Assessment of the biocompatibility of Epiphany root canal sealer in rat subcutaneous tissues

Objective. The aim of this study was to evaluate the biocompatibility of the root canal sealer Epiphany in rat subcutaneous tissues.

Study design. Polyethylene tubes were filled with the sealer (I: Epiphany; II: photoactivated Epiphany; III: Epiphany associated with self-etch primer; IV: photoactivated Epiphany associated with primer; and V: control group) and later implanted into 4 different regions of the dorsum of 15 adult male rats (Rattus novergicus, Albinus Wistar). After 7, 21, and 42 days, 5 animals were killed, obtaining 4 samples per group, in addition to the control group, at each analyzed time.

Results. In all periods, Epiphany induced a mild inflammatory reaction. However, in group II, in which the primer was not used, extensive necrosis and a moderate to intense inflammatory reaction were observed, mainly after 7 and 21 days.

Conclusion. Epiphany sealer appears biocompatible when tested on rat subcutaneous tissues.

Several studies have been conducted to assess the biocompatibility of root canal sealers, essential for ensuring their good performance and successful endodontic treatment. To evaluate the biologic response and the irritant potential of new endodontic materials introduced to the market, preliminary studies using in vivo experimental methods, such as implanting these materials in the connective tissue of laboratory animals, are commonly performed.

Among the new root canal obturation systems recently introduced is Resilon-Epiphany. Resilon is a synthetic polycaprolactone polymer, based on a polyester-based polymer that contains dimethacrylates, which can bond to methacrylate-based resin sealers, such as Epiphany, a resin-based dual-cure root canal sealer. According to the manufacturer, Resilon also contains bioactive glass, calcium hydroxide, and radiopaque filler. Some investigators have reported greater sealing capacity of this system than other materials and that it is associated with less apical periodontitis. However, in another study the interfacial strength obtained with this system to intraradicular dentin was not superior to that of gutta-percha associated with an epoxy-resin sealer. The Resilon-Epiphany system is also followed by a self-etch primer that contains sulfonic acid–terminated functional monomer, HEMA, water, and polymerization initiator. The use of Resilon associated with the Epiphany sealer provides one of the main characteristics of this system, which has been suggested to have the capacity to produce a bonded monoblock filling.

Although the Epiphany root canal sealer has been approved for endodontic use by the Food and Drug Administration (FDA) in the USA, there are very few published studies evaluating its biocompatibility and cytotoxicity and its association with the self-etch primer that accompanies it. Therefore, the aim of the present study was to evaluate the biocompatibility of the Epiphany root canal sealer, either associated with the primer or not, in rat subcutaneous connective tissues and to compare the results with those of previous studies.

Material and methods

This study was evaluated and approved by the Animal Research Ethics Committee of the University of Ribeirao Preto, Sao Paulo, Brazil, and the ethical concepts for use of laboratory animals were observed in all phases of the experiment. The material assessed was the Epiphany root canal sealer (Pentron-Clinical Technologies, Wallingford, CT), which was associated or not with self-etch primer. According to this association of the tested sealer, the following 4 experimental groups (and 1 control group) were established: group I: Epiphany; group II: photoactivated Epiphany; group III: Epiphany associated with self-etch primer; group IV: photoactivated Epiphany associated with self-etch primer; and group V: control, with empty tube.

For the study, 15 male adult rats (Rattus novergicus, Albinus Wistar) weighing between 200 and 250 g were used. They were kept in an acclimatized room and received a balanced diet and water ad libitum. The animals were anesthetized by intramuscular injection of ketamine chlorhydrate (0.1 mg/mL) associated with xylazine (0.05 mg/mL), followed by shaving of dorsal fur, disinfection, incision, and dissection of the subcutaneous tissue to insert the test material. To implant the root canal sealer into the rat subcutaneous tissues, sterilized polyethylene tubes 1.2 mm in diameter (0.8 mm internal diameter) and 10 mm long were used. To prevent the sealer from escaping, one of the ends of the tubes was closed by heat. The Epiphany root canal sealer was manipulated in accordance with the manufacturer’s recommendation for clinical use, in a dark room, because it is a dual-cure sealer. In groups III and IV, a brush was used to coat the internal surface of the polyethylene tubes with the primer supplied with the sealer. The manipulated sealer was carefully put into polyethylene tubes with aid of a paper cone compatible with the diameter of tubes, ensuring that there were no empty spaces and that the sealer did not overflow. Next, the sealer in the tubes was light activated (groups II and IV) and immediately implanted in the subcutaneous tissue in the rat dorsa. The surgical wounds were sutured.

Each animal received 4 tubes with material, 2 being in the scapular region and 2 in the pelvic region (one on the right side and the other on the left), each tube representing a distinct group. After 7, 21, and 42 days, the rats were killed by anesthetic overdose and the tubes removed for histologic analysis. Thus, there were 5 rats for each study period, 4 of them representing the experimental groups and 1 the control group (group V), in total, 4 samples for each experimental group in each of the periods. The specimens were fixed in 10% buffered formalin solution for 24 h and processed for conventional histologic examination.

The connective tissue adjacent to the open end of each tube was sectioned at a microtome setting of 5 µm, stained with hematoxylin and eosin, and evaluated for the presence or absence of neutrophilic leukocytes, macrophages, lymphocytes, plasma cells, giant foreign body cells, dispersed material, and necrotic tissue. Each criteria evaluated as absent (—), slight (+), moderate (++) and intense (+++). Depending on these features, except the presence of dispersed material, the inflammatory reaction of the connective tissue was classified as none to slight (grade I), moderate (grade II), and severe (grade III), according to criteria described by Shahi et al.

Results

In addition to the 1 specimen used as control in each study period, the connective tissue response along the lateral wall outside the polyethylene tubes of all specimens served as a negative control for the technique. It was possible to observe a thin layer of connective tissue, without inflammatory reaction, between the tube wall and the adjacent tissue in all specimens, in all study periods. The connective tissue adjacent to the open end of the polyethylene tube was evaluated, and a descriptive analysis of the histologic findings was made. The data obtained are summarized in Table I.

Seven-day results

Group I (Epiphany). In the evaluated area, a layer of cellularized connective tissue that presented slight chronic inflammatory reaction, predominantly formed by lymphocytes and hyperemic blood vessels, was observed (Fig. 1). Scarce neutrophilic leukocytes and small focus of necrosis were also noted. No macrophages or foreign body multinucleated giant cells were observed. In addition, there was dispersed material characterized by an amorphous, diffuse, granular, black-colored material.

Group II (photoactivated Epiphany). After 7 days, the subcutaneous tissue in this group presented moderate to intense chronic inflammatory reaction, with extensive necrosis (Fig. 2). No phagocytic activity was observed.

Group III (Epiphany associated with primer). Histologic features were similar to those found in group I (Epiphany).

Group IV (photoactivated Epiphany associated with primer). Histologic features were similar to those found in group I (Epiphany).

21-day results

Group I (Epiphany). In the connective tissue adjacent to the open end of the tube, there was a mild chronic inflammatory reaction, predominantly lymphocytic. No foreign body multinucleated giant cells and macrophages were observed. Dispersed material was found in great quantity. Scarce foci of necrosis were also noted.

Group II (photoactivated Epiphany). In contrast with all the other groups of this same period, the connective tissue adjacent to the root canal sealer presented an intense chronic inflammatory reaction, associated with several hyperemic blood vessels. Extensive necrosis was observed next to the dispersed material, which was seen in abundance.

Group III (Epiphany associated with primer). The connective tissue located at the open end of the tube showed a mild chronic inflammation, predominantly formed by lymphocytes. Foreign body giant cells and macrophages were absent. Residual dispersed material was noted in great quantity. No necrosis or any degenerative changes were observed.

Group IV (photoactivated Epiphany associated with primer). This group presented histologic features similar to group I in this same period, with mild chronic inflammation, small foci of necrosis, and residual dispersed material in extensive areas (Fig. 3).

42-day results

Group I (Epiphany). In this group, there was connective tissue that presented a mild to absent chronic inflammatory reaction, with little residual dispersed sealer. There was no necrosis or any other changes.

Group II (photoactivated Epiphany). Differing from the features found in the group I in this same period, there was a moderate chronic inflammation, associated with a few congested blood vessels. Foci of necrosis were still observed adjacent to residual dispersed sealer.

Group III (Epiphany associated with primer). There was a slight to absent chronic inflammatory reaction, without foreign body giant cells or macrophages. Some dispersed material was observed. Similar to group I in this same period, no necrosis or other degenerative changes were observed.

Group IV (photoactivated Epiphany associated with primer). The histologic features were similar to group III in this same period.

Discussion

Gutta-percha has been the most commonly used material for obturating the root canal system, although it is not considered to be the ideal root canal filling material. One of its disadvantages is the need to use a root canal sealer with good sealing properties. Moreover, when the coronal restoration is inadequate or absent, saliva may dissolve the root canal sealer, leading to reinfection and consequently to unsuccessful endodontic treatment. Among the root canal sealers now used in dental practice, there are some composed of calcium hydroxide, glass ionomer, zinc oxide– eugenol, and silicones, and some are resin based.

Recently, a new root canal obturation system was introduced to the market and has become popular among endodontic and general dentists. This system is composed of a synthetic polycaprolactone polymer, Resilon, associated with a resin-based dual-cure root canal sealer, Epiphany. The Resilon-Epiphany system is said to quickly form a “monoblock” filling that bonds to the dentin wall in the root canal. According to the manufacturer, Epiphany is not cytotoxic, is bio-compatible and nonmutagenic, and has been approved by the FDA for endodontic use.

In any root canal filling system the sealer may escape into the periapical region in cases of overfilling. If the sealer causes a foreign body chronic inflammatory reaction, it could induce the persistence of apical periodontitis. Although the manufacturer postulates that Epiphany is biocompatible, there are few studies that evaluated its biocompatibility and cytotoxicity. Furthermore, experiments present conflicting results. Al- though there are no earlier studies evaluating the biocompatibility of the Epiphany root canal sealer associated with self-etch primer, a recent study assessed the cytotoxicity of the primer only. In the present study, the results obtained in most of the groups showed a good biologic response. Some authors have reported similar findings, but they did not use the self-etch primer. The root canal sealer Epiphany has a high calcium release. This feature causes a more alkaline pH of the tissue site, which may favor tissue repair. Other authors, however, observed that Epiphany is more cytotoxic than other conventional sealers. In the present study the biologic response was less favorable in group II (photoactivated Epiphany) compared with the other groups: the connective tissue adjacent to the material showed moderate to intense chronic inflammatory reaction and extensive necrosis after 7 and 21 days. This intense inflammation decreased after 42 days. The sealer was light activated in this group, but the self-etch primer was not used. Under this condition, Epiphany could eventually present irritant reaction products, possibly free monomers, at the surface in contact with the connective tissue. Thus, it appears that when the primer is not used, these possibly toxic reaction products promote a less favorable biologic response.

In spite of the good results obtained in most groups in the present study, there was severe tissue irritation observed in group II. Because few studies have been conducted so far, further research is required to fully assess the biologic response and the cytotoxicity of this sealer. From a biologic standpoint, the response after the use of the self-etch primer requires further investigations.

Authors: Mônica Maria Denadae de Campos-Pinto, Danilo Alessandro de Oliveira, Marco Aurélio Versiani, Yara Teresinha Correa Silva-Sousa, Manoel Damião de Sousa-Neto, Danyel Elias da Cruz Perez

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