Assessment of calcium ion loss and its effect on microhardness of root canal dentin using 1% phytic acid and 17% EDTA: An in vitro study

Abstract

Background:

Irrigation is an essential component of successful endodontics. Various chemical irrigants have been assessed for their efficacy. Newer alternatives are being currently investigated to overcome the shortcomings of the currently used irrigants.

Aims:

The aim of the study was to compare the effectiveness of phytic acid in calcium ion removal and its effect on the microhardness of the root canal dentin during endodontic procedure.

Subjects and Methods:

This was an in vitro study conducted on 45 recently extracted single-rooted human mandibular premolars. They were decoronated and sectioned. One half of the tooth was used to evaluate calcium ion loss, while the other half was used to analyze microhardness.

Statistical Analysis:

The data were analyzed using analysis of variance and Tukey’s post hoc analysis was used. P <0.001 was considered significant.

Results:

There was a less significant decrease in calcium level of root dentin after treatment with phytic acid as compared with ethylenediaminetetraacetic acid (EDTA). There was no significant difference between phytic acid and EDTA in relation to calcium ion loss and microhardness with their respective control group.

Conclusion:

1% Phytic acid is a suitable irrigating solution compared to 17% EDTA, due to its less demineralizing effect on radicular dentin.

INTRODUCTION

Endodontic therapy consists of access cavity preparation, biomechanical preparation, and three-dimensional root canal obturation. The anatomical complexities of the root canal harbor persistent biofilms. Therefore, achieving root canal disinfection and avoiding re-infection must be the main objectives of endodontic treatment. Use of chemicals and mechanical instrumentation is essential to help clean and remove necrotic tissue from the root canal.[1]

Chemical disinfection is performed using various agents such as root canal irrigants and antimicrobial medications, which flushes debris from root canal, while also preventing the extrusion of infectious material into the periapical region. If irrigation is not performed during instrumentation, the leftover debris within the root canal may lead to an endodontic failure.[2]

Many substances have been employed as root canal irrigants, such as acids (citric and phosphoric acid), proteolytic enzymes, alkaline solutions (NaOCl, NaOH₂, Urea, and KOH₂), chelating agents (Ethylenediaminetetraacetic acid [EDTA] and EGTA), oxidative agents (H₂O₂), biguanides, and normal saline. However, there is no solution that satisfies all the criteria for an ideal irrigant.[3] Sodium hypochlorite (NaOCl), owing to its bactericidal activity, is widely used in endodontics. However, its use is linked to undesirable effects such as risk of emphysema when forcibly extruded beyond the apex, potential for allergy, unpleasant odor and taste, biodegradability, and inability to eliminate smear layer.[1] Citric Acid and EDTA and among the other adjuvants recommended in endodontic treatment.[3]

To counter the ineffectiveness, potential side effects, and safety concerns of synthetic drugs, herbal alternatives are taking glam. The past decade has seen a paradigm shift toward the use of phytochemicals in endodontics due to the growing public interest in herbal medicines, which owing to their ability to combat the inefficiency, adverse effects, and safety issues have an edge over conventional root canal irrigants.[1] Phytic Acid (IP6) is a herbal product that strongly binds to important minerals such as iron, calcium, and zinc.[4] When compared to EDTA, IP6 allowed effective removal of the smear layer from NaOCl-treated dentin and did not affect the chemical composition.[5]

Endodontic irrigants can alter dentin’s chemical composition by removing calcium ions from hydroxyapatite, leading to changes in microhardness, permeability, and solubility, characteristics of dentin. This in turn affects their interaction with materials used for obturation and coronal restoration as well as inhibiting resistance to bacterial ingress and permitting coronal leakage. Measuring microhardness can indirectly indicate mineral changes in dental tissues.[6] To the best of our knowledge, there is a dearth of literature regarding the effect of phytic acid on the calcium ion loss and microhardness in radicular dentin. Thus, this study was designed to examine and assess the effectiveness of phytic acid on the removal of calcium ions and how it affects the root canal dentin’s microhardness during endodontic treatment.

SUBJECTS AND METHODS

This was an in vitro study conducted on 45 recently extracted single-rooted human mandibular premolars. These were extracted for orthodontic and periodontal reasons. The teeth satisfying the following inclusion criteria were selected: teeth with mature root apex and patent canals, without anatomical variations, caries, cracks, or restorations. Multirooted teeth were excluded from the study.

Sample preparation

Forty-five recently extracted single-rooted mandibular premolars were collected and stored in 0.9% saline until use. Decoronation was done using a high-speed diamond disc, following which longitudinal sectioning into two parts was performed using a low-speed micromotor with a carborundum disc under water cooling. Following evaluation of the 90 specimens under a stereomicroscope (Lawrence and Mayo), cracked teeth were replaced. A water-cooled carborundum wheel was used to polish them. The final polishing was done with a felt cloth and a buff containing 0.05 μm-sized aluminum oxide powder and distilled water. To maintain standardization, one half of the tooth was used to evaluate calcium ion loss, while the other half was used to analyze microhardness.

Forty-five specimens were divided into three subgroups of fifteen each, depending on the irrigating solution used.

Group A – For calcium ion loss

The specimens were isolated by applying nail varnish on the external surfaces and then immersed in a plastic jar with irrigation solutions as follows:

• Group A₁ – 5 ml, 1% Phytic Acid (TCI chemicals, Chennai) for 1 min

• Group A₂ – 5 ml, 17% EDTA solution (Prime Dental Products Pvt. Ltd.) for 1 min

• Group A₃ – 5 ml, distilled water for 1 min.

These were analyzed for calcium ion loss using an Atomic Absorption Spectrophotometer (AAS) [Figure 1].

Figure 1.

Atomic Absorption Spectrophotometer (AA-240FS, Varian Techtron, Pty Ltd)

Group B – For microhardness

The samples were inserted horizontally in autopolymerizing resin, exposing dentin. They were submerged in a plastic jar filled with test solutions.

• GroupB₁ – 5 ml, 1% Phytic Acid for 1 min

• GroupB₂ – 5 ml, 17% EDTA solution for 1 min

• GroupB₃ – 5 ml, distilled water for 1 min.

At the conclusion of the active treatment period (1 min), the samples were rinsed with distilled water, dried, and mounted on Vicker’s microhardness tester [Figure 2] to evaluate microhardness.

Figure 2.

Vicker’s Microhardness Tester (Metatech)

Statistical analysis

The data were entered into an Excel sheet and analyzed using SPSS version 21 (SPPSSInc Chicago, IL). Analysis of variance and Tukeys post hoc analysis were applied.

RESULTS

Phytic acid showed a less significant calcium ion loss of root dentin post-treatment as compared to EDTA, and EDTA showed the greatest percent decrease in the microhardness of root dentin compared to the phytic acid and distilled water [Table 1 and Graph 1]. Tukey’s post hoc multiple comparison test revealed that nonsignificant difference between phytic acid and EDTA in relation to calcium ion loss and microhardness with their respective control groups [Table 2 and Graph 2].

Table 1.

Comparison of the mean(SD) among the 3 groups using one-way ANOVA test

Test	Group (n=15)	Mean=SD	F	P*
Calcium ion loss (ppm)	A1 (1% phytic acid)	1.65±0.2	732.47	<0.001**
	A2 (17% EDTA)	2.86±0.3
	A3 (distilled water)	0.19±0.04
Vickers micro-hardness values	B1 (1% phytic acid)	61.61±0.6	751.149	<0.001**
	B2 (17% EDTA)	58.13±0.7
	B3 (distilled water)	69.21±0.9

*P<0.05 significant, **P<0.001 highly significant. SD: Standard deviation, EDTA: Ethylenediaminetetraacetic acid

Graph 1.

Comparison of the Calcium ion loss (ppm) among the 3 groups

Table 2.

Comparison of calcium ion loss and microhardness between all the groups using Tukey post hoc analysis

I group	Mean difference (I−J)	SE	Significant	95% CI
				Lower bound	Upper bound
Variable: Calcium loss
Distilled water
17% EDTA	−3.058600*	0.080489	0.000	−3.25415	−2.86305
1% phytic acid	−1.848067*	0.080489	0.000	−2.04361	−1.65252
17% EDTA
Distilled water	3.058600*	0.080489	0.000	2.86305	3.25415
1% phytic acid	1.210533*	0.080489	0.000	1.01499	1.40608
1% phytic acid
Distilled water	1.848067*	0.080489	0.000	1.65252	2.04361
17% EDTA	−1.210533*	0.080489	0.000	−1.40608	−1.01499
Variable: Microhardness
1% phytic acid
17% EDTA	3.48133*	0.29254	0.000	2.7706	4.1921
Distilled water	−7.60467*	0.29254	0.000	−8.3154	−6.8939
17% EDTA
1% phytic acid	−3.48133*	0.29254	0.000	−4.1921	−2.7706
Distilled water	−11.08600*	0.29254	0.000	−11.7967	−10.3753
Distilled water
1% phytic acid	7.60467*	0.29254	0.000	6.8939	8.3154
17% EDTA	11.08600*	0.29254	0.000	10.3753	11.7967

*The mean difference is significant at the 0.05 level. EDTA: Ethylenediaminetetraacetic acid, SE: Standard error, CI: Confidence interval

Graph 2.

Comparison of the Vickers micro-hardness values among the 3 groups

DISCUSSION

The major objectives of root canal therapy include the eradication of pulpal tissue components, bacteria, and biofilm, creation of a fluid-tight barrier to stop reinfection, and promotion of adjacent tissue healing, which is accomplished by efficient irrigation and the use of appropriate instrumentation. About 50% of the root canal walls remain unprepared when using the current nickel–titanium instrumentation system and conventional stainless-steel hand instruments.[7,8]

A smear layer is formed over dentin after biomechanical preparation of the canal occludes the dentinal tubules.[9] Through its ability to penetrate mechanically unreachable places, flush debris, and remove the smear layer from the root canal system, irrigation helps achieve effective root canal debridement.[10]

The trace element composition of digested teeth has been measured using many analytical techniques, including ICP-AES, mass spectrometry, AAS, neutron activation analysis, and anodic stripping voltammetry.[9] The technique employed in this work to measure the demineralization effect of the chelators and ascertain the calcium concentration in each sample is atomic absorption spectroscopy.[11] It calculates the concentration of an object element in a sample by using the principle that atoms in the ground state absorb light of a specific wavelength that travels through an atomic vapor layer of the element.[12]

In prior studies of Das et al., Taneja et al., and Dhawan et al. microhardness estimation using Vicker’s microhardness test as evaluated[6,11,13] have verified that it is appropriate and useful for assessing significant surface alterations of dental hard tissues following the use of irrigating solutions. As a result, the Vickers microhardness test was chosen for this investigation.

Previous studies of Thangaraj et al. and Patil and Uppin revealed that the loading force was selected to be in the range of 50–300 g while the dwell time was 10–20 s.[12,14] To reduce the impact of the structural differences between various teeth and create a fair baseline for assessment, we investigated the mid-root area, which is about halfway between the cementum and central lumen, and where the root’s dentin surface was consistent as in the study by Patil and Uppin.[14]

Studies of Ulusoy and Görgül and Aranda-Garcia et al. have demonstrated that that in addition to benefits like disinfection, debris flushing, smear layer removal, and dentinal wall lubrication, irrigating solutions like NaOCl, EDTA, and hydrogen peroxide can modify the Ca/P ratio and significantly lower the microhardness of radicular dentin.[15,16] According to Ambareen and Chinappa, researchers are searching for herbal alternatives in endodontics due to the ongoing rise in antibiotic-resistance and adverse effects brought on by synthetic medications.[17] Herbal and natural products are superior to conventional irrigants, owing to their high antimicrobial activity, biocompatibility, as well as their ease of availability, cost-effectiveness, extended shelf life, low toxicity, and lack of microbial resistance. This has grown their popularity across centuries.[18]

When EDTA and calcium ions in dentine interact, soluble calcium chelates are created. Mohammadi et al. report EDTA decalcifies dentin in 5 min to a depth of 20–30 μm,[19] facilitating lubrication, emulsification, smear layer removal, and the enlargement of small, blocked, or calcified canals. Concerns have been raised over EDTA’s cytotoxicity and the potential to seep into the periapical tissue due to its slow rate of biodegradation. It does not possess any antibacterial properties. Given these facts, a different smear layer removal agent is necessary, and efforts are ongoing to find a more biocompatible substance to replace EDTA.[20] Studies of Thangaraj et al., Patil and Uppin, and Sayin et al. have proved that EDTA showed significant changes in radicular dentin.[12,14,21] Hence, in this study, EDTA was chosen as a positive control group.

Distilled water, another popular irrigation option, is biocompatible and does not alter the dentin’s physical characteristics. Nevertheless, it cannot dissolve, disinfect, remove smear layers, and have antibacterial qualities. As a result, it can be used as a final rinse to get rid of any chemical irrigant that was left over after root canal preparation and as an adjunct to chemical irrigant. Prior research by Nassar et al. and Sayin et al. demonstrated that distilled water had no discernible effects on radicular dentin. As a result, it was selected as the study’s negative control group.[20,21]

We observed that 1% phytic acid less effectively chelated calcium ions and affected the microhardness of radicular dentin than 17% EDTA. However, no significant calcium ion loss or microhardness reduction was observed in the distilled water group (P > 0.05). EDTA showed significant calcium ion loss (2.86 ± 0.3, P < 0.05) and reduction in radicular dentin microhardness (58.13 ± 0.7, P < 0.05) compared to the control group. This could be attributed to its decalcification qualities and acidic pH. Following EDTA irrigation, the organic component of dentin is depleted, which could alter the Ca/P ratio and, consequently, the microhardness. These results are in agreement with those obtained in the literature of research by Nikhil et al., Cobankara et al., Dhawan et al., Patil and Uppin, and Sayin et al.[4,9,13,14,21]

Distilled water, a negative control group, did not significantly affect the Ca/P ratio (0.19 ± 0.04, P < 0.05) and microhardness (69.21 ± 0.9, P < 0.05) of radicular dentin. This could be attributed to the fact that the ratio of the organic to inorganic portion of radicular dentin was not considerably changed by distilled water with a pH of around 7, which in turn did not change the dentin’s microhardness. Similar results were obtained in previous studies by Patil and Uppin, Nassar et al., and Sayin et al.[14,20,21] Taneja et al.[11] observed that the mean calcium ion loss was higher in the EDTA group compared to the distilled water group.

A major limitation of the current research would be that the coronal reservoir of the irrigant is eliminated by decoronation of the samples. Further research on in vivo models is required to validate the findings.

CONCLUSION

The present study suggests that phytic acid leads to structural changes in radicular dentin due to demineralizing effect on root canal dentin. This softening effect on the dentinal walls could be beneficial and facilitates the quick preparation and negotiation of complex root canals, proving advantageous in clinical practice.

Further in vitro and in vivo studies are needed to evaluate the efficacy of phytic acid to be used as an endodontic irrigant clinically.

Within the limitations, it can be safely concluded that 1% phytic acid appears to be a suitable irrigating solution, because of it is less demineralizing effect as compared to 17% EDTA on radicular dentin.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.