Researchers have established that bacteria are the primary cause of root canal infections. Viridans streptococci (alpha-hemolytic streptococci) were the most often found bacterial group in the root canals of permanent teeth until 1970 [1, 2]. Still, studies of the microbiota found in the root canals of baby teeth have shown that anaerobic microorganisms are the most common type in these canals when the pulp is dead and there are sores around the edges of the tooth. In the root canals of primary molars that have undergone unsuccessful treatment, these anaerobic microorganisms comprise over 70% of the microbiota [3]. Anaerobic microorganisms are the dominant bacteria found in primary teeth that require extraction [1, 4]. Due to the swift advancement of caries in primary teeth and the resulting pulpal damage caused by bacteria and their toxins that infect the tissue, endodontic therapy is required. Controlling infection is critical for two main reasons: first, the extensive medullary bone gaps facilitate the spread of infection, and second, the developing permanent tooth germ is in close proximity to the roots of the primary teeth. Many often view instrumentation as the fundamental aspect of endodontic treatment for caries. Still, the complicated structure of the root canal system in primary teeth, which includes dentinal tubules, auxiliary canals, and root tip bifurcation, makes it hard to clean them completely with instruments alone. As a result, root canal irrigation is a crucial component of chemomechanical preparation. Clinical practice uses different irrigation agents based on their ability to kill microorganisms, clean well, and be compatible with biological systems. Despite being considered an optimal solution for irrigating root canals, children cannot use sodium hypochlorite (NaClO) at the necessary concentrations due to its unfavorable effects or restrictions. These include unpleasant taste and odor, toxicity, ineffectiveness in eliminating the smear layer, and incomplete eradication of microbes from infected canals. The unintentional release of NaClO results in immediate intense pain, swelling of nearby soft tissues, bruising, and abnormal sensations, all due to the body's tissue reaction [5]. People commonly use Chlorhexidine digluconate, an alternative irrigant, for disinfection because of its strong antibacterial properties. However, it does not have the potential to dissolve tissue. Both hydrogen peroxide and regular saline do not possess optimal antibacterial activities [6]. As a result, it is critical to continue the search for an optimal root canal irrigant for primary teeth. Ozone, with its antibacterial, antiviral, and antifungal properties, can be considered a promising agent to address these limitations. You can deliver it in both gaseous and liquid forms. When applied in any form, they exhibit potent antibacterial effects by rapidly oxidizing the cell walls and cytoplasmic membranes of microorganisms. It can also get into the periapical tissue through the apical foramen, which helps tissue grow back by increasing blood flow and oxygenation in the area. A recent study found that ozonated water is less harmful to mouse fibroblasts compared to NaClO, indicating that using ozonated water may be beneficial for endodontic therapy [7]. In vitro studies have demonstrated that aqueous ozone does not cause harm to oral cells. It is necessary to examine whether the antibacterial efficacy of aqueous ozone is equivalent to that of NaClO. Additional findings indicate that ozone could serve as a viable substitute for disinfection in root canals. Currently, there is a global movement towards the use of non-toxic botanical substances that have a history of traditional medical usage. As a result, we began searching for plant-based products that may not have the harmful impacts seen in synthetic items. Our hypothesis suggests the potential use of green tea as an irrigant for primary teeth. Green tea has several significant benefits, including its widespread availability, economic efficiency, extended shelf life, minimal toxicity, and absence of microbial resistance. Furthermore, epigallocatechin-3-gallate (EGCG), the primary polyphenol found in green tea, possesses anti-inflammatory, antibacterial, anticarcinogenic, and antioxidant characteristics [8]. Hence, the objective of this study was to evaluate and compare the antibacterial effectiveness of aqueous ozone, green tea, and normal saline in eradicating anaerobic bacteria during pulpectomy procedures performed on primary teeth.

This study was conducted on 60 patients between the ages of 4 and 8 years, indicated for pulpectomy in primary teeth, who visited the Department of Dentistry, Darbhanga Medical College and Hospital, Darbhanga, Bihar (India). Written consents were obtained from the parents/guardians of each participant after providing them with relevant information. The subjects were chosen based on the following criteria for inclusion and exclusion:

Inclusion Criteria:

• Patients aged 4 to 8 years with a deciduous tooth with a single root that is suitable for pulpectomy.
• Teeth having over two-thirds of the root length remaining.
• Patients who express a willingness to take part in the study.
• Otherwise, patients who are in normal and healthy condition and do not have any systemic disease.

Exclusion Criteria:

• Severely deteriorated dental condition.
• Teeth exhibiting advanced internal or exterior resorption.
• Tooth displaying the development of a sinus tract.
• Patients who have had antibiotics during the last 3 months before the reporting date.
• Patients with glucose-6-phosphate-dehydrogenase deficiency (favism), hyperthyroidism, severe anemia, severe myasthenia, and any congenital heart disease.
• Individuals who have an allergic reaction to ozone.
• Patients who are not willing to engage in the study.

The 60 infected teeth of the chosen participants were assigned at random to one of three treatment groups, depending on the irrigating substance employed during the endodontic therapy.

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

The endodontic therapy, which involved root canal irrigation, was conducted in a double-blinded manner.

Preparation of root canal irrigants and transport media:

Preparation of ozonated water: The preparation of ozonated water for root canal irrigation involved the use of a GE OZONE G™ machine. This machine produced water with an ozone concentration of 20 mg/l within 8 minutes. The process utilized a 1-litre input of distilled water, which was treated in a glass container. To maintain concentration, the water that was made had to be utilized within 20 minutes of its production.

Process of preparing green tea: The tea extract was prepared using commercially available green tea. A quantity of 25 grams of tea was combined with 50 milliliters of water using conventional preparation methods. Subsequently, the concoction was subjected to a 2-minute boiling process, followed by straining, filtering, cooling, and finally, preservation in an airtight receptacle. The extract was utilized within a 24-hour timeframe after it was prepared.

Preparation of transport media: A total of 3.8 grams of concentrated Clostridial HiVeg Agar media (HIMEDIA M154), along with 10 ml each of dithiothreitol and resazurin, were suspended in 100 ml of distilled water while ensuring the concentration was maintained. The media underwent autoclaving. After preparing, 2 milliliters of this medium were placed into the vacutainers for transportation. The medium was ready to be used 48 hours after preparation and was stored at a temperature range of 2°C to 8°C. Typically, the transportation medium has a pale yellow hue, but it changes to a pink shade in the presence of oxygen. This inherent characteristic aided in verifying the preservation of sufficient anaerobic conditions throughout transportation.

Endodontic procedure and bacterial culture:

The patient was sedated and placed in isolation using a rubber dam. The tooth and the rubber dam next to it were sterilized using a solution of iodine tincture. The root canal access was performed using a high-speed air-rotor handpiece and a round bur. After obtaining access, a sterile broach was used to extract the root canal substance, which was promptly transferred to a tube containing a transport medium. The tube was securely closed in preparation for its transmission to the microbiological laboratory. This served as the specimen for the "preirrigant culture" for all three groups. On the same day, the process of determining the working length and performing biomechanical preparation was carried out using files that were three times larger than the first instrument. During the process of instrumentation, the root canal was repeatedly flushed with 2 ml of a specific irrigant that is suitable for the particular group, after each stage. Measurement and control of variables using instruments. An aseptic paper point was inserted into the root canal and remained in position for 1 minute. The paper tip was extracted using a sterilized tweezer and promptly transferred to a tube filled with a transport medium. This specimen was representative of the "postirrigant culture". The access opening was closed using a temporary restorative material, creating a layer that was roughly 4 mm thick. The patient's second appointment was planned for the third day after the surgery.

During the second appointment, the tooth was isolated and the surrounding was disinfected, just like in the previous appointment. The provisional dental filling was extracted. A subsequent microbiological specimen was collected. This specimen is referred to as the "third-day culture". The microbiological specimen was thereafter delivered to the laboratory to quantify the bacterial population. The key stage in obtaining an anaerobic bacterial culture was ensuring a completely sterile anaerobic environment throughout the procedure. This was achieved by utilizing the laminar airflow system. This method involved placing all the equipment and material inside the laminar air flow chamber utilizing the two utility windows provided for this purpose. The windows were subsequently fully sealed using tape. An anaerobic environment was established within this laminar airflow through the interaction of Na2CO3 with HCl, resulting in the liberation of a substantial quantity of CO2. After creating the anaerobic environment, the sample was streaked onto the sheep agar plates using a sterile toothpick and a cotton swab. Once streaking was completed on a specific sample, the lid was securely closed and sealed using paraffin tape. Subsequently, CO2 was pumped into these sealed agar plates to establish an appropriate anaerobic atmosphere. The identical process was repeated until all samples were infected. The agar plates were enclosed in a polythene bag, together with pyrogallol stored in a plastic tube. The bag was then sealed and placed in a gaspak, ensuring a tight closure. The entire assembly was stored in a 3.5-liter anaerobic jar and placed in an incubator at a temperature of 37°C for 24 hours. After 24 hours, the samples were carefully extracted and the quantity of colony-forming units (CFUs) was determined using a digital colony counter.

Statistical Analysis: The collected data was organized into a table using Microsoft Excel 2019. Subsequently, the data was transferred to GraphPad version 8.4.3 for further statistical analysis. The mean and standard deviation (SD) were used to evaluate the average and spread of quantitative variables.

For the normal saline irrigation group, the mean CFU count was lower after initial irrigation when compared with preirrigation counts, with a mean difference of 43.25, and the mean CFU count after final irrigation was also decreased, with a mean difference of 114.07. The mean difference of the CFU count comparison between initial irrigation and final irrigation was 70.89. The P value for this reduction was <0.0001, indicating statistical significance. These findings suggest that irrigation with normal saline has limited antimicrobial efficacy [Table 1].

In the green tea extract irrigation group, the mean CFU count was lower after initial irrigation when compared with preirrigation counts, with a mean difference of 37.09, and the mean CFU count after final irrigation was also decreased, with a mean difference of 141.47. The mean difference of the CFU count comparison between initial irrigation and final irrigation was 104.38. The P value for this reduction was <0.0001, indicating statistical significance. These findings suggested that although green tea showed no antimicrobial activity after initial irrigation, it exhibited considerable activity after final irrigation [Table 1].

In the ozonated water irrigation group, the mean CFU count was lower after initial irrigation when compared with preirrigation counts, with a mean difference of 106.39, and the mean CFU count after final irrigation was also decreased, with a mean difference of 156.67. The mean difference of the CFU count comparison between initial irrigation and final irrigation was 49.79. The P value for this reduction was <0.0001, indicating statistical significance. These findings suggested that the ozonated water also exhibited considerable antimicrobial efficacy after both initial and final irrigation [Table 1].

Table 1: Showing the antimicrobial efficacy of normal saline irrigation, green tea extract irrigation, and ozonated water irrigation

Intergroup comparison of antimicrobial efficacy of three irrigants:

After initial irrigation: The mean CFU count after the initial irrigation with ozonated water was significantly lower compared to both green tea and normal saline. This indicates that ozonated water demonstrated the highest antimicrobial efficacy after the initial irrigation, while the antimicrobial effects of green tea and normal saline were comparable [Table 2].

After final irrigation: The mean CFU count after the final irrigation with ozonated water was significantly lower than that with both green tea and normal saline. Additionally, the mean CFU count after the final irrigation with green tea was significantly lower than that with normal saline. These findings suggest that ozonated water had the highest antimicrobial efficacy after the final irrigation. Furthermore, the antimicrobial activity of green tea after the final irrigation was significantly better than that of normal saline [Table 3].

Table 2: Showing the Intergroup comparison of antimicrobial efficacy after initial irrigation

Table 3: Showing the Intergroup comparison of antimicrobial efficacy after final irrigation (on Day 3)

DISCUSSION:

Despite over a hundred years of technological advancements in root canal treatments, clinical investigations show that bacteria still persist in the canal even when standardised cleaning and shaping processes and irrigants are used. Therefore, we assessed the antimicrobial effectiveness of normal saline, ozonated water, and green tea in reducing the number of colony-forming units (CFUs) in anaerobic bacterial culture, as part of our investigation to find the best irrigant for paediatric root canal treatments. The study comprised children between the ages of 4 and 8, who were deemed suitable for performing pulpectomy procedures [9,10]. The study focused exclusively on teeth with a single root in order to examine three aspects of root canal infections in primary teeth: (1) variations in necrosis and pulp vitality between different root canals within the same tooth; (2) differences in bacterial microbiota composition between different root canals within the same tooth; and (3) significant variations in the quantity of microbiota present in root canals within the same tooth. Considering these factors, it was hypothesized that teeth with many roots could have contributed to inaccurate findings. The use of blinding and randomization in the study ensured that the groups were more comparable and reduced the potential for bias and confounding [11,12]. This investigation involved analyzing samples of three distinct irrigants (normal saline, ozonated water, and green tea) at three specific time intervals: before irrigation, after the initial irrigation, and after irrigation on the third day after the operation. The samples were tested for microbiological culture. The average colony-forming units (CFUs) in all three groups of irrigants were identical during the preirrigation stage. This outcome was predictable given that the samples were assigned to groups in a random manner and no irrigation was conducted prior to the collection of the samples. The results we obtained were in line with other studies that have shown a bacterial count within the range of 102–108 [1,13,14]. Green tea possesses remarkable therapeutic properties [8]. Furthermore, it has been noted that green tea exhibits a distinct antibacterial impact on Enterococcus faecalis [15]. The antioxidant capacity of green tea polyphenols is closely correlated with the arrangement of aromatic rings and hydroxyl groups in their structure. The antibacterial activity occurs due to the binding and neutralization of free radicals by the hydroxyl groups, resulting in the breakdown and disintegration of the bacterial cell wall. In our study, we employed a concentration of 50 mg per ml of green tea for irrigation. This concentration was determined based on the research conducted by Araghizadeh et al., who found that a concentration of 50 mg per ml of green tea polyphenols effectively targets anaerobic bacteria such as Porphyromonas gingivalis, Prevotella intermedia, and Actinomyces actinomycetes [15]. In addition, research has discovered that EGCG, the predominant polyphenol found in green tea (Camellia sinensis), has proven to be a potent antimicrobial agent against both the planktonic and biofilm forms of E. faecalis. It effectively hinders bacterial growth and suppresses the expression of particular genes associated with virulence and biofilm formation [16]. Consistent with these observations, our findings indicate that green tea extract demonstrates notable antibacterial efficacy, particularly when used as a last irrigation on the third day. The antimicrobial activity of Green tea, Triphala, 5% NaOCl, and a Mixture of Tetracycline, acid, and detergent (MTAD) was assessed by measuring the reduction in CFUs per ml during irrigation. The study found that 5% NaClO exhibited the highest antibacterial effectiveness against E. faecalis biofilm [17]. Additionally, Triphala, green tea, polyphenol, and MTAD also demonstrated statistically significant antibacterial activity, which aligns with our own findings. Additionally, it was determined that while standard irrigants are commonly utilized, they have limitations in their ability to kill germs. On the other hand, extracts from neem leaves or green tea exhibit notable antimicrobial properties against E. faecalis, comparable to chlorhexidine (CHX) [18]. All of the previous reports are consistent with our current investigation. Al-Azzawi conducted a study to assess the efficacy of 5% green tea extract in inhibiting the growth of E. faecalis. The antibacterial effect of the tested irrigants was measured by calculating the zones of inhibition. The results showed that the mean zone of inhibition for the green tea extract was 8.88 mm, which was the smallest among the various irrigants tested, including NaOCl, CHX, and Miswak. A study demonstrated that NaClO exhibited the highest level of antibacterial activity, followed by extracts of Neem, Triphala, and green tea, in that order [19]. The study explains the inconsistent results observed when using green tea as an irrigant by suggesting that the antibacterial properties of green tea may be due to its low level of fermentation [20]. Through the process of fermentation, catechins like EGCG undergo destruction, resulting in a decrease in the antibacterial capabilities of the tea. Nevertheless, there is no known singular technique that is universally superior for all varieties of tea, and the standardized approach proved suboptimal for most sorts. Therefore, it is likely that the preparation method did not facilitate the complete release of all chemical compounds included in the tea. Alternative approaches to preparation may have yielded divergent clinical outcomes. Furthermore, a prolonged duration of tea exposure may have been required to provide a more potent antibacterial effect. Ozone is a discerning oxidant that specifically impacts particular molecules. However, when it is dissolved in water, it becomes exceedingly unstable and swiftly breaks down through a convoluted sequence of chain reactions. Consequently, the production of hydroxyl (HO.) radicals occurs, which are highly reactive oxidizing agents. Ozone undergoes two distinct and simultaneous ways of reactivity with different chemical substances in aqueous systems. One mode involves direct reactions between ozone molecules, while the second mode involves reactions mediated by free radicals [21]. Both of these strategies may contribute to the eradication of germs by ozone. Within living organisms, the contents of root canals and decayed teeth include a variety of substances, including iron. These compounds can enhance the antibacterial properties of ozone in teeth. Additionally, they can facilitate the production of potent hydroxyl radicals within the body, further augmenting the antimicrobial effects of ozone [22]. Ozone exerts a highly harmful impact on microaerophilic and anaerobic bacteria. A comparison was made between the antibacterial efficacy of the Endox Endodontic System, MTAD, 3% NaOCl, and heal ozone. The study determined that ozone has significant potential for application as an antibacterial agent in endodontics. Additionally, it was found that both MTAD and heal ozone are equally efficient as a 3% NaOCl solution in reducing mixed bacterial infection in the root canal system [23]. Cardoso et al. found that the use of ozonated water as an irrigant effectively decreased the presence of Candida albicans and E. faecalis in the root canals of human teeth [24]. Comparatively, the effectiveness of sterile physiologic sodium chloride solution, 3% hydrogen peroxide solution, 0.2% CHX solution, 1.5% NaClO solution, and 3% NaClO solution was assessed by measuring the reduction of colony-forming units (CFUs). The study concluded that ozonized oxygen is a viable option for disinfecting root canal systems when the use of NaOCl is not recommended [24,25]. In our study, we conducted a comparison between ozonated water, green tea extract, and normal saline in terms of their antibacterial activity. We measured the reduction of colony-forming units (CFUs) and determined that ozonated water was the most effective among the three irrigants. Research has demonstrated that both highly concentrated gaseous ozone (ranging from 1 to 53 g/m3) and aqueous ozone (ranging from 1.25 to 20 µg/ml) were effective against the studied microorganisms in suspension and the biofilm. The effectiveness of ozone variesd depending on the dosage, strain of microbe, and duration of exposure. The researchers determined that a concentration of 20 µg/ml of aqueous ozone had the same level of effectiveness as 5.25% NaOCl and 2% CHX, as stated in reference [26]. In our investigation, we employed a comparable level of aqueous ozone concentration and discovered that ozonated water was more efficient in combating anaerobic organisms when compared to green tea and normal saline. It is reasonable to hypothesise that the antibacterial impact of ozone would have been more pronounced if it had been employed in the presence of a lower amount of organic matter. Their opinion is that traditional irrigation methods, including the use of sodium hypochlorite (NaOCl), should be employed for cleaning and shaping. They suggest using ozonated water as the final irrigant, along with ultrasonication. In contrast to the reports and our findings, Müller et al. discovered that a 5% solution of sodium hypochlorite (NaOCl) is more effective than gaseous ozone in eradicating bacteria that are arranged in a cariogenic biofilm [27]. Furthermore, research has demonstrated that the use of ozonated water at a concentration of 0.68 ppm, 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), or gaseous ozone is ineffective in eliminating E. faecalis from infected human root canals [28]. Research has indicated that the amount of ozone absorbed in water increases in a nearly straight line with time, starting at 5 seconds and reaching around 60 seconds. However, ozone is not stable in water and soon evaporates at room temperature [21,29].

Ozonated water is a favorable choice for irrigation due to its neutral taste and strong antimicrobial effectiveness, particularly against anaerobic bacteria. Scientific research suggests it is well-received among pediatric patients. Green tea also showed considerable antimicrobial activity, though not as potent as ozonated water. Its affordability, accessibility, and longer shelf-life make it a practical option for irrigation in various cases. Our study findings suggest that both ozonated water and green tea could serve as promising alternatives to commonly used irrigants.

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