Sealer Systems on Apical Endotoxin Penetration: A Coronal Leakage Evaluation

Sealer Systems on Apical Endotoxin Penetration: A Coronal Leakage Evaluation

Basic Research–Technology Effect of Root Canal Filling/Sealer Systems on Apical Endotoxin Penetration: A Coronal Leakage Evaluation Anne E. Williamso...

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Basic Research–Technology

Effect of Root Canal Filling/Sealer Systems on Apical Endotoxin Penetration: A Coronal Leakage Evaluation Anne E. Williamson, DDS, MS, Deborah V. Dawson, BA, ScM, PhD, David R. Drake, MS, PhD, Richard E. Walton, DMD, MS, and Eric M. Rivera, DDS, MS Abstract Endotoxin, elaborated by gram-negative organisms, is an important factor in apical periodontitis. The objective of this study was to evaluate the magnitude of endotoxin penetration through root canal treated teeth using a dual chamber model system. Forty-four maxillary anterior teeth were prepared endodontically and canals filled either by lateral condensation or a warm thermoplasticized technique in combination with either Roth’s 801 or AH 26 sealer. Teeth were suspended in the model system with a mixed anaerobic bacterial suspension in the upper chamber and HBSS in the lower chamber. The QCL-1000 LAL assay was used to measure endotoxin at 0, 1, 7, 14, and 21 days. Response feature analysis using trapezoidal area under the curve was performed; the four treatment groups were compared using nonparametric methods. Groups differed (p ⫽ 0.028), with thermoplasticized root canal filling/ Roth’s 801 sealer permitting the least apical endotoxin penetration. Results suggest that Roth’s 801 sealer may have a role in inhibiting endotoxin penetration.

Key Words Endotoxin, apical periodontitis, coronal leakage

Address requests for reprints to Dr. Williamson, Department of Endodontics, College of Dentistry, University of Iowa, Iowa City, IA 52242; E-mail: [email protected] Copyright © 2005 by the American Association of Endodontists

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ndotoxin has an important role in the development, progression, and pathogenicity of periradicular lesions. Endotoxin, a highly potent immunologic mediator, is an integral component of the outer cell membrane of gram-negative bacteria, which is released in conjunction with cell wall turnover. Recent studies (1–3) have shown that endotoxin is a rapid and powerful agent in lesion development as well as bone destruction in the periradicular area. Additionally, endotoxin has been shown to inhibit early development of tensile strength in surgical wounds (4). Endotoxin has also been shown to stimulate IL-6 production, a proinflammatory cytokine that stimulates osteoclastic activity in pulpal fibroblasts, particularly in the presence of methyl mercaptan produced by Gram negative anaerobes (5). Conversely, endotoxin induces IL-10 receptor gene expression in pulpal fibroblasts. IL-10 has been shown to inhibit expression of proinflammatory cytokines, IL-6 and IL-8(6). Thus, IL-10 may play an advantageous role in wound healing by decreasing the inflammatory response after pulpal injury. The process whereby endotoxin penetrates the root canal system to gain access to the periradicular tissues is important to better understand how and why some root canal treatments become contaminated or re-contaminated with bacteria. Several bacterial species have been shown to penetrate root canal systems after root canal filling because of leakage from the coronal or apical (7, 8), or via lateral or accessory canals and fracture events. Bacterial byproducts (endotoxins) will also pass through filled canals. Both time of exposure and number of infecting organisms as well as quantity of endotoxin, influence the severity of the pulpal/periapical response (9). Utilization of new molecular techniques (DNA-DNA hybridization, Polymerase Chain Reaction, etc.) to detect bacteria in pulpal and periradicular infections, has resulted in identification of several new putative pathogens and numerous ‘old’ species have been reclassified taxonomically (10). This further illustrates the complexity of bacterial biofilms present in infected root canals. The amount of endotoxin present necessary to propagate periradicular disease in filled root canal systems is of interest; if certain techniques or materials limit or prevent the penetration of endotoxin to the periapical tissues, it may be possible to achieve even higher success rates with root canal treatment. Root canal filling materials must be taken into consideration as related to success. The effect of different materials is likely an important related factor to periradicular disease, particularly with respect to the ability, or inability, to prevent endotoxin contamination and passage through the root canal space. Probably the important root canal filling component in this respect is the sealer. It is unknown whether the incomplete set or delayed setting time in sealers has an impact on endotoxin penetration. Recent data have shown variable setting rate of sealers at 37°C. In a recent study, the slowest was Grossman’s type sealer (Roth’s 801) that did not set in the canal space 8 wk after application while AH 26, Sealapex and Tubliseal achieved complete set after 4 wk (11). This lack of set of the Roth’s 801 may contribute significantly to endotoxin to penetration, resulting in periradicular immune/inflammatory responses. Stability of the materials must also be questioned; unset or partially set sealer likely would be more prone to deterioration if exposed to oral or tissue fluids. This may contribute to early and rapid coronal leakage (7). If the apical canal has large areas of unset sealer exposed to periradicular tissue fluids, the sealer breakdown could lead to bacterial accumulation and growth in the canal space, thereby enabling endotoxin to filter through the canal to the apical tissues and predisposing to failure (11).

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Basic Research–Technology Root canal filling techniques may affect endotoxin passage. Guttapercha used in warm root canal filling techniques has been shown to shrink (12). Endotoxin may thus penetrate through the remaining space. With lateral techniques, sealer has been shown to provide incomplete coverage of canal walls and cones (13). This too, would allow endotoxin to penetrate through the filled root canal. Success is partially based on the ability of the root canal filling/ sealer system to block bacteria and bacterial by-products from leaking through the filled root canal system and gaining access to the periapex. Therefore, the purpose of this study was to compare two different root canal filling techniques, both lateral condensation (LC) and a warm thermoplasticized technique (WTT), in combination with either AH26 or Roth’s 801 sealer on apical endotoxin penetration.

Materials and Methods Preoperative Preparation of Teeth Forty-four human maxillary anterior teeth (22 central incisors, 18 lateral incisors, and 4 canines) were used for this study. Curvatures were less than 20°, confirmed radiographically. External root surfaces were debrided of bone, calculus, and soft tissue using a Gracey curette. Teeth were then wrapped in sterile gauze saturated with 2.6% sodium hypochlorite (NaOCl) for 5 min for surface sterilization and rinsed with two consecutive changes of sterile distilled water. The teeth were stored in 0.1% Thymol solution until initiation of the experiment. Canal Preparation Crowns of test teeth were sectioned at the CEJ. Canal patency was confirmed by placing a #10 K-Flex file (Kerr Sybron, Romulus, MI) into the canal and advancing the file until it could be visualized at the apical foramen. The corrected working length was established by subtracting 1 mm from this measurement. Passive step back was performed to a #25 K-Flex file. Standardized preparation to a master apical file size of 40 was completed in each canal. Successively larger files shortened by 0.5 mm, respectively, were used in a step back peripheral filing motion. Irrigation with 2 ml of 2.6% NaOCl was used between each file, as well as recapitulation with a #10 K-Flex file. Canal preparation was considered complete when canal walls were glassy smooth and a B finger plugger could be placed to the corrected working length passively. Initial endotoxin levels for each experimental model were obtained using a QCL-1000 Limulus Amebocyte Lysate Assay kit (BioWhittaker Inc, Walkersville, MD). The LAL test is a highly sensitive, quantitative analysis, utilizing a chromogenic substrate to detect gram-negative bacterial endotoxin.

with cyanoacrylate and wax. Positive Control Group: Experimental samples filled with single gutta-percha cone, no sealer. With the LC technique, the canal walls were coated with sealer (either Roth’s 801 or AH 26); the apical 5 mm of the master cone was coated with the same sealer and slowly placed in the canal to working length. Accessory cones coated with sealer were placed accordingly after insertion of the B finger plugger after each cone placement. With the WTT method, a tight ‘apical plug’ was fit with the master cone of gutta-percha (␤-phase), where the apical aspect of the guttapercha had a snug fit and the apical 5 mm was covered with either Roth’s 801 or AH26 sealer. All but the apical 5 mm of the master cone was seared off with a System B (SybronEndo, Orange, CA) set at 200°F and vertically compacted before backfilling. Canal walls were coated with the same sealer; canals were backfilled with ␣–phase gutta-percha with the Obtura II gun (Obtura Corporation, Fenton, MO). Excess guttapercha in both techniques was seared. No temporary or permanent filling was placed over the root canal fillings. Teeth were placed in endotoxin-free cryovials with sterile cotton moistened with endotoxin-free water; caps were placed loosely and samples were placed in a 37°C humidor for 4 wks to allow for sealer set of the AH 26, while allowing the Roth’s to remain unset.

Assembly of Model Systems The dual chamber model system has been used in other studies (7, 8, 14). The model system is comprised of an upper chamber, the subject tooth and the lower chamber containing the tooth root suspended in Hank’s Balanced Salt Solution (HBSS). The upper chamber is designed to hold the mixed bacterial suspension; endotoxin quantification is from the lower chamber. Model system assemblies were sterilized by gamma irradiation using 5000 cGrays from a (137) Cesium source. Setup and inoculation of the models was done under a sterile laminar hood. Upper chambers were adjusted so that 2 mm of the root extended into the HBSS. Model systems were placed into an anaerobic chamber (Coy, 5% CO2, 10% H2, 85% N2) at 37°C for 48 h to achieve reduced conditions. One milliliter aliquots of mixed bacterial suspension were added to each upper chamber and model systems were then incubated in the anaerobic chamber at 37°C.

Reducing Background Endotoxin Levels To reduce endotoxin to levels acceptable for the inclusion criteria of less than 10 pcg/ml, teeth were washed with 2 ml endotoxin-free water and placed into endotoxin-free cryovials containing 2 ml of saturated 2 mg/ml Ca(OH) 2 solution and incubated for 7 days, then replaced with endotoxin-free water. In samples where endotoxin levels obtained were less than 10 pcg/ml, vials were tightly capped and placed in a 2 to 8°C refrigerator until obturation.

Maintenance of the Mixed Bacterial Culture in the Upper Chamber To maintain viability of the mixed bacterial culture in the upper chambers, suspensions were replaced with fresh mixed bacterial suspensions from ⫺90°C stock every 3 days. Cell viability, culture purity and bacterial numbers were assessed from two randomly selected samples from each experimental and control group. These samples were diluted and spiral plated onto Crystal Violet Erythromycin (CVE), Campylobacter Rectus (CR), Maltose-Starch-Colistin-Novobiocin (MSCN), and blood agar plates, incubated under anaerobic conditions for 4 days, and enumerated using spiral plating technology (15, 16). Potential contamination of the model systems was monitored by morphologic and gram stain evaluation of organisms from the blood agar plates.

Root Canal Filling Teeth were randomly divided into one of four experimental (n ⫽ 8) and two control groups (n ⫽ 6). Teeth were filled as follows: group 1, LC, AH 26 sealer; group 2, LC, Roth’s 801 sealer; group 3, WTT, AH 26 sealer; group 4, WTT, Roth’s 801 sealer. Negative Control Group: Experimental samples filled by LC/AH 26 sealer, then sealed completely

Endotoxin Quantification From the Lower Chambers Samples (25 ␮l) from each lower chamber were taken and assayed for presence of endotoxin at 0, 1, 7, 14, and 21 days postinoculation, using a QCL-1000 LAL assay kit (BioWhittaker Inc, Walkersville, MD). In addition, model systems with detectable endotoxin in the lower chambers were also assayed for viable bacteria.

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Figure 1. (A) Example graph demonstrating trapezoidal area. (B) Mean endotoxin leakage of 0 to 21 days. Error bars represent SEM. LC, lateral condensation; WTT, Warm Thermoplasticized Technique.

Statistical Methods Response feature analysis using trapezoidal area under the curve to summarize time course data was performed (17, 18) providing a single measure for that unit (Fig. 1A). The use of trapezoidal area seemed appropriate given the observed patterns of the time courses, which were not amenable to modeling using smooth curves or polynomial regression. In addition to the response profile reflected in trapezoidal area, endotoxin levels observed at each of the individual time points were also analyzed. For trapezoidal area, and outcomes at day 14 and day 21, standard regression (two-way ANOVA) methods were used to assess the effects of sealer type and root filling method, as well as possible interaction between the two factors, since model assumptions, such as normality, appeared to be reasonable. However, outcomes at day 1 and day 7, clearly did not conform to normality assumptions, and were analyzed using a nonparametric approach. The distribution of each of these measures was compared for the four treatment groups defined by sealer type and root filling method, using the Kruskal-Wallis procedure.

Results Positive and negative controls behaved as expected; the positive controls leaked rapidly and consistently over time whereas the negative controls did not exhibit leakage. The time in which endotoxin leakage was detected in the model systems is shown in Table 1. At Day 1 postinoculation, 24 of 32 model systems leaked. By day 21, only an additional three models exhibited leakage for a total of 27/32 that had leaked endotoxin. At day 21, leakage profiles observed were different across the groups, group 1 (LC/AH26) exhibited the most endotoxin leakage over time compared to group 4 (WTT/Roth’s 801 sealer) which demonstrated the least amount of endotoxin leakage through the apex (Fig. 1B). Mean endotoxin leakage for all groups at each time point are given in Fig. 2A. The first analytic approach was to determine a summary measure of units/milliliter of endotoxin leakage over time via trapezoidal area. Overall results showed that group 1 (LC/AH26) leaked the most followed by group 3 (WTT/AH26), then group 2 (LC/ Roth’s) and finally group 4 (WTT/Roth’s) had the least endotoxin leakage over the time course (Fig. 1B). The data provided no evidence of interaction between root canal filling method and sealer type (p ⫽ 0.22) or of an effect because of obturation method (p ⫽ 0.89); but strong evidence of an effect because of sealer type (p ⫽ 0.009), with lower amount of endotoxin leakage seen among treatment combinations using Roth’s 801 sealer, and the least amount of endotoxin leakage with WTT and Roth’s 801.

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Results were also evaluated at each time point. There was no evidence of differences among the four treatment groups found at either day 1 (p ⫽ 0.23) or day 7 (p ⫽ 0.46). Results on day 14 paralleled those found for trapezoidal area: No evidence was found for interaction (p ⫽ 0.69), or for root canal filling method (p ⫽ 0.32), but strong evidence was found for an effect because of sealer type (p ⫽ 0.0008). Again, lower amount of endotoxin leakage seen among treatment combinations using Roth’s 801 sealer, although the least amount of endotoxin leakage was observed with LC and Roth’s 801 at day 14 (Fig. 2B). At day 21, there was a suggestion of an interaction between sealer type and root canal filling method (p ⫽ 0.074), corresponding to the somewhat different pattern of results seen in Fig. 2B. Data on day 21 show that the least amount of endotoxin leakage occurred in models filled with the WTT and Roth’s sealer (Fig. 2C) while the levels of leakage were similar for the other three groups.

Discussion The purpose of this study was to determine the effect of root canal filling technique and sealer type on the penetration of endotoxin from a mixed anaerobic bacterial community through the apical foramen of root canal treated teeth. We chose endotoxin penetration as the primary outcome variable in the present study because of the known role of endotoxin in periapical pathosis (2, 16, 19 –23). When analyzing endotoxin leakage at specific time points, several intriguing results arose. Specifically, at day 21, there appeared to be a trend suggesting interaction between variables of root canal filling and sealer type suggesting that the effect of these variables together was not strictly additive. Similarly, patterns of endotoxin leakage were not entirely consistent at individual time points or when the summary measures were considered: For example, a trend was observed at day 14 indicating that the LC technique with Roth’s 801 could result in less endotoxin penetration. However, at day 21 it was the WTT that allowed the least endotoxin penetration, the same treatment group with the least endotoxin penetration based upon the summary measure, trapezoidal area. This represents a reversal of pattern for root canal filling method relative to the results at day 14, although the Roth’s 801 sealer groups consistently showed the lowest endotoxin levels. Potentially, the alpha phase gutta-percha used in the WTT becomes unstable after exposure to a mixed bacterial culture for 14 days, which allows for more leakage. Perhaps the increasing size of the biofilm in the canal causes the alpha phase gutta-percha to pull from canal walls, thereby allowing more bacterial ingress. Even more difficult to explain is the shift at day 21 where the lowest endotoxin group was now Roth’s sealer and WTT.

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Basic Research–Technology TABLE 1. Number of model systems per group exhibiting endotoxin leakage over the entire time course of the study Group 1 LC/AH 26 Group 2 LC/Roth WTT/AH 26 Group 4 WTT/Roth

Day 0

Day 1

Day 7

Day 14

Day 21

0/8 (0%)

7/8 (87.5%) 8/8 (100%) 4/8 (50%) 5/8 (62.5%)

6/8 (75%) 4/8 (50%) 6/8 (75%) 4/8 (50%)

7/8 (87.5%) 2/8 (25%) 8/8 (100%) 8/8 (100%)

7/8 (87.5%) 8/8 (100%) 8/8 (100%) 4/8 (50%)

1/8 (12.5%) 0/8 (0%) 0/8 (0%)

Number in each group exhibiting endotoxin leakage for individual time points. There is variability within each group over time. LC, Lateral Condensation; WTT, Warm thermoplasticized technique.

The total number of models that showed bacterial leakage was 5/44 models, two of which were positive controls. Endotoxin leakage preceded bacterial leakage in every instance. Bacteria first appeared in the lower chambers at day 7, with three models demonstrating bacterial leakage. Two of these three were positive controls, while the other model was from group 2 (LC/ Roth’s 801). At day 14 an additional model demonstrated bacteria in the lower chamber and this model was from group 2 as well. By day 21 one more model, this from group 3 (WTT/AH26), demonstrated bacterial leakage in the lower chamber. Only one species, F. nucleatum, was recovered in each instance. Variation in leakage patterns as seen in Table 1, represent the dynamic nature of bacterial ecosystems. Bacterial counts in previous studies have been shown to exhibit periodicity in leakage profiles (8, 24). It seems reasonable that this phenomenon occurs with endotoxin as well; however, the mechanisms by which it occurs may be different. Whole organisms penetrated the root canal system in these previous studies, whereas with endotoxin, only a portion of a cell wall is required to release endotoxin. Variability in endotoxin levels may also be a result of variations in canal anatomy. We reduced as many variables as possible by using only maxillary anterior teeth with less than 20° curvatures and sectioning teeth at the CEJ to make the root length between 13 and 15 mm. There have been a number of model systems and methods described to assess endotoxin penetration through the canal system. Trope et al. (25) studied apical endotoxin penetration by using a dual chamber model system. Their approach was different from ours in that our design measured endotoxin emanating from mixed anaerobic bacterial suspensions and biofilms in the canals of root canal treated teeth, while their methodology included adding pure endotoxin (100 ␮g/ml) directly to the upper chamber. Studies have shown that endotoxin can be released as ‘blebs’ from matrix vesicles upon growth of gram negative bacteria (26, 27). Because of the ability of bacteria to release endotoxin during growth, endotoxin liberated from bacterial biofilms may better simulate a clinical endodontic situation. Carrutu et al. (28) used a dual chamber model system similar to ours except for lower chamber design specifications and addition of a lateral port. Like the Trope (25) study, pure endotoxin was added directly to the upper chamber. They found that bacteria penetrated the root canal system within 15 to 17 days, while there was no endotoxin penetration throughout a 60 day experiment. Without the presence of a bacterial biofilm within the root producing endotoxin, the endotoxin pipetted into the upper chamber of the system may simply have adsorbed to the lower chamber walls. The one time inoculation of endotoxin does not allow for the continual production of endotoxin from a live bacterial biofilm. Alves et al. (7) compared bacterial penetration to endotoxin penetration using a dual chamber model system similar to ours and the same organisms in the mixed bacterial community this study has employed. Although both bacteria and endotoxin were able to penetrate the root 602

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canal system, they concluded that endotoxin penetration preceded bacterial penetration that is consistent with the present findings. Alves et al. (7) used the same mixed bacterial community as the present investigation, as well as a similar model system to examine both bacterial and endotoxin penetration. It was concluded that endotoxin penetrated the root canal system in an average of 23 days, although some samples showed endotoxin penetration in eight days. Our study showed endotoxin penetration as early as 24 h; however, at very low levels, just over the detection limit of the assay, and present in 55% of samples after seven days. The more rapid penetration of endotoxin in the current study may be attributable to the amount of time the sealer was allowed to set. Sealers in the unset state are more cytotoxic than after achieving set (29 –31). It is plausible that the unset sealer killed the bacteria initially introduced into the model systems and with subsequent additions of viable bacteria, enough were able to survive and multiply until the sealer reached a less bactericidal state. Sealer set may play an important role in endotoxin penetration as possibly evidenced by the difference in time for endotoxin penetration in the current study and others discussed previously. A study by Trope et al. (25) demonstrated endotoxin penetration after 3 days using Roth’s 801 sealer in conjunction with gutta-percha for obturation; however, no mention was made of time frame for sealer set. Using Roth’s 801 sealer and gutta-percha for obturation, endotoxin was observed in the lower chambers after 8 days in a similar study by Alves (7); again however, no mention was made of the time frame for sealer set. Antimicrobial properties of sealer likely impact bacteria/endotoxin behavior. The liquid component of Roth’s 801 sealer is eugenol. Because ZOE-based sealers set very slowly (longer than 8 wk), free eugenol may exert a bactericidal effect. Research done by Hashieh et al. (32) evaluated the concentration of eugenol present in Phosphate Buffered Saline (PBS) immediately after sealing, at 1 day and 1 month. Eugenol levels were barely detectable, implying eugenol is not being released into periapical tissues. When maintained in an enclosed space, it remains uncertain what effect eugenol may have on the bacterial biofilm in the canal. Sealer distribution and adherence along canal walls likely impact endotoxin penetration. The gaps between canal walls and sealer/guttapercha would provide a portal for bacterial and endotoxin passage. Facer and Walton (34) found that no sealer provided complete coverage; sealer was often absent between gutta-percha and canal walls and between gutta-percha cones. This may provide an additional route between the gutta-percha cones and the sealer for endotoxin penetration. Our study was similar in part to that published by Barrieshi et al. (8). Both AH26 sealer and Roth’s 801 sealer were used in the current study, whereas Barrieshi used only Roth’s 801. It was found in the Barrieshi study, as in this study, that endotoxin preceded bacterial penetration of the canal system. It is currently not known whether this phenomenon is because of the sealer itself or other factors including the

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Figure 2. (A) Box-and-whisker plots of trapezoidal area summarizing endotoxin leakage over the entire time course for four treatment groups defined by root filling methods and sealer type. (B) Box-and-whisker plots of the levels of endotoxin leakage in EU/ml at day 14 postinitiation for each of the four treatment groups defined by root filling method and sealer type. (C) Box-and-whisker plots of the levels of endotoxin leakage in EU/ml at day 21 postinitiation for each of the four treatment groups defined by root filling method and sealer type. LC, lateral condensation; WTT, Warm Thermoplasticized Technique.

smear layer, composition of individual sealers, species in the mixed bacterial suspension, sealer set and consistency and possibly tooth type. There may be several reasons for these findings with respect to sealer: the eugenol in the unset sealer may impact the cells in the outermost layer of the biofilm adhering to the canal wall, hindering the ability of the bacteria to multiply for a time. The cytotoxicity of the sealer may inhibit elaboration of endotoxin within the canal for a period of time by killing or altering bacteria. Perhaps the unset nature of the sealer impedes the physical progress of the bacteria themselves. Our ongoing studies are focusing on a relationship between sealer set and bacterial penetration through filled root canal systems. Our study showed that endotoxin leaks more readily through the root canal system in root canal treated teeth using AH26 as the sealer. As with any in vitro model system, results must be interpreted and extrapolated to clinical situations with caution. What can be interpreted from these results, however, is the apparent lack of a perfect root sealing system utilizing the techniques in this study.

Acknowledgments This project was supported by an Endodontic Research Grant from the AAE Foundation. The authors wish to give special thanks to Bonny Olsen for her technical support. Drs. Williamson, Drake, and Walton are affiliated with the Department of Endodontics; Dr. Dawson is affiliated with the Department of Community and Preventive Dentistry at the University of Iowa, College

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of Dentistry. Dr. Rivera is affiliated with the Department of Endodontics, University of North Carolina School of Dentistry.

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