Antimicrobial efficacy of commercially available endodontic bioceramic root canal sealers: A systematic review

Background Recently, a new generation of bioceramic root canal sealers has been introduced onto the market. Many in vitro studies have investigated the antimicrobial properties of these sealers, but their comparative efficacy in antimicrobial activity is still unknown. Methodology Three electronic databases were searched: MEDLINE and Embase via the OvidSP platform, and Web of Science, up to June 25, 2019. Studies were included irrespective of study design, type of publication and language. Reporting quality was assessed by two authors independently. Meta-analysis was not performed due to studies being highly heterogeneous. Results We included 37 studies that analysed the antimicrobial effects of bioceramic sealers. Most of them used a planktonic cell model, with the exception of nine studies which used biofilms. It was not possible to make direct comparison of results from studies and to give a clear conclusion about the comparative antimicrobial activity of these materials because the studies used heterogeneous sources and ages of microorganisms, setting and contact times of sealers, and antimicrobial tests. Furthermore, some materials showed completely different results when tested with different methods. Conclusions In conclusion, multiple in vitro studies have shown that bioceramic sealers may have various degrees of antimicrobial activity. However, it is still impossible to make conclusions about their comparative efficacy and to recommend the use of one over another in clinical practice because the studies available were conducted in different ways, which makes meta-analysis futile. A uniform methodological approach, consistent definitions and studies on humans are urgently needed in this field of research so that recommendations for practice can be made.


Results
We included 37 studies that analysed the antimicrobial effects of bioceramic sealers. Most of them used a planktonic cell model, with the exception of nine studies which used biofilms. It was not possible to make direct comparison of results from studies and to give a clear conclusion about the comparative antimicrobial activity of these materials because the studies used heterogeneous sources and ages of microorganisms, setting and contact times of sealers, and antimicrobial tests. Furthermore, some materials showed completely different results when tested with different methods.

Conclusions
In conclusion, multiple in vitro studies have shown that bioceramic sealers may have various degrees of antimicrobial activity. However, it is still impossible to make conclusions about PLOS

Introduction
Microorganisms and their products are the main aetiologic factors responsible for pulpal diseases and periapical lesions [1]. Microorganisms found in root canals are commonly organized in biofilms, in which they are more resistant to antimicrobials than bacteria in the planktonic state [2]. Shen et al. [3] showed that biofilms aged 3 weeks and older are more resistant to chlorhexidine (CHX) than 2-week old and younger biofilms. Similar results were shown by Stojicic, Shen and Haapasalo [4], where 1% sodium hypochlorite (NaOCl) and 2% CHX were effective in killing 1-and 2-week old bacterial biofilms, whereas 3-week-old and older biofilms showed increased resistance to these agents. The objectives of root canal treatment are elimination of infection from the root canal and prevention of its reinfection by filling and sealing the root canal space [5][6][7]. Although chemomechanical preparation significantly reduces the number of microorganisms in the root canal, 40-60% of root canals still remain positive for bacterial presence after this treatment [8,9].
Thus, endodontic sealers play an important role in controlling endodontic infection by entombing residual bacteria and preventing leakage of nutrients and reinfection of the root canal [10]. Multiple commercial endodontic sealers, available on the market, are claimed to have antimicrobial properties. Many studies have reported that freshly prepared root canal sealers (resin-, zinc oxide eugenol-, calcium hydroxide-, silicate-and silicon-based sealers) are effective against Enterococcus faecalis (E. faecalis), but their antimicrobial effectiveness after 2 to 7 days of ageing has not been reported [11][12][13][14][15][16].
The first bioceramic material used for root canal obturation was described in 1984 [18]. The forerunners of modern bioceramic materials were calcium phosphate sealers like Sankin apatite root canal sealers (I, II and III) (Sankin Kogyo, Tokyo, Japan) and experimental sealers known as Capseal (I and II) [17].
A new era of bioceramic materials started in the mid-1990s when bioceramic materials based on MTA were introduced firstly as root repair cements [19]. Those were mainly Portland-derived cements like ProRoot MTA (Dentsply Tulsa, Tulsa, OK, USA), which have been used as root-end filling materials, and root repair and pulp capping materials [20][21][22]. Because of their dense consistency, these cements are not easy to place in root canals [23], therefore, bioceramic based root canal sealers have recently been developed [24,25]. The first sealer based on MTA was MTA Fillapex (Angelus, Londrina, Brazil), introduced onto the market in canal sealer (Innovative BioCeramix Inc., Vancouver, Canada) and Endosequence BC Sealer (Endosequence BC Sealer, Brasseler, Savannah, GA, USA) are available on the market. Bioceramic sealers have also attracted attention because of their alkaline pH, biocompatibility, bioactivity, non-toxicity, dimensional stability, sealing ability and potential to increase root strength after obturation [27,30].
However, the comparative antimicrobial effectiveness of sealers is unknown, and it is not known which models have been used to prove their antimicrobial activity.
Therefore, the aim of this systematic review of the literature was to provide knowledge synthesis of the available body of evidence regarding the antimicrobial properties of endodontic bioceramic sealers for differently aged microorganisms, regardless of study design, and to analyse their methods, results, conclusions and comparative effectiveness, as well as reporting the quality of the literature published on this subject.

Methods
We conducted a systematic review of literature according to the methods and guidelines from the Centre for Reviews and Dissemination [31] and the PRISMA Statement [32]. The protocol of the systematic review was registered and published in the PROSPERO database (registration number: CRD42018082375).

Clinical question
We defined the clinical question in the following way: i) we included studies that analysed the antimicrobial properties of bioceramic endodontic sealers, conducted on any type of patient or in vivo and in vitro experimental models; ii) eligible interventions were endodontic bioceramic sealers; iii) any type of comparator was eligible; iv) outcomes were size of the inhibition zone, number of microorganisms, percentage of dead cells in dentinal tubules, changes in microbial growth and biovolume of viable cells.

Inclusion criteria
Studies of any design that analysed the antimicrobial properties of endodontic bioceramic sealers, in vivo studies on both humans and animals and in vitro studies conducted on any type of laboratory model were considered for inclusion in this review.

Exclusion criteria
Studies were excluded if they evaluated the antimicrobial properties of other types of root canal sealers (calcium hydroxide, resin, zinc eugenol or silicone-based sealers). Likewise, studies that evaluated bioceramic-based cements such as MTA cements or Biodentin (Septodont, Saint-Maur-des-Fossés, France) were excluded because their use for the purpose of root canal filling is limited. Experimental sealers which are not commercially available on the market were also excluded. Studies that evaluated sealers developed before the MTA era were also excluded. related to bioceramic endodontic sealers with those for antimicrobial activity (S1 Appendix). There were no restrictions regarding the language or status of the publication. Search results were exported into EndNote X5 software (Clarivate Analytics, Boston, MA, USA). Duplicates were removed, at first by using the built-in EndNote feature for de-duplication and then manually. In order to find additional studies that might potentially fulfil the eligibility criteria for inclusion, the references of all included studies were searched, and studies that cited all included studies were also searched at the Web of Science; these were then screened to potentially find more relevant studies that were not found during the initial database search.

Study selection
Two authors (MŠM, TPP) independently screened titles and abstracts obtained via the database search. Full texts of studies that were considered relevant or potentially relevant in the first screening phase were obtained and thoroughly analysed for eligibility by two authors independently (MŠM, TPP). At both stages of the screening process, all discrepancies were resolved via discussion or by involving the third author (IB).

Outcomes
Outcome measures used in this systematic review were as follows: size of the inhibition zone, number of microorganisms (colony-forming units), percentage of dead cells in dentinal tubules, changes in microbial growth and biovolume of viable cells.

Data extraction
After screening the full texts, a data extraction sheet was developed, tested on two studies and refined accordingly. Two authors (MŠM, TPP) independently extracted data. All disagreements were resolved via discussion or by involving the third author (IB). The following data were collected from each study: (i) general information, including the first author's name, publication year, aim of the study, study design; (ii) general methods: evaluation methods, model used, microorganisms tested, duration of microorganism growth, sealer tested, setting time of sealer before contact with microorganisms, contact time between sealer and microorganisms; (iii) outcomes studied and (iv) experimental results. In the case of incomplete or unclear data, study authors were contacted for clarifications. If authors did not respond after the second email, we did not contact them further.
Studies were then divided into seven groups depending on which material they used. Young and mature biofilms were defined according to the study of Stojicic, Shen and Haapasalo [4] where young biofilms implied only microbial clusters up to 2 weeks old and mature biofilms as bacterial clusters of more than 2 weeks of maturation.

Data synthesis
For all included studies, narrative and tabular synthesis of data was performed. Meta-analysis could not be performed due to the heterogeneity of studies.

Results
The study selection flow chart representing the stages of the systematic review process is presented in Fig 1. The search strategy yielded 3651 results consisting of titles with or without abstracts. After software and manual de-duplication, 2217 were screened. For further inclusion, 109 titles were considered. Then, abstracts and full texts were searched. The final number of included studies, which met the established criteria, was 37 including three studies [33,34,35] of which two [34,35] were published only as a conference abstract, and another [33] which was found by searching other sources. Neither human nor animal in vivo studies were found. The authors had access to all full texts. One study [44] reported only qualitative results. Thus, data about the antimicrobial efficacy of each group could not be extracted precisely. We contacted the corresponding authors in cases when additional data were required [27, [44][45][46][47][48][49][50][51][52][53], but most of them did not reply after the second email. One author [47] replied and wrote that disclosure of raw data is against their policy. One message was returned as undelivered [44].
The antifungal efficacy of MTA Fillapex was studied in five studies [33, 45,46,48,54] and the results are shown in Table 2.
It was not possible to make a uniform conclusion for this group of studies about the comparative efficacy of MTA Fillapex in terms of its antibacterial and antifungal effects because the studies were highly heterogeneous-using different comparator materials, different research methods and different microbial species.

Endosequence BC Sealer, iRoot SP and Totalfill BC Sealer.
After the introduction in 2009 of Endosequence BC Sealer, also known as iRoot SP [63], to the North American market, Totalfill BC Sealer, a material with the same composition, was introduced in Switzerland  In three studies, [65][66][67] ADT was used to investigate the antimicrobial efficacy of Endosequence BC, and three studies used CLSM [37,39,42]. Also, three studies used DCT [35, 51,65] and one used scanning electron microscopy (SEM) evaluation [44].
Two studies investigated the antibacterial efficacy of iRoot SP. One of them [27] studied only efficacy against E. faecalis, and another against E. faecalis and Staphylococcus aureus (S. aureus) [47]. Antifungal activity was studied in two studies [45,47]. All studies were conducted on planktonic cells [27, 45,47].
DCT was used in the studies of Ozcan et al. [45] and Nirupama et al. [47] and a modified direct contact test (MDCT) was used in the study of Zhang et al. [27].
Five studies investigated the antimicrobial efficacy of Totalfill BC sealer [10,41,43,50,56]. Two studies [50,56] used planktonic cells in ADT and DCT. Kapralos et al [10] used planktonic cells in MDCT and young biofilms in DCT and the membrane restricted test (MRT).  [44] used SEM to explore bacterial growth, but there was no control group and the results were not clearly reported.
The results of all studies that analysed the antibacterial activity of Endosequence BC Sealer, iRoot SP and Totalfill BC Sealer are shown in Table 3.
It was not possible to make a uniform conclusion for this group of studies about the comparative efficacy of Endosequence BC Sealer, iRoot SP and Totalfill BC Sealer in terms of their antibacterial activity because the studies were highly heterogeneous-using different comparators, different research methods and different bacterial species.
Ozcan et al. [45] showed that freshly mixed iRoot SP produced a significant (p < 0.05) reduction in fungal growth which was not significantly different (p > 0.05) from that in the MTA Fillapex group. IRoot SP showed significantly better results when compared with freshly mixed GuttaFlow (Coltène-Whaledent, Langenau, Germany) (p < 0.05). Only freshly mixed AH Plus showed significantly higher antifungal efficacy than other sealers (p < 0.001). One and seven day old samples exhibited slight or no antifungal efficacy without significant differences between sealers and the positive control (p > 0.05). In the study of Nirupama et al. [47], iRoot SP was comparable with TubliSeal EWT and AH Plus and they had significant antifungal activity when compared to the positive control (p < 0.05).

BioRoot RCS.
Four studies [38,41,50,56] investigated the antibacterial efficacy of BioRoot RCS. All studies used E. faecalis. Two of them [50,56] studied efficacy on planktonic cells, and one [38] was conducted on planktonic cells and young biofilms while one studied efficacy on old biofilms [41]. As mentioned, Poggio et al. [56] and Colombo et al. [50] used DCT and ADT. In the study of Poggio et al. [56], BioRoot RCS exhibited a similar inhibition In the study of Colombo et al [50], BioRoot RCS showed the lowest antibacterial activity which was comparable with that of MTA Fillapex and Sealapex in ADT. Only EasySeal showed significantly higher efficacy compared to other sealers (p < 0.01). In DCT, BioRoot RCS showed the lowest activity after 6 min of contact, similar only to MTA Fillapex. Also, after 15 and 60 min, BioRoot RCS showed a significant increase in bactericidal effect (p < 0.05).
In the study of Arias-Moliz and Camilleri [38], ADT and intratubular infection tests (using CLSM) were used. Irrigation with ethylenediaminetetraacetic acid (EDTA) in combination with sealing with BioRoot RCS or AH Plus showed a significantly larger zone of inhibition against planktonic cells than MTA Fillapex in ADT. No inhibition zone was obtained when BioRoot RCS was exposed to phosphate-buffered saline (PBS) or water. In the intratubular infection test, BioRoot showed the highest antibacterial efficacy in all irrigation protocols. Irrigation with EDTA exhibited the highest number of dead cells, followed by water, without significant differences.
In the study of Alsubait et al [41], BioRoot RCS did not significantly differ from AH Plus and Totalfill after 1 day. After 7 days, BioRoot RCS showed the lowest antibacterial activity when compared with Totalfill and AH Plus. However, after 30 days, BioRoot RCS killed the highest percentage of bacteria which was significantly higher than in AH Plus (p = 0.000) and Totalfill groups (p = 0.04).
All studies used ADT while Morgental et al. [16] used DCT.
In the study of Morgental et al. [16], CPM Sealer in ADT was not able to inhibit E. faecalis as well as White MTA (Angelus, Londrina, Brazil). A greater inhibition zone was obtained in MTA Fillapex and Endofill (Dentsply, Petrópolis, Brazil) groups. As for DCT, all sealers were similar to the negative control in all experimental periods (p > 0.05).
Tanomaru et al [69] reported mean inhibition zones for six different materials, one of which was CPM. However, statistical analysis was not performed due to different degrees of diffusion in agar among the different materials. Thus, it was not possible to compare the sealers investigated.
The results of the study of Mohammadi et al. [68] are not clear because the data for two sealers that were not previously reported in the methodology are described in the results section.
3.2.5. Smartpaste Bio. Smartpaste Bio was studied in just one study [33]. The microorganisms tested were: E. faecalis (ATCC 29212), S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), Escherichia coli (E. coli) (ATCC 25922) and the fungus C. albicans (ATCC 10231). The test used in the study was ADT. Smartpaste Bio showed significant inhibition of bacterial growth (p < 0.05) at all time points, except on P. aeruginosa where AH plus showed better efficacy. MTA Fillapex showed significantly lower antimicrobial efficacy (p < 0.05) than Smartpaste Bio. All sealers tested showed decreased antimicrobial activity after a prolonged time period.

Discussion
We found 37 studies about the antimicrobial efficacy of bioceramic root canal sealers. However, despite this large number of studies, it was not possible to make conclusions about the comparative efficacy of bioceramic sealers because these studies were highly heterogeneous. Since these studies used different sources and ages of microorganisms, different setting and contact times of sealers and different antimicrobial tests, they could not be directly compared, even when they studied the same bioceramic sealers. We were unable to find two studies which used exactly the same experimental conditions, and therefore we were only able to conduct a narrative analysis. Even though this kind of evidence precludes making any conclusions for practice, that could help practitioners in choosing the best bioceramic sealer, our study has unearthed a number of issues that warrant further attention for researchers in this field.
Firstly, there are different classifications of bioceramic root canal sealers. Although many studies investigated these materials and described their compositions, we found only two reviews [17,70] where their classification was suggested, and in these two the classifications were different. Al-Haddad and Che Ab Aziz [17] divided bioceramic materials into three subgroups: calcium silicate-, MTA-and calcium-phosphate based materials, while Jafari and Jafari [70] described only two subgroups: calcium-silicate based (MTA-and non-MTAbased) and calcium-phosphate based materials. We recommend clear classification in order better understand bioceramic materials.
Moreover, although several systematic reviews [11,[70][71][72] discussed the antimicrobial efficacy of bioceramic sealers, none of them provided a broader view of antimicrobial activity. Alshwaimi et al. [11] included only studies on E. faecalis where DCT was used while Almeida et al. [71] included only studies which compared the antimicrobial activity of bioceramic and conventional materials. Also, Jafari and Jafari [70] and Donnermeyer et al. [72] provided little information about antimicrobial activity.
Secondly, most of the included studies investigated the antimicrobial efficacy of a single microorganism-E. faecalis, because of its ability to penetrate deep into dentin tubules, form biofilms, survive nutrition deprivation and resist commonly used disinfection agents [73][74][75][76][77][78]. Also, results from earlier studies suggest that fungi [79,80] could be associated with persistent apical periodontitis, but only a few studies investigated the influence of bioceramic sealers on fungi. Therefore, a recommendation for further studies is to investigate the efficacy of root canal sealers on fungi and on other bacteria lineage which may also be responsible for the failure of root canal treatment [10,43,44,46,47,55,[67][68][69].
Furthermore, despite recent recommendations from 2012. by De Deus [81], published as an Editorial in International Endodontic Journal, to use only mature biofilms in such studies, only six [37,39-43] of the included studies investigated the antimicrobial effectiveness of bioceramic sealers on mature biofilms. In this review, we defined young and mature biofilms according to the study of Stojicic, Shen and Haapasalo [4] but there remains need for general consensus on a suitable model of endodontic biofilm age still remains for future studies.
De Deus [81] also recommended that the conditions used should be similar to those in the filled root canal. Hence, older tests like ADT and DCT should be replaced with newer methodology.
It has already been shown that ADT has limitations-such as dependency on the solubility and diffusion characteristics of the test material and media, and it has been proposed that it is only used to test water-soluble materials [58]. However, it is still widely used, as shown in many studies we included [16,34,35,38,48,50,52,53,[56][57][58]60,61,65,66,68,69]. Another commonly used test was DCT. Its limitations are an inability to use freshly mixed sealers because they may adhere to substrate [40], and it does not allow evaluation of microorganisms in biofilms [47,82]. Recently, new technology using CLSM has been introduced [37-39,41,42]. Used with bacterial viability staining, this model might be suitable for measuring the antimicrobial activity of root canal sealers in infected dentin against microorganisms associated in biofilm [39].
It is also worth emphasizing that the included studies used different setting times of sealers and contact times between sealers and microorganisms. It would be worthwhile defining time points within different stages of material setting and important points during contact time. However, it is also disputable whether certain tests could be performed for a prolonged period when it is known that microorganisms could die spontaneously due to environmental conditions [37].
In conclusion, multiple in vitro studies have shown that bioceramic sealers may have various degrees of antimicrobial activity. However, it is still impossible to make conclusions about their comparative efficacy and to recommend the use of one over another in clinical practice because the studies available were conducted in different way, which makes meta-analysis futile. A uniform methodological approach, consistent definitions and studies on humans are urgently needed in this field of research so that recommendations for practice can be made.