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RESEARCH REPORT |
1 Dental Materials and Restorative Dentistry Department, University of Siena, Italy;
2 Restorative Dentistry Department, University of Padua, Italy;
3 Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA; and
4 Pediatric Dentistry and Orthodontics, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR, China;
* corresponding author, kfctay{at}netvigator.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: In vivo • root dentin • collagen fibrils • bacteria • matrix metalloproteinases
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Bacterial infection caused by coronal leakage remains the central issue in endodontic failure, but very few studies have examined the condition of root canal dentin in post-restored teeth. Zinc phosphate is an initially acidic, non-bonding cement that has the capacity to dissolve the smear layer and demineralize the underlying intact dentin (Shimada et al., 1999). Recent in vitro studies have reported the disappearance of denuded collagen fibrils within incompletely resin-infiltrated regions of dentin hybrid layers (De Munck et al., 2003). It is expected that teeth restored with zinc-phosphate-cemented posts and retrieved after long periods of intra-oral function are useful as in vivo models for examining the course of degradation of collagen fibrils.
Thus, the objective of this study was to test the hypothesis that denuded collagen fibrils that are exposed by zinc phosphate cement in dentin lining dowel spaces degrade with time. The null hypothesis tested was that clinical aging has no effect on the integrity of root dentin collagen matrices adjacent to metal posts that are luted with a conventional non-bonding cement.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The study protocol was approved by the Commission for Medical Ethics of Siena University, Italy. The patients were informed of the intent of the study, and their written consents were obtained. Five teeth had been endodontically treated 12 years previously, 7 teeth had been treated 10 years previously, 14 teeth 8 years previously, 13 teeth 5 years previously, and 3 teeth 2 years previously. During extraction, these teeth were fractured into 109 fragments. These fragments were grouped according to their years of clinical service and stored in Karnovsky’s fixative at 4°C until they were further processed for microscopy. In addition, 5 sound, recently extracted single-rooted lower premolars were root-treated in the same manner. Dowel spaces were prepared after the endodontic treatment, filled with zinc phosphate cement, and used as the control. They were used to compare the status of the intra-radicular collagen fibrils along the coronal part of the root canals (i.e. within the post spaces) with that of the specimens that had undergone different periods of intra-oral service. The crown of each tooth or fragment was removed by means of a slow-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). The roots were processed for scanning electron microscopic (SEM) and transmission electron microscopic (TEM) examination.
SEM Examination
The metal posts and luting cements were gently removed from the root dentin with an ultrasonic scaling tip under copious water-cooling. All specimens were dehydrated by being passed through an ascending series of aqueous ethanol solution of increasing concentration, until absolute ethanol was eventually reached. They were then immersed in hexamethyldisilazane (SPI Supplies, West Chester, PA, USA) that was allowed to evaporate slowly, following the dehydration protocol for SEM examination reported by Nation (1983). The processed specimens were sputter-coated with gold (Edwards Co. Ltd., London, UK), and the morphology of the demineralized collagen matrix along the post spaces was examined with a scanning electron microscope (Model 505, Philips, Eindhoven, The Netherlands) operating at 15 kV.
For statistical analysis, the 109 root fragments were classified according to their period of clinical service and the degree of severity of collagen degradation within the demineralized matrices. The three age periods were: 3–5 yrs, 6–9 yrs, and 10–12 yrs. The increasing order of severity of collagen degradation was based on the following parameters: (I) collagen fibrils clearly identified in the demineralized matrix above the mineralized dentin; (II) loss of integrity of the surface demineralized collagen matrix; and (III) partial disintegration of the demineralized collagen matrix, with exposure of the underlying mineralized dentin. The number of tooth fragments in which a demineralized collagen matrix could not be identified was also recorded but not included in the statistical analysis. The data were arranged into a 3x3 contingency table and analyzed with the Chi-square statistic at the 95% confidence level.
TEM Examination
Two specimens each from the control group, the 3–5-year group, and the 10–12-year group were selected for TEM examination. They were post-fixed with 1% osmium tetroxide and completely demineralized in buffered ethylene diamine tetraacetic acid (pH 7.0). The demineralized specimens were dehydrated, infiltrated with epoxy resin, and prepared for TEM examination according to the protocol reported by Tay et al.(1999). The status of the intertubular collagen fibrils along the post spaces derived from the coronal third of the root dentin was examined with 70- to 90-nm-thick sections that were double-stained with 1% phosphotungstic acid and 2% uranyl acetate for 20 min each. They were examined under a transmission electron microscope (EM208S, Philips) operating at 80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). In the 3–5-year group, many of the unetched specimens revealed a thin, demineralized collagen matrix in which the integrity of the fibrillar network was maintained, but with the collagen fibrils appearing more sparse than those from the control group (Fig. 1B). In addition, globular structures were found within the intertubular matrices and the dentinal tubules. The integrity of collagen matrix along the surface of the post space was lost in the 6–9-year group (Fig. 1C), with the presence of a mesh of collapsed, amorphous substances despite the use of the same dehydration technique for investigation. In the 10–12-year group, there were additional regions in which the demineralized matrices were absent, exposing the lateral branches of the dentinal tubules in the underlying mineralized dentin (Fig. 1D).
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. Statistical analysis revealed a significant relation between the clinical service period of these specimens and their severity of collagen degradation (
2 = 69.5 with four degrees of freedom; P < 0.001).
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). The surface collagen fibrils were more loosely arranged than those from the underlying intact root dentin (Fig. 2B). The subsurface collagen fibrils exhibited the loss of cross-banding and disruption of fibrillar arrangement (Fratzl et al., 1998), revealing microfibrillar strands. Taken together, these features were indicative of collagenolytic activity that resulted in denaturing of the collagen fibrils (Fig. 2C).
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). In some areas, a more severe collagenolytic activity could be observed, as evidenced by the breakdown of the collagen fibrils into a disorganized mesh of microfibrillar strands (i.e., gelatin; Bailey, 2000) (Fig. 3B). Additional regions could be identified wherein this denatured collagen (gelatin) was almost completely digested (Figs. 3A, 3C). Taken together, these features were indicative of the existence of a combination of collagenolytic and gelatinolytic activities in the long-term clinically aged specimens. Fig. 3D summarizes the percent of the dentin surface lining the post-space that exhibited the variations in collagen fibril content, as a function of years of service.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Results from the control group reinforced those from previous work that zinc phosphate cement is acidic enough to partially demineralize the root dentin (Shimada et al., 1999). However, we surmised that additional demineralization of the dowel spaces could also have occurred via ingress of acidogenic bacteria during the long periods of intra-oral function. Although root canal infections harbor a complex but selective microflora (Peters et al., 2002), infection through dentinal tubules occurs predominantly by Gram-positive bacteria, mainly the Streptococcus species (Love and Jenkinson, 2002). We speculate that bacterial growth occurred along the dowel spaces when coronal leakage progressed sufficiently to permit the continuous seepage of oral fluids. Since these bacteria are not motile, invasion is relatively slow. However, they may remain viable for prolonged periods (Svensäter et al., 2001).
Proteolysis of the demineralized collagen matrix can occur via several mechanisms. The general consensus is that intact native collagen fibrils are cleaved by collagenases that attach to individual triple-helical molecules approximately three-fourths of the way from the amino-terminal end of the molecule, and cleaving through all 3 polypeptide helical chains (Bailey, 2000). These 2 fragments from each denatured helical chain are further digested by gelatinases with intermediate releases of short amino acid moieties (Ottl et al., 2000). Such a cascade of events may be interpreted ultrastructurally as a progressive loss of order in the degrading collagen matrices. In the 3–5-year group, there was a loss of cross-banding within partially unraveled collagen fibrils. However, the helical structures of individual collagen fibrils were still maintained (Fig. 2C
). Degradation was further enhanced by the loss of helical structure in the 10–12-year group, with the fibrils uncoiling into microfibrillar elements (Fig. 3B), presumably by gelatinases. Finally, gelatinolysis resulted in almost complete digestion of these microfibrillar elements (Fig. 3C).
It is tempting to attribute the degradation of the root dentin matrices to the proteolytic activity derived from bacteria that were present within the dowel spaces. Collagenolytic activity is not expressed by Streptococcus mutans and Actinomyces species, although some Porphyromonas gingivalis strains that are involved in infected root canals have been shown to possess collagenolytic potential (Dung, 1999; Odell et al., 1999). Gelatinolytic activity has not been found in caries-related bacteria (Tjäderhane et al., 1998), but it has been identified from Enterococcus faecalis in root canals with persistent infections (Hubble et al., 2003). While these bacteria may contribute to at least part of the degradation process, it is doubtful if they could be present concomitantly in every retrieved fragment.
Conversely, recent studies revealed the contributions of host-derived matrix metalloproteinases (MMPs) to the breakdown of the collagen matrices in the pathogenesis of dentin caries (Tjäderhane et al., 1998; van Strijp et al., 2003). MMPs are a family of zinc-dependent proteolytic enzymes that are capable of degrading the dentin organic matrix after demineralization. Gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8 and MMP-20) activities are present in latent forms within the dentinal matrix (Tjäderhane et al., 1998; Martin-De Las Heras et al., 2000; Sulkala et al., 2002), and gelatinases are also present in saliva (Sulkala et al., 2001). Those that are present in the dentinal matrix may be released during demineralization under environmental (e.g., zinc phosphate cementation) or pathological (e.g., bacterial acid production) conditions, and activated by low pH to participate in the sequential degradation processes (Tjaderhane et al., 1998; Sulkala et al., 2001). Dentin collagen matrices produced by in vitro acid demineralization were found to have disappeared almost completely when these specimens were stored septically for 500 days (Hashimoto et al., 2003) or stored under aseptic conditions for up to 250 days (Pashley et al., 2004). Conversely, the degradation process was completely inhibited with the incorporation of protease inhibitors that halted the activities of host-derived MMPs (Pashley et al., 2004).
The retrospective nature of specimen retrieval in this study necessitates our reporting on the use of zinc phosphate cements as luting materials even though dentin-bonding resin cements are available. Nevertheless, the creation of a demineralized collagen matrix by the acidic zinc phosphate cement parallels the use of phosphoric acid as an etchant for the demineralization of dentin substrates, being the first step in generating micromechanical retention in contemporary total-etch adhesives. Since the release and activation of host-derived MMPs may be associated with the degradation of acid-demineralized, denuded collagen fibrils, these enzymatic activities may also occur within the incompletely resin-infiltrated, subsurface regions of hybrid layers created by contemporary adhesives, in spite of the better surface seal achieved with these adhesives. This may account for the partial disappearance of hybrid layers reported by De Munck et al.(2003) on crown dentin. The results of this study indicate that future investigations using molecular biology tools should be performed to delineate the contributions of host-derived MMPs in the hydrolytic degradation of root dentin-dowel post interfaces.
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ACKNOWLEDGMENTS |
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Received May 6, 2003; Last revision February 4, 2004; Accepted February 24, 2004
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REFERENCES |
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Alves J, Walton R, Drake D (1998). Coronal leakage: endotoxin penetration from mixed bacterial communities through obturated, post-prepared root canals. J Endod 24:587–591.[ISI][Medline]
Bachicha WS, DiFiore PM, Miller DA, Lautenschlager EP, Pashley DH (1998). Microleakage of endodontically treated teeth restored with posts. J Endod 24:703–708.[ISI][Medline]
Bailey AJ (2000). Perspective article: the fate of collagen implants in tissue defects. Wound Repair Regen 8:5–12.[ISI][Medline]
Barrieshi KM, Walton RE, Johnson WT, Drake DR (1997). Coronal leakage of mixed anaerobic bacteria after obturation and post space preparation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 84:310–314.[ISI][Medline]
De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. (2003). Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 82:136–140.
Dung SZ (1999). Effects of mutans streptococci, Actinomyces species and Porphyromonas gingivalis on collagen degradation. Zhonghua Yi Xue Za Zhi (Taipei) 62:764–774.
Fogel HM (1995). Microleakage of posts used to restore endodontically treated teeth. J Endod 21:376–379.[ISI][Medline]
Fox K, Gutteridge DL (1997). An in vitro study of coronal microleakage in root-canal-treated teeth restored by the post and core technique. Int Endod J 30:361–368.[ISI][Medline]
Fratzl P, Misof K, Zizak I, Rapp G, Amenitsch H, Bernstorff S (1998). Fibrillar structure and mechanical properties of collagen. J Struct Biol 122:119–122.[ISI][Medline]
Galvan RR Jr, West LA, Liewehr FR, Pashley DH (2002). Coronal microleakage of five materials used to create an intracoronal seal in endodontically treated teeth. J Endod 28:59–61.[ISI][Medline]
Hashimoto M, Tay FR, Ohno H, Sano H, Kaga M, Yiu C, et al. (2003). SEM and TEM analysis of water degradation of human dentinal collagen. J Biomed Mater Res 66(B):287–298.
Hubble TS, Hatton JF, Nallapareddy SR, Murray BE, Gillespie MJ (2003). Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin. Oral Microbiol Immunol 18:121–126.[ISI][Medline]
Love RM, Jenkinson HF (2002). Invasion of dentinal tubules by oral bacteria. Crit Rev Oral Biol Med 13:171–183.
Metzger Z, Abramovitz R, Abramovitz L, Tagger M (2000). Correlation between remaining length of root canal fillings after immediate post space preparation and coronal leakage. J Endod 26:724–728.[ISI][Medline]
Martin-De Las Heras S, Valenzuela A, Overall CM (2000). The matrix metalloproteinase gelatinase A in human dentine. Arch Oral Biol 45:757–765.[ISI][Medline]
Nation JL (1983). A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy. Stain Technol 58:347–351.[ISI][Medline]
Odell LJ, Baumgartner JC, Xia T, David LL (1999). Survey for collagenase gene prtC in Porphyromonas gingivalis and Porphyromonas endodontalis isolated from endodontic infections. J Endod 25:555–558.[ISI][Medline]
Ottl J, Gabriel D, Murphy G, Knauper V, Tominaga Y, Nagase H, et al. (2000). Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases. Chem Biol 7:119–132.[ISI][Medline]
Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. (2004). Degradation of dentin collagen by host-derived enzymes during aging. J Dent Res 83:216–221.
Peters LB, Wesselink PR, van Winkelhoff AJ (2002). Combinations of bacterial species in endodontic infections. Int Endod J 35:698–702.[ISI][Medline]
Reid LC, Kazemi RB, Meiers JC (2003). Effect of fatigue testing on core integrity and post microleakage of teeth restored with different post systems. J Endod 29:125–131.[ISI][Medline]
Saunders WP, Saunders EM (1994). Coronal leakage as a cause of failure in root-canal therapy: a review. Endod Dent Traumatol 10:105–108.[ISI][Medline]
Shimada Y, Kondo Y, Inokoshi S, Tagami J, Antonucci JM (1999). Demineralizing effect of dental cements on human dentin. Quintessence Int 30:267–273.[ISI][Medline]
Sulkala M, Wahlgren J, Larmas M, Sorsa T, Teronen O, Salo T, et al. (2001). The effects of MMP inhibitors on human salivary MMP activity and caries progression in rats. J Dent Res 80:1545–1549.
Sulkala M, Larmas M, Sorsa T, Salo T, Tjäderhane L (2002). The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth. J Dent Res 81:603–607.
Svensäter G, Björnsson O, Hamilton IR (2001). Effect of carbon starvation and proteolytic activity on stationary-phase acid tolerance of Streptococcus mutans. Microbiology 147:2971–2979.
Tay FR, Moulding KM, Pashley DH (1999). Distribution of nanofillers from a simplified-step adhesive in acid-conditioned dentin. J Adhes Dent 1:103–117.
Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T (1998). The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. J Dent Res 77:1622–1629.
van Strijp AJ, Jansen DC, DeGroot J, Ten Cate JM, Everts V (2003). Host-derived proteinases and degradation of dentine collagen in situ. Caries Res 37:58–65.[ISI][Medline]
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