Effect of passive ultrasonic irrigation on hard tissue debris removal: a systematic review and meta-analysis

: Accumulated hard tissue debris (AHTD) in root canal irregularities may negatively impact adequate root canal disinfection. In light of this, the efficacy of passive ultrasonic irrigation (PUI) to reduce AHTD has been largely studied in in vitro studies, which have adopted different analytic methods of varying accuracy to determine the extent of AHTD more correctly. Therefore, the aim of this study was to compare how well PUI and non-activated irrigation (NAI) systems perform in reducing AHTD during final irrigation protocols, based exclusively on studies whose analyses used microCT scanning. A systematic search of the studies published up to April 2020 was performed using MeSH terms and free terms, in the following databases: PubMed, Scopus, Web of Science, BVS (Lilacs and BBO) and Embase. The inclusion criteria consisted of laboratory studies that evaluated the amount of AHTD, and compared PUI with NAI protocols using microCT analysis. The risk of bias in the selected studies was assessed critically by two reviewers. A meta-analysis was performed using the RevMan software program (P<0.05), and included studies providing the standardized mean difference (SMD), using a fixed effect model, and adopting a confidence interval of 95%. In all, 3495 studies were identified, three of which met the inclusion criteria. All three were considered as having a low risk of bias. The meta-analysis comparing the ability of PUI and NAI protocols to remove hard tissue debris showed a higher percentage of AHTD reduction (P<0.01) for PUI, with a confidence interval of 1.41 [0.79, 2.02]. The heterogeneity among the studies was 82% (I 2 ). Considering the limitations of the present study, this systematic review and meta-analysis showed that PUI was more effective than NAI in removing hard tissue debris, based exclusively on studies that used microCT scanning to provide a more precise analysis of the two techniques used. The findings presented in the present study reinforce the concept that PUI can increase residue removal and improve the cleanliness of the root canal in endodontic treatments.


Introduction
The root canal system has a complex anatomy with areas that cannot be touched mechanically by endodontic instruments, such as isthmuses, fins Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.and recesses in oval-shaped canals. 1,2Apart from this, the use of endodontic instruments in contact with dentin walls produces dentin debris that accumulates in root canal irregularities and their complexities. 3,4Residues such as pulpal tissue, infected dentin debris and microbial biofilm may remain inside the root canal system, and may interfere in the overall quality of root canal filling procedures, potentially contributing to endodontic treatment failure. 5,6,7Nevertheless, instrumentation alone cannot provide the complete removal of debris, even more so in the apical areas, where the use of irrigation protocols is key to achieving satisfactory outcomes for debris removal.
The accumulated hard tissue debris (AHTD) within root canal irregularities may be inaccessible to conventional syringe-and-needle non-activated irrigation (NAI). 6,8Additional supplementary methods, such as passive ultrasonic irrigation (PUI) using sodium hypochlorite (NaOCl) and/or ethylenediaminetetraacetic acid (EDTA), have been proposed to drive irrigant solutions into these root canal complexities. 9,10PUI consists essentially of transmitting energy from an ultrasonically oscillating instrument to the irrigant solutions in the root canal. 11mong the different irrigant activation techniques, PUI is widely used. 9,12,13Some studies showed that PUI improves the penetration, circulation and flow of the irrigant, thus promoting a significant reduction in AHTD from complex areas of the root canal system. 13,14,15nalysis of AHTD removal can be performed with different evaluation methods, including microcomputed tomography (microCT), scanning electron microscopy (SEM), and photomicrography, which can make the comparison of results among the studies more difficult. 16Furthermore, many studies that compare PUI and NAI have methodological limitations, such as differences in the volume of irrigant solution, and time of contact of the irrigant inside the root canal.][19][20] A previous systematic review determined the ability of PUI and NAI to reduce AHTD and the smear layer. 13However, SEM was used as the analytical tool in this particular review, bearing in mind that this imaging technique has been widely considered a non-trustworthy and non-reproducible method for these purposes in other studies in the literature. 21,22Accordingly, the results for the review in question might not be as accurate as those obtained exclusively by microCT, which has proven to be the most precise tool for quantitative and qualitative analysis of hard tissue debris removal in the root canal system. 8,23,24Therefore, considering the limitations of the Virdee et al. 13 study, and the lack of systematic reviews that evaluated the reduction in AHTD using microCT technology, the objective of this study was to compare how well PUI and NAI are able to reduce AHTD using microCT.In this context, this systematic review aimed to answer the following focused question: "Is PUI comparable to NAI for AHTD removal in mature extracted human teeth?"

Methodology Protocol and registration
This systematic review protocol was registered on The Open Science Framework, and is available at the following link: osf.io/e2pux (DOI: 10.17605/OSF.IO/7G8XP).This study was performed following the guidelines of the 2009 PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) statement (http://www.prisma-statement.org).

Search strategy
T he sea rch procedu res were per for med independently by two examiners (A.F.A.B. and C.O.L.).The PubMed, Scopus, Web of Science, BVS (Lilacs and BBO) and Embase electronic databases were searched for studies published up to April 2020, with no restrictions on language or year.The electronic search strategy was developed using the most frequently cited descriptors in previous publications on this topic, combining Medical Subject Heading terms (MeSH) and text words (tw).The following terms were combined for each database: "Ultrasonic irrigation," "Ultrasonic activation," "Ultrasonic therapy," "Ultrasonics," "Ultrasonic*," "Debris," "Hard tissue debris," and "Smear layer."The Boolean operators "AND" and "OR" were applied to combine the terms and create the search strategy.The search strategies defined for each database are detailed in Table 1.No filters or limits were applied in the searches.The setup alerts were programmed in all the databases used.A complementary screening of the references of the selected studies was performed, and a hand search was performed in the Journal of Endodontics and the International Endodontic Journal to find any additional studies that did not appear in the primary database search.Articles from different sources were imported to the EndNote Web reference manager (EndNote™), to catalogue the references and automatically remove duplicate records.

Eligibility criteria
Studies that evaluated the amount of AHTD and compared the PUI with the NAI protocol were included.The eligibility criteria was based on the PICOS strategy (PRISMA-P 2016), as follows: Studies that did not use microCT analysis were excluded, as well as those that did not use NAI as a control group, that had no standardized root canal preparation, and/or that did not use the same volume, composition, concentration or contact time of irrigant solutions for the NAI and PUI groups.Reviews, letters, opinion articles, case reports, serial cases, and studies that did not perform the chemo-mechanical step were also excluded.

Selection of the studies
Two authors (A.F.A.B. and C.O.L.) examined the titles and abstracts of the studies independently, and made a selection among those retrieved.The full text was accessed when the abstract and titles alone were not enough to determine the inclusion or exclusion of a particular study.The second stage of the selection consisted of reading the full texts, and determining whether the respective study would be included, based on the eligibility criteria using the PICOS strategy.In the event of a disagreement between the two examining authors regarding the inclusion of certain studies, the issue would be resolved

# 1 AND # 2 63
Embase #1 "Ultrasonic irrigation":ab,ti OR "Ultrasonic activation":ab,ti OR "Ultrasonic therapy":ab,ti OR "Ultrasonic therapy"/exp OR Ultrasonic*:ab,ti OR "Ultrasonics"/exp 227,175 #2 Debris:ab,ti OR "Hard tissue debris":ab,ti OR "Smear layer":ab,ti OR "Smear layer"/exp 87,747 consensually, and, if the two authors still failed to reach an agreement, the matter would be decided by a third experienced author (E.J.N.L.S).Duplicate studies in the database search were considered only once.

Data extraction
Two authors (A.F.A.B. and C.O.L.) collected the data independently from the included studies.Eventual disagreements regarding data collection were resolved consensually between the two authors, and, whenever no agreement could be reached, a third experienced author would decide (E.J.N.L.S).Information regarding publication (author and publication year), financial support, sample size, control group, amount of irrigant solution and statistical analysis were extracted.In case of missing data, up to three attempts were made by email to contact the respective authors.

Quality assessment
Each study selected was evaluated independently by two authors (A.F.A.B. and C.O.L.), in relation to inner methodological risk of bias.
As for the laboratory studies, a quality assessment was adopted, with adaptations used in previous systematic reviews. 25,26,27The following parameters were considered for the quality assessment of the studies included: a) sample size calculation, b) samples with similar conditions, c) control group, d) standardization of procedures, e) statistical analysis, and f) other risks of bias.In domain 1, the study was considered as having a low risk of bias whenever the sample size was calculated and described in the methodology section.In domain 2, the samples were considered similar if the same group of teeth had been used for the AHTD evaluation.Domain 3 evaluated whether the procedures in the control group (NAI) were similar to those of the PUI group, such as root canal preparation, similar volume, type of irrigant and depth of needle insertion.In domain 4, the low risk of bias of a study indicated NAI and PUI groups using standardized procedures, such as having the same instrument for root canal preparation, the same irrigant, the same volume and concentration of irrigant, and the same needle depth for both groups.In domain 5, there was low risk of bias when the statistical analysis was performed with the proper tests.Finally, in domain 6, the study was deemed as having low risk of bias if the irrigant used was similar to that used in clinical conditions, and if there was an industry involved in the sponsorship.
During the quality assessment, each parameter for all studies included was judged as having "low," "high," or "unclear" risk of bias.If a given study failed to match the aforementioned respective criteria in any said domain, it would be considered as having a high risk of bias for that given domain.Two independent authors analyzed the manuscripts included (A.F.A.B. and C.O.L.).In the event of a disagreement between the authors, the issue would be resolved by a third author (E.J.N.L.S.).If one or more parameters were considered "unclear," up to three attempts were made to contact the author of that study by email to obtain the information needed to ascertain risk of bias of that domain or domains, on an adequate basis.
Each of the six previously described domains was evaluated one at a time in each study, and color coded as follows: low risk of bias (green); high risk of bias (red); unclear (yellow).The general determination of the individual risk of bias of each study was designed as follows: studies with 5 or more green markers were considered as having a low risk of bias; 4 green markers meant moderate risk of bias; 3 green markers or fewer involved high risk of bias.

Meta-analysis
Quantitative analysis was carried out on the studies that provided data on the percentual reduction of AHTD, and the number of specimens used per group.The meta-analysis was presented as a standardized mean difference (SMD), with a fixed effect model, and a confidence interval of 95%. 28The meta-analysis was performed using the RevMan software program (version 5.2; the Nordic Cochrane Centre, the Cochrane Collaboration, 2012, Copenhagen, Denmark).The number of samples and the differences in the percentual reduction of AHTD between the groups were obtained, and the results of the meta-analysis were presented using a forest plot.The heterogeneity among the studies was tested using the Higgs index (I 2 ), and a forest plot was generated for these comparisons.

Selection of studies
Figure 1 shows the flow diagram of the search strategy.Initially, the search resulted in 3,495 studies published in the relevant databases.Of these, 1,725 were excluded due to duplicity.Analysis of titles and abstracts of the remaining 1,770 eligible papers culminated in the selection of 171 suitable, published studies.The main reason for rejection was the failure to meet the inclusion criteria, especially in relation to the lack of microCT analysis of AHTD.After a comprehensive reading, 157 studies were excluded due to absence of a microCT analysis; 9 due to absence of a NAI group; and 2 due to absence of human teeth.At the end, a total of 3 studies were selected for the present systematic review.The references of the selected studies were searched electronically, and then hand-searched, but no further articles were found.Since the two independent reviewers agreed upon the studies that were included, at every step of the selection process, there was no need for the third author to intervene.

Characteristics of the included studies
The data collected from the 3 included studies [29][30][31] are summarized in Table 2. Sample size calculations were performed in two of the studies. 29,30The study by Zhao et al. 31 did not perform any sample size calculation, thus justifying the high risk of bias stipulated in domain 1.All studies were evaluated in relation to the percentual volume of AHTD using microCT analysis, in mandibular molars.Moreover, all the studies presented NAI and PUI groups, and all used the same procedures for both groups, such as using instruments of the same tip diameter and taper in root canal preparation, and the same volume of irrigant and depth of the needle.One of the three studies chose distilled water as the irrigating solution to increase AHTD formation in root canal preparation, and did not use EDTA during the final irrigation, 30 while the other two studies used sodium hypochlorite (NaOCl) during all the steps of the endodontic treatment, and also used EDTA during the final irrigation procedures. 23,29This may justify considering the study as having a high risk of bias in this particular domain (other risk of bias).Nonetheless, all three studies 29,30,31 were considered as having a low risk of bias.The results are described in Figure 2.

Summary measures and meta-analysis
The three studies selected [29][30][31] reported quantitative results that allowed a quantitative analysis to be conducted.The meta-analysis was performed on how well PUI and NAI were able to reduce AHTD in the root canals.This was determined by considering the means and standard deviations in the percentual reduction of AHTD. Figure 3 shows the forest plot of the pooled comparison between PUI and NAI, which demonstrated a higher percentage of AHTD reduction (p < 0.00001) for PUI, with an SMD, and a confidence interval of 1.41 [0.79, 2.02].The heterogeneity among the studies was 82% (I 2 ).

Discussion
The meta-analysis showed that the PUI irrigation protocol can reduce greater percentages of AHTD from root canals than the NAI group.This finding can be attributed to the studies' being performed on mandibular molars, which present isthmuses and/or "C-shaped" root canals, hence having a more complex anatomy.Studies on the anatomical complexities of the mesial roots of mandibular molars have shown a prevalence of isthmuses of approximately 80% between 3-6 mm from the apex. 32These anatomical structures can retain debris from instrumentation. 33he PUI protocol has been described as an excellent auxiliary activation method especially in root canal irregularities and isthmuses, 29,34 since the transmission of energy caused by ultrasonic files induces the stream and cavitation of the irrigation solution, thereby eliminating the vapor lock. 11This induction should be further researched to determine whether these results are similar when dealing with teeth having simpler anatomies, considering that most studies with these types of teeth have found that PUI has little or no impact on the general outcome of endodontic treatment. 35ll the studies selected in this systematic review 29,30,31 performed the root canal preparation with single-use instruments of similar taper and tip size, thus helping to make the comparison.Regarding chelating agents, previous studies showed an additional effect of EDTA in removing AHTD. 17,36In this systematic review, the irrigation with a chelating agent (EDTA) was found to be performed in two 29,31 of the three selected studies.
The study by Leoni et al. 30 showed a 94.1% reduction in AHTD in the root canals following instrumentation with PUI.This result is considerably higher than that of the other studies included. 29,31his difference may be attributed to the irrigation in root canal preparation performed with NaOCl and EDTA at 2 mm from the working length in two of the studies.On the other hand, the irrigant of choice in the study by Leoni et al. 30 was distilled water, and the irrigation was performed only at the orifice level, and done deliberately to provide greater formation of AHTD after root canal preparation, according to the authors.This methodological difference can explain the greater formation and accumulation of AHTD after root canal preparation, which can influence the calculation of the percentual reduction in AHTD after the final irrigation protocol.However, the percentage of AHTD reduction in all the studies included presented similar values in the NAI group. 29,30,31In this case, Leoni et al. 30 did not find greater AHTD reduction values in comparison with the other studies. 29,31This could be because NAI was not able to reach the isthmuses in mandibular molars completely. 30Although irrigation during root canal preparation was performed with distilled water, which does not simulate clinical conditions, all the procedures in the study by Leoni et al. 30 were standardized between the groups (same irrigant, same volume of irrigant, depth of the needle and root canal preparation procedures), a strategy which allows comparison of the evaluated groups (PUI and NAI).Therefore, only the other risk of bias domain was considered as having a high risk of bias.
Although these studies are very similar, as stated before, there are differences that may justify the heterogeneity in their results (82%), such as the concentration and type of irrigating solution (NaOCl and distilled water), and the power settings of the ultrasonic devices and tips used.This highlights the relevance of standardizing experimental protocols to allow a more precise analysis of the various techniques applied.Although the NaOCl concentrations during final irrigation protocols were different among the studies (ranging from 1 to 2.5%), it is noteworthy to point out that the removal of AHTD does not seem to be a purely chemical phenomenon, but rather one with a strong physical component. 37,38 previous systematic review that used SEM analysis 13 to evaluate the removal of debris with different activation techniques, demonstrated that PUI improved debris removal in the middle third only when compared with NAI.However, the conventional SEM is a non-trustworthy and non-reproducible method for evaluating AHTD removal.21,22 The process of mounting, sectioning and gold sputtering the teeth could potentially affect the remaining debris or smear layer on the root canal walls. Moeover, root canal areas not touched during instrumentation may have been erroneously scored in those studies as areas of removed smear layer.21 In addition, another clear limitation of this method is that the evaluation is always subjective, qualitative and operator-dependent.22 On the other hand, the present study was based exclusively on published studies that used microCT analysis to compare PUI and NAI in their methodology.This is why so few studies were included in this review.It is important to emphasize that all three selected studies were considered as having a low risk of bias, and were well designed.29,30,31 These studies presented some similar methodological conditions; namely, they all used the same tip diameter and taper of instruments in the root canal preparation, and were performed on mandibular molars.The methodology in many previous studies did not include an NAI group, in which the traditional technique with syringes and needles was applied and used as a reference for the comparison.18,20, This flaw actually compromises any possibility of realistically assessing what advantages PUI may offer over the traditional irrigation protocol, since the studies lacking this comparison are likely to find superior results for PUI simply because the total volume of irrigating solution is greater after agitation, regardless of any actual influence wielded by the traditional protocol.In light of this, studies that do not offer the proper basis for an accurate comparison, as stated previously, should be considered unreliable in ascertaining the superiority of PUI performance over the traditional irrigation protocol. This s a critical issue, since our search was finally left with only three studies that met the inclusion criteria, and one of the main reasons for exclusion of the rest was that there was no adequate control group.
One limitation of the present systematic review is the reduced number of eligible studies.Further studies with an improved study design and proper methods of analysis (microCT) are required to raise the power of the external validity.The heterogeneity among the studies was high (82%), and the forest plot demonstrates that high heterogeneity was attributed to the study by Leoni et al. 30 It is suggested that irrigation with distilled water chosen by Leoni et al.
-unlike NaOCl as the irrigating solution and EDTA as the chelating agent chosen by the other authorswas responsible for the overall heterogeneity among the studies.In relation to the microCT analysis, previous in vitro studies have found that this is the most precise tool for quantitative and qualitative evaluation of AHTD and apically extruded debris within the root canal system. 8,23,24,39tudies involving PUI are based mainly on its use during the final irrigation.Nevertheless, a new technique has recently been proposed for improving removal of smear layer and debris from the root canal, whereby the irrigant is activated each time a file is removed from the root canal, and an irrigant is used. 40This approach has shown good results, but more studies are needed to provide a more definite conclusion 40 .Moreover, other studies should be devised to assess possible dentin erosion caused by the action of the metal tip used for PUI procedures. 41,42ased on the findings obtained herein, it remains clear that more in vitro studies should be performed using microCT analysis, and that they should also include control and experimental groups with standardized protocols, so as to enable greater reliability in future comparisons.

Conclusion
Considering the limitations of the present study, although no irrigation protocols are able to completely free the root canal system of AHTD, this systematic review showed that final irrigation protocols with passive ultrasonic irrigation were more effective than non-activated irrigation in removing such debris.The findings presented in this study reinforce the concept that PUI can increase residue removal and improve the cleanliness of root canals in endodontic treatment.

Figure 1 .
Figure 1.Flow diagram of the literature search and included studies.

Figure 2 .
Figure 2. Quality assessment of the included studies.

Figure 3 .
Figure 3. Forest plots of the pooled analysis comparing PUI and NAI.

Table 1 .
Search strategy for the databases.

Table 2 .
Qualitative analysis and characteristics of the included studies.