheral rim of enamel.
The horizontal sectioned teeth were embedded into the rectangular aluminium blocks, exposing the cut dentin. All the prepared teeth were then stored in distilled wateruntil use.
| Method|| |
I. Pretreatment regimen
The stored embedded teeth were retrieved and the dentin surface polished with a wet 600 grit. Aluminium oxide abrasive paper.
The flattened surfaces were covered with an acid resistant nail varnish leaving only a dentin surface of 3 mm exposed. This was then immersed in artificial saliva for 24 hrs, in order to stabilize the ionic changes between the tooth and surrounding area, standardizing the mineral content of the dentin surfaces, before subjecting to demineralizing and remineralizing treatment.
The specimens were randomly assigned to the three subgroups.
1. Control subgroup
The teeth were immersed in artificial saliva during the experimental treatment period till bonding.
2. Demineralized subgroup:
Teeth were immersed in demineralizing solution for 32 hours.
3. Remineralized subgroup
Teeth were first immersed in demineralizing solution for 32 hours, removed, rinsed in deionized water and immersed in remineralizing solution for 8 days.
II. Bonding regimen and adhesive application
Acid etching 35% phosphoric acid for 15 seconds + wash with water for 30 seconds and blot dried.
Acid etching + NaOCI - 35% phosphoric acid for 15 seconds + wash with water for 30 seconds + 5.25 NaOCI applied for 1 minute + water washed for 30 seconds and blot dried.
All the bonded specimens of the 12 subgroups were colour coded and numbered for future reference. The test specimens were then stored in distilled water 30 days before subjecting to shear bond test analysis.
Testing the shear bond test
A knife edge shearing chisel was employed to debond the prepared buttons of composite resin. The chisel was mounted on to the upper member of the shear chuck. The cutting edge of the chisel was then engaged at the dentin-composite interface and force applied perpendicular to the long axis of the specimen.
The equipment was operated at a crosshead speed of 1 mm/min and the maximum load to debond the specimens were recorded in Newtons (N). Shear bond strength was calculated in Megapascals (Mpa) by the ratio of the maximum load in Newtons to the cross sectional area of debonded interface in square mm.
After the shear bond testing, the debonded surface of all groups were stained with 0.5% Basic fuschin for a duration of 2 mins and examined under a stereomicroscope at magnification of 6.5 X to define the location of bond failure.
The observations made were categorized into
I. Adhesive failure : Occurring purely at the restoration dentin interface.
2. Cohesive failure : Occurring purely within the composite resin material or purely within dentin.
3. Mixed failure : Combination of adhesive and any cohesive failure.
Scanning electron microscopic evaluation
Additional two (1 mm in thickness) horizontal sections of specimens were taken to evaluate the effect of both the remineralization and demineralization solution on normal dentin. Further six vertical sections (1 mm thickness) of the bonded composite dentin interface with both the bonding agents were taken after subjecting them to acid etching alone and acid etching followed by sodium hypochlorite treatment to study resin dentin interface.
Observations were made under a JEOL 100 CX-Il electron microscope at 20 KV. Vertical sections were imaged at a magnification of 800 x and 2000 x and horizontal sections were imaged at 2000x and 8000x.
At the end of the immersion period, the teeth were removed from the solution, rinsed with deionized water and dried prior to the bonding procedure.
| Results|| |
The shear bond strength of composite resins Adper Single Bond 2 and Excite were determined for the control, remineralized and demineralized groups both with and without sodium hypochlorite application. A comparison was drawn between the two bonding agents and between each of the pretreatments applied.
The 't' test has been ems ployed to analyse the results. The values were compared with 5% (0.05) level of significance. Significance value was set at < 0.05.
Predominantly Mixed failures - 84%, Adhesive failures -14%. and Cohesive failure - 2%
| Discussion|| |
Adhesion of restorative materials to enamel has become routine and is a reliable aspect of modern restorative dentistry, but dentinal adhesion has proved to be more difficult and less predictable. This is due to its complex structure, formation of smear layer as debris which is burnished on to the dentinal surface while dentin is cut or ground. If the substrate is dried out after acid etching and rinsing, the demineralized collagen might collapse and prevent adhesive resin from infiltrating into the substrate. On the other hand, if the substrate is too wet the excess water can interfere with the action of the adhesive resin. The presence of residual collagen phase of smear layer resists acid etching and consist of small particles of demineralized / denatured collagen which prevents the collagen network from being completely exposed and makes it less permeable. Acid etching seems to induce conformational modification on dentin collagen, characteristic of denaturation and fragmentation process. Silica remnants from the phosphoric acid thickener present on the exposed collagen fibres, cannot be removed by vigorous rinsing. These particles will not bond to infiltrating resin and hence, increase nanoleakage and lead to failure over time. Further the exposure of collagen fibrils on acid etching into which the overlying resin monomers infiltrate are shown to degrade over years Thus, NaOC1 a deproteinising agent is used to completely remove the collagen layer. Bond strength after NaOCI application to normal dentin has been evaluated earlier. Hence, this study was undertaken to assess the effect of NaOCI on demineralized and remineralized dentin surface which would more aptly mimic a clinical carious lesion.
Recently extracted intact premolars for orthodontic purposes was used in the study. This ensured that the teeth could be acquired fresh and the age factor acting as a variable could be avoided. Further, these teeth are caries free and had no sclerotic dentin, in which the tubules are generally occluded by mineral crystals and less etchable. Thus, the bond strength to such dentin are generally lower.  Hence, in this study the use of such virgin dentin subjected to either demineralization or remineralization protocols provided for a controlled substrate resulting in a more meaningful study.
Remineralization specimens were subjected to prior demineralization solution immersion for 32 hours to more closely mimic the clinical condition encountered in the oral cavity, wherein, after an acid challenge saliva brings about remineralization of tooth. 
Although, occlusal dentin tends to give lower bond strength than proximal / buccal dentin due to the regional variability of dentin wetness, occlusal dentin is the bonding substrate used in this study  . Further, with the composite resin becoming more popular as a restoration for occlusal surface of posterior teeth, the occlusal dentin was selected, to give a more predictable and clinical relevant bonding surface.
Present study employed superficial dentin rather than deep dentin because significant higher bond strength in superficial dentin was recorded compared to deep dentin. There are publications showing that the percentage of intertubular dentin is lower in deep dentin than in superficial dentin because of the increasing diameter of the dentinal tubules. Hence, in the present study the teeth were ground just below DEJ to expose superficial dentin. 
Studies have examined the dentin composite bond strength as a function of duration of storage. It was stated that, atleast 24 hours of storage is necessary to permit polymerization shrinkage of the composite to take place and the composite to equilibriate with water. Water equilibriation may take upto 7 days depending on the filler content of the composite. Further, composite / dentin bonds need to be exposed to water to simulate in vivo conditions. Thus, in this study a duration of 1 month of storage period was employed. A drop in strength value was seen when temperature increased to 55° C in earlier studies. Hence, the specimens were stored at room temperature. 
To produce a shear bond failure, the applied force must be located at the interface between the dentin and adhesive. In blunt edge shear testing device or wire loop method the applied force is located away from the interface and is more likely to produce a tensile failure rather than a shear failure. Hence, the knife edge shearing device was employed in the present study as recommended by American Dental Association. ,
The bonded surface area used in this study for shear bond test was 3 mm in diameter. This is in accordance with the earlier studies where 3, 5, 10 mm diameters of bonded surface area have been used. Larger specimens contain more defects than smaller ones, and hence, there is a decrease in bond strength. Thus, for optimal results while using the shear bond test, a surface area of3mm was chosen. 
Although, the ISO standard has recommended that in shear bond test, the load should be applied with a cross head speed of 0.75 0.3 mm/mins. In this study a cross head speed of 1 mm / min was used to minimize the effect of the deviation of applied force away from the adhesive interface and minimizing the effect of this variable on the shear bond strength. 
Two ethanol based adhesive were compared to avoid the gross differences in bond strength results. 
Both the fifth generation bonding agents Adper Single Bond 2 and Excite bonding agent employed the total Etch concept of bonding. Studies have revealed higher strength values with the total etch compared to the self etch approach, and higher bond strength values with the wet bonding technique. 
The percentage of NaOCI used in the study is 5.25%. Earlier studies have found 5.25% NaOCI for 60 secs to be effective in removal of underlying exposed collagen fibrils after acid etching for 30 secs. Thus in the study to maintain optimal results 5.25% NaOCI for 60 sets was chosen. 
NaOCI application was performed after acid etching and not prior to it. Use of NaOCI prior to etching was not found to be effective as there is insufficient exposure of underlying collagen fibrils. 
In the present study the bond strength values of both the bonding agents were much lower than test reported in product literature. This may be due to the fact that these bond strength values were recorded after a duration of 1 month after distilled water storage. It has been reported in earlier studies that bond strength decreases due to hydrolytic degradation with time. Secondly, the bonding was carried out invitro, wherein all the in vivo conditions of a hydrated dentin surface could not be simulated. This may have resulted in a dehydrated dentin surface on exposure to air after sample conditioning or NaOCI application. This may have contributed against the wet bonding criteria required to bond the present fifth generation bonding agents used in this study.
To assess the effect of application of the demineralizing and remineralizing solution, SEM photographs of the horizontal sections were taken. The samples subjected to demineralizing solution, revealed a regular smear layer comprising of smaller closely packed particles. On the other hand, in the remineralizing samples, a highly irregular surface made of uneven clumped larger size particles were seen. This may be due to the partial dissolution of the surface smear layer by the acidic pH of demineralizing solutions (pH 4.4). The alkaline pH of remineralizing solution (pH-9) and the NaF present in it may have resulted in a surface deposition of mineral crystals on the exposed dentin surface.
Longitudinal sections of bonded interface of demineralized specimens without NaOCI pretreatment showed variation in the appearance of hybrid layer between the two bonding agents. In Adper Single Bond 2 the hybrid layer was thicker, with longer resin tags and lateral branches. Whereas, in Excite, the hybrid layer was thinner with short and wide even funnel shaped resin tags. This might have accounted for the slightly lower bond strength of subgroup B2 when compared to B 1, but the values are not statistically significant.
Sections of bonded interface of remineralized specimen without NaOCI pre-treatment showed characteristic features which are in agreement with earlier studies. The hybrid layer in Adper Single Bond 2 (sub group C 1) was thinner with very short few, intact resin tags. Many resin tags were broken at their necks. Excite bonding agents (sub group C2) showed a similar picture but with a slightly thicker hybrid layer and longer resin tags. A gap was seen above the hybrid layer. A better penetration of resin tags in (sub group C2) may be the reason for slightly higher bond strength of this sub group over C 1, but this value is not statistically significant. Furthermore, the bond strength values of the remineralised sub group (C1 and C2) for both the bonding agents was lower than the demineralized sub group (BI and B2) and statistically significant : this is clearly evident in the SEM photographs and may be accounted for, by the tubule occlusion brought about by the remineralizing solution, and hence, effecting the marginal integrity and quality of hybrid layer.
Resin bonded interface of Excite bonding agents subjected to NaOCI treatment showed statistical differences in bond strength values for demineralized (B22) sub groups and remineralized (C22) sub groups. Higher bond strength values were obtained for the demineralized sub group (B22). SEM revealed the direct resin tag penetration into the underlying dentinal tubules (Reverse Hybrid layer) with absence of a true hybrid layer for both bonding agents. The total absence of any resin infiltrated collagen layer, may prevent any hydrolytic degradation from taking place, thus accounting for the overall increase in bond strength values of the control, demineralized and remineralized sub groups (A22, B22, C22), when subjected to NaOCI pre-treatment after acid etching. In the NaOCI pre-treated samples, B22 sub group showed better opening of dentinal tubules and an even resin tag formation. Whereas, the C22 sub group showed very few patent tubules and resin tags, due to blockage of tubules. Hence, the lower bond strength achieved with this sub group. Similar results were obtained for the other bonding agent Adper Single Bond 2 as well.
The results of the stereomicroscopic examination reveals that, the predominant mode of failure in all the sub groups were mixed (within the material) and few adhesive failures. This mode of failure is advantageous since, this results in less pulp irritation. Adhesive mode of failure is not preferred as it leaves dentin surfaces exposed and hence predisposes to irritation of pulp dentin organ.
It should be noted that, the bond strengths obtained in the study indicate short term, non thermally stressed values. Normal masticatory stress may have an entirely different effect on the resin bond that was not simulated in this in vitro study. Any deterioration of bond strength over long periods of time that may occurwas not taken into consideration.
Moreover, the effect of pulpal pressure, dentinal fluid composition, C-factor and other tooth dynamics such as flextural phenomenon were not taken into consideration. Therefore, further invitro studies are necessary in order to test the long term interaction of these other variables with the ones used in this study and its effect on bond strength stability.
| Conclusion|| |
Under the conditions of this invitro study
- The shear bond strength of bonding agents to demineralized dentin subjected to sodium hypochlorite treatment was more than the remineralized sub group.
- The de and remineralized subgroups subjected to NaOCI pretreatment showed higher bond strength values, though statistically not significant.
- There was no statistically significant difference in the bond strength values of the bonding agents Adper Single Bond 2 and Excite in the demineralized and remineralized subgroups when subjected to NaOCI or acid etching treatment alone.
- The main mode of failure observed in all the sub groups was mixed failure.
- SEM photographs of the bonded interface subjected to acid etching alone showed a distinct hybrid layer with resin tag penetration into the underlying dentinal tubules. The bonded interface subject to deproteinization treatment following acid etching showed absence of hybrid layer with direct resin tag penetration into underlying dentinal tubules (Reverse hybrid layer).,[Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7]
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Department of Conservative Dentistry and Endodontics, J.S.S Dental College and Hospital, Mysore -570015
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]