Evaluation of Microleakage of Nanoparticle-incorporated Cyanoacrylate Root Canal Sealer Using the Radioisotopic Method: An In Vitro Study

INTRODUCTION

The primary goal of conventional endodontic treatment is the prevention and/or elimination of apical periodontitis, a frequent complication encountered brought on by pathogenic bacteria in the root canal system. The management strategy is directed toward the eradication of micro-organisms from the root canal system and the prevention of re-infection. The most important phase of endodontic therapy is the chemomechanical preparation of the root canal using a combination of mechanical instrumentation and chemical disinfection, followed by sealing off the pulp space and the access cavity.¹

The successful outcome of endodontic treatment is determined by the establishment and maintenance of a hermetic seal of the root canal system, which necessitates the usage of intracanal sealers that can firmly adhere to the root canal walls and the materials used for root filling.² An ideal root canal sealer requires that it must provide an optimal seal when set; there should be an adequate setting time to ensure the proper working time, dimensional stability, appropriate adhesion with canal walls, and biocompatibility.³

However, the development of voids between the sealer and dentin, may let the passage of bacteria and their byproducts and jeopardize the efficacy of the treatment. In addition, microleakage at the sealer-dentin interface might also contribute to root canal failure owing to sealers.^3–6 This can be resolved by bonding the sealer to radicular dentin and creating a monoblock. Hence, the type of endodontic sealer plays a critical role in the extent of bacterial leakage, which is a frequent occurrence seen in endodontically treated teeth. This raises interest concerning the standard of obturation offered by the sealing materials currently in the global dental market.⁷ Thus, to overcome the shortcomings of conventional sealers, newer nanomaterials and tissue adhesives are being introduced to the field of endodontics with promising features and exceptional properties as far as surface chemistry and bonding are concerned.

In this study, nanoparticle-based (NPB) cyanoacrylate sealer and epoxy resin-based (ERB) sealer (AH-Plus; Dentsply, Germany) are evaluated for microleakage of the samples. The NPB sealer prepared using zinc oxide nanoparticles (ZnO-Np) and Chitosan nanoparticles (CS-Np) along with isoamyl cyanoacrylate which is expected to have better sealing properties and less microleakage is compared with conventional gold standard sealer (ERBS). The NPB sealer, comprising of chitosan and zinc oxide as nanoparticles were tested for microleakage and assessed and evaluated with ERB sealer. Numerous techniques, including bacterial penetration, fluid transfer, radioisotope clarifying penetration, electrochemical approaches, and gas chromatography, are available for assessing the effectiveness of root canal sealers. Clarification penetration of radioisotopes is a novel methodology introduced recently. The current study is designed to assess and analyze the microleakage of the newly introduced NPB sealer and the gold standard ERB sealer (AH Plus, Dentsply, Germany) using the radioisotopic dye penetration method and confocal laser scanning microscopic analysis. The null hypothesis tested was that there are no variations in the microleakage between the sealers groups.

MATERIALS AND METHODS

RESULTS

DISCUSSION

The microbial invasion along the root canals is prevented by a good three-dimensional obturation of the root canal and applying an endodontic sealer to fill the voids at the gutta-percha/dentin interface.¹⁵ The selection and manipulation of the sealer are very important criteria for apical microleakage in endodontically treated teeth. Even though several methodologies are present in evaluation of microleakage, most widely used was confocal laser microscopic technique. But the speed of image acquisition is a major drawback of point-scanning confocal microscopy for live cell imaging applications, as it may be insufficient to gather data on rapidly changing biological processes.¹⁶ In nuclear medicine, the radioisotopic dye penetration technique, is a very popular in vitro method for evaluation of microleakage as it is relatively easy and rapid by allowing the tracer agent to penetrate the filled canal.¹⁷ In this study, an attempt is done to include the newer methodology to compare the microleakage of novel endodontic sealer with the gold standard root canal sealer under standard laboratory conditions.

In the present study, the results revealed no leakage in the negative control group, confirming that two coats of nail varnish effectively prevented dye penetration, while the positive control group showed the greatest dye penetration, indicating the efficacy of dye penetration in leakage studies.

In the current study, the experimental NPB sealer was tested and compared with the ERB sealer. The ERB sealer is one of the most widely used ERB sealers to date and is regarded as the gold standard for evaluating new sealers due to its superior sealing capabilities, high strength, dimensional stability, and low solubility.¹² However, when compared with NPB sealer, it exhibited higher microleakage at all the segments from the apex. This might be attributed to the larger particle size in the resin-based sealer.¹⁴

The NPB sealer used in the present study is a cyanoacrylate-based cement incorporated with nanoparticles, chitosan, and zinc oxide. The cyanoacrylate can become a potential material for sealer since it can be used in the form of a dentin adhesive for bonding to intraradicular dentin. With the tooth as a substrate, cyanoacrylate exhibits a chemical adhesive mechanism. When compared with conventional dental cement, it has a unique bonding process. With a strong tendency for bonding to basic substances, it hardens by the anionic polymerization of calcium ions into dentin by cyano and carbonyl groups found in cyanoacrylate.¹⁸ The two nanoparticles, chitosan and zinc oxide were incorporated to improve the physiochemical and antimicrobial properties of a sealer. A natural polycationic linear polysaccharide, chitosan is chemically made up of randomly distributed ß-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. It is created by deacetylating chitin compounds, which make up the majority of crustacean animals’ shells, including crabs and shrimp.¹⁹ Nanoparticles of Chitosan (CS-NPs) have been developed recently, being extensively researched in the area of endodontics, with the ability to enhance physiochemical and antimicrobial characteristics. Based on other studies, the chitosan nanoparticles when used with a root canal sealer enhance sealer adaptation, thereby improving the apical sealing.²⁰ Also, it has been observed that adding CS-NPs to the zinc oxide-eugenol sealer prevents the formation of biofilm at the sealer-dentin interface.²¹

Additionally, since chitosan is hydrophilic, it can closely adhere to intraradicular dentine resulting in an effective absorption into the canal wall. Since it has many hydroxyl and amino groups, ionic interactions take place readily with dentinal calcium ions. The amino group in chitosan can be protonated, which allows it to penetrate deeper into the dentinal tubules and attract more molecules out for adsorption into the radicular dentine.²²

Another nanoparticle that was incorporated in NPB is zinc oxide (ZnO-Np). Based on the studies, this modification inhibits biofilm formation within the sealer dentin interface, reduces cytotoxicity, and improved the sealing ability and antibacterial properties of NPB sealer. This will reduce or stop the growth of microorganisms at the sealer and tooth interface.²³ Hence, compared with ERB, NPB demonstrated lesser microleakage. The results can be supported by another study where it was observed that in the apical third region of the root, ERB sealer with the inclusion of CS-NPs showed fewer gaps than the same without. The interaction between the ERB sealer and the dentine wall was improved by the incorporation of CS-NPs relative to other groups.²⁴ The usage of sealers with optimal qualities like adaptability, adhesion, and tubular penetration enables superior root canal sealing due to the increased sealer contact with the radicular dentine and encapsulation of remaining microbes in the tubules. Additionally, a study by Ramar et al.; demonstrated that CS-NPs are biocompatible with fibroblasts and that they can be utilized in conjunction with root canal sealants in primary teeth.²⁵

In the case of ERB, which is considered a gold standard, it is crucial to stress that AH Plus sealer penetrates more deeply into surface micro-irregularities and inside lateral root canals due to its resin composition, flow, and lengthy curing time.²⁶ These characteristics cause the sealer and dentin structure to become more intertwined, which, along with the cohesion among the cement molecules, increases the adhesion and resistance to dislodgment from dentin.²⁷ However, even after such bonding mechanisms, ERB exhibited greater microleakage than NPB sealer, indicating superior adaptation of NPB sealer due to cyanoacrylate and the incorporated nanoparticles.

In radioisotopic dye penetration method, the initial values have shown lesser radioactivity of NPB sealer (p < 0.0001) indicating better sealing of the same. The values were initially taken before the removal of nail varnish which indicates the combined radioactivity measured on the nail varnish and the restorative area of the samples. The values were finally taken after the removal of nail varnish which indicates the radioactivity on the restorative areas of the samples. After the removal of nail varnish, the radioactivity of the NPB sealer is lower than the ERB sealer (p < 0.0001), again indicating the better sealing of the NPB sealer. This concludes that the microleakage in NPB sealer is lesser than the microleakage observed in ERB sealer. This can be attributed to the composition of NPB sealer which includes cyanoacrylate and nanoparticles. Cyanoacrylate’s adhesive property aids in the sealer’s improved adhesion to the dentinal surfaces. A study found that when incorporated in root canal sealers, such as zinc oxide-eugenol, showed significant decrease in microleakage.²⁸ Another study done by Bernardes et al., suggested that the filler particle size affects the flow of sealer; the lesser the size, and the higher the flow. With the increase in the sealer flow, there is an increased penetration of sealer to uneven surfaces and root canals, as can be seen in the case of NPB.²⁴

In confocal laser microscopic method, NPB sealer exhibited the lesser microleakage when compared with the ERB sealer. This indicates the similarity of the results obtained from radioisotopic method. Upon considering the apical, middle, and coronal sections of confocal method; the apical section exhibited the least microleakage than the coronal section. This finding was along expected lines and could be ascribed to one or more of the following reasons: greater diameter and number of the dentinal tubules coronally, lesser tubule density apically, propensity of sclerotic dentin to form toward the apical region, the reduced effectiveness of irrigants at the apex of the root canal and greater lateral compacting forces coronally during obturation. These findings are in accordance with the study done by Mokashi et al.¹⁴

The method used for the detection of microleakage in the current study was the radioisotope penetration method. This had been recently used for the evaluation of microleakage of restorative materials. This methodology was compared and assessed with the confocal laser scanning electron microscopic analysis of the same samples to avoid bias. Several radioactive isotopes, including the markers⁴⁵ Ca and¹³¹I have been utilized in microleakage research. Since Technetium-99m (99mTc) is the radionuclide that is utilized the most in the area of nuclear medicine, it was chosen for this study. This radioactive technology allows for the quantitative evaluation of microleakage from the same samples at intervals over long periods without causing any damage to them.^29,30 This radionuclide was selected because it has a half-life of around 6 hours and is the cold kit that is most frequently used in nuclear medicine for single photon emission. To allow time for the above-mentioned procedures and the gamma camera radiation measurement, 3 hours here chosen as the duration for which the teeth were immersed in the sodium pertechnetate solution. To avoid any contamination after the immersion time, the samples were meticulously prepared for gamma camera quantification following immersion. Each sample’s radioactivity was accurately measured using a gamma camera. The results obtained in this study, comparing both methodologies are in the same frequencies. This indicates that the radioisotopic dye penetration method can be effectively used for the analysis of microleakage of root canal sealers.

The limitation of this study is that it is an in vitro study and performed under controlled laboratory conditions. Moreover, the present study is done on single-rooted teeth and further similar studies are required for multirooted teeth.

Within the limitations of this in vitro study, the NPB sealer tested demonstrated greater resistance to microleakage as compared with the ERB sealer. Considering the superior physiochemical and antibacterial properties of the NPB sealer, it seems close to fulfilling the ideal requisites of a root canal sealer. The radioisotopic dye penetration method supplemented with confocal laser scanning microscopy validates the results of the study. However, long-term clinical studies may have to focus on the precise determination of the degree of sealing by the obturation materials and the physiochemical properties utilizing more specific and innovative methods of evaluation.

CONCLUSION

The NPB sealer, exhibiting lesser microleakage when tested with both methodologies, can become a potential root canal sealer in future endodontics due to superior physiochemical properties attributed to the cyanoacrylate and incorporated nanoparticles.

ORCID

Chitharanjan Shetty https://orcid.org/0000-0001-5154-8429

Shazeena Qaiser https://orcid.org/0000-0001-6579-5594

Aditya Shetty https://orcid.org/0000-0002-8170-3845

Rashi Shroff https://orcid.org/0009-0002-2741-5081

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