Abstract

Connective tissue response and push-out bond strengths of a new (mineral trioxide aggregate) MTA based endodontic sealer were evaluated.Polyethylene tubes containing ProRoot ES and Endosequence BC sealers were subcutaneously implanted in 45 rats. After 3, 7, 30 days the animals were euthanized, and the specimens were evaluated histologically. Bond strengths of the sealers to root dentine were measured using a push-out bond strength test.Scores of the inflammatory tissue reaction for ProRoot® ES were significantly lower than BC sealer group at 3 days with a thinner fibrous capsule at 30 days. Also, they were not significantly different from the control group. The push-out bond strength of ProRoot® ES could not be evaluated due to handling problems, nevertheless the measured Endosequence BC bond strengths were within an acceptable range.ProRoot® ES had better biocompatibility, seen as more favorable tissue response, than the BC Sealer, although the latter had improved handling characteristics.

1. Introduction

A significant objective of the obturation stage in endodontic therapy is to provide a hermetic seal of the prepared root canal and its accessory spaces ¹. Endodontic sealers, along with core filling materials like guttapercha, have an important role for a successful root canal treatment. According to Grossman, an ideal endodontic sealer should provide a bacteria resistant seal with strong bonding to canal walls, possess a reasonable working time, exhibit low solubility in oral and tissue fluids and show a strong antimicrobial influence ². Since these materials may contact periapical tissues directly through their extrusion from the apex or indirectly due to leach out of their degradation products, their cytotoxicity may affect wound healing or result in cellular degeneration ³. Therefore, the root canal sealer’s biocompatibility remains a fundamental concern for choosing a suitable sealer.

ProRoot ES Endo Root Canal Sealer (Dentsply, Tulsa Dental, Tulsa, OK, USA) is a recently introduced mineral trioxide aggregate (MTA) based sealer manufactured from an enhanced formula of ProRoot MTA root repair material. MTAs are bioactive calcium-silicate hydraulic materials whose structure is mainly based on Portland cement constituents ⁴. They have been used in endodontic applications such as apexogenesis, pulp capping and as root repair materials due to their biocompatibility, bioactivity and sealing ability. These materials can stimulate the precipitation of apatite via a reaction between the calcium they release and the phosphate present in physiologic fluids ⁵. ProRoot MTA root repair materials are mainly composed of tri-calcium silicate, di-calcium silicate, and bismuth-oxide as a radiopacifier ⁶. ProRoot ES sealers are supplied as a powder and liquid system in which the powder is described to have a finer particle size that may contribute to a higher calcium ion release and an increased pH level ⁷. The liquid is a water based gel, formed of a viscid solution of water-soluble polymers. Mixing the powder and liquid yields a colloidal gel that hardens to form an impermeable barrier against microleakage.

EndoSequence BC Sealer (Brasseler, Savannah, GA, USA) is also a calcium-silicate based bioceramic material that is supplied as a readily mixed injectable paste. It consists of tricalcium silicate, dicalcium silicate, calcium phosphate, zirconium oxide, colloidal silica, and calcium hydroxide ⁸. The presence of water is essential for the hardening of this material, hence it utilizes dentin water content for its setting reaction ⁹. It shows powerful antimicrobial effect, biocompatibility, and osteoconductivity. Moreover, this sealer can produce hydroxyapatite during its setting resulting in a chemical bond with the dentin ¹⁰.

One of the most important causes of failure of root canal treatment is the apical and coronal escape of bacteria and their byproducts in a leaking endodontically treated root canal ¹¹. An ideal endodontic sealer should strongly cling to the root dentinal walls to fill in the spaces between dentin and filling materials through which percolation of fluids and bacteria may occur. A strong bond also ensures the stabilization of the apical seal during subsequent manipulation ¹². To determine the efficiency of bond between endodontic materials and dentin, bond strength methods such as the push out test, which utilizes dentin cylinders filled with sealers, have been used ¹³.

The aim of this study was to assess the biocompatibility and dentin bond strength of ProRoot ES sealer compared to Endosequence BC sealer. The following null hypotheses were tested:

1. There is no difference in the connective tissue reactions caused by subcutaneous implants of the tested materials in rats.

2. There is no difference in dentin bond strength between the tested materials.

2. Materials and Methods

The study was conducted in accordance with the guidelines of Mansoura University Research Ethics Committee (no. 11131118) according to NIH guide for the care and use of laboratory animals, Eighth edition 2011.

Forty-five adult male Sprague Dawley rats weighing between 200g - 250g were divided into three groups, fifteen animals each.

Control group: received empty polyethylene tubes (10 mm length x 3mm diameter).

Group 1: received polyethylene tube filled with ProRoot ES.

Group 2: received polyethylene tube filled with Endosequence BC Sealer.

The animals were intramuscularly injected with xylazine hydrochloride 0.33 mL/100 g (Bayer, Leverkusen, Germany) and zolazepam (Virbac SA, Carros, France). The dorsal fur was shaved, and the skin was disinfected then incised and divulged to insert the testing materials.

Each tube had one end closed with heat to prevent the sealer from extruding. The sealers were injected into the sterile tubes, then inserted into the rats’ dorsal subcutaneous tissue. The incisions were stitched by a 5-0 Vicryl suture material (Johnson & Johnson, Belgium).

Five animals from each group were sacrificed by overdose anesthesia at 3,7 and 30 days. Tissue biopsies containing the implanted tubes (with one cm safety margins) were excised and fixed in 4 % formaldehyde for 24 h. After removal of the tubes, the tissues were fixed in paraffin blocks and processed for histological examination using hematoxylin-eosin stains.

Quantitative assessments of inflammatory cells were done at ×400 magnifications, and the inflammatory reaction was scored as previously described ¹⁴;

0 - none or few inflammatory cells and no reaction;

1- less than 25 cells and mild reaction;

2- between 25 - 125 cells and moderate reaction;

3- equal or more than 125 cells and severe reaction.

Statistical methods

Data presented in mean and standard deviation and evaluated using (SPSS, Inc., Chicago, IL, USA) version 23. For comparison of different groups, one way Analysis of variance (ANOVA) and tukey were used. Two-way ANOVA was used to detect the effect of groups & time on the number of inflammatory cells. P value less than 0.05 was considered statistically significant.

Twenty human extracted for orthodontic purposes with mature single were selected from clinic, Faculty of Dentistry, Mansoura University. Only sound teeth with straight roots and a completely formed apex were chosen, while, defected roots were excluded. A radiograph was taken to exclude the presence of other canals and any other anatomical complexities. The roots were prepared using step-back technique up to master apical file #40. Root canals were irrigated after each file using 2 mL 5.25% sodium hypochlorite in a syringe with a 30-gage side vented needle (ProRinse, DENTSPLY, Tulsa Dental Specialties, TN, USA). To remove the smear layer, irrigation of each canal consisted of 2 mL EDTA (17%) (Inter Med-Vista Dental. Racine, WI, USA), which remained in the canal for 1 min, followed by 5 mL NaOCl (2.5%). All canals received a final rinse with 2 mL normal saline and then dried using paper points before receiving the sealers.

For the first group, all teeth were filled with "EndoSquence BC sealer using the BC tip and condensed incrementally a plugger and paper points under magnification. After obturation, teeth were radiographed to make sure their canals are fully-filled. For the second group, however, it was noticed that the ProRoot ES sealer was remarkably difficult in manipulation after mixing, although a syringe was used as a tribunal to facilitate injection into canals. After the material was injected and during condensation, it got pulled out during outward movement because of its high viscosity and stickiness, this prevented the canals from being fully obturated. All teeth were stored for one week at 37°C with 100% humidity to allow sealers to fully set.

Each tooth was poured in acrylic blocks and horizontally sectioned into 1 mm thick slices using a low speed diamond disk under water-cooling. Push-out bond strength test was performed using a universal testing machine ¹⁵. The load was applied in an apico-coronal direction at a crosshead speed of 0.5 mm/min. The test could not be done for the second group (the ProRoot ES) due to the incomplete filling of the canals.

The push-out bond strength was calculated using the formula:

Bond strength in MPa = maximum load in N / adhesion area in mm²

The adhesion area was gauged by using the formula (1):

(1)

Where =3.14, r₁, r₂ are the coronal and apical radii respectively, and h is the slice’ thickness

3. Results

A moderate inflammatory reaction was observed in all of the three groups (Figure 1). The tissues were less organized and infiltrated with remnants of neutrophils, lymphocytes and macrophages, however no areas of necrosis were detected. The score of the inflammatory reaction in ProRoot ES sealer group was significantly lower than that of EndoSquence BC group but not significantly different from the control group (Table 1).

The tissue began to be more organized with the beginning of the appearance of collagen fibers and fibroblast cells in between (Figure 2). The inflammatory cells were mostly lymphocytes with remnants of neutrophils and macrophages. Multinucleated giant cells were also spotted in specimens of the Endosequence BC. The Endosequence BC group showed significantly higher amounts of inflammatory cells relative to the control group, and was also higher than the ProRoot ES group, but not significant. The intensity of inflammation significantly decreased compared to that at 3 days in both the control and Endosequence BC groups, however, the decrease in the ProRoot ES group was not significant. The number of inflammatory cells in specimens of ProRoot ES group was not significantly different from the control or the Endosequence BC groups (Table 2).

The tissue was more organized showing a milder inflammatory reaction with fewer lymphocytes (Figure 3). The number of inflammatory cells significantly decreased in all of the groups compared to their numbers at 3 and 7 days (Table 2). Fibrous capsules were formed with collagen fibers and fibroblast cells in between in all of the specimens. The capsule observed in the ProRoot ES group was significantly thinner than that observed in Endosequence BC group which had more densely packed collagen fibers (Table 3). There was no significant difference between the scores of the inflammatory reactions for the three groups after 30 days. Additionally, there was no significant difference between the fibrous capsule thickness in control group and ProRoot ES group.

Means of push-out bond strength of EndoSquence BC sealer group in was 6.33, 6.5 and 5,17MPa for apical, middle and coronal sections respectively (Table 4).

4. Discussion

Materials used for root canal filling are critical to successful endodontic treatment. An important characteristic is biocompatibility which is the capability of the material to act with an appropriate host tissue response ¹⁶. In vivo tests are founded on clinical and histologic evaluation especially for the inflammatory response ¹⁶. Subcutaneous and intraosseous implantation have been widely accepted as one of the most dependable techniques that can be implemented in animal model studies ¹⁷. In this study, polyethylene tubes were utilized as they maintain the tested materials in contact with tissues in a controlled manner ¹⁸.

Both the Endosequence BC and the ProRoot ES sealers groups showed moderate inflammatory reactions with predominance of chronic inflammatory cells at days 3 and 7 that significantly decreased to a milder reaction at day 30 during the study. This was in accordance with the results of a study by Khalil et al. ¹⁹ who found that MTA and Endosequence materials provoked initial moderate to severe inflammatory reactions at 7 days that were significantly reduced by 30 days. The initial inflammatory reactionobserved on the third day in all of the tested groups may have been due to the surgical procedures and the tissue reaction to receiving a subcutaneous implant ²⁰. However, the inflammatory response was significantly higher with the Endosequence BC sealer than with ProRoot ES sealer at 3 days. The inflammation also extended deep in the subcutaneous tissue and was not limited to the end of the tube. Multinucleated giant cells were noted in the Endosequence specimens at 7 days and absent in those of ProRoot ES which means that the Endosequence induced a more irritable effect, which led us to reject the first null hypothesis. This was in agreement with Taha NA et al. who noted the presence of more inflammation and multinucleated giant cells in Endosequence specimens compared with specimens of MTA material ²¹. On the other hand, these results were in contrast with the study by Khalil et al. which reported a stronger inflammatory reaction to MTA repair material ¹⁹. Other studies showed longer tissue reactions up to 60 days after MTA materials implantation ^{22, 23}.

Throughout the study, the intensity of the inflammation in ProRoot ES group was not significantly different from the control group. When MTA comes into contact with soft tissue, it dissolves into calcium hydroxide, which has a high pH. This alkaline pH can promote apical obliteration with calcified tissue formation. It also interferes with osteoclastic activity and encourages alkalization of the adjacent tissue, which helps healing and explains the more favorable effect of the sealer ²⁴.

The fibrous capsule formation started early at 7 days in the Endosequence group till reaching a well formed mature one at 30 days. In the ProRoot ES group, the collagen fibers of the capsule were significantly thinner and not so packed at 30 days. The increase of the fibrous tissue capsule thickness may be accredited to a stronger inflammatory reaction and to the effect of mast cells on fibroblast proliferation and the fibrosis activity as a part of the inflammatory process. Mussel et al ²⁵ and Shahiet al ²⁶, suggested that the quality and thickness of the fibrous capsule around a material is inversely related to the biocompatibility of that material and is an indication of inflammation. Whereas, others suggested that the fibrous capsule deposition around an implanted material is a sign of tissue tolerance ²⁷. Undeniably, all dental materials produce some irritation once they interact with the living tissues ²⁸.

The second null hypothesis could not be verified as the ProRoot ES Sealer showed great limitation during canal filling, that prevented bond-strength measurements. This raises questions about its clinical feasibility and further sustainability. On the Other hand, Endosequence BC Sealer was much easier in handling and its mean bond-strength value was within the accepted range [29,30] ^29,

5. Conclusions

ProRoot ES Sealer has better biocompatibility by having favorable tissue cellular responses and thinner fibrous capsule, when surgically implanted into the subcutaneous tissue of rats, than the Endo sequence BC Sealer, although the later has much improved handling and manipulation characteristics.

Conflict of Interest

The authors deny any conflicts of interest in connection with this article.

References