Periapical Repair after Root Canal Filling with Different Root Canal Sealers

One of the goals of endodontic therapy is to allow apical and periapical repair. Endodontics is still searching for the ideal root canal filling material, which should have certain physical, chemical and biological properties (1). Zinc oxide and eugenol-based sealers are used worldwide. However, their biological properties are not satisfactory (2), since they induce the presence of chronic periapical inflammatory infiltrate (3) that may persist for long periods of time (4). Regarding epoxy resin-based sealers, AH Plus stands out due to its excellent biological properties (5). Leonardo et al. (5) histologically evaluated the response of apical and periapical tissues of dog teeth after pulpectomy and were able to demonstrate hard tissue formation in the periapical region when AH Plus sealer was used. The silicone-based material Roeko Seal (Roeko, Periapical Repair after Root Canal Filling with Different Root Canal Sealers


INTRODUCTION
One of the goals of endodontic therapy is to allow apical and periapical repair.Endodontics is still searching for the ideal root canal filling material, which should have certain physical, chemical and biological properties (1).
Zinc oxide and eugenol-based sealers are used worldwide.However, their biological properties are not satisfactory (2), since they induce the presence of chronic periapical inflammatory infiltrate (3) that may persist for long periods of time (4).
Regarding epoxy resin-based sealers, AH Plus stands out due to its excellent biological properties (5).Leonardo et al. (5) histologically evaluated the response of apical and periapical tissues of dog teeth after pulpectomy and were able to demonstrate hard tissue formation in the periapical region when AH Plus sealer was used.

Periapical Repair after Root Canal Filling with Different Root Canal Sealers
Langenau, Germany) has shown good physical-chemical properties, such as adequate flowability (6) and low occurrence of apical infiltration (7,8).Moreover, this sealer has low cytotoxicity (9,10).Huumonen et al. (11) evaluated radiographically the apical repair process in human teeth sealed with Grossman and Roeko Seal Automix sealers.One-hundred and ninety-nine teeth with apical lesions were sealed and radiographically evaluated 3 and 12 months after root canal filling.The authors highlighted the lack of significant difference between the 2 evaluated sealers.Resilon (Resilon Research LLC, Madison, CT, USA) is a root canal filling material based on a thermoplastic synthetic polymer with properties similar to gutta-percha, and is used in association with Ephiphany (Pentron Clinical Technologies, Wallingford, CT, USA), a methacrylate-based material.When Epiphany is used with Resilon, there is a strong bond between the dentin walls and the root canal filling material, increasing the resistance to bacterial penetration (12).
Several authors have studied the Resilon/Epiphany system (13,14).Onay et al. (14) evaluated in vivo the biocompatibility of Resilon, gutta-percha, and Epiphany after implantation of samples in rat connective tissue.After 1, 4, and 8 weeks, samples were removed.The authors concluded that all the materials tested presented acceptable biocompatibility.Souza et al. (15) evaluated the intraosseous biocompatibility of AH Plus, EndoREZ, and Epiphany in Guinea pigs, observing that AH Plus promoted moderate to severe response, while with Epiphany bone formation was observed, accompanied by mild or no inflammatory response.When the biocompatibility of Sealapex was compared with Epiphany sealer, Leonardo et al. (16) observed that, after root canal filling in dog teeth, Epiphany showed better results in the histological analysis compared to Sealapex.
New clinical studies became necessary to evaluate the biological properties of Roeko Seal and of the Resilon/Epiphany system in experimental animal models, especially studies that involve histopathological analysis.Accordingly, the present study aims to assess the biological properties of these materials, by evaluating the periapical repair after root canal filling in dog teeth.

MATERIAL AND METHODS
Four mongrel dogs, approximately one year old, were utilized in this study.The teeth selected were the second, third, and fourth mandibular premolars and the second and third maxillary premolars, totalling 64 roots, which were divided in 4 experimental groups.The experimental procedures were in accordance with the Institutional Committee of Animal Experimentation and the International Guiding Principles for Biological Research Involving Animals (Geneva, 1985).
After isolation of the teeth with a rubber dam and disinfection of the operative field with 2% gluconate chlorhexidine, access to the pulp chambers were made.The working length was determined to be 2 mm short of the radiographic apex using #25 K-type files (Dentsply/ Maillefer, Ballaigues, Switzerland).The pulp tissue was removed, and the root canals were irrigated with 2 mL of 1% sodium hypochlorite solution.
The apical cementum layer was then perforated with the sequential use of #15 to #30 K-files, thus creating standardized apical foramen openings.Thereafter, the root canals were instrumented to the working length up to #60 K-file under irrigation with 1% sodium hypochlorite solution at each instrument change.A #30 K-file was taken to the total root length to ensure apical patency.After final irrigation with sodium hypochlorite, the root canals were dried with sterile paper points and then filled with 14.3% EDTA (Odahcan-Herpo, Prod.Dent.Ltda., Rio de Janeiro, RJ, Brazil) for 3 min.Sterile saline was used to rinse out the EDTA, and the root canals were then dried with sterile paper points.
The canals were filled by lateral condensation of gutta-percha and one of the sealers.The sealers were taken the root canals using a #50 K-file up to the working length.For the teeth filled with Resilon/Epiphany, the primer was first applied to the root canal walls with sterile paper points and then, obturation was done by lateral condensation of the Resilon cones.In all groups, the coronal access preparations were restored with a glass ionomer cement base (Vitremer; 3M/ESPE, St. Paul, MN, USA) and silver amalgam (Velvalloy; S.S.White Artigos Dentários Ltda., Rio de Janeiro, RJ, Brazil).
After a 90-day experimental period, the animals were euthanized with an intravenous overdose of sodium pentobarbital.The maxillas and mandibles were dissected and sectioned to obtain individual roots that were fixed in sodium cacodylate solution with added sucrose and glutaraldehyde (17).The specimens were then washed and demineralized with an EDTA-based solution.Six-micrometer-thick serial sections (25-30 sections per specimen were obtained and stained with hematoxylin-eosin (HE) and Mallory trichrome.The sections were examined under optical microscopy by two calibrated examiners blinded to the treatment of each group.In case of disagreement, the specimen was re-evaluated and a consensus was reached between the examiners.Scores of A, B, C or D. from best to worst, were given to each of the following parameters (16,17): Intensity and extension of the inflammatory infiltrate, periodontal ligament thickness, bone and apical cementum resorption, and apical opening sealing (Table 1).
The intensity of periapical inflammatory infiltrate was evaluated by counting the number of inflammatory cells in all specimens using the Image Pro Plus v. 6.1 software (Media Cybernetics, Silver Spring, USA).The medians (50%) and quartiles (25% and 75%) were calculated and the values were distributed in 4 intervals.These values were used to establish intervals corresponding to scores A to D (Table 1).The apical periodontal ligament thickness was evaluated according to the distance (in mm) between the apical surface and the bone tissue, using the Image Pro Plus v. 6.1 software (Media Cybernetics).The medians (50%) and quartiles (25% and 75%) were calculated and the values were distributed in 4 intervals.These values were used to establish intervals corresponding to scores A to D (Table 1).
Results were calculated by adding the values of all histopathological parameters in each group and then comparing the groups.Statistical analysis was done with the nonparametric Kruskal-Wallis test and the 2-by-2 comparisons of the Dunn's method (p<0.05).

RESULTS
Significant difference between Intrafill, a zinc oxide and eugenol-based material) and the other materials (p<0.05) was detected.General analysis of the histopathological parameters demonstrated that Intrafill presented less favorable results in terms of periapical repair compared to the other materials (p<0.05).AH Plus, Roeko Seal and Resilon/Epiphany had similar periapical repair (p>0.05), and all showed consistently more favorable results than Intrafill (p<0.05).The results of the qualitative analysis for the tested materials are described in the following paragraphs.

Intrafill
Inflammatory cell count demonstrated the presence of severe/intense inflammatory reaction in most specimens evaluated in this group.Measurement of the apical periodontal space and assignment of scores demonstrated that in this group, the periodontium was intensely/severely thickened (Fig. 1).

AH Plus
The periapical inflammatory reaction in this group was less intense than that observed for Intrafill (p<0.05) (Fig. 2).Inflammatory cell count in the 16 cases in this group showed that 12 specimens had score  of A or B. The periapical periodontium was slightly or moderately thickened.

Roeko Seal
The degree of periapical inflammation was similar to that observed for AH Plus (Fig. 3).Inflammatory cell count showed that the cases in this group (n=16) had predominant scores of A or B. Apical foramen sealing was observed in most of the cases.

Resilon/Epiphany
Periapical inflammatory response was similar Table 1.Frequency of scores corresponding to each histopathological parameter for the ested materials.to that detected for AH Plus and Roeko Seal (p>0.05) (Fig. 4).The periapical periodontium showed, in most cases, mild or moderate thickening.Mineralized tissue deposition at the apical foramen was observed.

DISCUSSION
Comparative analysis of the histopathological results demonstrated better tissue repair when AH Plus, Roeko Seal, and the Resilon/Epiphany System were used.These experimental groups presented high frequency of partial biological sealing of the apices, predominance of mild or moderate inflammatory infiltrate, and no areas of cementum and bone resorption.
A zinc oxide and eugenol-based sealer was used as a control because this material is known to induce chronic periapical inflammatory infiltrate (3,18).In the specimens where Intrafill (zinc oxide and eugenol-based material) was used, intense/severe periapical inflammatory infiltrate was observed.Deposition of mineralized tissue at the apex occurred in a few cases and only partially covered the apical foramen.The results for this zinc oxide and eugenol-based sealer are in agreement with Leonardo et al. (2), who reported unsatisfactory periapical repair when Fill Canal (another zinc oxide and eugenol-based material) was used in root canal fillings in dog teeth.
The biocompatibility of AH Plus confirms previous results from Leonardo et al. (5), evaluated the performance of this material in dog teeth after pulpectomy reporting no inflammatory cells or areas of necrosis associated with AH Plus.They observed hard tissue formation with AH Plus in most of the specimens, and inflammatory response adjacent to the zinc-oxide and eugenol sealer.
These favorable results were supported by detection of only mild/slight periapical inflammatory infiltrate, along with deposition of mineralized tissue at the apex, which covered up to half or more than half of the apical foramen in the majority of cases.The favorable results of periapical healing obtained after root canal filling with AH Plus (5) allow using this material as parameter for comparison with new filling materials.
Acceptable results in terms of apical and periapical repair have also been observed for the Resilon/ Epiphany System.Our results showed mineralized tissue deposition at the apical foramen in all teeth, and in most cases the mineralized tissue covered more than half of the foramen.
Shipper et al. (19), evaluated periapical tissues of dog teeth filled with gutta-percha and AH 26 or  with the Resilon/Epiphany System, finding periapical inflammation in 82% of the teeth filled with AH 26 and gutta-percha, and in only 19% of the cases where Resilon/Epiphany was used.
Souza et al. (15) evaluated the intraosseous biocompatibility of AH Plus, EndoREZ, and Epiphany in Guinea pigs, and observed new bone formation when Epiphany was used, along with mild or no inflammatory response.Leonardo et al. (16) studied in vivo the biocompatibility of Sealapex and of the Resilon/Epiphany System in endodontically treated dog teeth.The results showed that roots canals filled with Epiphany/Resilon, with coronal restoration, had significantly less periradicular inflammation and more biological apical sealing than roots canals filled with gutta percha and Sealapex.
Epiphany sealer has calcium hydroxide and may release calcium and hydroxyl ions contributing to its biocompatibility.Rezende et al. (20) observed that the solubility of Epiphany was greater than other sealers, with higher amounts of calcium ion release.
Roeko Seal, a silicone-based sealer, induced periapical repair, with results similar to AH Plus and Epiphany.The inflammatory infiltrate detected in the specimens where Roeko Seal was used was predominantly slight/mild.Favorable results were also observed in terms of periapical repair: deposition of mineralized tissue at the apical foramen was detected and covered half or more than half of the foramen in the majority of teeth, in addition to one case of complete seal.
Silicone is a biocompatible material, which may explain the good results obtained with Roeko Seal.Lodiene et al. (10) performed a cytotoxicity study in which Epiphany root canal sealer was significantly more toxic to L-929 cells than the silicone-based Roeko Seal and the single methacrylate-based EndoREZ root canal sealers.Miletic et al. ( 9) evaluated the cytotoxicity of endodontic sealers at 1 h, 24 h, 48 h, 1 week, and 1 month after their manipulation, and reported that Roeko Seal did not present cytotoxic effects in any of the periods evaluated.
According to the present study, the Resilon/ Epiphany System, the silicone-based product Roeko Seal, as well as the AH Plus sealer presented good results in terms of periapical repair.Based on this biological property, it can be suggested that these materials are adequate choices for clinical application.

Figure 2 .
Figure 2. AH Plus.Image of the apex showing partial biological seal by deposition of mineralized tissue (A) and mild inflammatory cell infiltrate (B).HE (original magnification ×100).

Figure 3 .
Figure 3. RoekoSeal.Image of the apex showing partial biological seal by deposition of mineralized tissue (A) and mild inflammatory cell infiltrate (B).HE (original magnification ×100).