Many studies [
Although sodium hypochlorite is the most commonly used irrigant, chlorhexidine has recently been introduced as an alternative irrigating solution. However, it does not described any possible effect of the association between them.
Chlorhexidine is a cationic molecule, which can be used during treatment. It has a wide range of antimicrobial activity. Furthermore, because of its cationic structure, chlorhexidine has a unique property named substantivity [
Ozone is being presented as a possible alternative antiseptic agent due to its antimicrobial power [
Flow cytometry has been proven as an excellent tool in microbiology field to quickly demonstrate antimicrobial activity as well as to clarify about mechanism of action/resistance [
Our study purposes a novel approach to be used in endodontic treatment, leveraging the synergy achieved between two products with antibacterial action which is effective against
220 extracted human single rooted teeth were selected to this study. Working length was determined using a size 15 K-file (Maillefer Inc.), until its tip became visible from the apical foramen, 1 mm short of the apical foramen. Teeth were prepared up to a size 40 K-file (Maillefer Inc.). Teeth were irrigated with 2 mL of saline solution at each change of file. Smear layer was removed with 10% acid citric solution (10 mL) and a final irrigation of saline solution was made according to the method of Schäfer and Bössmann [
The teeth were randomly assigned to four experimental groups for
In the control group, the contaminated root canals of 10 teeth were irrigated with 10 mL of 0.9% (w/v) sterile saline solution.
In the sodium hypochlorite (NaOCl) group, teeth were divided into three subgroups of 10 teeth each. Root canals were irrigated with 10 mL of 1%, 3%, or 5% NaOCl solution.
In the chlorhexidine (CHx) group, teeth were divided into two subgroups of 10 teeth each. Root canals were irrigated with 10 mL of 0.2% or 2% CHx solution.
In the ozone group, teeth were divided into four subgroups of 10 teeth each. Gaseous ozone was applied with an ozone generator (Prozone, W&H) for 24, 60, 120, and 180 seconds.
Upon treatment, all teeth were washed with 10 mL of sterile saline solution. In order to assess the number of viable microorganisms, the root canals were incubated with Brain-Heart infusion broth at 37°C for 24 h, because if smeared immediately no growth was visible. Viable microorganisms were quantified by determining the number of colony-forming units (CFU) in Brain-Heart agar or Sabouraud dextrose agar for
The association between NaOCl or chlorhexidine and ozone was evaluated in 20 teeth (10 infected with
In order to clarify the mechanisms of action of CHx and ozone, the most effective agents, the following different fluorescent markers were used: propidium iodide (PI) binds to DNA but only if cell cytoplasmatic membrane is damaged, that is, death (Molecular Probes Europe BV, Leiden, Netherlands) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX) is a lipophilic and anionic fluorescent stain that accumulates intracellular when the cytoplasmatic membrane is depolarized (Molecular probes) and additionally only regarding yeast FUN-1, a fluorescent probe that is converted only by metabolically active cells. Acquisition was performed with cellQuest TM pro Software and based on light-scatter and fluorescence signals resulting from 15 mW laser illumination at 488 nm and 635 nm. Signals corresponding to forward and side scatter (FSC and SSC) and fluorescence were accumulated at FLI (530/30 nm) for DIBAC, FL2 (620 nm) for FUN-1 and FL3 (>670 nm) for PI. For sample preparation, 1 × 106 cells/mL of each strain was incubated with each fluorescent probe for 30 min in the dark at 1
Statistical analysis was performed using a
In the description of the database the percentages (%) and absolute value (N) were used, since the dependent variables were dichotomous (presence or absence of microorganisms and effectiveness or ineffectiveness of the formulations tested). The comparative analysis of different irrigation solutions and between different concentrations within the same solution was performed using the chi-square test. The significance level (
All the tested concentrations of NaOCl were ineffective to completely disinfect the roots, being the percentage of failure teeth inversely related to the concentration. Regarding
Percentage of teeth failed after different treatments.
The 5% NaOCl combined with ozone, even in the maximum time (180 s), did not show significant differences from the isolated treatments (Figure
The main findings of the present study indicate that the irrigation with 2% CHx and 24 s of gaseous ozone may be advantageous, particularly for postdisease treatment. This protocol involving the synergism between ozone gas and 2% CHx solution, never described before, seems to be effective and has potential to be used in the clinical practice. Furthermore, it is one of few protocols described with complete elimination of the microorganisms usually resistant to endodontic treatment, particularly in the tooth model, contaminated with a pure culture. In addition, flow citometry is shown to be an excellent tool to clarify the antimicrobial effect of the drugs [
In the present study the irrigating solutions tested, hypochlorite and CHx, showed antimicrobial activity particularly on
Similarly, 2% CHx was very efficient for
10% citric acid solution was used to finish the chemomechanical preparation in order to remove the smear layer formed during the root canal preparation [
The application of gaseous ozone in short periods of time as 24 s and 60 s, was not completely effective for any of the microorganisms.
Previous studies showed similar results. Gomes et al. 2001 [
Ozone is being presented as a possible alternative antiseptic agent due to its antimicrobial power [
In the present study, even with the highest periods (120 s, 180 s), ozone was not completely efficient.
The possible synergism between ozone gas and the irrigating solutions of sodium hypochlorite 5% and 2% CHx was investigated. These solutions were selected because they were the ones that had better antimicrobial activity, by themselves. A complete elimination of both
The synergism obtained can be explained by the distinct mode of action. Both ozone and chlorhexidine depolarize the cells in dose-dependent manner.
In conclusion, only the combined action of 2% CHx and ozone gas for short period promotes the complete elimination of both microorganisms tested in the tooth model. The results of this study could lead to redirect efforts towards new protocols for reducing microbial load in infected root canals looking for synergisms between new or already known antimicrobial products.
The authors declare that there is no conflict of interests regarding the publication of this paper.