Laboratory study of tissue repair of resin-based endodontic sealers in critical surgical defects

Abstract Filling materials should be restricted to the root canal space. However, sometimes it is impossible to control the apical extrusion, in this case, the fate of the filling material and the result of the treatment will depend on its physicochemical properties and biocompatibility. Objective To evaluate the tissue response and bone repair capacity of endodontic sealers that were implanted in the calvaria of Wistar rats, forming the groups (n=16): AH Plus and Sealer Plus, compared to the clot group. Methodology On days 30 and 60, the animals were euthanized, the calvaria was removed and processed for hematoxylin-eosin, immunohistochemistry for collagen type I, Picrosirus red and microtomographic analysis. Data were subjected to ANOVA and Tuckey tests (p<0.05). Results At 30 days, all groups showed an intense inflammatory reaction (p>0.05). At 60 days, the AH Plus and Sealer Plus maintained an intense inflammatory infiltrate compared to the clot group (p<0.05). We observed immunopositive areas for type I collagen in all groups at 30 days and 60 days (p>0.05). We observed more red collagen fibers for the Sealer Plus compared to the clot group at 30 days (p<0.05). Considering the total fibers, the clot group at 30 days compared to 60 days after surgery showed an increase in the amount of matrix (p<0.05). There were no statistical differences between groups for green and yellow fibers (p>0.05). Regarding morphometric parameters, at 30 days, the newly formed bone volume and number of bone trabeculae were higher in the groups with sealers compared to the clot group (p<0.05). At 60 days, AH Plus and Sealer Plus showed greater bone neoformation compared to the clot group (p<0.05). Conclusions Despite AH Plus and Sealer Plus induced an intense inflammatory reaction, they can be considered biocompatible materials, since they allowed bone repair.


Introduction
Endodontic treatment depends on infection control by cleaning, shaping and filling the root canal system. 1,2 The root canal filling materials must show biocompatibility and sealing capacity to prevent both colonization and reinfection by pathogenic microorganisms, preventing communication and irritation to the periapex. 2, 3,4 Filling materials must be restricted to the space of the root canal. 5,6 However, sometimes it is impossible to control the application of the material and an apical extrusion occurs. 7 In these cases, the fate of the filling material will depend on its solubility in tissue fluids, the susceptibility to phagocytosis, and its biocompatibility. 7 The overfilled material may act as a foreign body, inducing histological reactions ranging from simple periradicular inflammation to necrosis of the periodontal ligament. 8 AH Plus (Dentsply Maillefer, Ballaigues, Vaud, Switzerland) is an epoxy resin-based sealer and considered the gold standard endodontic sealer due to its physicochemical properties (setting time, flow, film thickness, dimensional stability, radiopacity and solubility), 9,10,11 biocompatibility, 12,13 sealing ability 14,15 and satisfactory antimicrobial activity. 16 AH Plus sealer is commonly used in clinical practice and experimental studies in animals that examined periapical repair against this material reported good biological activity. 17,18,19,20   After anesthesia, trichotomy was performed in the frontoparietal region, the animal was placed in the frontal decubitus, the area was sanitized with topical polyvinylpyrrolidone (PVPI) and a sterile field was applied. Then, surgical access was performed through a "V" shaped incision, the total flap was folded back and removed with the retractors to expose the parietal bone on both sides.

Microtomographic analysis
After euthanasia of the 48 specimens, the parietal bone was removed with a margin of three mm of bone tissue around the defect and immediately fixed in 10% buffered formalin (pH 7.2).
All specimens were scanned using a computerized microtomography (SkyScan 1146, Billerica, Massachusetts, New England, USA) at an energy level of 50 Kv and current of 800 µA. Images were captured with a 22.9 µm pixel camera, with 180° rotation around the vertical axis and 1.0° rotation step.
X-rays, filtered with a 0.05 mm aluminum filter and a flat field correction, were taken the day before the scan to correct variations in the camera's pixel sensitivity. to yellow and red (thick fibers) was measured using AxioVision software to define the corresponding area (pixel 2 ) of these fibers and the total birefringent fibers.

Immunohistochemical analysis to detect type I collagen
The immunoexpression of type I collagen in tissue in contact with the materials and the control group

Statistical analysis
All data are expressed as mean and standard deviation (SD) and statistically analyzed by one-way analysis of variance (ANOVA) and Tuckey test. All statistical analyses were performed using SigmaPlot 12.5 (Systat software, CA, USA). The p-value was considered significant at 5%.

Results
No animal died during the experiments and we did not notice side effects due to endodontics sealers. Two different blind investigators were involved in the data analyses for each specimen.

Immunohistochemical analysis
After 30 and 60 days, we observed immunopositive areas for type I collagen more frequently in the groups with AH Plus or Sealer Plus than in the control group ( Figure 2 and 3). However, this parameter did not show a statistically significant difference between the three groups in both periods (p<0.05) (Figure 2).

Microtomographic Analysis
At 30 days, the newly formed bone volume and number of bone trabeculae in the Sealer Plus and AH Plus groups were higher compared to the control group (p<0.05). At 60 days, Sealer Plus had more bone neoformation compared to the control group (p<0.05), but it did not differ from AH Plus (p>0.05).

Consequently, both sealers analyzed at 30 days
showed a gradual decrease in trabecular separation, with statistical difference compared to the control group (p<0.05). This same parameter showed a statistical difference in the Sealer Plus group at 60 days compared to the control group (p<0.05).
Regarding trabecular thickness, there was no statistical difference between the groups in both periods analyzed (p>0.05). (Figures 2 and 4).

Discussion
An ideal endodontic sealer must have adequate physicochemical properties, antimicrobial activity and biocompatibility, since they will be in contact with the periapical tissues. 13,33,34 Sealer extrusion may happen in some clinical situations, such as in apical root resorption. 7 In these cases, the material must not interfere or aid in the repair process, including bone remodeling. Overfilled material may act as a foreign body and the tissue response depends on its composition. 8

Standard deviations in parentheses
Different letters indicate differences between groups for the same period (ANOVA and Tuckey tests p<0.05)  Our data showed that, despite Sealer Plus and AH . The delineated area (white line) shows the region of the HE, Picrosirius red and Immunohistochemical sections in higher magnification. HE photomicrographs sections showed bone neoformation next to the defects of control group and sealers (asterisks); besides a fibrous capsule was juxtaposed to this bone (arrows); many fibroblasts and few inflammatory cells, especially plasmocytes and lymphocytes, were present in this tissue. Picrosirius red section shows a bundle of collagen fibers (red, orange, green) formed in the adjacent bone. Areas immunomarked (immunohistochemical sections) in brown showed the detection nex to the newly formed bone type I collagen that can be seen in the tissue in contact with the newly formed bone (asterisks)  We observed a significant amount of red collagen adjacent to the Sealer Plus and AH Plus after 30 days compared to the control group (p<0.05). Despite many inflammatory cells, the repair process in the adjacent tissue may have been initiated faster than the control group. This hypothesis is reinforced for the immunohistochemical analysis, since immunopositive areas for type I collagen were more frequent in the Sealer Plus and AH Plus groups. A previous study showed that cell proliferation and adhesion of extracellular matrix proteins, such as type I collagen, in the migration process are involved in the colonization of cells to tissues. Thus, the expression of these proteins is crucial for the repair process. 40 The repair process in the adjacent tissue occurs because both materials caused a reduced inflammatory reaction, culminating with bone neoformation as seen after 30 and 60 days.
During root canal filling, the materials used may come into contact with the periapical tissue. Among the properties of the materials, biocompatibility is an essential characteristic. 41,42 Endodontic sealers should allow or promote the resolution of periapical inflammatory and/or infections. 41 The healing process is dynamic and involves different stages. Initially, the inflammatory process decreases, followed by the remodeling of the tissue and the formation of collagen.
Finally, when bone loss occurs, there will be the neoformation of this tissue. According to our results, both Sealer Plus and AH Plus modulate the repair process, since they allow the formation of collagen and bone faster than the control group.
Among the limitations of our study, we emphasize that the results must be considered before being extrapolated to humans, since Rattus norvegicus is a different species. However, this study helps to further assess Sealer Plus participation in the bone repair process.

Conclusion
The results showed that Sealer Plus was similar to AH Plus in the inflammatory process, in the maturation of collagen fibers and in bone neoformation in both evaluated periods. Therefore, Sealer Plus and AH Plus can be considered biocompatible materials, modulating the bone repair process. Clinically, both materials are indicated in cases with risk of overfilling.