Journal of Conservative Dentistry

: 2023  |  Volume : 26  |  Issue : 3  |  Page : 344--348

Flexural strength and microhardness of human radicular dentin sticks after conditioning with different endodontic chelating agents

Ahmed El-Banna1, Maii Y Elmesellawy2, Mohamed Ahmed Elsayed3,  
1 Department of Biomaterials, Faculty of Dentistry, Ain Shams University, Cairo, Egypt
2 Department of Endodontic, Faculty of Dentistry, Beni-Suef University, Beni Suef, Egypt
3 Department of Endodontics, RAK College of Dental Sciences, RAK Medical and Health Sciences University, Ras Al-Khaimah, United Arab Emirates; Department of Endodontics, Faculty of Dentistry, Assiut University, Assiut, Egypt

Correspondence Address:
Dr. Ahmed El-Banna
Organization of African Unity St, El-Qobba Bridge, El Weili, Cairo


Introduction: The objective of this in vitro study was to examine the impact of different endodontic chelating agents on the flexural strength and microhardness of root dentin. Materials and Methods: Fourty dentin sticks of (1 mm × 1 mm × 12 mm) were obtained from 10 single-rooted premolars and divided into four groups (n = 10). One stick from each tooth was assigned to one of the experimental groups and was soaked in one of the experimental chelating solutions for 5 min 17% ethylenediaminetetraacetic acid (EDTA), 2.5% phytic acid (PA), 18% etidronic acid, or saline (control group). Following the 5-min soak, the sticks' flexural strength was evaluated using a 3-point loading test using the universal testing machine, and the surface microhardness was tested using a Vickers's microhardness tester. Results: PA (2.5%) and etidronic acid (18%) showed no significant detrimental effect on either the flexural strength or the surface microhardness of radicular dentin compared to the control. EDTA (17%) exhibited a significant drop in the flexural strength and microhardness of radicular dentin compared to the other groups. Conclusions: PA and etidronic acid chelators do not compromise the surface and bulk mechanical properties of radicular dentin.

How to cite this article:
El-Banna A, Elmesellawy MY, Elsayed MA. Flexural strength and microhardness of human radicular dentin sticks after conditioning with different endodontic chelating agents.J Conserv Dent 2023;26:344-348

How to cite this URL:
El-Banna A, Elmesellawy MY, Elsayed MA. Flexural strength and microhardness of human radicular dentin sticks after conditioning with different endodontic chelating agents. J Conserv Dent [serial online] 2023 [cited 2023 Sep 27 ];26:344-348
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Chelating agents decalcify radicular dentin and eliminate the smear layer formed after the mechanical preparation of the root canal. The smear layer consists of dentin debris, pulp tissue residues, and bacteria. A layer as such acts as a barrier that precludes irrigants from directly reaching and disinfecting the dentin surface, the dentinal tubules, and altering the sealing quality of obturation.[1]

Moreover, chelating agents, such as ethylenediaminetetraacetic acid (EDTA pH 8), is a used as a gold standard strong chelator with proven smear layer removal capacity. In addition, it is used in regenerative endodontic procedures of necrotic immature teeth. EDTA facilitates the release of growth factors from the dentin surface which in turn stimulates mesenchymal cell differentiation into odontoblast-like cells. In addition, chelating agents expose collagen fibers of the root dentin, promoting the attachment of the newly formed odontoblast-like cells to the dentin surface.[1],[2]

However, research has shown that the extended use of strong chelators as EDTA can have a detrimental effect on the biomechanical properties of root dentin, as evidenced by a drop in both microhardness and flexural strength.[2]

Recently, chelators, such as phytic acid (PA) (inositol hexakisphosphate pH 1.3) and etidronic acid (1-hydroxyethane-1,1-diphosphonic acid [HEDP] pH 11), have been considered potential alternatives to EDTA. PA, a plant-based extract has a strong affinity to cations such as calcium (Ca+2), iron, and zinc, granting effective leaching of the inorganic element which makes it highly effective in smear layer removal comparable to 17% EDTA with greater biocompatibility compared to EDTA.[3]

Etidronic acid is a soft biocompatible chelator that can be used alone or with sodium hypochlorite (NaOCl) displaying antibacterial capacity.[4] Currently, employed for clinical use as a continuous chelation-based irrigant known as Dual Rinse® HEDP (Medcem, Weinfelden, Switzerland) with highly efficient chelation capabilities and smear layer removal comparable to EDTA. The capsule contains 0.90 g of etidronic acid powder ready to mix with 10 mL NaOCl 1%–5% attaining HEDP 9%, or 5 mL distilled water to obtain HEDP18%.[5]

In a recent revascularization study, both PA and etidronic acid (HEDP) showed the potential for triggering growth factor release from root dentin and promoting cell migration.[6] To date, there is limited literature on the impact of PA and HEDP on the mechanical and physical properties of root dentin. Therefore, this study aimed to compare the effect of 2.5% PA and 18% HEDP compared to 17% EDTA on radicular dentin flexural strength and microhardness.

 Materials and Methods

Dentin sticks preparation

Ten sound single-rooted premolars extracted for orthodontic reasons were collected from the oral and maxillofacial surgery clinic following informed consent and ethical approval (RAKMHSU-REC-186-2022/23-F-D). Four radicular dentin sticks were obtained from each root as shown in [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d using a precision diamond saw (Isomet4000, Buhler, Germany) under copious water coolant. One stick from each tooth was assigned to one of the four experimental irrigants.{Figure 1}

Irrigant preparation

Four irrigant solutions were used for this study: Group 1 (control): saline (FIPCO, Egypt. Lot no. B12049#C), Group 2: 17% EDTA (Prevest DenPro Limited, India. lot no. PK2122161), Group 3: 2.5% PA (Sigma, Aldrich, Louis, MO, USA), and Group 4: 18% etidronate Dual Rinse® HEDP (Medcem GmbH, Switzerland. Lot no. DR190124).

Saline and EDTA solutions were used as received without any modifications. For Group 3, 0.5 mL of the PA was mixed with 9.5 mL of saline to obtain 10 mL of 2.5% PA. For Group 4, two capsules of dual rinse were mixed with 10 mL of saline to obtain 18% etidronate according to the manufacturer's instructions. Each stick was immersed separately in 1 mL of the irrigant solution in a sealed plastic tube and placed in an ultrasonic vibrator for 5 min. Then the stick was rinsed with saline then tested immediately [Figure 1]e.

Flexural strength testing

The flexural strength of the dentin sticks was evaluated using the universal testing machine (Instron 3365, Norwood, MA, USA). The dimensions of each stick were measured before testing to ensure accurate calculations. The sticks were placed on a metal support with a 1 mm diameter round support and 10 mm support distance and loaded with a round metal rod at a speed of 1 mm/min until failure [Figure 1]f. The Bluehill 3 software (Instron, Norwood, MA, USA) was used to calculate the flexural strength.

Microhardness testing

The fractured dentin sticks were collected and the Vickers hardness (HV) number was measured using a microhardness tester (Wilson® Tukon™ 1102, Buehler, Germany) [Figure 1]h. For each stick, three indentations were made with a minimum separation of 1 mm distance between the adjacent indentations [Figure 1]g. The load was applied smoothly and without impact, by pressing the square diamond pyramid shape indenter into the test specimen with a load of 50 g (HV 0.1) for 10 s. After removing the load, the impression diagonals were measured three times using three different magnification settings and averaged, usually to the nearest 0.1-μm using a micrometer. The HV was calculated using the formula HV = 1854.4 L/d2, where the load (L) is measured in gf and the average diagonal (d) is in μm, resulting in hardness number units of gf/μm2.

Statistical analysis

The numerical data were expressed in terms of mean and standard deviation values. Data normality and variance homogeneity were assessed using the Shapiro–Wilk and Levene's tests, respectively. Homogeneity assumption was met in the surface hardness data so they were analyzed using one-way ANOVA followed by Tukey's post hoc test. However, the assumption was violated in the flexural strength data so they were analyzed using Welch one-way ANOVA followed by Games-Howell post hoc test. The significance level was set at P < 0.01 within all tests. Statistical analysis was performed with R statistical analysis software version 4.1.3 for Windows.


Results for intergroup comparisons for flexural strength and surface hardness are presented in[Table 1]. EDTA revealed a statistically significant reduction in both the microhardness and flexural strength of radicular dentin compared to all the other groups (P < 0.001). Both PA and dual rinse did not significantly reduce neither the flexural strength nor the microhardness of radicular dentin when compared to the control group. There is a moderate positive correlation between surface hardness and flexural strength (rs = 0.584) as presented in [Figure 2].{Figure 2}{Table 1}


The chemical and structural composition of human dentin can be altered by root canal treatment, along with the various irrigants involved. EDTA is a widely used dentinal chelator, despite the fact that it is a significant environmental nonbiodegradable pollutant.[7] Irrespective of its popularity, a quest for similarly effective chelating agents and organic acids is in progress; hence, this study was conducted to compare the sole effect of promising gentle dentin chelators such as PA (pH:1.3)[3] and etidronate (pH11)[8] on the flexural strength and surface microhardness of radicular dentin.

The effectiveness of chelators in eradicating the smear layer and opening dentinal tubules is determined by their concentration and contact time. For clinical practice, 17% EDTA is typically used, however, etidronate is recommended to be used at 18% when mixed with saline.[9] PA beneficial in smear layer eradication at a much lower concentration of 2.5% compared to EDTA, which interprets its biocompatibility.[3],[7]

Various treatment times of 1, 5, and 15 min were proposed in the literature for optimal results;[10] still, it is agreeable that prolonged decalcifying agents' application has proven to be deleterious on root dentin.[11] Accordingly, in this study, all solutions were used for a contact time of 5 min, as this allows a completed action of smear layer elimination, simulating the clinical application time of these solutions and standardizing the contact time between all agents used.[12],[13]

When assessing a new agent, it is crucial to examine its impact on the mechanical properties of dentin after application. The flexural strength and microhardness characteristics are of great clinical significance, flexural strength is an effective approach for assessing the mechanical properties of bulk dentin, whereas microhardness is a commonly used method for evaluating surface dentin mechanical properties.[14],[15],[16] Both tests were used to indirectly evaluate mineral content and structural changes simultaneously following various treatments on radicular dentin,[17] and their subsequent impact on the hard tooth structure, the likelihood of root fracture, as well as adhesion and sealing ability to the root canal wall.[18]

To control the variability of results, every four radicular dentin sticks were acquired from the same root for evaluating flexural strength and surface hardness. Each of these sticks was assigned to one of the different experimental chelators tested and then compared to the control (saline). The same stick that was used for testing the flexural strength, was also used for testing of the surface microhardness to ensure better correlation and standardization of the specimens and hence of the results; without any structural changes during sectioning after being subjected to the irrigating solutions. The only limitation of this method is that it does not simulate the clinical condition of the application of chelating agent.

It is well-known that the inorganic element of the hard tooth structure contributes to the strength and elastic modulus, whereas the organic element is accountable for its toughness.[19] In the current study, it was noticed that none of the tested irrigants as PA and etidronate significantly decreased the mechanical properties of dentin; yet, EDTA caused a significant drop in its flexural strength compared to the untreated radicular dentin sticks. This could be attributed to the decalcifying influence of EDTA on the mineral content of dentin mainly in the calcium-to-phosphorus ratio, owing to the consequent extraction of calcium ion from mineralized dental tissues resulting in a weaker substrate, thus predisposing endodontically treated teeth to root fracture. This study's results are in agreement with Nassar et al.[7] and Baruwa et al.,[13] noted that EDTA can cause depletion of calcium cations from the surface of radicular dentin to a depth of almost 150 um. However, our results are conflicting with Bosaid et al.,[20] who claimed that EDTA did not affect the dome of the dentin bulk properties as the flexural strength analysis. This can be attributed to the difference in the methodology of specimen preparation, testing, and treatment protocol.

The dentin surface microhardness was reduced significantly upon the EDTA application, which could be credited to the substantial loss in the mineral content and hydroxyapatite in the intertubular dentin which negatively impacts the hardness of the human dentin structure. This came in accordance with Poggio et al.,[21] De-Deus et al.[22] Nikhil et al.,[23] and Bosaid et al.[20] Though PA and etidronate have both demonstrated minimal effect on the surface microhardness of root dentin, which came in agreement with Dineshkumar et al.,[24] and De-Deus et al.[25] confirming that both are weak calcium-depleting agents that do not cause detrimental change on dentin compared to EDTA.


Considering the limitations of this study, it can be concluded that both 2.5% PA and 18% etidronic acid chelators do not compromise the surface and bulk mechanical properties of radicular dentin.


We would like to thank Dr. Bassam Ahmed for his meticulous support in the statistical analysis for this article.

We would like to thank Amira Abo Zaid designs for her artistic graphical designs used in this research.

Financial support and sponsorship

This project is partially supported for laboratory testing in the biomaterials research lab, Faculty of Dentistry, Ain Shams University.

Conflicts of interest

There are no conflicts of interest.


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