Comparison of Cytotoxicity between Mineral Trioxide Aggregate Mixed with Chlorhexidine and Common Endodontic Regeneration Medicaments on Periodontal Ligament Stem Cells: an in Vitro Study
Abstract
Objectives: The combination of mineral trioxide aggregate (MTA) and 2% chlorhexidine (CHX) has been recently introduced as an intracanal medicament. The aim of this study was to evaluate the potential cytotoxic effects of MTA mixed with 2% chlorhexidine gel on human periodontal ligament stem cells (PDLSCs) and compare it with other common endodontic regeneration medicaments.
Materials and Methods: Minimum inhibitory concentration and minimum bactericidal concentration of six experimental groups against Enterococcus faecalis was determined. The study groups consisted of RetoMTA mixed with 2% chlorhexidine gel (MTA+CHX), calcium hydroxide (CH), CH mixed with CHX gel, two concentrations of double antibiotic paste, and 2% CHX. The direct cytotoxic effect of minimum bactericidal concentration was evaluated by MTT on PDLSCs on days 1, 3, and 7. One-way ANOVA and post hoc tests were used for data analysis (P<0.05).
Results: The viability of cells treated with MTA+CHX decreased significantly over time (P<0.05) making this group the most cytotoxic intracanal medicament on the 3rd and 7th days of treatment. On day one, the highest viability percentage was detected in the CH+CHX group followed by the CHX group. On day 3, CH+CHX and CHX groups displayed the highest viability percentage. On day 7, the highest viability was observed in the CHX group, which showed no significant difference with the control group (P=0.12).
Conclusion: Regarding the antimicrobial potency of intracanal medicaments at minimum bactericidal concentration levels, CHX gel appears to be the least cytotoxic drug, while MTA+CHX shows the highest reduction in viability percentage
1. Fouad AF, Khan AA. Etiology and pathogenesis of pulpitis and apical periodontitis. In: Ørstavik D, (editor). Essential endodontology: prevention and treatment of apical periodontitis, 3rd ed. Hoboken, NJ: John Wiley & Sons Ltd; 2019. p. 59–90.
2. El Karim I, Kennedy J, Hussey D. The antimicrobial effects of root canal irrigation and medication. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007 Apr;103(4):560-9.
3. Nakashima M, Akamine A. The application of tissue engineering to regeneration of pulp and dentin in endodontics. J Endod. 2005 Oct;31(10):711-8.
4. Fouad AF. Microbial Factors and Antimicrobial Strategies in Dental Pulp Regeneration. J Endod. 2017 Sep;43(9S) :S46-S50.
5. Galler KM. Clinical procedures for revitalization: current knowledge and considerations. Int Endod J. 2016 Oct;49(10):926-36.
6. AAE Clinical Considerations for a Regenerative Procedure. Available at: https://www.aae.org/specialty/wp-content/uploads/sites/2/2017/06/currentregenerativeendodonticconsiderations.pdf. Accessed Feb 11, 2023.
7. Galler KM, Krastl G, Simon S, Van Gorp G, Meschi N, Vahedi B, et al. European Society of Endodontology position statement: Revitalization procedures. Int Endod J. 2016 Aug;49(8):717-23.
8. Siqueira Jr J, Lopes H. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J. 1999;32(5):361-9.
9. Qadiri SY, Mustafa S. Role of calcium hydroxide in root canal therapy: A comprehensive review. JAMDSR. 2019;7(7):1-3.
10. Neelakantan P, Sriraman P, Gutmann JL. Removal of calcium hydroxide intracanal medicament by different irrigants and irrigating techniques: a cone beam computed tomography analysis. Gen Dent. 2017 Nov-Dec;65(6):45-49.
11. Jenks DB, Ehrlich Y, Spolnik K, Gregory RL, Yassen GH. Residual antibiofilm effects of various concentrations of double antibiotic paste used during regenerative endodontics after different application times. Arch Oral Biol. 2016 Oct;70:88-93.
12. Manochehrifar H, Parirokh M, Kakooei S, Oloomi MM, Asgary S, Eghbal MJ, Mashhadi Abbas F. The Effect of Mineral Trioxide Aggregate Mixed with Chlorhexidine as Direct Pulp Capping Agent in Dogs Teeth: A Histologic Study. Iran Endod J. 2016 Fall;11(4):320-324.
13. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review--part II: leakage and biocompatibility investigations. J Endod. 2010 Feb;36(2):190-202.
14. Estrela C, Bammann LL, Estrela CR, Silva RS, Pécora JD. Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal. Braz Dent J. 2000;11(1):3-9.
15. Miyagak DC, de Carvalho EM, Robazza CR, Chavasco JK, Levorato GL. In vitro evaluation of the antimicrobial activity of endodontic sealers. Braz Oral Res. 2006 Oct-Dec;20(4):303-6.
16. Bidar M, Naderinasab M, Talati A, Ghazvini K, Asgari S, Hadizadeh B, Gharechahi M, Mashadi NA. The effects of different concentrations of chlorhexidine gluconate on the antimicrobial properties of mineral trioxide aggregate and calcium enrich mixture. Dent Res J (Isfahan). 2012 Jul;9(4):466-71.
17. Mahmoud O, Al-Meeri WA, Farook MS, Al-Afifi NA. Calcium Silicate-Based Cements as Root Canal Medicament. Clin Cosmet Investig Dent. 2020 Feb 24;12:49-60.
18. Alarbeed MS, Ahmed GM, Elkhodary S, Shaker OG. Effect of combination of calcium hydroxide and chlorhexidine gel 2% as intracanal medication in comparison to calcium hydroxide paste as intracanal medication on postoperative pain and bacterial endotoxin in necrotic teeth: a randomized controlled trial. Acta Scien Dent. 2019;3:124-30.
19. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001 Jul;48 Suppl 1:5-16.
20. Sabrah AH, Yassen GH, Gregory RL. Effectiveness of antibiotic medicaments against biofilm formation of Enterococcus faecalis and Porphyromonas gingivalis. J Endod. 2013 Nov;39(11):1385-9.
21. Khoshkhounejad M, Sobhi Afshar M, Jabalameli F, Emaneini M, Sharifian M. Cytotoxicity Evaluation of Minimum Antibacterial Values of Different Medicaments Used in Endodontic Regenerative Procedures. Eur J Dent. 2019 Oct;13(4):514-520.
22. Markossian S, Grossman A, Brimacombe K, Arkin M, Auld D, Austin C, et al. Assay guidance manual (Internet). Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences;2004.
23. Conde MCM, Chisini LA, Sarkis-Onofre R, Schuch HS, Nör JE, Demarco FF. A scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation. Int Endod J. 2017 Sep;50(9):860-874.
24. Shimizu E, Jong G, Partridge N, Rosenberg PA, Lin LM. Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure. J Endod. 2012 Sep;38(9):1293-7.
25. Lin LM, Rosenberg PA. Repair and regeneration in endodontics. Int Endod J. 2011 Oct;44(10):889-906.
26. Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet. 2006 Apr;7(4):319-27.
27. Farhad Mollashahi N, Saberi E, Karkehabadi H. Evaluation of Cytotoxic Effects of Various Endodontic Irrigation Solutions on the Survival of Stem Cell of Human Apical Papilla. Iran Endod J. 2016 Fall;11(4):293-297.
28. Kruse CR, Singh M, Targosinski S, Sinha I, Sørensen JA, Eriksson E, Nuutila K. The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study. Wound Repair Regen. 2017 Apr;25(2):260-269.
29. Hernandez EP, Botero TM, Mantellini MG, McDonald NJ, Nör JE. Effect of ProRoot MTA mixed with chlorhexidine on apoptosis and cell cycle of fibroblasts and macrophages in vitro. Int Endod J. 2005 Feb;38(2):137-43.
30. Sumer M, Muglali M, Bodrumlu E, Guvenc T. Reactions of connective tissue to amalgam, intermediate restorative material, mineral trioxide aggregate, and mineral trioxide aggregate mixed with chlorhexidine. J Endod. 2006 Nov;32(11):1094-6.
31. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod. 2012 Oct;38 (10) :1372-5.
32. Saberi EA, Gholami F, Pirhaji A, Shahreki E. In vitro cytotoxic effects of different intracanal medicaments on the co-culture of dental mesenchymal stem cells using the MTT assay. Giornale Italiano di Endodonzia. 2021 Apr 7;35:10-19.
33. Soares Ade J, Lins FF, Nagata JY, Gomes BP, Zaia AA, Ferraz CC, de Almeida JF, de Souza-Filho FJ. Pulp revascularization after root canal decontamination with calcium hydroxide and 2% chlorhexidine gel. J Endod. 2013 Mar;39(3):417-20.
34. Jones CG. Chlorhexidine: is it still the gold standard? Periodontol 2000. 1997 Oct;15:55-62.
35. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009 Apr;42(4):288-302.
36. Hamed SJ, AL-Yasiri IK, Ali NT, Al-Feron MA. Antibacterial activity of calcium hydroxide combined with chlrohexidine or sodium hypochlorite against gram positive and gram negative bacteria. J Nat Sci Res. 2014;4(12):55-61.
37. de Souza LB, de Aquino SG, de Souza PP, Hebling J, Costa CA. Cytotoxic effects of different concentrations of chlorhexidine. Am J Dent. 2007 Dec;20(6):400-4.
38. Tu YY, Yang CY, Chen RS, Chen MH. Effects of chlorhexidine on stem cells from exfoliated deciduous teeth. J Formos Med Assoc. 2015 Jan;114(1):17-22.
39. Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicol In Vitro. 2008 Mar;22(2):308-17.
40. Widbiller M, Althumairy RI, Diogenes A. Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization. J Endod. 2019 Feb;45(2):156-160.
2. El Karim I, Kennedy J, Hussey D. The antimicrobial effects of root canal irrigation and medication. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007 Apr;103(4):560-9.
3. Nakashima M, Akamine A. The application of tissue engineering to regeneration of pulp and dentin in endodontics. J Endod. 2005 Oct;31(10):711-8.
4. Fouad AF. Microbial Factors and Antimicrobial Strategies in Dental Pulp Regeneration. J Endod. 2017 Sep;43(9S) :S46-S50.
5. Galler KM. Clinical procedures for revitalization: current knowledge and considerations. Int Endod J. 2016 Oct;49(10):926-36.
6. AAE Clinical Considerations for a Regenerative Procedure. Available at: https://www.aae.org/specialty/wp-content/uploads/sites/2/2017/06/currentregenerativeendodonticconsiderations.pdf. Accessed Feb 11, 2023.
7. Galler KM, Krastl G, Simon S, Van Gorp G, Meschi N, Vahedi B, et al. European Society of Endodontology position statement: Revitalization procedures. Int Endod J. 2016 Aug;49(8):717-23.
8. Siqueira Jr J, Lopes H. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J. 1999;32(5):361-9.
9. Qadiri SY, Mustafa S. Role of calcium hydroxide in root canal therapy: A comprehensive review. JAMDSR. 2019;7(7):1-3.
10. Neelakantan P, Sriraman P, Gutmann JL. Removal of calcium hydroxide intracanal medicament by different irrigants and irrigating techniques: a cone beam computed tomography analysis. Gen Dent. 2017 Nov-Dec;65(6):45-49.
11. Jenks DB, Ehrlich Y, Spolnik K, Gregory RL, Yassen GH. Residual antibiofilm effects of various concentrations of double antibiotic paste used during regenerative endodontics after different application times. Arch Oral Biol. 2016 Oct;70:88-93.
12. Manochehrifar H, Parirokh M, Kakooei S, Oloomi MM, Asgary S, Eghbal MJ, Mashhadi Abbas F. The Effect of Mineral Trioxide Aggregate Mixed with Chlorhexidine as Direct Pulp Capping Agent in Dogs Teeth: A Histologic Study. Iran Endod J. 2016 Fall;11(4):320-324.
13. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review--part II: leakage and biocompatibility investigations. J Endod. 2010 Feb;36(2):190-202.
14. Estrela C, Bammann LL, Estrela CR, Silva RS, Pécora JD. Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal. Braz Dent J. 2000;11(1):3-9.
15. Miyagak DC, de Carvalho EM, Robazza CR, Chavasco JK, Levorato GL. In vitro evaluation of the antimicrobial activity of endodontic sealers. Braz Oral Res. 2006 Oct-Dec;20(4):303-6.
16. Bidar M, Naderinasab M, Talati A, Ghazvini K, Asgari S, Hadizadeh B, Gharechahi M, Mashadi NA. The effects of different concentrations of chlorhexidine gluconate on the antimicrobial properties of mineral trioxide aggregate and calcium enrich mixture. Dent Res J (Isfahan). 2012 Jul;9(4):466-71.
17. Mahmoud O, Al-Meeri WA, Farook MS, Al-Afifi NA. Calcium Silicate-Based Cements as Root Canal Medicament. Clin Cosmet Investig Dent. 2020 Feb 24;12:49-60.
18. Alarbeed MS, Ahmed GM, Elkhodary S, Shaker OG. Effect of combination of calcium hydroxide and chlorhexidine gel 2% as intracanal medication in comparison to calcium hydroxide paste as intracanal medication on postoperative pain and bacterial endotoxin in necrotic teeth: a randomized controlled trial. Acta Scien Dent. 2019;3:124-30.
19. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001 Jul;48 Suppl 1:5-16.
20. Sabrah AH, Yassen GH, Gregory RL. Effectiveness of antibiotic medicaments against biofilm formation of Enterococcus faecalis and Porphyromonas gingivalis. J Endod. 2013 Nov;39(11):1385-9.
21. Khoshkhounejad M, Sobhi Afshar M, Jabalameli F, Emaneini M, Sharifian M. Cytotoxicity Evaluation of Minimum Antibacterial Values of Different Medicaments Used in Endodontic Regenerative Procedures. Eur J Dent. 2019 Oct;13(4):514-520.
22. Markossian S, Grossman A, Brimacombe K, Arkin M, Auld D, Austin C, et al. Assay guidance manual (Internet). Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences;2004.
23. Conde MCM, Chisini LA, Sarkis-Onofre R, Schuch HS, Nör JE, Demarco FF. A scoping review of root canal revascularization: relevant aspects for clinical success and tissue formation. Int Endod J. 2017 Sep;50(9):860-874.
24. Shimizu E, Jong G, Partridge N, Rosenberg PA, Lin LM. Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure. J Endod. 2012 Sep;38(9):1293-7.
25. Lin LM, Rosenberg PA. Repair and regeneration in endodontics. Int Endod J. 2011 Oct;44(10):889-906.
26. Solter D. From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet. 2006 Apr;7(4):319-27.
27. Farhad Mollashahi N, Saberi E, Karkehabadi H. Evaluation of Cytotoxic Effects of Various Endodontic Irrigation Solutions on the Survival of Stem Cell of Human Apical Papilla. Iran Endod J. 2016 Fall;11(4):293-297.
28. Kruse CR, Singh M, Targosinski S, Sinha I, Sørensen JA, Eriksson E, Nuutila K. The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study. Wound Repair Regen. 2017 Apr;25(2):260-269.
29. Hernandez EP, Botero TM, Mantellini MG, McDonald NJ, Nör JE. Effect of ProRoot MTA mixed with chlorhexidine on apoptosis and cell cycle of fibroblasts and macrophages in vitro. Int Endod J. 2005 Feb;38(2):137-43.
30. Sumer M, Muglali M, Bodrumlu E, Guvenc T. Reactions of connective tissue to amalgam, intermediate restorative material, mineral trioxide aggregate, and mineral trioxide aggregate mixed with chlorhexidine. J Endod. 2006 Nov;32(11):1094-6.
31. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod. 2012 Oct;38 (10) :1372-5.
32. Saberi EA, Gholami F, Pirhaji A, Shahreki E. In vitro cytotoxic effects of different intracanal medicaments on the co-culture of dental mesenchymal stem cells using the MTT assay. Giornale Italiano di Endodonzia. 2021 Apr 7;35:10-19.
33. Soares Ade J, Lins FF, Nagata JY, Gomes BP, Zaia AA, Ferraz CC, de Almeida JF, de Souza-Filho FJ. Pulp revascularization after root canal decontamination with calcium hydroxide and 2% chlorhexidine gel. J Endod. 2013 Mar;39(3):417-20.
34. Jones CG. Chlorhexidine: is it still the gold standard? Periodontol 2000. 1997 Oct;15:55-62.
35. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J. 2009 Apr;42(4):288-302.
36. Hamed SJ, AL-Yasiri IK, Ali NT, Al-Feron MA. Antibacterial activity of calcium hydroxide combined with chlrohexidine or sodium hypochlorite against gram positive and gram negative bacteria. J Nat Sci Res. 2014;4(12):55-61.
37. de Souza LB, de Aquino SG, de Souza PP, Hebling J, Costa CA. Cytotoxic effects of different concentrations of chlorhexidine. Am J Dent. 2007 Dec;20(6):400-4.
38. Tu YY, Yang CY, Chen RS, Chen MH. Effects of chlorhexidine on stem cells from exfoliated deciduous teeth. J Formos Med Assoc. 2015 Jan;114(1):17-22.
39. Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicol In Vitro. 2008 Mar;22(2):308-17.
40. Widbiller M, Althumairy RI, Diogenes A. Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization. J Endod. 2019 Feb;45(2):156-160.
| Issue | Vol 20 (Continuously Published Article-Based) | |
| Section | Original Article | |
| DOI | https://doi.org/10.18502/fid.v20i1.12024 | |
| Keywords | ||
| Mineral Trioxide Aggregate Chlorhexidine Calcium Hydroxide Anti-Bacterial Agents Regenerative Endodontics Stem Cells Cytotoxicity Tests, Immunologic | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Ghabraei S, Afkhami F, Shamshiri AR, Mohammadi Z. Comparison of Cytotoxicity between Mineral Trioxide Aggregate Mixed with Chlorhexidine and Common Endodontic Regeneration Medicaments on Periodontal Ligament Stem Cells: an in Vitro Study. Front Dent. 2023;20.
