Biocompatibility and biomineralization assessment of bioceramic-, epoxy-, and calcium hydroxide-based sealers

Obturation of the root canal system aims to fill empty spaces, promoting hermetic sealing and preventing bacterial activity in periapical tissues. This should provide optimal conditions for repair, stimulating the process of biomineralization. An endodontic sealer should be biocompatible once it is in direct contact with periapical tissues. The aim of this study was to evaluate the rat subcutaneous tissue response to implanted polyethylene tubes filled with Smartpaste Bio, Acroseal, and Sealapex and investigate mineralization ability of these endodontic sealers. Forty Wistar rats were assigned to the three sealers groups and control group, (n = 10 animals/group) and received subcutaneous implants containing the test sealers, and the control group were implanted with empty tubes. After days 7, 15, 30, and 60, animals were euthanized and polyethylene tubes were removed with the surrounding tissues. Inflammatory infiltrate and thickness of the fibrous capsule were histologically evaluated. Mineralization was analyzed by Von Kossa staining and polarized light. Data were tabulated and analyzed via Kruskal-Wallis and Dunn’s test. All tested materials induced a moderate inflammatory reaction in the initial periods. Smartpaste Bio induced the mildest inflammatory reactions after day 15. No difference was observed among groups after days 30 or 60. Von Kossa-positive staining and birefringent structures observed under polarized light revealed a larger mineralization area in Sealapex-treated animals followed by Smartpaste Bio-treated animals. At the end of the experiment, all tested sealers were found to be biocompatible. All sealers induced biomineralization, except Acroseal, which induced a mild tissue reaction.


Introduction
Efficient cleaning and shaping of the root canal system is essential to achieve the biological and mechanical objectives of endodontic treatment.This involves removal of the pulp tissue and remains as well as microorganisms and their by-products while providing the appropriate conical shape for subsequent root canal filling to achieve the desired three-dimensional obturation. 1 Gutta-percha alone as a filling material is not sufficient to provide adequate root canal system sealing.Adequate sealing requires the use of an endodontic sealer to fill gaps between the cone and root canal walls; this can be achieved by evenly applying the fluid sealer. 2,3As the sealer reaches the apical foramen, it comes in direct contact with the periapical tissue, and therefore should be biocompatible.Although the contact area is small, there is always concern regarding the adverse reactions that the sealer may cause on the tissues. 4ndodontic sealers are divided into different groups, according to their main components, such as zinc oxide and eugenol, resin, and calcium hydroxide. 5epending on these main components, local adverse effects may occur, which delay or hinder repair. 6ioceramic sealers are being introduced into the market.Their composition includes tricalcium and dicalcium silicates, calcium phosphates, calcium hydroxide, and zirconium oxide as a radiopacifier.All abovementioned components are applicable for biomedical and dental use and possess hydrophilic properties. 7,8The Smart Seal obturation system consists of a bioceramic sealer (Smartpaste Bio ® , CRD Ltd, Stamford, UK) identified as a hydroxyapatite-based cone polymer with an external layer of the expandable hydrophilic hydrogel Smartpoint. 5,9The manufacturer affirms that Smartpaste Bio produces hydroxyapatite and calcium hydroxide as by-products of a setting reaction consistent with alkaline pH, antibacterial activity, radiopacity, and biocompatibility. 10croseal (Acroseal ® Specialites-Septodont, Saint Maur-des-Fosses, France) is an endodontic sealer containing 28% calcium hydroxide, along with a radiopaque excipient and resin compound (epoxy resin).Previous studies have demonstrated antimicrobial activity against Enterococcus faecalis, low toxicity, and suitable physicochemical properties.11,12 According to the manufacturer, the sealer formulation has recently been modified with a reduction in the calcium hydroxide concentration and an increase in its resinous compound diglycidyl ether of bisphenol A (DGEBA).
Sealapex (Sealapex ® SybronEndo, Glendora, CA) is a sealer that contains calcium oxide and forms calcium hydroxide after being hydrated on contact with the tissue fluid.Sealapex is characterized by biocompatibility and an osteoinductive ability to stimulate the deposition of mineralized tissue, thus inducing apical sealing after endodontic treatment. 13,14his sealer was submitted to a reformulation, which has a 2-year shelf life rather than the 1-year shelf life of the previous formulation.One of the major alterations in Sealapex is the change in the radiopacifier from barium sulfate to bismuth trioxide. 15n ly limited data concern ing the Smart Seal obturation system are currently available, and there is a current lack of scientific studies regarding biocompatibility of Smartpaste Bio and its mineralization ability.Additionally, the components in the reformulated versions of Acroseal and Sealaplex need to be studied further.
The purpose of the present study was to analyze biocompatibility (inflammation response) of Smartpaste Bio, Acroseal, and Sealapex and investigate the mineralization ability of these endodontic sealers.The null hypothesis was that biocompatibility and mineralization were not induced by Smartpaste Bio, Acroseal, or Sealapex.

Methodology
Forty male 4-6-month-old Wistar rats, weighing 250-280 g, were used in the study.The animals were housed in temperature-controlled rooms and were provided with water and food ad libitum.Animal care was performed according to the Faculdade de Odontologia de Araçatuba, UNESP, Ethical Committee, which approved the experimental project.
One hundred and twenty polyethylene tubes (Abbott Laboratories of Brazil, Sao Paulo, SP, Brazil) with a 1.0 mm internal diameter, 1.6 mm external diameter, and 10.0 mm length were filled with the tested sealers.Acroseal and Sealapex were prepared according to the manufacturer's recommendations and inserted into the tubes with a lentulo spiral (Dentsply Maillefer, Tulsa, USA).Smartpaste Bio is available in a ready-to-use syringe and was directly inserted into the polyethylene tubes.Forty extra polyethylene empty tubes were used as controls, totaling to 160 tubes in the experiment.
After administration of xylazine (10 mg/kg Rhobifarma Indústria Farmacêutica Ltda, Hortolândia, Brazil) and ketamine (25 mg/kg União Química Farmacêutica Nacional S/A, São Paulo, Brazil) intramuscular anesthesia, the backs of the animals were shaved, antisepsis was obtained with 5% iodine solution, and a 2.0 cm incision was formed in a head-tail orientation with #15 Bard-Parker blade (BD, Franklin Lakes, USA), creating two pockets on each side of the incision.Three polyethylene tubes, containing the sealers, and an empty tube, as the control, were implanted in each animal in opposite directions (upper right, upper left, lower right, and lower left), and the skin was closed with a 4/0 silk suture (Johnson & Johnson Produtos Profissionais Ltda, São José dos Campos, Brazil).
On days 7, 15, 30, and 60 after implantation, the animals were euthanized by an anesthetic overdose.Polyethylene tubes, with the surrounding tissues, were removed and fixed in 10% buffered formalin at pH 7.0. 16The specimens were processed for glycol methacrylate embedding, 17 serially cut into 3 μm sections, and stained with hematoxylin-eosin (HE).The 10 μm sections were stained according to the Von Kossa technique or were not stained.
Polarized light (PL) was used to observe birefrigent structures and the Von Kossa (VK) technique was used to observe biomineralization, as it darkly stains mineralized structures. 18,19ssue reactions at the open end of the tubes were scored according to previous studies 14,19,20 as follows: 0, few inflammatory cells or no reaction; 1, less than 25 cells and mild reaction; 2, between 25 and 125 inflammatory cells and moderate reaction; and 3, 125 or more inflammatory cells and severe reaction (400 × magnification).Fibrous capsules were considered thin when < 150 μm and thick when > 150 μm.Calcification was recorded as positive or negative by Von Kossa staining and present or absent under PL (100 × magnification).
Data were statistically analyzed by Kruskal-Wallis and Dunn's test; p < 0.05 was considered significant.

Control group
A moderate chronic inflammatory reaction (median score 2) was observed at the first two time periods (Table 1), i.e., on days 7 and 15 (Figures 1 A and B).Inflammatory cell infiltration, comprising lymphocytes and macrophages, was present in the fibrous capsule, which was thick.In the 30-and 60-day samples, the fibrous capsule surrounding the tube was thin, with few inflammatory cells (median score 1).The control group was negative for Von Kossa stain and no birefringent structures were observed under PL at all time periods (Table 2).

Smartpaste Bio
Only on day 7, a moderate inflammatory cell infiltration, comprising lymphocytes and macrophages, was present in the thick fibrous capsule.On days 15, 30, and 60, the intensity of the inflammation was reduced (median score 1) and the fibrous capsule was thin, similar to that observed in the control group (Figure 1).Granulations birefringent to PL were present mostly on day 7. From days 15 to 60, the birefringent granulations started to disappear (Table 2).Von Kossa positive stains were observed at all time periods.

Acroseal
On days 7 and 15, a moderate inflammatory cell infiltration (median score 2), comprising lymphocytes and macrophages, was present in a thick fibrous capsule (Table 1).On days 30 and 60, the inflammation intensity reduced and the fibrous capsule was thin, similar to that observed in the control group (Figure 1 K,L).Birefringent granulations to PL were absent and Von Kossa was negative at all time periods (Figure 2 I-P)

Sealapex
On days 7 and 15, a moderate inflammatory reaction (median score 2), consisting mainly of lymphocytes and macrophages, was present in the thick fibrous capsule (Table 1).The intensity of the inflammation reduced from day 30 to day 60, exhibiting the remaining macrophages phagocyting extravasated sealer (median score 1).The fibrous capsule near the tube opening was thin (Figure 1 G, H) and granulations birefringent to PL and Von Kossa positive staining were observed near the tube opening at all time periods (Figure 2 A-H).

Comparison among groups
Data were compared for each time period as shown in Table 1 and 2. After day 7, HE staining revealed a tissue response similar to that of an inflammatory reaction, and there were no statistically significant differences among the inflammation scores of the experimental groups.On day 15, there was a statistically significant difference between inflammatory cell numbers in the Smartpaste Bio and other groups; the inflammatory score was lower than that of the other groups (p < 0.05) and it also presented a thin fibrous capsule.On days 30 and 60, there was no statistically significant difference among scores of the different groups, with respect to the inflammation tissue response.
PL and Von Kossa analysis revealed that Acroseal had no mineralization induction at any time periods, unlike Sealapex, which presented birefrigent structures under PL and Von Kossa positive staining at all time periods (Table 2).Smartpaste Bio samples presented birefrigent structures and Von Kossa positive staining in 100% of the specimens until day 15 and decreased over time, differentiating from each time period along the experiment, but maintaining the already mineralized structures (Table 2).

Discussion
In our study, all tested sealers, except Acroseal, promoted mineralization, and all sealers demonstrated biocompatibility; therefore, we rejected the null  hypothesis.The mild-to-moderate inflammatory reactions induced in all groups during the initial experimental periods subsequently decreased and the fibrous capsule became thinner.The use of subcutaneous implants was initiated by Torneck. 21His pioneering research, which aimed to evaluate the reaction of the subcutaneous connective tissue of rats to implanted polyethylene tubes, became a commonly used preliminary method to evaluate biocompatibility. 22,23Here the reactions to empty tubes were similar to those that were previously reported. 16,24revious studies reported that a glycol methacrylate technique was an excellent alternative for the evaluation of biocompatibility of endodontic sealers; this technique is easy to execute and reproducible and thereby provides a better definition of the degree of the inflammatory process. 17The specimens were cut into 3 μm sections and stained with HE to analyze the inflammatory infiltrate.Scores used to analyze inflammatory reactions were consistent with those reported in previous studies. 14,16,19The 10 μm sections were stained according to the Von Kossa technique to observe mineralized structures or unstained to allow observation of birefringent structures under PL which are related to calcium carbonate crystals. 18,25he reaction of calcium ions from Ca(OH) 2 and the ionic dissociation of carbon dioxide from the tissues results in the formation of calcite crystals that are birefringent to PL, and induce the formation of calcified areas.Moreover, the use of calcium reduces the presence of carbon dioxide, which is used by bacteria for anaerobic respiration, 26 whereas the high pH provided by the hydroxyl favors the tissue restoration process and antimicrobial properties. 27,28ndodontic sealers containing calcium oxide have been suggested as obturating materials because of their capacity to dissociate into calcium and hydroxyl ions, resulting in a higher pH that favors repair and induces mineralized tissue formation. 13,26,35,39he setting time could favor a calcium ion release that generally occurs before the setting of materials. 36lthough Acroseal has a long setting time, 12 its calcium release was inferior to that with Sealapex because of the relative insolubility of its epoxy base.This finding illustrates the need for calcium hydroxide-based sealers to be minimally soluble and liberate hydroxyl and calcium ions, 35 which explains the absence of mineralization observed in this study.
With respect to Smartpaste Bio, the use of a pre-mixed sealer eliminates the potential of heterogeneous consistency during on-site mixing.Studies involving other bioceramic endodontic sealers already revealed hard tissue deposition 29 and favorable tissue response to partial pulpotomy. 30onobasic calcium phosphates are included in the sealer to facilitate the reaction with calcium hydroxide to produce water and hydroxyapatite upon activation of the sealer by water. 31revious studies on bioceramic sealers have revealed lower inflammatory mediators and better osteoblast expression, thus indicating that the bioceramic is biocompatible. 32,33These earlier findings on the biocompatibility of bioceramic sealers also match the results of this study.
Bioceramics are sealers that are recently used in endodontic treatment, with a restricted literature concerning its physical and chemical properties.These calcium silicate sealers have been claimed to be excellent in endodontic obturation with an alkaline pH and solubility.An in vitro study conducted in 2015 revealed that the solubility of Smartpaste Bio promotes alkalinity and calcium release superior to that with a MTA-based sealer after setting time. 40irefrigent granulations were present in all tissues (100%) at initial time periods, and they decreased over time in the Smartpaste Bio group.This is probably related to a reduction in calcium release after day 30, but the mineralized structures stained by Von Kossa were positive for all time periods.
Even with the reformulation, Acroseal maintained biocompatibility, inducing a low inflammatory response and no mineralized areas were observed in any time period, similar to previous results. 34n a calcium release study, Sealapex presented high calcium and hydroxyl release, particularly after longer time intervals, which were verified in this in vivo study by the presence of birefrigent granulations under PL from day 7 to day 60, and biomineralization at all time periods. 36revious studies with the original formulation of Sealapex presented mild inflammation during initial periods, which reduced over time. 37Analyzing the new formulation of Sealapex, inflammatory infiltrate increased at day 14. 38and at day 90 of the experimental period, 15 suggesting that the alterations might have affected tissue compatibility.The results in this research reveal a moderate initial inflammatory response, decreasing after day 30.

Conclusion
At the end of the experiment, all tested sealers were biocompatible, with a statistical difference observed with Smartpaste Bio at day 15.All sealers induced biomineralization, except for Acroseal.Further studies are necessary to better analyze the behavior of this bioceramic material and its physicochemical properties and to confirm the findings of the present study.

Table 1 .
Percentage of samples in each group categorized according to the inflammatory score and the rating thickness of fibrous capsule.

Table 2 .
Percentage of samples in each group categorized according to Von Kossa positive to mineralization and presence of birefrigents crystals under polarized light.