| Abstract|| |
Aim: The purpose of this study was to evaluate the sealing ability of mineral trioxide aggregate (MTA) and EndoSequence with chitosan and carboxymethyl chitosan (CMC) as retrograde smear layer removing agents using scanning electron microscopy (SEM).
Materials and Methods: Forty human single rooted teeth were taken. Crowns were decoronated and canals were obturated. Apically roots were resected and retrograde cavities were done. Based on the type of retrograde material placed and the type of smear layer removal agent used for retrograde cavities, they were divided into four groups (N = 10): Group I chitosan with EndoSequence, group II chitosan with MTA, group III CMC with EndoSequence, and Group IV CMC with MTA. All the samples were longitudinally sectioned, and the SEM analysis was done for marginal adaptation.
Statistical Analysis: Kruskal-Wallis and Mann-Witney analysis tests.
Results: SEM images showed the presence of less gaps in group III, i.e., CMC with EndoSequence when compared to other groups with statistically significant difference.
Conclusion: Within the limited scope of this study, it was concluded that EndoSequence as retrograde material showed better marginal sealing ability.
Keywords: Carboxymethyl chitosan (CMC); chitosan; EndoSequence; mineral trioxide aggregate (MTA)
|How to cite this article:|
Nagesh B, Jeevani E, Sujana V, Damaraju B, Sreeha K, Ramesh P. Scanning electron microscopy (SEM) evaluation of sealing ability of MTA and EndoSequence as root-end filling materials with chitosan and carboxymethyl chitosan (CMC) as retrograde smear layer removing agents. J Conserv Dent 2016;19:143-6
|How to cite this URL:|
Nagesh B, Jeevani E, Sujana V, Damaraju B, Sreeha K, Ramesh P. Scanning electron microscopy (SEM) evaluation of sealing ability of MTA and EndoSequence as root-end filling materials with chitosan and carboxymethyl chitosan (CMC) as retrograde smear layer removing agents. J Conserv Dent [serial online] 2016 [cited 2020 Jan 27];19:143-6. Available from: http://www.jcd.org.in/text.asp?2016/19/2/143/178693
| Introduction|| |
The presence of endodontic microbiota and biofilms in inaccessible areas cause failure in the endodontic treatment.  When nonsurgical root canal treatment fails to treat periradicular lesions of endodontic origin, surgical endodontic treatment is preferred.  The important factor in the success of surgical endodontic treatment is to achieve a good seal between the tooth and root-end filling material.
Several root-end filling materials have been introduced such as amalgam, Cavit, composite resins, gutta-percha, intermediate restorative material (IRM), zinc oxide and eugenol cement, super ethoxy benzoic acid (EBA), mineral trioxide aggregate (MTA), biodentine, and EndoSequence.  Marginal adaptation of retrograde materials is very important in endodontic surgery success. This property will determine whether the material is suitable for clinical use,  so the purpose of this study was to compare the marginal adaptation of the MTA and EndoSequence.
During retrograde cavity preparation, the smear layer is formed over the cut dentinal surfaces. The smear layer contains organic, inorganic material, bacteria, and their by-products.  It acts as a barrier between filling materials and the canal wall, thus compromising the formation of a satisfactory seal that may further lead to microleakage. 
Various agents, such as sodium hypochlorite, ethylenediaminetetraacetic acid (EDTA), mixture of tetracycline acid detergent (MTAD), organic acids (e.g., citric acid), have been introduced for smear layer removal, regarding orthograde and retrograde root preparation.  The alternating use of EDTA and sodium hypochlorite has been recommended for the efficient removal of the smear layer.  However, the use of these solutions may cause periapical inflammatory reactions and reduce periapical healing. To minimize their harmful effects on periapical tissues the use of biocompatible solutions is essential.
Chitosan is a natural polysaccharide that is biocompatible, biodegradable, nontoxic, having chelating property but limited solubility.  Carboxymethyl chitosan (CMC) has been introduced to overcome limited solubility of chitosan that is formed by carboxymethylation of chitosan. 
Hence, in this study, chitosan and CMC solutions were used for the smear layer removal that are more biocompatible alternatives to EDTA and other chelating agents at the surgical site.
The treatment outcome is negatively affected by the failure of materials in achieving marginal adaptation and also due to the occurrence of cracks and spaces in the interface between the material and the dentin walls.  Although there are inherent limitations in in vitro studies conducted in laboratories, scanning electron microscopy (SEM) has been used in this study to evaluate the marginal adaptation of root-end filling materials. 
| Materials and Methods|| |
Forty human single rooted teeth, free of caries, crack, or fracture, were taken. The teeth were preserved in 2% formalin before use. The crowns were decoronated to prepare a standardized 16-mm tooth length from the root apex. Cervical third of each root canal was enlarged using Gates-Glidden drills (Dentsply Maillefer, Switzerland) sizes 1 to 3. Canals were prepared using K-Files (Dentsply Maillefer, Switzerland) up to size #40, 1 mm short of the apical foramen. During instrumentation, the root canals were irrigated with 3 mL of 3% sodium hypochlorite and saline at each change of files and dried with paper points. The root canals were obturated with standard 2% gutta-percha points coated with AH plus sealer using the conventional lateral compaction technique. Now the apical 3 mm of roots was sectioned perpendicular to the long axis of the tooth with a sectioning disc. Then, a 3-mm deep root-end cavity was prepared using no. 2 round carbide bur. The materials were prepared according to the manufacturer's instruction, and then the root-end cavities were filled. Based on the type of smear layer removing agent used for root-end cavities, i.e., 5 mL of 0.2% of chitosan [Indian overseas (P) LTD, Kerala] and CMC [Indian overseas (P) LTD, Kerala] and type of root-end filling material placed, i.e., MTA and EndoSequence (Brasseler, USA) samples were divided into four groups (N = 10), in group I chitosan as smear layer removing agent and cavities filled with EndoSequence, group II chitosan as smear layer removing agent and cavities filled with MTA, group III CMC as smear layer removing agent and cavities filled with EndoSequence, group IV CMC as smear layer removing agent and cavities filled with MTA. Materials are allowed to set and after that all the samples were sectioned longitudinally into two halves with the help of hard tissue microtome for the SEM evaluation. The distance between the root-end filling materials and dentinal walls was measured under scanning electron microscope. Kruskal-Wallis and Mann-Whitney U test analyses were used to determine statistical difference between various groups.
| Results|| |
The SEM examination of root-end-filled teeth showed a mean value of marginal gap of 1.81 μm in group I [Figure 1]a, 2.60 μm in group II [Figure 1]b, 1.40 μm in group III [Figure 1]c, and 2.16 μm in group IV [Figure 1]d. The marginal gap between dentinal walls and filling material is less in group III showing statistically significant difference when compared with other groups [Table 1].
|Figure 1: (a) Chitosan with EndoSequence, (b) Chitosan with MTA, (c) Carboxymethyl chitosan with EndoSequence, (d) Carboxymethyl chitosan with MTA|
Click here to view
|Table 1: The mean ± SDs (ìm) of gaps between the experimental filling materials and dentinal walls|
Click here to view
| Discussion|| |
Endodontic treatment failure necessitates other treatment options such as retreatment, endodontic surgery, and even tooth extraction. Achieving adequate seal prevent microorganisms from reaching apical and periapical tissues. So the success of endodontic treatment or surgery depends on the coronal and apical sealing. 
This study evaluated the marginal adaptation of root-end filling materials such as MTA and EndoSequence. MTA aggregate is composed of tricalcium aluminate, tricalcium silicate, tricalcium oxide, and silicon oxide. It has excellent biocompatibility and good sealing ability.  Torabinejad et al. evaluated the sealing ability of super-EBA, amalgam, and intermediate restorative materials with dye leakage methods. The results showed that MTA leaked significantly less when compared to other materials. 
Another root-end filling material used was EndoSequence, which is a bioceramic material. It is available as Premixed syringe form and is composed of zirconium oxide, calcium silicates, tantalum oxide, calcium phosphate monobasic thickening agents, and proprietary fillers. 
Shi et al. compared the apical sealing ability of a bioceramic based root canal repair filling material (i Root FS, Innovative BioCeramix Inc, Vancouver, Canada) with that of MTA by means of dye penetration method and found that the samples filled with iRoot FS showed less leakage compared with the samples filled with MTA as root-end filling material. 
In many studies, dye penetration method was used for the assessment of microleakage; however, there are certain limitations for traditional dye leakage methods such as dissolution during the process, and it is also difficult to observe the maximum dye penetration depth.  So in the present study, SEM examination was used to determine the marginal adaptation of root-end filling materials. It should be noted that SEM examination is a suitable method for the assessment of marginal adaptation because of its high magnification and good resolution. 
The results of this study showed that marginal gap was less in samples filled with EndoSequence as root-end filing material when compared with samples filled with MTA with statistically significant difference. This might be due to certain drawbacks of MTA such as difficulty in handling, slow setting reaction that might contribute to leakage, surface disintegration leading to loss of marginal adaptation, and continuity of the material. 
Endosequence is directly applied over the prepared cavity and the by-products formed in the setting reaction of are hydroxyapatite and water. According to the manufacturers of EndoSequence, water formed in this reaction is important in controlling hydration rate and setting reaction of this material. 
Jeevani et al. evaluated the sealing ability of MICRO-MEGA Mineral Trioxide Aggregate (MM-MTA), EndoSequence, and biodentine as furcation repair materials using a dye extraction leakage method. They observed that EndoSequence showed less dye absorbance when compared with MM-MTA and biodentine. 
Ghattas et al., observed that EndoSequence™ Root Repair material showed sealing ability similar to that of white mineral trioxide when used as a root-end filling material. 
The presence of smear layer may inhibit or significantly delay the penetration of irrigating solutions, sealers, and medicaments into the dentinal tubules.  There is a controversy regarding the presence and removal of smear layer. It is now generally advocated that the smear layer should be removed prior to the root canal obturation to facilitate better adaptation of the filling material to the root canal wall and to improve adhesion. 
Alternate use of EDTA and NaOCL as retro smear layer removing agents may cause periapical inflammatory reactions at surgical site.  Calt et al. observed that usage of EDTA for prolonged periods caused excessive tubular and intertubular dentin erosion.  So the use of biocompatible retro smear layer removing agents is essential. Chitosan and CMC are more biocompatible and used as retro smear layer removing agents in this study. Chitosan is natural polysaccharide obtained by the deacetylation of chitin. Chitin and chitosan do not cause any biological hazard and are inexpensive. 
Chitosan exhibits many biological actions such as antimicrobial, wound healing, mucoadhesive, sustained drug releasing property, chelating agent, and also as irrigating solution.  CMC obtained by carboxymethylation of chitosan having good solubility when compared to chitosan.  Irrespective of the type of smear layer removing agent used, when results were observed they showed negligible effect on retro smear layer removal.
| Conclusion|| |
Within the limited scope of this study, it is concluded that irrespective of the type of retrograde smear layer removing agent used, groups filled with EndoSequence showed less marginal gap with statistically significant difference when compared with groups filled MTA as root-end filling material.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Shahi S, Yavari HR, Eskandarinezhad M, Kashani A, Rahimi S, Sadrhaghighi H. Comparative investigation of marginal adaptation of mineral trioxide aggregate (MTA) and Portland cement as root-end filling materials: A scanning electron microscopy (SEM) study. Afr J Biotechnol 2011;10:16084-8.
Bidar M, Moradi S, Jafarzadeh H, Bidad S. Comparative SEM study of the marginal adaptation of white and grey MTA and Portland cement. Aust Endod J 2007;33:2-6.
Oliveira HF, Gonçalves Alencar AH, Poli Figueiredo JA, Guedes OA, de Almeida Decurcio D, Estrela C. Evaluation of marginal adaptation of root-end filling materials using scanning electron microscopy. Iran Endod J 2013;8:182-6.
Almeida BM, Abad ED, Filho HR, Zoffoli JD. In vitro
evaluation of dentin marginal adaptation of three root-end filling materials inserted with and without surgical microscope. Dental Press Endod 2012;2:20-5.
Violich DR, Chandler NP. The smear layer in endodontics - A review. Int Endod J 2010;43:2-15.
Kuruvilla A, Jaganath BM, Krishnegowda SC, Ramachandra PK, Johns DA, Abraham A. A comparative evaluation of smear layer removal by using EDTA, Etidronic acid, and Maleic acid as root canal irrigants: An in vitro
scanning electron microscopic study. J Conserv Dent 2015;18:247-51.
Dechichi P, Christian C. Smear layer: A brief review of general concepts. Part II. The most common agents to remove endodontic smear layer. RFO UPF 2006;11:100-4.
Madison S, Krell KV. Comparason of ethylenediaminetetraacetic acid and sodium hypochlorite on the apical seal of endodontically treated teeth. J Endod 1984;10:499-503.
Dutta PD, Dutta J, Tripathi VS. Chitin and chitosan: Chemistry, properties and applications. J Sci Ind Res 2004;63:20-31.
Mourya VK, Inamdar NN, Tiwari A. Carboxymethyl chitosan and its applications. Adv Mat Lett 2010;1:11-33.
Jeevani E, Jayaprakash T, Bolla N, Vemuri S, Sunil CR, Kalluru RS. Evaluation of sealing ability of MM-MTA, EndoSequence, and biodentine as furcation repair materials: UV spectrophotometric analysis. J Conserv Dent 2014;17:340-3.
Torabinejad M, Smith PW, Kettering JD, Pitt Ford TR. Comparative investigation of marginal adaptation of mineral trioxide aggregate and other commonly used root-end filling materials. J Endod 1995;21:295-9.
Shokouhinejad N, Nekoofar MH, Razmi H, Sajadi S, Davies TE, Saghiri MA, et al
. Bioactivity of EndoSequence root repair material and bioaggregate. Int Endod J 2012;45:1127-34.
Shi S, Zhang DD, Chen X, Bao ZF, Guo YJ. Apical sealing ability of bioceramic paste and mineral trioxide aggregate retrofillings: A dye leakage study. Iran Endod J 2015;10:99-103.
Tawil SB, Dokkyl NA, Hamid DA. Sealing ability of MTA versus Portland cement in the repair of furcal perforations of primary molars: A dye extraction leakage model. J Am Sci 2011;7:1037-43.
Ghattas SM. A comparative evaluation of the sealing ability of two root end filling materials: An In Vitro
leakage study using enterococcus faecalis. J Endod 2011;37:1025-9.
Bystrom A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60 cases of endodontic therapy. Int Endod J 1985;18:35-40.
Shenoy A, Ahmaduddin, Bolla N, Raj S, Mandava P, Nayak S. Effect of final irrigating solution on smear layer removal and penetrability of the root canal sealer. J Conserv Dent 2014;17:40-4.
Shaik J, Garlapati R, Nagesh B, Sujana V, Jayaprakash T, Naidu S. Comparative evaluation of antimicrobial efficacy of triple antibiotic paste and calcium hydroxide using chitosan as carrier against Candida albicans and Enterococcus faecalis: An in vitro
study. J Conserv Dent 2014;17:335-9.
Ballal NV, Shavi GV, Kumar R, Kundabala M, Bhat KS. In vitro sustained release of calcium ions and pH maintenance from different vehicles containing calcium hydroxide. J Endod 2010;36:862-6.
Department of Conservative Dentistry and Endodontics, Sibar Institute of Dental Sciences, Takkellapadu, Guntur - 522 509, Andhra Pradesh
Source of Support: None, Conflict of Interest: None