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RESEARCH REPORT |
1 Department of Orthodontics and Pediatric Dentistry, University of São Paulo State, Araraquara Dental School, São Paulo, Brazil;
2 Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA;
3 Institute of Dentistry, University of Helsinki, Finlandm and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital (HUCH); and
4 Pediatric Dentistry and Orthodontics, University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Pokfulam, Hong Kong SAR, China;
* corresponding author, franklintay{at}gmail.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: primary dentin • hybrid layer • collagenolysis • gelatinolysis • MMPs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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A potential bond degradation mechanism that is derived from beneath the bonded interface is the instability of the demineralized dentin collagen matrix. This was manifested as the thinning or disappearance of collagen fibrils from aged, unbonded, or bonded dentin (Hashimoto et al., 2003a,b; Ferrari et al., 2004; Yoshida et al., 2004), or the failure of aged hybrid layers to take up heavy metal stains (De Munck et al., 2003; Armstrong et al., 2004). The issue of collagen instability has potential consequence in the pathogenesis of dentinal caries, with the breakdown of acid-demineralized collagen matrices by host-derived matrix metalloproteinases (MMPs), a class of zinc- and calcium-dependent endopeptidases (Tjäderhane et al., 1998; Sulkala et al., 2001; van Strijp et al., 2003).
In the context of dentin bonding, residual collagenolytic activity was observed in mineralized dentin powder produced from extracted teeth that accounted for the disintegration of collagen fibrils from unbonded, aged acid-etched dentin, in the absence of the contribution from bacterial or salivary MMPs. This low but persistent endogenous collagenolytic activity was completely inhibited by the use of protease inhibitors, the incorporation of which preserved the structural integrity of the collagen fibrils (Pashley et al., 2004). In that study, phosphoric-acid-etching reduced, but did not completely inhibit, the inherent collagenolytic activity of mineralized dentin, while the use of chlorhexidine, in even very low concentrations, strongly inhibited such activity. Thus, apart from being a commonly known disinfectant, chlorhexidine also functions as a potent MMP inhibitor (Gendron et al., 1999).
These reported results, however, are of clinical significance only if proteolysis of the resin-infiltrated collagen network can be demonstrated in vivo. To ascertain that endogenous enzymatic activities are involved, we must identify such activities from aged, adhesive-bonded dentin that is completely surrounded by enamel (De Munck et al., 2003). Such an in vivo study should also demonstrate that such activities can be arrested via the use of potential non-toxic MMP inhibitors, such as chlorhexidine or doxycycline (Grenier et al., 2002), that can be safely applied intra-orally, unlike the highly toxic protease inhibitors that were utilized in the previous in vitro study (Pashley et al., 2004). Thus, this study tested the null hypothesis that there is no difference between the ultrastructure of vital human dentin in primary molars bonded with an etch-and-rinse adhesive only and that of dentin bonded in conjunction with the use of chlorhexidine as an MMP inhibitor applied after phosphoric-acid-etching but before adhesive application.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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For each recruited subject, the contralateral pair of primary molars was rendered caries-free under local anesthesia, rubber dam isolation, and the use of a caries detector dye. The control tooth was acid-etched with phosphoric acid gel for 15 sec, bonded with Single Bond (3M ESPE, St. Paul, MN, USA) with a moist-bonding technique, and restored incrementally with a microfilled resin composite (EPIC-TMPT, Parkell Inc., Farmingdale, NY, USA). A layer of hybrid resin composite (Z250, 3M ESPE) was used as the last increment. The experimental tooth was subjected to a similar treatment, with the exception that 2% chlorhexidine digluconate solution (Cavity Cleanser, Bisco Inc., Schaumburg, IL, USA) was applied generously to the cavity after acid-etching. The solution was gently blot-dried after a dwell time of 30 sec prior to the placement of the same adhesive and resin composite. Post-operative bitewing radiographs of the restored teeth were taken as records.
Following the six-month period of intra-oral functioning, the primary molars of the three subjects who completely satisfied all criteria were evaluated for their marginal integrity, according to USPHS criteria. All the restorations were intact, with no associated clinical symptoms, no chipping of the composites, and no recurrent caries, and their marginal integrities were graded as ‘alpha’. These teeth were extracted under local anesthesia and immediately fixed in 2.5% glutaraldehyde. Each pair of contralateral specimens was stored in 0.2% sodium azide solution in coded glass vials, so that subsequent specimen-processing was performed blindly with respect to the group designations for the control and experimental teeth of each respective subject.
Laboratory Procedures
When the specimens were received, each tooth was sectioned buccolingualy into 4 1-mm-thick serial sections, each containing a part of the restoration. Two sections from each tooth were randomly selected and immersed in a 50 wt% ammoniacal silver nitrate solution for 24 hrs, according to the tracer protocol for nanoleakage examination reported by Tay et al.(2002). These specimens were processed for TEM examination without further laboratory demineralization. The other 2 sections were completely demineralized in ethylene diamine tetra-acetic acid. Both undemineralized and demineralized, epoxy-resin-embedded, 90-nm-thick sections were prepared according to the TEM protocol of Tay et al.(2002). Only the resin-dentin interfaces from the cavity floors of the control and experimental teeth were examined, since they represented the parts of the restorations that were the most secluded from the oral environment.
Undemineralized sections were examined without being stained further. Demineralized sections were stained with 2% uranyl acetate and Reynolds’ lead citrate for examination of the characteristics of the resin-dentin interfaces, and with 1% phosphotungstic acid and 2% uranyl acetate for examination of the status of the collagen fibrils. The sections were examined in a TEM (Philips EM208S, Philips, Eindhoven, The Netherlands) operating at 80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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–3. All restorations were placed on Class I cavities with enamel cavosurface margins (Figs. 1a, 1b, 2a, 2b, 3a, 3b). In general, more extensive silver deposits could be identified from unstained, undemineralized, silver-impregnated sections of the control teeth (Figs. 1c, 2c, 3c). The heavy silver deposits in the control teeth had a tendency to obscure the additional electronlucent, silver-free zones that were concomitantly present within these hybrid layers (Figs. 3d, 4f). These electronlucent silver-free zones were absent from hybrid layers of the experimental teeth (Figs. 1d, 2d, 3d).
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, 2f, 3f). In contrast, all specimens from the control teeth exhibited evidence of degradation within the hybrid layers. Different patterns could be seen, ranging from nearly complete disintegration of the entire collagen matrix (Fig. 1e), to partial degradation along the surface part of the hybrid layer (Fig. 2e), to partial, vertical degradation of parts of the hybrid layer, with these zones appearing abruptly among intact, non-degraded regions (Fig. 3e).
Figs. 4a–4e depict the variation in the extent of degradation from different regions of the control teeth. They ranged from discontinuous (Fig. 4a) to coalesced voids (Fig. 4b), to larger patches with microfibrillar strands (Fig. 4c), to regions with complete absence of collagen fibrils (Fig. 4d), and, ultimately, to the loss of the bulk of the stainable portion of the hybrid layer (Fig. 4e). No TEM evidence of bacteria was seen in the bonded dentin or within the restorations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It is disturbing to observe that subclinical deterioration of the hybrid layers occurred so quickly in vivo, in the absence of adjunctive clinical signs and symptoms. Unlike previous in vitro studies (De Munck et al., 2003; Armstrong et al., 2004), the resin-dentin interfaces examined were protected by bonded enamel cavosurface margins and were not sectioned into beams for an accelerated aging effect. Under the conditions used in this study, it appeared that clinical degradation of hybrid layers by endogenous, host-derived MMPs, at least in exfoliating primary molars, occurred at a rate much faster than the 3–5 years of accelerated aging required for such conditions to be identified in vitro. Initially, we speculated that degradation of hybrid layers by endogenous MMPs would occur more rapidly under in vitro conditions, due to the possible disturbance of the balance between MMPs and their natural inhibitors, TIMPs (tissue inhibitors of metalloproteinases), when extracted teeth are used for aging experiments. It is known that both TIMP-1 and TIMP-2 can complex with active MMP-2 and inhibit proteolytic activity (Ward et al., 1991; Palosaari et al., 2003), and both of them (especially TIMP-1) are expressed by human odontoblasts (Palosaari et al., 2003). Since these natural MMP inhibitors have shorter half-lives (Bode et al., 1999) than MMPs, prolonged interruption of MMP-TIMP interaction, such as the cessation of dentinal fluid flow (i.e., in vitro conditions), may prevent the replenishment of pulpal TIMPs out into peripheral dentin. Apparently, these initial assumptions were not fully supported by our in vivo results.
The reason for the rapid degradation compared with in vitro studies may be that bonding was performed on caries-affected dentin. Caries-affected dentin demonstrates decreased collagen cross-linking (Kuboki et al., 1977). Since carious dentin exhibits increased collagenolytic activity compared with intact dentin (Dayan et al., 1983), the speed of degradation may partially be caused by the increased MMP activity of caries-affected dentin. Moreover, the observations in the control group vs. the experimental group may, in part, be the result of chlorhexidine, being an effective antimicrobial agent, in controlling remnant bacteria that were present within the caries-affected dentin. Unlike previous in vitro studies that demonstrated the almost complete absence of staining from hybrid layers, due to the longer period of accelerated aging, a series of progressively advancing patterns of degradation could be identified from the present in vivo results (Fig. 4), probably due to the shorter period between intra-oral bonding and specimen retrieval. The differences in the degradation patterns may also reflect the local differences in the extent of the caries process, thus affecting the local MMP activity.
We demonstrated, in control specimens, the existence of discontinuous patches of grossly disintegrated microfibrillar fragments that remained silver-free despite the loss of the electron density which is characteristic of the normal hybrid layer. These fragments probably represented remnant 3/4- and 1/4-length fragments that resulted from collagenolysis (Gross and Nagai, 1965), but were retained by the adhesive resins within the hybrid layer. The additional moderate to severe nanoleakage (i.e., silver uptake) in the hybrid layers of the control specimens probably represented regions wherein leaching of the degraded fragments occurred with concurrent hydrolysis of the hydrophilic resin components within the hybrid layers. Conversely, the silver-free electronlucent areas in the undemineralized control sections that corresponded to regions with a complete lack of stainable fibrillar components in demineralized sections suggested that, in these regions, the degenerated microfibrillar fragments have further been degraded beyond detection. Such a process may occur via gelatinolytic MMPs released from the underlying mineralized dentin, with the gelatin breaking down into peptides of lower molecular weight (kDa). This phenomenon is analogous to the appearance of clear bands in Coomassie-blue-stained gels, when the cleavage products of gelatin were subjected to Western blotting after treatment with MMP-2 (Gelatinase A) or MMP-9 (Gelatinase B) (Gendron et al., 1999; Smith et al., 2004). Subsequent to the report of collagenolytic activity in mineralized dentin powder (Pashley et al., 2004), we have also identified separate gelatinolytic activity from the same substrate, via the use of fluorescein-labeled gelatin derived from porcine skin (Pashley et al., unpublished results).
The ultrastructural nature of the present study precluded the identification of the exact classes of MMPs involved in in vivo degradation of dentin hybrid layers. Collagenase MMP-8 has been shown to exist in carious human dentin (Tjäderhane et al., 1998), and it seems to be also present in intact dentin (Sulkala et al., unpublished results). The gelatinase MMP-2 has been shown to be present in human dentin (Martin-De Las Heras et al., 2000). Apart from its gelatinolytic activity, MMP-2 is also capable of collagenolysis (Aimes and Quigley, 1995), albeit at a slower rate (Patterson et al., 2001). Thus, even in the absence of an endogenous collagenase source, endogenous MMP-2 from the dentin matrix can apparently result in the slow but complete cleavage of the entire resin-infiltrated collagen network.
Since conservative dentistry does not involve only the use of etch-and-rinse adhesives on primary dentin, further studies are urgently needed to validate these results using both etch-and-rinse and self-etch adhesives on vital normal and non-vital coronal, extraradicular, and intraradicular adult dentin. Although the results of the present study are clinically provocative, it is reassuring that the use of chlorhexidine as an MMP inhibitor resulted in the arrest of the in vivo degradation of the hybrid layers. Such a protocol may also be of clinical value to prevent collagen degradation in root canals (Ferrari et al., 2004), in light of the recent upsurge of interest in the application of bondable root-filling materials to intraradicular dentin (Shipper et al., 2004; Zmener, 2004).
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ACKNOWLEDGMENTS |
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Received February 20, 2005; Last revision April 28, 2005; Accepted April 28, 2005
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REFERENCES |
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