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Collagen Degradation in Endodontically Treated Teeth after Clinical Function
1 Dental Materials and Restorative Dentistry Department, University of Siena, Italy; Correspondence: * corresponding author, kfctay{at}netvigator.com
Endodontically treated teeth restored with posts are susceptible to coronal leakage after long-term function. We hypothesize that demineralized collagen matrices (DCMs) created in dentin by acidic zinc phosphate cement within the dowel spaces degrade with time. Forty-two post-restored teeth were extracted after three periods of clinical service and were examined, by means of scanning and transmission electron microscopy, for the status of the DCMs. SEM revealed a progressive degradation of the DCMs, becoming less dense after 3 to 5 years, losing structural integrity after 6 to 9 years, and partially disappearing after 10 to 12 years. TEM revealed evidence of collagenolytic activity within the DCMs, with loss of cross-banding and unraveling into microfibrils, and gelatinolytic activity that resulted in disintegration of the microfibrils. Bacterial colonization and the release of bacterial enzymes and of host-derived matrix metalloproteinases may contribute to the degradation of collagen fibrils in root dentin after clinical function.
Key Words: In vivo • root dentin • collagen fibrils • bacteria • matrix metalloproteinases
It is well-established that coronal leakage is a potential cause of failure in endodontically treated teeth (Saunders and Saunders, 1994). This problem can be more pronounced after post preparation, when only relatively short (ca. 4–5 mm) lengths of obturating materials remain in the canals (Barrieshi et al., 1997; Fox and Gutteridge, 1997; Metzger et al., 2000; Abramovitz et al., 2001). Although coronal leakage has been substantially reduced with the use of adhesive secondary resin seals (Galvan et al., 2002) and resin cements (Fogel, 1995), zinc phosphate cement is still used in some parts of the world for luting of metal posts and crowns. Most adhesive resin cements cannot guarantee a fluid-tight seal that prevents the ingress of oral fluids, bacteria, and endotoxins (Alves et al., 1998), particularly after long periods of simulated intra-oral function (Reid et al., 2003). It is also unlikely that zinc phosphate cement, that exhibited the most severe initial microleakage (Bachicha et al., 1998), can sustain a long-term seal over years of clinical service. 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.
Forty-two clinically asymptomatic, endodontically treated teeth that were scheduled for extraction because of prosthodontic or periodontal reasons were selected. These teeth were all previously root-filled with gutta-percha and Grossman’s sealer by means of a lateral condensation technique, with additional dowel spaces prepared and with metal posts cemented with zinc phosphate cement. Half of these teeth were restored with metal crowns, while the remaining functioned as abutments of fixed partial dentures. The metal posts inserted into these teeth included 9 tapered self-threading posts, 15 parallel-sided threaded metal posts, and 18 cast dowel-core that were all cemented with zinc phosphate cements. 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 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
SEM evaluation revealed a progressive deterioration of the structural integrity of the collagen matrix that corresponded with the period of clinical function of the specimens. The control specimens (i.e., teeth that were endodontically treated in vitro) exhibited a three-dimensional network of dense collagen fibrils on top of the dentin surface (Fig. 1A  ). 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).
The distribution of the 109 fragments that were retrieved after different periods of clinical service is shown in the Table . 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).
TEM micrographs from the control group revealed a dense collagen matrix that contained intact, banded collagen fibrils (not shown). In the 3–5-year group, electron-dense, remnant luting cement could be observed along the dentin surface and within the dentinal tubules (Fig. 2A ). 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).
In the 10–12-year group, zones of grossly denatured collagen fibrils could be identified adjacent to bacteria that were present along the surfaces of the root dentin (Fig. 3A ). 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.
Since there was a significant difference in the status of the collagen fibrils from different clinical function periods, we have to reject the null hypothesis, and assert that progressive degradation of the collagen fibrils occurred in root canals in which metal posts were luted with zinc phosphate cement. 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 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.
This study was based on the work partially performed by Cecilia Goracci for the fulfillment of the degree of Doctor of Philosophy, University of Siena, Italy. This study was supported, in part, by grants DE 014911 and DE 015306 from the National Institute of Dental and Craniofacial Research (DHP) and by grant 20003755/90800/08004/400/01 from the Faculty of Dentistry, University of Hong Kong. We thank Amy Wong of the Electron Microscopy Unit, the University of Hong Kong, for technical assistance. The authors are grateful to Zinnia Pang and Michelle Barnes for secretarial support. Received for publication May 6, 2003. Revision received February 4, 2004. Accepted for publication February 24, 2004.
Journal of Dental Research, Vol. 83, No. 5,
414-419 (2004)
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