Bioactivity of Bioceramic Materials Used in the Dentin-Pulp Complex Therapy: A Systematic Review

Dentistry-applied bioceramic materials are ceramic materials that are categorized as bioinert, bioactive and biodegradable. They share a common characteristic of being specifically designed to fulfil their function; they are able to act as root canal sealers, cements, root repair or filling materials. Bioactivity is only attributed to those materials which are capable of inducing a desired tissue response from the host. The aim of this study is to present a systematic review of available literature investigating bioactivity of dentistry-applied bioceramic materials towards dental pulp stem cells, including a bibliometric analysis of such a group of studies and a presentation of the parameters used to assess bioactivity, materials studied and a summary of results. The research question, based on the PICO model, aimed to assess the current knowledge on dentistry-based bioceramic materials by exploring to what extent they express bioactive properties in in vitro assays and animal studies when exposed to dental pulp stem cells, as opposed to a control or compared to different bioceramic material compositions, for their use in the dentin-pulp complex therapy. A systematic search of the literature was performed in six databases, followed by article selection, data extraction, and quality assessment. Studies assessing bioactivity of one or more bioceramic materials (both commercially available or novel/experimental) towards dental pulp stem cells (DPSCs) were included in our review. A total of 37 articles were included in our qualitative review. Quantification of osteogenic, odontogenic and angiogenic markers using reverse transcriptase polymerase chain reaction (RT-PCR) is the prevailing method used to evaluate bioceramic material bioactivity towards DPSCs in the current investigative state, followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential. Mineral trioxide aggregate and Biodentine are the prevalent reference materials used to compare with newly introduced bioceramic materials. Available literature compares a wide range of bioceramic materials for bioactivity, consisting mostly of in vitro assays. The desirability of this property added to the rapid introduction of new material compositions makes this subject a clear candidate for future research.

The aim of this study is to present a systematic review of available literature investigating bioactivity of dentistry-applied bioceramic materials towards dental pulp stem cells; including a bibliometric analysis of such group of studies and a presentation of the parameters used to assess bioactivity, materials studied and summary of results.

Materials and Methods
This systematic review was conducted in accordance with the PRISMA guidelines or preferred reporting items for systematic reviews and meta-analyses [18]. Our review was not eligible for registration with PROSPERO, as it did not involve health studies in which participants were people nor animal research studies exclusively.
In terms of the research question, based on the PICO model, our review aimed to assess the current knowledge on dentistry-based bioceramic materials by exploring to what extent they express bioactive properties in in vitro assays and animal studies when exposed to dental pulp stem cells, as opposed to a control or compared to different bioceramic material compositions, for their use in the dentin-pulp complex therapy.

Inclusion and Exclusion Criteria
Studies assessing bioactivity of one or more bioceramic materials (both commercially available or novel/experimental) towards DPSCs were included in our review. We established bioactivity assessment as any test or measurement for odontogenic, osteogenic, angiogenic and/or mineralization potential of DPSCs exposed both directly or indirectly to bioceramic materials. Studies assessing cytotoxicity and/or biocompatibility alone i.e., cell viability or proliferation were excluded. Studies assessing any other type of stem cell apart from DPSCs were also excluded.
The series of inclusion and exclusion criteria were established by a consensus reached from all authors after discussion, considering the research question and the objectives of the study while aiming for an ample range of results to be provided from the search.

Sources of Information
To identify potentially relevant studies, a thorough electronic search was made in PubMed, Web of Science, Scopus, Embase, Cochrane, and Lilacs databases. Study search was performed during October, November and December 2018. In particular cases, the authors of the articles were contacted by email to request missing information. The structured search strategy and data extraction were conducted by an individual examiner.

Study selection
Articles identified using the search terms were exported to RefWorks (ProQuest, MI, USA) to check for duplicates. Once duplicates were discarded, a first screening of record titles and abstracts

Study Selection
Articles identified using the search terms were exported to RefWorks (ProQuest, MI, USA) to check for duplicates. Once duplicates were discarded, a first screening of record titles and abstracts was carried out according to the previously described inclusion and exclusion criteria. Remaining studies were assessed for eligibility and qualitative synthesis by full-text screening.

Study Data
For the bibliometric analysis, the following variables were recorded for each article: author and year of publication, journal, country, and institution. For the synthesis of study methodology, a summary of the materials and methods of included studies was transcribed by listing the following variables: study type, bioceramic materials used, bioactivity analysis and duration of the analysis. For the synthesis of results, studies were categorized in terms of the significant results found, the duration in which these significant results were found, and their significance level.

Quality Assessment
The quality of the studies was assessed using a modified CONSORT checklist of items for reporting in vitro studies of dental materials [19] and the ARRIVE guidelines for reporting animal research [20].

Study Selection and Flow Diagram
The search identified 1023 preliminary references related to the bioactivity of bioceramic materials towards dental stem cells, of which 355 were found in PubMed, 473 in Web of Science, 179 in Embase, 15 in Scopus, and 1 in Cochrane databases. Search made in LILACS produced no results. After excluding 203 duplicates, the remaining 820 were screened. Of these, 783 were excluded on reading the title and abstract as they did not fulfil our inclusion criteria. The resulting 37 articles were examined at full-text level, and all of them resulted to be eligible for our review (Figure 2).

Bibliometric Analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.

Bibliometric analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.

Bioactivity Analysis
A wide range of analyses of bioactivity were presented from the included studies. The most common analysis was the quantification of the expression of odontogenic, osteogenic and/or angiogenic markers or genes using reverse transcription polymerase reaction (RT-PCR), followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential.
Other analyses include western blot, micro-computed tomography (micro-CT), scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), transmission electron microscopy (TEM), histological analysis, immuno-fluorescence, and immuno-histochemical assays. Bioactivity analyses alongside with their duration and a description of the study associated with them are presented in Table 1.

Bibliometric Analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.

Study Type
Articles included fell into two main categories in terms of type of study: in vitro, or animal study. In some cases, articles presented both an in vitro and an animal study [26,27,37]. There were two studies which analyzed bioactivity of bioceramic materials towards hDPSCs ex vivo [33,42].

Cell Variant
All studies included used dental pulp stem cells (DPSCs) as their cell variant to assess bioceramic material bioactivity.

Bioceramic Materials Used
Bioceramic materials studied ranged from commercially available ( Table 2) to novel or experimental materials (Table 3). A separate category was presented for bioceramic materials which were combined with an additive for their analysis (Table 4).

Quality Assessment
All in vitro studies analyzed using the modified CONSORT checklist [19] (Table 5) presented a structured abstract (item 1) and an introduction which provided information about the background of the bioceramic material and/or bioactivity analysis studied (item 2a). Within the introduction, the majority of studies presented clear objectives and hypotheses (item 2b). Description of methodology as well as of the variables studied was sufficiently clear to allow for replication in all studies (items 3 and 4), but none of them presented a detailed report of the calculation of sample size or random allocation sequence (items 5-9). All studies indicated the statistical method used (item 10), but presented significance level as p values, and not confidence intervals (item 11). Discussions generally included a brief synopsis of the key findings and comparisons with relevant findings from other published studies, but often failed to address the limitations of the studies, which we considered as a reason for non-fulfillment (item 12). Sources of funding (if any) were indicated in the majority of studies (item 13), and indications for access to full trial protocols were obviated in all studies (item 14). Table 5. Results of the assessment of in vitro studies by the use of the modified CONSORT checklist [19]. Cells marked with an asterisk "*" represent study fulfilment for the given quality assessment parameter. Cells left blank represent non-fulfilment.

Studies
Modified Only three out of the five animal studies analyzed using the ARRIVE guidelines [20] (Table 6) were headed with a sufficiently descriptive title (item 1), but all of them provided a detailed abstract (item 2). All studies provided sufficient scientific background (item 3a) and established clear objectives (item 4) in the introduction, but failed to justify the use of the animal species studied to address the scientific objectives (item 3b). Ethical statements were clear in all studies (item 5), and study design, experimental procedures were detailed enough in all except one (items 6 and 7). Details about the experimental animals and how they were distributed in the study design were included in every study (items 8-11 and 14), but housing and husbandry information was obviated in all cases (item 9). Both experimental outcomes and statistical methods were described in all studies (items 12 and 13). All studies reported the results for each analysis carried out with a measure of precision (item 15), but all of them failed to report baseline data about health status of the animals studied and any adverse effects they could have suffered after the experiment (items 14 and 17). Lastly, items referring to the discussion were fulfilled by all studies (items 18-20). Table 6. Results of the assessment of animal studies by the use of the ARRIVE guidelines [20]. Kyung-Jung et al. [26] * * * * * * * * * * * * * * * * Wongsupa et al. [27] * * * * * * * * * * * * * * * * * * Atalayin et al. [29] * * * * * * * * * * * * * * * Zhu et al. [37] * * * * * * * * * * * * * * * Daltoé et al. [50] * * * * * * * * * * * *

Study Tesults
Significant results from included in vitro studies are presented in Tables 7-17, and significant results from included animal research studies are presented in Table 18.
Studies comparing a bioceramic material and a non-bioceramic material did not show positive significant results for the bioceramic materials studied. One of the studies showed that DDM produced a greater bioactivity-related gene expression than HA-CPC [26]; and the other one showed mixed results for Ca 3 SiO 3 , which produced a greater expression of some markers but not others compared to Ca(OH) 2 [57] (Table 10).
Both studies comparing a bioceramic material and an additive with the bioceramic material itself showed positive significant results for the bioceramic material in combination with the additive (γION-CPC and αION-CPC, [24]; GNP-CPC, [25]) (Table 12).

Results for ALP Activity
There was only one study comparing a bioceramic material with MTA in terms of ALP activity, and it produced negative results for the bioceramic material studied (Quick-Set2, [31]). The rest of the studies compared two different biomaterials or different concentrations of the same bioceramic material (Table 14).

Results for Other Bioactivity-Related Analyses
Western blot analyses showed mixed results for Zn0/1/2/3 compared to a control [28], and a higher expression of bioactivity-related markers by PR-MTA compared to Quick-Set2, and by both of them compared to a control [31]. ATR-FTIR showed positive results for PR-MTA compared to Quick-Set2, and for both of them compared to a control [31]. ELISA showed mixed results for MTA and CEM [39]. Assessment of the level of grey in mineralization nodules using Gene Tool showed positive significant results for PLGA/TCP compared to PLGA/HA and PLGA/CDHA [42]. Lastly, both the TRACP & ALP assay kit (Takahara, Shiga, Japan) and the OC and DSP emzyme-linked immunosorbent assay kit (Thermo Fisher Scientific, Waltham, MA, USA) showed that the addition of polydopamine to PR-MTA produced better results than PR-MTA itself.   Table 11. Summary of the results of included studies showing significant differences between various bioceramic materials or different concentrations of the same bioceramic material for ARS staining.

Discussion
The attractiveness of bioceramic materials for their desirable properties added to their constant development, the demand for new advances and the ampliation of treatment indications results in an overflow of related literature over time. Therefore, it seems convenient to establish an updated and organized vision of the commercially available and experimental dentistry-applied bioceramic materials' characteristics. With this in mind, the aim of this study was to present a systematic review of available literature investigating bioactivity of these materials towards dental pulp stem cells.
In terms of results, it can be highlighted that the most common method used to assess bioactivity in the included studies was the expression of bioactivity-related markers using reverse transcriptase polymerase chain reaction or RT-PCR. A recent systematic review illustrates this tendency by assessing gene expression of dental pulp cells in response to tricalcium silicate cements [58]. Studies also tended to compare new bioceramic materials with the established mineral trioxide aggregate or the more recently introduced Biodentine, as shown in Table 2, in which they appear as the most studied materials.
The use of additives in combination with bioceramic materials looks promising, in some cases enhancing or positively influencing the material's results in bioactivity assays in comparison with the bioceramic material itself. For example, positive significant results have been shown for iron oxide [24], gold [25], and bioactive glass [32] nanoparticles in combination with calcium phosphate. However, we need to interpret these results with caution, being able to extrapolate them to clinical practice only when a clear dosage or ratio for the additive and bioceramic material has been established in controlled clinical trials.
New material compositions being studied also need to be taken into consideration for future investigations, as some of them have shown positive significant results in bioactivity assays. Novel materials like Exp. PPL [22], Gelatin-HA-TCP [23] and Zinc Bioglass (Zn0/1/2/3) [28] have all shown positive significant results for ARS staining and ALP activity assay compared to a control, and more specifically, Exp. PPL has shown a greater expression of DSPP and OCN compared to MTA and a control; Gelatin-HA-TCP has shown a greater expression of RUNX2, OSX and BSP compared to a control; and Zinc Bioglass (Zn0/1/2/3) has shown a greater expression of RUNX2, ON, CON, MEPE, BSP, and BMP-2 compared to a control. So again, in order to extrapolate these results to clinical practice, it would be interesting to carry out further studies investigating these biomaterials in different conditions. When assessing quality and risk of bias, included studies referred a similar structural pattern. They reported essential data like a sufficient abstract, a clear objective or objectives, a detailed description of methodology, a mention of the statistical tests used and relevant conclusions; but often failed to justify the sample size used, to describe the randomization process used (if any), and most importantly to address the study's limitations in the discussion. It may be worth noticing for future reviews that a checklist for reporting in vitro studies or "CRIS" guideline is under development [59] to address the need for uniform methodology in the assessment of this type of studies.
The introduction of new bioceramic materials and the use of additives in combination with them calls for updated research in the field. At the current state, bioactivity assessment of these materials towards dental pulp stem cells centers on in vitro assays or animal research at most. For future studies, it could be interesting to explore the mechanisms with which this bioactivity is achieved and move on towards in vivo trials.

Conclusions
Quantification of osteogenic, odontogenic and angiogenic markers using reverse transcriptase polymerase chain reaction or RT-PCR is the prevailing method used to evaluate bioceramic material bioactivity towards DPSCs in the current investigative state, followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential. Mineral trioxide aggregate and Biodentine are the prevalent reference materials used to compare with newly introduced bioceramic materials. Available literature compares a wide range of bioceramic materials for bioactivity, consisting majorly of in vitro assays. The desirability of this property added to the rapid introduction of new material compositions makes this subject a clear candidate for future research.