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ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 5  |  Page : 73-78  

A comparative evaluation of intraradicular smear removal efficacy of 2% chitosan (low molecular weight), 4% Chitosan Citrate, and 10% Citric Acid when Used as Final Rinse in Irrigation Protocols: A Field Emission Scanning Electron Microscopic Study


1 Department of Conservative Dentistry, RVS Dental College and Hospital, Coimbatore, Tamil Nadu, India
2 Department of Conservative Dentistry, Vinayaka Missions Sankarachariyar Dental College, Salem, Tamil Nadu, India
3 Department of Oral Medicine, JKK Nataraja Dental College and Hospital, Tiruchengode, Tamil Nadu, India
4 Department of Conservative Dentistry, JKK Nataraja Dental College and Hospital, Tiruchengode, Tamil Nadu, India

Date of Web Publication27-Nov-2017

Correspondence Address:
M Praveen
Department of Conservative Dentistry, RVS Dental College, Sulur, Coimbatore - 641 005, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.JPBS_158_17

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   Abstract 


Aim: This study aims to compare the smear layer removal efficacy of 2% chitosan (low molecular weight) (C-LMV), 4% chitosan citrate (CC), and 10% citric acid (CA) when used in specific irrigant protocols. Materials and Methods: A total of 60 single-rooted maxillary incisors and canines were decoronated, standardized to a root length of 15 mm, and prepared with rotary files up to protaper F3 size. Sodium hypochlorite was used as initial rinse [8 ml]. The samples were divided into experimental (Group III, IV, V, and VI) (n = 10) and control groups (I – 17% EDTA, II – normal saline) (n = 5) based on the type of final rinse solution (5 ml) used, that is, 2% C-LMV, 4% C-citrate, 10% CA, and 1% acetic acid. Samples were dehydrated, split buccolingually, gold sputter coated, and examined in field emission scanning electron microscope. Results: Overall, the Group IV, V, and III presented the least amounts of smear, debris, and erosion among the experimental groups at the apical, middle, and coronal one-thirds of the root canal with a mean value of 1.53 ± 0.42, 1.33 ± 0.42, and 1.60 ± 0.46, respectively, and there was no statistically significant difference (P > 0.05). Conclusion: The use of CC as final rinse solution during biomechanical preparation seems promising.

Keywords: 10% citric acid, 17% ethylenediaminetetraacetic acid, chelation, chitosan, field emission scanning electron microscope, final rinse, smear layer


How to cite this article:
Praveen M, Aarthi G, Meenapriya P K, Kumar S S, Mohan Kumar N S, Karunakaran J V. A comparative evaluation of intraradicular smear removal efficacy of 2% chitosan (low molecular weight), 4% Chitosan Citrate, and 10% Citric Acid when Used as Final Rinse in Irrigation Protocols: A Field Emission Scanning Electron Microscopic Study. J Pharm Bioall Sci 2017;9, Suppl S1:73-8

How to cite this URL:
Praveen M, Aarthi G, Meenapriya P K, Kumar S S, Mohan Kumar N S, Karunakaran J V. A comparative evaluation of intraradicular smear removal efficacy of 2% chitosan (low molecular weight), 4% Chitosan Citrate, and 10% Citric Acid when Used as Final Rinse in Irrigation Protocols: A Field Emission Scanning Electron Microscopic Study. J Pharm Bioall Sci [serial online] 2017 [cited 2022 Jul 6];9, Suppl S1:73-8. Available from: https://www.jpbsonline.org/text.asp?2017/9/5/73/219288




   Introduction Top


Eick et al. in 1970[1] first reported the presence of a smear layer as made up of particles of size from 0.5 to 1.5 μm achieving an overall thickness of 2-5 μm. The intraradicular smear layer was first described by Mc Comb and Smith in 1975,[2] as consisting not only of dentin but also the fragments of odontoblastic processes, pulpal tissue remnants, microorganisms, and their byproducts. An efficient irrigation protocol to remove the debris and smear layer is needed. The presence of a smear layer prevents the penetration of intracanal medicaments into dentinal tubules and interferes with sealer adaptation to radicular dentin.[3],[4],[5] Evaluation of effect of smear layer on the apical and coronal seal suggests that smear layer being a loosely adherent structure should be completely removed from the surface of the root canal as it can harbor microorganisms and cause microleakage. Efficient irrigant delivery and agitation methodologies are mandatory for successful outcome of endodontic procedures. Chemicals, sonic technologies, ultrasonics, and LASERS have been used either individually or in combination with appropriate canal preparation techniques to achieve the objective of smear removal.[6]

Chelating agents have played an important role as final rinses in irrigation protocols and have been shown to effectively remove the smear layer.[7] A review of literature highlights the limited effectiveness of irrigating solutions in apical one-third of the root canal regardless of instrumentation and irrigation techniques. Chelating agents such as ethylenediaminetetraacetic acid (EDTA), citric acid (CA), and phytic acid have been effectively used for removal of smear as final rinse solutions. EDTA is considered a pollutant, as it is not found originally in nature.[8] Chitosan is a natural polysaccharide with molecular weight ranging from 1000,000 to 3000,000 and excellent properties of biocompatibility, biodegradability, bioadhesion, and nontoxic to human body. It is obtained by deacetylation of chitin which is the most abundant substance in nature after cellulose. It is available in different forms also shows remarkable chelating capacity for different metallic ions, which justifies its use in various sectors of industry.[9]

This study aims to compare intraradicular smear layer removal ability of 2% chitosan (low molecular weight), 4% chitosan citrate (CC), and 10% CA when used in specific irrigant protocols.


   Materials and Methods Top


Intact teeth devoid of anomalies, defects, carious lesions, restorations, and endodontic treatment were selected, analyzed using digital radiography to ensure that they had a patent single canal and standardized by decoronating them at a distance of 15 mm from the apical foramen by sectioning with a water-cooled diamond disc. The teeth were then dried, coded, wax applied at apical third of the root, and embedded in transparent small plastic containers filled with soft polyvinyl siloxane impression material. The aim was to prevent irrigants from extruding the apex to simulate in vivo closed apex conditions. The samples were then randomly divided into two control groups (n = 5) and four experimental groups (n = 10) [Table 1]. The instrumentation was initiated with hand files (Dentsply Maillefer, Ballaigues, Switzerland) up to size 20 followed by protaper rotary files from size S1-F3. The root canals of the samples were prepared using protaper rotary instruments with X-smart plus endomotor (Dentsply Maillefer, Ballaigues, Switzerland) as per the manufacturer instructions. About 1 ml of the irrigant was used for canal irrigation after using each instrument and before proceeding to the next. A total of 8 ml of the irrigant was used during biomechanical preparation procedure. The irrigant was delivered using a 30-gauge side vent prorinse needle (Dentsply, Tulsa Dental) at the working length. The samples were then irrigated with a final rinse of 5 ml of irrigant solution as per the respective group using a 30-gauge side vent prorinse needle for three minutes.
Table 1: Irrigant grouping

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During the first minute delivery of irrigant, the needle was withdrawn to 5 mm inserted back to working length followed by rotation of the needle by 180° three times alternatively. During the second minute, a F3 size gutta-percha cone (Dentsply Maillefer, Ballaigues, Switzerland) was inserted to working length and withdrawn three times (Manual Dynamic Activation). This was done to improve the irrigant delivery and replacement to the apical third of the canal space. The remaining irrigant was left in the canal for three minutes. After the completion of five minutes, a postfinal rinse irrigation of 10 ml of distilled water was done to flush out the remaining final rinse irrigant from within the canal. The teeth were then carefully split longitudinally in a buccolingual plane dividing them into two halves using a mallet and a chisel. For each tooth, the half containing the most visible part of the apex was selected, stored, and coded. The teeth were then placed in a 10% neutral buffered formalin solution at 18°C for 24 h. They were then postfixed in osmium tetroxide (1%w/v) for two hours before being dehydrated in graded solutions of isopropyl alcohol (S.V. Drugs and chemicals, Faridabad, India). The teeth were then placed in a filter paper for 24 hours after which separation markings of 5 mm made for the apical, middle, and coronal thirds, respectively, on the split half of the root using a custom-made former. The prepared samples were then irradiated with UV light in a UV light sterilization chamber and stored in sterile pouches. The coded samples of each group were mounted on to aluminum stubs with carbon tape (Royal tapes Pvt Ltd., Chennai, India) with the entire root canal visible and facing upward. Each of the specimens was coated with a 20–30 nm thin layer of gold in a gold sputter coating machine (Quorum, United Kingdom). The samples were then examined using a field emission scanning electron microscope with a high resolution (SIGMA 0336 FESEM, ZIESS, Munchen, Germany). The SEM photomicrographs were obtained at X2000 magnification using digital image analysis software and stored appropriately for subsequent analysis. The most representative micrographs were taken for each millimeter of the specimen and were recorded for apical, middle, and coronal thirds, respectively. The smear, debris, and erosion were evaluated using criteria developed by Caron et al.,[10] Dadresanfar et al.,[11] and Torabinejad et al.,[12] respectively. The results which were scored by the independent operators were compared and tabulated for their respective score values of smear layer, debris, and erosion in the apical, middle, and coronal thirds of the root canal.


   Results Top


The coronal third presented least amount of smear with a mean value of 2.02 ± 1.28 followed by the middle third with a mean value of 2.34 ± 1.15, and the most amount of smear was in the apical third of the canals with a mean value of 2.90 ± 0.98 [Figure 1] and [Figure 2], [Chart 1],[Chart 2],[Chart 3].
Figure 1: Scanning Electron Microscopic (or) SEM image comparison Groups I and II

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Figure 2: Scanning Electron Microscopic (or) SEM image comparison Groups III to VI

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Overall, the Groups IV, V, and III presented the least amounts of smear, debris, and erosion among the experimental groups at the apical, middle, and coronal one-thirds of the root canal and there was no statistically significant difference (P > 0.05) [Figure 2].


   Discussion Top


Researchers have reported the thickness of the smear layer to be generally in the range of 1–2 μm.[7] The smear produced during a motorized preparation of the canal is much more in volume when compared to hand preparation of canals.[13] Smear may vary depending on the nature of dentin, the type, sharpness, and geometry of cutting instruments used to prepare the radicular space. During the early stages of biomechanical instrumentation, smear layer formed on the walls of the root canals can have a relatively high organic content because of necrotic and/or viable pulp tissue present in the root canals.[14]

Smear layer removal is fairly easy in the occlusal and middle one-third of the root canal, but it is relatively more difficult to remove in apical third of the canals. The apical third of the root presents challenges with regard to curvature, size of canal, taper and diameter, ramifications, deltas, isthmuses, and permeability of dentin.[15] The capillary action between the dentinal tubules and smear material, the cutting action of the rotary tools possibly explain the tubular packing phenomenon during which strong adhesive forces come into play (capillary action hypothesis). Addition of surface active agents to irrigants increases the depth of penetration.[16]

Shahravan et al.[4] in their meta-analysis in 2007 observed that removal of smear from the radicular dentin significantly improves the apical and coronal seal of the obturated root canal and is independent of the type of the sealer type of obturation, the type of dye used for testing, site of leakage, and the duration of the test. A number of reasons have been proposed to support the idea of smear layer removal, that is, the presence of microorganisms, tissue debris, the unpredictable diameter and volume of the canal system, prevents the penetration of irrigants and intracanal medicaments into the dentinal tubules, acts as a bacterial substrate, loosely adherent structure which possibly leads to microleakage, and affects the bond between the sealer material and radicular dentinal structure.

In this study, Group IV presented the least amounts of smear among all experimental groups at the apical, middle, and coronal third levels with mean value of 1.53 [Chart 4]. On statistical comparison and analysis, there was no statistically significant difference between experimental Groups III to V (P > 0.05). The coronal third presented the least amount of smear followed by the middle third, and the most amount of smear was in the apical third of the canals [Chart 5].



The concept of a working solution and an irrigant solution was proposed by Kaufman and Greenberg in 1986,[17] where the working solution was the one which was first used to cleanse the canal during the preparation procedure, and the irrigant solution was the one which was essential to remove the debris and smear layer. Researchers tried out new methodologies and found that combinations of irrigants were the most effective at smear removal in the root canal. As there was no single solution which could dissolve the organic tissues and demineralize the inorganic layer, the sequential use of organic and inorganic solvents were advocated.[18] Various combinations of sodium hypochlorite and other chelating agents have been tried for effective smear removal.

This study used a side-vented needle with the vent at 1 mm from the tip in a customized irrigant protocol. Computational dynamic fluid flow has demonstrated the limitations of a side vent needle on irrigant replacement, and suitable modifications were made in this study to enhance irrigant replacement. The volume of the irrigant is vital, and in this study, a volume of 8 ml during the initial rinse and 5 ml during the final rinse was used. The duration of the exposure of the final rinse is important and a final rinse exposure time of 5 minutes was used in this study.

The effectiveness of irrigant protocols is dependent on how effectively it can bring the irrigant solution in contact with the contents of the root canal space. Manual dynamic activation where a well-fitting Gutta-percha point is placed to the working length and moved up and down in 2-3 mm strokes, can sufficiently improve the displacement and exchange of the irrigant. This method was incorporated into the custom protocol used in this study in the second minute of the final rinse as it effectively negates vapor lock effect, especially in the curved apical thirds of the root. This study adopted a closed-ended root canal system.

Chitosan has excellent qualities of biocompatibility, biodegradability, lack of toxicity, and bioadhesion and is abundantly available in nature. It is a polysaccharide from the shells of crustaceans such as shrimp and crab and includes pandalus borealis and the cell walls of fungi. It is derived from chitin which is insoluble in water, alcohol, and is a limitation in the use of the material. In spite of the limitations, they have found many applications which include, sutures which are absorbable, wound dressings, raw material for man-made fibers, and as chelating agents. The different applications of chitosan and chitin require different properties, which are achieved with the degree of deacetylation and the variations in molecular weight. They have found applications in different industries such as medical, engineering, food processing, textiles paper, agriculture photography, biomedical, and tissue engineering.[19]

Chitosan is only soluble in dilute acid solutions. Water soluble chitosan is also available. The high cost of extraction and purification of chitin chemically limits the use of this polymer to high-value applications. Biomedical and pharmaceutical are the most promising fields of application. Chitosan has been shown to have an antibacterial effect against Gram-positive and Gram-negative organisms and fungi. The covalent immobilization of chitosan on collagen has been proposed to induce the remineralization of the dentin surface. The calcium ions on the dentinal surface bind to functional phosphate groups of the chitosan molecule. This leads to the formation of a favorable surface for crystal nucleation. The antibacterial mechanism of chitosan has been attributed to its polycationic nature. It interacts with microorganisms altering their cell permeability and subsequent leakage of intracellular constituents. It also improves the resistance of collagen of radicular dentinal surface to degradation by collagenase. The use of chitosan has been shown to alter the bacterial adhesion mechanisms, thereby preventing biofilm formation.[20]

Various theories have been put forward for chelation mechanism of chitosan to dentinal structure. The first is the bridge model theory, which states that two or more amino groups of a chain of chitosan bind to the same metal ion. The second theory states that only one amino group of the chitosan is involved in the binding.[21]. Chitosan polymer is composed of several units of dimer of chitin which has got nitrogen atoms with free pairs of electrons which lead to ionic interaction between the metal and the chelating agent. In an acid medium, this forms an ionic form which results in the amino groups being protonated which is responsible for attraction to other molecules and results in adsorption. The process of formation of complexes between chitosan and metal ions occurs as a result of ion exchange, adsorption, and chelation. The prevalent conditions such as the pH of the solution, the chemical structure of chitosan, and the type of ions determine the type of interaction which takes place. Chitosan has been used to repair bone and has been shown to be one of the most promising dental biomaterials. It improves bone regeneration in dental bone loss.[22] It has also been shown to increase salivary secretion when incorporated in chewing gums. It also exerts an antibacterial effect and suppresses the growth of oral microorganisms.[23],[24] A reduction in the microhardness of dentin clinically facilitates the negotiation of narrow and curved canals. The reduction in microhardness facilitates easy instrumentation procedures. Chitosan has been shown to reduce the microhardness of dentin when used as an irrigating solution within the root canal and its effects greater in higher concentrations.[25]

CA has been tried out as an irrigant in endodontics successfully in varying concentrations. Chitosan has excellent antibacterial properties and is a chelating agent. It is soluble fully in dilute acids. The mixture of chitosan and CA would be beneficial in improving the antibacterial and smear clearing efficacy. In the present study, the mixture of chitosan and CA as CC solution has been evaluated as an irrigant solution in the apical, middle, and coronal thirds of the root canal.

Although the result of this study appears promising and chitosan is available abundantly the processing and commercial deacetylation process makes the chitosan very expensive which is a limitation for a material which is available in plenty.


   Conclusion Top


The incorporation of CA with chitosan seems a step in the right direction. The biocompatibility of this material, antibacterial property, lack of toxicity, and excellent biodegradability makes this material an ideal choice as a final rinse solution.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Eick JD, Wilko RA, Anderson CH, Sorensen SE. Scanning electron microscopy of cut tooth surfaces and identification of debris by use of the electron microprobe. J Dent Res. 1970;49:Suppl:1359-68.  Back to cited text no. 1
    
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McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975;1:238-42.  Back to cited text no. 2
    
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Shahravan A, Haghdoost AA, Adl A, Rahimi H, Shadifar F. Effect of smear layer on sealing ability of canal obturation: A systematic review and meta-analysis. J Endod 2007;33:96-105.  Back to cited text no. 4
    
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Spanó JC, Silva RG, Guedes DF, Sousa-Neto MD, Estrela C, Pécora JD. Atomic absorption spectrometry and scanning electron microscopy evaluation of concentration of calcium ions and smear layer removal with root canal chelators. J Endod 2009;35:727-30.  Back to cited text no. 8
    
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Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233-9.  Back to cited text no. 12
    
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Czonstkowsky M, Wilson EG, Holstein FA. The smear layer in endodontics. Dent Clin North Am 1990;34:13-25.  Back to cited text no. 13
    
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Cameron JA. The use of ultrasound for the removal of the smear layer. The effect of sodium hypochlorite concentration; SEM study. Aust Dent J 1988;33:193-200.  Back to cited text no. 14
    
15.
Ribeiro EM, Silva-Sousa YT, Souza-Gabriel AE, Sousa-Neto MD, Lorencetti KT, Silva SR. Debris and smear removal in flattened root canals after use of different irrigant agitation protocols. Microsc Res Tech 2012;75:781-90.  Back to cited text no. 15
    
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Aktener BO, Cengiz T, Piskin B. The penetration of smear material into dentinal tubules during instrumentation with surface-active reagents: A scanning electron microscopic study. J Endod 1989;15:588-90.  Back to cited text no. 16
    
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Kaufman AY, Greenberg I. Comparative study of the configuration and the cleanliness level of root canals prepared with the aid of sodium hypochlorite and bis-dequalinium-acetate solutions. Oral Surg Oral Med Oral Pathol 1986;62:191-7.  Back to cited text no. 17
    
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Baumgartner JC, Brown CM, Mader CL, Peters DD, Shulman JD. A scanning electron microscopic evaluation of root canal debridement using saline, sodium hypochlorite, and citric acid. J Endod 1984;10:525-31.  Back to cited text no. 18
    
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Silva PV, Guedes DF, Nakadi FV, Pécora JD, Cruz-Filho AM. Chitosan: A new solution for removal of smear layer after root canal instrumentation. Int Endod J 2013;46:332-8.  Back to cited text no. 19
    
20.
Del Carpio-Perochena A, Kishen A, Shrestha A, Bramante CM. Antibacterial properties associated with chitosan nanoparticle treatment on root dentin and 2 types of endodontic sealers. J Endod 2015;41:1353-8.  Back to cited text no. 20
    
21.
Blair HS, Ho TC. Studies in the adsorption and diffusion of ions in chitosan. J Chem Technol Biotechnol 1981;31:6-10.  Back to cited text no. 21
    
22.
Ezoddini-Ardakani F, Azam AN, Yassaei S, Fatehi F, Rouhi G. Effects of chitosan and dental bone repair. Health 2011;3:200-5.  Back to cited text no. 22
    
23.
Hayashi Y, Ohara N, Ganno T, Ishizaki H, Yanagiguchi K. Chitosan-containing gum chewing accelerates antibacterial effect with an increase in salivary secretion. J Dent 2007;35:871-4.  Back to cited text no. 23
    
24.
Pimenta JA, Zaparolli D, Pécora JD, Cruz-Filho AM. Chitosan: Effect of a new chelating agent on the microhardness of root dentin. Braz Dent J 2012;23:212-7.  Back to cited text no. 24
    
25.
Suzuki S, Masuda Y, Morisaki H, Yamada Y, Kuwata H, Miyazaki T The study of chitosan-citrate solution as a root canal irrigant – A preliminary report Oral Hyg Health 2014; 2:142.  Back to cited text no. 25
    


    Figures

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