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Year : 2022  |  Volume : 40  |  Issue : 2  |  Page : 201-207

Comparative evaluation of chelating efficacy of nano-chitosan, pomegranate extract, and ethylenediaminetetraacetic acid on primary radicular dentin: An in vitro study

1 Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
2 Department of Oral and Maxillofacial Surgery, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India

Date of Submission26-Jun-2021
Date of Decision31-Jul-2021
Date of Acceptance27-Sep-2021
Date of Web Publication15-Jul-2022

Correspondence Address:
Dr. A J Sai Sankar
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jisppd.jisppd_231_21

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Background: Irrigation is a crucial aspect of root canal treatment, and it is imperative to employ chelating agents to eliminate the smear layer during biomechanical preparation. They in turn react with mineral content of dentin, leading to decreased strength and increased susceptibility to fracture. Aims: This study aimed to assess and compare mineral loss and microhardness from primary root canal dentin following the usage of different irrigating solutions and determine the least detrimental irrigant among the tested solutions. Materials and Methods: Sixty-six primary anterior teeth were divided into three groups with 22 in each– Group I: 17% ethylenediaminetetraacetic acid (EDTA) Group II: 0.2% Nano chitosan Group III: Pomegranate extract. The decoronated teeth were split longitudinally. Half of it was directly subjected to Vickers test, and the other half was immersed in a magnetic stirrer bath containing test solution to record the mineral loss from solution. Postmicrohardness values were recorded on the specimen and compared with initial values. Statistical Analysis Used: Statistical analysis was done using SPSS software (Version 20, SPSS, IBM, Armonk, NY, U. S. A). Results: Descriptive statistics were calculated, and the groups were compared using analysis of variance test and post hoc Tukey test. Pomegranate extract showed least effect on mineral content and microhardness compared to 17% EDTA and 0.2% nano chitosan. P ≤ 0.05 was considered statistically significant. Conclusion: Pomegranate aril extract showed better results with selected parameters.

Keywords: Chelating agents, chitosan nanoparticles, microhardness, mineral loss, pomegranate extract, primary root dentin

How to cite this article:
Nirmal G D, Sai Sankar A J, Sridevi E, Sridhar M, Sankar K S, Satish P R. Comparative evaluation of chelating efficacy of nano-chitosan, pomegranate extract, and ethylenediaminetetraacetic acid on primary radicular dentin: An in vitro study. J Indian Soc Pedod Prev Dent 2022;40:201-7

How to cite this URL:
Nirmal G D, Sai Sankar A J, Sridevi E, Sridhar M, Sankar K S, Satish P R. Comparative evaluation of chelating efficacy of nano-chitosan, pomegranate extract, and ethylenediaminetetraacetic acid on primary radicular dentin: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2022 [cited 2022 Oct 5];40:201-7. Available from: http://www.jisppd.com/text.asp?2022/40/2/201/351047

   Introduction Top

A clean root canal system along with a three-dimensional seal is the clinician's path to success.[1] Biomechanical preparation is of paramount importance where endodontic instruments are used in association with auxiliary chemical substances like irrigants, which help clean the root canal system areas that cannot be reached directly by instrumentation. Regardless of the instrument and technique employed, the maneuver of endodontic instruments against dentinal walls consequently promotes the formation of smear layer.[2],[3] The complete removal of smear layer requires chelating agents, followed by tissue solvents because no single solution can furnish both effects alone. Therefore, ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite solutions have been jointly advocated as an effective irrigation regimen.[4]

Chelating agents were introduced to endodontics by Nygaard-Ostby[5] as an adjunct to prepare narrow and calcified root canals and smear layer removal. They increase the penetration of irrigants into dentinal tubules, thus allowing adequate disinfection and a reduction in dentin microhardness that facilitates the access and action of endodontic instruments in the root canals.[6],[7] However, usage of endodontic irrigants like EDTA causes dentinal cracks and alterations in the chemical composition of dentin particularly, the calcium and phosphate ratio which in turn changes the microhardness, permeability, and solubility characteristics of dentin, thereby adversely affecting sealing ability and adhesion of dental materials to dentin.[8]

Recently, the global scenario is inclining towards natural products like Chitosan which is a polysaccharide biopolymer produced by the alkaline deacetylation of chitin, the principal component of crustacean and shrimp exoskeletons.[9] Chitosan contains ideal properties including biocompatibility, biodegradability, bioadhesion, high chelating ability for various metal ions in acidic conditions, lack of toxicity, and even antibacterial activity. Owing to these properties, proposed uses of chitosan in dentistry are diverse. It was used as an irrigant in several studies[10],[11] and Bastawy et al.[9] reported that even at the lowest concentration, chitosan removed the smear layer as effective as or more than EDTA. Ikono et al.[12] found that chitosan nanoparticles (Cs-NPs) have smaller size and better penetration than chitosan, and this property makes it unique with superior qualities.

Among the myriad of herbal alternatives available, pomegranate (botanical name: Punica granatum) finds a noteworthy mention of its several antioxidant activities, including radical scavenging ability, ferrous ion chelating, and ferric ion reducing power.[13] Pomegranate is now finding prime uses in the dental arena as well. Prior studies[14],[15] employing the pomegranate rind or extract of pomegranate suggest that pomegranate has effective antimicrobial activity, but the chelating potency of pomegranate extract in endodontics has been left unexplored. Hence, an attempt is made to study pomegranate aril extract's effect on the primary radicular dentin.

Even though plentiful studies regarding the effect of root canal irrigants on permanent root dentin are available in the literature,[3],[8],[11] their impact on primary root canals is quite limited. Hence, the results obtained cannot be pertinent to primary teeth due to morphological differences such as dentinal tubules, accessory canals, root tip bifurcation, and compositional variations.[16] Henceforth, the present study was aimed to evaluate the effect of 17% EDTA, 0.2% Nano-chitosan and pomegranate aril extract as irrigating agents on the microhardness and mineral content of primary radicular dentin.

   Materials and Methods Top

The present laboratory study investigated the effect of various root canal irrigants on the calcium and phosphate content and microhardness of primary root dentin using the Atomic Absorption Spectrophotometer (AA-6300, Shimadzu, Kyoto, Japan); Vandomolybdophosphoric acid method, and Vickers Hardness Test, respectively. After obtaining clearance from the institutional ethical committee, the present in vitro study was planned with a sample size of 70 teeth which was calculated using G * POWER software with a power of 80%, effect size of 0.4, and an alpha error of 5%.

The sample included human primary anterior teeth which were un-restorable due to caries or trauma or over-retained. These teeth were collected from the tooth bank in our institute and stored in distilled water at room temperature (37°C) until use. The soft and hard debris on the root surfaces was cleaned using hand scaler, decoronated at the cementoenamel junction, and at the root apex to obtain 10 mm standard-sized specimens using a low-speed diamond disc. Roots were sectioned longitudinally into two halves, and pulp tissue was removed using a brush. Four samples were discarded due to improper sectioning, and the remaining 66 samples were randomly divided into three groups of 22 specimens in each using computer randomization method depending on the irrigating solution used. Group I: 17% EDTA, Group II: 0.2% nano-chitosan and Group III: Pomegranate aril extract.

Before commencing the experiment, 17% EDTA solution (Prevest Denpro Ltd, Jammu) was procured. 0.2% nano chitosan solution was made by dissolving 0.2 g of chitosan powder (size 395 ± 98.5 nm) (Aura Biotechnologies, Chennai) in 1% acetic acid with the volume of 100 ml. To obtain a homogenous solution, the mixture was stirred with a magnetic agitator for 2 h. Chitosan was synthesized from shrimp shells (degree of deacetylation >75%) using ionic gelation method and polyanion tripolyphosphate (TPP) as crosslinker.[11]

Fresh pomegranate fruits were brought to the laboratory, cleaned in running tap water; surface sterilized using 70% alcohol, and then rinsed with sterile distilled water. Pomegranate aril juice was freshly prepared after removing the rind and separating the arils. The arils were blended in a domestic mixer without dilution. The blend was first filtered through a sieve followed by centrifugation of the obtained extract to precipitate the residual waste, and the supernatant clear solution obtained was again passed through a filtration assembly using a series of membrane filter papers of 0.45 microns.[17] The final extract thus obtained was placed in the ultraviolet (UV) sterilizer and used in the study. Freshly prepared undiluted and filtered pomegranate aril extract has been used to know its indisputable effect on human radicular dentin and to avoid any bias that might occur with the use of preservatives. The pH of the test solutions of different groups I, II, and III were 7.2; 3.5; and 3.4, respectively, which was determined using a pHmeter with an accuracy of ±0.002.

In each group, one-half of the specimen was immersed in a magnetic stirrer bath containing 10 ml of test solution for 5 min. Accordingly, 1 ml of solution was aspirated from the bath after 5 min for Calcium (Ca) analysis using Atomic Absorption Spectroscopy technique.

The phosphate level of the solution was calculated using the vanadate-molybdate method which is based on the APHA Standard Method 4500-P C. 0.25 ml of vanadate-molybdate reagent was added to 1 ml of sample irrigating solution from the magnetic stirrer bath and the solution was mixed by pipetting up and down for several times till yellow color is developed in the cuvette. This sample was placed in the UV-visible spectrophotometer (Shimadzu UV-1700 Pharma Spec) to get the absorbance value of phosphate converted to phosphate concentration in parts per million.[18]

The other half of the specimen was mounted horizontally on an acrylic block and the dentin surface was polished with silicon carbide sandpaper with three progressively increasing grit sizes (400, 600, and 1200, respectively) to obtain a smooth surface for analyzing surface microhardness using digital microVickers hardness tester under an indentation load of 150 g with an indentation time of 15 s. The mean average of the three indentations taken on the middle third of the root dentin was considered as baseline hardness value.

After immersing in the respective irrigating solutions, the part of the specimen used for mineral loss analysis was flushed with saline, air dried, mounted on an acrylic block, dentin surface was polished using silicon carbide sand papers (400, 600, and 1200) and subjected to Vickers test. The difference between initial and final microhardness was noted.

A single operator performed the entire procedure. However, to avoid bias, a second operator who was unaware of the prior results evaluated the samples randomly. Interexaminer reliability was assessed by the kappa test. As an interexaminer reliability statistic of 0.93 was achieved indicating excellent agreement (Intraclass correlation coefficient = 0.93), the scores given by the first investigator were only considered for the analysis. Statistical analysis was done using SPSS Version 20 (SPSS, IBM, Armonk, NY, U. S. A), and the groups were compared using analysis of variance test and post hoc Tukey test as necessary. The P ≤ 0.05 was considered statistically significant.

   Results Top

The mean calcium and phosphate loss in the three groups after placing in the respective irrigants ranged from 1.9 ppm to 24.26 ppm and 2.6 ppm to 29.3 ppm respectively, with highest loss seen in group II followed by I and III [Table 1].
Table 1: Mean calcium and phosphate loss following the usage of different irrigating solutions

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The preimmersion microhardness values of the samples ranged between 56.44 and 56.97 Vickers hardness numbers (VHN) which were not statistically significant (P > 0.05). However, when the pre and post- immersion microhardness values were compared, groups I and II showed statistically significant change from their baseline values ranging from 19.74 to 20.26 VHN. Whereas in group III samples, not much difference (1.23) was noticed between pre and postmicrohardness values, although the change was statistically significant (P < 0.05) [Table 2].
Table 2: Pre-and post-immersion microhardness values

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On intergroup comparison, the mean difference in calcium mineral loss varied between 17.75 and 22.36 with least variation recorded between groups I, II and the highest change observed between groups II, III. The amount of loss noticed was statistically significant (P < 0.05) among all the three groups [Table 3]. The mean difference in phosphate loss ranged between −23.73 and 26.67 with least change recorded between groups I, III and highest change observed between groups II, III. A statistically significant difference in phosphate loss was noticed on an intergroup comparison between I, II and II, III with no significant difference in phosphate loss between I and III, the value was 2.93 [Table 3]. The microhardness values span from −17.78 seen between groups I and III to 0.34 observed between groups I and II. As shown in [Table 4], it was evident that intergroup comparison of microhardness postimmersion showed no significant difference between groups I, II but these solutions have significant difference with group III [Table 4].
Table 3: Inter group comparison of calcium and phosphate mineral loss

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Table 4: Inter group comparison of post immersion microhardness values

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Thus, it can be hypothesized that pomegranate aril extract demonstrated minimal effect on calcium, phosphate loss and microhardness values. Although 17% EDTA showed less mineral loss than 0.2% nano chitosan, the variation in microhardness was analogous with it. Novel irrigating solution nano chitosan had the most significant effect on calcium and phosphate loss and microhardness values.

   Discussion Top

The root canal system is remarkably complex and variable, limiting the ability to clean and disinfect it predictably. Peters et al.[19] stated that irrespective of the instrumentation technique used, 35% or more of the root canal surfaces, including canal fins, cul-de-sacs, and isthmi remain un-instrumented with the evidence derived from their study on permanent teeth using micro-computed tomography scans before and after mechanical instrumentation. Thus, the use of chemical adjuncts is considered a sine qua non in root canal treatment.[20],[21] Concerning primary teeth, in particular, more ictus is on chemical means with limited mechanical debridement.[22] Therefore, irrigation is an indispensable part of root canal debridement.

An ideal irrigant should have many prerequisites.[23],[24] In addition, the choice of a cleanser in the pulpal therapy of primary teeth should have auxiliary requirements such as: They must not irritate the periapical tissues to avoid harming the germ of the permanent successor tooth, should facilitate easy instrumentation in the ribbon shaped canals, increase root dentin permeability without altering the microhardness of primary teeth.[25]

The most common chelating solutions are based on EDTA, which reacts with the calcium ions in dentin and forms soluble calcium chelates.[10] Chelating solutions remove dentin calcium ions, favoring smear layer removal and altering the microhardness. Baldasso et al.[26] have hypothesized that these changes could increase the tooth's susceptibility to fracture. Biomaterials like Cs-NPs reduce the possibility of bacterial penetration, prevents dentinal micro-fractures, and improves the mechanical properties of the root dentin.[27] Its high chelating capacity for different metallic ions and low cost, made it a preferable irrigant for the study. Natural phytochemical pomegranate has active components such as organic acids and bioactive compounds like phenolics and flavonoids, principally anthocyanins that inhibits the plaque-forming microorganisms and effectively removes smear layer in the root canals.[28]

This study quantified the calcium ions chelated by different solutions from the root canal dentin by atomic absorption spectroscopy, a susceptible procedure popular for its elemental selectivity.[29] Among the various phosphate determination methods, spectrophotometry involving molybdovanadate and ammonium molybdate is most widely used for biological applications as it is more sensitive, and the preparation of reducing agent is easy.[30]

Microhardness determination is sensitive to composition and surface changes of the tooth structure; hence, it can provide indirect evidence of mineral changes, either loss or gain in dental hard tissues.[9] Previous investigations have demonstrated that Vickers microhardness test is an appropriate and practical tool for evaluating dentin surface changes treated with chemical agents.[9],[31] The fact that there were no statistically significant differences between the pretreatment microhardness values of all the tested groups proved that the specimens were standardized. The resulted difference in microhardness values was because of the different irrigating solutions used. In the current study, the longitudinal sectioning of the roots was preferred as it could show an accurate representation of clinical situation,[6] and as the irrigants first contact the most superficial layer of root canal dentin, its microhardness was measured.[32] In the present study, the root canals were not prepared before analysis; thus, no smear was present on the dentin surface. This step was omitted so that calcium and phosphate loss from intact root dentin could be measured, avoiding any possible contamination of readings that could result from calcium and phosphate being incorporated into loosely bound dentin.[8]

The rationale behind using 17% EDTA was to test a low yet commonly used EDTA concentration on calcium and phosphate removal. 0.2% chitosan application for 5 min is the most viable combination for its use on root dentin and smear layer removal.[33],[34] Hence, a similar concentration of nano-chitosan has been used for equitable comparison. In the present study, a 5-min exposure time was advocated for each chelating solution as it may simulate the clinical application time of the irrigant solution.[35]

Atomic absorption spectrophotometric analysis of calcium and vanadomolybdophosphoric acid method for phosphate evaluation exposed that 0.2% nano-chitosan produced the highest release of calcium and phosphate followed by 17% EDTA and pomegranate aril extract. Almost similar results were obtained in the studies done on permanent teeth by Pedro et al.[2] However, contrast findings were seen in studies by Spanó et al.[36] and Bastawy et al.[9] in which 17% EDTA and 0.2% chitosan groups contained the highest concentration of calcium ions extracted from root canals, without significant difference between them. Nonetheless, study by Berastegui et al.[37] exhibited the highest calcium loss from root dentin with EDTA than chitosan. The discrepancy in the results may be attributed to factors influencing the agents' demineralization capacity such as contact time, pH, concentration and the amount of available solution, assessment of calcium and phosphate ion concentration in the specimen rather than in solution, irrigation protocol followed coupled with difference in methodology.[7] Furthermore, permanent dentin is richly mineralized, and concentration of calcium and phosphorus in both peritubular and intertubular dentin is also high. Even dentinal micromorphology studies showed potential differences between primary and permanent teeth.[38]

It is believed that adsorption, ionic exchange, and chelation properties of chitosan are responsible for eliminating dentin calcium ions.[39] There was a significant decrease in calcium and phosphate levels in dentin following EDTA treatment which was less than nano-chitosan. This might be attributed to the fact that EDTA forms a stable complex with dentin's calcium ions. Correspondingly, EDTA molecule's carboxyl groups ionize, releasing hydrogen atoms that compete with calcium ions. After all available ions are bound, equilibrium is formed, and no further dissolution occurs. It is also possible that dentin's organic matrix may act as a barrier in the dissolution of the inorganic component. Thus decalcifying action of EDTA stops.[9] Pomegranate aril extract displayed least mineral loss, and possible reasons accredited to this may be weak organic acids such as ascorbic acid, citric acid, and malic acid in the arils, which have a weak chelating ability.[28],[40]

Notable variation in VHN values following irrigation protocol indicates the potential effect of these chemical solutions on radicular dentin components. The postimmersion VHN values in the present study suggest that dentin microhardness was significantly reduced by irrigating solutions compared to baseline values. As compared to pomegranate aril extract group, both 0.2% nano chitosan and 17% EDTA showed an identical reduction in root canal dentin's microhardness. The chelating activity of EDTA produces softening of calcified components of dentin, and subsequently, a reduction in the microhardness of the root canal dentin is noticed.[29] The chelating property of chitosan responsible for calcium and phosphate extraction from radicular dentin might also be accountable for reduction in microhardness of dentin which was analogous with that of 17% EDTA. The results are in accordance with Pimenta et al.[33] and Silva et al.[34] who found that 0.2% chitosan is equally effective as 15% EDTA in microhardness reduction. On the other hand, Bastawy et al.[9] reported that 0.2% chitosan caused less reduction in dentin microhardness than 17% EDTA. In case of pomegranate aril extract, it may be hypothesized that the presence of weak acids could have altered dentin's microstructure that is liable for a minimal reduction in root dentin VHN values. Nevertheless, it may be theorized that the time-dependent demineralization effect of the irrigation regimens tested herein may be reduced in the clinical settings by the presence of smear layer, volume of irrigant used, contact period, and irrigation protocol followed.

   Conclusion Top

All the irrigants used in the study demonstrated mineral loss from the root canal dentin with maximum loss observed in 0.2% nanochitosan and least with pomegranate aril juice. A significant difference in the VHN values was recorded in all the tested groups, suggesting that microhardness is directly proportional to mineral loss. Thus, the present study's observations demonstrate the need to incorporate herbal agents such as pomegranate extract as a valuable adjunct during endodontic procedures; however, further research to enhance its clinical applicability and establish its capability as a root canal irrigant needs to be evaluated.


The authors would like to thank Dr. Manoj Kumar Mallela, Professor and HOD, Department of Pediatric and Preventive Dentistry, Panineeya Institute of Dental Sciences, Hyderabad and Dr. Srikanth Mallineni, Faculty, Narayana Dental College and Hospital, Nellore for their valuable suggestions on improvising the manuscript.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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Mallya L, Shenoy R, Mala K, Shenoy S. Evaluation of the antimicrobial efficacy of 20% Punica granatum, 0.2% chlorhexidine gluconate, and 2.5% sodium hypochlorite used alone or in combinations against Enterococcus faecalis: An in-vitro study. J Conserv Dent 2019;22:367-70.  Back to cited text no. 40


  [Table 1], [Table 2], [Table 3], [Table 4]


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  2005 - Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow 
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