Effect of sodium hypochlorite solution and gel with/without passive ultrasonic irrigation on Enterococcus faecalis, Escherichia coli and their endotoxins

Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics . The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.


Introduction
Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics (Iqbal, 2012). It has been used since the second half of the 18 th century (Sedgley, 2004) because it has antimicrobial action (Luebke, 1967) and dissolves necrotic tissues (Taylor & Austin, 1918).
Enterococcus faecalis is a Gram-positive bacterium found in the root canal system (RCS) and can be disinfected by NaOCl (Siqueira et al., 2000). It may also be found in secondary infections of endodontically treated teeth (Machado et al., 2020). In addition, Escherichia coli, a Gram-negative bacterium, is also found in endodontic infections (Narayanan & Vaishnavi, 2010). The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without PUI on E. faecalis, E. coli, and their endotoxins, LTA and LPS, respectively.

Methods
This study was approved by the research ethics committee of São Paulo State University, Institute of Science and Technology (n o 1.504.995). The teeth used in this study were obtained from clinics where teeth are donated during routine procedures and following authorization of the patients. The research team presented the terms of donation by the clinics from which the teeth where obtained to the research ethics committee when submitting the study methodology. A total of 40 human lower premolars were collected (based on dimensional and morphological similarities).

Specimen preparation
To standardize root canal diameter, the teeth were initially instrumented with a #30 K-file (Maillefer, Ballaigues, Switzerland) and irrigated with 5 mL of NaOCl 1% for each file used. The canals were dried with sterile paper points (Dentsply Ind Com LTDA, RJ, Petrópolis, Brazil) and the apical region was sealed

Irrigation protocol (repeated three times in each third of the root canal) Final wash
NaOCl 5 mL of NaOCl 2.5% during instrumentation without PUI and then 5 mL remained in the canal without any activation.
NaOCl + PUI 5 mL of NaOCl 2.5% during instrumentation without PUI and then 5 mL activated with PUI 10 mL of 2.5% NaOCl solution activated with PUI.

NaOCl gel
Filled with 2 mL of 3% NaOCl gel and irrigated with 10 mL of saline solution during instrumentation without PUI.
Filled with 2 mL of 3% NaOCl gel and irrigated with 10 mL of saline solution without PUI activation.

NaOCl gel + PUI
Filled with 1 mL of 3% NaOCl gel and irrigated with 10mL of saline solution during instrumentation without PUI and then filled with 1 mL of 3% NaOCl gel and irrigated with 10mL of saline solution activated with PUI Filled with 2 mL of 3% NaOCl gel and irrigated with 10 mL of saline solution activated with PUI.

Sample collection
Three samples were collected during the experiment, as in (Maekawa et al., 2013): S1, at baseline to confirm biofilm formation; S2, immediately after treatment; and S3, after EDTA application. LTA was assessed using enzyme-linked immunosorbent assay using ELISA 96-well plates (Nunc Thermo Scientific, Waltham, MA, USA) sensitized with anti-LTA monoclonal antibody (manufacturer) and kept overnight at relative humidity. Next day, the plates were washed with a wash buffer (PBS with 0.05% Tween 20) and incubated with a blocking buffer (PBS with 2% bovine serum albumin, BSA) for 1 h at room temperature. Then, they were washed with a wash buffer and received 100 µL of the samples collected and 100 µL of the LTA standard followed by serial 2-fold dilutions (standard curve) and maintained for 2 hours at room temperature. Afterwards, the plates were washed again and 100 µL of anti-LTA antibody was added for 1 hour at room temperature. The plates were washed again and 100 µl of horseradish peroxidase HRP conjugated rabbit IgG antibody was added for 1 hour at room temperature. Lastly, the plates were washed, and the reaction was developed using tetramethylbenzidine (TMB). After 20 min under the light, 50 µL of stop solution (2 N sulfuric acid) was added to each well of the plate and optical densities were read in the microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm absorbance (Machado et al., 2020).

Results
All experimental groups were effective in reducing the microbial load (CFU/mL) of E. coli ( Figure 1) and E. faecalis ( Figure 2) in S2 (from S1 levels). There was no significant  NaOCl gel has been suggested as an alternative endodontic irrigant because theoretically it has the same antimicrobial action of NaOCl solution, but with less apical extrusion and could thus be safer (Nesser & Bshara, 2019). It is effective in reducing the microbial load, but has been shown to be less effective when compared to NaOCl solution of a lower concentration (Poggio et al., 2010; Zand et al., 2016). In the present study, NaOCl gel was shown to be just as effective as NaOCl solution in reducing microbial load.
To the best of our knowledge, there are no studies in the literature evaluating the effect of NaOCl gel over endotoxins. In this study it was statistically as effective as the solution. But both were more effective when combined with PUI as it increases NaOCl penetration into dentinal tubules (Faria et al., 2019) Table 2).
NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA at S2. Groups with PUI showed a statistically increased reduction in LPS and LTA compared with groups without PUI (Figure 3). As a limitation of this study, the LPS and LTA levels were not assessed in S3.

Discussion
Well endodontically treated teeth fail mainly because of secondary intraradicular infection (Siqueira, 2001). E. faecalis and E. coli, among other bacteria, are the most detected microorganisms in periapical lesions (Geibel et al., 2005).
The use of NaOCl as an irrigant cannot be over-emphasized. In this study, it was effective in disinfecting E. faecalis and E. coli (Table 2) In the present study, NaOCl was not effective in detoxifying LPS and LTA completely (Figure 3). In support of this, the     In conclusion, our study showed that NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli formation and their endotoxins (LPS and LTA).  The introduction: It presented briefly the current literature and a historic background about the use of sodium hypochlorite. As a suggestion for the researchers in their future projects, I think that it is more appropriate to test another Gram-negative bacterium than e.coli. Even e.coli was found in the root canal system, however, I think that there are a variety of microorganisms that may be tested and have more relevance in the literature as example: Porphyromonas gingivalis (P. gingivalis) as in the study of Wang et al (2019) 1 and others in the study of Lukic et al (2020) 2 .

Data availability
The methods: The study design was carefully planned, I think that the study in its current version is accepted to be indexed, all the cited articles in the methodology section have more details about the execution of this study. I am just wondering why the researchers did not use the sodium hypochlorite in the same concentration in both solution and gel forms? It is not a big deal here as the concentration is almost the same (2.5 and 3 %), however, I think this should be explained in the discussion section, or at least mentioning a previous study that used different concentrations.
The discussion: Why did the authors not take advantage of the positive results obtained from using PUI in this study to be discussed furtherly in the discussion section or even in the introduction section? I think another paragraph will improve the discussion section. The conclusions of this paper drawn adequately supported by the results.