The comparative of chitosan and chitosan nanoparticle versus ethylenediaminetetraacetic acid on the smear layer removal: A systematic review and meta‐analysis of in vitro study

The purpose of this systematic review of meta‐analysis was to compare the effectiveness of removing the smearing layer using EDTA versus Chitosan (Ch) and Chitosan nanoparticles (Ch‐NPs). A search was performed in four electronic databases (Web of Science, PubMed, Scopus, and Cochrane). The included studies were assessed by two reviewers using Joanna Briggs Institute's critical appraisal checklist for the quasi‐experimental studies. Outcomes obtained by scanning electron microscopy (SEM) and conventional methods were presented as standardized mean differences alongside 95% confidence intervals. Seven investigations employed 212 single‐root teeth. In the apical section (p = .317, 95% CI = −0.820 to 0.266, Tau2 = 0.387), middle segment (p = .914, 95% CI = −1.019 to 0.912, Tau2 = 1.027), and coronal segment (p = .277, 95% CI = −1.008 to 0.289, Tau2 = 0.378). This meta‐analysis found no difference between Ch, Ch‐NPs, and EDTA in removing the smear layer in the three segments. This systematic review is designed to show evidence related to the PICO question, in which our outcome is smear layer removal and not the clinical success of such a treatment.

comparing the effects of chitosan or chitosan nanoparticles with EDTA on smear layer removal.
• The results encourage further exploration of chitosan and chitosan nanoparticles for clinical use in dentistry, while considering their specific applications and longterm effects.

| INTRODUCTION
Several factors influence the success of root canal therapy, including biomechanical preparations, chemical root canal cleansing using irrigation solutions, intracanal medicaments, and obturation of the root canal.In root canal treatment, one of the most important things to do is to use irrigation solutions to clean the root canal.The goal of root canal irrigation is to help with the cleaning of the root canals and to get rid of microorganisms and their products (Buldur & Kapdan, 2017;Haapasalo et al., 2014).
The endodontic tools create smear layers and smear plugs on the dentinal tubules, whether they are employed manually or automatically.Thin smear layers of varying thicknesses are generated in 0.5-2 μm (Ratih, Sari, et al., 2020).The smear layer compromises disinfection by harboring microorganisms and necrotic debris, restricting irrigant penetration and providing avenues for microleakage following root canal obturation (Shahravan et al., 2007;Virdee et al., 2018).It consists of organic and inorganic components.The clinicians prefer the combined use of sodium hypochlorite (NaOCl), an organic tissue dissolving agent, and ethylenediaminetetraacetic acid (EDTA), a chelating agent, because of sufficient lack of a single irrigant for its removal (Virdee et al., 2018;Yamada et al., 1983).
NaOCl at concentrations of 2.5%-5% is a standard irrigation solution in endodontics.Because NaOCl may remove organic tissues but not inorganic tissues from the smear layers on the root canal walls, additional irrigation solutions must be used, such as the last irrigation solution (Darrag, 2014).EDTA is often used in clinics and is considered the gold standard for the final irrigation solution because it can bind to calcium ions in dentin and dissolve the inorganic part of smear layers (Koga et al., 2015).Dentin erosion and diminished microhardness may occur if EDTA is applied to the dentinal wall for an extended period (Ratih, Enggardipta, & Kartikaningtyas, 2020).In addition, EDTA is a pollutant and does not possess any antibacterial characteristics (Berástegui et al., 2017).
Various irrigation solutions have recently been introduced to overcome the disadvantages of conventional irrigation solutions.
These are maleic acid and citric acid.The disadvantage of these solutions is that they are insufficient on their own to remove the smear layer.They need extra irrigation solutions such as NaOCl to remove the smear layer.Additionally, the antibacterial feature is limited.MTAD (mixture of tetracycline citric acid and detergent) was introduced to eliminate these limitations.MTAD has disadvantages such as being toxic and limited antibacterial effects.In addition, QM _ IX (mixture of EDTA and chlorhexidine) is introduced currently as an irrigation solution.It has good antibacterial properties and the ability to remove the smear layer.Ch is presented as an alternative to all these irrigation solutions (Küçük et al., 2016).
Chitosan, derived from deacetylated chitin, is a natural antibacterial biopolymer with non-toxic properties.It holds potential in dentistry for its antimicrobial nature and ability to form bonds with dentin's calcium, aiding remineralization.As a biocompatible, biodegradable agent, chitosan acts broadly against bacteria.Its antimicrobial potency is influenced by solvent activity and molecular weight, intensifying at lower pH (No et al., 2002;Xu et al., 2011).Chitosan has been utilized as an irrigation solution in root canal treatment, effectively removing smear layers while displaying antimicrobial effects.Chitosan's chelating properties have two competing explanations.The "chemical chain bridge model" suggests multiple amino groups in a chitosan chain bind to a metal ion, while the "hook or free-arm model" proposes the metal ion attached to an amino group is the sole engaged one.Chitosan, composed of chitin dimers, uses free electrons for metal interaction in this nitrogen-based dimer.
Recent research indicates that nanoparticles, defined as particles below 100 nm, exhibit broad antibacterial activity (Samuel & Guggenbichler, 2004).Biocompatibility, as described by Williams (2008), pertains to nanoparticles' capacity to function biologically without causing harm to patients, locally or systemically.It refers to nanoparticles behaving compatibly with living organisms.

| Chitosan solution preparation
To prepare the chitosan solution with a concentration of 0.2%, 0.2 g of chitosan with a degree of deacetylation of 90% was diluted in 100 mL of acetic acid with a concentration of 1%, and the mixture was stirred with a magnetic stirrer for a period of 2 h.

| The characterization of chitosan nanoparticles
Ch-NPs are described as follows: Ultraviolet-visible spectroscopy (UV-Vis): To verify nanoparticle production, the solution is scanned in the range of 200-600 nm with water as the reference.
Scanning electron microscopy (SEM): The size and shape of dried chitosan nanoparticles are analyzed in the SEM instrument.
Dynamic light scattering (DLS): It is the method that is used to determine the average particle size of nanoparticles.Through dynamic light scattering (DLS) and using a zetasizer, the particle size distribution and zeta potential of chitosan nanoparticles are determined.
Despite an abundance of literature reporting on the smear layer removal efficiency of EDTA versus Ch outcomes are often conflicting.
Additionally, to the best knowledge, there had been no previous systematic reviews addressing the question of whether using Ch as the final irrigation solution leads to more favorable outcomes than using EDTA.Therefore, the initial aims of this study were to use metaanalysis to examine the effectiveness of eliminating the smear layer of Ch-NPs against EDTA.Thus, the second purpose of this study was to compare the effectiveness of Ch against EDTA in eliminating the smear layer.
The first tested null hypothesis was that there was no difference between Ch-NPs versus EDTA in the efficacy of removing the smear layer by using meta-analysis.With meta-analysis, the second null hypothesis was that there was no difference between Ch and EDTA in how well they eliminated the smear layer.

| Protocol
The systematic review protocol was carried out according to the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) statement (Page et al., 2021).The systematic review protocol was registered in the PROSPERO database under no.CRD42022353699.

| Study design and eligibility criteria
This systematic review was to analyze the experimental trial that aimed to answer the following guiding question: Are there differences in efficiency on smear layer between chitosan, chitosan nanoparticle, and EDTA used as a chelating agent during the root canal treatment?
The questions were structured according to the PICOS format.
P: Extracted fully formed (mature) human teeth.
I: chitosan and chitosan nanoparticles.
S: SEM studies in the laboratory.
Studies with missing or unclear data were excluded.The search strategy was limited to English and Turkish articles published before February 17, 2022.S1 presents all search strategies with the yields.

| Selection process
The searching process was performed in three different stages.In the first stage, the results obtained through the search were managed systematically using EndNote X9 software for Windows.Then, duplications were removed by the EndNote X9 software and manually.In the second stage, the two independent authors (EBC, HI) calibrated based on the inclusion criteria and scrutinized the titles and their respective abstracts.Unrelated studies were removed.In the final stage, the authors scrutinized the data to confirm whether they fulfilled the inclusions criteria.The authors were not blinded to the names of authors, countries, and journals.Agreement between the authors was calculated by using the Cohen kappa statistics.Any discrepancies or disagreements were resolved by discussion with a senior reviewer (FC).

| Process of data collection and extraction
The analysis process was carried out on all studies that met the inclusion criteria.It was limited to those where outcomes were quantitatively presented as p values, means, and standard deviations, or in a form allowing for a manual calculation.

| Data item
Data were extracted according to the following information: author, year of publication, country, type, and the total number of samples magnification range of SEM, control and intervention groups, irrigation regime, scoring system, canal segments, and results.As several different smear scoring systems were used in included studies, standardized mean differences (SMD) were calculated to allow direct comparisons among these studies.

| Risk of bias in individual studies
Quality of individual studies was used to categorize the level of evidence.Joanna Briggs Institute's critical appraisal checklist for the quasi-experimental studies was used for evaluating the quality of individual studies.It consists of nine questions requiring a yes or no/unclear response which equated to 1 and 0 points, respectively.
Scores of ≥7 were deemed the low risk of bias, 5-6 medium risk, and ≤4 high risks.Egger tests were used to evaluate bias across studies.

| Data synthesis
All analyses were performed using the Comprehensive Meta-Analysis (CMA version 2.0).The standardized mean difference (SMD) was calculated for the continuous variable with a 95% confidence interval (CI).If I 2 ≤ 50%, then the fixed-effect model was used; otherwise, the random-effect model was used.The forest plots were calculated considering 95% of CI and p-values.

| Study selection
The reviewers obtained a total of 186 articles from four databases (PubMed, Scopus, Web of Science, and Cochrane library) and manual searching.These articles were exported to endnote software and then 13 duplicated studies were removed.The remaining studies were exported to Microsoft excel 2013 for windows and then 8 duplicated studies were removed.Thus, 165 articles remained.Two independent reviewers read to the title of them and removed 138 irrelevant studies.The reviewers read the abstract of the remaining studies and excluded 12 studies because of dentin microhardness, sealer dentin tubules penetration (Antunes et al., 2019;Aydin et al., 2016;Aydın et al., 2019;Bayram et al., 2017;Choudhury et al., 2020;Gokturk & Ozkocak, 2022;Gyulbenkiyan et al., 2020;Kesim et al., 2018;Prasanthi et al., 2019;Ratih, Sari, et al., 2020), has not EDTA group (Silva et al., 2012), and lack of a SEM study (Mathew et al., 2017).
Finally, 15 studies for the full-text assessment were analyzed and only seven studies (Darrag, 2014;_ Ilhan, 2019;Kamble et al., 2017;Kaur et al., 2020;Madhusudhana et al., 2015;Penumaka et al., 2019;Sari et al., 2018) were considered eligible.The reasons for excluding four studies were missing data sets (Gusiyska et al., 2016;Praveen et al., 2017;Sarkees & Al-Maarrawi, 2020;Silva et al., 2013).Although the effect of Ch-NPs and EDTA on the smear layer was examined in two studies (del Carpio-Perochena et al., 2015;Ratih, Enggardipta, & Kartikaningtyas, 2020), they did not have the adequate data set to do a meta-analysis.They were excluded for this reason.We attempted to connect by email with authors whose studies have missing data.For this process, we sent two separate emails at an interval of 15 days.
None of these authors replied with any positive or negative responses.One of the excluded studies relied only on qualitative analysis (Pimenta et al., 2012).Another study compared EDTA alone with a mix of Ch and EDTA in removing the smear layer.We did not include this study because the effect of each could not be determined (Geethapriya et al., 2016) (Figure 1).Inter-reviewers agreement abstracts κ = 0.84 ± 0.03; full-texts: κ = 0.68 ± 0.06.

| Study characteristics
Two hundred twelve single root teeth (n = 106 in Ch groups and n = 106 in EDTA groups) were used in seven studies.The magnification range was selected between 1000Â and 3000Â to evaluate intra canal cleanliness under SEM.While EDTA concentration was preferred 17% all of them, Ch concentration was employed 0.2% in six studies (Darrag, 2014;Kamble et al., 2017;Madhusudhana et al., 2015;Penumaka et al., 2019;Sari et al., 2018) and 1% in one study (Kaur et al., 2020).The scoring system developed by Hülsman (Hülsmann et al., 1997) in three studies ( _ Ilhan, 2019;Penumaka et al., 2019;Sari et al., 2018) the scoring system developed by Torabinejad et al. (2003) in two studies (Kaur et al., 2020;Madhusudhana et al., 2015), and the scoring system developed by Kanter et al. (2011) in two studies (Darrag, 2014;Kamble et al., 2017) were used.The NaOCl was used when the root canals were prepared.Ch as a final irrigation solution has used a range of volume 1-5 mL and range of time 1-5 min.EDTA as a final irrigation solution has used a range of volume 1-5 mL and range of time 30 s 5 min.The root canals were prepared up to size 40.The evaluation of the smear layer was carried out on apical segments in two studies (Kamble et al., 2017;Penumaka et al., 2019).The others in this evaluation were performed on apical, middle, and coronal segments (Darrag, 2014;_ Ilhan, 2019;Kaur et al., 2020;Madhusudhana et al., 2015;Sari et al., 2018) in Table 1.

| Risk of bias in studies
The quality of included studies in the meta-analysis was scored independently based on the standardized Joanna Briggs Institute (JBI) critical appraisal checklist for quasi-experimental studies (Institute, 2017).

| Results of individual studies
Single root and single canal teeth were selected and randomly distributed in all included studies.However, it was unclear how the other properties (root canal curvature, oval or not, etc.) that affect the removal of the smear layer of the teeth were distributed among the groups.There was no control group in one study (Sari et al., 2018).The sample size was calculated as preoperative in one study (Penumaka et al., 2019).The evaluation of interobserver was carried out in one study ( _ Ilhan, 2019).
We used the random effect model since the apical to coronal I 2 values were 73.055, 85.398, and 69.617, respectively.The effect of Ch and EDTA did not have a statistical significance impact on the smear layer in the apical segment (p = .317,95% CI = À0.820 to 0.266, Tau 2 = 0.387) Figure 2, in the middle segment ( p = .914,95% CI = À1.019 to 0.912, Tau 2 = 1.027) Figure 3, and coronal segment p = .277,95% CI = À1.008 to 0.289, Tau 2 = 0.378) in Figure 4.The results of this meta-analysis showed high heterogeneity for each segment.We removed the studies one by one to determine the source of the heterogeneity.Then, the meta-analysis was rerun.I 2 was less than 50 when only one study ( _ Ilhan, 2019) was excluded in the coronal segment.We found I 2 = 2.91 and Tau = 0.073 when this study was excluded.We selected a fixed effect model because I 2 = 2.9.Metaanalysis was rerun and found p = .725,95% CI (À0.486 to 0.338).
The study of Sehitoglu and Cakici was the study that most affected heterogeneity in the middle segment.We found I 2 = 72.089,Tau = 0.638, and p = .205,CI (À1.217 to 0.261) when it was excluded.The study of Kaur et al. was the study that most affected heterogeneity in the apical segment.We found I 2 = 54.90,Tau = 0.42, and p = .717,CI (À0.546 to 0.375) when it was excluded.
Although studies causing heterogeneity were removed, the relationship between Ch and EDTA did not change in all segments.Egger's (Egger et al., 1997) test was conducted to assess publication bias, indicating no significant publication bias in the apical to coronal segment p = .74714;p = .07437;p = .32831,respectively.

| DISCUSSION
The first null hypothesis of the meta-analysis was not tested due to missing data.p-values apical to coronal are p = .317;p = .914;p = .277,respectively.Therefore, the second null hypothesis of the meta-analysis was accepted.The use of conventional SEM methods has some restrictions.When the smear layer is examined with the SEM method, the process of mounting, sectioning, and gold sputtering teeth is done.These processes could potentially affect the remaining debris or smear layer on root canal walls.Results may vary depending F I G U R E 2 Forest plots for smear layer removal in the apical.
F I G U R E 3 Forest plots for smear layer removal in the middle.
on the area to take an image.If the area selected to take the image was not touched by the instruments, the results may give a false representation.For example, if the dentinal tubules are closed and/or destroyed for physiological or pathological reasons, the results of the study may be affected (Paqué et al., 2005;Peters et al., 2003).These factors need to be considered in the planning of the study, but it was not considered in the included studies.A lack of pre-and postoperative measurements could potentially cause introduce bias (Virdee et al., 2018).Additionally, a variety of images were taken, ranging from 1000Â to 3000Â magnification.For this reason, interpreting the results of studies is difficult.
Two hundred twelve teeth were used in included seven studies.
The sample size in these studies was not calculated.The experimental procedures were carried out with a small number of samples.A small Unfortunately, we were unable to include any Ch-NPs studies because they did not have suitable data sets for meta-analysis.Additionally, a lack of knowledge of nanomaterial characterization in dental studies is a particularly problematic issue.For these kinds of investigations, the researchers need to have a much deeper understanding of these nanomaterials.As an illustration, the nanoparticle synthesis process may produce several sizes of chitosan, and surface modification each with its own unique properties.As a result, all chitosan nanoparticles cannot display the same characteristics, and you may not get the same results in other studies.
As a result of the meta-analysis of the included studies, there was no difference between the Ch and EDTA effect on the removal of the smear layer in the three segments.Chitosan is a natural final irrigation solution, which has attracted attention in endodontics because of its biodegradability, biocompatibility, antibacterial, bio adhesion, and lack of toxicity.Ch and Ch-NPs can be an alternative option instead of EDTA with these properties (Akıncıbay et al., 2007;Senel et al., 2000).
Calculating sample size, using a curved canal, using an oval canal, preferring the appropriate rate and time for final irrigation solutions, and calculating agreement between the authors must be considered in future research.
Four databases were searched in this meta-analysis.There were seven studies corresponding to the inclusion criteria.The quality of them was medium and high risk.Low-risk studies are needed for a high level of evidence in the future.Therefore, when planning laboratory studies, the guides prepared by experts should be followed.

| CONCLUSION
Within this meta-analysis limitations, there was no difference between the Ch and EDTA effect on the removal of the smear layer in the three segments.Therefore, Ch and Ch-NP may be used as an F I G U R E 4 Forest plots for smear layer removal in the coronal.
alternative material to EDTA in terms of the effectiveness of eliminating the smear layer in root canal treatment.The purpose of this systematic review is to demonstrate evidence related to the PICO question, in which the elimination of the smear layer serves as the result rather than the treatment's clinical efficacy.

Four
databases, relevant to formally published research in endodontics, were searched systematically by two independent authors (EBC, HI) up to February 17, 2022, using the broad keywords and MeSH terms.The PubMed (including MedLine), Web of Science, Scopus, and Cochrane databases were searched.Furthermore, we manually searched with the assistance of the references of the eligible articles and previous reviews.All steps were performed to minimize selection and publication biases.Table sample size does not suitable to represent the whole population, although it has advantages such as lower costs and faster results.This situation results in a limitation in the studies.Single root teeth were used in included studies.They have no information about the root canal ovality.Whereas an oval root canal may have an unprepared area after the root canal preparation.It may affect the results.This should be considered when creating a research question.The effectiveness of irrigation solutions is important in removing the smear layer.Root canals must be of sufficient diameter for irrigation solutions to rinse effectively.Since the root canal preparation is made in a conical shape, the narrowest diameter of the root canal is the apical and the widest diameter is the coronal part of the root.Therefore, the part where the smear layer is removed the least is apical.Our data set showed that the smear layer removal score was the biggest in the apical part of the root.Although EDTA causes erosions and a decrease in dentin microhardness in more than 1 min of use,Silva et al. (2012) reported that 0.2% Ch for 3 min was efficient in removing the smear layer while causing little erosion of dentin.This contrasted with EDTA, which causes erosions and a decrease in dentin microhardness in more than 1 min of use.The usage time of final irrigation solutions must be considered when planning the future studies.
Methodological characteristics and critical appraisal of all studies included.Quality of the included studies.
F I G U R E 1 Flow diagram of the systematic literature search according to PRISMA 2020 guidelines.T A B L E 1