Biocompatibility and immunolabeling of fibronectin and tenascin of resinous root canal sealers.

Aims: The aim of this study was to evaluate the biocompatibility of resinous root canal sealers: Sealer 26, AH plus, and SK Seal Root Canal Sealer in the subcutaneous tissue of rats. Subjects and
Methods: Twenty-four Wistar rats received polyethylene tubes containing the sealers and empty tubes as control (n = 6). After 7, 15, 30, and 60 days, animals were killed and polyethylene tubes were removed with the surrounding tissues. The specimens were embedded in paraffin, processed for hematoxylin-eosin and immunohistochemistry assessed for fibronectin (FN) and tenascin (TN). Statistical Analysis Used: Data were tabulated and analyzed via Kruskal-Wallis and Dunn's test (P < 0.05).
Results: All groups induced a moderate inflammatory reaction after 7 and 15 days (P > 0.05); after 30 days, a mild inflammatory infiltrate was observed in control groups, and moderate in sealers groups (P > 0.05); all groups showed mild inflammatory infiltrate at 60 days (P > 0.05). Overall, the fibrous capsule was considered thick only on the 7th day and became thin over time. All groups had expression for FN and TN in all analyzed periods, with high immunolabeling in sealers groups when comparing with the control group (P < 0.05).
Conclusion: All sealers demonstrated biocompatibility and induced FN and TN expression. Copyright:

INTRODUCTION

The success of endodontic therapy is directly related to infection control by cleaning, shaping and filling of the root canal system.[1] The obturation phase completes the treatment and must be performed associating gutta-percha and an endodontic sealer, to reduce gaps between the filler material and root canal walls.[123] The aim is to fill anatomically difficult access areas, preventing or minimizing infiltration or even sealing remaining microorganisms.[123]

There is a diversity of endodontic sealers available and their main components – such as zinc oxide and eugenol, glass ionomer, epoxy resinous, calcium hydroxide, among others – are responsible for the biological and physicochemical properties of the material.[4] Among these properties, an ideal endodontic sealer should be biocompatible, due to the direct contact with periapical tissues.[4] In addition, if the material induces biomineralization, the repair process is also improved.[5]

Schröeder introduced resinous endodontic sealers, and their long use is related to the excellent adhesion to root canal walls and marginal sealing ability, reducing apical and coronary infiltration.[6] The Sealer 26 (Dentsply, RJ, Brazil) is a resinous endodontic sealer containing 37% of calcium hydroxide in the powder composition, while its paste has epoxy bisphenol resinous.[7] The great advantage of this sealer is that besides inducing low cytotoxicity,[7] it has the ability to induce repair through the deposition of mineralized tissue,[8] which is not observed in other resinous sealers.[9] In addition, studies have shown that Sealer 26 prevents bacterial infiltration.[8]

AH Plus (Dentsply, OK, USA), an epoxy resin-based sealer, has been considered the gold standard by its satisfactory physico-chemical properties,[10] sealing ability,[11] and antimicrobial activity.[12] However, some studies report its cytotoxicity,[13] which is related to the release of small amounts of formaldehyde or through the amine and epoxy resinous components.[14]

Endodontic sealers with different compositions are in constant development, with the objective of improving physicochemical and biological properties, aiming the ideal sealer. The SK Seal Root Canal Sealer (SK Seal; Skada Limited, Harrow, UK) is a resinous sealer available in the double syringe. The desirable characteristics of resinous endodontic sealers, such as low solubility, suitable flow, long-term dimensional stability, satisfactory bond strength with dentin, and seal ability.[151617] were added to this sealer, along with calcium oxide, to promote enhanced repair.[8] The base paste consists of the epoxy oligomer, ethylene glycol, bismuth subcarbonate, calcium, zirconium phosphate, and calcium oxide. The catalyst paste contains polyaminobenzoate, triethanolamine, calcium phosphate, bismuth subcarbonate, zirconium oxide, and calcium oxide. The syringe automatically provides the two pastes in the ratio of 2:1, facilitating the manipulation. According to the manufacturer, this sealer follows the requirements for root canals sealers with sealing capacity, not staining dental structure, insoluble in liquids, and excellent radiopacity. However, there are no studies investigating the biological properties of SK Seal Root Canal Sealer.

The biocompatibility evaluation of endodontic sealers, are usually performed in the subcutaneous tissue of rats,[46918] which are limited to histological aspects of tissue reactions induced by the material's components and studies on the expression of extracellular matrix proteins during tissue repair are scarce. In this repair process occurs the participation of two glycoproteins present in the extracellular matrix: Tenascin (TN) and fibronectin (FN). TN has antiadhesive activity, which, along with the FN adhesiveness and scattering activity, allows cellular movement,[19] and promotes healing.[20] FN regulates adhesion, migration, and cells differentiation.[21] Furthermore, FN facilitates platelet aggregation through its deposition on collagen and/or fibrin,[22] also contributing to healing.[19] In addition, both extracellular matrix components stimulate the formation of mineralized tissue.[2324] Both glycoproteins have already been evaluated during pulp repair induced by Ca(OH)2[24] and retrofilling materials as ProRoot MTA and Portland cement.[25] However, to date, the expression of these proteins has not yet been analyzed in endodontic sealers AH Plus, Sealer 26 and SK Seal Root Canal Sealer.

Therefore, this study evaluated the tissue response of SK Seal, Sealer 26, and AH Plus sealers regarding biocompatibility and immunolabeling of TN and FN, in rats' subcutaneous tissue. The null hypothesis was that these sealers would exhibit no differences in biocompatibility and immunolabeling.

SUBJECTS AND METHODS

Twenty-four male 4–6-month-old Wistar rats 250–280 g were used in the study. The sample size was established based on a previous study involving the analysis of materials in the subcutaneous tissue of the rats.[4] The animals were housed in a temperature-controlled environment (22°C ± 1°C, 70% humidity) with a 12-h light-dark cycle and received water and food “ad libitum.” All experimental procedures of this study were approved by the institutional Ethics Committee on the Use of Animals (CEUA protocol 2015-00452) at UNESP-Universidade Estadual Paulista (São Paulo, Brazil) in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD, USA).

Subcutaneous implant

Seventy-two polyethylene tubes (Abbott Laboratories of Brazil, SP, Brazil) with a 1.0 mm internal diameter, 1.6 mm external diameter, and 10.0 mm length were filled with the sealers (Sealer 26, AH Plus, and SK Seal), which were manipulated according to the manufacturer recommendations and inserted into the tubes with a syringe. Twenty-four empty tubes were used as control.[9]

After administration of intramuscular anesthesia with xylazine (10 mg/kg; Rhobifarma Indústria Farmacêutica Ltda, São Paulo, Brazil) and ketamine (80 mg/kg; União Química Farmacêutica Nacional S/A, São Paulo, Brazil), the back of the animals were shaved, antisepsis was achieved with 5% iodine solution, and a 2.0 cm incision was performed in a head-to-tail orientation with #15 Bard-Parker blade (Becton-Dickinson, Franklin Lakes, NJ), creating two pockets on each side of the incision. Three polyethylene tubes containing the sealers and an empty tube were randomly 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.

Histologic analysis

After the periods of 7, 15, 30, and 60 days, the animals were euthanized by an anesthetic overdose. The polyethylene tubes were removed with the surrounding tissue and fixed in 10% buffered formalin at pH 7.0. The specimens were embedded in paraffin, serially cut into 5 μm sections, and stained with hematoxylin-eosin or submitted to immunohistochemistry by using an indirect immunoperoxidase technique for FN (primary antibody rabbit, SC-9068, Santa Cruz Biotechnology, CA, USA) and TN (primary antibody rabbit, SC-20932, Santa Cruz Biotechnology, CA, USA). The specimens were also submitted to the procedures suppressing the use of primary antibodies to the negative control.

The inflammatory infiltrate at the open end of the tubes was scored according to previous studies[49] as follows: 0, few inflammatory cells or no reaction; 1, <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. Immunolabeling for FN and TN was defined as the presence of brownish color in the extracellular matrix. The criteria for establishing the adopted scores were 0, absence of immunolabeling; 1, low immunolabeling standard; 2, moderate immunolabeling standard; and 3, high immunolabeling standard (×1000 ).

Statistical analysis

Data were statistically analyzed by Kruskal–Wallis and Dunn's test. The P value was considered significant at 5%.

RESULTS

Group control

Moderate chronic inflammatory reaction was observed after 7 and 15 days [Figure 1A and B]. Inflammatory cells, such as lymphocytes and macrophages, were present in the fibrous capsule, classified as thick. On days 30 and 60 [Figure 1C and D], a mild inflammatory infiltrate was observed in a thin fibrous capsule. Regarding the analysis of FN and TN, this group has low immunolabeling in all periods [Figure 2A-D; a-d].

Figure 1

Representative images of tissue reaction of groups. Control Group: (A-D) (days 7, 15, 30 and 60, H and E, ×100); AH Plus: (E-H) (days 7, 15, 30 and 60, H and E, ×100); Sealer 26: I-L (days 7, 15, 30 and 60, H and E, ×100) and SK Seal Root Canal Sealer: M-P (days 7, 15, 30 and 60, H and E, ×100)

Figure 2

Representative images of immunolabeling for fibronectin (A-P) and tenascin (a-p). Control Group: A-D; a-d (days 7, 15, 30 and 60); AH Plus: E-H; e-h (days 7, 15, 30 and 60); Sealer 26: I-L; i-l (days 7, 15, 30 and 60); SK Seal Root Canal Sealer: M-P; m-p (days 7, 15, 30 and 60, ×1000)

AH plus

On days 7, 15, and 30, a moderate inflammatory reaction was observed, with the presence of macrophages and lymphocytes, reducing until day 60, where shows mild inflammatory infiltrate. The fibrous capsule was considered thick until 15 days, reducing after 30 days and became thin by the end of the experiment [Figure 1E-H]. In immunohistochemistry analysis, a moderate standard of immunolabeling was observed at all periods for both markers [Figure 2E-H; e-h].

Sealer 26

Moderate inflammatory reaction was observed on days 7, 15, and 30 [Figure 1I-K], exhibiting lymphocytes and macrophages. On days 7 and 15, the fibrous capsule was considered thick. After 30 days, inflammation decreased, and the fibrous capsule became thin. On day 60, the inflammatory reaction became mild [Figure 1L].

In the immunohistochemistry analysis, on days 7, 30, and 60, there was moderate immunolabeling of FN; on days 15, this sealer has moderate to high immunolabeling [Figure 2I-L]. For TN, moderate immunolabeling was present throughout all periods [Figure 2i-l].

SK seal root canal sealer

After 7 days, a mild inflammatory reaction was present in half of the specimens; the other had moderate-to-severe inflammatory reaction. On days 15 and 30, a mild-to-moderate inflammatory reaction was present, with a predominance of lymphocytes and macrophages. On day 60, there was a reduction of the inflammatory reaction. The fibrous capsule was considered thin in all periods [Figure 1M-P].

On days 7, 30, and 60, there was moderate immunolabeling for FN; on day 15, moderate-to-high immunolabeling were detected [Figure 1M-P]. This response was similar for TN on day 7; on day 15, there was moderate immunolabeling for this protein, decreasing over time [Figure 2m-p].

Comparison among groups

Data were compared for each analysis period as shown in Tables 1 and 2. On the 7th day, only specimens of SK Seal had a mild inflammation, but with no significant difference when compared to other groups (P > 0.05). On day 15, the specimens of Sealer 26 showed moderate inflammation, as well as most of the specimens of the groups control and AH Plus, while SK Seal showed mild and moderate inflammation, with no significant difference among groups (P > 0.05). At 30 days, the control group had mild inflammation, SK Seal had mild and moderate inflammation, and others sealers, moderate inflammation in most of the specimens. After 60 days, all sealers showed mainly mild inflammation, with the same specimens showing the absence of inflammation. SK Seal showed a thin fibrous capsule in all periods, and the other groups showed fibrous capsules initially thick and became thin over time.

Table 1

Percentage of samples in each group according to the inflammation score and fibrous capsule thickness

Groups	Scores					P	Capsule
	0	1	2	3	Med
7 days
Control	0	0	6	0	2a	0.601	Thick
AH plus	0	0	6	0	2a		Thick
Sealer 26	0	2	4	0	2a		Thick
Sk Seal	0	3	1	2	1a		Thin
15 days
Control	0	1	5	0	2a	0.235	Thick
AH Plus	0	2	4	0	2a		Thin
Sealer 26	0	0	6	0	2a		Thick
Sk Seal	0	3	3	0	1/2a		Thin
30 days
Control	0	6	0	0	1a	0.074	Thin
AH Plus	0	2	4	0	2a		Thin
Sealer 26	0	2	3	1	2a		Thin
Sk Seal	0	3	3	0	1/2a		Thin
60 days
Control	2	4	0	0	1a	0.901	Thin
AH Plus	2	4	0	0	1a		Thin
Sealer 26	2	4	0	0	1a		Thin
Sk Seal	1	5	0	0	1a		Thin

*Different letters in the same column indicate that there is statistical difference between the groups in each analysis period (P<0.05)

Table 2

Percentage of scores attributed to fibronectin and tenascin immunolabeling in each group

Groups	Score fibronectin					P	Score tenascin					P
	0	1	2	3	Med		0	1	2	3	Med
7 days
Control	0	5	1	0	1a	0.001	0	4	2	0	1a	0.006
AH Plus	0	0	5	1	2b		0	0	5	1	2ab
Sealer 26	0	0	6	0	2ab		0	0	6	0	2ab
Sk Seal	0	0	2	0	2ab		0	0	3	3	2/3b
15 days
Control	0	5	1	0	1a	0.005	0	5	1	0	1a	0.005
AH Plus	0	0	4	2	2ab		0	0	4	2	2b
Sealer 26	0	0	3	3	2/3b		0	2	4	0	2ab
Sk Seal	0	0	3	3	2/3b		0	0	4	2	2b
30 days
Control	0	5	1	0	1a	0.002	0	6	0	0	1a	0.002
AH Plus	0	0	6	0	2ab		0	0	4	2	2b
Sealer 26	0	0	5	1	2ab		0	1	5	0	2ab
Sk Seal	0	0	4	2	2b		0	4	2	0	1ab
60 days
Control	0	6	0	0	1a	<0.001	0	5	1	0	1a	0.006
AH Plus	0	0	4	2	2b		0	0	4	2	2b
Sealer 26	0	0	5	1	2b		0	1	5	0	2ab
Sk Seal	0	0	2	0	2ab		0	4	2	0	1ab

*Different letters in the same column indicate that there is statistical difference between the groups in each analysis period (P<0.05)

Regarding immunohistochemistry analysis for FN [Table 2], on days 7, all sealers showed a moderate immunolabeling, with significant difference for AH Plus when compared to the control group (P < 0.05). On day 15, Sealer 26 and SK Seal were difference when compared to the control group (P < 0.05), with a higher immunolabeling. On days 30, all the sealers had moderate immunolabeling, but SK Seal was statistically different compared to the control group (P < 0.05). Sealer 26 and AH Plus had statistical difference, compared with the control group, on days 60 (P < 0.05).

For TN [Table 2], AH Plus had moderate immunolabeling at all periods, with the statistical difference on days 15, 30, and 60 when compared to the control group (P < 0.05). Sealer 26 had moderate immunolabeling in all periods of analysis, without difference with the other groups (P > 0.05). SK Seal had higher immunolabeling compared to the control group on days 7 and 15 (P < 0.05), and mild immunolabeling after (P > 0.05).

DISCUSSION

This study evaluated the biocompatibility and ability of FN and TN expression of resinous endodontic sealers. The null hypothesis was partially accepted since all the materials showed biocompatibility, but there was a difference in protein expression. The moderate inflammatory reactions in all groups during the initial experimental periods subsequently decreased and the fibrous capsule became thin. The empty tubes used as control had similar reactions to the results already reported previously.[49] The initial moderate inflammation in the control group (days 7 and 15) is due the surgical trauma and is considered a normal reaction of the tissue by subcutaneous implantation.[49]

The AH Plus and Sealer 26 showed biocompatibility, corroborating previous studies.[2627] A moderate inflammatory reaction was present in the initial periods of both groups, decreasing over time, along with the capsule around the tube. The most prominent initial inflammation with subsequent reduction observed in the reaction with AH Plus was already expected, as other studies have also pointed to the in vivo biocompatibility of this material.[4]

After 30 days, the moderate inflammatory reaction observed in Sealer 26 group may be explained due to its composition. Although biomineralization was not evaluated in this investigation, the presence of calcium hydroxide [Ca(OH)2] induces mineralization by dissociation of calcium and hydroxyl ions, resulting in elevated pH, activating the alkaline phosphatase.[9] Therefore, the alkalinity can be induces damage on subcutaneous tissue, as observed in previous results of materials with Ca(OH)2in its formulations,[9] followed of decrease over time to a not significant level.

Regarding SK Seal, there are no previous studies to support or contradict our findings, since this may be the first research to assess this sealer. The SK Seal also demonstrated biocompatibility when compared to the other sealers and the control group, showing a moderate to mild tissue response in initial time periods, decreasing until the end of the experiment. According to the manufacturer, the radiopacifier of this sealer is bismuth subcarbonate, the same radiopacifier found in Acroseal (Specialités-Septodont, Saint Maur-des-Fossés, France),[28] which was biocompatible in a previous study.[9] This sealer also has zirconium oxide in its composition, which was related to the inhibition of discoloration normally caused by MTA-based materials.[29] The other sealer components, including epoxy resinous-such as AH Plus-are usually related to the good biocompatibility of materials, but physical-chemical tests need to be performed.

As the dynamics of the extracellular matrix in the repair of the subcutaneous tissue of rats, the expression of glycoproteins FN and TN were evaluated. This study showed that the sealers showed a higher FN expression in relation to the control group, mainly at 15 days for Sealer 26 and SK Seal. For TN, at 7 and 15 days, SK Seal showed a higher immunolabeling in relation to the control group, and AH Plus at 15 up 60 days.

Previous studies reported the adhesive properties of these glycoproteins with direct participation in tissue repair processes.[20] Martinez et al.[30] identified, through immunolabeling technique, the distribution of TN, FN, and type III collagen in the human pulp, and observed a strong expression of TN and FN. Piva et al.[24] studied the expression of FN and TN during pulp repair induced by Ca(OH)2and verified that both extracellular matrix glycoproteins were expressed during the healing process; in addition, TN was evidenced in advanced stages of dentin barrier formation.

The FN glycoprotein was evaluated by Fayazi et al.[25] in response to fibroblasts of the human periodontal ligament with retrofilling materials (ProRoot MTA, Portland cement, and amalgam) and it was observed that after 1 week, Portland cement and MTA groups showed higher expression of FN, but there was no significant difference between these two groups. All studies emphasized the importance of understanding the cells and elements of the extracellular matrix participation in the repair of the involved tissues, when in contact with endodontic materials.

As previously stated, only few studies investigated expression of extracellular matrix proteins during tissue repair in contact with endodontic sealers, which hinders comparing our results on the dynamics of the extracellular conjunctive matrix with scientific literature. Thus, more studies are required to understand the reflexes caused by the root canals sealers when in contact with the extracellular matrix.

CONCLUSION

Endodontic sealer SK Seal was biocompatible as Sealer 26 and AH Plus, and all materials induced FN and TN expression, representing an alternative among resinous root canal sealers. However, since this is the first study evaluating the biological properties of SK Seal, additional research is necessary to confirm the present findings.

Financial support and sponsorship

FAPESP (no 2015/08251-8).

Conflicts of interest

There are no conflicts of interest.