- Email: [email protected]
In Vitro Evaluation of the Cytotoxic Effects of Acid Solutions Used as Canal Irrigants
Basic Research—Technology
In Vitro Evaluation of the Cytotoxic Effects of Acid Solutions Used as Canal Irrigants C. F. Malheiros, MDS, M. M. Marques, MDS, DDS, and G. Gavini, MDS, DDS Abstract Solutions of EDTA and citric acid have been used as canal irrigants. These substances must be compatible with apical periodontal tissue. The aim of this study was to evaluate comparatively the cytotoxicity of a 17% EDTA solution and that of three solutions with different concentrations of citric acid (10, 15, and 25%) on cultured fibroblasts. The solutions were diluted to 0.1% and 0.5% in culture medium and then applied to NIH 3T3 cells. After 0, 6, 12, and 24 h (short-term assay; viability) and 1, 3, 5, and 7 days (long-term assay; survival), the cells were counted. The data were compared by ANOVA. In the short-term experiments, all solutions presented a percentage of cell viability similar to that of control cells, except for the 17% EDTA solution diluted to 0.5%. After the long-term assay, all groups presented a continuous and progressive cell growth except for the 17% EDTA solution and for the 25% citric acid solution at a 0.5% dilution. The citric acid solution did not impair cell growth and viability, proving to be noncytotoxic in vitro.
Key Words Root canal irrigants, cell culture, cytotoxicity
From the Department of Endodontics, School of Dentistry, University of Sa˜o Paulo, Cidade Universita´ria, Sa˜o Paulo, SP, Brazil. Address requests for reprint to Dr. Giulio Gavini, Disciplina de Endodontia, Departamento de Dentı´stica, Faculdade de Odontologia, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 2227, Cidade Universita´ria Sa˜o Paulo SP, 05508-900, Brazil. E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
746
Malheiros et al.
A
cid solutions have been recommended for endodontic treatment purposes since 1957 (1). These irrigating solutions promote smear-layer removal, dentine wall cleaning, and root canal disinfection (2–5). More recently, irrigating solutions have been analyzed under different point of views by several authors (6 –11). Some of them are still looking for solutions capable of desinfecting the dentin, removing the smear layer, opening dentinal tubules and allowing the antibacterial agents to penetrate the entire root canal system. Among of them, Torabinejad et al. (11) presented a new solution, a mixture of a tetracycline isomer, an acid, and detergent (MTAD) with good results. Scelza et al. (6) showed a high cleansing capacity of 10% citric acid solution. We have previously shown that citric acid used in different concentrations is an effective dentin demineralizer (5). Some authors have focused their studies on the effect of irrigating solutions such as 17% EDTA and 50% citric acid solutions on the antibacterial effect of root canal medication (9). Siqueira et al. (8) analyzed the effect of several irrigating solutions, including the combined irrigation with 2.5% NaOCl and citric acid showing important reduction of infection within the root canal. The acid solutions when used as irrigating because of the removal of smear layer of the root canal walls are able to improve the post retention (7). On the other hand, although these acid solutions promote the penetration of the endodontic sealers into the dentinal tubules, the sealer bond strength is not increased (10). There are some studies on the biological effects of citric acid solutions (12–14), and EDTA solutions (13–17). However, there are only few studies comparing the in vivo cytotoxic effects of citric acid and EDTA solutions, and no data on the comparative in vitro cytotoxic effects of these two acid solutions were found in the literature. There is a relation between the cytotoxcity of a given substance in vitro and its irritant effect in vivo (18). The aim of this study was to evaluate comparatively the cytotoxicity of a 17% EDTA solution and that of three different concentration solutions of citric acid (10, 15, and 25%) on cultured fibroblasts using the Trypan blue dye exclusion assay.
Materials and Methods The cytotoxicity of a 17% EDTA solution, at 0.1% and 0.5% dilutions, and three different concentration solutions of citric acid (10, 15, and 25%) was measured in vitro. NIH-3T3 cells (CRL 1658) obtained from American Type Culture Collection (Rockville, MD) were grown at 37°C in Dulbecco⬘s modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented by 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil) and 1% antibiotic-antimycotic solution (Sigma) in a 5% CO2 atmosphere with 95% humidity. The experimental groups were: (I) control, cultures grown on fresh DMEM; (II) DMEM containing 17% EDTA solution diluted to 0.1%; (III) DMEM containing 25% citric acid solution diluted to 0.1%; (IV) DMEM containing 15% citric acid solution diluted to 0.1%; (V) DMEM containing 10% citric acid solution diluted to 0.1%; (VI) DMEM containing 17% EDTA solution diluted to 0.5%; (VII) cultures treated with a 25% citric acid solution diluted to 0.5%; (VIII) DMEM containing 15% citric acid solution diluted to 0.5%; (IX) cultures treated with a 10% citric acid solution diluted to 0.5%. Both an immediate or short-term response and a long-term survival that measured the retention of the self-renewal capacity of the cells were analyzed.
JOE — Volume 31, Number 10, October 2005
Basic Research—Technology Short-Term Assay (cell viability) Cells (2 ⫻ 104) were plated on 60 mm Petri dishes. Three days later, the experimental culture medium was exchanged for medium containing the dilutions of the testing solutions (II through IX) and the control cultures (I) received fresh medium. The media inside the dishes were discarded and replaced by the irrigating solutions diluted in fresh DME medium containing 10% fetal bovine serum. After 0, 6, 12, and 24 h the cells of three dishes per group were counted, and viability curves were plotted. Because cultured cells can be prepared in a uniform suspension in most cases, the provision of large numbers of replicates is often unnecessary. Usually three replicates are sufficient, and for many simple observations, e.g. cell counting, duplicates may be sufficient (19). Long-Term Assay (cell survival) Cells (1 ⫻ 104) were plated on 60 mm diameter culture dishes. After 6 h, when the cells were all attached to the bottom of the Petri dishes, the culture medium was exchanged for culture medium containing the dilutions of the test solutions (II through IX) and, the control cultures (I) received fresh DMEM. On 1, 3, 5, and 7 days after seeding, the cells were counted and growth curves were constructed. Maintenance of cell viability was obtained by exchanging half of the culture medium by fresh DMEM every 3 days. With this procedure during the experimental time the testing solutions were equally diluted in all experimental groups, simulating what would occurs at the in vivo tissue where the circulatory system (blood and lymphatic vessels) dilutes foreign substances. Growth and Viability Cell Curves Growth curves were constructed as described in other studies (13, 19, 20). Briefly, the cell number was determined by counting the viable cells in a hemocytometer using the Trypan blue dye exclusion assay. For each time period, three dishes of each group were counted. The number of viable cells harvested from each Petri dish was obtained by the following mathematical equation: UC ⫻ D ⫻ 104/#SQ, where UC ⫽ unstained cell count (viable cells), D ⫽ the dilution of the cell suspension, and #SQ ⫽ number of squares of the hemocytometer counted. The viability percentage of the cell population of each Petri dish was obtained by the following mathematical equation: UC/TC ⫻ 100, where UC ⫽ unstained cell count (viable cells) and TC ⫽ total cell count (stained plus unstained cells). Statistical Analysis Each data point corresponded to the mean ⫾ SEM of either cell count or percentages of cell viability from 3 dishes. The data were compared by ANOVA complemented by the Tukey’s test. The level of significance was 5% (p ⱕ 0.05).
Figure 1. Growth curves of NIH 3T3 cells treated with acid solutions diluted up to 0.1% and control. All cultures present progressive cell growth. Cultures treated with the 17% EDTA solution (A) present significantly smaller cell counts than the cultures in the other groups (p ⱕ 0.05).
Figure 2. Growth curves of NIH 3T3 cells submitted to acid solutions diluted up to 0.5% and control. There is no cell growth in cultures treated with the 17% EDTA solution. Growth in cultures treated with the 25% citric acid solution (a) is significantly smaller than that of control cultures and cultures treated with 10% and 15% citric acid solutions (p ⱕ 0.05).
through V) from day 1 to day 7 (Fig. 1). Cultures treated with 17% EDTA diluted up to 0.1% (II) presented a significantly smaller cell count than did other groups (I, III, through V). Cell viability in these groups was maintained between 92% and 98%. When diluted up to 0.5%, the 17% EDTA (VI) solution obstructed cell growth (Fig. 2) and there were no viable cells in the dishes at any experimental time. The groups treated with 25% citric acid diluted to 0.5% (VII) presented a significantly smaller cell growth than did the other groups (I, VII, IX) (Fig. 2), but cell viability was similar to that of these groups. In GVII, the cell growth rate increased 5 days after seeding (Fig. 2). Growth and viability in groups I, VII, and IX were similar to each other, with no statistical differences.
Results Short-Term Assay Throughout the experimental time (0 –24 h), control (I) and treated cultures (II through V) maintained stable cell viability (87.67– 96.53%), when the testing solutions were diluted to 0.1%. When the dilution was 0.5%, the cultures treated with 17% EDTA (GVI) displayed a striking drop in cell viability (%) reaching total cell death within 6 h. The other groups (VII through IX) maintained cell viability of approximately 95%. Long-Term Assay There was a progressive cell growth in the control cultures (I) and in the cultures treated with the testing solutions diluted up to 0.1% (II JOE — Volume 31, Number 10, October 2005
Discussion Cytotoxicity can be defined as the in vitro effects of some substance or procedure at the cellular level. These effects can be measured using a cell culture technique that is cheap, reproducible and allows easy quantification. Moreover, this technique allows the study of cell behavior in a controlled environment, free from the organism’s complex interactions (19). Using the Trypan blue dye exclusion assay we compared the cytotoxicity of a 17% EDTA solution and that of three different concentration solutions of citric acid (10, 15, and 25%) on cultured fibroblasts (NIH3T3 cell line). Our results showed that the 17% EDTA solution caused higher cytotoxic effects than did the tested citric acid solutions.
Cytotoxic Effects of Acid Solutions
747
Basic Research—Technology This finding is in agreement with the previous studies developed by Souza (14). Cultures treated with 17% EDTA diluted to 0.1% presented significantly smaller cell counts than did the other groups. When the 17% EDTA solution was diluted to 0.5%, cell growth did not occur and there were no viable cells at any experimental time. Atalla and Calvert (21) previously observed a similar cytotoxic aggressiveness of the EDTA solution, as also did Koulaouzidou (17). The acid solutions applied on the fibroblasts were diluted to 0.1% and 0.5% because cultured cells are more susceptible to drug toxic effects than the periapical tissue. In the body, the phagocytic cells and the lymph and blood channels all help to dilute and carry away the drug (18). The cell viability of cultures treated with citric acid solutions at all tested concentrations (10, 15, and 25%) and dilutions (0.1% and 0.5%) were similar to that of control cultures. Additionally, cells treated with citric acid solutions do not lose their ability to enter in cell cycle; they retain their proliferation activity, as demonstrated by the long-term assay. These results clearly demonstrate that the citric acid solution is noncytotoxic to fibroblasts in culture. However, other of our results were intriguing, because although the growth of cultures treated with 0.5% dilution of 25% citric acid solution was impaired, the cell viability was not affected. These results could be a result of an inhibition of proliferative activity of the cells, not by killing them but by having a reversible effect on the cell cycle rhythm. The cells, although alive, were not capable of proliferating. However, this effect was terminated when, after two medium changes, when the irrigating solution were more diluted, the growth rate increased. Although EDTA and citric acid solutions at several different concentrations are efficient irrigating solutions (1– 6) clinicians must be aware of the fact that these solutions will be in direct contact with periodontal tissues. They must also know that higher concentrations of these acids lead to a higher cytotoxicity (14, 15). Even though it is an organic acid that takes part in the Krebs cycle, present in the cells of every superior animal, and is also biologically acceptable (14), in our study we have demonstrated that citric acid, when applied at high concentrations, leads to a delay in cell growth. The measurement of cytotocxicity, as carried out in the present study, is a purely cellular event. Thus, it does not reflect in vivo situations where a complex pharmacokinetics of drug exposure takes place (19). New studies are needed to assess the tissue reactions that occur when irrigants are applied in the root canal. Moreover, these studies should focus on how these solutions act, aiming at improving their effectiveness.
References 1. Nygaard-O¨stby B. Chelation in root canal therapy: Ethylenediamine Tetracetic Acid for cleansing and widening root canals. Odontol Tskr 1957;65:3–11.
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2. Patterson S. In vivo and in vitro studies of the effect of the disodium salt of EDTA on human dentine. Oral Surg Oral Med Oral Pathol 1963;16:83–103. 3. McComb D, Smith DC. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1975;1:238 – 42. 4. Yamada RS, Armas A, Goldman M, Lin PS. A scanning electron microscopic comparison of a high volume flush with several irrigating solutions. Part III. J Endod 1983; 9:137– 42. 5. Malheiros CF, Gavini G. Desmineralizing effect of citric acid in different concentrations over different periods of time. Rev Odontol Univ Sa˜o Paulo 1998;5:64 – 8. 6. Scelza MFZ, Antoniazzi JH, Scelza P. Efficacy of final irrigation: a scanning electron microscopic evaluation. J Endod 2000;26:355– 8. 7. Mayhew JT, Windchy AM, Goldsmith J, Gettleman L. Effect of root canal sealers and irrigation agents on retention of preformed posts luted with a resin cement. J Endod 2000;26:341– 4. 8. Siqueira JF Jr, Roˆc¸as IN, Santos SRLD, Lima KC, Magalha˜es FAC, Uzeda M. Efficacy of instrumentation techniques and irrigation regimens in reducing the bacterial population within root canals. J Endod 2002;28:181– 4. 9. Portenier I, Haapasalo H, Yamauchi M, Haapasalo M. Inactivation of the antibacterial activity of iodine potassium iodide and chlorexidien digluconate against Enterococcus faecalis by dentin, dentin matrix, type-I collagen, and heat-killed microbial whole cells. J Endod 2002;28:634 –7. 10. Saleh IM, Ruyter IE, Haapasalo MP, Orstavik D. Adhesion of endodontic sealers: scanning electron microscopy and energy dispersive spectroscopy. J Endod 2003; 29:595– 601. 11. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 12. Chan CP, Jeng JH, Hsieh CC, Lin CL, Lei D, Chang MC. Morphological alterations associated with the cytotoxic and cytostatic effects of citric acid on cultured human dental pulp cells. J Endod 1999;25:354 – 8. 13. Scelza MFZ, Daniel RLDP, Santos EM, Jaeger MMM. Cytotoxic effects pf 10% citric acid and EDTA-T used as root canal irrigants: an in vitro study. J Endod 2001;27: 741–3. 14. Souza SMG. Evaluation of EDTA, EGTA and citric acid subcutaneously injected in mice. (Master’s thesis), Faculdade de Odontologia, Universidade de Sa˜o Paulo, Bauru, 1999, p 47. 15. Salomon JP, Franquin JC. Cytotoxicite´ osseuse d’une solution che´latante utilise´e en endodontie. Rev Fr Endod 1988;7:29 –36. 16. Segura JJ, Calvo JR, Guerrero JM, Jimenes Planas A, Liamas R. EDTA inhibits the adherence capacity of macrophages: endodontic implications. J Endod 1997;23: 205– 8. 17. Koulaouzidou EA, Margelos J, Beltes P, Kortsaris A. Cytotoxic effects of different concentrations of neutral and alkaline EDTA solutions used as root canal irrigants. J Endod 1999;25:21–3. 18. Wall GL, Dowson J, Shipman C Jr. Antibacterial efficacy and cytotoxicity of three endodontic drugs. Oral Surg Oral Med Oral Pathol 1972;33:230 – 41. 19. Freshney, RI. Measurement of cytotoxic and viability. In: Culture of animal cells: a manual of basic technique, 2nd ed. New York, Wiley-Liss; 1990:245–56. 20. Azevedo CL, Marques MM, Bombana AC. Cytotoxic effects of cyanoacrylates used as retrograde filling materials: an in vitro analysis. Pesqui Odontol Bras 2003;17:113– 8. 21. Attalla MN, Calvert JM. Irritational properties of root canal medicaments. J Can Dent Assoc 1969;35:76 – 82.
JOE — Volume 31, Number 10, October 2005
In Vitro Evaluation of the Cytotoxic Effects of Acid Solutions Used as Canal Irrigants C. F. Malheiros, MDS, M. M. Marques, MDS, DDS, and G. Gavini, MDS, DDS Abstract Solutions of EDTA and citric acid have been used as canal irrigants. These substances must be compatible with apical periodontal tissue. The aim of this study was to evaluate comparatively the cytotoxicity of a 17% EDTA solution and that of three solutions with different concentrations of citric acid (10, 15, and 25%) on cultured fibroblasts. The solutions were diluted to 0.1% and 0.5% in culture medium and then applied to NIH 3T3 cells. After 0, 6, 12, and 24 h (short-term assay; viability) and 1, 3, 5, and 7 days (long-term assay; survival), the cells were counted. The data were compared by ANOVA. In the short-term experiments, all solutions presented a percentage of cell viability similar to that of control cells, except for the 17% EDTA solution diluted to 0.5%. After the long-term assay, all groups presented a continuous and progressive cell growth except for the 17% EDTA solution and for the 25% citric acid solution at a 0.5% dilution. The citric acid solution did not impair cell growth and viability, proving to be noncytotoxic in vitro.
Key Words Root canal irrigants, cell culture, cytotoxicity
From the Department of Endodontics, School of Dentistry, University of Sa˜o Paulo, Cidade Universita´ria, Sa˜o Paulo, SP, Brazil. Address requests for reprint to Dr. Giulio Gavini, Disciplina de Endodontia, Departamento de Dentı´stica, Faculdade de Odontologia, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 2227, Cidade Universita´ria Sa˜o Paulo SP, 05508-900, Brazil. E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
746
Malheiros et al.
A
cid solutions have been recommended for endodontic treatment purposes since 1957 (1). These irrigating solutions promote smear-layer removal, dentine wall cleaning, and root canal disinfection (2–5). More recently, irrigating solutions have been analyzed under different point of views by several authors (6 –11). Some of them are still looking for solutions capable of desinfecting the dentin, removing the smear layer, opening dentinal tubules and allowing the antibacterial agents to penetrate the entire root canal system. Among of them, Torabinejad et al. (11) presented a new solution, a mixture of a tetracycline isomer, an acid, and detergent (MTAD) with good results. Scelza et al. (6) showed a high cleansing capacity of 10% citric acid solution. We have previously shown that citric acid used in different concentrations is an effective dentin demineralizer (5). Some authors have focused their studies on the effect of irrigating solutions such as 17% EDTA and 50% citric acid solutions on the antibacterial effect of root canal medication (9). Siqueira et al. (8) analyzed the effect of several irrigating solutions, including the combined irrigation with 2.5% NaOCl and citric acid showing important reduction of infection within the root canal. The acid solutions when used as irrigating because of the removal of smear layer of the root canal walls are able to improve the post retention (7). On the other hand, although these acid solutions promote the penetration of the endodontic sealers into the dentinal tubules, the sealer bond strength is not increased (10). There are some studies on the biological effects of citric acid solutions (12–14), and EDTA solutions (13–17). However, there are only few studies comparing the in vivo cytotoxic effects of citric acid and EDTA solutions, and no data on the comparative in vitro cytotoxic effects of these two acid solutions were found in the literature. There is a relation between the cytotoxcity of a given substance in vitro and its irritant effect in vivo (18). The aim of this study was to evaluate comparatively the cytotoxicity of a 17% EDTA solution and that of three different concentration solutions of citric acid (10, 15, and 25%) on cultured fibroblasts using the Trypan blue dye exclusion assay.
Materials and Methods The cytotoxicity of a 17% EDTA solution, at 0.1% and 0.5% dilutions, and three different concentration solutions of citric acid (10, 15, and 25%) was measured in vitro. NIH-3T3 cells (CRL 1658) obtained from American Type Culture Collection (Rockville, MD) were grown at 37°C in Dulbecco⬘s modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented by 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil) and 1% antibiotic-antimycotic solution (Sigma) in a 5% CO2 atmosphere with 95% humidity. The experimental groups were: (I) control, cultures grown on fresh DMEM; (II) DMEM containing 17% EDTA solution diluted to 0.1%; (III) DMEM containing 25% citric acid solution diluted to 0.1%; (IV) DMEM containing 15% citric acid solution diluted to 0.1%; (V) DMEM containing 10% citric acid solution diluted to 0.1%; (VI) DMEM containing 17% EDTA solution diluted to 0.5%; (VII) cultures treated with a 25% citric acid solution diluted to 0.5%; (VIII) DMEM containing 15% citric acid solution diluted to 0.5%; (IX) cultures treated with a 10% citric acid solution diluted to 0.5%. Both an immediate or short-term response and a long-term survival that measured the retention of the self-renewal capacity of the cells were analyzed.
JOE — Volume 31, Number 10, October 2005
Basic Research—Technology Short-Term Assay (cell viability) Cells (2 ⫻ 104) were plated on 60 mm Petri dishes. Three days later, the experimental culture medium was exchanged for medium containing the dilutions of the testing solutions (II through IX) and the control cultures (I) received fresh medium. The media inside the dishes were discarded and replaced by the irrigating solutions diluted in fresh DME medium containing 10% fetal bovine serum. After 0, 6, 12, and 24 h the cells of three dishes per group were counted, and viability curves were plotted. Because cultured cells can be prepared in a uniform suspension in most cases, the provision of large numbers of replicates is often unnecessary. Usually three replicates are sufficient, and for many simple observations, e.g. cell counting, duplicates may be sufficient (19). Long-Term Assay (cell survival) Cells (1 ⫻ 104) were plated on 60 mm diameter culture dishes. After 6 h, when the cells were all attached to the bottom of the Petri dishes, the culture medium was exchanged for culture medium containing the dilutions of the test solutions (II through IX) and, the control cultures (I) received fresh DMEM. On 1, 3, 5, and 7 days after seeding, the cells were counted and growth curves were constructed. Maintenance of cell viability was obtained by exchanging half of the culture medium by fresh DMEM every 3 days. With this procedure during the experimental time the testing solutions were equally diluted in all experimental groups, simulating what would occurs at the in vivo tissue where the circulatory system (blood and lymphatic vessels) dilutes foreign substances. Growth and Viability Cell Curves Growth curves were constructed as described in other studies (13, 19, 20). Briefly, the cell number was determined by counting the viable cells in a hemocytometer using the Trypan blue dye exclusion assay. For each time period, three dishes of each group were counted. The number of viable cells harvested from each Petri dish was obtained by the following mathematical equation: UC ⫻ D ⫻ 104/#SQ, where UC ⫽ unstained cell count (viable cells), D ⫽ the dilution of the cell suspension, and #SQ ⫽ number of squares of the hemocytometer counted. The viability percentage of the cell population of each Petri dish was obtained by the following mathematical equation: UC/TC ⫻ 100, where UC ⫽ unstained cell count (viable cells) and TC ⫽ total cell count (stained plus unstained cells). Statistical Analysis Each data point corresponded to the mean ⫾ SEM of either cell count or percentages of cell viability from 3 dishes. The data were compared by ANOVA complemented by the Tukey’s test. The level of significance was 5% (p ⱕ 0.05).
Figure 1. Growth curves of NIH 3T3 cells treated with acid solutions diluted up to 0.1% and control. All cultures present progressive cell growth. Cultures treated with the 17% EDTA solution (A) present significantly smaller cell counts than the cultures in the other groups (p ⱕ 0.05).
Figure 2. Growth curves of NIH 3T3 cells submitted to acid solutions diluted up to 0.5% and control. There is no cell growth in cultures treated with the 17% EDTA solution. Growth in cultures treated with the 25% citric acid solution (a) is significantly smaller than that of control cultures and cultures treated with 10% and 15% citric acid solutions (p ⱕ 0.05).
through V) from day 1 to day 7 (Fig. 1). Cultures treated with 17% EDTA diluted up to 0.1% (II) presented a significantly smaller cell count than did other groups (I, III, through V). Cell viability in these groups was maintained between 92% and 98%. When diluted up to 0.5%, the 17% EDTA (VI) solution obstructed cell growth (Fig. 2) and there were no viable cells in the dishes at any experimental time. The groups treated with 25% citric acid diluted to 0.5% (VII) presented a significantly smaller cell growth than did the other groups (I, VII, IX) (Fig. 2), but cell viability was similar to that of these groups. In GVII, the cell growth rate increased 5 days after seeding (Fig. 2). Growth and viability in groups I, VII, and IX were similar to each other, with no statistical differences.
Results Short-Term Assay Throughout the experimental time (0 –24 h), control (I) and treated cultures (II through V) maintained stable cell viability (87.67– 96.53%), when the testing solutions were diluted to 0.1%. When the dilution was 0.5%, the cultures treated with 17% EDTA (GVI) displayed a striking drop in cell viability (%) reaching total cell death within 6 h. The other groups (VII through IX) maintained cell viability of approximately 95%. Long-Term Assay There was a progressive cell growth in the control cultures (I) and in the cultures treated with the testing solutions diluted up to 0.1% (II JOE — Volume 31, Number 10, October 2005
Discussion Cytotoxicity can be defined as the in vitro effects of some substance or procedure at the cellular level. These effects can be measured using a cell culture technique that is cheap, reproducible and allows easy quantification. Moreover, this technique allows the study of cell behavior in a controlled environment, free from the organism’s complex interactions (19). Using the Trypan blue dye exclusion assay we compared the cytotoxicity of a 17% EDTA solution and that of three different concentration solutions of citric acid (10, 15, and 25%) on cultured fibroblasts (NIH3T3 cell line). Our results showed that the 17% EDTA solution caused higher cytotoxic effects than did the tested citric acid solutions.
Cytotoxic Effects of Acid Solutions
747
Basic Research—Technology This finding is in agreement with the previous studies developed by Souza (14). Cultures treated with 17% EDTA diluted to 0.1% presented significantly smaller cell counts than did the other groups. When the 17% EDTA solution was diluted to 0.5%, cell growth did not occur and there were no viable cells at any experimental time. Atalla and Calvert (21) previously observed a similar cytotoxic aggressiveness of the EDTA solution, as also did Koulaouzidou (17). The acid solutions applied on the fibroblasts were diluted to 0.1% and 0.5% because cultured cells are more susceptible to drug toxic effects than the periapical tissue. In the body, the phagocytic cells and the lymph and blood channels all help to dilute and carry away the drug (18). The cell viability of cultures treated with citric acid solutions at all tested concentrations (10, 15, and 25%) and dilutions (0.1% and 0.5%) were similar to that of control cultures. Additionally, cells treated with citric acid solutions do not lose their ability to enter in cell cycle; they retain their proliferation activity, as demonstrated by the long-term assay. These results clearly demonstrate that the citric acid solution is noncytotoxic to fibroblasts in culture. However, other of our results were intriguing, because although the growth of cultures treated with 0.5% dilution of 25% citric acid solution was impaired, the cell viability was not affected. These results could be a result of an inhibition of proliferative activity of the cells, not by killing them but by having a reversible effect on the cell cycle rhythm. The cells, although alive, were not capable of proliferating. However, this effect was terminated when, after two medium changes, when the irrigating solution were more diluted, the growth rate increased. Although EDTA and citric acid solutions at several different concentrations are efficient irrigating solutions (1– 6) clinicians must be aware of the fact that these solutions will be in direct contact with periodontal tissues. They must also know that higher concentrations of these acids lead to a higher cytotoxicity (14, 15). Even though it is an organic acid that takes part in the Krebs cycle, present in the cells of every superior animal, and is also biologically acceptable (14), in our study we have demonstrated that citric acid, when applied at high concentrations, leads to a delay in cell growth. The measurement of cytotocxicity, as carried out in the present study, is a purely cellular event. Thus, it does not reflect in vivo situations where a complex pharmacokinetics of drug exposure takes place (19). New studies are needed to assess the tissue reactions that occur when irrigants are applied in the root canal. Moreover, these studies should focus on how these solutions act, aiming at improving their effectiveness.
References 1. Nygaard-O¨stby B. Chelation in root canal therapy: Ethylenediamine Tetracetic Acid for cleansing and widening root canals. Odontol Tskr 1957;65:3–11.
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JOE — Volume 31, Number 10, October 2005