Efficacy of Ozone to Eliminate Endopathogenic Microorganism in Rootcanal Biofilm

Abstract

Objective:

The researchers wanted to see whether ozonated water with ultrasonication and sodium hypochlorite can destroy Enterococcus faecalis bacteria in root canals.

Materials and Methods:

A total of 40 single-rooted human teeth were used. A total of 100 roots were harvested and mechanically prepared. The root canals were randomly divided into four classes (n = 10) after being infected with E. faecalis for 24 h. Each sample's MTT value was calculated.

Conclusions:

NaOCl and aqueous ozone provide antibacterial effects in in-vitro conditions in root canals.

INTRODUCTION

Effective eradication of the causative microorganism during the root canal treatment is needed for successful treatment of primary and secondary endodontic infections. The bacteria must be able to withstand the chemo-mechanical preparation, treatment, and low-nutrient environment to remain viable in a treated root canal system.

Endodontic irrigants must have antimicrobial activity and must be low in cytotoxicity to the periapical and oral mucosal tissues. An anti-inflammatory effect, particularly in cases of recurrent apical periodontitis, may be beneficial.[1] Chemical debridement is especially important for teeth with complicated inner anatomies, such as fins or other abnormalities that instrumentation can miss.[2]

Enterococcus faecalis is usually found in root canal systems of chronic disease and may withstand the antimicrobial effects of sodium hypochlorite (NaOCl) and other medications.

Because of its potent antibacterial action, aqueous ozone is one of the newer irrigating solutions that has shown promising results when used as a root canal irrigant. As compared to other antiseptics, aqueous ozone has no cytotoxicity and is extremely biocompatible.[2]

The aim of this in-vitro research was to see how effective aqueous ozone is against a single strain of Enterococcus faecalis as a root-canal disinfectant along with the comparison of its effectiveness in eradicating E. Faecalis with 3% NaOCl.

MATERIALS AND METHODS

Methodology

For the analysis, a total of 40 recently extracted permanent single-rooted teeth were obtained and stored according to OSHA guidelines.

Selection of Teeth

Inclusion criteria

• Permanent single-rooted tooth

• Tooth having a length of 20 mm and above

• Tooth with well-formed roots

Exclusion criteria

• Single-rooted tooth that is less than 20 mm in length

• Teeth with developmental root defects and malformed teeth

• On all teeth, a typical access cavity was prepared. The number 2 and 3 Gates Glidden drills were used for coronary preflaring. Hand instrumentation was used to plan the entire working length of the canals up to the size of 40 k file.

• Recapitulation was done after every file used to check for patency and NaOCl was used to irrigate the canal prepared thoroughly. To remove the smear layer, 17% ethylene diamine tetraacetic acid (EDTA) and 3% NaOCl were used, followed by sterile distilled water irrigation. To ensure specimen standardization, the teeth were then decoronated at 15-mm length from the apical area.

Sticky wax was used to seal the apical area. Nutrient broth (NB) was made by dissolving 13 g of commercially available nutrient medium (HI Media, India) in 1000 mL of distilled water and boiling to fully dissolve the medium. The medium was dispensed as required and autoclaved for 15 min at 15-lbs pressure (121°C).

A standard E. Faecalis strain (NCTC 12697) was obtained, and nutrient agar was used to keep it alive (NA). Fresh inoculum was made by inoculating single colonies in 25 mL of NB and adjusting the count to 10 5 cells per mL with sterile saline, which was also used in the experiment.

Ozonated water preparation

For 20 min, the ozone sterilizer's outlet ball was submerged in the bottom of a conical flask containing 0.5 L of ordinary water. Ozone gas was created and released via this ozone sterilizer as a result of electrical discharge. As ozonated water has a 12-h half-life, each sample's root canal was irrigated with freshly prepared ozonated water.

Four groups of ten teeth each were chosen at random and were then irrigated with the following irrigants and 35-gauge needles.

Grouping of teeth

GROUP I: Saline (negative control) group:

Infected root canals were irrigated for 300 s with saline solution.

GROUP II: NaOCl (positive control) group:

Infected root canals were irrigated for 300 s with 3% NaOCl.

GROUP III: Aqueous ozone with manual irrigation technique group:

Aqueous ozone was obtained from the commercially available ozone generator

Infected root canals were irrigated for 300 s with aqueous ozone.

GROUP IV: Aqueous ozone with passive ultrasonic irrigation technique

A commercially available ozone generator was used to produce aqueous ozone (Beijing M Fresh High Tech Co, Ltd., China). Infected root canals were irrigated with aqueous ozone for 300 s while also being treated with a passive ultrasonic system. An ISO 15 K-file was mounted passively in the aqueous ozone-filled canal and triggered for 300 s with an ultrasonic system at 70 kHz and 200 mW/cm.

MTT (Sigma Aldrich, USA) was diluted to a concentration of 5.0 g/L in distilled water and deposited at 20°C in 1.5-mL centrifuge tubes. E. faecalis was isolated from dental chips by vortexing them in 1 mL PBS for 30 min. The MTT stock solution was applied to fresh cell dilutions (1/10, v/v) to start the reduction reaction, and the mixtures were incubated at 37°C with the tube cap open to initiate the reduction reaction. The formazan crystals were obtained by centrifuging the 1.5-mL centrifuge tubes at 10,000 g for 20–30 s and pipetting off the medium without shaking. 500 mL of DMSO (Himedia, India) was applied to dissolve the pellets, and the suspension was moved into a new 96-well plate (Nunc, ThermoScientific, USA) and measured at 550 nm by using DMSO as the blank in a microplate reader (ERBA, Germany). A550 value of 1.0 formed by the dissolved formazan crystals from the cells in the LBG medium at 37°C in 20 min is known as one MTT reduction unit (MRU). E. Faecalis MTT reduction activities were averaged over four measurements with the corresponding standard.

The suspension was moved into a new 96-well plate (Nunc, ThermoScientific, USA) and measured at 550 nm by using DMSO as the blank in a microplate reader (ERBA, Germany). A550 value of 1.0 formed by the dissolved formazan crystals from the cells in the LBG medium at 37°C in 20 min is known as one MTT reduction unit (MRU). E. Faecalis MTT reduction activities were averaged over four measurements with the corresponding standard.

Each sample's MTT value was calculated. Following that, the MTT values for each sample were compared using a parametric one-way ANOVA test. Posthoc tests were used to compare the antimicrobial effects of different irrigants.

RESULTS

The raw data collected pertaining to the MTT values observed in different samples of various groups are given below [Table 1].

Table 1.

MTT values observed in various groups

Group 1 (saline)	Group 2 (NaOCl)	Group 3 (ozone)	Group 4 (ozone with ultrasonic agitation)
301.4	143.8	71.2	41.4
192.2	73.8	121.3	34.4
333.4	146.6	87.1	42.5
237.1	38.3	38.1	41.9
139.2	39.4	51.2	43.9
362.1	40.8	38.1	44.4
249.6	46.2	40.1	41.8
327.5	38.6	96.1	41.2
255.8	53.6	79.7	38.6
302.6	41.8	91.1	98.1

An analysis of variance (ANOVA) was used to observe the statistical significance [Table 2]. The following observations were made from the statistical analysis:

Table 2.

Antibacterial efficacy of various irrigants against E. faecalis

Groups	Mean	Std. Deviation	P	Significance
SALINE	271.8	67.1	0.000	HS
HYPO	68.1	45.5
OZONE	73.7	29.2
OZONE + ULTRASONIC	46.1	18.1

• Group 1 had a mean value of 271.8

• Group 2 had a mean value of 68.1

• Group 3 had a mean value of 73.7

• Group 4 had a mean value of 46.1

Statistical analysis revealed that there is a difference in the antibacterial efficacy of various irrigants, which is highly significant.

All groups showed antibacterial efficacy against E-Faecalis, among which group 4 (ozone with ultrasonic agitation) with a mean value of 47.1 exhibited maximum antibacterial efficacy, followed by group 2 (NaOCl) with mean value of 68.1, group 3 (ozone) with mean value of 72.7, and group 1 (saline) with a mean value of 271.8.

Posthoc analysis revealed a substantial difference in the antibacterial efficacy of saline when compared to the other groups Table 3 {NaOCl (P = 0.00), ozone (P = 0.00), and ozone with passive ultrasonic agitation (P = 0.00)}, all of which were found to be highly statistically significant. However, on comparing the antibacterial efficacy of NaOCl with ozone (P = 0.995) and ozone with passive ultrasonic agitation (P = 0.713), the results were found to be statistically insignificant. Similar statistically insignificant results were found among the groups using ozone and ozone with passive ultrasonic agitation (P = 0.567). Hence, it can be concluded that ozone and NaOCl are equally effective.

Table 3.

comparative antibacterial efficacy of various irrigants against E. faecalis

Multiple Comparisons Tukey HSD
Group	Compared with	Mean Difference (I-J)	Sig.
SALINE	Hypo	203.8*	0.00
	Ozone	199.1*	0.00
	Ozone + Ultrasonic	224.8*	0.00
HYPO	Ozone	4.6	0.99
	Ozone + Ultrasonic	20.9	0.71
OZONE	Ozone + Ultrasonic	25.6	0.56

^*P value <0.05 is significant

DISCUSSION

Miller[3] identified microorganisms found in and retrieved from polluted root canals in 1894.

Bacterial pulpal penetration is often asymptomatic, though pain and discomfort may develop later. The consequences of bacterial penetration of the pulp[4] are of greater concern.

The attainable target is to lower bacterial populations below the levels required to cause or maintain disease.[5]

Enterococci faecalis is responsible for 80% of all Enterococci infections, while Enterococcus faecium is responsible for the remaining 20%.[6]

It was observed that the risk of E. Faecalis from food products triggered a transient oral infection thus infecting the root canal and hence it was proposed the theory that the food-derived microbiota may reach the root canal system through microleakage.[7]

A biofilm is a microbial community known as cells attached to a substratum, encased in an extracellular polymerase material matrix, and exhibiting altered growth phenotypes.[8] Bacteria make up a variable percentage of the overall biofilm volume, ranging from 5% to 35%. Extracellular matrix[9] makes up the remainder of the volume.

The root canal system's complexity is a roadblock to achieving the primary goals of full cleaning and shaping of root canal systems.[10]

An irrigant should have no negative effects on the dentin or the filling material's ability to seal.[10] Because of its bactericidal activity and capacity to dissolve essential and necrotic organic tissue, NaOCl is the most commonly used endodontic irrigant.[11,12] NaOCl solutions, on the contrary, have no impact on the inorganic components of the smear sheet. Chelant and acid solutions, such as EDTA, citric acid, and phosphoric acid,[13,14] have been suggested for extracting the smear layer from instrumented root canals.

Within the study's limitations, it can be concluded that ozonated water (mean value: 73.71) has antibacterial efficacy comparable to NaOCl (mean value: 69.02) (P = 0.995 in a posthoc test with NaOCl-3). Better results were obtained when ozonated water was combined with ultrasonic agitation (mean value: 48.02) (posthoc test with NaOCl: P = 0.567). To determine the feasibility of using ozonated water as a root canal irrigant, further in-vivo testing is needed.

CONCLUSION

Under the limitations of this review, the following conclusions can be drawn:

1. Aqueous ozone has potent antibacterial activity.

2. It has antibacterial properties equal to 3% NaOCl.

3. When aqueous ozone was combined with passive ultrasonic agitation, antibacterial effectiveness was higher than when aqueous ozone or NaOCl was used alone.

More research is needed to determine the effects of contact time and different ozone concentrations on antibacterial efficacy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.