| Abstract|| |
Aim: The purpose of this study was to evaluate the effect of phytic acid, ethylenediaminetetraacetic acid (EDTA), and chitosan solutions on the microhardness of human radicular dentin.
Materials and Methods: Thirty dentin specimens were randomly divided into three groups of 10 specimens each according to the irrigant used: G1 - 1% phytic acid, G2 - 17% EDTA, and G3 - 0.2% chitosan. A standardized volume of each chelating solution was used for 3 min. Dentin microhardness was measured before and after application at the cervical, middle, and apical levels with a Vickers indenter under a 200-g load and a 10-s dwell time. The results were analyzed using one-way analysis of variance (ANOVA) and Student's t test.
Results: Microhardness of the radicular dentin varied at the cervical, middle, and apical levels. EDTA had the greatest overall effect, causing a sharp percentage reduction in dentin microhardness with a significant difference from phytic acid and chitosan (P = 0.002). However, phytic acid and chitosan differed insignificantly from each other (P = 0.887).
Conclusion: All tested chelating solutions reduced microhardness of the radicular dentin layer at all the levels. However, reduction was least at the apical level. EDTA caused more reduction in dentin microhardness than chitosan while phytic acid reduced the least.
Keywords: Chelator; chitosan; ethylenediaminetetraacetic acid (EDTA); microhardness; phytic acid
|How to cite this article:|
Nikhil V, Jaiswal S, Bansal P, Arora R, Raj S, Malhotra P. Effect of phytic acid, ethylenediaminetetraacetic acid, and chitosan solutions on microhardness of the human radicular dentin. J Conserv Dent 2016;19:179-83
|How to cite this URL:|
Nikhil V, Jaiswal S, Bansal P, Arora R, Raj S, Malhotra P. Effect of phytic acid, ethylenediaminetetraacetic acid, and chitosan solutions on microhardness of the human radicular dentin. J Conserv Dent [serial online] 2016 [cited 2019 Aug 20];19:179-83. Available from: http://www.jcd.org.in/text.asp?2016/19/2/179/178705
| Introduction|| |
The smear layer produced during preparation of the root canal should be removed to achieve a three-dimensional seal.  Chelates are stable complexes of metal ions with organic substances as a result of ring-shaped bonds. Nygaard-Ψstby proposed the use of ethylenediaminetetraacetic acid (EDTA) for chemically softening the root canal dentin, and allowing the instrumentation of calcified, narrow canals. 
Chelation can cause changes in the calcium to phosphorus ratio of the dentin and in turn, may reduce the microhardness and increase the permeability and solubility of the root canal dentin. This also inhibits resistance to bacterial ingress, thus permitting coronal leakage and adversely affecting the sealing ability and adhesion of dental materials such as resin-based cements and root canal sealers. ,,, Indirect evidence of mineral loss or gain in dental hard tissues can be provided by the microhardness determination. 
EDTA is not originally found in nature and is therefore, considered to be a pollutant. , Overuse of this compound has considerably increased its concentration in rivers and lakes. Considering these facts, an alternative agent is warranted and the search for more biodegradable material to replace EDTA still continues.
Chitosan, a natural polysaccharide is biocompatible, biodegradable, and bioadhesive. It lacks toxicity and has chelating ability. , It has become ecologically interesting for various applications due to its abundance in nature and low production costs.  In dentistry, its addition to calcium hydroxide paste as an intracanal medication has been shown to promote prolonged calcium ion release.  0.2% chitosan in combination with ultrasonics performed better than 17% EDTA in the removal of oil-based calcium hydroxide.  Silva PV et al. found 0.2% chitosan to be equally effective to 15% EDTA and 10% citric acid in smear layer removal and microhardness reduction. ,
Phytic acid (known as inositol hexakisphosphate, IP6), a saturated cyclic acid, is the principal storage form of phosphorus in many plant tissues, especially bran and seeds.  It can be found in cereals and grains. Phytic acid has a strong binding affinity to important minerals, such as calcium, iron, and zinc, although the binding of calcium with phytic acid is pH-dependent.  Nassar et al. reported the ability of phytic acid to remove the smear layer from instrumented root canals and flat coronal dentin surfaces and found that phytic acid was less cytotoxic and biocompatible compared to EDTA. 
To date, there is no study evaluating the effect of phytic acid on the microhardness of radicular dentin. The purpose of this in vitro study was to evaluate the effect of 1% phytic acid, 17% EDTA, and 0.2% chitosan solutions on the microhardness of human radicular dentin.
| Materials and methods|| |
Preparation of samples
Fifteen recently extracted human canine teeth free from caries, cracks, and dilacerations were selected and decoronated at the cementoenamel junction. The roots were split longitudinally into two parts, making a total of 30 segments and the root halves were horizontally embedded in autopolymerizing acrylic resin so that their dentin was exposed. The dentin surfaces of the mounted specimens were grounded flat and smooth on a digital polishing machine (Sumitra enterprises, Delhi, India) with a series of ascending grades of silicon carbide abrasive papers (600-grit, 1,200-grit, and 2,000-grit) under distilled water to remove any surface scratch and finally polished with 0.1-mm alumina suspension on a rotary felt disk.
Determination of microhardness
Three separate indentations were made at the cervical, middle, and apical levels of the root dentin in each sample with a Vickers diamond indenter (Mitutoyo Corporation, Yokohama, Kanagawa, Japan) microhardness tester at X 10 parallel to the edge of the root canal lumen at a depth of 0.5 mm from the pulp-dentin interface, each using a 200-g load and a 10-s dwell time and the pretreatment microhardness value was calculated [Figure 1].
|Figure 1: (a) The schematic representation of the measurement point from the different root level (b) Photomicrograph of the diamond shaped indentation 0.5 mm from the pulp dentin interface|
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For the preparation of 0.2% chitosan solution (pH 3.2) and 1% phytic acid (pH 3.2), 0.2 g of chitosan (Acros Organics, Belgium, europe) with 90% degree of deacetylation was diluted with 100 mL of 1% acetic acid and 1 g of phytic acid (Wako Pure Chemical Industries, Osaka, Japan) in 100 mL of distilled water and the mixture was stirred for 2 h using a magnetic stirrer (Remi, Mumbai, India).
Specimens were randomly divided into three groups (n = 10) and were prepared as follows: G1 - the phytic acid group, in which the specimens were treated with 50 μL of 1% phytic acid (pH 3.2) for 3 min. This was applied on the flattened smooth dentinal surface; G2 - the EDTA group, in which the specimens were similarly treated with 50 μL of 17% EDTA (pH 7.3) for 3 min; and G3 - the chitosan group, in which the specimens were treated with 50 μL of 0.2% chitosan (pH 3.2) for 3 min. All solutions were applied with agitation by using a microbrush. Specimens were rinsed in distilled water, and blotted dry. Vickers hardness number was again recorded as described before for each specimen and the decreases in microhardness were calculated as the percentage (%) in microhardness values.
The data were analyzed statistically using one-way analysis of variance (ANOVA) (P = .05) and Student's t-test.
| Results|| |
The pretreatment Vickers microhardness values (mean ± standard deviation) for all tested specimens at the cervical, middle, and apical levels were 50.02 ± 7.34, 49.11 ± 6.84, and 48.14 ± 6.34, respectably. All the chelators on application reduced the dentin microhardness at all the levels [Table 1]. Although the percentage reduction in microhardness was less at the apical level than the cervical and middle level, the difference was statistically insignificant (P > 0.05) [Table 1].
|Table 1: Mean Vickers microhardness values of radicular dentin specimens at different levels with respect to the type of treatment|
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EDTA caused the greatest percentage reduction in dentin microhardness (8.27 ± 3.05) and the pairwise comparison indicated that this reduction was significantly more (P < 0.05) than phytic acid (6.49 ± 1.90) and chitosan (6.54 ± 1.96), which were similar to each other (P > 0.05) and were least effective in terms of their effect on dentin microhardness [Table 2].
|Table 2: Percentage decrease in Vickers microhardness values (mean ± standard deviation) of the radicular dentin after use of the tested chelating solutions|
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| Discussion|| |
Vickers and Knoop microhardness tests are suitable and practical methods for evaluating surface changes of dental hard tissues treated with chemical agents and have been used previously for the same.  The Vickers microhardness test was preferred in this study because of suitability of the method.
The microhardness of the radicular dentin varied at different levels  and declined when the dentin was tested from the superficial to deep regions. Thus, in the present study, to measure the Vickers hardness values for the dentin, indentations were made in the cervical, middle, and apical thirds of the radicular dentin and were done at the 0.5-mm level from the root canal walls for standardization. For evaluation of a reduction in hardness, standardization was done by estimating the pretreatment hardness of every sample and then comparing with the posttreatment hardness.
In the present study, the hardness of radicular dentin varied at the cervical, middle, and apical levels (50.02 ± 7.34, 49.11 ± 6.84, and 48.14 ± 6.34, respectably), which were well within the range documented in the literature and the difference between the different levels was statistically insignificant (P > 0.05). This confirmed the finding of Patterson  and Panighi et al.
Application of all the chelating agents decreased the dentin microhardness. The results obtained were similar to previous studies ,, where application of chelating agents resulted in reduction of microhardness. The significant alteration in dentin hardness after the irrigation treatment indicated potent direct effects of these chemical solutions on the components of dentin structure.
All the chelators reduced the hardness at all the three levels; however, the percentage microhardness reduction was found least at the apical level (P > 0.05). This could be attributed to the composition of the apical region. Chelating solution reduces the mineral and noncollagenous protein (NCP) component of the dentin, leading to surface softening.  Therefore, along with calcium ions it removes calcium, which is bonded to the extracted fraction of NCP. Since the content of noncollagenous organic matrix decreases in the apical part of the root dentin, this may explain the lower degree of decalcification in this part of the root. 
In the present study, percentage dentin microhardness reduction was significantly more in the EDTA group (P < 0.05) than chitosan and phytic acid, which was contradictory to the results of the study of Pimenta et al. that concluded that chitosan, EDTA, and citric acid reduced root canal microhardness with no statistically significant difference among the solutions.
The effect of EDTA on reducing dentin microhardness has been reported by Poggio et al., Ari et al., and De-Deus et al.,, EDTA efficiently reduces dentinal microhardness due to its chelating property. The degree of mineral content and the amount of hydroxyapatite in the intertubular substance are considerable factors in determining the intrinsic hardness profile of dentin structure. 
Phytic acid is highly negatively charged molecule that has affinity to Ca ++ . Nassar et al. found phytic acid to be more effective in removing the smear layer from NaOCl-treated flat coronal dentin surfaces and instrumented root canals than EDTA. The pH of 1% phytic acid solution was around 1.2 in that study and this acidity, along with chelation ability, attributed to effective smear layer removal and Ca ++ extraction.
Chitosan also resulted in significantly lesser microhardness reduction of the radicular dentin than EDTA. Pimenta et al. showed that application of the 0.2% of chitosan solution for 3-5 min was the most viable combination for use on the root dentin. They found 0.2% chitosan to be equally effective in removing smear layer as 15% EDTA. Darrag  on comparison found that final irrigation with 0.2% chitosan solution was more efficient in smear layer removal than 17% EDTA. The mechanism of action of chitosan is not fully known. It is believed that chitosan polymer being hydrophilic favors intimate contact with root canal dentin and gets adsorbed to root canal walls. Its cationic nature helps in ionic interaction between the dentin calcium ions and the chelating agent.  As chitosan is insoluble in water and 1% acetic acid is used to form a solution, the acid might enhance the chelating efficacy of chitosan.
Some chelating solutions also cause erosion of the root dentin  and may decrease the resistance to penetration of bacteria and coronal leakage. Erosion can be avoided by using chelators at low concentrations  or shorter chelating times; therefore, chelators that are effective at low concentrations and in a lesser time should be preferred, i.e., 1% phytic acid and 0.2% chitosan are better.
The similar chelating effect of 0.2% chitosan and 1% phytic acid compared to EDTA, associated with its lower percentage reduction in microhardness of the dentin as found in this study and lower concentration required for removing smear layer without alteration of the intertubular dentin indicate that this chelating solution should be preferred for dentin decalcification. As described in the Materials and Methods section, chitosan was diluted in 1% acetic acid for preparation of the solution. Thus, it could be speculated that the chelating effect observed in this study was due to the acid and not chitosan. However, previous studies have shown that the capacity of 5% acetic acid for reducing dentin microhardness, and removing the smear layer and chelating calcium ions in the root canal was insignificant in relation to 15% EDTA and 10% citric acid.  In this way, it is highly evident that the effect caused by chitosan on dentin microhardness is exclusively due to the substance and not to the acid. Prior to the clinical use of a new substance or product, further studies are needed to investigate in detail its physical, chemical, and biological properties in order to verify the benefits and consequences to humans.
It is essential to conduct further studies to check for the effect of 1% phytic acid and 0.2% chitosan on the microhardness and surface roughness of the root dentin, adhesion of bacteria and root canal fillings to root dentin, and also the fracture resistance of roots after treatment with 1% phytic acid and 0.2% chitosan solutions at different time intervals.
| Conclusion|| |
Despite being structurally different, when subjected to the same chelators the root thirds behaved similarly. All tested chelators reduced microhardness of the human radicular dentin. 17% EDTA reduced the dentin microhardness more significantly than 1% phytic acid and 0.2% chitosan. However, microhardness reduction was same for 1% phytic acid and 0.2% chitosan.
The authors deny any financial affiliation. The authors thank Mr. Arvind Kumar Shukla for his statistical support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jhamb S, Nikhil V, Singh V. An in vitro
study to determine the sealing ability of sealers with and without smear layer removal. J Conserv Dent 2009;12:150-3.
Østby NB. Chelation in root canal therapy. Ethylenediamine tetra-acetic acid for cleansing and widening of root canals. Odont Tidskrift 1957;65: 3-11.
Poggio C, Dagna A, Colombo M, Rizzardi F, Chiesa M, Scribante A, et al
. Decalcifying effect of different ethylenediaminetetraacetic irrigating solutions and tetraclean on root canal dentin. J Endod 2012;38:1239-43.
Ari H, Erdemir A, Belli S. Evaluation of the effect of endodontic irrigation solutions on the microhardness and the roughness of root canal dentin. J Endod 2004;30:792-5.
De-Deus G, Paciornik S, Mauricio MH. Evaluation of the effect of EDTA, EDTAC and citric acid on the microhardness of root dentin. Int Endod J 2006;39:401-7.
Qing Y, Akita Y, Kawano S, Kawazu S, Yoshida T, Sekine I. Cleaning efficacy and dentin micro-hardness after root canal irrigation with a strong acid electrolytic water. J Endod 2006;32:1102-6.
Panighi M, G′Sell C. Influence of calcium concentration on the dentin wettability by an adhesive. J Biomed Mater Res 1992;26:1081-9.
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.
Silva PV, Guedes DF, Nakad 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.
Akncbay H, Senel S, Ay ZY. Application of chitosan gel in the treatment of chronic periodontitis. J Biomed Mater Res B Appl Biomater 2007;80:290-6.
Kurita K. Chemistry and application of chitin and chitosan. Polym Degrad Stab 1998;59:117-20.
Peter MG. Applications and environmental aspects of chitin and chitosan. J Macromol Sci 1995;32:629-40.
Vineeta N, Gupta S, Chandra A. Retrievability of calcium hydroxide intracanal medicament with Chitosan from root canals: An in vitro
CBCT volumetric analysis. J Conserv Dent 2014;17:454-7.
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.
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.
Raboy V. myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry 2003;64:1033-43.
Dendougui F, Schwedt G. In vitro
analysis of binding capacities of calcium to phytic acid in different food samples. Eur Food Res Technol 2004;219:409-15.
Nassar M, Hiraishi N, Tamura Y, Otsuki M, Aoki K, Tagami J. Phytic acid: An alternative root canal chelating agent. J Endod 2015;41:242-7.
Meredith N, Sherriff M, Setchell DJ, Swanson SA. Measurement of the microhardness and Young′s modulus of human enamel and dentin using an indentation technique. Arch Oral Biol 1996;41:539-45.
Patterson SS. In vivo
and in vitro
studies of the effect of the disodium salt of ethylenediamine tetra-acetate on human dentine and its endodontic implications. Oral Surg Oral Med Oral Pathol 1963;16:83-103.
Kawasaki K, Ruben J, Stokroos I, Takagi O, Arends J. The remineralization of EDTA-treated human dentine. Caries Res 1999;33:275-80.
Verdelis K, Eliades G, Oviir T, Margelos J. Effect of chelating agents on the molecular composition and extent of decalcification at cervical, middle and apical root dentin locations. Endod Dent Traumatol 1999;15:164-70.
Darrag AM. Effectiveness of different final irrigation solutions on smear layer removal in intraradicular dentin. Tanta Den J 2014;11:93-9.
Zhang J, Xia W, Liu P, Cheng Q, Tahirou T, Gu W, et al
. Chitosan modification and pharmaceutical/biomedical applications. Mar Drugs 2010;8:1962-87.
Sen BH, Ertürk O, Piºkin B. The effect of different concentrations of EDTA on instrumented root canal walls. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;1084:622-7.
Subharti Dental College, NH-58, Delhi-Haridwar Bypass Road, Meerut - 250 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]