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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 2  |  Page : 102-107

Antimicrobial efficacy of commercially available ozonated olive oil and sodium hypochlorite with and without ultrasonic activation in primary endodontic infections: A randomized clinical trial

Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana, India

Date of Submission18-Aug-2021
Date of Decision13-Nov-2021
Date of Acceptance17-Jan-2022
Date of Web Publication01-Jul-2022

Correspondence Address:
Dr. Garima Kaushik
Department of Conservative Dentistry and Endodontics, Sudha Rustagi College of Dental Sciences and Research, Faridabad, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_57_21

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Aim: This study compared the antimicrobial efficacy of commercially available ozonated olive oil and sodium hypochlorite (NaOCl) with and without ultrasonic activation in primary endodontic infections.
Materials and Methods: Fifty-six patients fulfilling the inclusion criteria were selected and randomly assigned to four groups (n = 14) according to the irrigant and irrigation technique employed during biomechanical preparation. Group 1: NaOCl, Group 2: NaOCl with passive ultrasonic irrigation (PUI), Group 3-Ozonated olive oil, and Group 4-Ozonated olive oil with PUI. Bacteriological samples were taken from the canals before (S1) and after (S2) preparation using sterile paper points. Microbiological samples (S1, S2) were incubated and plated on Brain Heart Infusion agar. Colonies were counted after 24 h using the classic bacterial counting method. Collected data were statistically analyzed.
Results: Statistically significant reduction (P < 0.05) of bacterial counts was found from S1 to S2 in all four experimental groups. The mean percentage reduction of bacterial counts of Group 1 and Group 3 was found to be lower than that of Group 2 and Group 4. The highest mean percentage bacterial reduction was seen in Group 2 (P < 0.05).
Conclusion: PUI significantly enhanced the antimicrobial activity of the experimental groups and ozonated olive oil can be used as an adjunctive irrigant in primary endodontic infections. The antibacterial activity of ozonated olive oil with PUI was found to be comparable with that of NaOCl with PUI.

Keywords: Antibacterial activity, ozonated olive oil, passive ultrasonic irrigation, sodium hypochlorite

How to cite this article:
Mittal R, Tandan M, Kaushik G. Antimicrobial efficacy of commercially available ozonated olive oil and sodium hypochlorite with and without ultrasonic activation in primary endodontic infections: A randomized clinical trial. Endodontology 2022;34:102-7

How to cite this URL:
Mittal R, Tandan M, Kaushik G. Antimicrobial efficacy of commercially available ozonated olive oil and sodium hypochlorite with and without ultrasonic activation in primary endodontic infections: A randomized clinical trial. Endodontology [serial online] 2022 [cited 2022 Aug 8];34:102-7. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/2/102/349575

  Introduction Top

Successful endodontic therapy relies on chemomechanical preparation, irrigation, microbial control, and complete obturation of the root canal system. Hand and rotary instrumentation mechanically eliminate microorganisms from most of the accessible parts of the primary root canal. However, the intricacy of root canal morphology provides areas such as narrow isthmus, curved apical third, apical deltas, ribbon-shaped, and oval canals, which cannot be cleaned even after meticulous mechanical preparation.[1] Residual pulp tissue, bacteria, and dentin debris may persist in these irregularities of the root canal system and may lead to treatment failure or persistent periapical pathosis.[1],[2] To assist in cleaning and debridement of root canal system, a range of irrigating and disinfecting solutions have been used such as saline, hydrogen peroxide, sodium hypochlorite (NaOCl), chlorhexidine, ethylenediaminetetraacetic acid, ethylene, Q-mix, MTAD, etc.[1],[3] During and after instrumentation, the irrigants facilitate the removal of any residual tissue, microorganisms, and dentin debris from the root canal.[2]

NaOCl is the “gold standard” irrigant and is used in concentration ranging from 0.2% to 5%.[3],[4] It is a strong base (pH >11) and acts as an organic solvent.[4] The antibacterial activity and the tissue dissolution capacity of NaOCl are dependent on its concentration.[5] One of the biggest disadvantages of NaOCl is its strong toxicity to the periapical tissues upon extrusion through the apex.[6] Other side effects are unpleasant to taste and odor, inability to remove the smear layer, allergic potential, discoloration of clothes, and weakening effect on dentin on long-term exposure.[5],[7],[8] Thus, due to the potential side effects, safety concerns, and limitations in instrumentation methods there is difficulty in complete cleaning of root canals, making it necessary to develop alternative procedures to optimize the cleaning process.[9]

Ozone, a powerful oxidizing agent,[2] has strong bactericidal, virucidal, and fungicidal effects making it a potential agent for root canal disinfection.[10] Ozone is nontoxic to oral cells[6] and very efficient against antibiotic-resistant strains.[2] Its effect increases in acidic pH unlike NaOCl whose rate of decomposition rapidly decreases from pH 11-7.[2],[11] Three fundamental forms of ozone application are: – (1) ozonated water, (2) ozonated olive oil, and (3) oxygen/ozone gas. Ozonated water and olive oil have the capacity to entrap and then release oxygen/ozone, constituting an ideal delivery system.[12] Ozone, when dissolved in water, becomes highly unstable and rapidly decomposes, so it cannot be stored. In contrast, when it is dissolved in an oil base, it has a life span that could be measured in years. It chemically reacts with oil and forms long complex molecules.[13] Hydrolysis of ozonized oil can generate hydrogen peroxide, aldehydes, and acetones. Kishore et al. evaluated the antibacterial activity of the ozonized oil, calcium hydroxide, and their combination against Enterococcus faecalis and concluded that ozonized oil was the most effective medicament.[14] Pratyusha et al. evaluated the antibacterial activity of the ozonated olive oil and cold pressed neem oil against E. faecalis using the agar well diffusion method and concluded that ozonated olive oil was more effective.[13]

Apart from irrigant, irrigation technique also plays a crucial role in successful endodontic treatment. Ultrasonic activation has often been combined with conventional irrigants to obtain a higher antibacterial effect.[6] During ultrasonic activation, ultrasonic waves induce two physical phenomena: Acoustic streaming and cavitation of the irrigant solution.[15] Passive ultrasonic irrigation (PUI) relates to the activation of an irrigant by an ultrasonically activated file or tip that is not used for canal preparation.[16] It has shown promising results in the removal of debris from root canals and enhancing the antimicrobial activity of the irrigant.[16],[17] Bago Juričet al. compared the antibacterial efficacy of (Er, Cr: YSGG) laser-activated irrigation, PUI, RinsEndo, and conventional syringe irrigation against E. faecalis and found that there was a significant reduction in the number of colony-forming units after PUI.[18]

The available literature has not evaluated the combined effect of ozone and PUI in clinical situations. Therefore, this in vivo study was undertaken to evaluate the antimicrobial efficacy of ozonated olive oil with and without ultrasonic activation in clinical settings and compare it with NaOCl.

  Materials and Methods Top

This randomized clinical trial was approved by the Institutional Ethical Committee of SudhaRustagi College of Dental Sciences and Research, Faridabad, (9315/2018). The present study followed the Consolidated Standards of Reporting Trials guidelines and was registered at www.ctri.nic.in (Clinical Trials Registry-India Reg. No: CTRI/2019/08/020553). To determine the sample size, a pilot study was conducted. The sample size was calculated using G Power Software (version 3.0.10) (Chicago, USA). Based on the calculated effect size of 0.59, 5% level of precision, 95% confidence level, and 80% power of the study. The required sample size was 56, with 14 per group. The study and associated risks were explained to the patients, and written informed consent was obtained from each patient. This study took place in the postgraduate clinic of the Department of Endodontics, SudhaRustagi College of Dental Sciences and Research, Faridabad from August 2019 to January 2020, and the entire treatment was carried out by a single operator.

Patients with the noncontributory medical history, intact permanent teeth without any previous restoration, teeth with necrotic or infected pulp as diagnosed clinically and radiographically, teeth with the adequate coronal structure for proper isolation, temporization, and restoration were included.

Patients with systemic conditions, pregnant patients, acute periapical abscess, retreatment cases, patients on antibiotic therapy within 3 months, teeth with calcified canals, presence of intraoral or extraoral sinus, immature apex, internal or external resorption, teeth with periodontal pockets >5 mm were excluded.

The oral cavity along with the experimental tooth were disinfected with the 0.2% chlorhexidine solution (Hexidine Mouthwash, Icpa Health Products Ltd.). Each tooth was anesthetized (Biocaine-ADR Local Anaesthetic solution, Biochem Pharmaceutical Industries Ltd., India) and isolated with rubber dam (HygenicR Dental Dam Kit, ColteneWhaledent, Switzerland). Before preparation of the access cavity, disinfection of the crowns and surrounding structures (dam and clamp) was done with 2.5% NaOCl (Bharat Chemical, India) for 30s, subsequently, 5% sodium thiosulfate was used to deactivate the effect of the NaOCl. Sterility control samples were taken with sterile paper points (Metabiomed, India) from the rubber dam, clamp, and coronal surface of the tooth. Caries was removed and access cavity was prepared using a high-speed endo access bur #2 (DentsplyMaillefer, Switzerland) underwater spray with an air rotor handpiece (NSK Pana Air, Japan). The working length was determined using an electronic apex locator (Morita Root ZX Mini) and confirmed radiographically by using an RVG (Kodak 5100, Eastman Kodak Company, France). Pretreatment Sample (S1) was obtained by injecting normal saline (5 ml) (0.9% v/w, Lifusion™, India) into the root canal and circumferentially pumping a #10 K-file (Mani, Inc. MDCI Ltd., Japan) (1 mm short of working length). A sterile paper point was placed into the canal for 60 s after immersing into the transport media (Peptone water, HiMedia Laboratories Pvt. Ltd., India). It was then immediately transported to the test tube containing transport media. Three samples were taken for each tooth at each stage (i.e., S1 and S2). The patients were then randomly divided into four experimental groups:

  • Group I: NaOCl (n = 14)
  • Group II: NaOCl with ultrasonic activation (n = 14)
  • Group III: Ozonated olive oil (DentOzone, India) which is composed of medical-grade ozonized herbal olive oil (n = 14)
  • Group IV: Ozonated olive oil with ultrasonic activation (n = 14).

The teeth were prepared using step-back technique up to master apical size #40 K-file and stepped back up to size #60. Irrigation was done with the respective irrigants during biomechanical preparation. In Groups I and III, the canals were irrigated with 5 ml of respective irrigants. In Groups II and IV, the canals were irrigated with 5 ml of respective irrigants and passively activated by U-files (Mani, Inc. MDCI Ltd., Japan), 2 mm short of the working length.

Postinstrumentation sample (S2) was collected in a similar manner as S1. The investigator was blinded and the microbiological samples (S1, S2) were preincubated for 30 min and shaken vigorously in a vortex mixture for 60 s. Samples were then were plated on brain heart infusion agar (HiMedia Laboratories Pvt. Ltd., India). Colonies were counted after 24 h using the classic bacterial counting method. The data collected were subjected to statistical analysis.

Patients were recalled after 7 days. The tooth was isolated using a rubber dam and disinfection was done using 0.2% chlorhexidine solution (Hexidine Mouthwash, Icpa Health Products Ltd.). The temporary restoration was removed using a round diamond bur and an air rotor handpiece. The patients were asymptomatic. Teeth were obturated with lateral condensation technique using #40 guttapercha (Metabiomed, India) as master cone (after radiographic verification) and AH Plus sealer (Dentsply, Germany). The access cavities were then restored with composite (Spectrum, Dentsply India Pvt. Ltd., India).

Statistical analysis

Data were statistically analyzed using SPSS (21.0 version) (IBM Corp., Armonk, NY). The normality of data was checked using Shapiro–Wilk test. Inferential statistics were performed using one-way ANOVA. Post hoc Tukey analysis and paired t-test was done for pair-wise comparison. The level of significance was set at P < 0.05.

  Results Top

None of the teeth were positive for bacteria in the sterility control sample.

In intragroup comparison, a statistically significant reduction was found from S1 to S2 in all the four experimental groups as shown in [Table 1].
Table 1: Comparison of mean and standard deviation of bacterial colony count (colony forming units/ml ×106) of each group at S1 and S2 stages for various groups

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In intergroup comparison, the mean percentage reduction of bacterial counts of Group 1 (NaOCl) (55.08 ± 7.14) and Group 3 (Ozonated olive oil) (54.17 ± 6.83) were found to be lower than that of Group 2 (NaOCl with ultrasonic activation) (77.65 ± 4.97) and Group 4 (Ozonated olive oil with ultrasonic activation) (71.67 ± 3.74) as shown in [Table 2].
Table 2: Comparison of mean percentage reduction of bacterial count (S1-S2/S1*100) for various groups

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However, the difference was not statistically significant between Group 1 (NaOCl) and Group 3 (Ozonated olive oil), but the difference was statistically significant between Group 2 (NaOCl with ultrasonic activation) and Group 4 (Ozonated olive oil with ultrasonic activation) with highest mean percentage bacterial reduction seen in Group 2 (NaOCl with ultrasonic activation) (77.65 ± 4.97).

  Discussion Top

Irrigation is an integral part of chemo-mechanical preparation and it plays a major role in the cleaning and disinfection of the root canal system.[19] Irrigation renders the canal system free of necrotic pulp tissue, bacteria and dentinal debris.[1]

NaOCl is currently the most widely used irrigating solution due to its superior antibacterial efficacy.[19],[20] NaOCl is highly toxic at high concentrations and tends to induce tissue irritation on contact. Its accidental injection beyond the root apex causes tissue reaction characterized by pain, swelling, hemorrhage, and in some cases the development of secondary infection and paresthesia.[10] Other drawbacks of NaOCl comprise its unpleasant taste, ability to bleach clothes, corrosion of endodontic instruments, inability to remove the smear layer, and weakening effect on dentine.[8],[21],[22]

Ozone is an effective antioxidant associated with the low level of hazards and a high level of biocompatibility.[23],[24] Studies have proven its ability to interact effectively with microbiota in the root canal system and eliminate them.[23] Ozone has a high oxidation potential, being 1.5 times more effective than chlorine as an antimicrobial agent against several microorganisms.[25] Ozonated oils are obtained from the chemical reaction between ozone and unsaturated fatty acids of vegetable oils.[26] The reaction of ozone with oil occurs exclusively with the carbon-carbon double bonds present in the unsaturated fatty acids and produce different toxic products such as several oxygenated compounds, hyperoxides, ozonides, aldehydes, peroxides, diperoxides, and polyperoxides. These compounds are responsible for the wide antibacterial activity of ozonized oil.[13]

PUI can be an important supplement for cleaning the root canal system and when compared with traditional syringe irrigation, it removes more organic tissue, planktonic bacteria, and dentine debris from the root canal. PUI relies on the transmission of acoustic energy from an oscillating file or smooth wire to an irrigant in the root canal.[27]

In the present study, NaOCl activated by ultrasonics showed the highest bacterial count reduction, followed by ozonated olive oil activated by ultrasonics, then NaOCl and least reduction were seen with ozonated olive oil.

PUI enhanced the antibacterial activity of both NaOCl and ozonated oil when compared to manual syringe irrigation. The possible reasons could be the activation of the irrigant because of the implosion of microcavitation bubbles and hydrodynamic effects.[17] The active streaming of the irrigant enhances its potential to contact a greater surface area of the canal wall and increased wall shear stress resulting in greater removal of bacteria from the root canal.[17],[27]

The significant increase in antibacterial efficacy of NaOCl when activated with ultrasonics could be due to the temperature rise. Part of the ultrasonic energy is transformed into heat energy, which leads to an effective intracanal temperature of 45°C. The thermal effect might be advantageous when ultrasonics is used in combination with NaOCl over ozonated olive oil.[17] The bactericidal activity of NaOCl is more than doubled for every 5° increase in temperature in the range of 5°C–60°C due to increased content of free available chlorine.[11] Cunningham et al. evaluated the influence of ultrasonics on the antibacterial efficacy of NaCl and NaOCl. It was found that ultrasonics was better at reducing bacterial counts with both NaCl and NaOCl than manual syringe irrigation. NaOCl with ultrasonics was found to be the most effective.[28] Huque et al. evaluated intracanal irrigation procedures in eradicating bacteria from the surface, shallow and deep layers of root dentine using extracted human teeth and observed that ultrasonic irrigation with 5.5% and 12% NaOCl eradicated bacteria from the artificial smear layer, whilst 12% NaOCl irrigation with a syringe was also insufficient.[29]

Ozonated olive oil activated by PUI resulted in comparable antimicrobial activity to NaOCl with ultrasonic activation as ultrasonication allowed deeper penetration and effective agitation of the irrigant in dentin tubules resulting in its enhanced antibacterial activity.[6] Case et al. evaluated the effects of gaseous ozone on biofilms of E. faecalis with and without the use of passive ultrasonic agitation. It was found that ultrasonics increased the antibacterial efficacy of ozone as compared to ozone alone.[30] Hubbezoglu et al. assessed the antibacterial efficacy of aqueous ozone in root canals infected by E. faecalis with and without PUI. It was found that a higher concentration of ozone with PUI possessed superior antibacterial efficacy than manual syringe irrigation.[6]

Bacterial count reduction obtained by NaOCl with manual syringe irrigation was greater as compared to ozonated olive oil with manual syringe irrigation. These findings are in accordance with the results obtained from several in vitro studies.[20],[21],[31] Tuncay et al. compared the antibacterial effects of gaseous ozone and photoactivated disinfection (PAD) with 2.5% NaOCl against E. faecalis biofilms and found that both PAD and gaseous ozone have a significant antibacterial effect on infected root canals. However, 2.5% NaOCl was superior in terms of its antimicrobial abilities compared with the other disinfection procedures.[32]

In the current study ozonated olive oil showed the least bacterial count reduction. The bactericidal efficacy of ozone is based on its ability to formoxidated radicals, as a result of which the cell membranes get damaged by altering the osmotic stability and permeability.[20] During the ozonolysis reaction, there is typically an increase in peroxide and acid values and a decrease in iodine values. Ozonated olive oil showed the least bacterial reduction as the ozonolysis reaction is meaningless if peroxide released by ozonated oil could not be quantified. These considerations bring us to two important conclusions. First, ozonated oil manufacturers should describe the peroxide, acid, and iodine values on the product label. Second, further studies considering the standardization of ozonolysis procedures for vegetable oils are warranted.[33] Another limitation could be that the known endodontic disinfectants are provided in liquid form, presumably to facilitate penetration into the accessory canals and tubules whereas ozonated olive oil is more viscous in nature thereby limiting the penetration.[34] Müller et al. assessed the antimicrobial potential of gasiform ozone and photodynamic therapy (PDT) on a multispecies oral biofilm and found that gasiform ozone and PDT had a minimal effect on the viability of microorganisms organized in a cariogenic biofilm. Only 5% NaOCl solution was able to totally eliminate the microorganisms in the biofilm.[31]

  Conclusion Top

Further in vivo studies are required to better evaluate the effectiveness of ozonated olive oil with and without ultrasonic activation as an intracanalirrigant. Therefore, within the limitations of this study, it can be concluded that PUI significantly enhances the antimicrobial activity of the experimental groups, and the antibacterial activity of ozonated olive oil with ultrasonic activation was found to be comparable with that of NaOCl with ultrasonic activation. Hence, ozonated olive oil with ultrasonic activation can be recommended as a potential endodontic disinfection regimen.

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Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2]


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