|Year : 2018 | Volume
| Issue : 3 | Page : 189-195
Comparative evaluation of cytotoxic effects of MTAD and sodium hypochlorite using lactate dehydrogenase and trypan blue assays: An in vitro study
Anant Singh1, Pooja Kakkar1, AB Pant2
1 Department of Conservative Dentistry and Endodontics, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India
2 Sr. Scientist, In Vitro Toxicology, Indian Institute of Toxicology Research-CSIR, Lucknow, Uttar Pradesh, India
|Date of Web Publication||25-Jul-2018|
Dr. Anant Singh
Department of Conservative Dentistry and Endodontics, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow - 226 025, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Aim: This study aims to examine the cytotoxic effects of BioPure MTAD and three clinical concentrations of sodium hypochlorite (NaOCl) solutions (1%, 3%, and 5%) on cultured human periodontal ligament (PDL) cells.
Materials and Methods: Human PDL cells were grown on cell culture plates and were placed in contact with various concentrations of tested irrigants. Cytotoxicity of both BioPure MTAD and different concentrations of NaOCl was assessed for immediate short-term intervals (1, 5, 10, and 15 min) and midterm intervals as well (24, 48, 72, and 96 h). Different parameters of toxicity such as lactate dehydrogenase assay and trypan blue dye exclusion assay were studied along with morphological changes at all the different time points. The mean zones of inhibition were calculated for each group and were statistically analyzed.
Results: BioPure MTAD was least toxic of all test irrigants in short-term and midterm time intervals. 5% NaOCl showed the maximum cytotoxicity at short-term time intervals. All cells died within the first 24 h for all three NaOCl concentrations.
Conclusions: BioPure MTAD showed minimal toxicity and the three concentrations (1%, 3%, and 5%) of NaOCl showed detrimental effects on the PDL cells and its toxicity was dose dependent.
Keywords: BioPure MTAD, cytotoxicity, lactate dehydrogenase assay, periodontal ligament cells, trypan blue dye exclusion assay
|How to cite this article:|
Singh A, Kakkar P, Pant A B. Comparative evaluation of cytotoxic effects of MTAD and sodium hypochlorite using lactate dehydrogenase and trypan blue assays: An in vitro study. Saudi Endod J 2018;8:189-95
|How to cite this URL:|
Singh A, Kakkar P, Pant A B. Comparative evaluation of cytotoxic effects of MTAD and sodium hypochlorite using lactate dehydrogenase and trypan blue assays: An in vitro study. Saudi Endod J [serial online] 2018 [cited 2018 Aug 17];8:189-95. Available from: http://www.saudiendodj.com/text.asp?2018/8/3/189/237563
| Introduction|| |
During the microbial control phase of endodontic treatment, irrigating solutions assist in the dissolution of the pulp tissue and antimicrobial activity inside all aspects of the root canal system. However, endodontic irrigants may inadvertently come in contact with periradicular tissue. Tissue toxicity is therefore an important consideration to have when deciding an endodontic irrigation solution for root canal therapy. Minimal toxicity to the surrounding tissues is required for irrigants because they can be accidentally extruded beyond the apical foramen as a result of poor operator technique and/or anatomical variations at the root apex resulting in periapical damage.
Sodium hypochlorite (NaOCl) as an endodontic irrigant is considered as the gold standard in chemomechanical cleansing of the root canal. Previous literature is replete with evidence of its antimicrobial activity and tissue-dissolving capacity.,, In endodontic practice, several concentrations of NaOCl ranging from 0.5% to 5.25% are routinely used for irrigation. It is also well known that the antimicrobial effectiveness and tissue dissolution properties of NaOCl are strongly influenced by the concentration of the solution.,
It has been reported that the cytotoxic effects of NaOCl were observed at lower concentrations. Furthermore, effective concentrations of this solution are known to exhibit cytotoxicity to vital tissues resulting in hemolysis, inhibition of neutrophil migration, and damage to endothelial and fibroblast cells. Clinically, this could manifest as pain, ulceration, and delayed wound healing in the periradicular tissues.
An ideal endodontic irrigant should, therefore, be nontoxic and nonirritating to the periodontal tissues.
In 2003, a new endodontic irrigant was introduced by Torabinejad and Johnson, which was later marketed under the trade name BioPure MTAD (Dentsply, Tulsa Dental, Tulsa, OK, USA). BioPure MTAD is an aqueous mix of a broad-spectrum antibiotic (3% doxycycline), demineralizing agent (4.25% citric acid), and detergent (0.5% polysorbate 80). A cytotoxic study conducted by Zhang et al. comparing MTAD to commonly used irrigants and medications using methyl thiazol tetrazolium (MTT)-tetrazolium assay concluded that it could fulfill the ideal requirement of minimal toxicity while retaining its antibiotic and demineralizing potential.
In the past, colorimetric and luminescence-based assays have been employed for in vitro cytotoxic evaluation of endodontic irrigants based on one parameter: the viable cell metabolic activity. However, the cytotoxicity of MTAD and different clinical concentrations of NaOCl (1%, 3%, and 5%) on the parameter of cell membrane integrity has not yet been analyzed. Hence, the aim of this study was to determine the cytotoxic potential of MTAD and NaOCl in its various clinical concentrations of 1%, 3%, and 5% on cultured human PDL cells using two cell membrane integrity assays: lactate dehydrogenase (LDH) assay and trypan blue assay.
| Materials and Methods|| |
The materials tested in this study were BioPure MTAD (Dentsply Tulsa Dental, Tulsa, OK) and three concentrations (1%, 3%, and 5%) of NaOCl (Hyposol, Jammu and Kashmir, India). Diluted NaOCl solutions were prepared by adding distilled water (Minitube, New Delhi, India) to 5% NaOCl. Test solutions were divided into respective groups for evaluation (Group I: 1% NaOCl, Group II: 3% NaOCl, Group III: 5%, Group IV: BioPure MTAD, and Control group: distilled water).
All patients who volunteered were briefed about the nature of the study and informed consent was taken for the experimental procedures. Approval for the study protocol was obtained from the institutional review board. Healthy PDL tissue was taken from freshly extracted human premolar teeth extracted atraumatically for orthodontic reasons from adult male/female patients. All teeth used for the study were free from any evidence of caries, root resorption, periapical pathology, or root canal therapy. The PDL tissue collected was immediately stored in Hanks' balanced salt solution containing an antibiotic (penicillin/streptomycin) and antifungal (amphotericin B) agent. The specimens were then stored at 4°C for 6 h.
The primary cultures of human PDL fibroblasts were obtained by following the methods of Pant et al. with desired modifications. The teeth were washed in Dulbecco's minimum essential medium (MEM) (Gibco BRL, Grand Island, NY, USA) containing antibiotic-antimycotic, and then, adherent soft tissues were removed from the crown and the coronal one-third of the root and discarded. The crown and coronal one-third of the root were then placed in 5.25% NaOCl for 2 min to reduce the bacterial contamination, as well as to kill any remaining gingival epithelial cells. The middle 1/3rd of the root was then scraped to obtain PDL tissue specimen. The tissue specimens were placed in sterile Petri dish More Details containing a thin layer of MEM containing 10% fetal bovine serum (Medox biotech, Chennai, Tamil Nadu, India). The PDL tissue was disaggregated using 0.2% collagenase (Roche, Mumbai, India) and 0.125% trypsin (Sigma-Aldrich, MI, USA) for 30 min at 37°C, and the cells were collected by centrifugation at 1000 rpm for 5 min. The pellet of packed cells was then resuspended in the 6-well culture plates (Corning Glass Works, Corning NY, USA) in complete MEM and incubated at 37°C with an atmosphere of 95% air and 5% CO2 for the attachment. Growth was permitted to continue until the cell attained the confluent monolayer, at which time they were trypsinized (trypsin 0.05%–ethylenediaminetetraacetic acid 0.53 mM) and passaged into T-25 culture flasks (Nunc, Denmark) to expand the cell population ( first cell passage). The cells of third and fourth passages were trypsinized and pooled for experimentation (to control the cell variability). Cell number for experimentation was determined using an Electronic Coulter Counter (Model Zf, Coulter Electronics, Hialeah, Florida, USA). The numbers of viable cells in each batch were measured by trypan blue dye exclusion (TBDE) test (Sigma-Aldrich, MI, USA) before each experiment and batches showed that cell viability >95% was used for the experiment.
Lactate dehydrogenase release assay
The LDH assay is a quantitative analysis designed to measure the intactness of the cell membrane on damaged cells by the amount of cytoplasmic LDH released into a commercially available medium kit (LDH Assay Kit, Sigma-Aldrich, MI, USA), following the exposure of various concentrations of test irrigants at different time intervals. After exposure to experimental irrigants, the plates were incubated as per the experimental schedule in CO2 incubator and centrifuged at 250 x g for 4 min. Then, supernatant of each well was transferred to a fresh flat bottom 96-well culture microtiter plates and proceeded further for enzymatic analysis as per the instructions given in the kit. The untreated sets that served as positive controls were also run under identical conditions
Trypan blue dye exclusion assay
The TBDE assay test was conducted to study the cell viability by assessing the loss of membrane integrity following the protocols of Siddiqui et al. with desired modifications. The cells (1 × 106) were seeded in T-25 cm 2 culture flasks and allowed to grow for 24 h in 5% CO2–95% atmosphere at 37°C under high humid conditions. Then, the medium was replaced with serum-free medium supplemented with different dilutions of experimental irrigant to test for various time intervals. The treated cells were then allowed to incubate for intervals of 24–96 h. Immediately after the completion of respective incubations, cell suspensions were aspirated and centrifuged at 600 rpm for 5 min and washed with sterile phosphate-buffered saline (pH 7.4) twice, before stain with trypan blue dye. The cell suspension was then mixed with trypan blue dye (0.4% solution) at a ratio of 1:5 vol/vol (dye: cell suspension) and placed in a hemocytometer (Sigma-Aldrich, MI, USA). The live (unstained transparent) and dead (blue stained) cells were counted under ×100 magnification in a phase-contrast inverted microscope (Leica, Wetzler, Germany). The untreated sets were also run simultaneously under the identical conditions and served as control.
Primary human PDL fibroblast cells were seeded in 12-well culture plates, incubated to confluency and subjected to exposure of different concentrations (1%, 3%, and 5%) of NaOCl and MTAD for the time interval of 1, 5, 10, and 15 min and 24 h. The effect of the irrigants on cell morphology and cell proliferation was documented under ×100 magnification.
All assays were done in triplicate. The mean and standard deviations of zones of inhibition were calculated for each group. The data were analyzed using two-way analysis of variance (ANOVA). When a significant difference was found, Tukey's honest significant difference test was used to determine the significance among the means. P < 0.05 was considered statistically significant.
| Results|| |
The results of these assays are expressed in percentage cell viability. The cell growth and proliferation values of the negative and positive control groups obtained from each experiment were within the historical data range of the laboratory. The untreated batches of cells were designated as positive controls and run simultaneously under exact conditions. To make the data comparable among the test materials, which induced different and a wide range of cytotoxic responses, the experiments were carried out at immediate short-term intervals (1, 5, 10, and 15 min) and at midterm (24, 48, 72, and 96 h) intervals for BioPure MTAD alone. In the pilot study we conducted, all cells were found dead within 24 h for the different concentrations of NaOCl used; hence, the time period for NaOCl was limited to short-term intervals only [Figure 1].
|Figure 1: (a) Untreated periodontal ligament cells (b) All the periodontal ligament cells dead after 24 h of exposure to sodium hypochlorite|
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The time-related inhibition of cell proliferation by BioPure MTAD and three concentrations of NaOCl (1%, 3%, and 5%) is discussed in our results. It is evident from the data that there was a minimum percentage of LDH release activity at short-term time intervals for BioPure MTAD compared to all NaOCl groups for the same time intervals [Figure 2].
|Figure 2: Comparison of % cell viability as an indicator of lactate dehydrogenase release activity of BioPure MTAD compared with 1%, 3%, and 5% sodium hypochlorite at 1, 5, 10, and 15 min time intervals|
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As expected, there was a strong correlation between the cytotoxicity of NaOCl and its concentration [Figure 3]. For 1% NaOCl, the LDH release activity at 1 and 15 min was between 100% and 150%. Whereas, 5% NaOCl showed a significant percentage release of 125%–225% at the same time interval. Five percentage NaOCl could induce the maximum percentage of LDH release even at 15 min of exposure. The results showed that the cytotoxicity of BioPure MTAD in comparison to NaOCl groups of various concentrations at different time intervals was the least toxic.
|Figure 3: Dose-related inhibition of periodontal ligament cell proliferation by BioPure MTAD compared with 1%, 3%, and 5% sodium hypochlorite using lactate dehydrogenase assay at 1, 5, 10, and 15 min time intervals|
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A similar trend in the percentage cell viability was observed in both exposures using TBDE assay. In BioPure MTAD group, the percentage of cell viability was maximum at all the short-term time intervals [Figure 4] and [Table 1]. Overall, the response of NaOCl was dose-time dependent. 1%, 3%, and 5% NaOCl remained toxic even after 5 min and onward exposure periods [Figure 5] and [Table 2]. For midterm evaluation, MTAD showed statistically significant (P< 0.001) increases in the LDH release even at 24 h, but this increase reached near to control levels by 96 h [Figure 6] and [Table 3].
|Figure 4: Dose-related inhibition of periodontal ligament cell proliferation by BioPure MTAD compared with 1%, 3%, and 5% sodium hypochlorite using trypan blue dye exclusion assay at 1, 5, 10, and 15 min time intervals|
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|Table 1: Comparison of sodium hypochlorite solutions (1%, 3%, and 5%) and BioPure MTAD at different time intervals using trypan blue dye exclusion assay|
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|Figure 5: Decreased cell viability and altered morphology of exposed periodontal ligament fibroblast cells exposed to the indicated concentrations of sodium hypochlorite and MTAD for 1, 5, 10, and 15 min|
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|Table 2: Comparison of NaOCl (1%, 3%, and 5%) and BioPure MTAD at different time intervals using lactate dehydrogenase assay|
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|Figure 6: Altered morphology of exposed periodontal ligament cells to BioPure MTAD. The primary periodontal ligament cells were seeded in 96-well culture plates and exposed to MTAD for 24, 48, 72, and 96 h |
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|Table 3: Comparison of cytotoxicity parameters at different time intervals of BioPure MTAD at midterm (24, 48, 72, and 96 h) time interval|
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As evident from [Figure 5], NaOCl exposure resulted in concentration and time-dependent alterations in morphology as well as cell number. When the cells were exposed for 1 min to NaOCl, slight differences in the cell morphology were noted. In sharp contrast, for 5, 10, and 15 min of NaOCl exposure, the cells were seen to be less healthy with disruption of plasma membrane and release of cytosolic contents [Figure 5]. When cells were exposed for 24 h to NaOCl, all the cells were found to be dead with no viable cells [Figure 1].
In case of MTAD, there was no significant difference noted in the cell morphology at 1, 5, 10, and 15 min [Figure 6]. At 24 h, the cell number and proliferative capacity were significantly affected when compared to unexposed control groups with the increase in exposure periods. Cell rupture and damage with release of cytoplasmic contents into the cell medium can be observed [Figure 6].
| Discussion|| |
One of the main goals of root canal therapy is to completely disinfect root canals. The use of irrigants is to essentially facilitate intracanal cleansing by washing out debris, disinfecting, and dissolving any tissue tags that may remain within the complex canal anatomy.
The physical, chemical, and biologic properties are important factors to consider when deciding which irrigating solution to be used. The toxic effects of irrigating solutions used in endodontics are of particular interest because their untoward extrusion into the periapical area may be detrimental to healing capacity of the periradicular tissues.
It is a well-known fact that the worldwide use of NaOCl irrigation is because of its inherent antibacterial and pulp-dissolving capacity.,, Both these properties are, however, strongly influenced by their concentration., Therefore, biocompatibility plays a pivotal role in the choice of an endodontic irrigant during root canal preparation.
Studying the cytotoxic properties of an endodontic irrigant on PDL cells at various concentrations is on the way to determine the choice of irrigant. The primary PDL cell cultures have shown to retain their metabolic state when compared to their original tissue and therefore are commonly used for cytotoxicity evaluations. Due to this nearly unchanged metabolic state,in vitro studies closely approximate the in vivo situation. For these reasons, these cell cultures have been commonly used for cytotoxicity evaluation.,
Several modern assays are available to study the viability and proliferative capacity of cells in culture. The advantage of using microtiter plates (96-well format) is that the analysis of samples is quick and simultaneous. One parameter to evaluate cell death is to measure the cell membrane integrity. In this study, two assays were used to evaluate the viability and cytotoxicity of PDL cells by the intactness of their cell membrane.
The LDH is a stable cytoplasmic enzyme present in all cells. It is rapidly released into the cell culture supernatant on damage of the plasma membrane. The LDH release assay is a simple, accurate, and reproducible assay that determines the integrity of the cell membrane by measuring the cytoplasmic enzyme activity released by damaged/necrotic cells.
TBDE assay is a diazo dye used to color dead tissues or cells a distinctive blue color under a microscope. Live cells are excluded from staining.
The results of the present study have shown minimal cytotoxicity of BioPure MTAD against PDL cells when compared to the conventional gold standard of irrigation, NaOCl. The cytotoxicity of dental materials has been evaluated on the basis of changes in their cell number, intracellular metabolism, and morphology.
In the case of BioPure MTAD, maximum reduction in cell viability was recorded in the first 5 min of exposure, but these values were significantly less when compared to 1% concentration of NaOCl used for the same time period. Yasuda et al. have stated that BioPure MTAD has minimal cytotoxicity against MC3T3-E1 and PDL cells compared with conventional irrigants. Zhang et al. compared BioPure MTAD to the most commonly used intracanal irrigants using MTT-tetrazolium method on L929 fibroblasts and concluded that BioPure MTAD showed the least cytotoxic effects.
Regarding the cytotoxicity of various concentrations of NaOCl, Chang et al., with the help of PI fluorescence assay, reported that the toxic effects of NaOCl increased with concentration. PDL cells were destroyed at 3 and 24 h time interval when 5.25% NaOCl was diluted.
Our results concur with those obtained by Silva et al. and Spangberg et al. The results of their studies also concluded that 5% NaOCl showed the maximum cytotoxicity at short-time intervals  and various concentrations of NaOCl are dose dependent. Similar findings were observed by Essner et al. using CellTiter-Glo luminescent cell viability assay to determine the viability of the pulp cells. They found that as NaOCl concentration increased from 0.04% to 0.33%, the pulpal cells viability decreased correspondingly.
For midterm evaluation, BioPure MTAD showed a significant increase in cell viability. All cells were dead within 24 h when exposed to all the three concentrations of NaOCl.
| Conclusions|| |
The present study showed that in both cytotoxic assays, BioPure MTAD had a lower toxicity against PDL compared to NaOCl. Overall response of NaOCl was concentration dependent with 1%, 3%, and 5% NaOCl remained toxic even after 5 min and onward exposure periods. Further studies will be needed to determine the long-term toxic effects of BioPure MTAD on cells in vivo.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.
Mehdipour O, Kleier DJ, Averbach RE. Anatomy of sodium hypochlorite accidents. Compend Contin Educ Dent 2007;28:544-6, 548, 550.
Spencer HR, Ike V, Brennan PA. Review: The use of sodium hypochlorite in endodontics – Potential complications and their management. Br Dent J 2007;202:555-9.
Ruddle CJ. Endodontic disinfection: Tsunami irrigation. Saudi Endod J 2015;5:1-12. [Full text]
Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis
: An ex
study. Saudi Endod J 2012;2:70-4. [Full text]
Siqueira JF Jr., Batista MM, Fraga RC, de Uzeda M. Antibacterial effects of endodontic irrigants on black-pigmented gram-negative anaerobes and facultative bacteria. J Endod 1998;24:414-6.
Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod 2003;29:334-7.
Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ, et al. In vitro
evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84.
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.
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.
Shetty KP, Satish SV, Kilaru K, Ponangi KC, Venumuddala VR, Ratnakar P. Comparative evaluation of the cytotoxicity of 5.25% sodium hypochlorite, 2% chlorhexidine and mixture of a tetracycline isomer, an acid and a detergent on human red blood corpuscles: An in vitro
study. Saudi Endod J 2014;4:1-6. [Full text]
Gatot A, Arbelle J, Leiberman A, Yanai-Inbar I. Effects of sodium hypochlorite on soft tissues after its inadvertent injection beyond the root apex. J Endod 1991;17:573-4.
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.
Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J Endod 2003;29:654-7.
Pant V, Dixit J, Agrawal AK, Seth PK, Pant AB. Behaviour of human periodontal ligament cells on CO2
laser irradiated dentinal root surfaces: An in vitro
study. J Periodontal Res 2004;39:373-9.
Siddiqui MA, Singh G, Kashyap MP, Khanna VK, Yadav S, Chandra D, et al.
Influence of cytotoxic doses of 4-hydroxynonenal on selected neurotransmitter receptors in PC-12 cells. Toxicol In Vitro
Barnhart BD, Chuang A, Lucca JJ, Roberts S, Liewehr F, Joyce AP, et al.
An in vitro
evaluation of the cytotoxicity of various endodontic irrigants on human gingival fibroblasts. J Endod 2005;31:613-5.
Al-Shaher A, Wallace J, Agarwal S, Bretz W, Baugh D. Effect of propolis on human fibroblasts from the pulp and periodontal ligament. J Endod 2004;30:359-61.
Cook JA, Mitchell JB. Viability measurements in mammalian cell systems. Anal Biochem 1989;179:1-7.
Weyermann J, Lochmann D, Zimmer A. A practical note on the use of cytotoxicity assays. Int J Pharm 2005;288:369-76.
Korzeniewski C, Callewaert DM. An enzyme-release assay for natural cytotoxicity. J Immunol Methods 1983;64:313-20.
DeRenzis FA, Schechtman A. Staining by neutral red and trypan blue in sequence for assaying vital and nonvital cultured cells. Stain Technol 1973;48:135-6.
Yasuda Y, Tatematsu Y, Fujii S, Maeda H, Akamine A, Torabinejad M, et al.
Effect of MTAD on the differentiation of osteoblast-like cells. J Endod 2010;36:260-3.
Silva FT, Barcelos R, Petrópolis DB, Azevedo BR, Primo LG, Silva-Filho FC. Cytotoxicity of different concentrations of sodium hypochlorite on human osteoblasts. Rev Gaúcha Odontol 2009;57:317-21.
Spangberg L, Engström B, Langeland K. Biologic effects of dental materials 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro
. Oral Surg Oral Med Oral Pathol 1973;36:856-71.
Essner MD, Javed A, Eleazer PD. Effect of sodium hypochlorite on human pulp cells: An in vitro
study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:662-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]