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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 34  |  Issue : 2  |  Page : 76-79

Cytotoxicity of two different intercanal medicaments on human gingival fibroblasts - A Laboratory study


1 Private Practice of Endodontic, Isfahan University of Medical Sciences, Isfahan, Iran
2 Dental Research Center, Department of Endodontics, Dental Research Institute, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Endodontics, School of Dentistry, UCSF Preventive & Restorative Dental Science, San Francisco, California, USA
4 Post Graduate Student of Prosthodontics, Student Research Committee, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
5 Dentist, Private Practice, Isfahan, Iran
6 Department of Prosthodontics, School of Dentistry, Shahrekord University of Medical Sciences, Iran

Date of Submission13-May-2021
Date of Decision01-Jun-2021
Date of Acceptance12-Aug-2021
Date of Web Publication01-Jul-2022

Correspondence Address:
Dr. Amin Davoudi
Department of Prosthodontics, School of Dentistry, Shahrekord University of Medical Sciences, Resalat Square, Shahrekord, 038-322322400
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/endo.endo_107_20

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  Abstract 


Aim: Intracanal medicaments are often recommended during endodontic sessions to eliminate the necrotic debris and microorganisms. The aim of the present study was to observe the cytotoxicity of calcium hydroxide (CH) and colchicine (COL), at different concentrations, on human gingival fibroblast cells.
Materials and Methods: Human gingival fibroblasts were cultured on plastic flasks containing RPMI 1640 media and fetal calf serum 10% supplemented with antibiotic agents. Trypsin/ethylenediaminetetraacetic acid 0.2% enzyme was used to isolate the cells, and the suspension was transferred to tubes for centrifuging. Conventional CH and COL were separately mixed with sterile saline solution to prepare a stock media. By serial dilution of stock media, desired concentrations were prepared at 2, 1.75, 1.5, and 1.25 mg/ml, separately. After considering a control group, the cells were exposed to test materials. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted at 24 h, 72 h, and 7 days later. Optical density (OD) was evaluated to attain cell viability percentage. Finally, the recorded data were analyzed by Kruskal–Wallis and Mann–Whitney tests using SPSS software version 19 at a significant level of 0.05.
Results: The highest (1.40 ± 0.66) and lowest (0.15 ± 0.00) ODs were observed in CH 1.25 mg/ml and COL 1.5 mg/ml after 72 h, respectively. All of the concentrations of both CH and COL showed significant OD differences with the control group (all P = 0.001).
Conclusion: Both CH and COL manifested similar cytotoxicity on human gingival fibroblast cells.

Keywords: Calcium hydroxide, colchicine, cytotoxicity test, fibroblast


How to cite this article:
Barakatein B, Farhad A, Shadmehr E, Sharifi HM, Mohamad Sharif MM, Davoudi A. Cytotoxicity of two different intercanal medicaments on human gingival fibroblasts - A Laboratory study. Endodontology 2022;34:76-9

How to cite this URL:
Barakatein B, Farhad A, Shadmehr E, Sharifi HM, Mohamad Sharif MM, Davoudi A. Cytotoxicity of two different intercanal medicaments on human gingival fibroblasts - A Laboratory study. Endodontology [serial online] 2022 [cited 2022 Aug 17];34:76-9. Available from: https://www.endodontologyonweb.org/text.asp?2022/34/2/76/349577




  Introduction Top


An ideal intracanal medicament must impose the least cytotoxicity to the periapical tissue providing optimum antibacterial activities.[1],[2] Therefore, to reduce necrotic remnants and micro-organisms, the administration of different intracanal medications is often recommended between the treatment meeting.[3] An ideal intracanal medicament must impose the least cytotoxicity to the periapical tissue providing optimum antibacterial activities.[4] It has been stated that irrigants and intracanal medicaments have the potent to endanger the survivability of stem cells from human exfoliating deciduous teeth, and stem cells from the apical papilla, periodontal ligament cells within the root canal environment.[5] Therefore, evaluation of cytotoxicity of any endodontic materials is necessary before clinical application. Calcium hydroxide (CH) has been introduced as an effective antimicrobial agent with pH of 12.5. CH could neutralize the biological activity of bacterial lipopolysaccharide and make necrotic tissue more susceptible to the solubilizing by sodium hypochlorite (NaOCl) at the next appointment.[6] However, because of high pH, CH is potentially toxic and tends to dissolve soft tissues.[7] Moreover, released evidences mentioned that some microorganisms are resistant to CH such as Enterococcus faecalis (4), Candida species (4), and Actinomyces radicidentis.[8] Hence, some clinicians suggest to reinforce the antimicrobial efficacy of CH by adding some other agents to eliminate the viable microorganisms. Colchicine (COL), extracted from genus Colchicum autumn and Colchicum autumnale plants, is used for treating acute gout and familial Mediterranean fever.[9] some implications of COL are rheumatism treatment complaints, pericarditis and Behce's disease. It binds to tubulin, which is one of the main constituents of microtubule and essential for mitosis process, and inhibits its polymerization.[10] Therefore, COL can effectively act as a mitotic poison and stops cell division.[11] Besides that, COL has anti-inflammatory and analgesic properties.[12] Ahmad et al. claimed that COL had mild-to-moderate antibacterial and moderate-to-excellent antifungal properties.[13]

As evaluation of the cytotoxicity of any endodontic materials is critical before clinical application, the aim of the present study was to observe the cytotoxicity of CH and COL, at different concentrations, on human gingival fibroblast cells.


  Materials and Methods Top


This study was approved by the Research Ethics Committee of Torabinejad Research Center and Dental Implant Research Center, Isfahan University of Medical Sciences, Protocol # 393408.

Culture preparation

In this analytical-observational in vitro study, human gingival fibroblasts (C165, Pasteur Institute, Tehran, Iran) were allowed to attach to the bottom of plastic culture flasks containing RPMI 1640 media (Roswell Park Memorial Institute, NY, USA) and fetal calf serum 10% (Sigma-Aldrich, NY, USA) supplemented with antibiotic agents (penicillin 100 U/ml and streptomycin 100 U/ml). The plastic culture flasks were incubated at 5% CO2, 37°C, and 98% humidity for 24 h to prepare monolayer cells. When the cultured cells reached out to 80%, 3 cc of trypsin/ethylenediaminetetraacetic acid 0.2% enzyme (GIBCO, Grand Island, NY, USA) was used for 3 min at 37°C to isolate the cells. After neutralizing the enzyme by adding RPMI 1640 media, the suspension was transferred to tubes for centrifuging (Hettich, Kirchlengern, Germany) at 1400 cycles for 5–10 min. The supernatants were removed and replaced by RPMI 1640 media to prepare a cell suspension (VELP Scientifica, Usmate, Italy).

Preparing extract

Conventional CH (Merck, Whitehouse Station, NJ, USA) and COL (Sigma-Aldrich, NY, USA), both in the powder form, were separately mixed with sterile saline solution (0.2 mg of powder with 10 ml of saline solution) and incubated for 48 h to prepare a stock media of test materials. 0.2-micron filters were used to sterilize and purify the prepared extract. Then, serial dilution of stock media, by RPMI 1640 media, was done to prepare desired concentrations of 2 mg/ml, 1.75 mg/ml, 1.5 mg/ml, and 1.25 mg/ml of both CH and COL, separately. All the prepared concentrations were autoclaved (Faraz Mehr, Tehran, Iran) at 121°C for 30 min.

Cytotoxicity evaluation

After ensuring cell viability by trypan blue dye, 1.0 ml of RPMI 1640 media was transferred to an individual well of a 12-well plate in which 2.5 × 104 monolayer cells were seeded previously with the aid of Neubauer slides and cell-counting assay as follow:



Each three separate wells were exposed to the prepared concentrations of test materials (totally 24 wells), and three wells were designated as a control group without adding any test materials.

To evaluate the viable cells, an 3- (4,5-dimethylthiazol-2-yl)- 2,5 -diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, NY, USA) was performed at 24 h, 72 h, and 7 days later. The cytotoxicity of test materials was evaluated by MTT assay in which tetrazolium reagent is reduced by mitochondrial dehydrogenases into blue formazan molecules, and can be measured by spectrophotometry.[14] Hence, the amount of produced formazan is directly related to the total remained viable cells. Therefore, after elapsing time period, the exposed mediums were rinsed by phosphate-buffered saline. Then, 400 μl of medium plus 40 μl of MTT was added to the specimens. After 4-h incubation, the mediums were replaced by 400 μl dimethyl sulfoxide and stored for 2 h at darkness. In the next step, 100 μl of each colored media was transferred to the ELISA reader (Anthos 2020, Biochrom, Cambridge, UK) adjusted at 570 nm wavelength with 620 reference filters. Optical density (OD) was evaluated to attain cell viability percentage. Finally, the recorded data were analyzed by Kruskal–Wallis and Mann–Whitney tests using SPSS software version 19 at a significant level of 0.05.


  Results Top


[Table 1] represents the mean OD of test materials in different intervals. The highest (1.40 ± 0.66) and lowest (0.15 ± 0.00) ODs were observed in CH 1.25 mg/ml and COL 1.5 mg/ml after 72 h, respectively. Intragroup comparison was executed by the Kruskal–Wallis test, and no significant difference was found among the tested concentrations of each material, except for CH after 24 h (P = 0.03) and COL after 72 h (P = 0.01). Intergroup comparison, by Mann–Whitney, revealed no significant differences among CH and COL 2 mg/ml at different time intervals (all P = 0.10). The same results were found for the other concentrations of CH and COL (1.75 mg/ml, 1.5 mg/ml, and 1.25 mg/ml) at different time intervals (all P = 0.10). However, all of the concentrations of both CH and COL reflected significant OD differences with the control group (all P = 0.001).
Table 1: The mean optical density of tested materials at different concentrations and time period

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  Discussion Top


The cytotoxicity of each newly discovered dental material should be assessed in vitro and in vivo The in vitro examinations offer simplicity, reproducibility, and better handling of testing materials.[15] Fibroblasts are the major cells of the connective tissue that are responsible for producing and persevering the connective tissue matrix. Scientifically, the fibroblast cells uptake the inflammatory toxins and bacterial byproducts to diminish the inflammation phase. Hence, fibroblast cells seem to be appropriate for measuring the cytotoxicity of dental materials.[16] In the present study, monolayer fibroblast cells were cultured and minimum number of cell passage was ordered to preserve the cells' function as much as possible. According to the results of the present study, the CH caused significant cytotoxicity, compared to the control group, at all of the tested intervals.

Most of the published studies evaluated the CH cytotoxicity indirectly, in the way that CH was one of the compositions of tested dental materials.[17],[18],[19],[20],[21] Therefore, controversial result might be noticed during reviewing literatures.[19],[22] Beltes et al. surveyed the cytotoxicity of three endodontic sealers containing CH (Apexit, calcibiotic root canal sealer, and Sealapex). They observed that all of the studied materials showed toxic effects on L929 murine fibroblasts.[15] In another study, Key et al. evaluated the cytotoxicity of different endodontic sealers on gingival fibroblasts. The final results manifested that the sealer which contained CH caused the highest percentage of cell death after 24 h.[22] In contrast, Labban et al. observed the viability of dental pulp cells after exposure to CH 0.3–5 mg/ml and different antibiotic pastes. They reported that only CH 5 mg/ml caused significant cytotoxicity, moreover CH 2.5 mg/ml was the highest nontoxic concentration.[19] In the present study, despite significant cytotoxicity of CH compared to the control group, the CH 1.25 mg/ml showed promising results after 24 h in which it caused significantly higher cell viability than other concentrations. Labban et al. cultured pulpal cells, which were derived from healthy impacted teeth, and used different methods of cytotoxicity assay (lactate dehydrogenase assay). Different methodologies and the high proliferative potential of pulpal cells, which can overcome the toxicity of CH,[19] might explain the different statements between two studies. Another study, by Barbosa et al., was conducted to determine the cytotoxicity of CH 2 mg/ml and sodium lauryl sulfate solution at concentration of 5%, 10%, 20%, and 50% on human fibroblast.[20] Their final result revealed that only CH 50% (meaning 1 mg/ml) had toxic impacts on cultured fibroblasts. Apparently, higher concentration of CH was administered in the present study which might be the main reason of different results. The cytotoxicity of CH might be ascribed to its high alkalinity and interaction with hydroxyl ions adjacent to cells, resulting in cell necrosis and apoptosis.[23]

The biocompatibility of COL, as an intracanal medication, has not been evaluated until now. Biological effects of COL and its derivatives are strictly bound to their inhibitory activities on polymerization of tubulins. The majority of side effects of COL are demonstrated in proliferating cells, for example, intestinal mucosa (nausea and vomiting) or hairs (alopecia). Its major side effects mostly influence the proliferation of cells, for example, intestinal mucosa or hairs.[24] The result of the current study showed similar cytotoxicity effects between COL and CH. Mansour et al. evaluated the COL on collagen synthesis and secretion of liver fibroblasts. They observed that fibroblast proliferation was reduced after COL treatment, dose dependently.[25] In another study, Dvorak et al. tried to alleviate the cytotoxicity of COL. Hence, they examined 17 COL derivatives on human hepatocytes. Their final results reflected that replacing COL with a derivative with lower antitubulin activity might provide lower side effects. They stated that 10-O-demethylated derivatives of COL represented remarkably lower cytotoxicity on human hepatocytes.[26]


  Conclusion Top


With considering common limitations of in vitro studies, such as sample preparation and handlings, preserving the specimens during study and preparation of more derivatives,[27] the study concludes that both CH and COL manifested similar cytotoxicity on human gingival fibroblast, however, not much studies have reported the scope of COL, in endodontics. Nevertheless, few points of concern are in a document. Other derivatives of COL at other concentrations are recommended for future studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Gomes BP, Ferraz CC, Vianna ME, Rosalen PL, Zaia AA, Teixeira FB, et al. In vitro antimicrobial activity of calcium hydroxide pastes and their vehicles against selected microorganisms. Braz Dent J 2002;13:155-61.  Back to cited text no. 1
    
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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.  Back to cited text no. 5
    
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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.  Back to cited text no. 7
    
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Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579-86.  Back to cited text no. 8
    
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Larsson S, Rønsted N. Reviewing Colchicaceae alkaloids – Perspectives of evolution on medicinal chemistry. Curr Top Med Chem 2014;14:274-89.  Back to cited text no. 9
    
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Andreu JM, Timasheff SN. Tubulin bound to colchicine forms polymers different from microtubules. Proc Natl Acad Sci U S A 1982;79:6753-6.  Back to cited text no. 10
    
11.
Farrell KW, Wilson L. Proposed mechanism for colchicine poisoning of microtubules reassembled in vitro from Strongylocentrotus purpuratus sperm tail outer doublet tubulin. Biochemistry 1980;19:3048-54.  Back to cited text no. 11
    
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Schlesinger N. Management of acute and chronic gouty arthritis: Present state-of-the-art. Drugs 2004;64:2399-416.  Back to cited text no. 12
    
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Ahmad B, Khan H, Bashir S, Ali M. Antimicrobial bioassay of Colchicum luteum Baker. J Enzyme Inhib Med Chem 2006;21:765-9.  Back to cited text no. 13
    
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Sumantran VN. Cellular chemosensitivity assays: An overview. Methods Mol Biol 2011;731:219-36.  Back to cited text no. 14
    
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Beltes P, Koulaouzidou E, Kotoula V, Kortsaris AH. In vitro evaluation of the cytotoxicity of calcium hydroxide-based root canal sealers. Endod Dent Traumatol 1995;11:245-9.  Back to cited text no. 15
    
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Mozayeni MA, Milani AS, Marvasti LA, Asgary S. Cytotoxicity of calcium enriched mixture cement compared with mineral trioxide aggregate and intermediate restorative material. Aust Endod J 2012;38:70-5.  Back to cited text no. 16
    
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Sarigol CG, Cogulu D, Oncag O, Deliloglu IG. Cytotoxic effects of primary tooth root canal filling materials on L929 cell line. J Dent Child (Chic) 2010;77:72-6.  Back to cited text no. 17
    
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Miranda RB, Fidel SR, Boller MA. L929 cell response to root perforation repair cements: An in vitro cytotoxicity assay. Braz Dent J 2009;20:22-6.  Back to cited text no. 18
    
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Labban N, Yassen GH, Windsor LJ, Platt JA. The direct cytotoxic effects of medicaments used in endodontic regeneration on human dental pulp cells. Dent Traumatol 2014;30:429-34.  Back to cited text no. 19
    
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Barbosa SV, Barroso CM, Ruiz PA. Cytotoxicity of endodontic irrigants containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from mouse L929 cell line. Braz Dent J 2009;20:118-21.  Back to cited text no. 20
    
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Dianat O, Azadnia S, Mozayeni MA. Toxicity of calcium hydroxide nanoparticles on murine fibroblast cell line. Iran Endod J 2015;10:49-54.  Back to cited text no. 21
    
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Key JE, Rahemtulla FG, Eleazer PD. Cytotoxicity of a new root canal filling material on human gingival fibroblasts. J Endod 2006;32:756-8.  Back to cited text no. 22
    
23.
Silva EJ, Herrera DR, Almeida JF, Ferraz CC, Gomes BP, Zaia AA. Evaluation of cytotoxicity and up-regulation of gelatinases in fibroblast cells by three root repair materials. Int Endod J 2012;45:815-20.  Back to cited text no. 23
    
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Bibas R, Gaspar NK, Ramos-e-Silva M. Colchicine for dermatologic diseases. J Drugs Dermatol 2005;4:196-204.  Back to cited text no. 24
    
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Mansour MM, Dunn MA, Salah LA. Effect of colchicine on collagen synthesis by liver fibroblasts in murine schistosomiasis. Clin Chim Acta 1988;177:11-20.  Back to cited text no. 25
    
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Dvorak Z, Ulrichova J, Weyhenmeyer R, Simanek V. Cytotoxicity of colchicine derivatives in primary cultures of human hepatocytes. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2007;151:47-52.  Back to cited text no. 26
    
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