Demineralization effect of EDTA, EGTA, CDTA and citric acid on root dentin: a comparative study

Efeito do EDTA, EGTA, CDTA e ácido cítrico na desmineralização da dentina radicular: estudo comparativo

ORIGINAL ARTICLES

ENDODONTICS

Demineralization effect of EDTA, EGTA, CDTA and citric acid on root dentin: a comparative study

Efeito do EDTA, EGTA, CDTA e ácido cítrico na desmineralização da dentina radicular: estudo comparativo

Simone Maria Galvão de Sousa^I; Thelma Lopes Silva^II

^IAssociate Researcher, PhD, Department of Metallurgy and Material Science, Pontifical Catholic University of Rio de Janeiro

^IIBiochemist, MS, Department of Biochemistry, School of Dentistry of Bauru, University of São Paulo

ABSTRACT

The purpose of this study was to biochemically compare the decalcifying effects of 1% EDTA (pH 7.4), 1% EGTA (pH 7.4), 1% CDTA (pH 7.4), 1% citric acid solutions (pH 1.0 and 7.4) and saline solution (control) on root dentin. Forty-eight single-rooted teeth were used in this study. The canals were instrumented by the step-back technique and the roots were randomly divided into six equal experimental groups (n = 8) according to the irrigating agent tested. A total of 30 µL of each solution was pipetted into the root canal and allowed to set undisturbed for 5 minutes. After this time, 15 µL of the solutions were removed from each canal using a Hamilton syringe and placed in a container with 5 mL of deionised water. The µg/mL concentration of calcium ion (Ca²⁺) extracted from the root canal samples was determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Data were analysed by means of the Kruskal-Wallis and Mood's median tests. Citric acid solution at pH 1.0 removed more calcium than at pH 7.4 and than the other chelating solutions tested (p < 0.05). No differences were observed between EDTA and EGTA. Both EDTA and EGTA removed significantly more calcium than CDTA and citric acid at pH 7.4 (p < 0.05). There were no differences between citric acid at pH 7.4 and saline solution, which had the least efficacy for Ca²⁺ extraction (p > 0.05). These results indicate that citric acid at pH 1.0 is a good alternative as an irrigating solution to remove the smear layer and facilitate the biomechanical procedures.

Descriptors: Chelating agent; Citric acid; Edetic acid; Egtazic acid.

RESUMO

Este trabalho teve como objetivo comparar o efeito desmineralizante do EDTA (pH 7,4), EGTA (pH 7,4), CDTA (pH 7,4), ácido cítrico (pH 1,0 e 7,4) e da solução salina (controle) sobre a dentina radicular. Todas as soluções teste foram preparadas na concentração de 1%. Quarenta e oito dentes unirradiculares recém-extraídos foram utilizados neste experimento. Após a instrumentação dos canais radiculares pela técnica "step-back", as raízes foram aleatoriamente divididas em 6 grupos experimentais (n = 8) de acordo com a solução teste utilizada na irrigação final. Em cada grupo, 30 µL da solução teste foram pipetados no interior de cada canal radicular e mantidos estáveis por 5 minutos. Decorrido esse período, 15 µL da solução foram removidos do canal e depositados em frasco contendo 5 mL de água deionizada. A concentração de Ca²⁺ (µg/mL) extraída dos espécimes foi determinada pela espectrometria de absorção de massa (ICP-AES); e os dados foram submetidos à análise estatística pelos testes de Kruskal-Wallis e de mediana de Mood. O ácido cítrico em pH 1,0 foi a solução mais efetiva na remoção de Ca²⁺ comparativamente às demais soluções-teste (p < 0,05). Nenhuma diferença estatística foi observada entre a ação do EDTA e a do EGTA. Ambos os quelantes removeram significantemente mais Ca²⁺ que o CDTA e o ácido cítrico em pH 7,4 (p < 0,05). Não houve diferença significante entre ácido cítrico em pH 7,4 e solução salina (p > 0,05). Os resultados deste estudo indicam que o ácido cítrico em solução de pH 1,0 apresenta-se como uma boa opção para remover a "smear layer" e facilitar o preparo biomecânico do sistema de canal radicular.

Descritores: Quelantes; Ácido cítrico; Ácido edético; Ácido egtázico.

INTRODUCTION

Mechanically prepared dentin surfaces are always covered with the so-called smear layer, a loosely bonded amorphous layer of organic and inorganic debris. Thus, several chelating agents and acids have been used to remove this layer, with varying degrees of success.

These irrigating solutions have been chosen due to their direct action over calcium ions. The calcium present in hydroxyapatite crystals is one of the main inorganic elements of dentin. Any change in the calcium ratio can significantly alter the original proportion of organic and inorganic components, which can alter dentin permeability, microhardness and solubility.

Although EDTA has a long-standing history as the agent of choice in Endodontics, researchers have reported its irritating potential^7,9-11. Recently, EGTA has also been recommended for root canal biomechanical preparation, since it pre-sents a more precise action on calcium ions than EDTA^1,2,9,12,16. Another chelating agent, CDTA, has also been reported to significantly bind the divalent cation. EGTA and CDTA have been used in medicine to obtain calcium-free environments. Recent studies have investigated the application of both chelating agents as an alternative solution to remove the smear layer. Çalt, Serper¹ (2000) and Viswanath et al.¹⁶ (2003) compared the effect of EDTA and EGTA on removal of the smear layer by using scanning electron microscopy. Cruz-Filho et al.² (2001) and Sousa-Neto et al.¹² (2002) evaluated the influence of 15% EDTA, 1% CDTA and 1% EGTA on root dentin microhardness, and dentin adhesiveness and microleakage, respectively. In the study of Cruz-Filho et al.² (2001), EDTA was associated with a surfactant (Cetavlon). It is important to note that, in these investigations, the EDTA concentration tested was between 15-17%, and 1% was the concentration of EGTA and CDTA solutions. No study has yet compared all chelating agents using the same concentration.

Citric acid has also been studied for its ability as a decalcifying and cleansing solution in root canal irrigation^8,13,14,17. However, in some scanning electron microscope studies, it was observed that citric acid left precipitated crystals in the root canal walls^3,4. It is a biological and organic acid, with sufficient tissue compatibility^6,7,10,11,15.

Studies have demonstrated that the pH and concentration of demineralising solutions are important factors to be considered in their action in removing surface smear layer^4,5. A literature review has revealed that different mixtures, concentrations, pH, working time and methods have been used to evaluate the irrigating solutions.

Thus, considering these factors, this study was conducted to compare in vitro the decalcifying effect of 1% citric acid, 1% EDTA, 1% EGTA, 1% CDTA and saline solution (control) on root dentin, by using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The pH of the test solutions was 7.4 for all chelating agents and 1.0 and 7.4 for citric acid. The reason to evaluate two different pH values for the citric acid group was based on the long-standing studies about its best action at lower pH^4,5, and inclusion of a second value (pH 7.4) was intended to obtain a more accurate comparative result.

MATERIAL AND METHODS

Test materials

The materials used were: 1% EDTA pH 7.4 (ethylene-diaminetetra-acetic acid; Merck, Darmstadt, Germany); 1% EGTA pH 7.4 (ethylene-glycol-ether-diaminetetra-acetic acid; Sigma, St. Louis, MO, USA); CDTA (1,2 cyclohexane-diaminetetra-acetic acid, Sigma, St. Louis, MO, USA); 1% citric acid pH 1.0 and 7.4 (Merck, Darmstadt, Germany) and saline solution (control; Merck, Darmstadt, Germany). All test solutions were freshly prepared. The salts were weighed and diluted in deionised water (Permution E.J. Krieger & Cia. Ltda., Curitiba, Brazil), and their pH was adjusted (pH meter B371, Micronal, São Paulo, Brazilian Manufacturing) by addition of hydrochloric acid (HCl – Merck S.A. Indústria Química, Rio de Janeiro, Brazil). The molar concentrations of the acid solutions were: EDTA = 0.0263 mol/L, EGTA = 0.0213 mol/L, CDTA = 0.0274 mol/L, and citric acid = 0.0520 mol/L.

Procedure

Forty-eight single-rooted freshly extracted human teeth with straight root canals and similar length were used in this study. The anatomical crowns were removed transversally at the cemento-enamel junction, and the pulp tissue was extirpated. The working length of all teeth was established by passing a #15 K-file through the apical foramen and then reducing the length by 1.0 mm. The canals were instrumented by the step-back technique to #70 K-file (Dentsply Maillefer, Ballaigues, Switzerland) with the minimum master apical file #45. During instrumentation, each canal was copiously irrigated with 20 mL of 1% NaOCl (Biodinâmica, Ibiporã, PR, Brazil). Then, the roots were randomly divided into six equal experimental groups (n = 8) according to the irrigator tested. For all groups, 30 µL of each solution were pipetted into the root canal and allowed to set undisturbed for 5 minutes. After this time, 15 µL of the solutions were removed from the root canals using a Hamilton syringe (Hamilton Company, Reno, Nevada, USA) and placed in a container with 5 mL of deionised water. The concentration of Ca²⁺ (µg/mL) was determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES, Spectrometer plasma 400, Pelkin Elmer, Shelton, CT, USA). The data were statistically analysed by using the nonparametric Kruskal-Wallis test and Mood's median test for multiple comparisons, at the significance level of 5% (p < 0.05).

RESULTS

The means, standard deviations (SD), medians (semi-interquartile range) and minimum and maximum values of Ca²⁺ concentrations (µg/mL) in all experimental groups are shown in Table 1.

The Kruskal-Wallis test shows that there were statistical differences between the acids in terms of Ca²⁺ concentration. Mood's median test (Table 2) showed statistically significant difference between citric acid solution at pH 1.0 and the other groups (p < 0.05). This solution removed more calcium than citric acid solution at pH 7.4 and the other chelating solutions tested. Moreover, no differences were noted between EDTA and EGTA. Both chelating agents removed significantly more calcium than CDTA and citric acid at pH 7.4 (p < 0.05). There were no differences between citric acid at pH 7.4 and saline solution, which had the least efficacy for Ca²⁺ extraction (p < 0.05).

DISCUSSION

In this in vitro study, it was noteworthy that citric acid solution is not effective for Ca²⁺ removal at neutral pH. While EDTA, EGTA and CDTA act as chelating agents at neutral pH, citric acid acts better at lower pH values.

The statistical data showed that 1% citric acid at pH 1.0 was the best solution to remove calcium from the root dentin. Furthermore, considering the concentration factor, even though the 1% citric acid solution (pH 1.0) was employed, i.e., at a lower concentration, better results were obtained when compared to the EDTA, EGTA, and CDTA solution groups. The present results are in agreement with those of Hennequin et al.⁵ (1994) and Haznedaroglu⁴ (2003).

According to both previous studies, the pH of citric acid solution has been shown to be a more important factor to demineralisation than concentration. Sterrett et al.¹⁴ (1991) reported that this phenomenon may be due to a balance between the decrease in pH and the increase in viscosity of the solution caused by the increase in the constituent concentration. At high concentrations, citrate monopolises such a large portion of the solvent that the amount of solvent available for Ca²⁺ diffusion is dramatically reduced.

Furthermore, the self-limiting effect associated with pH changes during the demineralisation process of EDTA, EGTA and CDTA can be considered. These chelating agents react with calcium ions in the hydroxyapatite crystals to produce a metallic chelate, and this process is characterised by protonation and complex formation reaction occurs⁹. As the pH decreases during this process, both the rate of dentin demineralisation and the amount of dentin dissolved decrease.

The irrigating solutions used in this experiment were of low concentrations. It should be mentioned that EDTA is currently studied or used in clinical practice with concentrations between 15-17%. In fact, this is the first study comparing EDTA with EGTA, CDTA and citric acid at the same concentration and pH. Comparison between two different pH values of citric acid and the chelating agents was essential to confirm its action at lower pH and concentration values. Few studies have evaluated its action using these parameters so far.

Even though EGTA is reported to bind Ca²⁺ more specifically than EDTA, in some studies, although different analyses and concentrations have been used, EGTA was similar or less potent than EDTA. Çalt, Serper¹ (2000) indicated that the action of 17% EDTA is stronger than that of 17% EGTA for removal of smear layer. However, EGTA did not cause erosion of the intertubular and peritubular dentin. Cruz-Filho et al.² (2001) reported that 1% EGTA, 1% CDTA and 15% EDTAC reduced root dentin microhardness similarly. Viswanath et al.¹⁶ (2003), in a SEM study, demonstrated that both EGTA and EDTA completely removed the smear layer.

There was a molar concentration difference between all test solutions, but this difference was smaller among chelating agents. The present study revealed that EDTA and EGTA presented the same effect on dentin Ca²⁺ extraction. However, CDTA was statistically less effective, though its molar concentration was slightly higher than that of EDTA and EGTA. Note that the citric acid solution had almost double the molar concentration when compared with the other solutions.

Previous reports have shown that 15% EDTAC presented similar action when compared with 1% EGTA and 1% CDTA², and that EDTA-T was less effective than EDTA⁸ as a chelating agent. The present data demonstrated that 1% EDTA alone was as efficient as 1% EGTA and better than 1% CDTA. The concentration and association with other substances can be an important factor on the chelating agents' action process.

Considering the present results on the similar effect of 1% EDTA and 1% EGTA for removal of smear layer, the previous literature^1,2,16, and their cytotoxic effect^{2,6,7,9-11,16}, EGTA should be preferred when a chelating agent is required.

All acid solutions tested in this experiment are considered complexants and chelating agents. Under the conditions of this study, 1% citric acid (pH 1.0) demonstrated to be a good alternative as an irrigating solution to remove the smear layer and facilitate the biomechanical procedures. Scelza et al.⁸ (2003) also suggested the use of citric acid as an irrigating solution despite the lack of statistical difference between citric acid and 17% EDTA in terms of their efficacy in Ca²⁺ extraction from root dentin. Citric acid was reported to be inexpensive, effective on anaerobic microorganisms¹⁷ and more biocompatible than EDTA and EGTA^7,9-11. Sousa¹¹ (2000) and Soares, Sousa¹⁰ (2003) reported that, even at 15% and pH 1.0, citric acid was less cytotoxic than 15% EDTA. The 15% EGTA solution was more biocompatible than EDTA.

Combinations of decalcifying agents and -NaOCl solution have been recommended, because no single irrigator is capable of dissolving organic pulpal material and predentin as well as demineralising the inorganic calcified portion of the root canal wall. The authors suggested that the acidity yielded by utilisation of chemical solutions on endodontic therapy could be minimised by final flushing with water and use of calcium hydroxide sealers, which could neutralize this residual effect. Besides, the application time should also be well controlled in the etching procedure. Attention to these factors will lower the risk of damage to the dental tissue during root canal cleaning and thus increase the success of endodontic therapy.

CONCLUSION

According to this in vitro study, the 1% citric acid at pH 1.0 proved to be the most effective solution for root dentin Ca²⁺ extraction. Lower concentrations of EDTA and EGTA were found to be more effective than CDTA; 1% EDTA and 1% EGTA had similar demineralisation effect on dentin. Citric acid at neutral pH did not significantly change the calcium content of root dentin.

Received for publication on Sep 17, 2004

Sent for alterations on Oct 25, 2004

Accepted for publication on May 27, 2005

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Publication Dates

• Publication in this collection
  21 Nov 2005
• Date of issue
  Sept 2005

History

• Reviewed
  25 Oct 2004
• Received
  17 Sept 2004
• Accepted
  27 May 2005