Journal of Indian Society of Pedodontics and Preventive Dentistry
Journal of Indian Society of Pedodontics and Preventive Dentistry
                                                   Official journal of the Indian Society of Pedodontics and Preventive Dentistry                           
Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 164--170

Comparison of antimicrobial efficacy of aqueous ozone, green tea, and normal saline as irrigants in pulpectomy procedures of primary teeth


Suchi Agarwal1, Parimala Tyagi2, Ashwini Deshpande3, Saurabh Yadav4, Vipul Jain5, Kuldeep Singh Rana6,  
1 Department of Pedodontics and Preventive Dentistry, Bhabha College of Dental Sciences, Bhopal, India
2 Department of Pedodontics and Preventive Dentistry, People's College of Dental Sciences and Research Centre, Bhopal, India
3 Department of Oral Medicine and Radiology, GSL Dental College and Hospital, Rajahmundry, Andhra Pradesh, India
4 Department of Pedodontics and Preventive Dentistry, Children Dental College, Azamgarh, Uttar Pradesh, India
5 Department of Orthodontics and Maxillofacial Orthopedics, Bhabha College of Dental Sciences, Bhopal, India
6 Department of Conservative Dentistry and Endodontics, Government College of Dentistry, Indore, Madhya Pradesh, India

Correspondence Address:
Dr. Ashwini Deshpande
H. No . 4, SBI Officers Colony, 1st Main, Basaveshwarnagar, Bengaluru - 560 079, Karnataka
India

Abstract

Aim:Sodium hypochlorite, though considered an ideal root canal irrigant, cannot be used at required concentrations in children, due to its undesirable effects. Hence, it is imperative to search for an ideal root canal irrigant to avoid these undesirable effects which we hope to achieve with this study. The antimicrobial efficacy of aqueous ozone, green tea, and normal saline as irrigants in pulpectomy procedures of the primary teeth has been compared. Materials and Methods: Sixty patients between 4 and 8 years of age with a single-rooted deciduous tooth indicated for pulpectomy were included. The infected teeth were randomly allocated to one of the three treatment groups based on the irrigating agents used, namely normal saline, green tea extract, or ozonated water. Specimens for anaerobic culture were collected three times from the teeth: before irrigation, after initial irrigation, and on the 3rd day after final irrigation. Results and Conclusion: Mean colony forming unit (CFU) count after both initial and final irrigation with ozonated water was significantly lower when compared with green tea and normal saline. Further, it was observed that the mean CFU count with green tea was significantly lower than the counts obtained with normal saline on the 3rd day after final irrigation. Hence, both ozonated water and green tea could be considered a good alternative to conventional root canal irrigants in the primary teeth. Larger sample sizes with a larger variety of irrigants are recommended.



How to cite this article:
Agarwal S, Tyagi P, Deshpande A, Yadav S, Jain V, Rana KS. Comparison of antimicrobial efficacy of aqueous ozone, green tea, and normal saline as irrigants in pulpectomy procedures of primary teeth.J Indian Soc Pedod Prev Dent 2020;38:164-170


How to cite this URL:
Agarwal S, Tyagi P, Deshpande A, Yadav S, Jain V, Rana KS. Comparison of antimicrobial efficacy of aqueous ozone, green tea, and normal saline as irrigants in pulpectomy procedures of primary teeth. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2022 Jul 3 ];38:164-170
Available from: https://www.jisppd.com/text.asp?2020/38/2/164/288214


Full Text



 Introduction



It is well recognized that bacteria are the primary etiologic factor in the development of root canal infections. Until 1970, the most common bacterial group isolated from the root canals of permanent teeth was Viridans Streptococci (alpha-hemolytic streptococci).[1],[2] However, studies concerning root canal microbiota of the primary teeth reported that the most prevalent microorganisms in the root canals of the primary teeth with necrotic pulp and periapical lesions were anaerobic microorganisms, representing over 70% of the microbiota in the root canals of the primary molars that had been treated unsuccessfully.[3] Anaerobic microorganisms are also the most prevalent bacteria in the primary teeth indicated for extractions.[1],[4] Considering the rapid progress of caries in primary teeth, and the consequent pulpal damage by bacteria and their derived toxins that contaminate the tissue, the endodontic treatment becomes necessary. Control of infection is of fundamental importance for the following reasons: (1) the ample medullary bone spaces favor dissemination of infection and (2) the developing permanent tooth germ is very close to the roots of the primary teeth.

Instrumentation has been considered the cornerstone of endodontic treatment of caries. However, the structural complexities of the root canal system in the primary teeth, such as dentinal tubules, accessory canals, and root tip bifurcation, make it inaccessible to clean thoroughly by pure instrumentation. Therefore, the root canal irrigation becomes an essential part of chemomechanical preparation. Varied irrigating agents are employed in clinical practice, depending upon their antimicrobial efficacy, cleaning efficiency, and biological compatibility. Sodium hypochlorite (NaClO), though considered an ideal root canal irrigant, cannot be used at required concentrations in children due to its undesirable effects or limitations, such as unpleasant taste/odor, toxicity, ineffectiveness in removing smear layer, and incompletely eradicating microbes from the infected canals. Inadvertent extrusion of NaClO causes immediate severe pain, edema of neighboring soft tissues, ecchymosis, and paresthesia, due to tissue response.[5] Chlorhexidine digluconate, an alternate irrigant, although widely used in disinfection because of its excellent antimicrobial activity, completely lacks tissue-dissolving capability. Neither hydrogen peroxide nor normal saline offers ideal antimicrobial properties.[6] Hence, it remains critical to search for an ideal root canal irrigant for the use in the primary teeth.

To overcome these drawbacks, ozone may be considered a potential antibacterial, antiviral, and antifungal agent. It may be administered in both gaseous and aqueous forms. When administered in either form, they have powerful antimicrobial properties that are strong and are fast oxidizers of the cell walls and cytoplasmatic membranes of microorganisms. It has also been known to penetrate into the periapical tissue through the apical foramen, where it encourages tissue regeneration by oxygenating the tissue and increasing blood supply. A recent study demonstrated ozonated water to have much lower cytotoxicity against mouse fibroblasts when compared to NaClO, suggesting that ozonated water application may be useful for endodontic therapy.[7] Aqueous ozone also essentially shows no toxicity to oral cells as was observed in vitro. Although there is a need to investigate whether the antimicrobial efficacy is comparable to NaClO, the other observations suggest that ozone may be used as an alternative disinfectant in root canals.

The global scenario is now showing a trend toward the use of nontoxic plant products that have been of traditional medicinal use. This prompted us to look for plant products that might not have the deleterious effects observed with synthetic products. We hypothesized that green tea could have the potential to be used as an irrigant in primary teeth. The major advantages of using green tea are easy availability, cost-effectiveness, increased shelf-life, low toxicity, and lack of microbial resistance. In addition, epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has anti-inflammatory, antimicrobial, anticarcinogenic, and antioxidant properties.[8] Therefore, in the present study, we intended to compare the antimicrobial efficacy of aqueous ozone, green tea, and normal saline against anaerobes during pulpectomy procedures of the primary teeth.

 Materials and Methods



A cohort of 60 subjects between 4 and 8 years of age, indicated for pulpectomy in primary teeth, were selected from the outpatients visiting the Department of Pedodontics and Preventive Dentistry of People's College of Dental Sciences and Research Centre, Bhopal. The study was approved by the institutional ethical committee. Informed written consents were obtained from parents/guardians of each participant. The selection of subjects was made using the following inclusion and exclusion criteria

Inclusion criteria

Patients between 4 and 8 years of age with a single-rooted deciduous tooth indicated for pulpectomyTeeth with more than two-third of root length intactPatients willing to participate in the studyOtherwise normal and healthy patients with no systemic disease.

Exclusion criteria

Grossly decayed teethTeeth with advanced internal or external resorptionTooth with formation of sinus tractPatients who have been administered antibiotics in the past 3 months before the date of reportingPatients with glucose-6-phosphate-dehydrogenase deficiency (favism), hyperthyroidism, severe anemia, severe myasthenia, and any congenital heart diseasePatients with ozone allergyPatients unwilling to participate in the study.

The 60 infected teeth of the selected participants were randomly allocated to one of the following three treatment groups based on the irrigating agent used during the endodontic therapy:

Group 1: Normal saline (0.9 g/100 ml) (control group) (n = 20)Group 2: Ozonated water (20 mg/l) (n = 20)Group 3: Green tea (25 mg/50 ml of water) (n = 20).

The endodontic therapy including the root canal irrigation was double blinded.

Preparation of root canal irrigants and transport media

Ozonated water preparation

Ozonated water for root canal irrigation was prepared using GE OZONE G™ machine that generated water with an ozone concentration of 20 mg/l in 8 min with a 1 l input of distilled water which was treated in a glass container. To ensure maintenance of concentration, the prepared water was used within 20 min of preparation.

Green tea preparation

Commercially available green tea was used to prepare the tea extract. 25 g of tea was added to 50 ml of water under standard preparation conditions in the department of pharmacology. This mixture was then boiled for 2 min, strained, filtered, cooled, and stored in an airtight container. The extract was used within 24 h of its preparation.

Transport media preparation

3.8 g of reinforced Clostridial HiVeg Agar media (HIMEDIA M154) concentrate was suspended in 100 ml of distilled water, to which 10 ml of dithiothreitol and resazurin each was added while maintaining the concentration. The media were autoclaved. 2 ml of this medium was then dispensed into the vacutainers for transport which was ready to use after 48 h of preparation and stored at 2°C–8°C. Normally, the transport medium is pale yellow in color but turns pink in an oxygenated environment. This inherent property helped confirm the maintenance of adequate anaerobic conditions during transport.

Endodontic procedure and bacterial culture

Under strict aseptic conditions, the procedural tooth was anesthetized and isolated with a rubber dam. The tooth and adjacent rubber dam were disinfected with tincture of iodine solution. Root canal access was done with a high speed air-rotor hand-piece and a round bur. On gaining the access, a sterile broach was inserted to obtain root canal content which was immediately transferred to a tube containing transport medium. The tube was tightly sealed for onward transfer to the microbiological laboratory. This constituted the “preirrigant culture” specimen for all three groups.

On the same day, working length determination and biomechanical preparation were done with files 3 sizes beyond the first instrument. During instrumentation, the root canal was alternately irrigated with 2 ml of group specific irrigant, after every step of instrumentation. A sterile paper point was introduced into the root canal and was left in place for 1 min. The paper point was removed with a sterilized tweezer and then immediately transferred to a tube containing transport medium. This constituted the “postirrigant culture” specimen. Access opening was sealed with temporary restorative material to a thickness of approximately 4 mm. The patient was scheduled for the second appointment on the 3rd postoperative day.

On the second appointment, the tooth was isolated and the area was disinfected as before. Temporary restorative material was removed. A microbiological specimen was taken again. This constituted the “third-day culture” specimen. This microbiological specimen was then transported to the laboratory for bacterial counting.

To obtain anaerobic bacterial culture, the most important step involved was the creation and maintenance of a sterile anaerobic environment throughout the process, which was accomplished using the laminar airflow system. This process comprised of placing all the equipment and material inside the laminar air flow chamber using the two utility windows provided for the same. The windows were then sealed completely using tape. An anaerobic environment was created inside this laminar airflow by the reaction of Na2 CO3 and HCl, which releases large amount of CO2. Once the anaerobic environment is established, streaking of sample on the sheep agar plates was done with the help of sterile tooth pick with a cotton swab.

After streaking was done on a particular sample, the lid was closed and sealed with paraffin tape, and then, CO2 was injected in these sealed agar plates to create a suitable anaerobic environment. The same procedure was repeated until all samples were inoculated. All these agar plates were packed in a polythene bag along with pyrogallol in a plastic tube, then placed in gaspak, and tightly closed. This whole assembly was kept in a 3.5-l anaerobic jar in an incubator at 37°C for 24 h. After 24 h, the samples were cautiously taken out and the number of colony forming units (CFUs) was counted using a digital colony counter.

 Results



A total of 60 patients, between 4 and 8 years of age including 23 boys and 19 girls, were selected for the study after obtaining informed written consent from the parents/guardians. The antimicrobial efficacy of aqueous ozone, green tea, and normal saline as root canal irrigants was compared at different time intervals that are after initial irrigation and after final irrigation. Results were tabulated and the data obtained were then quantitatively analyzed using the Mann–Whitney U-test.

Antimicrobial efficacy of normal saline at different time intervals

Mean CFU count was lower after initial irrigation when compared with preirrigation counts, with a mean difference of 42.92, which was statistically significant (P < 0.0001). It was also observed that the mean CFU count after final irrigation was significantly (P < 0.0001) lower than at preirrigation, with a mean difference of 113.72. The mean difference of the CFU count comparison between initial irrigation and final irrigation was 70.80, which was statistically significant with P < 0.0001. These results clearly suggest that irrigation with normal saline has limited antimicrobial efficacy. The results are suggestive of normal saline having limited antimicrobial efficacy [Table 1].{Table 1}

Antimicrobial efficacy of green tea at different time intervals

Mean CFU count after initial irrigation was lower when compared with preirrigation counts, with a mean difference of 36.97. Furthermore, it was observed that the mean CFU count after final irrigation was significantly lower (P < 0.0001) than at preirrigation, with a mean difference of 140.32. When the mean CFU count after initial irrigation was compared with final irrigation, the mean difference was 103.35, which was statistically significant (P < 0.0001). The results suggested that although green tea showed no antimicrobial activity after initial irrigation, it exhibited considerable activity after final irrigation [Table 2].{Table 2}

Antimicrobial efficacy of ozonated water at different time intervals

Mean CFU count was lower after initial irrigation when compared with preirrigation counts with a mean difference of 106.12, which was statistically significant (P < 0.0001). It was also observed that the mean CFU count after final irrigation was remarkably lower (P < 0.0001) than counts taken at preirrigation, with a mean difference of 155.32. The difference in mean CFU counts after initial irrigation and final irrigation was 49.2 and was statistically significant (P < 0.0001). It could be inferred that ozonated water also exhibited considerable antimicrobial efficacy after both initial and final irrigation [Table 3].{Table 3}

Intergroup comparison of antimicrobial efficacy of three irrigants

After initial irrigation

Mean CFU count after initial irrigation with ozonated water (65.0) was significantly lower than that with both green tea (134.15, P < 0.0001) and normal saline (128.2, P < 0.0001). Mean CFU count after initial irrigation with green tea (134.15) was not significantly different from normal saline (128.2). This indicated that ozonated water exhibited the best antimicrobial efficacy after initial irrigation while the activity of both green tea and normal saline was at par [Table 4].{Table 4}

After final irrigation

Mean CFU count after final irrigation with ozonated water (15.8) was significantly lower than that of both green tea (30.8, P < 0.0001) and normal saline (57.4, P < 0.0001). In addition, it was observed that mean CFU count after final irrigation with green tea (30.8) was significantly lower (P < 0.0001) than that of normal saline (57.4). These results suggest that of the three irrigants, ozonated water had the best antimicrobial efficacy after final irrigation. Further, the antimicrobial activity of green tea after final irrigation was significantly better than that of normal saline [Figure 1] and [Table 5].{Figure 1}{Table 5}

 Discussion



Despite more than a century of technological improvements in root canal procedures, clinical studies indicate that bacteria remain in the canal even when standardized cleaning/shaping procedures and irrigants are employed. Hence, in the search for an ideal irrigant for pediatric root canal procedure, we evaluated normal saline, ozonated water, and green tea for their antimicrobial efficacy on the basis of their ability to reduce the number of CFUs in anaerobic bacterial culture.

Children aged between 4 and 8 years were considered an appropriate age group and were included in the study to carry out pulpectomy procedures.[9],[10] Only single-rooted teeth were included considering three aspects of root canal infections in primary teeth: (1) the presence of necrosis and pulp vitality in distinct root canals of the same tooth are different; (2) different composition of bacterial microbiota in distinct root canals of the same tooth; and (3) very different quantities of microbiota exist in root canals of the same tooth with statistically significant differences. Keeping these aspects in mind, it was assumed that, multi-rooted teeth could have led to distorted results. Blinding as well as randomization made the study groups more comparable as well as minimized the bias and confounding.[11],[12]

In this study, samples of three irrigants (normal saline, ozonated water, and green tea) at three different time intervals, viz., preirrigation, after initial irrigation, and after irrigation on the 3rd postoperative day, were subjected to microbial culture. The mean CFUs in all three irrigant groups were the same at preirrigation stage. This was expected since samples were randomly allocated to groups and no irrigation was done before sample collection. Our findings were also consistent with previous reports of a normal range of bacteria of 102–108.[1],[13],[14]

Green tea has been known to have excellent medicinal value.[8] It has also been observed that green tea has specific antibacterial effect against Enterococcus faecalis.[15] The antioxidant potential of green tea polyphenols is directly related to the combination of aromatic rings and hydroxyl groups that make their structure. The antimicrobial activity is a result of binding and neutralization of free radicals by the hydroxyl groups, leading to destruction and dissolution of the bacterial cell wall. In our study, we used 50 mg per ml of green tea for irrigation which is based on the findings of Araghizadeh et al., who had concluded that concentration of 50 mg per ml of green tea polyphenols is highly efficacious against anaerobes such as Porphyromonas gingivalis, Prevotella intermedia, and Actinomyces actinomycetes.[15] Besides, it was found that EGCG, which is the most abundant polyphenol in green tea (Camellia sinesis), is an effective antimicrobial agent against both the planktonic and biofilm forms of E. faecalis, inhibiting bacterial growth and suppressing the expression of specific genes related to virulence and biofilm formation.[16] In agreement with these reports, we found green tea extract to exhibit significant antimicrobial activity, especially after final irrigation on the 3rd day. Green tea, Triphala, 5% NaOCl, and Mixture of Tetracycline, acid and detergent (MTAD) were evaluated for antimicrobial activity on the basis of reduction in CFUs per ml on irrigation and had concluded that 5% NaClO showed maximum antibacterial efficacy against E. faecalis biofilm while Triphala, green tea, polyphenol, and MTAD also showed statistically significant antibacterial activity,[17] which is in agreement with our findings.Further, it was concluded that although conventional irrigants are more frequently used, they have shortcomings in antibacterial efficacy, and that neem leaf or green tea extracts have significant antimicrobial effect against E. faecalis similar to chlorhexidine (CHX).[18] The above reports are all in concurrence with our present study.

However, studies done by Al-Azzawi, who evaluated effectiveness of 5% green tea extract against E. faecalis, by calculating the zones of inhibition of the bacterial growth to measure the antibacterial effect of the tested irrigants, had obtained a mean of 8.88 mm of zone of inhibition, which was the least of the different irrigants tested including the NaOCl, CHX, and Miswak. It was also proved in a study that NaClO showed maximum antimicrobial activity, followed by extracts of Neem, Triphala, and green tea, respectively.[19] Such contradictory results using green tea as irrigant was explained in a study which suggested that green tea's effectiveness as an antimicrobial agent can in part be attributed to its low degree of fermentation.[20] During the fermentation process, catechins such as EGCG are destroyed, reducing the tea's antimicrobial properties. However, there is no documented single preparation method that is best for all tea types, and the uniform preparation method was not optimal for many of the types. Thus, the preparation style probably did not allow all types of to release their maximum chemicals during tea preparation. A different method of preparation could have produced different clinical results. In addition, a longer length of exposure to the tea may have been necessary for better antimicrobial effect.

Ozone is a selective oxidant and affects only certain compounds; however, when dissolved in water, it becomes highly unstable and rapidly decomposes through a complex series of chain reactions. As a result, hydroxyl (HO.) radicals are generated, which are among the most reactive oxidizing species. Ozone reacts with various chemical compounds in aqueous systems in two different and coexisting modes; one involving direct reactions of molecular ozone and the other a free radical-mediated reaction.[21] Both these mechanisms may be involved in the destruction of bacteria by ozone.In vivo root canal contents and caries, unlike artificial biofilms, contain many molecules such as iron, which can increase the antimicrobial effectiveness of ozone in teeth and can help produce the powerful hydroxyl radicalsin vivo to further increase the antimicrobial effectiveness of ozone.[22]

Ozone has a dramatically toxic effect to microaerophilic and anaerobic bacteria. The antimicrobial performance of Endox Endodontic System, MTAD, 3% NaOCl, and heal ozone was compared. It was concluded that ozone has great potential in endodontic antimicrobial use and that MTAD and heal ozone seem to be as effective as 3% NaOCl in reducing mixed bacterial infection in the root canal system.[23] In agreement, Cardoso et al. concluded that ozonated water, when used as an irrigant, significantly reduced the number of Candida albicans and E. faecalis in the root canals of the human teeth.[24] Similarly, the efficacy of sterile physiologic sodium chloride solution, 3% hydrogen peroxide solution, 0.2% CHX solution, 1.5% NaClO solution, and 3% NaClO solution was compared on the basis of reduction of CFUs and concluded that ozonized oxygen appears to be suitable for disinfecting root canal systems in cases where NaOCl is not indicated.[24],[25] In our study too, we had compared ozonated water with green tea extract and normal saline for antimicrobial efficacy on the basis of reduction of CFUs and have found that ozonated water was the most effective among the three irrigants.

It has been shown that highly concentrated gaseous (1–53 g/m3) and aqueous ozone (1.25–20 μg/ml) was dose-, strain-, and time-dependently effective against the tested microorganisms in suspension and the biofilm. They concluded that 20 μg/ml of aqueous ozone is as effective as 5.25% NaOCl and 2% CHX.[26] A similar concentration of aqueous ozone was used in our study, and we found ozonated water more effective against anaerobes as compared to green tea and normal saline. It can be speculated that the antimicrobial effect of the ozone would have been even greater if it had been used with less organic debris remaining. They opined that conventional irrigation (including NaOCl) should be used during cleaning and shaping, and ozonated water should be used as the final irrigant with ultrasonication.

Contrary to these reports and our own findings, Müller et al. found that 5% NaOCl is superior to gaseous ozone in eliminating microorganisms organized in a cariogenic biofilm.[27] Similarly, it was proved that the irrigation of infected human root canals with ozonated water 0.68 ppm, 2.5% NaOCl, 2% CHX, or the application of gaseous ozone was not sufficient to inactivate E. faecalis.[28] It has been stated that the absorbance of ozone in the water increases almost linearly with time, from 5 to approximately 60 s, but the stability of ozone in water is low and dissipates quickly at room temperature.[21],[29]

 Conclusion



Ozonated water can be opted for, as an irrigant of choice, owing to its neutral taste and potent antimicrobial efficacy, especially against anaerobes. Scientific literature points toward better acceptance of this irrigant in pediatric patients. Green tea also exhibited sufficient antimicrobial activity. Although this effect is less potent than ozonated water, the low cost, easy availability, and longer shelf-life could help in choosing it as the irrigant in many cases. Our study results indicated that ozonated water and green tea were potentially good alternatives to conventionally used irritants. Further research with larger sample sizes is required to corroborate these findings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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