A comparison of the sealing abilities between Biodentine and MTA as root-end filling materials and their effects on bone healing in dogs after periradicular surgery

ABSTRACT Objectives: To compare the sealing ability and biocompatibility of Biodentine with mineral trioxide aggregate (MTA) when used as root-end filling materials. Methodology: The Cell Counting Kit-8 (CCK-8) assay was used to compare the cytotoxicity of MTA and Biodentine. Twenty-one extracted teeth with a single canal were immersed in an acidic silver nitrate solution after root-end filling. Then, the volume and depth of silver nitrate that infiltrated the apical portion of the teeth were analyzed using micro-computed tomography (micro-CT). Seventy-two roots from 3 female beagle dogs were randomly distributed into 3 groups and apical surgery was performed. After six months, the volume of the bone defect surrounding these roots was analyzed using micro-CT. Results: Based on the results of the CCK-8 assay, MTA and Biodentine did not show statistically significant differences in cytotoxicity (P>0.05). The volume and the depth of the infiltrated nitrate solution were greater in the MTA group than in the Biodentine group (P<0.05). The volume of the bone defect was larger in the MTA group than in the Biodentine group. However, the difference was not significant (P>0.05). The volumes of the bone defects in the MTA and Biodentine groups were smaller than the group without any filling materials (P<0.05). Conclusions: MTA and Biodentine exhibited comparable cellular biocompatibility. Biodentine showed a superior sealing ability to MTA in root-end filling. Both Biodentine and MTA promoted periradicular bone healing in beagle dog periradicular surgery models.


Introduction
Root canal treatment is generally performed to treat dental pulp and periapical disease. When the root canal treatment and retreatment fail, periradicular surgery is the last hope for the affected teeth. 1 The aim of periradicular surgery is not simply to remove infected apical tissue or the root tip, but most importantly is to reseal the root canal system. 2 For this purpose, a root-end filling material must be applied to fill the root-end cavity and seal the exposed dentine during periradicular surgery. Hence, this material must possess sealing ability and biocompatibility. In addition, it should have antibacterial qualities and be easy to manipulate. 3 Mineral trioxide aggregate (MTA), due to its superior characteristics compared to other traditional root-end filling materials such as Amalgam, guttapercha and glass ionomer cement, 4 is one of the most ideal root-end filling materials. It is also indicated in direct capping, apexification and perforation repair in dentistry. Its good biocompatibility and sealing ability have been verified in long-term clinical practice since the mid-1990s. 5 However, the drawbacks of MTA, such as the potential for tooth discoloration, difficult handling characteristics and long setting time, limit its applications. 5,6 Current research is actively seeking alternative materials to MTA.
Biodentine is a new tricalcium silicate-based cement material that was introduced in 2009. It has better handling properties and a shorter setting time than MTA. In addition, it possesses very similar physical properties to dentine and poses low risk of tooth discoloration. The powder mainly consists of tricalcium silicate, zirconium, and calcium carbonate. The liquid is composed of water, calcium chloride and hydrosoluble polymer. 7 The Biodentine manufacturer claims that its applications are similar to MTA. For example, as a direct capping agent, Biodentine produces good results in asymptomatic vital permanent teeth with cariously exposed pulp. [8][9][10][11] Moreover, it has also been applied in pulpotomy and apexification. 12,13 Actually, the induction of mineralization of this bioactive material has been verified at the cell and molecular levels. 14 As shown in the study by Daltoe, Biodentine induces similar levels of mineralization markers in the pulp compared with MTA. Together, animal models in which Biodentine has been employed in furcation perforation repair show that both Biodentine and MTA are excellent perforation repair materials, but MTA results in a greater frequency in complete sealing of the furcation perforation. 15 However, the effect of Biodentine on the periapical tissue has not been completely elucidated.
The apical circumstance is more complex than tooth furcation and root canals. Hence, the sealing ability and biocompatibility of Biodentine as a root-end filling material must be studied. This study aims to compare citocompatibility in human periodontal ligament cells and sealing ability of Biodentine to MTA when used as root-end filling materials and their effects on apical bone healing after periapical surgery in dogs.

Cell Culture and Material Preparation
Three healthy, impacted third molars were extracted from three healthy male patients aged 18-22 years old. Informed consent was obtained from all patients before extraction. Briefly, the periodontal ligament was The 21 teeth were divided into three groups: the Biodentine group (n = 7), the MTA group (n = 7), and the blank control group (n = 7). Teeth in the blank control group were not prepared or filled. Root-ending cavities were filled with Biodentine and MTA, accordingly. All teeth were incubated at 37°C for 7 days. Nail varnish was used to coat the teeth's apical surface to determine microleakage. All teeth were placed in a 50% silver nitrate solution (Guangzhou Chemical Reagent Factory, China) at room temperature for 12 hours. The silver nitrate solution volumes and depth that infiltrated the root canal were calculated to assess the apical leakage in the MTA and Biodentine groups. Next, the ProTaper nickel-titanium system was used to prepare the root canal using an F4 file. During this procedure, saline and 3% hydrogen peroxide were used to rinse the root canal. The entire canal was filled with gutta-percha supplemented with AH Plus, and the root orifices were sealed with glass ion. X-rays were used to assess the root canal filling.
The procedures used for periradicular surgery were performed according to the method described by Apaydin. 18 Briefly, a full-thickness mucoperiosteal buccal flap with two releasing incisions was created.
The bone from the buccal side was removed with a round bar, and the bone defects were shaped with a high-speed handpiece with saline irrigation; the procedure stopped when the diameter of the defects

Statistical Analyses
Statistical analyses were performed using one-way analysis of variance followed by the Student-Newman-Keuls test with SPSS 13.0 software. 95% confidence intervals were determined. A P-value less than 0.05 was considered statistically significant.

Toxicity of the Materials
The RGR was set to 100% in the NC group. At 24 hours, a lower RGR was observed for PDLCs in the MTA group than in the negative control group

Discussion
In the present study, Biodentine possessed a similar sealing ability and biocompatibility to MTA.
Thus, Biodentine has great potential as an alternative to MTA in periradicular surgery.
According to the results of the CCK-8 assay, both MTA and Biodentine exhibited low levels of cytotoxicity at 48 hours, but not at 72 hours. The low levels of cytotoxicity induced by both materials in the early stage can be due to a hydration reaction. MTA and Biodentine contain calcium silicate, so calcium hydroxide would be created in a hydration reaction when calcium silicate reacts with water. Calcium hydroxide can increase the pH of the culture media, which damages PDSCs.
Other biocompatibility studies showed that MTA and Biodentine exhibited slight cytotoxicity, but the toxic effects were less than in other materials, such as octacalcium phosphate and IRM. [19][20][21] Sealing ability is a key characteristic of root-end materials that determines the success of periapical surgery. The common methodologies used to assess apical and coronal leakage include the dye penetration and extraction method, the fluid filtration or transportation method, and the bacteria and toxin infiltration method. 22 Due to its low cost and simplicity, the dye penetration and extraction method has been the technique preferred by most researchers.
However, the teeth must be sectioned longitudinally or transversely to examine the extent of dye penetration.
With this technique, the sample is destroyed and only Interestingly, a study by Kakaboura, et al. 25 (2007) evaluated the 3D-marginal adaptation between dentine and resin composites using computerized X-ray microtomography. This method possesses unique advantages such as full 3D-fidelity, enabling a quantitative evaluation of the interfacial adaptation at any site and direction. 26 We evaluated the microleakage between dentine and root-end filling materials with a similar method described by Kakaboura, et al. 25 (2007).
The entire leakage volume and depth were imaged, with no destruction of the samples. In the present study, a greater volume and depth of the infiltrated nitrate solution was observed in the MTA group than in the Biodentine group. However, according to Soundappan, et al. 27 (2014), the marginal adaptation of MTA and IRM was superior to Biodentine when used as root-end filling materials. These divergent results may be attributed to the differences in the accuracy of these methods. We measured microleakage with 3D imaging, which was more accurate than a simple qualitative evaluation of microleakage in transverse sections. Based on our 3D evaluation, we conclude that the sealing ability of Biodentine is adequate when applied in root-end fillings. The limitation of this study is that we only scanned the teeth after immersion. The results might be more convincing if we obtain the scan data before immersion in silver nitrate solution and compare them with the scanned data after immersion.
Furthermore, we explored the biological effect of A comparison of the sealing abilities between Biodentine and MTA as root-end filling materials and their effects on bone healing in dogs after periradicular surgery The beagle dog model has been widely used to evaluate the characteristics of dental materials, including Biodentine. For example, Silva, et al. 15 (2017). showed that Biodentine can be used as a furcation perforation repair material in the beagle dog model, with comparable results to MTA. According to De Rossi et al. 29 (2014), Biodentine and MTA both facilitate mineralized tissue bridge formation after pulpotomy.
However, no study has compared Biodentine and MTA when applied as root-end filling materials in vivo. In the present beagle dog periradicular surgery model, micro-CT was used to evaluate the bone healing process and the results showed that both materials exhibited good effects on periradicular bone healing.
As shown in the study by Daltoe, et al. 14 (2016), the ability of Biodentine to upregulate mineralization markers was similar to MTA. In addition, Ho,et al. 30 (2018) confirmed the potential utility of Biodentine in dental and bone regeneration using threedimensional printed Biodentine/polycaprolactone composite scaffolds. These studies have confirmed that Biodentine promotes bone healing.

Conclusions
Biodentine and MTA showed comparable cell biocompatibility. Biodentine was superior to MTA in terms of sealing ability when used as a root-end filling material. Both Biodentine and MTA promoted periradicular bone healing after periradicular surgery in beagle dog models.

Conflict of Interest
The authors have no conflicts of interest to declare.

Ethical Approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Informed Consent
Informed consent was obtained from all individual participants included in the study.