The Journal of Contemporary Dental Practice

Register      Login



Volume / Issue

Online First

Related articles

VOLUME 15 , ISSUE 4 ( July-August, 2014 ) > List of Articles


An in vitro Evaluation of the Cytotoxicity of Varying Concentrations of Sodium Hypochlorite on Human Mesenchymal Stem Cells

Zeeshan H Ahmad, Sarah M Alkahtany, Sukumaran Anil

Citation Information : Ahmad ZH, Alkahtany SM, Anil S. An in vitro Evaluation of the Cytotoxicity of Varying Concentrations of Sodium Hypochlorite on Human Mesenchymal Stem Cells. J Contemp Dent Pract 2014; 15 (4):473-481.

DOI: 10.5005/jp-journals-10024-1565

Published Online: 01-01-2015

Copyright Statement:  Copyright © 2014; The Author(s).



To evaluate and compare the cytotoxicity of various concentrations of sodium hypochlorite on immortalized human bone marrow mesenchymal stem cells (MSCs).

Materials and methods

The 5.25 percent sodium hypochlorite (NaOCl) at concentrations of 0.5, 0.1, 0.025, 0.0125, and 0.005 mg/ml were used to assess the cytotoxic effect on MSCs. Immortalized human bone marrow mesenchymal stem cells (hTERT-MSCs) were exposed to NaOCl at 5 different concentrations. Cell viability was assessed by 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alamarBlue assays. The cell morphology changes were assessed with scanning electron microscopy (SEM) after exposure to 2, 4, and 24 hour incubation. The ethidium bromide/acridine orange (EB/ AO) fluorescent stain was applied to the cells in the 8-chamber slides after they were incubated with the testing agents for 2 and 4 hours to detect live and dead cells. The observations were quantitatively and qualitatively analyzed.


The cell viability study using MTT assay and AB assay showed significant reduction with varying concentration at 2 and 4 hours incubation period. The cell viability decreased with the higher percentage of NaOCl. The exposure time also revealed an inverse relation to the cell viability. The SEM analysis showed reduction in the number of cells and morphological alterations with 0.5 mg/ml at 2 and 4 hours compared to 0.025 mg/ml NaOCl. Destruction of the cells with structural alterations and lysis was evident under fluorescence microscope when the cells were exposed to 0.5 mg/ml NaOCl.


Within the limitations of this in vitro study it can be concluded that NaOCl is toxic to the human bone marrow MSCs. The cell lysis was evident with higher concentration of sodium hypochlorite. From the observations, it can be concluded that a lower concentration of NaOCl may be used as endodontic irrigant due to its cytotoxic properties. Further studies are man datory to evolve a consensus on the optimal concentration of sodium hypochlorite to be used as endodontic irrigant.

How to cite this article

Alkahtani A, Alkahtany SM, Anil S. An in vitro Evaluation of the Cytotoxicity of Varying Concentrations of Sodium Hypochlorite on Human Mesenchymal Stem Cells. J Contemp Dent Pract 2014;15(4):473-481.

PDF Share
  1. Sodium hypochlorite in endodontics: an update review. Int Dent J 2008;58(6):329-341.
  2. Irrigation in endodontics. Dent Clin North Am 2010;54(2):291-312.
  3. Review: the use of sodium hypochlorite in endodontics—potential complications and their management. Br Dent J 2007;202(9):555-559.
  4. Root canal irrigants. J Endod 2006;32(5):389-398.
  5. Comparison of the antimicrobial activity of six irrigants on primary endodontic pathogens. J Endod 2005;31(6):471-473.
  6. Cytotoxic effects of NaOCl on vital tissue. J Endod 1985;11(12):525-528.
  7. Solvent action of sodium hypochlorite on bovine pulp and physico-chemical properties of resulting liquid. Braz Dent J 2001;12(3):154-157.
  8. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004;37(1):38-41.
  9. Effect of temperature on collagen-dissolving ability of sodium hypochlorite endodontic irrigant. Oral Surg Oral Med Oral Pathol 1980;49(2):175-177.
  10. The effects of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. J Endod 2005;31(9):669-671.
  11. Soft tissue dissolution capacity of currently used and potential endodontic irrigants. J Endod 2004;30(11):785-787.
  12. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37(7):438-446.
  13. Effects of sodium hypochlorite on soft tissues after its inadvertent injection beyond the root apex. J Endod 1991;17(11):573-574.
  14. Response of the human eye to accidental exposure to sodium hypochlorite. J Endod 1990;16(5):235-238.
  15. Accidental injection with sodium hypochlorite: report of a case. Int Endod J 2009;42(2):175-182.
  16. Facial atrophy following accidental subcutaneous extrusion of sodium hypochlorite. Br Dent J 2009;206(5):263-264.
  17. Tissue damage after sodium hypochlorite extrusion during root canal treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108(1):e46-49.
  18. Comparison of tetraacetylethylendiamine + sodium perborate and sodium hypochlorite cytotoxicity on L929 fibroblasts. J Endod 2010;36(9):1516-1520.
  19. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nature biotechnology 2002;20(6):592-596.
  20. Morphological cell changes due to chemical toxicity of dental material: an electron microscopic study of human periodontal ligament fibroblasts and L 928 cells. J Endod 1990;16(3):129-134.
  21. Attachment and morphological behavior of human periodontal ligament fibroblasts to mineral trioxide aggregate: a scanning electron microscope study. J Endod 2004;30(1):25-29.
  22. Apoptosis: identification of dying cells. Cell Biol Toxicol 1998;14(2):111-120.
  23. A simple technique for quantifying apoptosis in 96-well plates. BMC biotechnology 2005;5:12.
  24. Evaluation of the use of human Mesenchymal Stem Cells for acute toxicity tests. Toxicol in vitro 2011;25(8):1989-1995.
  25. Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines. J Biomed Sci 2010;17:64.
  26. Immortalized gingival fibroblasts as a cytotoxicity test model for dental materials. J Mater Sci Mater Med 2012;23(3):753-762.
  27. Histological study of the effect of some irrigating solutions on bacterial endotoxin in dogs. Braz Dent J 2004;15(2):109-114.
  28. An in vitro evaluation of the antimicrobial efficacy of irrigants on biofilms of root canal isolates. Int Endod J 2001;34(4):300-307.
  29. Toxicity of concentrated sodium hypochlorite used as an endodontic irrigant. Int Endod J 2004;37(4):272-280.
  30. Neurological complications following extrusion of sodium hypochlorite solution during root canal treatment. Int Endod J 2005;38(11):843-848.
  31. Severe tissue damage and neurological deficit following extravasation of sodium hypochlorite solution during routine endodontic treatment. Br Dent J 2005;198(12):749-750.
  32. Reactions of guinea pig subcutaneous connective tissue following exposure to sodium hypochlorite. Oral Surg Oral Med Oral Pathol 1980;49(5):460-466.
  33. Penetration of sodium hypochlorite into dentin. J Endod 2010;36(5):793-796.
  34. Chemical parameters, antimicrobial activities, and tissue toxicity of 0.1 and 0.5% sodium hypochlorite solutions. Antimicrob Agents Chemother 1985;28(1):118-122.
  35. Effect of sodium hypochlorite on human pulp cells: an in vitro study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112(5):662-666.
  36. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod 2011;37(8):1109-1115.
  37. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13(2):113-117.
  38. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicology in vitro 2008;22(2):308-317.
  39. Comparison of alamar blue and MTT assays for high throughput screening. Toxicology in vitro 2004;18(5):703-710.
  40. Adhesive resin induces apoptosis and cell-cycle arrest of pulp cells. J Dent Res 2003;82(8):592-596.
  41. Cell death modalities: classification and pathophysiological implications. Cell death and differentiation 2007;14(7):1237-1243.
  42. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 2004;16(6):663-669.
  43. Morphological and cytochemical determination of cell death by apoptosis. Histochem Cell Biol 2008;129(1):33-43.
  44. Anatomy of sodium hypochlorite accidents. Compend Contin Educ Dent 2007;28(10):544-550.
  45. Alguns aspectos do uso do hipoclorito de sódio em Endodontia. Rev. Fac Odontol Porto Alegre 1991;32(1):7-10.
  46. Effects of dentin on the antimicrobial properties of endodontic medicaments. J Endod 2007;33(8):917-925.
PDF Share
PDF Share

© Jaypee Brothers Medical Publishers (P) LTD.