Nanoparticles modified bioceramic sealers on solubility, antimicrobial efficacy, pushout bond strength and marginal adaptation at apical-third of canal dentin

Study design, consent, and inclusion criteria

The current comparative lab-based investigation followed the checklist for reporting in-vitro studies (CRIS) guidelines. The analyses centered on the dependent variable, with the experimental groups serving as the independent variable. The study was approved by the Ethical Committee of King Saud University under IRB # FC-5676-24. Written consent was taken from the patient whose teeth were used in the present study for experimental purposes. Forty-four human lower premolars that were extracted due to orthodontic or periodontal reasoning were utilized. To maintain consistency within the groups, digital periapical radiographs were taken from two different angles. Teeth with fully formed apex, single canal with straight roots, absence of caries no prior endodontic treatment, and no pathological modifications were included. Teeth that exhibited internal root resorptions, calcifications, fractures, or had more than one canal per root were excluded. All samples were scaled of any attached tissue and calculus using an ultrasonic scaling device (Newtron Booster, Satelec-Acteon, Merignac, France). This was followed by submerging them in 1% chloramine T solution for 24 h for disinfection (Al-Kheraif et al., 2022; Alkahtany et al., 2021).

Nanoparticles characterization

NPs in the present study were obtained commercially (Sigma-Aldrich GmbH, Berlin, Germany) and sputter-coated using gold (Electron Microscopy Sciences, Hatfield, PA, USA) for 3 min. These NPs were then observed under a scanning electron microscope (SEM) (Mira3-XMU model–Brisbane, Queensland, Australia) at 20 KeV to identify the surface morphology. Energy-dispersive X-ray spectroscopy (EDX) (JED-2300 Analysis Station, JEOL, Japan) was used for elemental analysis (Alfawaz et al., 2020; Aljamhan et al., 2021a).

Preparation of the NPs-modified BC sealer

The root canal sealer utilized in the existing exploration was the commercially available Total Fill BC sealer (FKG, Berlin, Germany). A pre-blended substance that was modified using different NPs. All the NPs were obtained commercially (Sigma-Aldrich GmbH, Berlin, Germany). The commercially obtained solid NPs in the concentration of 2 wt% were injected and mixed into a sealer using a precisely measured syringe and a micropipette.

Sample size calculation, cleaning, and shaping of the canal

By using the WHO sample size calculator, the following were the calculations; confidence level = 95%, absolute precision required = 9%, population mean = 8.40, population standard deviation = 0.14, sample size = 11 cases in each group (Al Ahdal et al., 2020; Habib et al., 2021). The coronal section of the samples was sectioned using a diamond saw (Auto-Instrument Co., Shenyang, China) along with constant water irrigation till the cementoenamel junction to standardize the root length to 15 mm. Subsequently, endodontic treatment was initiated, and the final canal length was established using #15 K file (Dentsply-Maillefer, Ballaigues, Switzerland) till anatomic foramen. The final working length (WL) was kept one mm shorter than the recorded length. For disinfection purposes, 2 ml 5.25% NaOCl solution (Cloraxid; Cerkamed, Stalowa Wola, Poland) was administered using a 30-gauge needle after each file till final cleaning and shaping. The root canals were then finally prepared utilizing a Protaper Gold rotary instrument (Dentsply Maillefer) starting with file S1 till F2 as a final finishing file. All the teeth were subsequently distributed into four groups based on the use of modified and unmodified sealers used for root canal obturation (n = 11) (Al Deeb et al., 2020; Aljamhan et al., 2021b).

Group 1 (Control): In this group, an unmodified Totalfill BC sealer was used to obturate the canal. Using paper points, the instrumented canals were dried. A single cone gutta percha size F2 along with sealer was used to fill the canal. To ensure that the obturation was adequate, radiographs of every obturated tooth were taken at two different angulations (Alrahlah et al., 2020). Group 2 (AgNP-modified BC sealer): In this group, AgNP-modified BC sealer was used to obturate the canal in the same way as Group 1. Group 3 (CHNP-modified BC sealer): In this group, CHNP-modified BC sealer was used to obturate the canal in the same way as Group 1. Group 4 (HApNP modified BC sealer): In this group, HApNP modified BC sealer was used to obturate the canal in the same way as Group 1. To ensure a complete setting, specimens were incubated (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h at 37 °C and 95% humidity.

Solubility testing of BC sealers

Solubility (S) was assessed following the International Standards Organization (ISO) 6876 specification. Five samples of each sealer in measurements of 5 ± 0.1 mm diameter and 2 ± 0.1 mm height were prepared using a stainless-steel ring mold and subsequently incubated at 37 °C in 100% humidity. The discs were extracted from the molds and were subjected to three weight measurements utilizing a precision balance (Ohaus Corp. Pine Brook, NJ USA). The initial mass (I) was documented as the average weight. The same discs were then subsequently immersed in 50 mL of distilled water set at a temperature of 37 °C and humidity of 95% for 24 h. Following that, discs were dried using absorbent paper, and positioned in a dehumidifying chamber until the mass reached stabilization and re-evaluated for their weights three additional times, and the mean weight was documented as the final mass (F) (Abu Zeid & Alnoury, 2023). The S of all investigated sealers was calculated using the formula ${\displaystyle S=\left[\left(I-F\right)/I\right]\times 100.}$

Testing antibacterial property

The antimicrobial efficiency of the modified and unmodified sealers was assessed against E. faecalis (ATCC 29212) using an agar diffusion test (ADT). For this, 200 µL of the bacterial suspension, containing approximately 10$\stackrel{ˆ}{}$ 6 cells, was utilized. The suspension was inoculated onto agar plates containing BHI broth. Perpendicular wells with a diameter of five mm were bored and subsequently filled with each material. The plates underwent incubation at a temperature of 37 °C for 24 h. The samples were analyzed for bacterial proliferation through the assessment of the inhibition halo present on the agar medium. Repetitions were conducted to evaluate the test and the outcome of the specified material after 24 h (Castillo-Villagomez et al., 2022).

Root slicing of specimens following PBS testing

The roots of 40 samples were divided into three segments, each measuring two mm in height using a diamond saw under constant water irrigation. The root slices (cervical, middle, and apical) were subsequently affixed onto an acrylic block measuring 1.5 × 1.5 mm. The PBS was assessed utilizing a universal testing machine (UTM) (Instron 3367; Instron Co., Canton, China). The cylindrical piston having a diameter of 0.7 mm and 0.4 mm and a height of four mm was used to apply continuous compressive force from the apical to the cervical direction at a speed of one mm/min until the bond between the sealer and root dentin failed. The PBS was measured in MegaPascals (MPa) by dividing the force in Newtons by the area of the bonded interface using the following formula (Alkhudhairy et al., 2019; Vohra et al., 2020) ${\displaystyle \text{Bond Strength}\left(\text{Mpa}\right)=\frac{\text{Load In Newton}}{\text{Area of Bonded Surface}}.}$

Failure analysis

The failure mode was assessed using a stereomicroscope (Leica Microsystems, Wetzlar, Berlin, Germany) at a magnification of 25×. Failures were categorized as adhesive, cohesive, and admixed.

Marginal adaptation via SEM

For marginal adaptation, one sample from each group was sectioned vertically with a hard tissue microtome. Samples were fixed to an aluminum stub, underwent sputter coating with gold, and were analyzed under SEM. The maximum gap width, characterized as the largest distance between the root filling with the root dentin, was assessed visually by various examiners at the apical-3rd to mitigate any possible bias (Enggardipta et al., 2020; Habib et al., 2021).

Statistical analysis

The normality of the data was assessed using Kolmogorov–Smirnov Test. SPSS Version 23 (IBM, Chicago, IL, USA) was used for data analysis. The results of sealer solubility, antibacterial efficacy, and PBS were assessed statistically using one-way ANOVA and post hoc Tukey multiple comparison tests. p < 0.05 for a level of significance.

Surface characterization of NPs and EDX

Figure 1: Chitosan nanoparticles exhibit an irregular morphology. Energy-dispersive X-ray (EDX) analysis reveals the elemental composition predominantly consists of carbon (C) and oxygen (O₂). Figure 2: Hydroxyapatite nanoparticles demonstrate agglomeration with a heterogeneous distribution of particle sizes. EDX analysis identifies calcium (Ca), oxygen (O₂), phosphorus (P), and carbon (C) as the major constituents. Figure 3: Silver nanoparticles display a spherical morphology and show signs of agglomeration. The major elements detected through EDX include carbon (C), oxygen (O₂), phosphorus (P), and silver (Ag).

Solubility

Solubility assessment of BC root canal sealer after modifying using various nanoparticles is demonstrated in Table 1. Group 1 (Control) demonstrated the highest solubility rate. In contrast, sealers modified with NPs exhibited significantly reduced solubility in comparison to the control group yet comparable to each other (p > 0.05) (Fig. 4).

Antimicrobial testing

The antimicrobial potency of different nanoparticle-modified BC sealers against E. faecalis is presented in Table 2. All the modified sealers exhibited the largest zone of inhibition with no significant difference i.e., AgNPs modified BC sealer (4.413 ± 1.3 mm), CHNPs modified BC sealer (4.336 ± 1.5 mm) and HApNPs modified BC sealer (4.321 ± 1.0 mm) (p > 0.05). Whereas the unmodified control group presented the smallest zone of bacterial inhibition (3.343 ± 1.2 mm) significantly lower than the modified sealer groups (p < 0.05) (Fig. 5).

PBS assessment

PBS of BC root canal sealers modified with different nanoparticles are demonstrated in Table 3. The strongest PBS was presented by a cervical third of Group 4 (HApNPs + BC sealer) specimens. However, the lowest bond scores were witnessed in the apical third of Group 1 (BC sealer) samples. Comparative analysis recognized that Group 2 (AgNPs + BC sealer), Group 3 (CHNPs + BC sealer) and Group 4 exhibited no significant disparity in their bond scores (p < 0.05). Nevertheless, Group 1 presented significantly lower scores than other tested groups (p < 0.05). Comparison within different sections of the same group discovered that the cervical and middle slices of the obturated roots established no significant difference in their PBS (p > 0.05). Nonetheless, apical sections showed markedly reduced bond scores (p < 0.05) (Fig. 6).

Fracture pattern assessment

Results indicated that specimens from Groups 2, 3, and 4 predominantly exhibited cohesive fractures. However, adhesive fractures were most observed in Group 1 samples (Fig. 7).

Marginal adaptation of sealer to radicular dentin at apical-third via SEM

Figure 8A: SEM illustrates a persistent gap between the unmodified BC sealer and canal dentin, which remains uniform across the entire interface. Additionally, instances of tearing within the sealer are also observed. Figure 8B: The SEM image of the BC sealer modified with silver reveals a pronounced gap between the canal dentin and the silver-modified BC sealer. Nonetheless, areas of intimate contact between the sealer and dentin are also evident. Figure 8C: SEM analysis of the BC sealer modified with chitosan demonstrates a strong interface between the dentin and the chitosan-modified sealer, with no observable gap. Figure 8D: The SEM image presents the interface of the BC sealer modified with hydroxyapatite nanoparticles. The interface exhibits uneven characteristics, characterized by significant erosion and cracks in the sealer, as well as a gap between the sealer and dentin.