- Email: [email protected]
The demineralizing efficiency of EDTA solutions on dentin
The demineralizing efficiency of EDTA solutions on dentin I. Influence
of pH
Jaime A. Cury, M.Sc.D., * Claudio Bragotto, ** and Luiz Valdrighi, Sco Paula, Brazil
D.D.S., ***
STATE UNIVERSITY OF CAMPINAS, DENTISTRY SCHOOL OF PIRACICABA The demineralizing efficiency of a sodium salt solution of EDTA was determined biochemically. EDTA solutions (0.3M) at pH 4.60, 5.00, 6.00, 7.00, 6.00, and 9.00 were tested on purified radicular dentin derived from ninety-one permanent human teeth. The results showed that the optimum pH for demineralization is between 5.00 and 6.00.
hemical substances capable of facilitating the inC strumentation of root canals are of considerable importaqce in endodontics. In addition to their demineralizing properties, such substances should be tolerated by the periapical tissues, be noncorrosive, be easily applied, and have antiseptic properties. The sodium salt of ethyl&ne diamine tetra-acetic acid (EDTA) has these properties and was introduced clinically by &tbys in 1957. Seidberg and Schilde? made an evaluation of EDTA in endodontics, but the relationship between its demineralizing efficiency and pH was not established. The American Dental Association’ suggested the use of EDTA at pH 11.05 in 10 to 15 percent concentration; however, Nikiforuk and Sreebny4 showed that the demineralization of the mandible of rats required less time if the pH of EDTA solutions was lowered. According to Bersworth Chemical Company,5 the solubility of calcium carbonate in EDTA solutions can be increased by increasing the pH and it reaches a maximum at pH 7.30. Because the sodium salt of EDTA is used in endodontics to remove dentin the effect of pH on this process has been examined.
*Assistant professor of Biochemistry, Dentistry School of Piracicaba, State University of Campinas. **Research Assistant, supported by a grant from CNPq (Proc. 215/ 76). *** Professor and Chairman of Endodontics, Dentistry School of Piracicaba, State University of Campinas.
446
MATERlAL AND h@TttODS Preparation of dentin
A pool of ninety-one human one-root teeth was used. After being cleaned, the crowns were separated from the roots at the cementoenamel junction level. The roots were dehydrated in an oven for 2 hours at 100” C. and pulverized in a Microyeur Quantitatif Dangomau (No. 0749902, Prolabo, Paris) apparatus. The resulting powder was passed through Telatest sieves and particles of 0.074 to 0.149 mm. were collected and purified by the use of bromoform-acetone. l The dentin thus obtained was kept in an oven at 50” C. for 48 hours and then stored in a vacuum desiccator. Preparation of EDTA
EDTA solutions (0.3M) were prepared at pH 5.00, 6.00, 7.00, 8.00, and 9.00. The solutions were adjusted with NaOH at the pH meter. The EDTA” used, when dissolved in water, presented a pH of 4.60. The concentration of 0.3M was chosen because it is the solubility limit for the disodium salt of EDTA in water.’ Determination of demineralzation
A 2.50 ml. quantity of EDTA solution was added to 2.50 mg. of dentin in a centrifuge tube. The tubes were agitated at room temperature for 5 minutes and immediately centrifuged at 2,345 x g for 30 seconds. Supematant aliquots (0.025 ml.) were removed and analyzed for phosphorus content. 3 Six determinations were made for each PH. The calorimetric analysis was made 003@-4220/81/100446+03$00.30/00 1981 The C. V. Mosby Co
447
Demineralizing ejficiency of EDTA
Volume 52 Number 4
in a spectrophotometer Lomb).
Spectronic
20 (Bausch &
RESULTS AND DISCUSSION
The quantities of phosphorus liberated from samples of dentin in EDTA solutions between pH 4.60 and pH 9.00 are shown in Fig. ,l. The phosphorus liberated increased with a decrease in pH from pH 9.00 to pH 6.00, and decreased between pH 5.00 and pH 4.60: Thus the maximum efficiency of phosphorus liberation by EDTA within the range pH 4.60 to 9.00 was between pH 5.00 and 6.00. In solution the sodium salt of EDTA is in the disodium form at pH 4.50 and in the tetrasodium form at pH 11.05. Thus it might be expected that the efficiency with which calcium is chelated would increase with an increase in pH, since the greater the dissociation of the EDTA the greater would be the attraction for calcium ions. However, for calcium salts which are only weakly dissociated the rate of chelation may be limited by the availability of calcium ions. Dentin is formed by hydroxyapatite which, in an aqueous medium, is weakly dissociated in the following way: WO’QMOH)2
* lOCaa+ + 6PG3,- + 20H-.
(1)
The concentration of calcium and phosphate ions in dentin solutions is very low with a pKsp of approximately 1 15.2.g When EDTA is present the calcium ions in solution are sequestrated, and to satisfy the solubility product more hydroxyapatite will dissolve, as shown: CA,,(PQ),(OH),
= lOCa*+ + 6PG$- + 2OH-. + EDTA 4 EDTA-Ca
The EDTA and the calcium ions form a stable complex and the reaction proceeds until an equilibrium is reached. This should be the basis of the EDTA action on dentin in a neutral pH. However, as the pH increases, the hydroxyl ion concentration in solution increases. When this happens a suppression in the dissociation of Eq. (1) will occur, and the amount of Ca2+ in solution will decrease. On the other hand, as the pH decreases, the proton predominating in solution will neutralize OH- ions and the equilibrium will be changed, increasing the amount of Ca*+ in solution. However, as the pH decreases the efficiency of the action, EDTAacid is restricted also, due to the suppression. of the dissociation of EDTA. Thus, at pH 5.50, the EDTA is present as the trisodium salt (20.4%) and the disodium salt (79.6%). *The disodium salt of EDTA, Carlo Erba.
I
1
5.0
6.0
I
7.0
1
I
8.0
9.0
PH
Fig. 1. Phosphorus liberated from radicular human dentin by
EDTA solutions (0.3M) at different pH values. Each Point representsthe mean of six determinations. The results presented here are in agreement with the data of Nikiforuk and Sreebny,4 who analyzed the pH effect (from 6.02 to 12.01) in EDTA solutions (0.25M) and demonstrated that at pH 6.02, 8 days were needed for the total demineralization of rats’ mandibles, whereas at pH 12.01 18 days were required. Although the maximum solubilization of calcium carbonate by EDTA solution can be reached at pH 7.30,5 this difference in behavior may be explained by different solubility products of calcium carbonate and hydroxyapatite with respect to pH. CONCLUSIONS
The following conclusions can be inferred: (1) The efficiency of EDTA solutions on the demineralization of dentin is influenced by pH. (2) The greatest demineralizing efficiency of EDTA solutions (0.3M) can be achieved between pH 5.00 and 6.00. We wish to thank Dr. Henrique V. Amorim (ESALQUSP) for the use of the pulverizer apparatus. REFERENCES 1. Asgar, L.: Chemical Analysis of Human Teeth, J. Dent. Res. 35: 742-748, 1956. 2. Council of Dental Therapeutics: Accepted Dental Remedies, ed. 3 1, Chicago, 1966, American Dental Association, p. 207, op.cit. ref. 8. 3. Colowick, S. P., and Kaplan, N. 0.: Methods in Enzymology, vol. III, New York, 1957, Academic Press, Inc., pp. 840-850. 4. Nikiforuk, G., and Sreebny, L. M.: Demineralization of Hard Tissues by Organic Chelating Agents at Neutral pH, J. Dent. Res. 32: 859-867, 1953.
448
Cury, Bragotto, and Valdrighi
Powerful Organic Chelating Agents: Technical Bulletin No. 2, Framingham, Mass., Bersworth Chemical Company, opcit. ref. 4. ijstby, B. N.: Chelation in Root Canal Therapy, Odont. Tidskr. 65: 3-11, 1957. Sand, H. F.: The Dissociation of EDTA and EDTA-sodium salts, Acta Odont. Stand. 19: 469-482, 1961. Seidberg, B. H., and Schilder, H.: An Evaluation of EDTA in Endodontics, ORAL SURG. 37: 609-620, 1974.
Oral Surg. October, I98 1 9. Lazzari, E. P.: Dental Biochemistry, ed. 2, Philadelphia, Lea & Febiger, Publishers, pp. 120-121. Reprint requests to: Dr. Jaime A. Cury Dentistry School of Piicicaba State University of Campinas S~O Paulo. Brazil
1976.
of pH
Jaime A. Cury, M.Sc.D., * Claudio Bragotto, ** and Luiz Valdrighi, Sco Paula, Brazil
D.D.S., ***
STATE UNIVERSITY OF CAMPINAS, DENTISTRY SCHOOL OF PIRACICABA The demineralizing efficiency of a sodium salt solution of EDTA was determined biochemically. EDTA solutions (0.3M) at pH 4.60, 5.00, 6.00, 7.00, 6.00, and 9.00 were tested on purified radicular dentin derived from ninety-one permanent human teeth. The results showed that the optimum pH for demineralization is between 5.00 and 6.00.
hemical substances capable of facilitating the inC strumentation of root canals are of considerable importaqce in endodontics. In addition to their demineralizing properties, such substances should be tolerated by the periapical tissues, be noncorrosive, be easily applied, and have antiseptic properties. The sodium salt of ethyl&ne diamine tetra-acetic acid (EDTA) has these properties and was introduced clinically by &tbys in 1957. Seidberg and Schilde? made an evaluation of EDTA in endodontics, but the relationship between its demineralizing efficiency and pH was not established. The American Dental Association’ suggested the use of EDTA at pH 11.05 in 10 to 15 percent concentration; however, Nikiforuk and Sreebny4 showed that the demineralization of the mandible of rats required less time if the pH of EDTA solutions was lowered. According to Bersworth Chemical Company,5 the solubility of calcium carbonate in EDTA solutions can be increased by increasing the pH and it reaches a maximum at pH 7.30. Because the sodium salt of EDTA is used in endodontics to remove dentin the effect of pH on this process has been examined.
*Assistant professor of Biochemistry, Dentistry School of Piracicaba, State University of Campinas. **Research Assistant, supported by a grant from CNPq (Proc. 215/ 76). *** Professor and Chairman of Endodontics, Dentistry School of Piracicaba, State University of Campinas.
446
MATERlAL AND h@TttODS Preparation of dentin
A pool of ninety-one human one-root teeth was used. After being cleaned, the crowns were separated from the roots at the cementoenamel junction level. The roots were dehydrated in an oven for 2 hours at 100” C. and pulverized in a Microyeur Quantitatif Dangomau (No. 0749902, Prolabo, Paris) apparatus. The resulting powder was passed through Telatest sieves and particles of 0.074 to 0.149 mm. were collected and purified by the use of bromoform-acetone. l The dentin thus obtained was kept in an oven at 50” C. for 48 hours and then stored in a vacuum desiccator. Preparation of EDTA
EDTA solutions (0.3M) were prepared at pH 5.00, 6.00, 7.00, 8.00, and 9.00. The solutions were adjusted with NaOH at the pH meter. The EDTA” used, when dissolved in water, presented a pH of 4.60. The concentration of 0.3M was chosen because it is the solubility limit for the disodium salt of EDTA in water.’ Determination of demineralzation
A 2.50 ml. quantity of EDTA solution was added to 2.50 mg. of dentin in a centrifuge tube. The tubes were agitated at room temperature for 5 minutes and immediately centrifuged at 2,345 x g for 30 seconds. Supematant aliquots (0.025 ml.) were removed and analyzed for phosphorus content. 3 Six determinations were made for each PH. The calorimetric analysis was made 003@-4220/81/100446+03$00.30/00 1981 The C. V. Mosby Co
447
Demineralizing ejficiency of EDTA
Volume 52 Number 4
in a spectrophotometer Lomb).
Spectronic
20 (Bausch &
RESULTS AND DISCUSSION
The quantities of phosphorus liberated from samples of dentin in EDTA solutions between pH 4.60 and pH 9.00 are shown in Fig. ,l. The phosphorus liberated increased with a decrease in pH from pH 9.00 to pH 6.00, and decreased between pH 5.00 and pH 4.60: Thus the maximum efficiency of phosphorus liberation by EDTA within the range pH 4.60 to 9.00 was between pH 5.00 and 6.00. In solution the sodium salt of EDTA is in the disodium form at pH 4.50 and in the tetrasodium form at pH 11.05. Thus it might be expected that the efficiency with which calcium is chelated would increase with an increase in pH, since the greater the dissociation of the EDTA the greater would be the attraction for calcium ions. However, for calcium salts which are only weakly dissociated the rate of chelation may be limited by the availability of calcium ions. Dentin is formed by hydroxyapatite which, in an aqueous medium, is weakly dissociated in the following way: WO’QMOH)2
* lOCaa+ + 6PG3,- + 20H-.
(1)
The concentration of calcium and phosphate ions in dentin solutions is very low with a pKsp of approximately 1 15.2.g When EDTA is present the calcium ions in solution are sequestrated, and to satisfy the solubility product more hydroxyapatite will dissolve, as shown: CA,,(PQ),(OH),
= lOCa*+ + 6PG$- + 2OH-. + EDTA 4 EDTA-Ca
The EDTA and the calcium ions form a stable complex and the reaction proceeds until an equilibrium is reached. This should be the basis of the EDTA action on dentin in a neutral pH. However, as the pH increases, the hydroxyl ion concentration in solution increases. When this happens a suppression in the dissociation of Eq. (1) will occur, and the amount of Ca2+ in solution will decrease. On the other hand, as the pH decreases, the proton predominating in solution will neutralize OH- ions and the equilibrium will be changed, increasing the amount of Ca*+ in solution. However, as the pH decreases the efficiency of the action, EDTAacid is restricted also, due to the suppression. of the dissociation of EDTA. Thus, at pH 5.50, the EDTA is present as the trisodium salt (20.4%) and the disodium salt (79.6%). *The disodium salt of EDTA, Carlo Erba.
I
1
5.0
6.0
I
7.0
1
I
8.0
9.0
PH
Fig. 1. Phosphorus liberated from radicular human dentin by
EDTA solutions (0.3M) at different pH values. Each Point representsthe mean of six determinations. The results presented here are in agreement with the data of Nikiforuk and Sreebny,4 who analyzed the pH effect (from 6.02 to 12.01) in EDTA solutions (0.25M) and demonstrated that at pH 6.02, 8 days were needed for the total demineralization of rats’ mandibles, whereas at pH 12.01 18 days were required. Although the maximum solubilization of calcium carbonate by EDTA solution can be reached at pH 7.30,5 this difference in behavior may be explained by different solubility products of calcium carbonate and hydroxyapatite with respect to pH. CONCLUSIONS
The following conclusions can be inferred: (1) The efficiency of EDTA solutions on the demineralization of dentin is influenced by pH. (2) The greatest demineralizing efficiency of EDTA solutions (0.3M) can be achieved between pH 5.00 and 6.00. We wish to thank Dr. Henrique V. Amorim (ESALQUSP) for the use of the pulverizer apparatus. REFERENCES 1. Asgar, L.: Chemical Analysis of Human Teeth, J. Dent. Res. 35: 742-748, 1956. 2. Council of Dental Therapeutics: Accepted Dental Remedies, ed. 3 1, Chicago, 1966, American Dental Association, p. 207, op.cit. ref. 8. 3. Colowick, S. P., and Kaplan, N. 0.: Methods in Enzymology, vol. III, New York, 1957, Academic Press, Inc., pp. 840-850. 4. Nikiforuk, G., and Sreebny, L. M.: Demineralization of Hard Tissues by Organic Chelating Agents at Neutral pH, J. Dent. Res. 32: 859-867, 1953.
448
Cury, Bragotto, and Valdrighi
Powerful Organic Chelating Agents: Technical Bulletin No. 2, Framingham, Mass., Bersworth Chemical Company, opcit. ref. 4. ijstby, B. N.: Chelation in Root Canal Therapy, Odont. Tidskr. 65: 3-11, 1957. Sand, H. F.: The Dissociation of EDTA and EDTA-sodium salts, Acta Odont. Stand. 19: 469-482, 1961. Seidberg, B. H., and Schilder, H.: An Evaluation of EDTA in Endodontics, ORAL SURG. 37: 609-620, 1974.
Oral Surg. October, I98 1 9. Lazzari, E. P.: Dental Biochemistry, ed. 2, Philadelphia, Lea & Febiger, Publishers, pp. 120-121. Reprint requests to: Dr. Jaime A. Cury Dentistry School of Piicicaba State University of Campinas S~O Paulo. Brazil
1976.