INTRODUCTION:

Root canal treatment aims to ensure root canal disinfection and prevent reinfections^1,2. Successful root canal treatment depends on good cleaning, shaping, and obturation^3,4. The smear layer can influence the penetration and adaptation of filling materials to canal walls; smear layer removal is imperative. Proper root canals are cleaned with irrigants; activatingirrigants helps increase the effectiveness^5,6.

Gold standard irrigants in endodontics nowadays are sodium hypochlorite, which can dissolve necrotic tissue and organic components of the smear layer, and EDTA, which acts as a chelating agentand can dissolve the inorganic smear layer^2,4.

Although it's the most recommended irrigant, this combination can cause dentinal erosion of peritubular and intertubular areas of dentine^7,8.

Chitosan is a natural biomaterial with broad-spectrum antibacterial properties, a high chelating effect⁹, and a similar structure with extracellular matrix components^10-12. Chitosan is more studied in drug delivery systems because it shows better stability simple and mild preparation methods, and it includes the various routes of administration such as oral, nasal, ocular and IV¹³. Chitosan oligosaccharides are degradation products with different physical properties, such as water solubility^14,15. In their research, Kaur et al. used Calcium hypochlorite 5% and chitosan oligosaccharide 1% as irrigants and proved that they could effectively remove the smear layer from root canals with minimal erosion¹¹. Irrigant activation, cavitation, and acoustic streaming can enhance the cleaning of root canal systems^4,16,17. The study aimed to investigate the effect of the chitosan oligosaccharide as final irrigants on smear layer removal and fracture resistance of endodontictreated teeth.

Material and Methods:

Extracted mandibular premolar for orthodontic purposes was obtained from the Department of Oral and Maxillofacial Surgery Universitas Sumatera Utara with the following inclusion criteria: no caries, one root canal, no crack, the teeth with closed apex, with no restoration and no root canal treatment was done before.The experimental design was a post-test-only control group with 56 teeth. For the smear layer removal test, 24 teeth were used and divided equally into four groups: Group A (irrigation with sodium hypochlorite 2.5% and EDTA 17%), Group B (irrigation with sodium hypochlorite 2.5% and chitosan oligosaccharide 0.5%), Group C (irrigation with sodium hypochlorite 2.5% and chitosan oligosaccharide 1%) and Group D (irrigation with sodium hypochlorite 2.5% and chitosan oligosaccharide 2%). For the fracture resistance test, 32 teeth were divided equally into two groups: Group E (irrigation with sodium hypochlorite 2.5% and EDTA 17%) and Group F (irrigation with sodium hypochlorite 2.5% and chitosan oligosaccharide 2%).

Preparation of irrigant solution:

The chitosan oligosaccharide 0.5% solution was made with 0.5grams of chitosan oligosaccharide powder with 100ml of aquadest, then mixed until completely dissolved. The chitosan oligosaccharide 1% solution was made with 1gram of chitosan oligosaccharide powder with 100ml of aquadest, then mixed till completely dissolved. The chitosan oligosaccharide 2% solution was made with 2 grams of chitosan oligosaccharide powder with 100mL of aquadest, then mixed until completely dissolved.

Sample Preparation:

Fifty-six teeth were stored in saline and randomly divided into groups A, B, C, D, E, and F. The teeth used in groups A,B,C, and D were de-coronated at the cementoenamel junction using a disk with a straight handpiece. Meanwhile,all the teeth were subjected to access cavities preparation in groups E and F. Then, in all groups, initial apical files were done until the working length with K-file #10 (M-access, Dentsply).

Root Canal Preparation:

Root canal preparation in Groups A, B, C, D, E, and F was done with S-One Blue rotary file (Fanta) according to the manufacturer's instructions until #25.06 file with speed 500rpm and torque 2.5 N. Irrigation was done with 30G side vented needle and passive ultrasonic irrigation (Ultra X, Eighteenth) with different kind of irrigans every change of instrument with 5ml of 2.5% sodium hypochlorite, saline, and final irrigation was done with EDTA 17% for 1 minute and chitosan oligosaccharide for 3 minutes according to the group.

SEM Sample Preparation:

A sterile paper point was left inside the root canal to protect the prepared channel from contamination by dentinal chips or debris during the splitting process. The canal orifices were closed with adhesive tape to prevent the insertion of dentin particles during the root notching by the diamond disc. Each root was notched carefully longitudinally buccal and lingual without penetrating the canal using a disc with a straight handpiece. The tooth was split, and one half was used for the SEM examination in the third apical area. The magnification used for smear layer analysis was 2000x. SEM images were then divided into nineregions. The smear layer on the canal wall was evaluated using the Torabinejad scoring system: Score 1: no smear layer on the canal's surface, and all tubules were clean and open.Score 2: moderate smear layer. No smear layer was on the canal's character, but the tubules contained debris.Score 3: Heavy smear layer. The smear layer covered the root canal surface and the tubules.

Root canal obturation:

Teeth in groups E and F were then dried with the paper point, and cone fitting was done. After that, obturation was done with a single cone technique with #25/.06 gutta percha and bioceramic sealers (Ceraseal, MetaBiomed). Then, thermocycling was done to all of the teeth samples.

Preparation for the mechanical test:

After obturation, the teeth roots in Groups E and F were covered with wax to 1mm below the cervical teeth with a thickness of about 0.3mm, which acts as the periodontal ligament. Teeth were then mounted in the molds with auto-polymerizing acrylic resin. After the acrylic was polymerized, wax was removed, and elastomer was used to fill the wax-cleaned area to stimulate the periodontal ligament. Samples were then placed in the Universal Testing Machine; the tip was directed parallel to the long axis of the tooth, using the vertically compressive load at the speed of 0.5mm/min. The shipment was vertically applied along the longitudinal axis of the tooth. The program automatically recorded the maximum force applied to the teeth that caused fracture and was recorded in Newton's (N).

Statistical Analysis:

Statistical analysis used was the t-independent test,one‑way analysis of variance, and LSD Post Hoc test, and the significance was tested at 0.05.

RESULTS AND DISCUSSION:

Figure 1 shows a representative SEM image at the magnification of 2000x in the apical third of the root canal. The image shows that group A has a large diameter of tubules compared with groups B, C and D. Table 1 shows how the ANOVA analysis of irrigants' solutions affects the cleanliness of the smear layer. (p=0.000). Table 2 reported the Post hoc LSD analysis indicates that there is a significant difference in smear layer cleanliness in group A and group B, as C (p=0.000). Still, there is no significant difference in smear layer cleanliness in groups A and D(p=0.084). Table 3 shows that T-independent test analysis indicates aconsiderable difference in fracture resistance in groups E and F (p=0.000).

Fig. 1: SEM representative image of smear layer in the apical third of root canal (magnification 2000x) (a) sodium hypochlorite 2.5% and EDTA 17% (b) sodium hypochlorite 2.5% and chitosan oligosaccharide 0.5% (c) sodium hypochlorite 2.5% and chitosan oligosaccharide 1% (d) sodium hypochlorite 2.5% and chitosan oligosaccharide 2%

Table 1: Mean of smear layer score and p-value

Group	Sample (n)	smear layer score (X̄ ± SD)	p-value
A	6	1,28±0,116	0.000
B	6	2,56±0,281
C	6	2,41±0,287
D	6	1,52±0,195

Group A – sodium hypochlorite 2.5% and EDTA 17%; Group B – sodium hypochlorite 2.5% and chitosan oligosaccharide 0.5%; Group C – sodium hypochlorite 2.5% and chitosan oligosaccharide 1%; Group D – sodium hypochlorite 2.5% and chitosan oligosaccharide 2%

Table 2: Post Hoc LSD analysis and p value between group

Group		P value
Group A	Group B	0.000*
Group A	Group C	0.000*
	Group D	0.084
Group B	Group C	0.279
Group B	Group D	0.000*
Group C	Group D	0.000*

*Significance

Table 3: Mean of fracture resistance of teeth and p-value

Group

Sample

(n)

Fracture resistance

(X̄ ± SD)

P value

518,81± 67,56

0.000

676,47 ±38,03

Group E – sodium hypochlorite 2.5% and EDTA 17%; Group F – sodium hypochlorite 2.5% and chitosan oligosaccharide 2%

Root canal treatment consists of a chemo-mechanical process that removes the smear layer. An irregular infected bacterial layer consists of organic, inorganic tissue and debris like saliva, blood, and dentinal chips¹⁸. Gold standard irrigation solutions are sodium hypochlorite, commonly used as an antibacterial effect and dissolving organic smear layer; ethylenediamine tetra-acetic acid (EDTA) 17%, a chelating agent that removes inorganic smear layer by decalcification^1,8,18.

Studies show the importance of using EDTA to remove inorganic components of the smear layer. Despite effectively removing the smear layer, EDTA has an erosive effect on dentin¹⁹. It has been proven from the SEM image in Figure 1 that group A,which used EDTA,has a larger diameter of dentine tubules, and intratubular dentine was destroyed. Sayin et al. also proved that using EDTA with or without sodium hypochlorite reduced the microhardness of root dentin²⁰. A combination of EDTA and sodium hypochlorite destroyed peritubular and intratubular dentin. Pimenta et al. stated that chitosan has a chelating effect, but intratubular dentin is safe without erosion; this research was also proven, as shown in SEM image (Figure 1)^21,22. In this research, the ANOVA analysis test shows different irrigants' effects on smear layer removal. Chitosan also has a chelating effect based on two theories. The first theory stated that two or more amino groups interact with the same metal ion, referred to as the bridge model. In the second theory, only one amino group is involved in the binding: the metal ion "anchored" to the amino group^11,19,21.

Factors that affectirrigants are volume, concentration, and time. In this research, we want to evaluate the concentration of chitosan oligosaccharide in removing the smear layer in 3 minutes, between 0, 5%, 1%, and 2 %. The most influential group is the 2% chitosan oligosaccharide group. In Post Hoc LSD analysis, there are also no differences between control group A (EDTA 17%) and group D (chitosan oligosaccharide 2%) in smear layer removal. Sodium hypochlorite can dissolve amino groups from dentine collagen²³. Erosion and collagen degradation can be fixed with donor amino from chitosan. It is stated that collagen dentin crosslinked with chitosan will be more stable and increase mechanical properties. Combining mechanical and chemical removal with 12% sodium hypochlorite solution effectively removes debris from nickel-titanium (Ni-Ti) instruments²⁵.

In the fracture resistance test, the chitosan oligosaccharide 2% group shows higher significance different from the EDTA 17% as final irrigants. Ernani et al. also stated that high chitosan molecule 0, 2% has more excellent fracture resistance than EDTA 17% group, in which chitosan prevents macro and micro-cracks and opens dentine tubule without changing intertubular dentin so that erosion is little²².

Fracture resistance in the chitosan group is higher than the EDTA group because chitosan has a structure similar to the extracellular matrix of glycosaminoglycans that provides mechanical stability and compressive strength to collagen due to their intertwined with a fibrous structure. Chitosan and derivates also interact and neutralize MMPS or bacterial collagenase, increasing dentin's resistance to degradation. Chitosan can also improve the mechanical properties of demineralized dentin by crosslinking and infiltration into the collagen ultrastructure²⁶.

This research is also using bioceramic sealers when doing obturation. Yaman et al. show that bioceramic sealer can enhance fracture resistance of teeth²⁷. Zhang et al. reported several mechanismsthat explain sealer penetration to root dentin²⁸. It is a mechanical interlocking bond through the dispersion of the sealer molecules into the dentinal tubules. Han and Okiji stated that penetration of the sealer's mineral content into the intertubular dentine denatures collagen fibers and formsa mineral infiltration zone²⁹. Others suggested that hydroxyapatite is formed along the mineral infiltration zone due to the partial reaction of phosphate with calcium silicate hydrogel and calcium hydroxide^30-33. Bioceramic sealer forms a specific interfacial layer at the dentin walls known as the mineral infiltration zone. The sealer's hydration products alter the collagen of the interfacial dentin due to their alkaline effects. This alteration leads to favoring mineralization in this area. This chemical and micromechanical interaction (tag-like structures) represents the main reason for bioceramic sealer adhesion and root dentin reinforcing fracture resistance of endodontic-treated teeth³⁴.

Conclusion:

Chitosan oligosaccharide 2% can be the alternative for final irrigants besides EDTA 17%; in smear layer removal, it doesn't have a significant difference and has better fracture resistance.

Acknowledgment:

The author would like to thank the Department of Conservative Dentistry, Universitas Sumatera Utara, for providing research facilities to conduct our research work.

Conflict of Interest:

There is no conflict of interest.

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Received on 23.12.2022 Modified on 12.06.2023

Research J. Pharm. and Tech 2024; 17(3):1212-1216.

DOI: 10.52711/0974-360X.2024.00189