Approaching spectrophotometric analysis in apical sealing ability performed with three different sealers mixed with chitosan nanoparticles: An in vitro study

Abstract

Aim:

The study aimed to evaluate and compare the apical sealing ability of three different sealers, i.e., NT Biocera Flo, AH Plus, and Calapex modified with chitosan nanoparticles using spectrophotometric evaluation.

Methodology:

Thirty-six extracted single-rooted mandibular premolars were selected. Access opening and biomechanical preparation were done using the step-back technique, and samples were randomly divided into three groups with three different sealers (n = 12): Group 1 – NT Biocera Flo with chitosan nanoparticles, Group 2 – AH Plus with chitosan nanoparticles, and Group 3 – Calapex with chitosan nanoparticles. Following obturation, samples were immersed in 2% methylene blue for 72 h. Teeth were then dissolved in 35% nitric acid, and dye penetration was quantified using a spectrophotometer at 670 nm. Data were statistically analyzed using Kruskal–Wallis and Mann–Whitney U tests.

Results:

Group 1 (NT Biocera Flo + chitosan nanoparticles) demonstrated the lowest mean dye penetration (1.56 ± 0.48), followed by Group 2 (AH Plus + chitosan nanoparticles) (2.02 ± 0.57), whereas Group 3 (Calapex + chitosan nanoparticles) showed the highest leakage (2.53 ± 0.34).

Conclusions:

NT Biocera Flo with chitosan nanoparticles demonstrated superior apical sealing ability, followed by AH Plus and Calapex.

Context:

Achieving an effective apical seal is critical to prevent microleakage and ensure the long-term success of root canal therapy. This study assesses the effect of chitosan nanoparticle incorporation on the apical sealing ability of commonly used endodontic sealers.

Settings and Design:

This in vitro experimental study was conducted in the Department of Conservative Dentistry and Endodontics, using extracted human mandibular premolar teeth to evaluate the apical sealing ability of different chitosan-modified endodontic sealers. A randomized laboratory design was employed, with samples allocated into three groups based on the sealer used, followed by standardized preparation, obturation, dye penetration, and spectrophotometric analysis.

INTRODUCTION

Successful root canal therapy depends largely on the establishment of an effective three-dimensional seal that prevents the ingress of microorganisms and their by-products into the periapical tissues. Among the various factors influencing treatment outcome, the quality of the apical seal is considered critical, as microleakage at this level remains a principal cause of endodontic failure. Even with advances in instrumentation and obturation techniques, the sealing ability of root canal sealers continues to play a decisive role in long-term clinical success.[1]

Endodontic sealers are designed to fill irregularities within the root canal system, enhance adaptation between the core material and dentinal walls, and entomb residual microorganisms. Conventional sealers such as epoxy resin-based and calcium hydroxide-based materials have been widely used due to their favorable handling properties and clinical performance. However, limitations such as polymerization shrinkage, solubility, and inadequate bonding to dentin have prompted continuous research into material modifications aimed at improving their sealing efficacy.[2,3] More recently, bioceramic sealers have gained popularity owing to their bioactivity, dimensional stability, hydrophilicity, and ability to form a chemical bond with dentin through hydroxyapatite formation.[4]

The incorporation of nanotechnology into endodontic materials has opened new avenues for enhancing their physical, chemical, and biological properties. Nanoparticles, due to their small size and high surface area, can improve sealer penetration into dentinal tubules, reduce porosity, and enhance interfacial adaptation.[5] Chitosan nanoparticles, derived from the natural biopolymer chitin, have attracted particular interest in dentistry because of their biocompatibility, biodegradability, antimicrobial activity, and ability to interact with dentin collagen.[6] Studies have demonstrated that chitosan nanoparticles can improve the sealing ability and antibacterial properties of endodontic sealers when incorporated into their formulation.[7,8]

Chitosan nanoparticles have found wide applications in dentistry and endodontics. Their inherent antimicrobial activity against endodontic pathogens such as Enterococcus faecalis has led to their use as intracanal medicaments, irrigant adjuncts, and sealer additives. Chitosan nanoparticles have also been incorporated into dental adhesives and restorative materials to enhance bond strength, improve collagen stabilization, and reduce enzymatic degradation of dentin.[9] In addition, their bio-adhesive and chelating properties have been exploited in smear layer removal and dentinal tubule occlusion.[10] In endodontic sealers, chitosan nanoparticles have demonstrated potential to improve sealing ability, enhance dentinal tubule penetration, and provide sustained antibacterial effects, thereby contributing to improved apical sealing and periapical healing.[11,12]

Evaluation of apical sealing ability has traditionally been performed using dye penetration, fluid filtration, bacterial leakage, and radioisotope methods. Among these, spectrophotometric dye analysis offers a quantitative, sensitive, and reproducible approach for assessing microleakage by measuring dye concentration objectively, thereby minimizing examiner bias.[13] This method has been widely accepted for comparative in vitro evaluation of sealing performance among different endodontic materials.[14]

Given the promising role of chitosan nanoparticles and the need for objective assessment techniques, the present in vitro study aims to evaluate and compare the apical sealing ability of three different endodontic sealers (NT Biocera Flo, AH Plus, and Calapex) incorporated with chitosan nanoparticles using spectrophotometric analysis. The findings of this study may contribute to the development of enhanced sealer formulations with improved apical sealing potential and long-term clinical predictability.

Aim

This in vitro investigation was to use spectrophotometric analysis to quantitatively assess the sealing capacity of three distinct sealers when combined with nanoparticles.

Objective

• By using spectrophotometric analysis to assess the sealing capacity of NT BioCera FLO in combination with chitosan

• Using spectrophotometric analysis to assess the sealing capacity of AH Plus combined with chitosan

• To assess the capacity to seal of Cal apex combined chitosan using spectrophotometric analysis

• To assess and contrast the sealing capacity of NT Biocera FLO, Ah Plus, Cal apex mixed with chitosan using spectrophotometric analysis.

METHODOLOGY

Before being used, 36 recently extracted human mandibular premolars with single, straight root canals were collected, adherent soft tissue debris and calculus were removed using ultrasonic scalers, and the teeth were disinfected and stored in 10% neutral buffered formalin until the time of experimentation. Teeth with cracks, resorptive defects, or previous endodontic treatment were excluded from the study. Standard access cavity preparation was performed, and canal patency was established using a #10 K-file. The working length was determined 1 mm short of the apex. The step-back technique was used for root canal instrumentation. The coronal section was sequentially flared after the apical portion was prepared up to a #50 master apical file. Canals were prepared by irrigating them with 2.5% sodium hypochlorite and then flushing them with saline.

Three groups (n = 12) were randomly selected from the produced samples.

According to the sealer used, each was modified with chitosan nanoparticles:

• Group 1: NT Biocera Flo + chitosan nanoparticles

• Group 2: AH Plus + chitosan nanoparticles

• Group 3: Calapex + chitosan nanoparticles.

Chitosan nanoparticles were incorporated into NT Biocera Flo, AH Plus, and Calapex sealers at a concentration of 0.2% (w/w). A measured quantity of 15 mg of chitosan nanoparticles was added to 100 mg of each sealer. According to the manufacturer, as all sealers are premixed, 15 mg of chitosan nanoparticle is dispensed on the mixing pad, and 100 mg of each sealer is mixed using an agate spatula, i.e., manual mixing is performed for 2 min to obtain the homogenous mixture.

The lateral compaction technique was used to obturate gutta-percha cones. Temporary restorative material was used to seal the coronal cavities. The sealers were allowed to set under controlled conditions before microleakage testing: NT Biocera Flo sealer was given approximately 4 h for initial setting consistent with typical bioceramic formulations; AH Plus sealer was permitted 24 h at 37°C for complete setting per manufacturer specifications; and Calapex sealer was allowed to set for approximately 40–60 min following mixing, as recommended for calcium hydroxide-based materials. Two layers of nail polish were applied to each tooth’s exterior, leaving the apical 2 mm untreated to allow for dye penetration. For 72 h at room temperature, every sample was immersed in 2% methylene blue dye.

Teeth were cleaned, dried, and then submerged in 35% nitric acid to breakdown the tooth structure and liberate the dye that had been absorbed. After centrifuging the resultant solutions, dye penetration as a sign of apical microleakage was measured in the supernatant using a ultraviolet-visible spectrophotometer set to 670 nm [Figure 1].

Figure 1.

(a) indicate that the resultant solution is obtained after preparation with 2% methylene blue dye and nitric acid solution (b) spectrophotometer machine

The data were statistically analyzed using the Mann–Whitney U test for pairwise group comparison and the Kruskal–Wallis test for overall comparison. Statistical significance was defined as a P < 0.05.

Statistical analysis

Spectrophotometric readings were utilized to gather data, which was then statistically analysed. Nonparametric tests were used since the dataset did not have a normal distribution. The Mann–Whitney U test was used for pairwise comparisons, and the Kruskal–Walli’s test was used to evaluate the overall differences between the three groups. The cutoff point for statistical significance was set at P < 0.05.

RESULTS

Samples showed varying degrees of apical dye penetration, which was quantified using spectrophotometric analysis. The mean absorbance values, representing the extent of microleakage, differed significantly among the three groups. Group 1 (NT Biocera Flo + chitosan nanoparticles) demonstrated the lowest mean dye penetration (1.56 ± 0.48), followed by Group 2 (AH Plus + chitosan nanoparticles) (2.02 ± 0.57), whereas Group 3 (Calapex + chitosan nanoparticles) showed the highest leakage (2.53 ± 0.34).

The Kruskal–Walli’s test revealed a statistically significant difference among the three groups (P < 0.05) [Table 1]. Subsequent Mann–Whitney U tests confirmed significant pairwise differences between all groups, indicating that the sealing ability varied distinctly depending on the sealer used.

Table 1.

Mean spectrophotometric dye absorbance values (µg/mL) for the three groups

Group	Sealer used (with chitosan nanoparticles)	Mean±SD	Median	P
Group 1	NT biocera Flo	1.56±0.48	1.48	<0.05
Group 2	Ah plus	2.02±0.57	1.96	<0.05
Group 3	Calapex	2.53±0.34	2.51	<0.05
Overall comparisons	-	-	-	<0.05

*Significant difference compared with other individual groups, *Significant overall variation between the groups. SD: Standard deviation

DISCUSSION

Applying an endodontic sealer to seal the spaces at the gutta-percha/dentin interface and properly obturating the root canal in three dimensions avoids microbial infiltration along the root canals. When it comes to endodontically treated teeth, the choice and application of the sealer are crucial factors.[15] The practitioners are searching for a sealer that can produce a robust apical seal. According to Grossman (1988),[16] sealers must not promote bacterial growth, maintain the seal apically, or cause tissue deterioration. Lateral and supplementary canals, as well as voids, are filled with sealers. Microleakage could lead to treatment failure if it is ineffective.

This study aimed to evaluate and compare the apical sealing ability of three different sealers mixed with chitosan nanoparticles using the spectrophotometric dye penetration method. There are various methods for calculating apical leakage. However, in terms of the type and degree of leakage, the in vitro methods for evaluating leakage cannot be applied to an in vivo setting. However, the only reliable preclinical screening method that can predict or predict clinical outcomes is still laboratory testing. A variety of experimental techniques, including radioisotopes, dyes, bacteria, proteins, endotoxins, glucose penetration, and computerized fluid filtration, have been used by researchers to assess microleakage after obturation.[17]

Although there are various sealers available in the market, in this study, three sealers used are NT Biocera Flo, AH Plus, and Calapex. NT Biocera Flo is a calcium silicate-based bioceramic sealer that exhibits bioactivity through the release of calcium and hydroxyl ions during setting, creating an alkaline environment favorable for antimicrobial action and periapical healing.[18] The material promotes hydroxyapatite formation at the sealer–dentin interface, enabling chemical bonding and improved interfacial adaptation. Its fine particle size and high flow facilitate penetration into dentinal tubules and canal irregularities, contributing to enhanced apical sealing. The moisture-activated setting reaction ensures reliable hardening under clinical conditions, whereas the premixed injectable formulation reduces operator variability. In addition, low solubility, dimensional stability, and adequate radiopacity further support the long-term integrity of the apical seal.[19]

AH Plus is an epoxy resin-based root canal sealer widely regarded for its favorable physicochemical properties and long-standing clinical performance. The material demonstrates excellent adhesion to dentinal walls through covalent interaction with collagen amino groups, contributing to effective interfacial adaptation and reduced microleakage. Its low solubility and minimal polymerization shrinkage enhance dimensional stability after setting, which is essential for maintaining the integrity of the apical seal. AH Plus exhibits adequate flow and penetration into dentinal tubules, allowing uniform distribution within the canal system. The sealer also shows good radiopacity and extended working time, facilitating controlled placement. However, its hydrophobic nature and lack of bioactivity limit chemical bonding with dentin, distinguishing it from newer bioceramic sealers.[20,21]

Calapex is a calcium hydroxide-based root canal sealer formulated to combine antimicrobial activity with biological compatibility. The material releases hydroxyl ions during setting, resulting in a high alkaline pH that is unfavorable for residual endodontic microorganisms and supportive of periapical tissue repair. Calcium ion release further contributes to mineralized tissue formation and may aid in hard tissue induction. Calapex demonstrates acceptable flow and handling properties, allowing adaptation to canal walls and accessory anatomy. However, calcium hydroxide-based sealers are known to exhibit comparatively higher solubility and lower dimensional stability than resin-based and bioceramic sealers, which may compromise long-term sealing ability. These characteristics may explain the relatively increased microleakage observed with Calapex in comparative in vitro studies.[22]

When 50% or more of the particles are in the range of 1–100 nm in terms of number, size, distribution, or at least one external dimension, the term “nano material” refers to natural, incidental, or manufactured material that contains particles in an unbound state or as an aggregate or agglomerate. Chitosan nanoparticles diffuse from the sealer to penetrate anatomical complexity. Because of its hydrophilic properties, which enable it to be in close contact with root dentine, chitosan is more readily absorbed into the canal wall.

Sealers incorporated with chitosan nanoparticles demonstrated improved apical sealing ability when compared to their unmodified counterparts. This enhancement can be attributed to the nanoscale particle size and bioadhesive nature of chitosan, which facilitates deeper penetration into dentinal tubules, improved interfacial adaptation, and reduced microgaps at the sealer–dentin interface. In addition, chitosan nanoparticles interact with dentin collagen and provide antimicrobial activity, contributing to reduced microleakage. In contrast, sealers without chitosan nanoparticles rely solely on their intrinsic physicochemical properties, which may be insufficient to overcome dentinal irregularities, resulting in comparatively inferior sealing performance.[23]

In this study, a spectrophotometer was used. Since 2% methylene blue dye exhibits leaking properties and a lower molecular weight than bacterial toxins, it was utilized. Considering the sealing property of the sealers combined with chitosan nanoparticles the result of this study showed that Group 1 (NT Biocera Flo + chitosan nanoparticles) demonstrated the lowest mean dye penetration (1.56 ± 0.48), followed by Group 2 (AH Plus + chitosan nanoparticles) (2.02 ± 0.57), whereas Group 3 (Calapex + chitosan nanoparticles) showed the highest leakage (2.53 ± 0.34).

With the limitations, the present in vitro study does not completely replicate clinical conditions, as factors such as thermal changes, occlusal stresses, and periapical tissue response were not simulated. In addition, the use of spectrophotometric dye penetration assesses microleakage quantitatively but does not represent bacterial leakage or long-term clinical performance of the sealers.

CONCLUSIONS

The findings of this investigation unequivocally show that NT Biocera Flo, combined with chitosan nanoparticles, provides superior apical sealing compared to AH Plus and Calapex. The markedly reduced microleakage observed in this group highlights the strong potential of chitosan-enhanced bioceramic sealers to significantly improve the quality and longevity of root canal therapy. These findings support their advancement toward broader clinical application.

Clinical significance

The findings highlight the strong potential of chitosan-enhanced bioceramic sealers, particularly NT Biocera Flo, to improve the apical seal and reduce microleakage — factors directly linked to long-term endodontic success. Using such materials may minimize postoperative complications, enhance healing, and increase the predictability of root canal outcomes in everyday clinical practice.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.