Evaluation of surface analysis of gutta-percha after disinfecting with sodium hypochlorite, silver nanoparticles, and chitosan nanoparticles by atomic force microscopy: An in vitro study.

AIMS AND
OBJECTIVES: The purpose of this study is to evaluate the surface topography of gutta-percha (GP) after disinfecting with sodium hypochlorite (NaOCl) (5.25%), silver nanoparticles (AgNPs) (70 µg/ml), and chitosan nanoparticles (ChNPs) (1.5 mg/ml) by atomic force microscopy (AFM).
MATERIALS AND METHODS: Forty GP cones were taken in this in vitro study. These samples were divided into four different groups such as Group I - control group (untreated GP Points) and Group II, III, and IV were treated with 5.25% NaOCl, 70 µg/ml AgNPs, and 1.5 mg/ml ChNPs, respectively. The surface topography analysis of the samples was performed using AFM. STATISTICAL ANALYSIS: Root mean square (RMS) and surface roughness parameters were used to compare the structure of GP points with contact mode imaging. These values were tested by IBM SPSS-20.0 version statistical software using one-way ANOVA and post hoc (Tukey's honestly significant difference) tests. They were considered statistically significant when P < 0.05.
RESULTS: The RMS and surface roughness values are significantly higher for NaOCl group (5.25% NaOCl) when compared with AgNPs group (70 µg/ml AgNPs) and ChNPs group (1.5 mg/ml ChNPs).
CONCLUSION: This study has shown more surface topography deterioration of GP treated with NaOCl and lesser deterioration with AgNPs and ChNPs which affects postoperative prognosis. Copyright:

INTRODUCTION

The success of root canal therapy mainly depends on thorough disinfection and the use of aseptic techniques. This remains true even for obturating materials also.[1] As per the contemporary concepts of infection control, every instrument and material when placed in root canal needs to be sterilized.[2] Gutta-percha (GP) is one of the widely used and accepted root canal filling materials (Monga P et al., 2018).[1] Although these GP cones are manufactured under aseptic conditions, they possess some antimicrobial activity due to its zinc oxide component.[3] It has been shown that GP cones which are taken from sealed package, when exposed to the dental operatory environment, become contaminated by a variety of microorganisms such as cocci, rods, and yeasts.[1] GP cones do not lend themselves to be readily sterilized by moist or dry heat because these high temperature sterilization methods were found to damage the cones.[1345]

In order to prevent contamination of these cones, the dentist must treat these cones either with decontaminating agent or ethylene oxide gas before using them for obturation. The latter process was found to be time consuming and clinically impractical.[6] Various chemical disinfectants such as sodium hypochlorite (NaOCl), glutaraldehyde, alcohol, iodine compounds, and hydrogen peroxide have been used for GP cone disinfection. The appropriate disinfectant is the one that can be routinely used in dental clinics which can provide a fast disinfection without modifying the structure of GP cones (Lalkrishna R et al., 2015).[17]

In spite of wide variety of disinfectants, the most commonly used is NaOCl in the concentration of 5.25% due to its broad spectrum of action against a variety of Gram-positive, Gram-negative, and spore-forming bacteria.[28] Several studies have shown that 5.25% NaOCl as a strong oxidizing agent causes topographic alterations in the cones.[35]

The problems which are caused by NaOCl have urged the scientists to look forward to new antimicrobial agents with minimal side effects on the material and tissue.[8] In this context, nanoparticles were being used due to their antimicrobial properties. Among them, silver nanoparticles (AgNPs) and chitosan nanoparticles (ChNPs) have emerged as effective antimicrobial agents due to their broad-spectrum effect.[89]

Nowadays, atomic force microscopy (AFM) is commonly used due to its scanning probe technique for characterization of the material. The key principle on which it works is probing of a sample surface with a small tip attached to a flexible cantilever.[810]

The objective of this study is to analyze the topographical changes of GP after disinfection with 5.25% NaOCl, 70 μg/ml (0.007%) AgNPs, and 1.5 mg/ml of ChNPs using atomic force microscope.

MATERIALS AND METHODS

Forty GP cones (ISO size 30 6% taper, DENTSPLY MAILLEFER) were taken in this in vitro study.

Samples preparation

GP cones were randomly selected from the same batch (ISO size 30) for the study before expiration date. All the samples were sectioned 3 mm from their tip and stick to a glass slide with fast setting cyanoacrylate glue. Following this procedure, the samples were divided into one control and three treatment groups as following:

Group I – 10 untreated GP points (control group)

Group II – 10 GP points immersed in 5.25% NaOCl (Modi Surgico Healthcare Pvt. Ltd, Maharashtra, India) for 1 min

Group III – 10 GP points immersed in 70 μg/ml (0.007%) AgNPs (Nano Wings Pvt. Ltd, Khammam, Telangana, India) for 1 min

Group IV – 10 GP points immersed in 1.5 mg/ml ChNPs (AURA Biotechnologies Pvt. Ltd, Chennai, India) for 1 min.

Fresh liquid (5 ml) of 5.25% NaOCl, AgNPs, and ChNPs were used for each period of immersion. After the immersion, the samples were thoroughly rinsed with 5 ml of deionized water, and specimens were dried with filter paper. Each glass slide was viewed under an AFM (JPK NanoWizard® ULTRA Speed 2) using contact mode imaging.

Atomic force microscopy

Typical AFM probes PPP-CONTSCR having resonance frequency 25 kHz and force constant of 0.2 N/m (curvature radius 10 nm) were used. The images were processed using Scanning Probe Microscope lab 4.0 Software (Santa Barbara, California, USA).

For the purpose of comparison, root mean square (RMS) and surface roughness were chosen to investigate structure of GP points.

Statistical analysis

Statistical analysis has been performed using statistical software IBM SPSS-20.0 IBM Corp., Armonk, New york, USA version using one-way ANOVA and post hoc (Tukey's honestly significant difference [HSD]) tests.

RESULTS

The AFM images [Figure 1] reveal that 5.25% NaOCl-treated GP (Rq = 62.90 nm, Ra = 45.41 nm) [Figure 1b] exhibits maximum surface alterations, followed by ChNPs (Rq = 24.46 nm, Ra = 18.46 nm) [Figure 1d], AgNPs (Rq = 23.29 nm, Ra = 17.57 nm) [Figure 1c], and control group (Rq = 10.56 nm, Ra = 0.30 nm) [Figure 1a]. The one-way ANOVA shows that there is statistical significant difference (P < 0.05) for the RMS and surface roughness between the control (untreated GP) and experimental groups (5.25% NaOCl, AgNPs, and ChNPs treated GP).

Figure 1

Atomic force microscope images of gutta-percha. (a) Control group, (b) sodium hypochlorite group, (c) silver nanoparticles group, and (d) chitosan nanoparticles group

The post hoc (Tukey's HSD) test shows that there is no statistical significant difference (P > 0.05) for both RMS and surface roughness [Table 1] between AgNPs and ChNPs, but there is significant difference from 5.25% NaOCl and control groups. 5.25% NaOCl group had shown highest surface alterations (Rq = 62.90 nm, Ra = 45.41 nm) and lowest was recorded in control group (Rq = 10.56 nm, Ra = 0.30 nm), while it was similar statistically in ChNPs (Rq = 24.46 nm, Ra = 18.46 nm) and AgNPs (Rq = 23.29 nm, Ra = 17.57 nm) [Table 1].

Table 1

Mean (standard deviation) of roughness values of gutta-percha treated with different disinfectants (n=10)

Disinfectant	Root mean square (Rq)	Surface roughness (Ra)
Untreated GP (control group)	10.56 (0.82)a	0.30 (0.66)a
5.25% NaOCl	62.90 (2.11)b	45.41 (2.88)b
Silver nanoparticles	23.29 (2.16)c	17.57 (0.91)c
Chitosan nanoparticles	24.46 (1.84)c	18.46 (1.33)c

Different letters in the column indicate a statistically significant difference. GP: Gutta-percha, NaOCl: Sodium hypochlorite

DISCUSSION

The dentists are occasionally facing the problem of infections that occur after obturation of root canal space. One of the possible explanations of this phenomenon may be the introduction of contaminated GP cones into the root canal. In a study done by Gomes et al.,[11] it was concluded that 5.5% of the cones taken from their boxes were contaminated.[12] As the placement of GP is the final step in obliteration of root canal space, so accordingly its sterility is of utmost importance in order to prevent the contamination of root canal space.[113] Due to thermoplastic nature of these GP cones, a quick chair-side disinfection is needed. In endodontic practice, NaOCl is one of the most common disinfectants which is used in the concentrations that range from 0.5% to 5.25%.[2] Senia et al. were the first to propose that the most efficient and reliable technique for disinfection of GP cones is by placing them into 5.25% NaOCl for at least 1 min.[514]

The findings of the present study showed that, according to the CMI parameter, 5.25% NaOCl causes aggressive topographic alterations in the cones which in turn lead to deterioration and also some crystal formation on GP which were in accordance with previous studies. Some studies reported that these crystal formations were more seen after rapid sterilization with 2.5% and 5.25% NaOCl.[5] In a study by Valois et al., it was concluded that 5.25% NaOCl had resulted in surface deterioration of GP cones because of loss of GP cone components by the oxidizing agent (NaOCl).[1516]

Deep irregularities which are formed through deterioration of GP cones can create interfacial gaps between the GP cones and the root canal wall, increasing the risk of leakage.[17] The cracks that were created after disinfecting the GP cones with 5.25% NaOCl are filled with resin sealers when coated in root canals, but once set, it shrinks leaving behind empty spaces which may increase the risk of leakage.[8] The present study also showed the similar results which effect the postoperative prognosis.

As the nanoparticles possess smaller size having the larger surface, they show the antimicrobial activity even at low levels. Silver is the frequently used nanoparticle, which is used at various concentrations in different forms. At low concentrations, AgNPs are nontoxic to human body and also have broad spectrum of antibacterial actions. The positive charge on the Ag+ ion is important for its antimicrobial activity. These AgNPs are accumulated in the bacterial cell membrane and disturb their membrane permeability, resulting in the cell death.[818] In regard to cytotoxicity of AgNPs, HeLa cells were assessed by being exposed to different concentrations of AgNps and had shown that 80 μg/ml concentration is harmful. Mishra P and Tyagi S et al., have introduced the standard GP that is coated with AgNPs which showed a significant antimicrobial against variety of pathogens.[8]

In the present study, 1.5 mg/ml ChNPs is taken, as it showed better antimicrobial activity in previous studies.[19] Chitosan-impregnated GP points showed higher antimicrobial activity than the commercial ones and the antimicrobial activity increased as the amount of chitosan used for the impregnation increased. This antibacterial nature of chitosan is due to its polycationic nature that interacts with negatively charged surface of bacteria which alters cellular permeability. These particles inhibit bacterial enzymatic degradation, thereby reducing the possibility of bacterial penetration. Impregnation of GP points with chitosan may be a good alternative to obtain GP points with improved antimicrobial properties.[9]

In the present study, AFM is used which provides three-dimensional imaging of molecular surfaces of nanometer resolution and does not require any special treatment (metal coating) which may lead to irreversible damage as compared to scanning electron microscope (SEM). In the SEM, vacuum environment is needed for functioning, but whereas in AFM, it can be operated effectively even in air or liquid.[10]

In this study, NaOCl-treated GP had shown highest surface alterations which is in accordance with studies done by Valois et al.,[15] Mishra and Tyagi,[8] John et al.,[20] and Tilakchand et al.[2] Both chitosan and AgNPs had shown less surface alterations when compared to NaOCl. Hence, these nanoparticles can be used as an alternative for disinfecting GP cones.

CONCLUSION

Within the limitations of this study, NaOCl (5.25%) showed greater irregularity on the surface of GP cones, whereas lesser deterioration was observed with AgNPs and ChNPs. Further research in this area should be conducted to investigate the surface energy and wetting ability of these nanoparticles to analyze its compatibility with sealers when used as irrigating solutions.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.