ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2314
World Journal of Dentistry
Volume 14 | Issue 10 | Year 2023

Evaluation of the Effect of Pretreatment with Ozone and Sodium Hypochlorite on Pit and Fissure Sealant in Primary Molars

Mehek Gandhi1, Laxmi Lakade2, Chetana Jagtap3, Sanket Kunte4, Rohan Shah5, Sudhir Dole6

1–5Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Bharati Vidyapeeth (Deemed to be University,) Pune, Maharashtra, India

6Independent Ozone Dentistry Practitioner, Mumbai, Maharashtra, India

Corresponding Author: Mehek Gandhi, Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Bharati Vidyapeeth (Deemed to be University,) Pune, Maharashtra, India, Phone: +91 9765000465, e-mail: mehekgandhi18@gmail.com

Received on: 06 September 2023; Accepted on: 08 October 2023; Published on: 06 November 2023

ABSTRACT

Aim: The aim of the study was to evaluate the effectiveness of pretreatment with 5.25% sodium hypochlorite (NaOCl) and ozone gas from microleakage on pit and fissure sealants in primary molars.

Materials and methods: A total of 36 noncarious primary molars were decoronated at the cementoenamel junction. These crowns were divided into three groups—NaOCl (n = 12), ozone (n = 12), and control (n = 12). Pretreatment of pits and fissures was done with 5.25% NaOCl for 60 seconds, ozone gas at 100 ppm for 30 seconds, and no pretreatment in the control group. Then, etching, bonding, and sealant application were done. Later, these crowns were subjected to thermocycling. A dye penetration test was done to assess the extent of microleakage using a stereomicroscope. Statistical analysis was done using the Kruskal–Wallis test in G*Power software.

Results: The mean microleakage score in the NaOCl group was 2.58, ozone 2.67, and control was 2.75. Comparison between groups showed no significant difference (p = 0888).

Conclusion: Pretreatment with ozone gas at 100 ppm and 5.25% sodium showed no statistically significant reduction in microleakage compared to the control group.

Clinical significance: Pretreatment of enamel or dentine before sealant placement is hypothesized to remove the hybrid layer, which decreases microleakage and improves the intraoral longevity of sealants. Various mechanical and chemical methods have been used and showed certain promising outcomes. However, in the present study, a comparison of newer material ozone gas against conventional material NaOCl showed no demonstratable reduction in microleakage in primary molars.

How to cite this article: Gandhi M, Lakade L, Jagtap C, et al. Evaluation of the Effect of Pretreatment with Ozone and Sodium Hypochlorite on Pit and Fissure Sealant in Primary Molars. World J Dent 2023;14(10):907–912.

Source of support: Nil

Conflict of interest: None

Keywords: Microleakage, Ozone, Pretreatment, Pit and fissure sealant, Primary molars, Sodium hypochlorite

INTRODUCTION

Dental caries is the most chronic disease of the oral cavity. The prevalence of primary teeth caries worldwide is 46.2%, while the prevalence of dental caries in permanent teeth in children around the world is 53.8%.1 Pandey et al. stated that the overall prevalence of dental caries in India is 54.16%.2 According to a meta-analysis of cross-sectional studies using the World Health Organization criteria, the prevalence of early childhood caries is 48%.3 Devan et al.’s comprehensive review and meta-analysis stated the prevalence of early childhood caries in India is 46.9%.4

Despite medical and therapeutic breakthroughs, early childhood caries remains one of the most important challenges for pediatric dentists. This is because the teeth lose their safety net during emergence into the oral cavity, rendering them more vulnerable to the assault of cariogenic food and microorganisms. As a result, it is vital to adopt all preventive treatments available to arrest the progression of childhood dental caries.

Pits and fissures of primary and young permanent teeth, owing to their complex morphology and retentive nature, are more susceptible to food lodgment, cariogenic bacteria, plaque retention, and, therefore, caries. According to Carlos et al., pit and fissure caries account for 80% of carious lesions and arise within the first 3 months after tooth eruption.5 Pits and fissures account for just 12.5% of total tooth surfaces,6 but they account for >85% of overall caries burden in school-aged children.7 Hence, pit and fissure protection is crucial, based on the data presented above. As a result, pit and fissure sealants are usually regarded the most effective therapy for preventing cavities in these locations. A Cochrane systematic review published in 2017 found that pit and fissure sealants decreased caries by 11–51% compared to no sealant when evaluated after 24 months.8 Up to 48 months, particular times revealed comparable results. Wright et al. showed an up to 80% decrease after 2 years in a systematic review.9 Despite their well-known advantages, pit and fissure sealants remain an underutilized preventative measure.

The determining element for the prevention of pit and fissure caries is sealant retention, resistance to microleakage, and dislodgement. Microleakage can degrade the sealant material and increase the likelihood of caries formation and progression on occlusal surfaces.10 The duration of sealant retention is determined by several parameters, including fissure kind, sealant type, adherence to the tooth surface, enamel condition, method sensitivity, and operator capabilities. Sealant failure is mostly caused by poor adhesion and occlusal wear.

The quality of adhesion depends on the etching of the enamel surface. Phosphoric acid is the conventional method used to create microporosities on enamel. The use of only phosphoric acid often leads to inadequate adhesion as phosphoric acid cannot etch the organic content of aprismatic enamel. Therefore, the advent of several methods, both mechanical and chemical, has been advocated before the application of the sealant for better adhesion and, in turn, retention.

Several authors have suggested that pretreatment of any kind helps increase the microporosities in the etched enamel and improve sealant adhesion.

Pretreatment of the enamel surface can be done mechanically using burs, air abrasion, laser ablation, pumice prophylaxis, etc. Recently, chemical methods of using various agents like sodium hypochlorite (NaOCl), chlorhexidine, and ozone as pretreatment agents have also been hypothesized.11 Ozone has been used in medicine for its therapeutic benefits. However, in dentistry, it has gained popularity in recent years due to its antibacterial, immunostimulating, anti-inflammatory, and analgesic effect on the oral tissues.12

Thus, the aim of this study was to evaluate the effect of pretreatment with 5.25% NaOCl and ozone gas at 100 ppm on microleakage in pit and fissure sealants in primary teeth.

MATERIALS AND METHODS

This study was approved by the research and ethical committee of Bharati Vidyapeeth Dental College and Hospital, Pune, Maharashtra, India (EC/NEW/INST/2019/329). The study duration was 5 months. A total of 36 noncarious primary molars extracted due to pathological or physiological mobility and over-retained deciduous teeth were included in the study. Carious primary molars, teeth with structural defects, cracks, and developmental anomalies were excluded. The sample size for microleakage was calculated using G*Power software, keeping the level of significance at 5%, the power of the study was kept at 80%, and an effect size of 1.18; the sample size estimated was 12 per group.

Specimen Preparation

A total of 36 noncarious extracted primary molars were washed, cleaned with pumice and water, autoclaved, and stored in saline at room temperature until the study procedure. Further, primary molars were decoronated at the cementoenamel junction to retain the crowns. They were divided randomly into three groups— NaOCl, ozone gas, and control (Flowchart 1).

Flowchart 1: Methodology

Sodium hypochlorite (NaOCl) group (n = 12): Occlusal pits and fissures of crowns were pretreated with 5.25% NaOCl (Irrisol) using a microtip brush for 60 seconds. They were rinsed with water, followed by acid etching with 37% phosphoric acid (Avue 37%) for 20 seconds. Later, a dentin bonding agent (3M ESPE Adper Single Bond) was applied, and sealant (Protec) was placed.

Ozone gas group (n = 12): Occlusal pits and fissures of crowns were pretreated with ozone gas (ozone GM/HR). This ozone gas was delivered from the ozone gas production machine at 100 ppm using a narrow gas delivery nozzle for 30 seconds. Following this, acid etching with 37% phosphoric acid (Avue 37%) for 20 seconds was done. Then, a dentin bonding agent (3M ESPE Adper Single Bond) was applied, and sealant (Protec) was placed (Figs 1A to C).

Figs 1A to D: (A) Pretreatment with ozone gas at 100 ppm; (B) Acid-etching following pretreatment; (C) Sealant placement; (D) Microleakage analysis under a stereomicroscope

Control group (n = 12): Occlusal pits and fissures of crowns were not pretreated with any agent. Hence, crowns were directly acid etched with 37% phosphoric acid (Avue 37%) for 20 seconds, followed by the application of dentin bonding agent (3M ESPE Adper Single Bond), and sealant (Protec) was placed.

All crowns were stored in distilled water separately in three different containers. Thereafter, the crowns were subjected to thermocycling (500× cycles for 1 day) to simulate the conditions of the oral cavity. Crowns were sealed with two coats of nail varnish and immersed in 2% methylene blue solution for 24 hours. Thorough rinsing with distilled water and air-drying of the crowns was done. Each crown was sectioned longitudinally in a buccolingual direction using a water-cooled diamond disk. The sections were kept dry until microscopic examination. The depth of penetration of methylene blue within the sealant helps in the evaluation of microleakage.

Microleakage Test

The methylene dye penetration test was evaluated using a stereomicroscope of prepared sections from each specimen. The degree of penetration of methylene blue dye on photographs (10× magnification) was observed to determine the depth of microleakage (Fig. 1D).

Microleakage was assessed using the scoring system given by Demirci et al., 2013.13

  • No dye leakage.

  • Dye leakage up to half of the cavity walls.

  • Dye leakage in the entire cavity wall.

  • Dye leakage in the cavity wall and floor.

  • Dye leakage partly or fully extends to the pulp.

Statistical Analysis

Data obtained was analyzed using SPSS v23 software, keeping the level of significance at 5%. Kruskal–Wallis test was used to compare the microleakage values, and a p-value of <0.05% was considered significant.

RESULTS

The microleakage scores were evaluated using a scoring system by Demirci et al., which ranges from score 0 to 4. Score 0 signifies no microleakage, whereas score 4 shows maximum microleakage.

The mean values of microleakage for the NaOCl group, ozone group, and control group were 2.58, 2.67, and 2.75, respectively. By applying the Kruskal–Wallis test, the mean values within the three groups were compared, which showed no statistically significant difference (p = 0.888) (Tables 1 and 2).

Table 1: Distribution of microleakage scores
Groups Number of samples with a score of 0 Number of samples with a score of 1 Number of samples with a score of 2 Number of samples with a score of 3 Number of samples with a score of 4 Total number of samples
NaOCl (n = 12) 0 (0%) 2 (16.7%) 4 (33.3%) 3 (25%) 3 (25%) 12 (100%)
Ozone (n = 12) 0 (0%) 3 (25%) 1 (8.3%) 5 (41.7%) 3 (25%) 12 (100%)
Control (n = 12) 0 (0%) 4 (33.3%) 0 (0%) 3 (25%) 5 (41.7%) 12 (100%)
Table 2: Comparison of microleakage between three groups
Groups Mean Standard deviation Mode p-value
NaOCl 2.58 1.08 2 0.888 (NS)
Ozone 2.67 1.15 3
Control 2.75 1.36 4

Kruskal–Wallis test; NS, nonsignificant difference

Mann–Whitney U test was applied for intergroup comparison of microleakage scores showing control vs ozone 0.715 [nonsignificant (NS)], control vs NaOCl 0.675 (NS), and ozone vs NaOCl 0.811 (NS) (Table 3).

Table 3: Pairwise comparison of microleakage scores
Groups p-value
Control vs ozone 0.715 (NS)
Control vs NaOCl 0.675 (NS)
Ozone vs NaOCl 0.811 (NS)

Mann–Whitney U test; NS, nonsignificant difference

DISCUSSION

Pit and fissure sealants provide dual protection against pit and fissure caries. Sealants used in primary prevention render pits and fissures self-cleansable and, in secondary prevention, slow the spread of noncavitated lesions. The determining element for the prevention of pit and fissure caries is sealant retention and resistance to microleakage and dislodgment. The introduction of acid etching by Buonocore14 caused a paradigm shift in improving and increasing the adhesion of the resin material to the enamel surface. Phosphoric acid is a well-known and widely used technique for creating microporosities in the enamel for sealant retention. Although, using phosphoric acid before sealant application does not always result in a uniformly and adequately etched enamel surface due to organic residues, fissure morphology, and aprismatic enamel structure (Garcia-Godoy and de Araujo; Garcia-Godoy and Gwinnett).

Tooth cleaning, enamel preparation, and tooth surface treatment (Geiger et al.) before etching and sealant application can help overcome these problems, thus improving the marginal seal and retention of sealants. Several techniques, such as pumice prophylaxis (Ellis et al.), air-abrasion with aluminum oxide particles delivered by air pressure (Kanellis et al.), slow or high-speed bur enameloplasty (Hatibovic-Kofman), laser (Feigel et al.), NaOCl (Espinosa et al.) etc. have been proposed and investigated to increase the quality and quantity of etched enamel with debatable outcomes. Therefore, in this study, we chose to evaluate the pretreatment of pits and fissures with NaOCl and ozone based on their properties.

Pit and fissure pretreatment evaluations have been majorly carried out in permanent molars compared to primary molars. Hence, primary molars were used for this study due to the difference in the enamel structure and content in permanent and primary teeth. This aided in eliminating anatomical differences and allowed for study standardization. Thermocycling is a standardized approach that is used to replicate the aging process in vitro. The samples are exposed to periodic cold and high-temperature exposures, and the bonded materials are assessed. When there is a difference in the coefficient of thermal expansion between the filling material and tooth structure, leakage occurs marginally. Due to its affordability, simplicity, and deeper staining, the dye penetration approach was chosen for the current investigation. The dye molecules’ low weight allows them to enter spaces that are inaccessible to microbiological organisms.

In this study, we evaluated the pretreatment of pits and fissures with NaOCl and ozone gas based on their properties. NaOCl is widely used in day-to-day dentistry.15 In the early 1900s, it was utilized as a wound irrigant and was brought into endodontics. It has low viscosity and nonspecific proteolytic and antibacterial activity. Espinosa et al.16 introduced NaOCl as a deproteinization agent in the pretreatment of pits and fissures. Around 1–5% concentration is commonly used as a pretreatment agent which removes enamel and dentinal organic materials. Ozone has piqued the interest of dentists since Dr EA Fisch initially reported on the use of ozonized water as a disinfectant and wound-healing agent following dental surgery in 1930. Today, the most common methods of administering ozone are gaseous ozone (produced by an ozone generator that converts oxygen into ozone), ozonated water (used as a mouthwash), and ozonated oils (produced by a chemical reaction between ozone and pure plant extracts to produce an oil or a jelly-like product).17,18 In pediatric dentistry, its use is attributed to its quick, easy, effective, and painless administration. Ozone is also used as a cavity disinfectant to treat open carious lesions, on extraction sockets, and in reimplantation of avulsed teeth. Thus, there is a need to use NaOCl as a conventional against ozone gas as a contemporary pretreatment agent to aid in achieving a reduction in microleakage in comparison to phosphoric acid alone.

In the present study, pretreatment with 5.25% NaOCl showed no significant reduction in microleakage (Tables 1 and 2). On application of the Mann–Whitney U test comparison with the control group, the p-value was 0.675, which is statistically insignificant (Table 3). Similar statistically insignificant results were observed by Bayrak et al.19 and Roopa et al.,20 who compared NaOCl with the control group, whereas Yamada et al.21 compared NaOCl to CariSolv. In contrast to our study, NaOCl pretreatment showed a statistically significant reduction in microleakage in studies done by Garrocho-Rangel et al.22 and Mubeena et al.23 They concluded that pretreatment with NaOCl was an effective method to control microleakage and increase the retention of pit and fissure sealants. A study done by Ahmed et al.24 evaluated etching patterns on pretreatment with NaOCl in primary hypocalcified teeth. They concluded that pretreatment with NaOCl significantly improved the etching pattern in primary teeth. However, as these studies were conducted on permanent molars, a direct comparison cannot be made with our study. Ahuja et al.25 evaluated the etching pattern followed by pretreatment with NaOCl in permanent molars and concluded that there was no significant difference with or without pretreatment. Arslan et al.26 investigated surface preparation with NaOCl on the shear bond strength of primary and permanent teeth and concluded that there was no significant difference in shear bond strength with or without the use of NaOCl in both primary and permanent teeth.

Ozone has various mechanisms of action when used as a pretreatment agent. During the pretreatment of enamel, ozone gas acts on the pits and fissures by causing a reversible condition of enamel dehydration. As a result, eliminating any leftover moisture may help the hydrophobic fissure sealant material penetrate deeper into the enamel surface and cracks.27 It also functions as a disinfectant, removing minute remnants of organic components in the fissure that typical fissure cleaning procedures24 do not eradicate, resulting in a cleaner enamel surface and increased material adhesion. According to Dukić et al., removing microscopic debris from the fissure may allow the sealant material to penetrate deeper into the fissure.28 Gaseous form of ozone is readily accessed in the deeper portions of the pits and fissures and helps remove the smear layer, thereby remineralizing noncavitated pits and fissures.

In a clinical trial done by Castillo et al., sealants applied after ozone treatment had the greatest remineralization effect when compared to all other untreated groups.29,30 A split-mouth study in which 258 pit and fissure caries lesions in primary teeth were treated with ozone gas (2100 ppm) for 10 seconds before receiving xylitol and fluoride showed a similar trend. As the lesions were followed for 12 months using an electrical caries monitor (ECM), the ozone-treated group showed a substantial improvement in lesion reversal, as measured by ECM scores, when compared to controls.30 Over a 3-month period, Huth et al. found that ozone administration dramatically decreased noncavitated early fissure caries in individuals at high caries risk.31 Hence, the gaseous form of ozone was chosen as a pretreatment agent in the present study.

The study done by Guneş et al.32 was in accordance with our study, where they showed no statistically significant differences in ozone gas pretreated groups. They evaluated the disinfectant property of ozone on microleakage in class V cavities in permanent third molars. They concluded that there was no statistically significant difference in the microleakage, but the mean microleakage was the least in the ozone group.

The study done by Cehreli et al.33 was in contrast with the current study, where they concluded that ozone pretreatment significantly reduced the extent of microleakage. This may be because their study was conducted on permanent third molars.

Çelik et al.34 investigated shear bond strength in human third molars using pretreatment with ozone gas against laser and control group. They concluded that pretreatment with ozone gas did not affect the shear bond strength. Bezgin et al.35 evaluated sealant adaptation and microleakage using ozone treatment after acid etching and concluded that ozone treatment improved sealant retention. This could be attributed to the different concentrations of ozone used and the nonspecification of the teeth used. Jumanca et al.36 conducted an in vivo study to evaluate the effect of pretreatment with ozone gas in permanent molars and premolars. They assessed the sealant retention after 12 months and concluded that the pretreated group’s sealant retention was far superior to the control group.

Ozone treatment is safe when administered by a trained practitioner at the appropriate doses. Considering it is a novel therapeutic drug in dentistry, its negative effects on oral tissues have not been recorded. In an in vitro study, Huth et al.31 researched the toxic effects on the epithelium inside the mouth of a 1-minute application of ozone and compared these effects with those of chlorhexidine gluconate (0.2 and 2%), NaOCl (5.25 and 2.25%), and hydrogen peroxide (3%). According to the study results, in contrast to the various toxic effects shown by the disinfectants, ozone displayed no toxic effect on the live cells of the fluid. Nagayoshi et al.37 reported a decrease in the number of Enterococcus faecalis and Streptococcus mutans following the application of ozone to contaminated dentine tubules. In the same study, similar results were shown from sonication together with ozone and the application of 2.5% NaOCl. In people allergic to ozone, if not delivered appropriately, it can induce epiphora, upper respiratory irritation, rhinitis, cough, headache, occasional nausea, vomiting, shortness of breath, blood vessel swelling, and poor circulation.12

Intergroup comparison was done using the Kruskal–Wallis test as the data was nonparametric and had three groups. The p-value obtained was 0.88, which was statistically insignificant; hence, no material proved to be an effective pretreatment agent compared to the control group before sealant application (Table 2).

This can be attributed to the effect of morphological (such as shallow pits and fissures in primary molars) and histological (such as prismless enamel in primary teeth) variations between the two types of dentitions on sealant retention.38 Differential adhesion between the primary and permanent dentitions cannot be neglected. As a result, a direct comparison of most of the preceding research with the current study using primary teeth as a sample is challenging. The present study is one of the few studies carried out with pretreatment of enamel with 5.25% NaOCl and ozone gas before pit and fissure sealant application to evaluate microleakage primary molars in comparison to several other studies done on permanent molars.

However, the study has its limitations of smaller sample size and in vitro study. The outcomes of this study represent preliminary evidence of the use of 5.25% NaOCl and ozone gas as pretreatment agents for facilitating enhanced adherence and retention of fissure sealants. Further laboratory and clinical research are needed to explore the continued effectiveness of fissure sealants on both normal and pretreated enamel of primary teeth.

CONCLUSION

Sealants have been shown to decrease caries by 59–96% during a 1–9-year follow-up periods.39 The lifespan of the sealant within the oral cavity is determined by proper tooth surface preparation and sealant application method. Pretreatment is one such way to ensure enamel preparation and reduce microleakage even more. The pretreatment agent can be chosen based on evidence-based findings and material accessibility. There is a scarcity of such investigations in the literature. The results of this study showed statistically insignificant results, and hence, the use of pretreatment agents in primary prevention of the microleakage is questionable. Therefore, further in vitro research must be undertaken with a larger sample size to evaluate the effectiveness of the pretreatment agents. To build evidence-based practice, a large number of in vivo studies need to be encouraged.

REFERENCES

1. Kazeminia M, Abdi A, Shohaimi S, et al. Dental caries in primary and permanent teeth in children’s worldwide, 1995 to 2019: a systematic review and meta-analysis. Head Face Med 2020;16(1):22. DOI: 10.1186/s13005-020-00237-z

2. Pandey P, Nandkeoliar T, Tikku AP, et al. Prevalence of dental caries in the Indian population: a systematic review and meta-analysis. J Int Soc Prev Community Dent 2021;11(3):256–265. DOI: 10.4103/jispcd.JISPCD_42_21

3. Uribe SE, Innes N, Maldupa I. The global prevalence of early childhood caries: a systematic review with meta-analysis using the WHO diagnostic criteria. Int J Paediatr Dent 2021;31(6):817–830. DOI: 10.1111/ipd.12783

4. Devan I, Ramanarayanan V, Janakiram C. Prevalence of early childhood caries in India: a systematic review and meta-analysis. Indian J Public Health 2022;66(Supplement):S3–S11. DOI: 10.4103/ijph.ijph_1078_22.

5. Carlos JP, Gittelsohn AM. Longitudinal studies of the natural history of caries. II. A life-table study of caries incidence in the permanent teeth. Arch Oral Biol 1965;10(5):739–751. DOI: 10.1016/0003-9969(65)90127-5

6. Ahovuo-Saloranta A, Hiiri A, Nordblad A, et al. Pit and fissure sealants for preventing dental decay in the permanent teeth of children and adolescents. Cochrane Database Syst Rev 2004;(4):CD001830. DOI: 10.1002/14651858.cd001830.pub2

7. Hicks MJ, Flaitz CM. Epidemiology of dental caries in the pediatric and adolescent population: a review of past and current trends. J Clin Pediatr Dent 1993 Fall;18(1):43–49.

8. Ahovuo-Saloranta A, Forss H, Walsh T, et al. Pit and fissure sealants for preventing dental decay in permanent teeth. Cochrane Database Syst Rev 2017;7(7):CD001830. DOI: 10.1002/14651858.cd001830.pub5

9. Wright JT, Tampi MP, Graham L, et al. Sealants for preventing and arresting pit-and-fissure occlusal caries in primary and permanent molars: a systematic review of randomized controlled trials-a report of the American Dental Association and the American Academy of Pediatric Dentistry. J Am Dent Assoc 2016;147(8):631–645.e18. DOI: 10.1016/j.adaj.2016.06.003

10. Simonsen RJ. Retention and effectiveness of a single application of white sealant after 10 years. J Am Dent Assoc 1987;115(1):31–36. DOI: 10.14219/jada.archive.1987.0199

11. Ciucchi P, Neuhaus KW, Emerich M, et al. Evaluation of different types of enamel conditioning before application of a fissure sealant. Lasers Med Sci 2013;30(1):1–9. DOI: 10.1007/s10103-013-1333-2

12. Naik SV, K R, Kohli S, et al. Ozone- a biological therapy in dentistry- reality or myth? Open Dent J 2016;10:196–206. DOI: 10.2174/1874210601610010196

13. Demirci M, Tuncer S, Tekçe N, et al. Influence of adhesive application methods and rebonding agent application on sealing effectiveness of all-in-one self-etching adhesives. J Esthet Restor Dent 2013;25(5):326–343. DOI: 10.1111/jerd.12034

14. Buonocore MG. A simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. J Dent Res 1955;34(6):849–853. DOI: 10.1177/00220345550340060801

15. Pecora JD, Sousa-Neto MD, Estrela C. Soluções irrigadoras auxiliares do preparo do canal radicular. In: Estrela C, Figueiredo JAP (Eds). Endodontia - Princípios Biológicos e Mecânicos. São Paulo: Artes Médicas; 1999. pp. 552–569.

16. Espinosa R, Valencia R, Uribe M, et al. Resin replica in enamel deproteinization and its effect on acid etching. J Clin Pediatr Dent 2010;35(1):47–51. DOI: 10.17796/jcpd.35.1.u425308167271132

17. Suh Y, Patel S, Kaitlyn R, et al. Clinical utility of ozone therapy in dental and oral medicine. Med Gas Res 2019;9(3):163–167. DOI: 10.4103/2045-9912.266997

18. Pattanaik B, Pattanaik S, Naitam D, et al. Ozone therapy in dentistry: a literature review. J Interdiscip Dent 2011;1(2):87–92. DOI: 10.4103/2229-5194.85024

19. Bayrak GD, Gurdogan-Guler EB, Yildirim Y, et al. Assessment of shear bond strength and microleakage of fissure sealant following enamel deproteinization: an in vitro study. J Clin Exp Dent 2020:e220–e226. DOI: 10.4317/jced.56281

20. Roopa KB, Abraham AB, Poornima P, et al. Evaluation of deproteinization on clinical success and longevity of pit and fissure sealants on erupting permanent first molars – an in vivo study. Int J Community Dent 2019;7(2):42–48. DOI: 10.4103/ijcd.ijcd_1_20

21. Yamada Y, Hossain M, Kimura Y, et al. Removal of organic debris from occlusal fissures: advantage of Carisolv system over sodium hypochlorite. J Clin Pediatr Dent 2010;35(1):75–79. DOI: 10.17796/jcpd.35.1.84368262v2j4vu96

22. Garrocho-Rangel A, Lozano-Vázquez C, Butrón-Tellez-Girón C, et al. In vitro assessment of retention and microleakage in pit and fissure sealants following enamel pre-etching with sodium hypochlorite deproteinisation. Eur J Paediatr Dent 2015;16(3):212–216.

23. Mubeena V, Emmatty TB, Kavita K, et al. Effect of enamel pre-etching with sodium hypochlorite deproteinisation and bonding agent on retention and microleakage of pit and fissure sealants: an in-vitro study. J Clin Diagn Res 2021;15(4):ZC01–ZC05. DOI: 10.7860/jcdr/2021/46976.14667

24. Ahmed AM, Nagy D, Elkateb MA. Etching patterns of sodium hypochlorite pretreated hypocalcified amelogenesis imperfecta primary molars: SEM study. J Clin Pediatr Dent 2019;43:257–262. DOI: 10.17796/1053-4625-43.4.6

25. Ahuja B, Yeluri R, Baliga S, et al. Enamel deproteinization before acid etching–a scanning electron microscopic observation. J Clin Pediatr Dent 2010;35(2):169–172. DOI: 10.17796/jcpd.35.2.9gw7147381836380

26. Arslan C, Üstün N, Cesur E. Effects of surface preparation with sodium hypochlorite on shear bond strength and residual adhesive of orthodontic brackets on primary and permanent teeth Eur Ann Den. Sci 2022;49(2):87–91. DOI: 10.52037/eads.2022.0032

27. Adalja C, Deshpande N, Dave D. Ozone: a future in periodontal therapy! Indian J Appl Res 2011;3(3):295–297. DOI: 10.15373/2249555x/mar2013/98

28. Dukić W, Dukić OL, Milardović S. The influence of Healozone on microleakage and fissure penetration of different sealing materials. Coll Antropol 2009;33(1):157–162.

29. Castillo A, Galindo-Moreno P, Avila G, et al. In vitro reduction of mutans streptococci by means of ozone gas application. Quintessence International 2008;39(10).

30. Unal M, Oztas N. Remineralization capacity of three fissure sealants with and without gaseous ozone on non-cavitated incipient pit and fissure caries. J Clin Pediatr Dent 2015;39(4):364–370. DOI: 10.17796/1053-4628-39.4.364

31. Rickard GD, Richardson R, Johnson T, et al. Ozone therapy for the treatment of dental caries. Cochrane Database Syst Rev 2004;(3):CD004153. DOI: 10.1002/14651858.CD004153.pub2

32. Huth KC, Jakob FM, Saugel B, et al. Effect of ozone on oral cells compared with established antimicrobials. Eur J Oral Sci 2006;114:435–440. DOI: 10.1111/j.1600-0722.2006.00390.x

33. Güneş Ş, Bahsi E, İnce B, et al. Comparative evaluation of the effects of ozone, diode laser, and traditional cavity disinfectants on microleakage. J Int Ozone Assoc 2014;36(2):206–211. DOI: 10.1080/01919512.2013.842888

34. Cehreli SB, Yalcinkaya Z, Guven-Polat G, Çehreli ZC. Effect of ozone pretreatment on the microleakage of pit and fissure sealants. J Clin Pediatr Dent 2010;35(2):187–190. DOI: 10.17796/jcpd.35.2.v2gn643k3774j014

35. Çelik N, Yapar MI, Karalar B, et al. Influence of laser and ozone pretreatment on the shear bond strength of fissure sealants: an in vitro comparative study. J Adv Oral Res 2020;11(2):189–195. DOI: 10.1177/2320206820944285

36. Bezgin T, Oz FT, Ozalp N. Efficacy of ozone pretreatment on fissure sealant adaptation and retention. J Int Ozone Assoc 2021;44(3):317–323. DOI: 10.1080/01919512.2021.1919054

37. Jumanca D, Anamaria M, Atena G, et al. Comparative study of acid etching on dental enamel. Revista de Chimie 2018;69(10):2913–2915. DOI: 10.37358/rc.18.10.6652

38. Nagayoshi M, Kitamura C, Fukuizumi T, et al. Antimicrobial effect of ozonated water on bacteria invading dentinal tubules. J Endod 2004;30(11):778–781. DOI: 10.1097/00004770-200411000-00007

39. Bhushan U, Goswami M. Evaluation of retention of pit and fissure sealants placed with and without air abrasion pretreatment in 6-8 year old children - an in vivo study. J Clin Exp Dent 2017:9(11):e211–e217. DOI: 10.4317/jced.53259

40. Schwimmer Y, Beyth N, Ram D, et al. Laser tooth preparation for pit and fissure sealing. Int J Environ Res Public Health 2020;17(21):7813. DOI: 10.3390/ijerph17217813.

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