Effect of Chelating Agents on Compressive and Push-Out Bond Strength of Calcium Silicate-Based Materials in Furcal Perforations

ABSTRACT

Objective:  To evaluate and compare the effects of 17% Ethylenediaminetetraacetic acid (EDTA) and 7% Maleic Acid (MA) on the push-out bond strength (POBS) and compressive strength (CS) of Biodentine and Bio-C Repair when used for furcal perforation repair.

Material and Methods:  Sixty extracted human mandibular molars were decoronated, and standardized furcal perforations were created. The teeth were mounted and randomly divided into two groups: Group I, Biodentine, and Group II, Bio-C Repair. Each group was irrigated with 2.5% Sodium Hypochlorite and then subdivided into three: Group A – 17% EDTA, Group B – 7% MA, and Group C – Distilled Water. For CS evaluation, eighteen cylindrical samples of each material were prepared, immersed in NaOCl, and similarly subdivided into three groups based on the irrigant used. All samples were tested using a universal testing machine.

Results:  Biodentine showed no significant difference in POBS among the irrigants (p=0.065). Bio-C Repair showed significantly higher POBS with MA and distilled water than with EDTA (p<0.001). Biodentine’s CS was significantly affected by the chelating agents (Control > EDTA > MA), whereas Bio-C Repair showed no significant difference; however, its CS remained lower than that of Biodentine.

Conclusion:  Chelating agents did not affect Biodentine's POBS, whereas MA improved Bio-C Repair's POBS over EDTA. Despite the reduction, Biodentine's CS remained higher than Bio-C Repair.

Keywords:
Chelating Agents; Compressive Strength; Root Canal Irrigants; Root Canal Filling Materials

Introduction

Root perforation is the second most common cause of endodontic failure [¹]. It can result from pathologic factors such as caries, resorptive process, or be caused iatrogenically, leading to complications during or after root canal treatment [²]. Failure to detect or treat them may result in the breakdown of the periodontium, potentially leading to tooth loss [²]. The prognosis of the tooth is associated with several key factors, including the perforation's location, size, timing of repair, and, most importantly, the choice of material used for the repair [³].

A wide variety of materials, including Glass Ionomer Cement, Composite, Mineral Trioxide Aggregate (MTA), Biodentine (BD), and EndoSequence Bioceramic Root Repair Material, have been used for perforation repair [⁴]. However, calcium silicates have become the preferred choice of material for sealing furcation perforations due to their biocompatibility and ability to induce the precipitation of calcium phosphate at the interface with periodontal tissues [⁵].

MTA has been regarded as an excellent material for perforation repair [⁶]. However, specific concerns have been raised regarding it, including the extended setting time and the prolonged maturation phase [⁷]. BD (Septodont, Saint-Maur-des-Fosses, France) is considered an alternative for MTA and is known for its excellent marginal adaptation, shorter setting time, superior bioactivity, and compressive strength (CS) [⁸].

Bio-C Repair (Angelus, Londrina, PR, Brazil) (BCR) is a relatively recent bioactive calcium silicate-based hydraulic cement available in a ready-for-use format [⁹]. The manufacturer claims that the material possesses multiple beneficial properties, including excellent consistency, antimicrobial activity, biocompatibility for tissue healing, and resistance to discoloration [⁹]. In addition, BCR demonstrates comparable cytocompatibility and biomineralization as MTA-HP and white MTA-Angelus [¹⁰].

The interaction between the root canal irrigants and repair materials used in furcation repair can indeed have an impact on the properties of the repair material [¹¹]. Several studies have investigated the effect of different irrigants on the properties of calcium silicate-based root repair materials [¹²-¹⁴]. The setting process of hydraulic tricalcium silicate cements involves hydrolysis and ion exchange, leading to the formation of calcium silicate hydrate and calcium hydroxide [¹⁵]. Previous studies have revealed that decalcifying agents can affect the setting reaction and damage the structure of tricalcium silicate-based cements by removing calcium from the cement structure [¹²,¹⁴]. The influence of irrigants on the characteristics of root repair materials depends on the compositional variations among the materials tested. With the ever-evolving development of root repair materials, it is essential to investigate the impact of root canal irrigants on these materials.

Currently, no studies have investigated the effects of chelating agents on the push-out bond strength (POBS) and CS of BCR as a material for furcal perforation repair. This study, therefore, aims to compare the POBS and CS of two commercially available Calcium Silicate-based Cements (CSCs) when exposed to chelators such as 17% Ethylenediaminetetraacetic acid (EDTA) and 7% Maleic Acid (MA). The null hypothesis tested is that these chelating agents will not affect the POBS and CS of BD and BCR as materials for root repair.

Material and Methods

Sample Size Calculation

For POBS: An effect size of 0.9 derived from a study by Alamoudi and Abu Zeid [¹⁶] was used for calculation. With a statistical power of 95% and an alpha of 5%, the calculated sample size was eight samples per group. The sample size was increased and rounded to 10 samples per group to account for the potential damage during handling and testing.

For CS: Based on the findings of Govindaraju et al. [¹⁴], an effect size of 1.2 was used. With a statistical power of 95% and an alpha level of 5%, the estimated sample size was five specimens per group. The sample size was rounded and adjusted to 6 samples per group to accommodate possible losses during specimen handling and testing.

Sample Preparation for POBS

The in vitro study adhered to the Declaration of Helsinki, with approval granted by the institutional ethics committee (IEC2:70/2022, March 11, 2022). Sixty intact human mandibular molars with divergent roots and teeth designated for extraction due to compromised periodontal health were included in the study. Teeth affected by caries, fractures, cracks, root canal-treated teeth, and teeth with fused roots, or any other developmental anomalies, were excluded.

The teeth were cleaned by removing superficial soft tissues with a curette and then stored in 0.2% sodium azide (KMC Pharmacy, Manipal, Karnataka, India) at 4 °C before use. The crowns were separated using a diamond disk under water spray at the cementoenamel junction (Horico Dental Hopf, Ringleb & Co. GmbH & Cie, Berlin, Germany) (Figure 1A). A perforation was made using the BR-45 MANI Dia-Bur (MANI, Inc., Takenzawa, Japan), having a diameter of 1 mm, positioned parallel to the long axis of the tooth and perpendicular to the furcal floor. This was followed by two complete penetrations of a #4 Peeso Reamer (MANI, Inc., Takenzawa, Japan) to increase the perforation to a diameter of 1.3 mm (Figure 1B). To ensure a consistent perforation wall height of 2 mm, a periodontal probe (Hu-Friedy Group, Chicago, IL, USA) was employed across all samples. Samples with inconsistent wall heights, i.e., greater than or less than 2 mm, were discarded. Subsequently, the teeth were mounted in acrylic molds (DPI-RR Cold Cure, DPI, Mumbai, India), ensuring a 2-mm gap between the furcation and acrylic mold surface (Figure 1C). This provided space for the insertion of Gelatamp (Coltène/Whaledent GmbH + Co. KG, Langenau, Germany), serving as a support during the placement of the root repair materials. Sixty samples were randomly divided into two groups of thirty samples each: Group I perforation to be restored with BD, while Group II perforation was to be restored with BCR.

Figure 1
Schematic illustration of sample preparation for push-out bond strength testing. A: Decoronated Mandibular first molar. B: Furcal perforation at the center of the pulp chamber. C: Space between the furcation and acrylic mold surface for placing the gelatamp. D: Perforation repaired with calcium-silicate-based repair material. E: Irrigation with chelating agents. F: Push-out bond strength testing using a Universal Testing Machine.

Manipulation of Materials

The composition of BD and BCR is presented in Table 1. BD was mixed according to the manufacturer's instructions. Subsequently, BD and BCR (ready-to-use putty form) were placed in small increments into the perforation site of their respective samples and compacted using a hand plugger (Hu-Friedy, Illinois, USA) (Figure 1D). The samples were subsequently wrapped in moist gauze and placed in an incubator (RIM-24HHDFSSOH, Rotek Instruments, Kerala, India) at 37 °C and 95% humidity to allow them to set for their respective setting times, i.e., 9-12 minutes for BD and 120 minutes for BCR.

Table 1
Composition of Biodentine and Bio-C Repair.

BIODENTINE: Powder and liquid to be mixed with an amalgamator for 30 s at 4000 rpm	BIO-C REPAIR: Premixed Bioceramic repair material
Powder
Tricalcium silicate	Tricalcium silicate
Dicalcium silicate	Dicalcium silicate
Iron oxide	Tricalcium aluminate
Zirconium oxide	Calcium oxide
Calcium carbonate	Zirconium oxide
Liquid
Calcium chloride	Silicon oxide
Hydrosoluble polymer	Polyethylene glycol
Water	Iron oxide

Irrigation Protocol

After the samples were set, the irrigation protocol was initiated. Samples from both Group I and II were first irrigated with 5 mL of 2.5%.

Sodium Hypochlorite (NaOCl) (KMC Pharmacy, Manipal, Karnataka, India) using an irrigation needle (PRIME, India) for 1 minute (Figure 1E). Subsequently, samples in both Groups were divided into three sub-groups, comprising 10 samples each, based on the chelating agent used:

• Group A was irrigated with 5 mL of 17% EDTA (KMC Pharmacy, Manipal, Karnataka, India) for 1 minute.

• Group B was irrigated with 5 mL of 7% MA (KMC Pharmacy, Manipal, Karnataka, India) for 1 minute.

• Group C served as control and was irrigated with 5 mL of Distilled water (DW) (KMC Pharmacy, Manipal, Karnataka, India) for 1 minute.

Following this, in both Groups A and B, a final irrigation using 5 mL of DW was performed to neutralize any residual effects of the chelating agents.

All the steps were carried out under magnification using dental loupes (2.5X, SurgiTel, Michigan, USA), which provided enhanced visibility and precision throughout the process.

Push Out Bond Strength Analysis

POBS was analyzed using a Universal Testing Machine (UTM) (Electropuls E3000 and Bluehill Universal Software, Instron Corp., Norwood, MA, USA) (Figure 1F). The restorative materials in the perforated area were pushed out in an apical direction, parallel to the long axis of the tooth, at a crosshead speed of 1 mm/min, using a plunger of 1 mm diameter until dislodgment. The maximum Force during dislodgment was recorded in Newtons (N). The dislodgement resistance was calculated by using the formula:

$\text{Bond strength}\left(\text{MPa}\right)=\frac{\text{Force necessary for dislodgment}\left(\text{N}\right)}{\text{Bonded surface area}\left({\text{mm}}^{\text{2}}\right)}$

Bonded surface area = 2 × π radius of the perforated area × height of perforation

Sample Preparation and Analysis for Compressive Strength

For the assessment of CS, a total of thirty-six cylindrical samples, eighteen of each material (BD and BCR), were prepared using a stainless-steel split mold with a diameter of 4 mm and a height of 6 mm. These samples were placed in an incubator (RIM-24HHDFSSOH, Rotek Instruments, Kerala, India) and wrapped in wet gauze to maintain a temperature of 37 °C and 95% humidity for 24 hours.

All samples were then immersed in 5 mL of 2.5% sodium hypochlorite (NaOCl) for 1 minute. Following the immersion, each set of samples of the respective restorative material was randomly divided into three sub-groups of 6 samples each, similar to the methodology for testing POBS. These groups were then immersed in 5 mL of the respective irrigants for 1 minute, namely 17% EDTA for Group A, 7% MA for Group B, and DW for Group C. The CS of all the samples was evaluated using the UTM at a crosshead speed of 0.5 mm/min. The maximum load value observed during the test, divided by the area of the sample, is considered as CS and is expressed in MPa.

Statistical Analysis

The statistical analysis was carried out using SPSS version 20 (IBM Corp., Armonk, NY, USA). A p-value of less than 0.05 was considered statistically significant. For the evaluation of the push-out bond strength the mean values were compared using ANOVA, accompanied by the post-hoc Games-Howell test for intra-group comparison and independent sample t-test for inter-group comparison. For the evaluation of compressive strength, the mean values were compared using ANOVA followed by the post-hoc Tukey’s test.

Results

Push-Out Bond Strength

Intra-group comparisons revealed no significant difference in mean POBS across various chelating agents in the BD group (p=0.065). However, significant differences were observed in the mean POBS for the BCR group (p<0.001). Post-hoc analysis revealed that the mean POBS was significantly higher for the DW (7.62 ± 1.61) and MA (8.19 ± 1.11) groups compared to the EDTA (4.82 ± 1.0) group (Table 2). An inter-group comparison of the POBS between the BD and BCR groups showed no significant differences after irrigation with the chelating agents used in the study (Table 3).

Table 2
Intra-group analysis of the mean POBS (in MPa) of BD and BCR after irrigation with DW, 17% EDTA, and 7% MA.

Groups	Irrigants			p-value	Post-hoc Test
	Distilled Water	EDTA	Maleic Acid
	Mean ± SD	Mean ± SD	Mean ± SD
Biodentine	6.41 ± 1.59	4.87 ± 2.04	8.08 ± 3.83	0.065	-
Bio-C Repair	7.62 ± 1.61	4.82 ± 1.0	8.19 ± 1.11	<0.001	DW, MA >EDTA

• SD: Standard Deviation; POBS: Push-out Bond strength; EDTA: Ethylenediaminetetraacetic acid.

Table 3
Inter-group analysis of the mean POBS (in MPa) of BD and BCR after irrigation with DW, 17% EDTA, and 7% MA.

Irrigants	Groups		p-value
	Biodentine	Bio-C Repair
	Mean ± SD	Mean ± SD
Distilled Water	6.41 ± 1.59	7.62 ± 1.61	0.111
EDTA	4.87 ± 2.04	4.82 ± 1.0	0.95
Maleic Acid	8.08 ± 3.83	8.19 ± 1.11	0.932

• SD: Standard Deviation; POBS: Push-out Bond strength; EDTA: Ethylenediaminetetraacetic acid.

Compressive Strength

The use of chelating agents affected the mean CS of the BD group, which was significantly higher with DW (49.13 ± 6.3), followed by EDTA (38.88 ± 5.24), and lowest in the MA group (30.46 ± 4.93). On the other hand, there were no significant differences in the mean CS of BCR among the irrigant groups (Table 4). Despite being affected by chelating agents, BD exhibited superior CS to BCR, regardless of the chelating agent used.

Table 4
Intra-group analysis of the mean CS (in MPa) of BD and BCR after irrigation with DW, 17% EDTA, and 7% MA.

Groups	Irrigants			p-value	Post-hoc Test
	Distilled Water	EDTA	Maleic Acid
	Mean ± SD	Mean ± SD	Mean ± SD
Biodentine	49.13 ± 6.3	38.88 ± 5.24	30.46 ± 4.93	<0.001	DW>EDTA>MA
Bio-C Repair	4.96 ± 2.0	2.55 ± 1.20	3.37 ± 1.13	0.203	-

• CS: Compressive Strength; SD: Standard deviation; EDTA: Ethylenediaminetetraacetic acid.

Discussion

POBS and CS are two significant properties that add to the function of a perforation repair material. Perforation repair materials need to possess sufficient POBS against the walls of root dentine to prevent their dislodgement from the repair site [¹⁷], along with adequate compressive strength to tolerate occlusal forces and forces due to subsequent placement of restorative material [¹⁸]. The current study revealed that chelating agents did not have an adverse effect on the POBS of BD, aligning with the findings of Ballal et al. [¹⁹]. This unaltered dislodgement resistance of BD can be attributed to its biomineralization ability [²⁰] and small particle size [²¹], which enhance its penetration into the dentinal tubules to form mineralized tags. This is further reinforced by crystal growth within the tubules, resulting in the formation of dentinal bridges between the repair material and root dentin [²²]. As indicated in previous studies by Han and Okiji [²³] and Atmeh et al. [²⁴], this mechanism can result in enhanced micromechanical retention. Despite the known undesirable effects of chelating agents on the properties of CSCs, it can be assumed that the cement-dentin interface remains less affected due to the micromechanical interlocking. Thus, the proposed null hypothesis that chelating agents would not affect the POBS of BD and BCR was partially rejected.

While the POBS of BD remained unaffected upon exposure to both the chelating agents, BCR exhibited a decreased bond strength with EDTA. According to the manufacturers [²⁵], when BCR comes in contact with tissue fluids and moisture, it releases active ions that interact with the inorganic and organic matrix of dentin, promoting the formation of an intermediate area known as the Mineral Infiltration Zone (MIZ). This zone in dentin is claimed to provide an excellent biological seal. Along with the setting expansion exhibited by BCR, these factors can contribute to displaying bond strength values comparable to BD. However, in the present study, EDTA decreased the bond strength of BCR. The exact cause of BCRs' improved dislodgment resistance with MA, while decreased bond strength with EDTA, is unknown. It may be due to the specific interaction of MA with BCR at the molecular level, posing an intriguing area for further investigation. This finding, also supported by Ulusoy et al. [²⁶], implies that the selection of chelating agents for removing the smear layer from root canal dentin following perforation repair should depend on the type of tricalcium silicate used for the repair.

Compressive strength serves as a key indicator for evaluating the setting and hydration processes in materials [²⁷]. The use of distilled water demonstrated the highest compressive strength in both materials compared to the chelating agents used. This indicates that the setting rate of Tricalcium silicate-based cement materials is not affected by the presence of distilled water [²⁶].

EDTA induced a notable reduction in the compressive strength of BD. This aligns with the findings of Govindraju et al. [¹⁴], suggesting that EDTA's chelating action interferes with the formation of calcium silicate hydrate gel. It's worth noting that the CS testing in the present study was conducted 24 hours after storage. In contrast, Govindraju et al. [¹⁴] performed it after 7 days, which may have impacted the values, as the compressive strength of BD is known to increase over time [²⁸]. Among the various irrigants applied to BD, MA demonstrated a higher reduction in compressive strength compared to EDTA. The difference can be attributed to the considerably lower pH of MA compared to the neutral pH of EDTA. A low pH can interrupt the setting reaction, affect adhesion, or increase the solubility of calcium silicate-based materials, thus affecting their mechanical properties [²⁹]. In a study by Ballal et al. [¹⁹] investigating the effects of chelating agents and acids on the structure of BD, the most visible changes were observed in the MA and Phosphoric acid groups. In the MA group, the crystalline structure of BD was altered, resulting in a porous appearance. These structural alterations could account for the reduced compressive strength of BD with MA in the current study. Thus, the null hypothesis regarding compressive strength was also partially rejected.

However, despite the above-described effects of the chelating agents on BD, BD exhibited a higher compressive strength than BCR, irrespective of the chelator. This enhanced strength of BD can be attributed to many factors, such as low liquid/powder ratio, low porosity due to additives in the mixing liquid of BD, finer particle size, and presence of calcium carbonate particles, which serve as nucleation sites for the formation of calcium silicate hydrate gel [²²,³⁰].In contrast, Bio-C Repair (BCR) demonstrated lower compressive strength (CS) compared to Biodentine (BD), with no significant differences observed among the various irrigant groups. The difference can be attributed to its compositional variation, as BCR lacks calcium carbonate and contains tri-calcium aluminate [²²]. These findings are in agreement with a study by Rodrigues et al. [³¹], which reported higher compressive strength for Biodentine compared to Bio-C Repair. The authors attributed this to the water-soluble polycarboxylate polymers in the liquid component of BDs, which act as water-reducing agents, resulting in a less porous and mechanically stronger material [³¹].

This study has some limitations. The methodology for CS testing involved the use of CSC discs fully immersed in irrigating solutions. However, this method does not accurately simulate clinical conditions, where only the coronal surface of the filling material is predominantly exposed to irrigating solutions after perforation repair. Another limitation is the absence of blood during sealing of perforation, a factor that is always present in clinical scenarios and can influence the behavior of a material [³²].

Conclusion

The choice of chelating agent after perforation repair should depend on the type of calcium silicate cement used. For Biodentine, 17% EDTA is a better option than 7% maleic acid, as maleic acid significantly reduced the compressive strength of Biodentine without affecting its push-out bond strength. On the other hand, for Bio-C Repair, 7% maleic acid is more suitable, as it improved the push-out bond strength compared to EDTA and did not affect its compressive strength. The variation in molecular interactions of chelating agents due to compositional differences in various upcoming calcium silicate cement can be a promising avenue for future research.

• Financial Support
  None.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

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Edited by

• Academic Editor:
  Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  23 Feb 2026
• Date of issue
  2026

History

• Received
  07 Apr 2025
• Reviewed
  29 Apr 2025
• Accepted
  12 May 2025