The effectiveness of nano-chitosan high molecular 0.2% as irrigant agent against Enterococcus faecalis with passive ultrasonic irrigant

Abstract

Context:

Enterococcus faecalis is the microorganism most frequently associated with failure of endodontic treatment. Chitosan is an irrigant in dentistry has the properties of biocompatibility, biodegradability, bioadhesion, and not toxic to human cells. Several studies have suggested the use of ultrasonics to enhancing the action of irrigants.

Aims:

The aim of the study was to investigate the bacterial growth and surface roughness of the root canal surface after irrigation and agitation with passive ultrasonic irrigant.

Subjects and Methods:

Experimental research with randomized block design obtained a sample size for each group of 9 samples with a total number of 27 teeth divided into three treatment groups. Statistical analysis used was one-way analysis of variance.

Results:

The irrigation material for nano-chitosan high molecular 0.2% with passive ultrasonic irrigation (PUI) activation was shown to cause lysis on surface of bacterial cell walls. There was no significant difference between the roughness values in all treatment groups.

Conclusions:

The irrigation of root canal treatment with 0.2% high molecular nano-chitosan with the addition of PUI activation had significant antibacterial activities against E. faecalis.

INTRODUCTION

Commonly, an endodontic treatment was considered as unsuccessful, if the treatment procedure failed to meet the standards for control and elimination of infection.[1,2,3] Enterecoccus faecalis is the microorganism most frequently associated with failure of endodontic treatment and persistent infection.[4] In vitro studies have shown that E. faecalis can invade peritubular dentin and not all bacteria have this ability.[1,5]

Irrigation has an important role in endodontic treatment.[6] Sodium hypochlorite (NaOCl) solution was recommended as an irrigants by Coolidge in 1919 because it has strong antimicrobial and proteolytic activity.[7] The use of NaOCl with high concentrations and for a long time can cause dentine collagen change and break.[8] The result of in vitro studies showed that 5.25% NaOCl irrigation for 40 min was the most effective, whereas 1.3% and 2.5% NaOCl at 40 min were inferior in eliminating E. faecalis from tubular dentine.[9] Based on the findings of this study, the American Association of Endodontists suggested using another irrigants to enhance the antibacterial effect during root canal cleaning and shaping.[10]

The side effects caused by the irrigation agent have prompted many researchers to explored alternative materials that are eco-friendly, safe, biocompatible, and cost effective. Among the alternative materials, chitosan appears to be one of the promising material.[8] The research conducted by Bhuva et al. found that nanoparticle chitosan can be used as the final irrigation in root canal treatment owing to its ability to remove smear layer and inhibit bacteria from re-colonizing the dentine at the root of the tooth.[11]

In general, it is difficult for the irrigants to reach the apical area. Due to the small diameter of the root canals and has ramification.[12] To optimize microbial reduction, several methods have been used, namely, sonic devices, ultrasonic, laser-activated irrigation systems, and electrochemical activations. One of the advantages of these methods is the generation of turbulence which aid in the delivery irrigants, during root canal treatments, into tubular dentin. The effective delivery of irrigants would result in significant removal of organic and inorganic materials and consequently, reduction of bacterial growth.[13] Several studies had demonstrated that ultrasound is effective in enhancing the action of irrigants, by aiding in cleaning complex anatomical areas.[12] Research conducted by Grundling et al. showed that passive ultrasonic irrigation (PUI) enhanced the ability of filtered water in significantly reducting in the number of E. faecalis bacteria colonies on bovine teeth.

The aim of this study was to investigate the effect of the combination of passive ultrasonic irrigant and chitosan on the bacterial growth and surface roughness of the root canal surface.

SUBJECTS AND METHODS

Extracted mandibular premolar teeth for orthodontic purposes were obtained from a dentist's practice with the following inclusion criteria: no root caries, one rooted tooth and one root canal, no root crack, the apex of the tooth has been completely closed, the length of the tooth selected between 20 and 25 mm. We ensured that the premolar teeth in this study were one root canal when we did the root canal treatment and when the teeth were examined during the roughness examination.

The experimental design was a randomized block design with a total number of 27 teeth divided equally into three treatment groups, namely, Group A: 0.2% high molecular nano-chitosan + PUI after remodeling treatment of root canals, Group B: 2.5% NaOCl with additional PUI after remodelling root canal treatment, and Group C: distilled water solution + PUI after remodeling root canal treatment.

Following the treatments, the growth of E. faecalis ATCC 29212 and the surface roughness of the tooth root canal were assessed.

Method

Sample preparation

Twenty-seven teeth were divided into three groups randomly and each treatment group amounted to 9 teeth. All the teeth were then subjected to access cavities preparation to obtain an Initial Apical File. Root canal preparation in Groups A, B, and C with the Mtwo file (VDW, Ballaigues, Switzerland) was carried out according to the manufacturer's instructions until #25.06 file with a torque of 2.3Ncm and a speed of 280 rpm.

Remodeling root canal treatment

The teeth which had been subjected to the root canal treatment were subsequently sterilized. One hundred microliters of Brain Heart Infusion medium was inserted into each tooth. It was incubated for 1.5 h. The tooth was rinsed with saline. Each tooth was subsequently injected with 25 μl of E. faecalis ATCC 29212 (1: 3). The teeth were incubated in an anaerobic atmosphere for 6 h. Furthermore, all teeth from all groups were prepared using the Mtwo # 30.05 file with a torque of 1.2 Ncm and a speed of 250 rpm (VDW, Ballaigues, Switzerland) to simulate retreatment. The teeth were then irrigated with 3 ml of test material each which was flowed through the cavity edge area slowly for 42 s and then activated with PUI (Endo Ultra, Vista) for 3 min. They were then incubated for 24 h, 48 h, and 72 h. After incubation, all the teeth that had been filled with the test material were then shaken for 5 min at 200 rpm.

Bacteria Enterecoccus faecalis isolation

The bacteria isolation process was performed using the T streak technique. E. faecalis was cultured on CHROMagar™ vancomycin-resistant enterococcus medium. The Petri dishes were divided into three parts using markers. The culturing process starts with heating the loop needle and letting it cool, then 1 pure culture loop was sampled and inoculated in area 1, with zigzag strokes. Then reheated the loop needle and wait for it to cool, then proceed with a zigzag stroke on area 2 which is slightly perpendicular to the first stroke, then followed by a zigzag stroke on area 3 with a second stroke. Petri dishes that have been scratched by bacteria are then closed tightly and incubated for 24 h at 37°C in an anaerobic atmosphere,[14] then equated with Mc Farland 0.5 or equivalent to a concentration of 1.5 × 10⁸ colony forming unit/ml.

Anti-Inhibition activity of Enterecoccus faecalis

A total of 25 μl of solution (test material and E. faecalis) from each group was put in chromagar medium, then spread with bent sticks throughout the media and cultured for 24, 48, and 72 h at 37°C and then examined the normal colony form which is compared with the colony with abnormal morphology. This is an indicator of the effect of the test material on E. faecalis to confirm E. faecalis cells, following which a gram stain examination was performed.

Roughness of root canal surface examination

Tooth specimen preparation

The treated mandibular premolar teeth were stored in glycerol solution, then rinsed with PBS solution for 10 s. The root of the tooth was cut transversely (distal mesial or vice versa) in the cementoenamel junction using a carborundum disc. When cutting, the teeth are rinsed with aquadest (One Med, Indonesia) so that they were wet, to facilitate the cutting. After that, the teeth specimens that were ready to be put into a sterile container containing glycerol solution.

Examination of specimens using the atomic force microscope

Specimens from each group were analysed using atomic force microscope (AFM) (Nanosurf easyScan 2 Controller, Switzerland), equipped with a piezoelectric scanner. The area examined was the surface area of the enamel with an AFM scanned area of 10 × 10 μm in the z-direction.

The data were analyzed using one-way analysis of variance. The level of significance for the analyses was set to P < 0.05.

RESULTS

Figure 1 shows that the 24 h group of the bacterial cells was still small, but there are cells that undergo lysis in the distilled water group. In the nano-chitosan 0.2% and NaOCl groups, many bacterial cells were analyzed with different numbers of lysed cells due to the influence of the experimental material. In the 48 h group it shows that were still many bacterial cells that undergo lysis, even the remaining dead bacterial cells can be seen in the distilled water group treatment. In the chitosan group, the number of bacterial cells decreased and tended to show lysis similar to the NaOCl group. In the 72 h group was explained that the distilled water group also still showed the number of dominant bacterial cells experiencing lysis even though some were still alive. This tendency is influenced by the workability of the chitosan and NaOCl test materials which lyse the surface of the bacterial cell walls.

Figure 1.

Profile of interaction activity between irrigation agents in the 24 h group (a) Distilled watert, (b) Chitosan, (c) Sodium hypochlorite), the 48 h group (d) Distilled watert, (e) Chitosan, (f) Sodium hypochlorite), the 72 h group (g) Distilled water, (h) Chitosan, (i) Sodium hypochlorite)

Figure 2 shows the AFM topography images of NaOCl 2.5%, nano-chitosan 0.2% and distilled water with various incubation time, whereas the surface roughness value was summarized in Figure 3 for clear comparison.

Figure 2.

Atomic force microscope topography images of (a) Sodium hypochlorite 2.5%, (b) nano-chitosan 0.2% and (c) distilled water with various incubation time

Figure 3.

Tooth surface roughness as a function of incubation time for 24, 48 and 72 h

DISCUSSION

Biofilms produced by bacteria are multicellular microbial clusters attached to surfaces and between surfaces. Biofilm formation and maintenance depends on the production of extracellular substances, proteins and exopolysaccharides which are part of the extracellular matrix. This extracellular matrix protects bacteria and is part of a multicellular collection.[4] Biofilms are attached to the host, have access to oxygen and nutrients, and provide protection for bacteria from the environment.[5] Biofilm maturity has an effect on an antimicrobial agent.[6]

High molecular chitosan nano can enter the cell membrane and cover the cell so that it interferes with the activity of the bacteria and over time the bacteria dies. Electronegative cells or polyanions in gram-positive bacteria are in their cell walls, namely lipoteichoic acid (LTA). This LTA reacts with polycation in chitosan.[7] NaOCl has the ability to dissolve necrotic tissue and organic components, low surface tension, an anti-bacterial effect, the ability to reduce endotoxicity and low viscosity which makes it ideal as a root canal irrigant.[8,9] The antibacterial activity of NaOCl is based on a high pH (hydroxyl ion action) (pH >11) which mimics the mechanism of action of calcium hydroxide. The high pH of NaOCl impairs the integrity of the cytoplasmic membrane by inhibiting enzyme action, altering biosynthesis in cellular metabolism and degradation of phospholipids. NaOCl also has the effect of damaging bacterial DNA by inducing the formation of chlorine derivatives.[10] According to Bhuva et al., 1% NaOCl and saline combined with PUI has a better effect and is statistically more significant when compared to conventional irrigation.[11] Ozok et al. reported that biofilms that were dual species or mature biofilms were more resistant to NaOCl than mono-species biofilms or early biofilm formation.[12]

Many studies have examined the antibacterial effects of PUI-assisted irrigation agents. PUI has the ability to be more effective in removing dentine debris, microorganisms (planktonic and biofilm) and organic tissue in root canals. The ultrasonic activation of the irrigants cause cavitation effects with the formation of bubbles that quickly explode and created a shock waves and lift the biofilm and acoustic streaming which produces shear forces that will help lift debris from the prepared root canals and provide the potential for irrigation materials to better contact the root canal walls. Near the instrument of ultrasonic activation, there is a boundary layer in which the fluid oscillates together with the file (oscillatory component). In the direction of oscillation, jets are formed (steady component) that may impact on a nearby root canal wall and flow back toward the file (steady component).[13,14,15]

Many antimicrobial theories of chitosan have been put forward, the most acceptable is the interaction between chitin/chitosan with a positive charge and microbial cell membranes with a negative charge. In this model the interaction is mediated by electrostatic forces between the protonated NH³⁺ groups and the negative residue, possibly by competing with Ca²⁺ for the electronegative site on the membrane surface. This electrostatic interaction produces two-fold disturbance, namely: (i) by changing the permeability of the membrane walls, and (ii) by hydrolyzing peptidoglycan on the walls of the microorganisms, which leads to leakage of intracellular electrolytes.[16]

The antibacterial activity of chitosan is influenced by a number of factors including the type of chitosan, the polymerization rate of chitosan and several other physicochemical properties.[17] Chitosan molecules can pass through the bacterial cell wall consisting of multilayer murein connected to each other, and reach the plasma membrane. This effect is due to electrostatic interactions between chitosan molecules and microbial cell membranes, which leads to protein leakage and increases the penetration of chitosan to the nucleus and binds to microbial DNA, which can inhibit mRNA and protein synthesis.[18] Furthermore, NaOCl also has the effect of damaging bacterial DNA by inducing the formation of chlorine derivative. However, it has disadvantages, such as unpleasant taste, toxicity and unable to remove the smear layer.[10,19] NaOCl is also unstable and prone to oxidation when exposed to oxygen, at room temperature, and under light, which significantly reduce its antibacterial activity[20] Moreover, the use of NaOCl with high concentrations and for a long period of time can damage dentine.[21]

One of the main advantages of chitosan in the endodontic is its ability to remove the smear layer. Pimenta et al. showed that chitosan has chelating properties when used as irrigation agent and can cause dentine erosion but not intertubular dentine.[22] The mechanism of chelating action is unclear, but it is believed through absorption, ion exchange and chelation properties. There are two theories explaining the chelating process of chitosan, namely: (i) Bridge model, two or more amino groups from one chain of chitosan will bind to the same metal ion (ii) Only one amino group of the structure is involved in the binding.[23] Disadvantage of NaOCl as an irrigants in root canal treatment are disable to dissolve the smear layer[19] and using it for long time can cause dentine collagen to change.[21]

In this study, there was no significant difference in the surface roughness value in all treatment groups (P > 0.05). Based on the incubation time there was also no significant difference between the 24 h, 48 h and 72 h groups (P > 0.05), but the impact of the preparation of the irrigation agent with the addition of ultrasonics in each treatment group with the incubation time was significantly different (P < 0.05). The results showed that the addition of chitosan did not significantly affect the surface roughness of the tooth root canals, but specifically it could be considered as an irrigation agent, especially at incubation time of 72 h [Figure 3].

CONCLUSIONS

In summary, the irrigation of root canal treatment with nano-chitosan high molecular 0.2% with the addition of PUI activation had significant antibacterial activities against E. faecalis. Accordingly, this work suggests the potential application of nano-chitosan for future biomaterial applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.