Evaluation of Cytotoxicity of Calcium Silicate-based Mineral Trioxide Aggregate Sealers: A Systematic Review of In Vitro Studies

INTRODUCTION

Endodontic therapy aims to sterilize the root canals and reduce pain and infection. The long-term objective for good clinical success is to seal and plug root canal systems to stop future microbial contamination. Endodontic materials with physical and chemical characteristics that are antimicrobial, hermetic, robust, and particularly biocompatible are used to achieve this goal.¹

During biomechanical preparation, the root canals are cleaned and simultaneously shaped to receive a standardized cone-shaped restorative material that would fit the canal, such as silver cones or gutta-percha. However, root canal anatomical irregularities such as accessory canals, voids, isthmus, and foramen prevent gutta-percha from completely sealing the canals.² This makes it necessary for a flowable sealing agent to bond with the canal walls and the restorative cone while adding anatomical irregularities to the restorative material. This flowable agent is called an “endodontic sealer.”³

Many endodontic sealers are available, but their use depends on multiple factors, such as the case to be treated, the cost of the sealer, etc.⁴ Table 1 provides details of all the commercially available endodontic sealers.

All endodontic sealers have advantages and disadvantages, but none meet all the criteria for an ideal sealer. Biocompatibility is an essential property of sealers because when a sealer comes in contact with periapical tissues, it can have undesired outcomes like lower healing effects, delayed sealer resorption in the periapical region, nerve injuries, and so on.⁵ Among all the sealers, mineral trioxide aggregate (MTA) sealers have been of profound interest to researchers.

Bioceramic sealers are classified into the following two types: Calcium silicate-based and calcium phosphate-based root canal sealers. The calcium silicate sealers are additionally classified into MTA-based and non-MTA-based sealers.⁶ The MTA-based sealers are ideal bioceramic materials that offer biocompatibility and biological activity in endodontics.^7,8 In the past two decades, it has emerged as one of the endodontic materials that have been studied most.^9,10 It was after first development at Loma Linda University (the 1990s) and acceptance by the US Food and Drug Administration (1993) as a root-end filling cement that it became commercially useful as ProRoot MTA^TM which is a calcium silicate-based root canal sealer either in white or grey color.¹¹ The MTA sealers’ composition varies from one manufacturer to another, such as Fillapex manufactured by Angelus, Londrina, Paraná, Brazil contains resins, such as salicylate resin, diluting, and natural resin, bismuth oxide, nanoparticles silica, MTA, pigments, and Endoseal MTA^TM manufactured by Maruchi, Wonju, Korea contains calcium silicate, calcium aluminoferrite, calcium aluminate, Calcium sulcate, radio pacifier, and a thickening agent (Table 1).

Bioactivity, biocompatibility, solubility in water, radiopacity, sealing ability, and low solubility are among MTAs most desired characteristics. The two most crucial qualities of MTA essential in dentistry are the ability to seal root canal spaces and biocompatibility. High biocompatibility reassures optimal healing responses, and optimal sealing provides a good prognosis.^12,13 The significant disadvantages of MTA are discoloration of teeth from releasing ferrous ions, extended setting time, improper handling time, etc. Furthermore, MTA’s physical, chemical, and hydration properties may be negatively impacted by any pH variation brought on by inflammatory changes in periradicular lesions.¹⁴ Several case reports and case series have reported that unintentional extrusion of MTA-based sealers didn’t cause any untoward effects or affect the prognosis of the tooth, yet extrusion of sealers is not recommended.¹⁵ While few studies have reported cytotoxicity associated with MTA-based root canal sealers.^16,17 In the present review, in vitro studies that tested the cytotoxicity of MTA were selected for the review as they help to study the individual components of the sealers.

METHODS

This systematic review was done according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines¹⁸ (Flowchart 1). Since the systematic review was non-clinical, the population, intervention, comparison, and outcome (PICO) research question was selected from the PICO framework¹⁹ (Table 2). It was framed as follows: Are MTA-based root canal sealers cytotoxic to human cells compared to non-MTA based sealers?

A thorough literature search was done from January 2018 to January 2023. This study span was considered because very few reviews were conducted on the studies published during this period.

The following keywords and Boolean operatives were applied in the search: Root canal sealers, MTA, Cytotoxicity, in vitro studies, cytotoxicity assays, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay.

The literature search was conducted in January 2023 utilizing the databases such as PubMed, Scopus, and Google India (http://www.google.co.in).

All citations were combined and uploaded to EndNote v.X9.3.3 (Clarivate Analytics, Pennsylvania, USA) to reject duplicate articles. Two reviewers separated all the headings and abstracts of the studies selected for full-text review. Two independent reviewers (NB and MAQ) autonomously assessed the full text of the articles chosen from the bibliography, which differed from the inclusion criteria.

Additional records were obtained by searching through the bibliography of the selected studies. The Medical Subject Headings (MeSH) and keywords were formed during the search. The focus of the inquiry involved in vitro experimental studies (evidence levels IIb). The level of evidence of selected studies was classified according to the guidelines of the Oxford Centre for Evidence-Based Medicine (http://www.cebm.net/levels_of_ evidence. asp).²⁰

In vitro studies, retrospective and prospective studies with placebo or blank control, and parallel group designs were combined in the review with the following inclusion and exclusion criteria:

RESULTS

In the present review, 115 articles on the cytotoxicity of MTA were found in databases. After filtering the articles for duplicates and eligibility, 19 articles were assessed. Studies that were done on animal-derived cells and in vivo studies, systematic reviews, randomized controlled trials (RCTs) were excluded and only in vitro studies on Human derived cells were selected for the review. Thus, in the present systematic review, six studies were included^22–28 (Table 4). It was observed that in these studies, along with MTA-based root canal sealers, calcium hydroxide-based sealers, resin-based sealers, zinc oxide eugenol-based sealers, silicate-based sealers, calcium silicate-based sealers, nanoceramic sealers, epoxy resin-based sealers, and other sealers were tested for cytotoxicity. In these studies, human periodontal ligament (PDL) stem cells, human PDL fibroblast cells and Human osteoblasts cells were used to assess these sealers’ cytotoxicity. During the study, the cells were in indirect contact with the sealers. The sealer extracts were diluted at a concentration of 1:1 to 1:32, depending on the studies. The cytotoxicity tests used in the studies were MTT assay, cytotoxicity by lactate dehydrogenase (LDH-assay), scanning electron microscopy, flow cytometry assay, real-time qPCR, alkaline phosphate staining, and methoxynitrosulfophenyl-tetrazolium carboxanilide (XTT) cytotoxicity, and micronucleus (MN) genotoxicity test.

DISCUSSION

The aim of using an endodontic sealer is to accomplish a three-dimensional obturation of the root canals.²⁹ Gutta-percha is traditionally used to obturate root canals, while the root canal sealers act as fillers between the root canal walls and gutta-percha. Many root canal sealers have been developed and marketed under various brand names (Table 1). Most of these root canal sealers demonstrate insufficient biological activity and have been cytotoxic when cultured immediately after being mixed.³⁰ Delayed wound healing and cellular degeneration have been reported when these sealers come in contact with periapical tissues.³¹ Furthermore, fluid or blood contamination at the apical third of the canals makes setting of hydrophobic sealers difficult.³² Thus, there was a necessity for a sealer that could set in a humid environment and overcome the drawbacks of the existing root canal sealers.

Mineral trioxide aggregate is one such material that can overcome the drawbacks of other sealers.³³ When MTA is applied as a root canal sealant and compressed on the dentin in the presence of phosphate, a dentin–MTA interface layer is formed. This layer, when examined, has a composition similar to hydroxyapatite. With this interstitial layer, marginal adaption is better than average. The MTA particles penetrate the dentinal tubules and impede them. This wards off harboring of microorganisms in the tubules.³³ Moisture is necessary for the setting reaction of MTA to occur and for the bioactivity process to be induced, which results in the formation of apatite precipitates.³⁴ Additionally, the hydration of MTA results in the formation of a tacky calcium silicate hydrate (CSH) gel which binds to the gutta-percha cone. Initially, the CSH is porous, solidifying to form a strong network in 4–6 hours. The complete setting of CSH occurs for several days. Thus, the long final setting time of MTA may prove to be advantageous for enhancing the sealing ability of MTA.³⁵

Various types of MTA-based root canal sealers are ProRoot Endo Sealer (Dentsply Tulsa Dental Specialties, Dentsply/Maillefer, Ballaigues, Switzerland), which contains major components such as tricalcium silicate and dicalcium silicate, with addition of calcium sulfate as setting retardant, bismuth oxide as radio pacifier and a minor amount of tricalcium aluminate.³⁶ It is known for biocompatibility and for having a relatable sealing ability to epoxy resin-based sealers superior to zinc oxide eugenol-based root canal sealers when evaluated using a fluid filtration system.³⁷ Also, MTA Fillapex®, Angelus Soluções Odontológicas, Londrina, Paraná, Brazil is another type of sealer made up of Salicylate resin, natural resin, bismuth, and silica. It is the first paste-based, MTA-based salicylate resin root canal sealer that may be used with any obturation technique. It has outstanding handling characteristics, delivers quickly and wasteless, and has a quick setting time.³⁸ The CPM Sealer (EGEO SRL, MTM Argentina S.A., Buenos Aires, Argentina) has a composition of tricalcium silicate, tricalcium oxide, tricalcium aluminate, and other oxides. It is known for its biocompatibility, antimicrobial activity, and less setting time.³⁹ Furthermore, MTAS Experimental Sealer was developed at Endo Araraquara Dental School UNESP, University of Estadual Paulista, Sao Paulo, Brazil. Approximately, 80% white Portland cement, zirconium oxide, which acts as a radioopacifying agent, calcium chloride as an additive, and resinous vehicle are its components. It has got a good capacity for the release of hydroxyl ions.⁴⁰ Moreover, MTA Obtura is a sealer developed by adding liquid resin as a curing initiator to replace saline. The development of MTA Obtura aimed to create an endodontic sealer compounding the biological and sealing features of MTA. This sealer has a good sealing ability.³⁹ In the present review, it was observed that five studies of the six studies that were selected assessed MTA Fillapex, while one study assessed EndoSeal MTA.^22–28

Before using any endodontic sealers clinically, it is necessary to test the material for genotoxicity, cytotoxicity, and biocompatibility, as these sealers can potentially interact with periapical tissues extruded due to condensation forces or overfilling the canals. The anatomy of the root canal also contributes to the extrusion of the sealer in the periapical space.⁴¹ To evaluate any biological and cytotoxic concerns associated with products, such as root canal sealants, in vitro assays are regarded as screening tests. The advantage of in vitro trials is that they are cost-effective, rapid assessment can be done, and predictivity of the material over a duration of time can be done.⁴² Thus, the present review selected all the in vitro studies on root canal sealers done on human cells published between 2018 and 2022.

Cytotoxic sealers cause the deactivation of enzymes such as zinc-dependent enzymes and alkaline phosphatase that are essential for bone formation, bone remodeling, wound healing, and collagen and gelatine degradation.⁴³ Four methods test the cytotoxicity of endodontic sealers:

1. MTT assay: This assay is also called Mosman’s tetrazolium toxicity assay. It is a colorimetric assay that checks the mitochondrial activity of the cells to know their viability. It is created on the conversion of MTT into formazan crystals by living cells.

2. Zygomorphy test: This test assesses the matrix metalloproteinases (MMPs) activity in cells based on which cytotoxicity is decided.

3. Cytotoxicity assay: This test assesses the toxicity of the sealers.

4. The XTT assay is a ready-to-use assay for the biological evaluation and screening of chemical, pharmaceutical, cosmetic, and medical device substances. It contains a tetrazolium-based compound sensitive to redox reactions and alive respiring cells. It is considered an early, straightforward, quick, and sensitive assay.

In the present review, five studies employed the MTT assay, while one study by Hilal Erdogan et al.²⁷ used the XTT assay. The difference between MTT and XTT assay is that in the MTT assay, cells must be lysed to dissolve formazan salt before measuring the absorbance. In contrast, XTT assay allows kinetic monitoring of the sample at different periods, and lysing of cells is not needed.⁴⁴

Studies on human-derived cells were only considered in this review due to the biochemical differences between human and animal-derived cells. When a root canal sealer extrudes from the canals, it comes in contact with PDL fibroblasts and other cells like osteoblasts and mesenchymal cells. When in communication, the sealers cause inflammation and necrosis of these cells, which could lead to delayed periapical healing.⁴⁵ Among the studies considered for this review, Susanne Jung et al. conducted a cytotoxicity test on human osteoblast cells.²³ Pritesh Jagtap et al.²² and Hilal Erdogan et al.²⁷ conducted cytotoxicity tests on human PDL fibroblast cells, while Ju Kyung Lee et al.,²⁴ Alexis Gaudin et al. and Bruna Barcelos Só et al.²⁸ conducted cytotoxicity tests on human periodontal ligament stem cells (hPDLSCs).

In the present review, MTA Fillapex was the most cytotoxic compared to other MTA-based sealers. In the study conducted by Pritesh Jagtap et al.,²² when human PDL fibroblast cells were bathed with freshly derived elute of the sealers within the first hour, MTA Fillapex displayed potent cytotoxicity, and during the first and second weeks, this toxicity did not lessen.²⁴ The results of this study were consistent with those of an earlier investigation done by TH Huang et al. that found that MTA Fillapex significantly reduced cell viability utilizing various techniques.⁴⁶ In a recent report demonstrating the long-lasting cytotoxic effects of sealers, similar outcomes were observed regarding MTA Fillapex cytotoxicity assessed using the MTT assay.⁴⁷

Conferring to a study by Jung et al.,²³ the solubility of sealers before and after setting greatly affected the cytotoxicity of the sealers. It was reported that sealers such as AH Plus were insoluble after setting the reaction. This explains the cause of the difference in cytotoxicity of AH Plus before and after the setting response.⁴⁸ Similarly, the solubility of MTA Fillapex increases after setting, which means that the cytotoxicity of MTA. Fillapex also increases, which is in contrast to AH Plus. This observation concluded that freshly mixed AH Plus and MTA Fillapex must be used cautiously, and contact of these sealers with cells like osteoblasts must be avoided.²³ In another study conducted by Ju Kyung Lee et al., epoxy resin-based sealers (AH Plus and AD Seal) and calcium silicate-based sealers (Endoseal MTA, Wellroot ST, and nanoceramic sealer) were tested for toxicity. It was observed that toxicity was highest when the sealers were mixed.²⁴ This initial toxicity of AH Plus can be explained by the fact that the sealer’s amine or epoxy resin components are released into the adjacent tissues, while the toxicity of calcium -silicate resins is due to the high pH.^49,50 The rise in pH is due to the formation of calcium hydroxide when these resins dissolve in the tissue fluid. Although the high pH of root canal sealers may harm cell viability, they may also have certain biological benefits. These sealers’ high alkalinity could alter the environment to encourage the growth of hard tissues and interfere with osteoclastic action, which promotes tissue healing.⁵⁰ Endoseal MTA and other calcium silicate-based sealers were reported to be more biocompatible than epoxy resins.²² Comparable outcomes were testified by Diomede et al.⁵¹

According to Gaudin et al., MTA Fillapex enhanced the proinflammatory mediators’ productions among all the calcium silicate-based sealers. These mediators trigger an innate immune response that adversely affects local wound healing.²⁵ This was proven by another study by Liu J et al.⁵² MTA Fillapex is a disalicylate-resin-based root canal sealer containing 13% MTA-like material. It is not a calcium silicate-based sealant or a “bioceramic” sealant. It has the physicochemical properties of resin-based sealers. Still, the biological properties of MTA.⁵³ The cytotoxic effect of MTA Fillapex can be attributed to its resinous components such as salicylate resins and silica. It is reported that salicylate resins that prolong the working time and setting time of MTA and render it better flowability also increase the cytotoxicity of MTA^54–56 According to Hilal Erdogan et al.,²⁷ MTA Fillapex displays time-dependent toxicity. It must be used with utmost care as it can cause an increase in micronuclei formation in cells that come in contact with MTA Fillapex.²⁷ Bruna Barcelos Só et al., in their study, mention that although MTA is present in MTA Fillapex, the salicylate resin component induces cell apoptosis by precipitating in the cell cytoplasm leading to fragmentation of DNA. However, in this study, the cytotoxicity of MTA Fillapex was comparatively lower than AH Plus.²⁸ This statement is in strong disagreement with other studies.^57–59 This disagreement can possibly be explained by the composition of the sealers. Thus, it can be mentioned that great caution needs to be exercised when using MTA Fillapex.

The present review is considerably significant since no recent reviews have analyzed the cytotoxicity and bioactivity of MTA-based root canal sealers. Furthermore, in the present study, a more laborious and objective defined procedure for the data collection, extraction, and compilation was followed, which helped disparagingly inspect the study methodologies and eliminate the ambiguous study designs and unclear protocols.

The limitation of the present review was that meta-analysis could not be accomplished due to the assortment in the selected studies, based on the assay used, cells used for testing cytotoxicity, etc. Since meta-analysis was not carried out, an overview of the study’s findings could not be given. The present review found very few studies in which MTA was tested as a root canal sealer. Most of the studies used MTA sealer for comparison. Thus, the available literature evidence was insufficient to conduct a more reliable review and reach a consensus. However, the present review confirms that caution must be exercised when using resin sealers, such as MTA Fillapex.

CONCLUSION

When a sealer is extruded into the periapical space beyond the apical foramen, cytotoxicity of endodontic sealers can lead to delayed healing of periapical lesions in a tooth that has undergone root canal therapy. Therefore, it is advisable to keep the obturant and sealer within the premises of the apical foramen and use a sealer that has the least proven cytotoxicity and is suitable for the selected case. The available literature suggests that MTA Fillapex has mild to moderate cytotoxicity and must be used cautiously. However, there is a need for more human cell-based studies to help arrive at a general agreement regarding the existing root-filling materials.

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