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Journal of Dental Research
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Biomaterials & Bioengineering

Effects of EDTA on the Hydration Mechanism of Mineral Trioxide Aggregate

Y.-L. Lee1, F.-H. Lin2, W.-H. Wang2, H.H. Ritchie3, W.-H. Lan1 and C.-P. Lin1,*

1 Graduate Institute of Clinical Dentistry,
2 Institute of Biomedical Engineering, College of Medicine, National Taiwan University and National Taiwan University Hospital, No. 1 Chang-Te Street, Taipei 10016, Taiwan, ROC; and
3 Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, Ann Arbor, MI, USA

Correspondence: * corresponding author, pinlin{at}ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA’s ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

Key Words: mineral trioxide aggregate • EDTA • hydration • cell adhesion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mineral trioxide aggregate (MTA) is considered to be a potentially ideal material for perforation repair, retrograde filling, apexification, and vital pulp therapy (Torabinejad and Chivian, 1999). Several in vitro and in vivo studies have demonstrated that the sealing ability and biocompatibility of MTA are superior to those of amalgam, IRM, and super EBA (Lee et al., 1993; Torabinejad et al., 1993; Osorio et al., 1998; Lin et al., 2004). However, some researchers have offered different opinions as to whether MTA is really superior to other materials in terms of sealing (Weldon et al., 2002; Tanomaru Filho et al., 2005). In addition, MTA is not easy to handle, and obtaining consistent results during clinical application can be difficult. Particle size, powder-to-liquid ratio, temperature, and the presence of air in the mixture may all influence the physical properties of MTA (Torabinejad et al., 1995). Another possible disadvantage of MTA is the fact that it takes a long time to set. Furthermore, an acidic environment has been shown to influence the hydration of MTA, resulting in a weakening of the material’s microstructure (Lee et al., 2004). Therefore, before it can be argued that MTA is an ideal material for restorative endodontics, more studies focusing on MTA’s setting mechanism are required for a full explanation of the behavior of MTA in clinical applications.

Ethylenediaminetetraacetic acid (EDTA) has 6 potential sites (4 carboxyl groups and 2 amino groups) available to bond with a metal such as calcium (Skoog et al., 1996). The structures of Ca–EDTA complexes, in which the calcium ion is effectively surrounded and isolated from the solvent, are highly stable. Due to its ability to form complexes with calcium ions, EDTA is commonly used to remove the smear layer in non-surgical endodontic treatment (Ingle and Bakland, 2002; Hulsmann et al., 2003). In addition, an EDTA-containing viscous chelator, RC-PrepTM, is often used to facilitate negotiation of the canal (Stewart, 1995; Burns and Herbranson, 2002), although this chelator is not easy to remove from the root canal or the perforation area. MTA is a tricalcium mineral complex (Torabinejad and White, 1995). When MTA dissolves in water, calcium ions are released (Sarkar et al., 2005) and precipitate with silica gel to form the solid structure of MTA. If, however, the final flushing after EDTA or RC-PrepTM use is inadequate, some EDTA may remain in the root canal system. We assume that the residual EDTA in the root canal system may chelate calcium ions released from MTA during hydration and disturb the precipitation of hydrated products. To our knowledge, the effect of EDTA on the hydration mechanism of MTA has not previously been investigated.

We conducted this study to test the hypothesis that EDTA may disrupt the hydration of MTA, leading to MTA with poor physical properties and biocompatibility.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of Physical Properties
MTA (ProRoot, Dentsply/Tulsa Dental, Tulsa, OK, USA) was mixed according to the manufacturer’s instructions and placed in a mold. Forty-five specimens were prepared and randomly divided into 3 groups of 15, and then stored in either distilled water (pH 7), normal saline (pH 7.4), or 17% EDTA solution (pH 7.4) (REDTA, Roth International, Chicago, IL, USA) at 37°C. Seven days later, the specimens were removed and processed for further analysis.

After being air-dried and gold-coated, 5 specimens from each group were examined with a scanning electron microscope (SEM) (Topcon ABT-60, Tokyo, Japan) equipped with the analySiS® 3.0 imaging system (Soft Imaging System, Münster, Germany). The Ca/Si molar ratio of 3 specimens from each group was measured by SEM (Hitachi S-800, Tokyo, Japan) with energy-dispersive x-ray spectroscopy (EDS) (EDAX PV9900, Mahwah, NJ, USA), with an accelerating voltage of 15 kV and a spot size of 100 nm. The x-ray intensities in counts per second were recorded. The unhydrated MTA powders were also prepared for EDS by the same procedure.

Another 5 specimens from each group were subjected to x-ray diffraction (XRD) anakysis by a Rigaku x-ray powder diffractometer (Geigerflex, Rigaku, Tokyo, Japan), with a Ni filter and CuK{alpha} radiation ({lambda} = 0.154 nm), at 30 kV and 20 mA. Samples were scanned at a range of 10° to 60°, and all data were collected in a continuous scan mode at a scanning rate of 4°/min. Crystalline formations were identified by a computerized auto-match system with a standard data file from the Joint Committee for Powder Diffraction Studies.

The remaining 5 specimens from each group were prepared for the microhardness test, which was conducted with a Knoop diamond indenter (Shimadzu HMV-2, Tokyo, Japan) under a load of 98.07 mN and a dwell time of 6 sec. Differences between groups in terms of microhardness were tested for statistical significance by one-way analysis of variance (ANOVA) and a multiple-comparison test (Tukey’s test). A probability level of p < 0.05 was defined as statistically significant.

Evaluation of Cell Adhesion
Ten MTA specimens were prepared according to the procedure described previously, and were randomly divided into 2 groups: the distilled water and EDTA groups. To remove the residual EDTA, and also to stop the hydration of MTA, we rinsed all specimens with 100% ethanol for 10 min twice (Bye, 1983), then with distilled water for 10 min three times. The specimens were transferred into 48-well culture plates, and 5 x 104 mineralizing rat pulpal cells (MRPC-1 cell line) (Lundquist et al., 2002) per well were seeded on top of the materials. After incubation for 4 hrs, the specimens were fixed with 2.5% glutaraldehyde at 4°C for 30 min, followed by serial dehydration with ethanol, CO2 critical-point-drying, and sputter-coating with gold. The morphology of the cells adhering to the MTA specimens was observed by SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Scanning Electron Microscopy–Energy-dispersive Spectroscopy (SEM-EDS)
The results of SEM demonstrated that the microstructures of both MTA stored in distilled water (Fig. 1AGo) and MTA stored in normal saline (Fig. 1BGo) were crystalline, being composed of cubic and needle-like crystals. In both groups, the main structure of hydrated MTA consisted of cubic crystals, while the needle-like crystals filled the inter-grain spaces. However, there were relatively more needle-like crystals in the normal saline group than in the distilled water group. By contrast, a granular structure, in which crystals had not formed, was observed in the EDTA group (Fig. 1CGo). Use of a higher magnification showed a domed structure with a slightly undulating surface texture (Fig. 1DGo).


Figure 1
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  		Figure 1. Scanning electron micrographs of hydrated mineral trioxide aggregate (MTA) in distilled water (A), in normal saline (B), and in ethylenediaminetetraacetic acid (EDTA) (C,D). The crystalline structure of hydrated MTA consisted of cubic and needle-like crystals in both the distilled water and normal saline groups. By contrast, the microstructure of MTA hydrated in EDTA consisted of an amorphous phase with a granular appearance and a domed structure. (A–C) Scale bar = 20 µm. (D) Scale bar = 3.33 µm.

 
The results of SEM-EDS revealed that the Ca/Si molar ratios of specimens stored in distilled water and normal saline (5.80 and 5.53, respectively) were close to that of MTA powder (5.37). However, the ratio for specimens stored in EDTA was much lower (1.04).

X-ray Diffraction Analysis
In the case of MTA powder before hydration, XRD analysis showed several sharp peaks of C3S (3CaO·SiO2), C2S (2CaO·SiO2), and C3A (3CaO·Al2O3), including a peak at 2{theta} = 27.3° ({diamondsuit}) and multiple peaks at 2{theta} = 32°–34° (•). After hydration, 3 phases of C3S, C2S, and C3A were identified at the same locations as those for the MTA powder. However, a clear decline in the intensities of these peaks, as well as an additional peak at 2{theta} =18° ({blacktriangleup}), were observed in the distilled water and normal saline groups. There was no significant difference in the XRD pattern between the EDTA-treated specimens and MTA powder (Fig. 2Go).


Figure 2
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  		Figure 2. X-ray powder diffraction (XRD) patterns for unhydrated mineral trioxide aggregate (MTA powder) and the hydrated MTA stored in distilled water (DW), normal saline (NS), and ethylenediaminetetraacetic acid (EDTA). Peaks at 2{theta} = 27.3° ({diamondsuit}) and 2{theta} = 32°–34° (•) are identified as the reactants in all MTA specimens. There was no significant difference in XRD pattern between the specimens stored in EDTA and the MTA powder itself. In the case of the distilled water and normal saline groups, an obvious decrease was noted in the intensities of the peaks of reactants; an additional peak was also observed at 2{theta} = 18° ({blacktriangleup}).

 
Microhardness Test
The mean microhardnesses (± SD) of MTA specimens stored in the distilled water, normal saline, and EDTA solutions were 52.248 ± 4.375 HKN, 48.690 ± 2.991 HKN, and 14.278 ± 6.426 HKN, respectively. One-way ANOVA showed significant differences among the 3 groups (p < 0.0001). The mean microhardness of the EDTA-treated specimens was significantly lower than those of the other two groups (p < 0.0001); however, there was no significant difference between the distilled water and normal saline groups (p = 0.098).

Cell Adhesion
After 4 hrs of co-culturing, MRPC-1 cells attached to, and began to spread on, the MTA specimens stored in distilled water (Figs. 3A, 3BGo); by contrast, the cells remained rounded on the surface of the MTA stored in EDTA (Fig. 3CGo). Bulbing of MRPC-1 cells was observed on the surfaces of the MTA specimens in the EDTA group (Fig. 3DGo).


Figure 3
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  		Figure 3. Scanning electron micrographs of MRPC-1 cells cultured on mineral trioxide aggregate (MTA) for 4 hrs. MRPC-1 cells show a flattened morphology (A) with filopodia (B) on the MTA specimens in distilled water. However, MRPC-1 cells remained rounded (C) and bulbing (D) on the MTA specimens in ethylenediaminetetraacetic acid. (A,C) Scale bar = 20 µm. (B,D) Scale bar = 3.33 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, the pH of all experimental environments was set to neutral, since the hydration of MTA could have been affected by an acidic environment (Lee et al., 2004). Using SEM-EDS, XRD, and the microhardness test, we demonstrated similarities in the microstructure, Ca/Si molar ratio, crystal phases, and microhardness of those MTA specimens hydrated in distilled water and those hydrated in normal saline. However, the normal saline environment offers more nucleation sites for the hydration of MTA, due to the higher Na+ and Cl– contents; this causes the formation of greater numbers of needle-like crystals. By contrast, no crystalline structures were found in those specimens stored in EDTA. Only amorphous phases with a granular appearance were observed in the EDTA group. The Ca/Si molar ratio of EDTA-treated MTA was also much lower than those of the other groups. These findings showed that no hydrated compound was produced when MTA was stored in the EDTA environment.

The chemical compositions of MTA and Portland cement are similar (Estrela et al., 2000). The hydration of MTA should resemble that of Portland cement, which produces calcium silicate hydrate (C-S-H) and portlandite (Bye, 1983; Cong and Kirkpatrick, 1996; Brown, 1999). MTA hydration proceeds in two stages: dissolution of the tricalcium complex in water, and precipitation and crystallization of the hydrated compounds. The hydration mechanism of MTA is illustrated in Fig. 4Go. First, the fine hydrophilic particles of the MTA powder dissolved; meanwhile, calcium ions hydrolyzed from the MTA diffused into the environment. Then, the silicate gel remaining at the MTA surface, which is bound with H2O, released OH– ions; this raised the concentrations of Ca2+ and OH– ions in the environment (Bye, 1983). As soon as the environment was supersaturated with respect to the hydration product, the surface of the material was covered by aggregations of small precipitates of C-S-H (Garrault-Gauffinet and Nonat, 1999), and the inter-grain spaces were filled with portlandite crystals (Bye, 1983). In the distilled water and normal saline groups, MTA powder dissolved and then precipitated to form a crystalline microstructure, which was composed of cubic and needle-like crystals. However, in the EDTA group, the non-hydrated fine particles dissolved, but no crystalline compound was produced. The residual elements merely aggregated to form a domed structure with a secondary undulating surface texture.


Figure 4
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  		Figure 4. Illustration of the hydration mechanism of mineral trioxide aggregate (MTA).

 
For the XRD analysis, we used the peak at 2{theta} = 18° ({blacktriangleup}) as the main peak when identifying the hydrated product, and the peak at 2{theta} = 27.3° ({diamondsuit}) and the multiple peaks at 2{theta} = 32°–34° (•) as the main peaks for the reactants, since most of the characteristic peaks corresponding to portlandite, C3S, C2S, and C3A were superimposed. After hydration in distilled water or normal saline, the intensities of the peaks of the reactants decreased, due to the dissolution of the C3S, C2S, and C3A. The peak corresponding to portlandite was present after hydration in these two environments, which indicated that the final Ca/Si molar ratio of MTA should have been greater than 1.5 (Cong and Kirkpatrick, 1996). In fact, SEM-EDS results consistently revealed that the Ca/Si molar ratio of those specimens was around 5.53–5.8. Due to the nano-scale of the C-S-H crystals (Richardson, 1999), only portlandite was identifiable in hydrated MTA when XRD was used. However, a totally different XRD powder pattern was observed in the EDTA group. Very slight decreases in the intensities of the peaks of the reactants, and the absence of the peak corresponding to portlandite, imply that the hydration of MTA may have been disrupted by EDTA.

SEM-EDS results showed that MTA specimens stored in EDTA had a much lower Ca/Si molar ratio (of 1.04), suggesting that no portlandite was formed. This was confirmed by the XRD analysis. Due to the high formation constant for Ca–EDTA (KCa-EDTA) of 5.0 x 1010(Skoog et al., 1996), as soon as the MTA powder dissolves in water, the Ca2+ ions released from MTA are chelated by EDTA to form the Ca–EDTA complex. The nucleation of C-S-H would then be blocked, because free calcium ions would be lacking (pathway I in Fig. 4Go). Furthermore, the nucleation of Ca(OH)2 would also be affected (pathway II in Fig. 4Go). This explains the absence of the characteristic peak of portlandite in the EDTA group. A drop in the Ca/Si molar ratio of the MTA stored in EDTA suggests that the C-S-H was poorly crystallized. This agrees with Cong and Kirkpatrick (1996), who reported that the type of C-S-H phase that develops is determined by the final Ca/Si molar ratio.

The results of the microhardness test showed that the EDTA-treated specimens had a hardness value significantly lower than those of the specimens in the other two groups; this may relate to the poorly crystallized C-S-H in the EDTA group, revealed by SEM-EDS. In addition, the MRPC-1 cells remained rounded, and bulbing of cells was observed on the MTA specimens stored in EDTA, although the cells were spread out and attached to the specimens stored in distilled water. Due to the poor hydration of MTA, high concentrations of toxic ions (such as aluminum, iron, and sulfur ions) were present on the surfaces of the EDTA-treated specimens, resulting in poor cell adhesion.

In conclusion, our hypothesis that EDTA may disrupt the hydration of MTA was proven. EDTA inhibits the hydration of MTA by chelating calcium ions released from the tricalcium complex, which is the principal ingredient of MTA. Furthermore, the physical properties of MTA were weaker, and cell adhesion to materials was poorer, after EDTA treatment. The EDTA solution used was detrimental to the hydration of MTA and the biocompatibility of MTA. Thus, before applying MTA to form an apical plug or repair a root canal perforation, the practitioner should ensure that EDTA has been completely removed by flushing the area with copious amounts of distilled water.


   ACKNOWLEDGMENTS
 
This work was supported by Grant NSC 93-2314-B-002-118 from the National Science Council, Taiwan.

Received for publication July 9, 2006. Revision received January 18, 2007. Accepted for publication February 8, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  • Brown PW (1999). Hydration behavior of calcium phosphates is analogous to hydration behavior of calcium silicates. Cem Concr Res 29:1167–1171.
  • Burns RC, Herbranson J (2002). Tooth morphology and preparation. In: Pathways of the pulp. 8th ed. Chapter 7. Cohen S, Burns RC, editors. St. Louis: Mosby, pp. 173–220.
  • Bye GC (1983). Portland cement: composition, production, and properties. New York: Pergamon Press.
  • Cong X, Kirkpatrick RJ (1996). 29Si MAS NMR study of the structure of calcium silicate hydrate. Adv Cem Based Mater 3:144–156.
  • Estrela C, Bammann LL, Estrela CR, Silva RS, Pecora JD (2000). Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal. Braz Dent J 11:3–9.[Medline] [Order article via Infotrieve]
  • Garrault-Gauffinet S, Nonat A (1999). Experimental investigation of calcium silicate hydrate (C-S-H) nucleation. J Cryst Growth 200:565–574.
  • Hulsmann M, Heckendorff M, Lennon A (2003). Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 36:810–830.[Medline] [Order article via Infotrieve]
  • Ingle JI, Himel VT, Hawrish CE, Glickman GN, Serene T, Rosenberg PA, et al. (2002). Endodontic cavity preparation. In: Endodontics. Chapter 10. Ingle JI, Bakland LK, editors. London: BC Decker, pp. 405–570.
  • Lee SJ, Monsef M, Torabinejad M (1993). Sealing ability of a mineral trioxide aggregate for repair of lateral root perforations. J Endod 19:541–544.[Medline] [Order article via Infotrieve]
  • Lee YL, Lee BS, Lin FH, Yun Lin A, Lan WH, Lin CP (2004). Effects of physiological environments on the hydration behavior of mineral trioxide aggregate. Biomaterials 25:787–793.
  • Lin CP, Chen YJ, Lee YL, Wang JS, Chang MC, Lan WH, et al. (2004). Effects of root-end filling materials and eugenol on mitochondrial dehydrogenase activity and cytotoxicity to human periodontal ligament fibroblasts. J Biomed Mater Res B Appl Biomater 71:429–440.[Medline] [Order article via Infotrieve]
  • Lundquist P, Ritchie HH, Moore K, Lundgren T, Linde A (2002). Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization. J Bone Miner Res 17:1801–1813.[Medline] [Order article via Infotrieve]
  • Osorio RM, Hefti A, Vertucci FJ, Shawley AL (1998). Cytotoxicity of endodontic materials. J Endod 24:91–96.[Medline] [Order article via Infotrieve]
  • Richardson IG (1999). The nature of C-S-H in hardened cements. Cem Concr Res 29:1131–1147.
  • Sarkar NK, Caicedo R, Ritwik P, Moiseyeva R, Kawashima I (2005). Physicochemical basis of the biologic properties of mineral trioxide aggregate. J Endod 31:97–100.[Medline] [Order article via Infotrieve]
  • Skoog DA, West DM, Holler FJ (1996). Complex-formation titrations. In: Fundamentals of analytical chemistry. 7th ed. Chapter 14. Skoog DA, West DM, Holler FJ, editors. Fort Worth: Saunders College Pub., pp. 278–301.
  • Stewart GG (1995). Gaining access to calcified canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endo 79:764–768.
  • Tanomaru Filho M, Figueiredo FA, Tanomaru JM (2005). Effect of different dye solutions on the evaluation of the sealing ability of mineral trioxide aggregate. Pesqui Odontol Bras 19:119–122.[Medline] [Order article via Infotrieve]
  • Torabinejad M, Chivian N (1999). Clinical applications of mineral trioxide aggregate. J Endod 25:197–205.[Medline] [Order article via Infotrieve]
  • Torabinejad M, White DJ, inventors (1995). Loma Linda University, assignee. Tooth filling material and method of use. US Patent 5,415,547. May 16.
  • Torabinejad M, Watson TF, Pitt Ford TR (1993). Sealing ability of a mineral trioxide aggregate when used as a root end filling material. J Endod 19:591–595.[Medline] [Order article via Infotrieve]
  • Torabinejad M, Hong CU, McDonald F, Pitt Ford TR (1995). Physical and chemical properties of a new root-end filling material. J Endod 21:349–353.[Medline] [Order article via Infotrieve]
  • Weldon JK Jr, Pashley DH, Loushine RJ, Weller RN, Kimbrough WF (2002). Sealing ability of mineral trioxide aggregate and super-EBA when used as furcation repair materials: a longitudinal study. J Endod 28:467–470.[Medline] [Order article via Infotrieve]

Journal of Dental Research, Vol. 86, No. 6, 534-538 (2007)
DOI: 10.1177/154405910708600609
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