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RESEARCH REPORT |
1 Department of Restorative Dentistry, Dental Materials, Piracicaba School of Dentistry, University of Campinas, Piracicaba, SP, Brazil;
2 Department of Prosthodontics, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil;
3 Dows Institute for Dental Research and Department of Operative Dentistry, University of Iowa, Iowa City, USA;
4 Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA; and
5 Institute of Dentistry, University of Helsinki, PO Box 41, 00014 University of Helsinki, and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland
* corresponding author, leo.tjaderhane{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: matrix metalloproteinase • hybrid layer • tooth • microtensile • adhesive
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In this context, it has been speculated that a decreasing concentration gradient of resin monomer diffusion within the acid-etched dentin, and a subsequent resin elution from hydrolytically unstable polymeric hydrogels within the hybrid layers (Wang and Spencer, 2003) leave the collagen fibrils unprotected and vulnerable to degradation by endogenous metalloproteinases (MMPs). MMPs are a group of 23 mammalian enzymes capable of degrading all extracellular matrix components. Human dentin contains at least collagenase (MMP-8), gelatinases MMP-2 and -9, and enamelysin MMP-20 (Martin-De Las Heras et al., 2000; Sulkala et al., 2002, 2006; Mazzoni et al., 2006). Dentin collagenolytic and gelatinolytic activities (Pashley et al., 2004; Mazzoni et al., unpublished observations) can be suppressed by protease inhibitors (Pashley et al., 2004), indicating that MMP inhibition could be beneficial in the preservation of hybrid layers. This was demonstrated in a recent in vivo study, in which the application of chlorhexidine, known to have a broad-spectrum MMP-inhibitory effect (Gendron et al., 1999), significantly improved the integrity of the hybrid layer in a six-month clinical trial (Hebling et al., 2005).
The aim of this in vitro study was to evaluate the effect of protease inhibition on resin-dentin bond strength after 6 mos of aging. The test hypothesis, based on the preservation of the hybrid layer in vivo (Hebling et al., 2005), was that MMP inhibition by chlorhexidine application prior to formation of the hybrid layer would decelerate the decrease of bond strength frequently seen in the microtensile model after aging (Hashimoto et al., 2003). The secondary hypothesis set that external MMP inhibition, previously seen to suppress the dentin MMP activity (Pashley et al., 2004), would further improve the preservation of the hybrid layer.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bonding Procedures
All preparations were etched with 35% phosphoric acid gel (Scotch Etchant, 3M ESPE, St. Paul, MN, USA) for 15 sec, rinsed for 30 sec with tap water, and vigorously dried with oil-/water-free air. The control preparations (n = 7) were re-hydrated with 1.5 µL of distilled water, while the experimental ones (n = 7) were re-hydrated with 1.5 µL of 2 wt% chlorhexidine digluconate solution (chlorhexidine). For both groups, after 60 sec, excess solution was removed with absorbent paper. Two consecutive coats of Single Bond primer/adhesive (3M ESPE, St. Paul, MN, USA) were applied to the entire preparation’s surface, and, after solvent evaporation, the preparation was light-cured for 10 sec. Five or 6 increments of resin composite (Z250, 3M ESPE) were obliquely added to the bonded surfaces and individually light-cured for 20 sec, under a halogen light-curing unit with an output of 700 mW/cm2. The teeth were stored in distilled water at 37°C for a wk.
Microtensile Bond Testing
Teeth were longitudinally sectioned across the bonded interface in sections perpendicular to the pulpal wall with a diamond saw, to produce a series of 0.9 mm x 0.9 mm x 8 mm beams. From 8 to 10 beams were obtained from each preparation. One-third of those specimens were immediately tested, while the remaining beams were randomly divided and stored at 37°C for 6 mos in artificial saliva (pH 7.1) containing (or lacking) proteolytic enzyme inhibitors. The artificial saliva and protease inhibitors cocktail was prepared as described previously (Pashley et al., 2004) (Table 1
). The storage medium was replaced weekly, since it has been shown to maintain the inhibitors’ activity (Pashley et al., 2004). Each specimen was individually fixed to a custom-made testing jig (Geraldeli’s device) with a cyanoacrylate glue (Model Repair II Blue, Dentsply-Sakin, Japan), and subjected to tensile load at a crosshead speed of 0.5 mm/min until failure (Instron 4411, Instron Corporation, Canton, MA, USA).
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). In cases of uncertainty, we used higher magnifications (500–4000x) (Fig. 1) to confirm the nature of fracture. The percentage of each fracture mode was then estimated for each specimen.
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= 0.05. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Six-month storage resulted in significant bond strength reduction of both chlorhexidine and control groups (p < 0.05). Storage in artificial saliva without added protease inhibitors reduced bond strength in the control group by 45.3%. In the chlorhexidine group, the reduction was 23.4%. The remaining bond strength was significantly higher in the chlorhexidine group (p < 0.05) (Fig. 2). Storage in artificial saliva containing protease inhibitors did not affect the bond strength when compared with that of those stored in artificial saliva without protease inhibitors (p > 0.05) (Fig. 2).
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. Of these, 93.5% (129/138) were mixed failures, with practically similar fracture patterns for control and chlorhexidine groups at the immediate testing period (Table 2). After 6 months’ storage, a significant increase in the percentage of failures in the bottom of the hybrid layer was observed for the control specimens, but not for chlorhexidine-treated specimens. The failures in the top of the hybrid layer were significantly less prevalent in the chlorhexidine group after 6 months’ storage, all for specimens stored with or without protease inhibitors, when compared with immediately tested specimens (p < 0.05) (Table 2). Also, after 6 months’ storage, significantly less failure occurred in the bottom of the hybrid layer, regardless of the storage medium, and in the top of the hybrid layer in specimens stored with protease inhibitors in the chlorhexidine-treated group when compared with respective controls (p < 0.05) (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Chlorhexidine has been widely used as an antimicrobial agent, including for disinfection before the placement of restorations. Previous studies have demonstrated that chlorhexidine application prior to acid-etching has no adverse effects on immediate composite-adhesive bonds in dentin (Perdigão et al., 1994; el-Housseiny and Jamjoum, 2000; de Castro et al., 2003), enamel (Filler et al., 1994; el-Housseiny and Jamjoum, 2000), or with resin-reinforced glass-ionomer cements (Cunningham and Meiers, 1997). The antimicrobial efficacy of chlorhexidine used as a preparation disinfectant may, however, be questioned if the surface is subsequently conditioned (Botelho, 2005). Recent studies have examined the use of chlorhexidine after acid-etching, demonstrating initial bond strengths comparable with those of the controls (Pilo et al., 2001; de Castro et al., 2003; Say et al., 2004), as was also observed in this study. While the evidence of chlorhexidine antimicrobial efficacy when used after acid-etching remains to be shown, analysis of the present data apparently indicates its beneficial effects on the preservation of dentin bond strength as an MMP inhibitor, when applied prior to bonding with no further rinsing. When applied in this manner, the naked collagen fibrils were exposed to chlorhexidine that was then sealed into the fibrils by adhesive resins. The reduction of bond strength with the chlorhexidine-treated group (23.4%) is most likely due to the hydrolytic degradation of the adhesive polymer (Carrilho et al., 2005a). The significantly higher cohesive failure rates within the adhesive layers and composite resin in chlorhexidine-treated group (Table 2) support the findings that lower reduction of bond strengths was due to a better preservation of the collagen fibrils.
The lack of effect of protease inhibitors incorporated into the storage solution in preventing reductions in bond strength indicates that the main effect was gained with direct inhibition of dentin-bound MMPs (Pashley et al., 2004). This assumption is supported by recent studies demonstrating that mineral oil used as a storage medium inhibited both the function of dentin-bound MMPs (Pashley et al., 2004) and the hydrolysis of polymerized matrix (Carrilho et al., 2005a), and preserved both the bond strength and the integrity of the hybrid layer (Carrilho et al., 2005b; García-Godoy et al., in press), suggesting the hydrolytic basis of the phenomenon. The possibility of bacteria being responsible for the loss of bond strength in this study is minimal, since the teeth were intact, and they were stored in artificial saliva containing an antimicrobial component (i.e., sodium azide), and no signs of biofilm formation were observed in SEM images. Comparable results with degradation of collagen fibrils in hybrid layers have been demonstrated in the absence of bacteria (Hashimoto et al., 2003; Pashley et al., 2004).
Few studies have examined the failure modes in an entire set of specimens. SEM analysis of the failure mode distribution revealed that 93.5% of the specimens demonstrated mixed failures, with the marked change in the site of failure between control and chlorhexidine groups after 6 months’ storage. Quantitative SEM analysis improved the accuracy of the failure mode distribution analysis compared with light microscopy (Hashimoto et al., 2003), and allowed for discrimination between the failures occurring in the top and bottom of the hybrid layer. This is important when one attempts to understand the mechanisms leading to the degradation of the hybrid layer and the reduction in dentin bond strengths with time. Previously, exposure of the collagen fibrils in the fracture site was demonstrated in aged specimens (Hashimoto et al., 2003), showing the shift of the failure site from the top to the bottom of the hybrid layer. The significantly lower percent failure mode in the hybrid layer, especially in the bottom part, after 6 mos with chlorhexidine treatment indicated that the higher bond strengths observed in this group reflected the preservation of hybrid layer collagenous matrix, especially in the bottom zone, where partially exposed collagen fibrils are most prone to initial enzymatic degradation. This may also reflect the better preservation of sub-hybrid layer dentin, in which both progressive demineralization and degradation of dentin collagenous matrix may occur with time (García-Godoy et al., in press).
The present in vitro study, as well as a previous in vivo study demonstrating preservation of hybrid layers with chlorhexidine treatment after acid-etching (Hebling et al., 2005), was performed with only one adhesive system. Thus, these studies merely provide the proof of concept, and the recommendation of the use of chlorhexidine after acid-etching must at this point be limited to the adhesive system in question. However, other in vitro experiments—with various bonding materials demonstrating that chlorhexidine application after acid-etching has no effect on immediate bond strength (Pilo et al., 2001; de Castro et al., 2003; Say et al., 2004)—encourage further experiments to evaluate the preservation of the hybrid layer with different bonding systems.
In conclusion, 2% chlorhexidine application after acid-etching preserves both the durability of the hybrid layer (as seen in the failure mode distribution analysis) and bond strength in vitro of aged specimens. The findings correlate well with the recent in vivo findings with similar experimental design (Hebling et al., 2005). The most plausible explanation would be the inhibition of dentin matrix-bound MMPs (Gendron et al., 1999), resulting in decreased degradation of hybrid layer and sub-hybrid layer collagen fibrils. While the improvement with chlorhexidine, both in bond strength and in hybrid layer durability in 6 mos, was significant compared with that in the controls, the mean reduction in bond strength was still 23.4% when compared with the initial values. Further in vitro and in vivo studies are needed to clarify the causes behind the remaining loss of bond strength, to optimize the MMP inhibitory effect (e.g., concentration of chlorhexidine, time of application), and to find the optimal MMP inhibitor that would result in the best time-related preservation of the dentin-adhesive interface.
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ACKNOWLEDGMENTS |
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Received May 19, 2006; Last revision September 30, 2006; Accepted October 5, 2006
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REFERENCES |
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