|Year : 2013 | Volume
| Issue : 1 | Page : 42-46
Cytotoxic evaluation of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts
Nagesh Bolla1, Sivavenkata Malati Nalli1, Sujana1, K Kiran Kumar2, Ranganathan1, Sarath Raj3
1 Department of Conservative Dentistry and Endodontics, Sibar Institute of Dental Sciences, Takkellapadu, Guntur, India
2 Department of Oral and Maxillofacial Pathology, Sibar Institute of Dental Sciences, Takkellapadu, Guntur, India
3 Department of Oral and Maxillofacial Pathology, Ragas Dental College, Chennai, India
|Date of Web Publication||13-Mar-2013|
Department of Conservative Dentistry and Endodontics, Sibar Institute of Dental Sciences, Takkellapadu, Guntur
Source of Support: None, Conflict of Interest: None
Objective: Endodontic irrigants solutions with anti-bacterial activity have been used in treatment of teeth with infected root canals; however, these solutions can irritate periapical tissues. The objective of this study was to evaluate the cytotoxicity of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts.
Materials and Methods: Periodontal ligament fibroblasts cells were cultured from orthodontically extracted premolars. The passage number between 3-6 was taken for further experiments. 2.5% sodium hypochlorite and 0.6% sodium dichloroisocyanuarate are the irrigating solution used for testing their cytotoxicity. The viability of cells after treating with irrigating solutions was evaluated by trypan blue dye exclusion.
Results: Results showed that 0.6% NaDCC solution was less cytotoxic on cultured human periodontal ligament fibroblasts compared with 2.5% NaOCl at 2 min, 30 min, and 1 hr.
Conclusion: This study suggested that sodium dichloroisocyanuarate is less cytotoxic than 2.5% NaOCl, suggesting its potential for use as root canal irrigant.
Keywords: Cytotoxicity, root canal irrigants, sodium dichloroisocyanuarate, sodium hypochlorite
|How to cite this article:|
Bolla N, Nalli SM, Sujana, Kumar K K, Ranganathan, Raj S. Cytotoxic evaluation of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts. J NTR Univ Health Sci 2013;2:42-6
|How to cite this URL:|
Bolla N, Nalli SM, Sujana, Kumar K K, Ranganathan, Raj S. Cytotoxic evaluation of two chlorine-releasing irrigating solutions on cultured human periodontal ligament fibroblasts. J NTR Univ Health Sci [serial online] 2013 [cited 2020 Apr 4];2:42-6. Available from: http://www.jdrntruhs.org/text.asp?2013/2/1/42/108512
| Introduction|| |
The goal of endodontic treatment is either prevention or elimination of a microbial infection in the root canal system providing an appropriate root canal filling and, consequently, the repair of the periapical tissues. Infiltrated micro-organisms are found in the root canals, which can be reached only by chemical agents used in biomechanical preparation or by intra-canal medications (Grossman and Meinmamn, 1941). , Irrigating solutions used in endodontic treatment not only present anti-microbial action, but they also clean the pulp chamber (Grossman and Meinmamn, 1941; Senia et al., 1971; Hand et al., 1978; Leonardo and Leal, 1991). The irrigating solution must have a combination of maximum anti-microbial action with minimum toxicity, physical and chemical properties associated with a feasible cost to the professional. When irrigating solutions are used in vital and non-vital teeth, besides in teeth with wide-open apex, irrigants can even be extruded in teeth with fully mature, intact apexes. Besides their antiseptic capacity, the cytotoxic potential of endodontic irrigants should also be analyzed. ,
Chlorine-releasing agents (CRA) are most commonly used as root canal irrigants.  For many years, the most frequently used irrigants have been sodium hypochlorite (NaOCl) alone or in combination. Their benefits, which include good tissue dissolution and disinfecting capability, have been demonstrated in several studies.  It also has minimal "clinical toxicity" when kept within the confines of the canals. However, NaOCl is extremely toxic to the periapical tissues if it is injected beyond the apex of the tooth.  Unfortunately, NaOCl has toxic effects on vital tissues, resulting in hemolysis, skin ulceration, and necrosis. 
In contrast, sodium dichloroisocyanurate (NaDCC) formulations were introduced relatively recently. NaDCC tablets possess some definite advantages over sodium hypochlorite (NaOC1), as well as some theoretical advantages awaiting experimental investigation. The microbicidal activity of solutions prepared from NaDCC tablets possibly may be greater than that of NaOC1 solutions containing the same total available chlorine (av.C1) for two reasons. First, the pH of solutions prepared from NaDCC tablets is much lower than that of NaOC1 solutions, and activity increases with acidity. In solution, free av.C1 is present either as hypochlorous acid or hypochlorite ion, depending on pH, and it has been postulated that hypochlorous acid is the predominantly active species whilst hypochlorite ion has little activity due to its negative charge impeding penetration of the microbial cell wall and membrane. The ratio of hypochlorous acid to hypochlorite ion increases with acidity. Secondly, in NaDCC solutions, only a proportion of the total av.C1 is free whilst the rest is combined; as free av.Cl is used up so the latter is released. This mechanism may serve to increase the microbicidal capacity of NaDCC solutions as found by Bloomfield (1973) and Bloomfield and Miles (1979), and possibly may also make them less corrosive, less toxic, and less prone to inactivation by organic matter than NaOC1 solutions of the same total av.C1.  The cytotoxic potential of NaDCC is still under research.
There are 3 levels of biological testing of dental materials (Tronstad and Wennberg 1980). The initial test is toxicity screening of the dental material. The second level of testing is performed on animals to evaluate the tissue or bone response of the host. The third test is to simulate clinical practice and is known as the usage test. Pre-clinical evaluation of the biocompatibility of medical devices and materials used in dentistry is necessary. As scientific knowledge advances the understanding of basic mechanisms, an in vitro model that simulates the in vivo test or clinical use and yields equally relevant information is advocated in cytotoxicity testing. Cell culture assay is one method of choice for toxicity screening of root canal irrigants. 
The purpose of the present study is to compare cytotoxicity of chlorine-releasing irrigants i.e. sodium hypochlorite and sodium dichloroisocynate on cultured human periodontal ligament fibroblasts by trypan blue dye exclusion method.
| Materials and Methods|| |
Human PDL cells were cultured by using an explants technique. Human PDL cells were cultured from the roots of freshly extracted premolars extracted for orthodontic reasons. After extraction, teeth were rinsed in phosphate-buffered solution and then placed in 60-mm Petri dishes containing alpha-modified Eagle's medium (aMEM) and 3 μg/ml amphotericin and 50 mg of streptomycin/mL. To avoid contamination from the gingiva, the periodontal ligament was carefully removed from the middle third of the root by scalpel. The fragments were grown in aMEM supplemented with 10% fetal calf serum (FCS) and antibiotics. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Experiments with PDL fibroblasts were conducted by using cells between the third and eighth passage.
Group I - control, fresh aMEM culture
Group II - aMEM containing 2.5% NaOCl
Group III - aMEM containing 0.6% NaDCC
Fibroblasts were seeded in 24-well culture plates and incubated for 24 hours [Figure 1]. After overnight attachment, the culture medium was replaced with fresh aMEM containing 5% FCS and the irrigation solutions. Concentrations of the solutions used are 2.5% NaOCl, 0.6% NaDCC.
After incubation for 10 min at room temperature, the effect of the different materials on cultured cells was evaluated by 0.2% (final concentration) trypan blue dye exclusion analysis. Briefly, the cell number was determined by counting the viable cells in a hemocytometer. The percentage of viable cells from each well after incubation with material extracts was obtained by applying the following equation:
% viable cells = (VC/TC) × 100,
Where VC = viable cells counted and TC = total cells counted (stained plus unstained cells).
| Results|| |
All the assays were repeated 3 times to ensure reproducibility. Mean and standard deviation were estimated for all the groups. The percentage of cell viability from different irrigating solutions was analyzed using by Wilcoxon-matched pairs test. The level of statistical significance was set at P < 0.05. The cytotoxic effects of the sodium dichloroisocyanuarate, sodium hypochlorite are shown in [Table 1]. At 2 min, sodium dichloroisocyanuarate showed 79% cell viability, whereas sodium hypochlorite showed 59% cell viability. At 30 min time period, NaDCC showed 81% cell viability while NaOCl showed 54% viability. At 1 hour also, NaDCC exhibited highest cell viability (84%) when compared with NaOCl, which exhibited 40% viability. [Figure 2] and [Figure 3].
|Figure 2: 40× shows PDL fibroblasts exposed to NaOCl for 1 hr with dead cells represented by black dots under dense trypan blue staining|
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|Figure 3: Comparison of two materials at 2 minute, half an hour and one hour exposure|
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|Table 1: comparison of 2 minute, half an hour, and 1 hour in sodium hypochlorite, sodium dichloroisocyante by wilcoxon-matched pairs test|
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| Discussion|| |
Irrigation has a central role in endodontic treatment. During and after instrumentation, the irrigants facilitate removal of micro-organisms, tissue remnants, and dentin chips from the root canal through a flushing mechanism. Irrigants can also help prevent packing of the hard and soft tissue in the apical root canal and extrusion of infected material into the periapical area. Some irrigating solutions dissolve either organic or inorganic tissue in the root canal. In addition, several irrigating solutions have anti-microbial activity and actively kill bacteria and yeasts when introduced in direct contact with the micro-organisms. However, several irrigating solutions also have cytotoxic potential, and they may cause severe pain if they gain access into the periapical tissues. An optimal irrigant should have all or most of the positive characteristics but none of the negative or harmful properties. 
Sodium hypochlorite is at present the most popular irrigant. It is broad-spectrum anti-microbial agent that has proven to be effective against bacteria, bacteriophages, spores, yeasts, and viruses.  Moreover, it has the ability to dissolve necrotic pulp tissue, organic remnants. , Higher concentrations increase its ability to dissolve the necrotic tissue and shorten the time needed for inhibition of bacterial growth, but at the same time, causes damage to periapical tissues.  Because irrigation with NaOCl is still not predictably effective, recent studies have focused on searching for an alternative solution.
Sodium dichloro isocyanuarate can be suggested for its use as an endodontic disinfectant because of its anti-microbial activity against different bacterial species when used at low concentrations.  NaDCC was found to be more effective in killing E. faecalis than NaOCl-a bacteria known to be resistant to calcium hydroxide dressings. 
Root canal irrigants cytotoxicity can be studied in vitro by means of several tests, being trypan blue dye exclusion assay chosen for the present study because it is easy to perform and allows distinguishing non-viable from viable cells by microscopic analysis. Trypan blue staining of non-viable cells is a common procedure used in cell culture research, and it relies on the premise that vital cells will not allow the stain to penetrate through cell membranes. The accuracy of trypan blue in distinguishing vital and non-vital cells has been verified using scanning electron microscopic. These authors showed that cells permeated by the stain (non-vital cells) presented disruption of organelles, whereas vital cells presented integrity of membranes and organelles, as confirmed by ultra structural analysis. ,
In the present study, our results showed that 0.6% NaDCC solution was less cytotoxic on cultured human periodontal ligament fibroblasts compared with 2.5% NaOCl. Our results were generally in agreement with that reported by Heling et al.  However, our results differed from those of Spangberg et al.  who recommended 0.5% NaOCl as an acceptable non-cytotoxic solution and Hegger et al. Who reported 0.025% NaOCl to be non-tissue toxicity.  Hidalgo et al, using an XTT assay, observed cell death in cultured skin fibroblasts after application of NaOCl at concentrations > 0.05% for 2-24 h. Chang et al,  using a PI fluorescence assay, found that 0.4% and 0.2% dilutions of a 5.25% NaOCl solution killed human periodontal ligament cells after 3 and 24 h of exposure, respectively. Esther et al, concluded that 0.1% and 0.5% dilutions of 2.5% NaOCl solution were less cytotoxic on 3T3-L1 cells, compared with 0.1% and 0.5% dilutions of 15% citric acid and 5% phosphoric acid solutions. It may result from different origins of the cells or different experimental protocols used in each laboratory. The cellular effects of NaOCl may not necessarily be comparable in all tissues.
Regeneration of the lesion and the periodontal connective tissue attachment apparatus after endodontic treatment is important. Toxic anti-microbial agents used in the root canal may impede periapical tissue healing. Studies have suggested that the cell growth, proliferation, and matrix synthesis of fibroblasts are necessary for regeneration. The detrimental effects of irrigation solutions might impair the reparative and regenerative potential of periapical tissues. 
It has been reported that periapical tissue damage is increased in necrotic pulp cases and in teeth with root canals with large apical foramina, because materials used in the canal will more readily leak beyond the apical foramen. This study suggests that these irrigation fluids may cause detrimental effects on vital tissues. Its clinical significance, however, needs to be evaluated further because concentration used, exposure time to the agent, and exposure surface area are important factors affecting the resulting effect. All this must be considered when selecting the appropriate agent.
| Conclusion|| |
By considering previous findings and the results obtained from present study, it may be concluded that 0.6% sodium dichloroisocyanuarate is less toxic when compared to 2.5% NaOCl solution. However, before its clinical application, more studies are warranted.
| Acknowledgement|| |
I/We sincerely thank Dr. Ranganathan MDS (professor) Ms. Jayanthi Sampath, Chennai research foundation, Chennai, for their co-operation, valuable support, encouragement, and guidance in completing my work.
| References|| |
|1.||Ferreira CM, Bonifácio KC, Fröner IC, Ito IY. Evaluation of the antimicrobial activity of three irrigating solutions in teeth with pulpal necrosis. Braz Dent J 1999;10:1-60. |
|2.||Kovac J, Kovac D. Effect of irrigating solutions in endodontic therapy. Bratisl Lek Listy 2011;112:410-5. |
|3.||Hülsmann M, Hahn W. Complications during root canal irrigation-literature review and case reports. Int Endod J 2000;33:186-93. |
|4.||Ercan E, Dulgergil T, Yavuz I. The effects of antibacterial solutions on microorganisms isolated from infected root canals In vivo. Biotechnol Biotechnol Eq 2006;20:149-56. |
|5.||Rosenthal RA, Schlitzer RL, McNamee LS, Dassanayake NL, Amass R. Antimicrobial acitivity of organic chlorine releasing compounds. J Br Cont Lens Assoc 1992;15:81-4. |
|6.||Johal S, Baumgartner JC, Marshall JG. Comparison of the antimicrobial efficacy of 1.3%NaoCl/Biopure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. J Endod 2007;33:48-51. |
|7.||Carson KR, Goodell GG, McClanahan SB. Comparison of the antimicrobial activity of six irrigants on primary endodontic pathogens. J Endod 2005;31:471-3. |
|8.||Hulsmann M, Hahn W. Complications during root canal irrigation: Literature review and case reports. Int Endod J 2000;33:186-93. |
|9.||Coates D. A comparison of sodium hypochlorite and sodium dichloroisocyanurate products. J Hosp Infec 1988;11:31-40. |
|10.||Tai KW, Huang FM, Chang YC. Cytotoxicity evaluation of root canal filling materials on primary human oral fibroblast cultures and a permanent hamster cell line. J Endod 2001;27:571-3. |
|11.||Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in Endodontics. Dent Clin N Am 2010;54:291-312. |
|12.||El Karim IE, Kennedy J, Hussey D. The antimicrobial effects of root canal irrigation and medication. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:560-9. |
|13.||Spanó JC, Barbin EL, Santos TC, Guimarães LF, Pécora JD. Solvent action of sodium hypochlorite on bovine pulp and physico-chemical properties of resulting liquid. Braz Dent J 2001;12:154-7. |
|14.||Khademi A, Usefian E, Feizianfard M. Tissue dissolvingability of several endodontic irrigants on bovine pulp tissue. Int Endod J 2007;2:65-8. |
|15.||Jeansonne MJ, White RR. A comparisonof 2.0% chlorhexidine gluconate and 5.25% sodium hypochloriteas antimicrobial endodontic irrigants. J Endod 1994;20:276-8. |
|16.||Bloomfield SF, Uso EE. Uso The antibacterial properties of sodium hypochlorite and sodium dichloroisocyanurate as hospital disinfectants. J Hosp Infect 1985;6:20-30. |
|17.||Estrela C, Pimenta FC, Ito IY, Bammann LL. Antimicrobial evaluation of calcium hydroxide in infected dentinal tubules. J Endod 1999;25:416-8. |
|18.||Correa GT, Veranio GA, Silva LE, Hirata Junior R, Coil JM, Scelza MF. Cytotoxicity evaluation of two root canal sealers and a commercial calcium hydroxide paste on THP1 cell line by trypan blue assay. J Appl Oral Sci 2009;17:457-61. |
|19.||Scelza MF, Oliveira LR, Carvalho FB, Faria SC. In vitro evaluation of macrophage viability after incubation in orange oil, eucalyptol and chloroform. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:24-7. |
|20.||Heling I, Rotstein I, Dinur T, Szwec-Levine Y, Steinberg D. Bactericidal and cytotoxic effects of sodium hypochlorite and sodium dichloroisocyanurate solutions In Vitro. J Endod 2001;27:278-80. |
|21.||Spångberg L, Engstrom B, Langeland K. Biologic effects of dental materials. III. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg Oral Med Oral Pathol 1973;36:856-71. |
|22.||Hegger JP, Sazy AJ, Stenberg BD, Strock LL, McCauley RL, Hernom DN, et al. Bacterial and wound-healing properties of sodium hypochlorite solutions: The 1991 Lindberg Award. J Burn Care Rehabil 1991;12:420-4. |
|23.||Hidago E, Bartolome R, Dominguez C. Cytotoxicity mechanisms of sodium hypochlorite in cultured human dermal fibroblasts and its bactericidal effectiveness. Chem Biol Interact 2002;139:265-82. |
|24.||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. |
|25.||Navarro-Escobar E, González-Rodríguez MP, Ferrer-Luque CM. Cytotoxic effects of two acid solutions and 2.5% sodium hypochlorite used in endodontic therapy. Med Oral Patol Oral Cir Bucal 2010;15:90-4. |
[Figure 1], [Figure 2], [Figure 3]