|Year : 2018 | Volume
| Issue : 1 | Page : 59-62
Cytotoxicity effects of endodontic irrigants on permanent and primary cell lines
Manikandan Ravinanthanan1, Mithra N Hegde2, Veena Shetty3, Suchetha Kumari4
1 Department of Conservative Dentistry and Endodontics, PMS College of Dental Science and Research, Thiruvananthapuram, Kerala, India
2 Department of Conservative Dentistry and Endodontics, AB Shetty Memorial Institute of Dental Sciences, NITTE University, Mangalore, Karnataka, India
3 Department of Microbiology, AB Shetty Memorial Institute of Dental Sciences, NITTE University, Mangalore, Karnataka, India
4 Department of Biochemistry, AB Shetty Memorial Institute of Dental Sciences, NITTE University, Mangalore, Karnataka, India
|Date of Web Publication||5-Mar-2018|
Dr. Manikandan Ravinanthanan
Department of Conservative Dentistry and Endodontics, PMS College of Dental Science and Research, Vattappara, Thiruvananthapuram, Kerala
Source of Support: None, Conflict of Interest: None
Background: Irrigants plays a vital role in disinfection of the root canal system. Although concentration dependent, a fine balance between antimicrobial efficacy and biocompatibility need to be maintained at all times. The aim of the present study was to evaluate the cytotoxicity of conventional irrigants on two different cell lines in a dose-dependent manner in vitro. Methods: Sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), and iodine potassium iodide (IKI) were prepared in concentrations of 5%, 2.5%, 2%, and 1%. About 0.9% saline served as negative control and Biopure MTADTM (100%) as positive control. Permanent (Henrietta Lacks [HeLa]) and primary (human gingival fibroblast [HGF]) cell lines were chosen to evaluate the cytotoxicity of the irrigants by trypan blue assay. A volume of 30 μl of the cell suspension was treated with 20 μl of irrigants. The cell suspension was loaded into Neubauer chamber after 5 min, and cell count was performed under inverted microscope and expressed as viability percentage. Results: NaOCl at all concentrations was cytotoxic on both cell lines; MTAD on HeLa had nonviable scores with limited viability on HGF. Only 1% IKI had better viability than higher concentrations. Although 1% CHX had higher viability on both cell lines, bactericidal concentration of 2% CHX showed promising results. Conclusion: Target cell line (HGF) appears to be more sensitive than the use of nontarget cell line (HeLa) for evaluating cytotoxicity. NaOCl and MTAD were cytotoxic and should be used with caution. Lower concentrations of CHX appear to be less cytotoxic than any irrigant and concentrations tested.
Keywords: Human gingival fibroblast, Henrietta Lacks, MTAD
|How to cite this article:|
Ravinanthanan M, Hegde MN, Shetty V, Kumari S. Cytotoxicity effects of endodontic irrigants on permanent and primary cell lines. Biomed Biotechnol Res J 2018;2:59-62
|How to cite this URL:|
Ravinanthanan M, Hegde MN, Shetty V, Kumari S. Cytotoxicity effects of endodontic irrigants on permanent and primary cell lines. Biomed Biotechnol Res J [serial online] 2018 [cited 2022 Jan 28];2:59-62. Available from: https://www.bmbtrj.org/text.asp?2018/2/1/59/226582
| Introduction|| |
Persistent intraradicular or secondary infections are the major causes of failure of both poorly treated and well-treated root canals. Chemomechanical preparation and three dimensional filling with an adequate seal are considered essential for long-term endodontic success. Intracanal irrigants play an essential role in removal of smear layer and disinfection. Combined use of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) has been advocated as the gold standard for the above purpose.
NaOCl (1%–5.25%) and EDTA (15%–17%) have been recommended for removal of organic and inorganic component of the smear layer, respectively. Although NaOCl is a potent antimicrobial irrigant, only higher concentrations are recommended in eradicating Enterococcus faecalis. Currently, E. faecalis biofilm were found to be resistant to 5.25% NaOCl. On the contrary, longer exposure times and higher concentrations are recommended for NaOCl to disinfect the canal system. EDTA has limited or no antimicrobial efficacy and thus serves as an adjunct rinse only.
Although clinically, 5.25% NaOCl and 17% EDTA has been advocated; this protocol has serious limitations that include marked reduction in mechanical properties of dentin and erosion of dentinal tubular microstructure. Recently, Marending et al. evaluated the effects of NaOCl on the structural, chemical, and mechanical properties of human root dentin and stated that NaOCl caused a concentration-dependent reduction of elastic modulus and flexural strength.
Most complications of the use of NaOCl appear to be the result of its accidental injection beyond the root apex which can cause violent tissue reactions characterized by pain, swelling, hemorrhage, and in some cases, development of secondary infection and paresthesia. A great deal of caution should therefore be exercised when using NaOCl during endodontic irrigation. Ehrich et al. suggested that a clinician should check, both clinically and radiographically for immature apices, root resorption, apical perforations, or any other conditions that may result in larger than normal volumes of irrigant being extruded from the root-canal system into the surrounding tissue.
After chemomechanical preparation with NaOCl has been accomplished, rinsing the canal with EDTA for at least 1 min using 5–10 ml is recommended. Prolonged exposure to strong chelators such as EDTA may weaken root dentin, as dentin hardness, and elastic modulus are functions of the mineral content of the dentin. About 0.12%–2% chlorhexidine (CHX) and 1%–2% iodine potassium iodide (IKI) are excellent antimicrobial irrigants but have limitations that restrict their use in clinical endodontics. Biopure MTAD (Tulsa, Dentsply) has gained endodontic significance and has been advocated as an alternative to EDTA. Most studies have recommended this regimen as it does not affect the dentin microstructure and hardness.
Toxicity of irrigants has been evaluated on various cell lines depending on their growth potential, metabolic characteristics, and DNA damage potential. Cell lines of target or nontarget tissues have been utilized and the results obtained have been mixed; thus no definite data exists to confirm simultaneously on both cell lines. Thus, the purpose of our study was to comparatively evaluate the cytotoxicity of conventional irrigants on continuous and primary cell lines based on their concentrations.
| Methods|| |
Preparation of irrigants and cell line
Thisin vitro study was performed in central research laboratory, A. B Shetty Memorial Institute of Dental Sciences, NITTE University. Conventional irrigants; NaOCl (Prevest Denpro Limited), CHX (Sigma) and IKI (Merk-Himedia) were obtained. Concentrations of 1%, 2%, 2.5%, and 5% of the irrigants were prepared for the study. Biopure MTAD (Tulsa Dentsply) and 0.9% normal saline served as control. Henrietta Lacks (HeLa) and human gingival fibroblast (HGF) cell lines were obtained from Manipal life sciences, Manipal and cultured at 37°C in a humidified atmosphere of 5% CO2/95% air.
Cell culture and storage
The cells were maintained in a growth medium containing the following constituents: Dulbecco's modified Eagle's medium (Himedia) with 25 mmol/L glucose, 1 mmol/L pyruvate, 4.02 mmol/L L-alanyl-glutamine, and 10% fetal calf serum (Sigma Aldrich). Confluent cells were detached with 0.15% trypsin (Himedia) for 5 min, following which 2 ml of complete medium was added, and the cells were centrifuged at 1000 rpm (180 g) for 5 min. Cell suspension was counted using a Neubauer chamber and seeded in 96 well microtiter plates (Himedia) at a density of 1 × 104 cells per well.
Cytotoxicity was assessed using trypan blue dye. Thirty microliter of the cell suspension was treated with 20 μl of irrigants (conventional irrigants and MTAD). Fifty microliter trypan blue dye (0.05%) was added and allowed for 5 min. The cell suspension was loaded into Neubauer chamber and cell count was performed under inverted microscope (Olympus, India). Nonviable cells appear blue stained. At least, 200 cells were counted per treatment. Vitality percentage was calculated using the formula (% viability = average number of viable cells/total number of cells × 100).
| Results|| |
In the NaOCl group, higher concentrations (5% and 2.5%) were extremely cytotoxic resulting in nonviable cells in both cell lines. About 2% NaOCl in HeLa accounted for nonviability, while in the HGF, a vitality count of 12.5% was noted. Viable cells in 1% NaOCl accounted for 13.6% (HeLa) and 35% (HGF), respectively as seen in [Figure 1].
|Figure 1: Viability percentage of cell lines with sodium hypochlorite, saline, and MTAD|
Click here to view
One percent CHX had mean viability scores of 94.9% on HeLa and 45% on HGF. There appeared to be little or no difference among 2% and 2.5% on both cell lines. Lowest viability was recorded in 5% CHX which accounted for 33.8% (HeLa) and 40% (HGF) as depicted in [Figure 2].
|Figure 2: Viability percentage of cell lines with chlorhexidine, saline, and MTAD|
Click here to view
IKI was similar to CHX and 1% IKI resulting in 63.7% (HeLa); 48% (HGF) cell viability. 2% and 2.5% IKI recorded near similar viable scores. 5% IKI accounted 32.2% (HeLa); 20% (HGF) viability, respectively, as seen in [Figure 3].
|Figure 3: Viability percentage of cell lines with iodine--potassium iodide, saline, and MTAD|
Click here to view
In the control groups, 0.9% saline (negative control) scored 93.5% (HeLa) and 66.25% (HGF) cell viability scores. 100% MTAD (positive control) on HeLa was extremely cytotoxic resulting in nonviable cells; on the contrary, 32.5% cell viability was noted in HGF [Figure 1], [Figure 2], [Figure 3].
| Discussion|| |
Irrigants play an essential role in chemomechanical root canal preparation. The antimicrobial efficacy is directly proportional to the concentration of the irrigants. On the contrary, it may seriously affect dentin characteristics and vital tissues if extruded periapically. Hence, a fine balance between antimicrobial efficacy and cytotoxicity need to be noted in clinical endodontics. Although commercial irrigants have been popular based on their antimicrobial efficacy, little is known about their cytotoxic potential. This study was done to evaluate the cytotoxicity of Biopure MTAD with conventional endodontic irrigants on two different cell lines.
Most studies have employed cell lines to evaluate the cytotoxicity of irrigants. The cell lines may reflect the target tissue or otherwise involving either a permanent or primary cell line depending on their study characteristics; however, the use of target tissue may reflect closely the clinical results. In this study, we evaluated the cytotoxicity of different concentrations of conventional irrigants on permanent (HeLa) and primary (HGF) cell lines to ensure uniform cell behavior and reproducibility of results, facilitating comparisons across studies, and test materials.,,
Trypan blue assay was chosen as it is easy to perform, allows qualitative and quantitative assessment of viable and nonviable cells by microscopic analysis. The underlying mechanism is that viable cells have clear cytoplasm while nonviable cells appear stained. The reliability of this test confirmed by ultrastructural analysis revealed nonviable cells allowed dye permeation through cell membrane with disruption of cellular organelles. The use of this assay on both cell lines ensures uniform cell behavior and reproducibility of results, facilitating comparisons across test irrigants.
Biopure MTAD (Dentsply, Tulsa) which consists of doxycycline, citric acid and tween 80; has been claimed to have superior antibacterial properties. All irrigants were treated for 5 minutes with the cell lines to standardize the protocol. Chang et al. reported NaOCl and CHX are cytotoxic  while Segura et al. and Trevino et al. reported CHX to be cytotoxic., Study data revealed 0.9% physiologic saline had lower viability on HGF (66.25%) in contrast to HeLa (93.5%). As stated by Eisenbrand et al., single-cell comet assays which are an integral part of cytotoxicity are not recommended on samples showing more than 30% cytotoxicity and thus were not performed. This suggests target cell lines are highly sensitive than nontarget cell lines.
The study results suggest that CHX was least cytotoxic followed by IKI and MTAD at any test concentration. NaOCl was most cytotoxic on both cell lines. These findings are in accordance with Missotten et al., who reported all test concentrations of NaOCl were practically cytotoxic with no surviving ocular cells after treatment with 3 min with 0.5% NaOCl. Ribeiro et al. concluded that CHX was not genotoxic on Chinese hamster ovary cells. Benjamin et al. reported 2% CHX was significantly less cytotoxic than 5% NaOCl on human periodontal ligament fibroblasts by trypan blue assay.
On the contrary, Sanchez et al. stated that bactericidal concentrations of CHX and IKI were cytotoxic on canine embryonic fibroblasts. Li et al. demonstrated that CHX could disrupt stable cellular redox balance promoting free radical generation and cell death; thus cytotoxic when extruded into the periradicular tissues. Marins et al. found MTAD to be genotoxic on murine fibroblast cell and did not cause cell death. NaOCl was cytotoxic and not genotoxic in a dose-dependent manner with viability scores 40% (5.25%) and 60% (2.5%) for a contact time of 5 min. The lower concentrations of MTAD used (in contrast to manufacturers instruction) could have accounted for higher cell viability scores. Zhang et al. stated that MTAD was not cytotoxic when assessed by MTT method.
| Conclusion|| |
The present study confirms that 1% CHX and 1% IKI had better viability on HGF. Two percent CHX which is clinically recommended shows promising results than any test irrigant. MTAD proved to be cytotoxic on HeLa with nonviable scores and had limited viability on HGF thus questioning its application for a contact time of 5 min as a final rinse. This study assessed the cell viability based on the principle of cell membrane as the target; further studies need to be done to assess the metabolic activity. Despite cytotoxicity, genotoxicity tests which assess the DNA damage need to be evaluated before a final conclusion can be drawn which limit the application of materials in clinical practice.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis
: Its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93-8.
Sedgley CM, Lennan SL, Clewell DB. Prevalence, phenotype and genotype of oral enterococci. Oral Microbiol Immunol 2004;19:95-101.
Kho P, Baumgartner JC. A comparison of the antimicrobial efficacy of NaOCl/Biopure MTAD versus NaOCl/EDTA against Enterococcus faecalis
. J Endod 2006;32:652-5.
Onçaǧ O, Hoşgör M, Hilmioǧlu S, Zekioǧlu O, Eronat C, Burhanoǧlu D, et al.
Comparison of antibacterial and toxic effects of various root canal irrigants. Int Endod J 2003;36:423-32.
Liu H, Wei X, Ling J, Wang W, Huang X. Biofilm formation capability of Enterococcus faecalis
cells in starvation phase and its susceptibility to sodium hypochlorite. J Endod 2010;36:630-5.
Retamozo B, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. Minimum contact time and concentration of sodium hypochlorite required to eliminate Enterococcus faecalis
. J Endod 2010;36:520-3.
Buck R, Eleazer PD, Staat RH.In vitro
disinfection of dentinal tubules by various endodontics irrigants. J Endod 1999;25:786-8.
Mohammadi Z. Sodium hypochlorite in endodontics: An update review. Int Dent J 2008;58:329-41.
Marending M, Luder HU, Brunner TJ, Knecht S, Stark WJ, Zehnder M, et al.
Effect of sodium hypochlorite on human root dentine – Mechanical, chemical and structural evaluation. Int Endod J 2007;40:786-93.
Verma S, Goel M, BalaS, Singh M. Issues of biocompatibility associated with commonly used endodontic irrigants: A review. Ind J Dent Sci 2012;4:109-13.
Ehrich DG, Brian JD Jr., Walker WA. Sodium hypochlorite accident: Inadvertent injection into the maxillary sinus. J Endod 1993;19:180-2.
Sceiza MF, Daniel RL, Santos EM, Jaeger MM. Cytotoxic effects of 10% citric acid and EDTA-T used as root canal irrigants: Anin vitro
analysis. J Endod 2001;27:741-3.
Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.
Torabinejad M, Shabahang S, Aprecio RM, Kettering JD. The antimicrobial effect of MTAD: Anin vitro
investigation. J Endod 2003;29:400-3.
Hauman CH, Love RM. Biocompatibility of dental materials used in contemporary endodontic therapy: A review. Part 1. Intracanal drugs and substances. Int Endod J 2003;36:75-85.
Aslantas EE, Buzoglu HD, Altundasar E, Serper A. Effect of EDTA, sodium hypochlorite, and chlorhexidine gluconate with or without surface modifiers on dentin microhardness. J Endod 2014;40:876-9.
Saghiri MA, Delvarani A, Mehrvarzfar P, Nikoo M, Lotfi M, Karamifar K, et al.
The impact of pH on cytotoxic effects of three root canal irrigants. Saudi Dent J 2011;23:149-52.
Merdad K, Pascon AE, Kulkarni G, Santerre P, Friedman S. Short-term cytotoxicity assessment of components of the epiphany resin-percha obturating system by indirect and direct contact Millipore filter assays. J Endod 2007;33:24-7.
Nishikiori R, Nomura Y, Sawajiri M, Masuki K, Hirata I, Okazaki M, et al.
Influence of chlorine dioxide on cell death and cell cycle of human gingival fibroblasts. J Dent 2008;36:993-8.
Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, et al.
Recommendations for conducting thein vivo
alkaline comet assay 4th
international comet assay workshop. Mutagenesis 2003;18:45-51.
Longo JPF, Valois CA, Tapajos ECC, Santos MFMA, Azevedo RB. Cytotoxicity and genotoxicity of human endodontic irrigants on human cells. Rev Clin Pesqui Odontol 2010;6:135-40.
Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al.
Anew solution for the removal of the smear layer. J Endod 2003;29:170-5.
Newberry BM, Shabahang S, Johnson N, Aprecio RM, Torabinejad M. The antimicrobial effect of biopure MTAD on eight strains of Enterococcus faecalis
: Anin vitro
investigation. J Endod 2007;33:1352-4.
Chang YC, Huang FM, Tai KW, Chou MY. The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:446-50.
Segura JJ, Jiménez-Rubio A, Guerrero JM, Calvo JR. Comparative effects of two endodontic irrigants, chlorhexidine digluconate and sodium hypochlorite, on macrophage adhesion to plastic surfaces. J Endod 1999;25:243-6.
Trevino EG, Patwardhan AN, Henry MA, Perry G, Dybdal-Hargreaves N, Hargreaves KM, et al.
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;37:1109-15.
Eisenbrand G, Pool-Zobel B, Baker V, Balls M, Blaauboer BJ, Boobis A, et al.
Methods ofin vitro
toxicology. Food Chem Toxicol 2002;40:193-236.
Missotten GS, Keijser S, de Keizer RJ. Cytotoxic effect of sodium hypochlorite 0.5% (NaOCl) on ocular melanoma cells in vitro
. Orbit 2008;27:31-5.
Ribeiro DA, Scolastici C, De Lima PL, Marques ME, Salvadori DM. Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:637-40.
Benjamin S, Nivedhitha MS, Kumar AG, Jayanthi S, Nandagopal S. Comparative evaluation of cytotoxicity of endodontic irrigants – Chlorhexidine, Sodium hypochlorite and Neem extract. J Pharm Res 2012;5:1273-5.
Sanchez IR, Nusbaum KE, Swaim SF, Hale AS, Henderson RA, McGuire JA, et al.
Chlorhexidine diacetate and povidone-iodine cytotoxicity to canine embryonic fibroblasts and Staphylococcus aureus
. Vet Surg 1988;17:182-5.
Li YC, Kuan YH, Lee TH, Huang FM, Chang YC. Assessment of the cytotoxicity of chlorhexidine by employing anin vitro
mammalian test system. J Dent Sci 2014;9:130-5.
Marins JS, Sassone LM, Fidel SR, Ribeiro DA.In vitro
genotoxicity and cytotoxicity in murine fibroblasts exposed to EDTA, NaOCl, MTAD and citric acid. Braz Dent J 2012;23:527-33.
Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-Tetrazolium method. J Endod 2003;29:654-7.
[Figure 1], [Figure 2], [Figure 3]
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