Bioactivity Potential of Bioceramic-Based Root Canal Sealers: A Scoping Review

Introduction: Bioceramic-based root canal sealers are novel materials with a bioactivity potential that stands out compared with conventional root canal sealers. However, the term bioactivity may be overused and is often misunderstood. Hence, the objective of this study was to synthesize and map key concepts related to the bioactivity analysis of bioceramic-based root canal sealers. Methods: The present scoping review is reported in accordance with the PRISMA-ScR Statement and is registered in the Open Science Framework. Two blinded reviewers carried out a comprehensive search in six databases up to January 10th, 2022: MEDLINE, Scopus, Embase, Web of Science, Cochrane Library, and Lilacs/BBO. Eligibility was considered for in vitro and in vivo studies that evaluated the bioactivity potential of bioceramic-based root canal sealers. Results: A total of 53 studies were included in the qualitative synthesis. In vitro bioactivity was evaluated through the mineralization potential, formation of carbonated apatite on the surface, and the gene expression related to proteins involved in the mineralization process. Meanwhile, for in vivo studies, staining techniques associated with immunohistochemical tests were mainly used to detect mineralization on the material–host tissue interface. Conclusions: According to the methodology used, the most prevalent methods to assess bioactivity in acellular form were the immersion of the material in Hank’s balanced salt solution, followed by surface observation with scanning electron microscopy and energy dispersive X-ray. In cell cultures, the chosen method was usually Alizarin Red staining, followed by the evaluation of alkaline phosphatase enzymatic activity and the use of molecular biology tests.


Introduction
The filling of the root canal system is an important step in endodontic treatment and has been attributed to the fact that it fosters the sealing of both the main root canal and its accessory ramifications, thus preventing the transit of microorganisms between the root canal and the periradicular tissues [1]. The most commonly used materials for filling root canals are gutta-percha associated with a root canal sealer [2]. Ideally, the root canal sealer should adhere to the root canal walls, promote adequate sealing, have an adequate radiopacity, have low setting contraction, have thin and small particles, not stain the dental tissues, have antimicrobial activity, be biocompatible, be insoluble to tissue fluids, have a workable setting time, and be easily removable from the inside of the root canal system if necessary [3,4].
Root canal sealers can be classified according to their composition as zinc-oxideeugenol-based, calcium-hydroxide-based, resin-based, glass-ionomer-based, silicon-based, and bioceramic-based [5]. In 2007, a calcium-silicate-based root canal sealer was developed and launched on the market under the commercial name iRoot SP ® (Innovate Bioceramix, Vancouver, BC, Canada) and was classified as a bioceramic-based root canal sealer [6].
Bioceramics are ceramic materials that contain silica, alumina, zirconia, bioactive glasses, ceramic glasses, calcium silicates, hydroxyapatite, and calcium phosphate [7]. For the filling of the root canal system, the goal of using hydraulic types of sealer is to achieve the most hermetic filling possible with inert materials. Additionally, the bioactivity potential and adhesion to the dentinal substrate of these types of materials are desirable characteristics [8].
Calcium-silicate-based root canal sealers show promising results when evaluated in vitro, surpassing conventional root canal sealers regarding some of their properties [9]. Bioceramic-based root canal sealers stand out for their biological characteristics when applied in various clinical situations, especially in cases where the risk of material extrusion into the periodontium is greater, such as in cases of root resorption, teeth with open apex, or overinstrumented canals [10].
When discussing biological properties in dental materials, some terminology should be considered. Biocompatibility is defined as "the ability of a material to function with an appropriate host response in a specific application" [11]. Meanwhile, the term bioactivity has been widely used in the market as a characteristic of bioceramic materials, showing a lack of standardization in the literature regarding the concept definition and the methodologies needed to assess this characteristic in dental materials. Bioactivity can be defined as the cellular response induced by the release of ions or biologically active substances from the biomaterial so that biomineralization occurs [12]. From this perspective, it was suggested to limit the term bioactive to materials that encourage biomineralization specifically, rather than the material in vitro performance [13]. However, this definition differs in a few studies [14,15].
For these reasons, the objective of this scoping review was to synthesize and map key concepts from studies that evaluated the bioactivity potential of bioceramic root canal sealers for better use of this concept in future studies. Thus, the following exploratory research question was asked based on the population-concept-context (PCC) framework for scoping reviews: What are the main methodologies (C) used to determine the bioactivity potential (C) of bioceramic-based root canal sealers (P)?

Protocol Registration
The protocol for this scoping review is available on the Open Science Framework through the link https://osf.io/3jdqu/ (acessed on 17 May 2021). The reporting of this study followed the recommendations of the Preferred Reporting Items for Systematic Review and Meta-analysis extension for Scoping Reviews (PRISMA-ScR) [16,17].

Eligibility and Exclusion Criteria
The inclusion and exclusion criteria were established by consensus among the reviewers after an in-depth discussion considering the research question, the study objectives, and possible methodological limitations.
Eligible studies were those that evaluated the bioactivity potential of bioceramic-based root canal sealers (commercially available or experimental) used in association with guttapercha. In vitro studies that evaluated the bioactivity potential of these root canal sealers were included. Furthermore, in vivo biocompatibility studies were also included. No restriction on the publication date of the evaluated studies was applied, but language restrictions were applied for English and Spanish.
Studies that evaluated only cytotoxicity, cell viability, or cell proliferation were excluded from this scoping review, as well as biocompatibility studies evaluating only the inflammatory response. In addition, studies that evaluated only root perforation or apical surgery were excluded.

Search Strategy
A search strategy was formulated using Medical Subject Headings terms (MeSH), Emtree terms, and Health Science Descriptors (DeCS) related to the research question, and is available in Supplementary Table S1. The terms used were selected to cover the largest number of relevant studies. An initial systematic search was performed by two independent and blinded reviewers (M.S.E. and L.P.A.) on 8 May 2021, in the following electronic databases: Cochrane Library, EMBASE, LILACS/BBO, PubMed/MEDLINE, SciVerse Scopus, and the Web of Science. This initial search aimed to verify whether the selected terms were in accordance with the study's objective and eligibility criteria.

Selection Process
After a systematic search in the databases, the studies' references were imported into Mendeley software (Elsevier, Amsterdam, The Netherlands) for duplicate removal. Then, they were exported into Rayyan online software (Qatar Computing Research Institute, Doha, Qatar) [18], where screening by title and abstract was performed by two independent and blinded reviewers (M.S.E. and L.P.A.). After the selection process, blinding between the reviewers was removed, and in the case of any disagreement, a third researcher with more experience in the field (E.P.) settled the issue. The selection was made according to the eligibility criteria and the articles that met the inclusion criteria or those with insufficient data in the title and abstract to make a clear decision were selected for full-text analysis. Additionally, references from the included studies were manually searched to identify potential studies that were not covered by the search strategy.

Data Collection Process
Data collection was performed by two independent and blinded reviewers (M.S.E. and L.P.A.) in an Excel spreadsheet (Microsoft Corporation, Redmond, WA, USA) with data regarding the author's name, year of publication, published journal, root canal sealer used, dilution medium (if applied), control group, bioactivity analysis method, sample number, duration (time of evaluation), and main findings. After the initial data collection, a third reviewer (F.I.) double-checked all of the retrieved data to avoid tabulation errors. This model was made in accordance with the recommendation for scoping reviews by the Joanna Briggs Institute [19] and was adjusted and previously tested by all of the reviewers involved in this study with at least two included articles. No quality assessment of the included evidence was carried out, as the aim of this review was to map the investigation techniques used to evaluate the bioactivity potential of bioceramic-based root canal sealers.

Results
The systematic search (last conducted on 10 January 2022) retrieved 3883 potentially relevant studies. Figure 1 is a schematic flowchart that synthesizes the article selection process according to the PRISMA 2020 Statement [17]. After removing duplicate records, 2218 studies were screened by title and abstract using the Rayyan online application (Qatar Computing Research Institute). A total of 2143 studies were excluded for not meeting the inclusion criteria and 75 studies were selected for full-text analysis. Of the 75 studies, 22 were excluded for the following reasons: 2 evaluated the bioactivity potential of restorative bioceramic materials; 8 studies evaluated the bioactivity of root canal sealers that do not have calcium silicate in their formulation; 1 was a narrative literature review not related to the bioactivity of root canal sealers; and, in 11 studies, the bioactivity potential was not evaluated. The remaining 53 studies met all inclusion criteria and were included in this scoping review.

Study Characteristics
A total of 53 studies evaluated the bioactivity potential of bioceramic-based root canal sealers, of which 37 were in vitro studies, and the remaining 16 were in vivo studies. The studies investigated 21 commercial and 17 experimental formulations among the different bioceramic-based root canal sealers. Although MTA Fillapex is based on salicylate resin, it was included in this analysis because it has 13% of MTA (mineral trioxide aggregate) in its composition, has a different biological and physicochemical behavior from conventional root canal sealers, and was classified as a bioceramic in a previous study [20].

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that do not have calcium silicate in their formulation; 1 was a narrative literature review not related to the bioactivity of root canal sealers; and, in 11 studies, the bioactivity potential was not evaluated. The remaining 53 studies met all inclusion criteria and were included in this scoping review.

Study Characteristics
A total of 53 studies evaluated the bioactivity potential of bioceramic-based root canal sealers, of which 37 were in vitro studies, and the remaining 16 were in vivo studies. The studies investigated 21 commercial and 17 experimental formulations among the different bioceramic-based root canal sealers. Although MTA Fillapex is based on salicylate resin, it was included in this analysis because it has 13% of MTA (mineral trioxide aggregate) in its composition, has a different biological and physicochemical behavior from conventional root canal sealers, and was classified as a bioceramic in a previous study [20].
The included studies had a divergence regarding the evaluation of the acellular and cellular bioactivity potential owing to the different methodologies used to investigate this property. This review classified acellular and cellular in vitro and in vivo studies The included studies had a divergence regarding the evaluation of the acellular and cellular bioactivity potential owing to the different methodologies used to investigate this property. This review classified acellular and cellular in vitro and in vivo studies separately. Among the included in vitro studies, 12 of them performed acellular in vitro investigations by immersing the hardened material in solutions that simulate body fluids (e.g., Hank's balanced salt solution, phosphate buffered saline, and simulated body fluid) followed by the material surface observation, with two of these studies evaluating the dentin-filling material interface. Among the solutions used in the methodologies, Hank's balanced salt solution (HBSS) was the most frequently used, with seven studies, followed by simulated body fluid (SBF) with three studies and phosphate buffered saline (PBS) solution with two studies. After immersing the specimens in these solutions, different methods were used to observe the surface morphology deposited on the materials' surface or the interface with the dentin. A swept emission field electron microscope (FE-SEM) was used in three studies, while eight used scanning electron microscopy (SEM) and one study used scanning electron microscopy with electron probe microanalysis (SEM-EPMA) for joint evaluation of microscopy and chemical or structural analysis. To evaluate the elemental composition of the material deposited on the surface of the bioceramic-based root canal sealers, it was also used in seven studies. Energy-dispersive X-ray spectroscopy (EDS or EDX) and X-ray diffraction (XRD) were used in three studies and Fourier transform infrared spectroscopy (FT-IR) was used in two studies. SEM-EPMA (scanning electron microscopy-electron probe microanalysis), micro-Raman spectroscopy, and confocal laser microscopy were used in one study each. Table 1 highlights the bioactivity analysis method used for each study.
All of the studies included in this review have observed material deposition along the filling surface or mineralization reactions at the interface with the root dentin, and these findings suggest the bioactivity potential of bioceramic-based root canal sealers.
The Endosequence BC Sealer was the bioceramic-based root canal sealer that obtained the most favorable results in five studies, along with iRoot ® SP and MTA Fillapex ® in three studies each; TotalFill BC and BioRoot RCS in two studies each; Ceraseal, Endoseal and Endosequence Hi-Flow in two studies each; and Bio-C sealer, Bio-C Sealer ION+, Well-Root ST, C-Root, Neo MTA Plus ® , and the experimental sealers showed positive results in one study each.
It is also relevant to acknowledge that one study [35] was carried out with the ex vivo use of rat's parietal bone to evaluate the bone tissue response to the tested bioceramic-based root canal sealers.
Of the 16 included in vivo studies shown in Table 3, nine evaluated only the reaction of rats' subcutaneous tissue o the materials, while four studies evaluated the bone implantation in rats, one concerned the evaluation of bone implantation in a rabbit, one study investigated both bone and subcutaneous tissue implantation in rats, and one investigated root canal treatment performed in dogs with radiographic and histological evaluation. One study evaluated bone implantation and the periapical tissue response by implanting bioceramic material in periapical tissues and not on the femur or tibia [58]. In 14 studies, the tested materials had a positive result for bioactive potential and, in 1 study, partial formation of bone tissue was observed in the period evaluated [59]. Additionally, nine studies used the von Kossa histochemical technique to detect mineralization activity [60][61][62][63][64][65][66][67].
In general, the analysis of the apatite deposition in vitro of materials can be carried out by evaluating the mechanism of apatite formation with immersion in phosphate-rich solutions and observation of the deposited material and further evaluating the materials reactivity, with this method being reported in some studies as a partial assessment of the bioactivity potential or acellular bioactivity [23].
The mineralized nodules' detection in cell cultures is mainly observed in a direct way by the staining technique using Alizarin red dye and indirectly by the enzymatic activity of the enzyme alkaline phosphatase and by the detection of genes or substances involved in biomineralization. The combination of these assessments at the cellular level also results in a partial assessment of the bioactivity potential, called cellular bioactivity [23].   For in vivo studies evaluating bioceramic-based root canal sealers, concerning the materials' subcutaneous implantation, the most mentioned detection methods for evaluating mineralized tissue formation were the use of histochemical hematoxylin-eosin techniques associated with the use of the Von Kossa staining technique [60,61,64,66,69], immunohistochemistry techniques to detect osteocalcin activity [65,66], osteopontin calcein detection with the use of Alizarin red dye [67], and usually a combination of those methods. When the in vivo evaluation was in bone tissue, histochemical analysis with the use of hematoxylin-eosin was preferred among the studies.

Discussion
Dental materials' bioactivity is a desired property for root canal sealers and is continually sought after by researchers and the dental materials industry to obtain a higher tissue repair rate, especially with the formation of mineralized tissue [70]. The term bioactivity has a broad definition, with the term bioactive referring to a material that was designed to induce a specific biological response [74]. However, depending on the area, a biomaterial can also be one that exhibits tissue adhesion as a result of the interaction with tissues at the interface [75]. Based on ISO 23317:2014 [76], for biomaterials that are implanted in a living body, a thin layer of calcium and phosphorous will form on its surface, then this apatite layer connects the implanted biomaterial to the living tissue without a distinct boundary.
Although the focus of this study is on the bioactivity of bioceramic-based root canal sealers, it is not just in the area of endodontics that bioactivity is a desired property. With the growth in the search for conservative and minimally invasive dentistry, several dentistry areas have sought bioactivity in their materials, such as bone implants [77], bone grafts [78], pulp capping agents [79], and restorative materials [80]. More recently, researchers have come to the concept that a bioactive material is one that acts upon or interacts with living cells and tissues to produce a specific response, such as biologically directed mineral formation [81]. This is a very desirable property in root canal sealers as dentin is vulnerable to hydrolyses, which can be catalyzed by stages of endodontic treatment, such as the use of sodium hypochlorite, calcium hydroxide dressings, and the fluids' penetration and diffusion [82].
There was significant variation in the parameters used to indicate bioactivity in the included studies. Several methodologies were found to assess this property in bioceramicbased root canal sealers, and the goal of most of them was to induce the carbonated apatite formation on the materials surface, mineralization nodules' formation in cells, and in vivo mineralized tissue formation.
Regarding the methodologies used to assess the potential for acellular bioactivity, immersion in phosphate-rich solutions refers to the material ability to induce in vitro apatite formation on the surface. This methodology is of interest because it is similar to in vivo tests and reduces the need for unnecessary animal testing [83]. Other solutions that simulate body fluids have been suggested for the same purpose, such as phosphatebuffered saline and Hank's balanced saline solution [26]. For the assessment of bioactivity in bioactive glasses, this methodology was referred to as an initial evaluation for the development of new materials and a partial bioactivity analysis because it assesses the material reactivity. The material reactivity is related to the release of calcium ions from the material, which react with the solution phosphate ions to form a calcium phosphate precipitate (similar to hydroxyapatite) on the material surface, not involving a biological reaction [27,84].
When evaluating the bioactivity potential in cells, the most frequent evaluation was using the enzyme alkaline phosphatase (ALP), followed by the technique using Alizarin Red (ARS) and molecular biology (RT-PCR and RT-qPCR). This is similar to another literature review that evaluated the bioceramic materials' bioactivity for pulp capping in human pulp stem cells (hDPSCs), in which the most frequent method was RT-PCR, followed by ALP and ARS [85]. In another study, it was reported that the bioactive potential of calcium-silicate-based materials could be assessed by osteogenic differentiation and mineralization by measuring ALP activity using ARS and the gene expression of genes linked to mineralization [86].
Furthermore, in studies with cells, the parameters to assess bioactivity were cell adhesion [15] and gene expression of proteins related to bone repair [37]. The ALP enzymatic activity was evaluated using dyes for evidence of mineralization, which is one of the main substances used to evaluate the bioactivity in cells according to the frequency presented in the included studies. Of the 20 studies evaluating this enzyme, 16 showed positive results, 2 were nonsignificant, and 2 had no positive effects.
In recent studies, the designation of material with bioactivity potential (acellular and cellular) suggests that it is a partial evaluation of this property, when in preliminary laboratory tests [23,66], as it was suggested that in vivo studies would be the best way to assess the real potential bioactivity of materials [73,87].
To prove mineralization in biocompatibility studies in subcutaneous tissue, the von Kossa technique was used in some studies [60,61]. However, it has already been reported in the literature that this technique detects calcium and not calcium phosphate, with a complementary evaluation through other markers that indicate mineralization being recommended in some studies [88], such as alkaline phosphatase enzyme or even by X-ray diffraction, if available [89]. In a previous review [83], only bone implantation tests were included and subcutaneous implantation tests were excluded. In the present review, it was chosen to also include these studies because more recent investigations have evaluated the materials' bioactive potential for their ability to stimulate mineralization in subcutaneous tissue using immunohistochemical techniques [65].
This study was carried out in an exploratory way to map the most commonly used methodologies to evaluate the bioactivity of bioceramic-based root canal sealers and obtain information on the materials' properties according to the methodology used. The search strategy used included the term angiogenic according to a previous study [85], which was shown not to be a property to assess bioactivity directly, but to contribute to starting the repair process of the affected region [35]. In addition, as reported, a material would be considered bioactive when used in its clinical use environment, stimulating mineralization by the organism, having completed the material development phases with the knowledge of physicochemical properties, ex vivo tests, with culture of cells, animal tests, and finally clinical trials [13,90].
In the present review, an evaluation was carried out with a focus on methodologies for laboratory studies that could better predict the clinical performance of bioceramic-based root canal sealers in terms of bioactivity, which is important for subsequent randomized clinical studies that aim to clinically prove the performance of these materials.

Conclusions
According to the methodology used, the most prevalent methods to assess bioactivity in acellular form were the immersion of the material in Hank's balanced salt solution, followed by surface observation with scanning electron microscopy and energy dispersive X-ray. In cell cultures, the chosen method was usually Alizarin Red staining, followed by the evaluation of alkaline phosphatase enzymatic activity and the use of molecular biology tests. In the animals' subcutaneous tissue, the von Kossa histochemical technique was the most commonly used method to detect calcium deposition and, when evaluating bone tissue, the most commonly used histochemical technique was the use of hematoxylin-eosin.