INTRODUCTION

The adhesive bonding of resin materials to etched enamel is a proven technique with long-term clinical success for the prevention of microleakage and the retention of esthetic restorative materials.1 A pertinent factor in achieving satisfactory adhesion of resin composites to dentin is the way the dentin surface is treated before an adhesive is applied. Fusayama and others suggested removal of the smear layer from dentin using phosphoric acid treatment to improve adhesion.2 Penetration of the adhesive resin into the exposed fibrillar collagen network of the demineralized dentin zone results in the formation of a micromechanical interlock known as the hybrid layer.3 The collagen fibril network of demineralized dentin represents a soft, delicate bonding substrate that may contribute to the technique sensitivity of bonding procedures. For proper formation of the hybrid layer, water is essentially required to prevent collapse of the collagen fibers. However, over-wetting may lead to dilution or deterioration of the monomers, reduced final degree of cure and formation of water-containing defects within the adhesive layer.4 If it is necessary to choose between over-drying or over-wetting of total-etched deep dentin, the former is to be preferred, since vital deep dentin is intrinsically wet after removal of the smear layer.5 The optimum level of moisture required to maintain the integrity of collagen without compromising bonding cannot be clinically ascertained.

The stress absorbing role performed by the hybrid layer in dentin bonding has also been the subject of controversy, as considerably higher bond strengths after elimination of the collagen network have also been reported.6–7 Spencer and Swafford have demonstrated that a demineralized, non-protected dentin zone remains beneath the hybridized layer due to incomplete resin infiltration.8 Subsequent hydrolysis of the exposed collagen peptides could lead to degradation of the dentin to resin bond, resulting in decreased bond strength and increased microleakage over time.9

Because of the limitations associated with the use of total-etch systems, research has been directed toward techniques that alter the chemical composition of dentin by removing the collagen from dentin to enhance resin bonding. The removal of unsupported collagen fibers could have a beneficial effect on the primer, adhesive spreading and diffusion through dentin.10 It may produce a more durable adhesion to the hydroxyapatite component of the dentin substrate.

NaOCl is a non-specific proteolytic agent that effectively removes organic compounds at room temperature and has been frequently applied for collagen removal with controversial results.11–12 Improvement in adhesion to demineralized and deproteinized dentin has been reported, but no interference and decrease in adhesion have also been reported.6–713–15 This decrease in bond strength after NaOCl application has been attributed to the loss of the shock absorbing effect of the hybrid layer or the reduction in the physical properties of dentin.

However, as the application time of NaOCl is minimal, some recent studies have related this interference mainly to the effect of the remaining NaOCl on the polymerization of resin and the specificity of different adhesive systems to the oxidizing effect of sodium hypochlorite.16–18 This residual NaOCl could be neutralized by the application of sodium ascorbate to the oxidized dentin, which acts as a reducing agent, restoring the redox potential of dentin and converting the microenvironment of the dentin from an oxidized substrate to a reduced substrate, thus facilitating complete polymerization. Lai and others demonstrated that a reduction in bond strength caused by NaOCl could be reversed by the application of 10% sodium ascorbate, a neutral biocompatible antioxidant, for one minute.16 However, sodium ascorbate, when applied directly on dentin without prior sodium hypochlorite treatment, has been found to reduce bond strength. End products formed by the reaction between sodium ascorbate and sodium hypochlorite are oxalic acid and L-threonic acid, both which are water soluble.19 It is possible that, by restoring the altered redox potential of the oxidized bonding substrate, sodium ascorbate allows free radical polymerization of the adhesive to proceed without premature termination and, hence, reverses the compromised bonding in NaOCl or H₂O₂ treated dentin.16

Although many in vitro studies have evaluated the effect of surface pre-treatments and compared the efficacy of moist and dry bonding techniques, the combined effect of moisture, collagen removal and reducing agent application on dentin bonding in vivo is still unclear. This study investigated the effect of collagen removal and sodium ascorbate treatment of phosphoric acid etched dentin on the microleakage and ultrastructure of resin tooth interface under moist and dry bonding conditions using an acetone-based 1 bottle adhesive system. The null hypothesis tested was that there is no significant effect of moisture level, NaOCl and sodium ascorbate treatment on resin-dentin bonding.

METHODS AND MATERIALS

This study was performed in 90 caries free premolars, scheduled for orthodontic extraction, after receiving the patient's informed consent. Class V cavities were made on the buccal surfaces of the premolars using an ISO 012 straight fissure diamond bur (Dentsply Detrey) in an air-water cooled high speed handpiece. No local anesthesia was used. The cavity preparations were standardized with a width of 3 mm mesiodistally, 2 mm occluso gingivally and 1.5 mm deep, measured at the gingival level, ensuring the axial wall to always be in dentin. The occlusal and gingival cavosurface margins were prepared butt joint in enamel without any bevel. No additional mechanical retention was placed. The teeth were divided into six groups of 15 teeth each according to different surface treatments and the degree of moisture of the bonding substrate.

Group 1 (Moist, M): The cavities were etched with 37% phosphoric acid for 15 seconds and rinsed with water for 30 seconds. The surface was blot dried with a dry cotton pellet, leaving it visibly moist. One coat of bonding agent Prime & Bond NT (Dentsply Detrey) was applied with a bristle brush and left undisturbed for 20 seconds, air-thinned for five seconds, then light cured for 10 seconds at a light intensity of 600 mW/cm².

Group 2 (Dry, D): After acid conditioning, the cavity surface was air dried for five seconds with compressed air from an air syringe, the tip of which was kept at a distance of 2 cm from the prepared tooth. Subsequently, Prime & Bond NT was applied, as for Group 1.

Group 3 (Moist Hypochlorite, MH): Three percent NaOCl was applied for two minutes to the acid conditioned cavity surface, followed by rinsing for 30 seconds. The surface was blot dried before bonding.

Group 4 (Dry Hypochlorite, DH): After NaOCl treatment, the surface was air dried for five seconds, followed by an application of the bonding agent.

Group 5 (Moist Hypochlorite Ascorbate, MHA): Ten percent sodium ascorbate was applied for one minute to the NaOCl-treated, acid conditioned tooth surface, then rinsed for 30 seconds. The surface was blot dried before bonding.

Group 6 (Dry Hypochlorite Ascorbate, DHA): After sodium ascorbate treatment as in Group 5, the cavity surface was air dried for five seconds before bonding.

The cavities were bulk filled with hybrid composite Spectrum TPH (Dentsply Detrey) and light cured for 40 seconds at 600 mW/cm². The restorations were polished using Enhance system disks (Dentsply Detrey). The teeth were extracted immediately after restoration, cleaned and stored in distilled water for between two to four weeks. Ten samples from each group were used for evaluation of microleakage and five for scanning electron microscopic examination.

RESULTS

Dye Leakage Study

Microleakage scores are presented in Table 1. Both the conventional acid-etched groups and the acid-etched NaOCl-treated groups demonstrated extensive leakage in both moist and dry conditions (p>.05). Sodium ascorbate treatment of the deproteinized dentin significantly reduced the microleakage as compared to the conventional acid-etched groups and acid-etched NaOCl-treated groups, with no statistically significant difference between moist and dry bonding.

Table 1 Leakage Scores Observed in Different Treatment Groups

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Scanning Electron Microscopy

SEM findings are summarized in Figures 1–6. For specimens that had been acid-etched but not deproteinized, there was little or no tubular penetration, with very few and short resin tags with the presence of generalized gap. For etched and NaOCl-treated samples, deeper tubular penetration and filled lateral tubular branches were observed, but some areas still showed an interfacial gap. Prime and Bond NT showed better interfacial adaptation, deeper tubular penetration and filled lateral tubular branches when used after NaOCl/sodium ascorbate treatment.

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DISCUSSION

In tooth colored restorative materials, early loss of restoration is no longer a clinical problem. However, marginal leakage and consequental marginal discoloration remains the most frequent reason to replace/repair an adhesive restoration.22 Additionally, a “retained” restoration is always assessed as “bonded” to the cavity walls, yet, retention and bonding are two different concepts. A composite restoration might be retained in a Class V cavity without being totally bonded at the resin dentin interface.23 Therefore, besides bond strength, testing of marginal sealing effectiveness of adhesive is needed.

In this study, the air drying protocol for Prime & Bond NT was used for experimental comparison, dissimilar in vitro conditions, as there is continuous transudation of dentinal fluid due to intrapulpal pressure in vital dentin that has a magnitude of 25 to 30 mm Hg or 34 to 40 cm of water.24–26 The presence of this intrinsic source of moisture may be all that is required to maintain the demineralized collagen matrix in its hydrated state.27 Moreover, as water is required to maintain the collagen in its expanded state, in groups with sodium hypochlorite treatment, removal of the collagen fibers with deproteinizing agents would facilitate the access of resin to a substrate that is more permeable and less sensitive to moisture.28

The results of this in vivo study demonstrated extensive leakage and generalized gap formation in samples restored with the hybrid composite Spectrum TPH and Prime & Bond NT without any surface treatment in both moist and dry conditions. These results are contrary to the previous in vitro study reporting a significant gap-free attachment of Prime & Bond NT to dentin following the manufacturer's instructions.29 Most of the previous studies, which have reported high bond strengths, have been performed in vitro on flat dentin surfaces.30–31 The current study has been performed in in vivo conditions in high C-factor cavities that could possibly explain the different results. Moreover, the environmental conditions might have also affected the results. It has been suggested that the hydrostatic pulpal pressure, the dentinal fluid flow and the increased dentinal wetness in vital dentin can affect the intimate interaction of certain dentin adhesives with dentinal tissue.32–34 These findings are supported by studies that demonstrated low bond strength in simulated clinical conditions maintaining intrapulpal pressure.35 Hannig and Friedrichs also observed that a completely gap-free adaptation between composite and dentin could not be achieved reproducibly and consistently, either in vivo or in vitro, in cavities with a high C-factor.36 They found that Prime & Bond 2.1 gave better results when applied in vitro, as compared to in vivo restorations. Median percentages for the in vivo/in vitro frequency of interfacial gap formation were 40% and 5.3%, respectively.36

No statistically significant difference in microleak-age between blot-drying and air-drying, as observed in this study, is contrary to studies that reported moist bonding was superior to dry bonding in vitro and in vivo.37–38 Other in vitro studies have also reported high bond strength to moist dentin than to air-dried acid etched dentin.39–40 The different results in these studies could be related to the difference in the degree of moisture of dentin due to either in vitro conditions or to the compromised blood supply of periodontally compromised teeth in vivo.

The results of this study, however, are consistent with studies reporting microleakage in all the groups at both the enamel and dentin margins in Class V cavities with no statistically significant difference between moist and dry bonding.41–42 Cardoso and others also reported similar bond strength to moist and dry dentin using an acetone-based adhesive system.43

In accordance with previous studies, specimens with NaOCl treatment showed deeper resin tag penetration into dentinal tubules as compared to specimens with no NaOCl treatment.644–45 Extensive leakage was still observed after NaOCl treatment for both moist and dry bonding groups. The results may be explained by assuming that whatever improvement in marginal sealing effectiveness was expected because deprotenization was probably negated because of partial inhibition of polymerization at the resin dentin interface due to the oxidizing effect of the residual NaOCl or altered chemical structure of dentin after NaOCl treatment. Pyridinoline crosslinks, which occur in collagen Type I and Type II, were found to be disrupted by NaOCl with the formation of chloramines and protein-derived radical intermediates.46–47 The presence of these reactive, residual, free-radicals in NaOCl-treated dentin may compete with the propagating vinyl-free radicals generated during light activation of the adhesive, resulting in premature chain termination and incomplete polymerization. It has been speculated that areas with unpolymerized resin may cause leakage.48 This, in conjunction with other factors, such as high C-factor and moisture level of in vivo conditions, might have resulted in extensive leakage. Perdigão and others reported that NaOCl treatment decreased bond strength in spite of deeper penetration of resin.14

Results of this dye leakage study support studies that reported no difference in microleakage, decreased marginal adaptation or increased leakage after NaOCl treatment.1549–50 However, the results of this study are contrary to studies that reported a decrease in microleakage and improved marginal adaptation after NaOCl treatment in Prime & Bond 2.1.1251 This may be because demineralization and deproteinization of dentin could be influenced by various factors present in different studies, such as adhesive system composition, depth (superficial vs deep dentin), type of dental substrate (vital vs non-vital dentin), cavity configuration, sensitivity of the adhesive systems to the oxidizing effect of NaOCl and variation in the concentration and duration of application of NaOCl. Although Tanaka and Nakai have recommended 10% NaOCl as ideal concentration for dentin treatment, Prati and others reported complete removal of collagen fibrils, with concentration as low as 1.5% NaOCl for two minutes, while Toledano and others used 5% NaOCl for two minutes.134952

Sato and others evaluated the effect of NaOCl treatment of etched air-dried dentin in vitro and reported that decreased bond strength of air-dried dentin increased after NaOCl treatment but observed the highest bond strength with wet bonding.53 The current study revealed no significant difference in dye leakage between moist and dry bonding after deproteinization with leakage in both groups. This could be attributed to the effect of NaOCl on the polymerization of resin, which seems to be a more crucial factor influencing bonding rather than the moisture level of the substrate.

The results of this study depicted a significant reduction in microleakage after sodium ascorbate treatment of the collagen depleted dentin. These findings could be related to reversal of the adverse effects of NaOCl on the polymerization of resin composite by sodium ascorbate.

Since Vitamin C (ascorbic acid) and its salts are non-toxic and widely used in the food industry as antioxidants, it is unlikely that their use on dentin will create any adverse biological effect or clinical hazard.16 Sodium ascorbate (pH 7) was used in place of ascorbic acid (pH 4) in this study to avoid the potential double etching effect of this mild acid on etched dentin.

This study observed no significant difference in microleakage with moist or dry bonding in deproteinized ascorbate-treated groups. As there would be no collagen network present on acid-conditioned NaOCl-treated dentin to interfere with resin infiltration, even air drying would not have adversely affected the resin dentin bonding. Acid-etching changes dentin to a hydrophobic surface with reduced surface energy.54 Collagen removal by NaOCl treatment increases the wettability of the dentin surface, because deproteinization results in a hydrophilic surface and chemical interactions between the resin and deproteinized dentin are more likely to occur.55 Elimination of the exposed collagen using NaOCl allows for the achievement of a rougher, porous surface similar to enamel, which allows for good mechanical retention and is less sensitive to water content, hence, reducing technique sensitivity.55

Within the limits of this in vivo study, it can be postulated that stability of the internal seal between resin composite and acid-conditioned dentin in cavities with a high C-factor is still questionable, and the optimum dentin surface pre-treatment still needs to be determined. The current study did not include caries affected dentin, effect of different concentrations of sodium hypochlorite and the long-term clinical evaluation of the restoration. In order to improve the longevity of adhesive resin composite restorations, further efforts should be directed to an improvement in the adhesive and bonding properties of filling materials placed on the dentin.

CONCLUSIONS

Restoration with Spectrum TPH and Prime & Bond NT does not prevent microleakage in Class V cavities in vivo. Sodium hypochlorite treatment, followed by sodium ascorbate treatment, significantly reduced microleakage. There was no statistically significant difference between blot drying and air drying in all the groups in this in vivo study. Sodium ascorbate/sodium hypochlorite treatment resulted in deeper tubular penetration and better interfacial adaptation at the resin dentin interface.