An In vitro Comparative Evaluation of Microleakage of ACP Containing Pit and Fissure Sealant and Moisture Tolerant Pit and Fissure Sealant

Background: Pits and fissure sealants are widely used to prevent caries in children. Microleakage is one of the most crucial factors resulting in sealant compromise. Factors pertaining to microleakage like, pre-treatment of occlusal surfaces, moisture control, bonding systems, and flowability of the sealant, will determine the longevity of the treatment. In vitro microleakage studies are useful in predicting the marginal sealing ability of pit and fissure sealants. Aim: This in vitro study was undertaken to evaluate and compare the microleakage of ACP (Aegis®) containing pit and fissure sealant and moisture tolerant pit and fissure sealant (Embrace Wetbond TM ). Study Design: In vitro study. Place and Duration of Study: Department of Pedodontics and Preventive Dentistry, Dr. D.Y. Patil Dental College and Hospital, Pune. between June 2020 and February 2021. Methodology: 26 permanent noncarious premolars extracted for orthodontic treatment were used. The sealants used for the analysis of microleakage were Aegis® and Embrace TM Wetbond TM pit and fissure sealants. The premolars were divided into two groups of 13 each. After sealant Original Research Article Mulani and Mathur; JPRI, 33(38A): 274-280, 2021; Article no.JPRI.71572 275 application, they were immersed in methylene blue dye. The teeth were subjected to thermocycling for 24 hours, after which The teeth were sectioned buccolingually and dye penetration was studied under a stereomicroscope (magnification 10X). Results: Both the groups were studied and statistically evaluated using MannWhitney U test. Aegis® showed higher microleakage in (46.2%) than Embrace (38%), though the results were not statistically significant. Conclusion: Both the materials showed microleakage, more In-vivo studies with longer followup periods are needed to evaluate and compare the clinical success of these pit & fissure sealants.


INTRODUCTION
Despite exhaustive research and implementation of numerous preventive strategies dental caries continues to be one of the most common diseases of mankind. Although the prevalence of dental caries is on a decline in the developed nations but in the developing countries it is still on the rise especially in children. Though fluoride's extensive use through community water fluoridation and topical application has given a significant relief from smooth surface dental caries 1 but caries in occlusal surface still remains a concern. Literature provides enough evidence on the effect of dental caries not just on oral health but also on the overall health of an individual [1,2,3].
Pit & fissure sealants constitute one of the important preventive strategies against the dental caries. Liu W et al. [4] conducted a study in Guangzhou City in 2019 and reported that the risk of caries development was decreased by 44% in the children whose fissure were sealed with sealants as compared to the ones left unsealed in a rural population while it was 35% amongst urban children. Splieth C et al. [5] also reported a significant benefit of fissure sealants in preventing dental caries on occlusal surface in primary as well as permanent dentition in children.
Pit and fissure not just help to seal the susceptible areas but also helps in remineralizing the demineralized surfaces near pits and fissures. The sealants pause the activity of the microbes, halts the caries process further acting as a preventive as well as a remineralizing agents. However, sealants come with few limitations like loss of retention and being technique sensitive. Resin sealants being hydrophobic demonstrate extremely sensitive nature to moisture in the oral cavity [6]. To overcome this moisture control challenge, Pulpdent in 2002 introduced a hydrophilic sealant EmbraceTM WetbondTM. Unlike other sealant materials that are hydrophobic and demand application of bonding agent for enhancing adaptation, the EmbraceTM WetbondTM material is a hydrophilic resin-based sealant that does not require the application of bonding agent. Aegis® pit & fissure sealant is another newly introduced sealant containing amorphous calcium phosphate that release calcium and phosphate to act as remineralizing agents. It is also known as a smart material [7]. Though newer sealants are marketed and all prove to be efficient in preventing dental caries but they all differ in their survival rate.
The marginal sealing ability of sealant materials is critical for effective treatment. Inadequate sealing leads to marginal leakage, i.e., seepage of bacteria, fluids, molecules and ions through the tooth-material interface , leading to caries progression below the sealant. In vitro microleakage studies can predict the marginal sealing ability of restorative materials Since there is a scarcity of data available on the micro leakage data comparing Aegis® and EmbraceTM WetbondTM pit and fissure sealants, this in-vitro study was undertaken to measure and compare the microleakage of Aegis® and EmbraceTM WetbondTM pit and fissure sealants.

MATERIALS AND METHODS
Before starting the study , approval from the institutional scientific and ethics committee was taken. 26 permanent premolars that were extracted for orthodontic treatment were included for the study. The sealants used for the analysis of microleakage were Aegis® pit and fissure sealant manufactured by Bosworth Companies, Keystone Industries and EmbraceTM WetbondTM pit and fissure sealant manufactured by Pulpdent. The samples were stored in aseptic medium in an accordance with CDC guidelines. Premolars with caries or developmental defects or fractures were excluded from this study. Manufacturer's instructions were followed for the application of sealants. Teeth were cleaned with pumice and water to remove debris from fissures and dried .Etching was done with 37% phosphoric acid using applicator tip, rinsing the tooth was done after 20 seconds to remove etchant material and teeth were dried to appreciate chalky white appearance to confirm adequate etching. Application of Aegis® pit and fissure sealant and EmbraceTM WetbondTM pit and fissure sealant on 13 extracted teeth of each of the two groups. The sealants were cured using a light cure gun for 40 seconds. All surfaces of the teeth were coated with a double layer of nail varnish except the occlusal surfaces, following with the teeth were then immersed in 1% methylene blue dye for 24 hours. Then the samples were removed from the dye and the excess was brushed off. Thermocycling (5 o -55 o C) was carried out for 500 cycles, with a dwell time of 30 seconds. The teeth were sectioned longitudinally in the mesio-distal section with the help of a diamond wheel measuring 0.2mm thickness. The root portions were removed after sectioning. The samples were studies under Stereomicroscope with 10X magnification for microleakage analysis.

Evaluation of Microleakage
For evaluating microleakage the sections were cleaned and examined under Stereomicroscope for penetration of dye independently by two examiners. Scoring criteria for penetration proposed by Theodoridou P et al. [8] was followed ( Table 1).
The data on microleakage were compared statistically between Aegis® and Embrace TM Wetbond TM pit and fissure sealants using the Man-Whitney test which was run in Statistical Package of Social Science (SPSS) software version 21. The significance was set at p<0.05.

RESULTS
This study was undertaken to compare the microleakage of Aegis® and EmbraceTM WetbondTM pit and fissure sealants through an in-vitro study.
The difference in microleakage scores between Aegis® and EmbraceTM WetbondTM sealant material for all the samples is represented in Table 2. After assessing the difference between the two groups it was found that the teeth sealed with Aegis® sealant displayed higher microleakage over Embrace TM Wetbond TM sealant (Graph 1) but the difference between them was not statistically significant with Man-Whitney test value of 54.00 and p=0.09 The microleakage with both the pit and fissure sealant materials was thus statistically comparable.

DISCUSSION
The success of pit and fissure sealants is governed by various factors. Few of them include bonding of sealant with the tooth surface, clinician's expertise, carious lesion in the pits and fissures, microleakage and penetration of sealant deep in the fissures. Microleakage is one of the biggest limitation of sealants, as it may lead to bacterial invasion and secondary caries. The present study used scoring criteria proposed by Theodoridou P et al. [8] which is widely accepted for the evaluation of microleakage.
As children are most benefited from sealants, preventing moisture contamination becomes challenging because of poor cooperation during the procedure leading to partial or complete sealant loss from the tooth surface and reporting a failure rate between 5 to 10% in a year [9]. Aegis® is a material containing amorphous calcium phosphate. The best property of this material is that the sealant releases calcium and phosphate ions and the ions will be released only when the pH drops down to 5.9 from 7.4 making it a smart material. The released calcium and phosphate ions help in remineralizing the demineralized areas of the fissures.
Aegis® was used on thirteen premolar samples and scanned under stereo-microscope for dye penetration to assess the microleakage associated with the material. It was found that none of the teeth showed complete adaptation and thus all presented with some amount of microleakage. Our results are comparable to studies conducted by Marks D et al. [7].The results of the present study were in contrast with another group of Selecman JB et al. [14]. In their study 90% of the molar samples presented no dye penetration while only one sample displayed dye penetration upto one-half of the sealants length. The variation in the results may be attributed to the pre-treatment of the enamel surface before sealant placement with air abrasion that was followed in the study by Selecman JB et al. [14] but not in the present study.
When the two sealants were compared for microleakage in the present study it was found that though higher scores of microleakage were observed in Aegis® as compared to Embrace TM Wetbond TM sealant material but the difference between them was not statistically significant. Both the materials were comparable in terms of microleakage. Though the literature does not mention any studies on the comparison of Aegis® with Embrace TM Wetbond TM for microleakage but they do report about the retention rates which can be associated with the microleakage. The success of the sealant depends on the adaptation of material to the tooth structure which will ultimately improve the retention rate thereby indicating less microleakage. with other flowable pit and fissure sealants too was reported to be significant for nanoleakage [16].
Pit and fissure sealants are becoming a part of the protocol for managing high risk carious groups. Several countries have developed their national programs of pit and fissure sealants for school children. India too has initiated a pilot program which will be conducted in all the districts of Maharashtra in government aided schools. The private as well as government dental Institutes in the state are a part of this program [17]. Therefore the clinical success of pit and fissure sealants is an integral step in maintaining the caries free state of the oral cavity.