Abstract

Background and aims. Since intracanal medicaments can affect the cell viability in periapical tissues, the aim of this

study was to evaluate the effect of calcium hydroxide and propolis on pulp fibroblasts.

Materials and methods. Two healthy third molars were used as a source to obtain fibroblasts. The fibroblasts were

cultured and subjected to 1 mg/mL of propolis and calcium hydroxide. This experiment was performed in six replicates and

cell viability was evaluated with MTT assay. Statistical analysis was performed by t-test.

Results. Comparison of cell viability with the use of 1 mg/mL of calcium hydroxide and propolis showed that cells subjected

to propolis were more viable when compared to calcium hydroxide (P < 0.05).

Conclusion. In this study, calcium hydroxide reduced fibroblast viability, significantly more than Iranian propolis. Other

properties should be evaluated before Iranian propolis could be indicated for use as intracanal medicament

Ranjbarian, Parisa.

Shiravi, Samaneh.

Zare Jahromi, Maryam.

PubMed

Background and aims. Since intracanal medicaments can affect the cell viability in periapical tissues, the aim of this study was to evaluate the effect of calcium hydroxide and propolis on pulp fibroblasts.

Materials and methods. Two healthy third molars were used as a source to obtain fibroblasts. The fibroblasts were cultured and subjected to 1 mg/mL of propolis and calcium hydroxide. This experiment was performed in six replicates and cell viability was evaluated with MTT assay. Statistical analysis was performed by t-test.

Results. Comparison of cell viability with the use of 1 mg/mL of calcium hydroxide and propolis showed that cells sub-jected to propolis were more viable when compared to calcium hydroxide (P < 0.05).

Conclusion. In this study, calcium hydroxide reduced fibroblast viability, significantly more than Iranian propolis. Other properties should be evaluated before Iranian propolis could be indicated for use as intracanal medicament

Maryam Zare Jahromi

Parisa Ranjbarian

Samaneh Shiravi

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Journal of Dental Research Dental Clinics Dental Prospects

Cytotoxicity Evaluation of Iranian Propolis and Calcium Hydroxide on Dental Pulp Fibroblasts

shahrekord university of medical scinces

  Journal of Dental Research, Dental Clinics, Dental Prospects Introduction ntracanal medicaments must be used at concentra-tions that are bactericidal while having minimal effects on cell viability in periapical tissues. The choice of this agent depends on biologic characteris-tics; such medicaments should be non-irritant, able to control the intensity and duration of inflammatory processes and infection, and have healing poten-tial.1,2 Presently, calcium hydroxide is the most commonly used medication for root canal disinfec-tion.1,2 This application of calcium hydroxide is based on its ability to neutralize microbial byprod-ucts; however, because of its high pH, calcium hy-droxide can lead to chronic inflammation and cell necrosis in vivo.3 In addition, calcium hydroxide has shown to be ineffective in destroying bacteria in vi-vo.3 The inefficiency of calcium hydroxide is sug-gested to be caused by failure of the medication to reach the site of action and by the buffering capacity of hydroxyapatite.4,5 There has been considerable discussion about finding alternate intracanal medica-tions as an adjunct to irrigation and mechanical cleaning for the elimination of bacteria with mini-mum side effect and maximum biocompatibility. Recently, propolis, a flavonoid-rich product of bee Original Article Cytotoxicity Evaluation of Iranian Propolis and Calcium Hydroxide on Dental Pulp Fibroblasts Maryam Zare Jahromi1 • Parisa Ranjbarian2 • Samaneh Shiravi3* 1Assistant Professor, Department of Endodontics, Faculty of Dentistry, Islamic Azad University Khorasgan (Isfahan) Branch, Isfahan, Iran 2Post-graduate Student, Department of Endodontics, Faculty of Dentistry, Islamic Azad University, Khorasgan (Isfahan) Branch, Isfahan, Iran 3Dentist, Private Practice, Isfahan, Iran *Corresponding Author; E-mail: samanehshiravi@ymail.com Received: 20 October 2013; Accepted: 4 October 2014 J Dent Res Dent Clin Dent Prospect 2014; 8(3):130-133 | doi: 10.5681/joddd.2014.024This article is available from: http://dentistry.tbzmed.ac.ir/joddd  This article is available from: http://dentistry.tbzmed.ac.ir/joddd © 2014 The Authors; Tabriz University of Medical Sciences This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract  Background and aims. Since intracanal medicaments can affect the cell viability in periapical tissues, the aim of this study was to evaluate the effect of calcium hydroxide and propolis on pulp fibroblasts. Materials and methods. Two healthy third molars were used as a source to obtain fibroblasts.  The fibroblasts were cultured and subjected to 1 mg/mL of propolis and calcium hydroxide. This experiment was performed in six replicates and cell viability was evaluated with MTT assay. Statistical analysis was performed by t-test. Results. Comparison of cell viability with the use of 1 mg/mL of calcium hydroxide and propolis showed that cells sub-jected to propolis were more viable when compared to calcium hydroxide (P < 0.05). Conclusion. In this study, calcium hydroxide reduced fibroblast viability, significantly more than Iranian propolis. Other properties should be evaluated before Iranian propolis could be indicated for use as intracanal medicament. Key words: Cytotoxicity, calcium hydroxide, fibroblast, MTT, propolis. I JODDD, Vol. 8, No. 3 Summer 2014 Iranian Propolis vs. Calcium Hydroxide Cytotoxicity    131 wax, has shown to possess antimicrobial and anti-inflammatory properties.7,8 The main chemical agents present in propolis are flavonoids, phenolics and various aromatic compounds.6 Flavonoids are well-known plant compounds that have antioxidant, antibacterial, antifungal, antiviral and anti-inflammatory properties; they also aid the immune system by promoting phagocytic activity and stimu-late cellular immunity.7 Other applications of propolis, other than as an in-tracanal medicament, in dentistry include medica-ment for wound healing, intermediate environment for avulsed teeth, its use as a pulp capping material, anti-cavity effect (due to antibacterial activity against Streptococcus mutans), treatment of denture stomatitis, tooth sensitivity control, effects and treatment of recurrent aphthous stomatitis and acute necrotizing ulcerative gingivitis. Commercially available forms of propolis are toothpastes, mouthwashes, rod tablets, beverages, cakes, powders, gels and soap. Awawdeh et al8 evaluated the efficacy of propolis and calcium hydroxide as a short-term intracanal medicament against Enterococcus faecalis. They concluded that propolis is very effective as intracanal medicament in rapidly eliminating E. faecalis ex vi-vo. Ahangari et al9 compared the antimicrobial activ-ity of propolis and calcium hydroxide and claimed that propolis is more effective against Enterococcus faecalis and Lactobacillus peptostreptococcus than calcium hydroxide in agar culture. Victorino et al10 produced a propolis paste formulation for endodontic use, with the lowest concentration of crude extract of propolis which retains its biological activity. Other authors have shown that propolis can be useful as a root canal dressing due to its low toxicity and broad antibacterial spectrum.11 Al-Shaher et al3 examined the resistance of fibroblasts of the periodontal liga-ment (PDL) and dental pulp to propolis compared with calcium hydroxide in their in vitro study. Data revealed that exposure of PDL or pulp fibroblasts to 4 mg/mL or lower concentrations of propolis re-sulted in more than 75% cells viability. On the con-trary, 0.4 mg/mL of calcium hydroxide was cyto-toxic and less than 25% of the cells were found to be viable. In conclusion, propolis can be recommended as a suitable transport medium for avulsed teeth.3 Further investigations may find propolis to be a pos-sible alternative for an intracanal antimicrobial agent. The aim of this study was to investigate the effect of Iranian propolis and calcium hydroxide on pulp fibroblasts.  Materials and Methods Preparation of ethanol extract of propolis (EEP) Propolis samples were obtained from the beehives of Esfahan countryside. Propolis was minced with little hand pressure into pieces with a thickness of 2-4 mm at 37°C and then transferred to an environment with a temperature of -20°C. After 24 hours the samples were ground in an electric mill. The resultant powder was transferred to and maintained in a -20°C envi-ronment for 24 hours and then was ground again.12 A total of 5 grams of propolis was dissolved in 50 mL of 96% ethanol at 37ºC and sonicated for 10 minutes. The solution was filtered using a 20-µ filter; EEP was obtained at a concentration of 100 mg/mL EEP has better effects than aqueous solution due to the easier release and isolation of flavonoids (the active component of propolis).11 To perform this experimental study, two healthy third molars were used as a source to obtain fibro-blasts; the extraction procedure was kept simple to prevent tooth damage. Immediately after extraction, the third molars were washed using gauze soaked in 70% ethanol (Zakariya Razi, Tehran, Iran), followed by a wash with sterile distilled water (Gibco,  Carls-bad, US). Holding the tooth with upper incisor for-ceps (Aesculap, Tuttlingen, Germany), a cut was made between the enamel and the cement using a cylindrical bur (Tizkavan, Tehtan, Iran). A fracture was made on the same line and the tooth fragments were placed in a Falcon flask containing sterile PBS 1X (Gibco, Carlsbad, US). The samples were rapidly transported to the laboratory and placed in Petri dishes in a laminar flow hood (Jal Tajhiz, Karaj, Iran). The tissues were isolated from the dental pulp using a #15 sterile endodontic file and forceps. Cel-lular separation was completed by digesting the di-vided pulp tissue with 3 mg/mL collagenase type I (Sigma, Seelze, Germany) for 60 minutes at 37°C. The cells were then separated using an insulin sy-ringe and centrifuged for 10 minutes at 1800 rpm. The cell fraction was washed twice with sterile PBS 1X and centrifuged again for 10 minutes at 1800 rpm at room temperature.13 The obtained fibroblasts were cultured in DMEM #Hams F12 (Gibco,  Carlsbad, US)  supplemented with 10% FBS (Sigma, Seelze, Germany), 2 ML-glutamine (Sigma, Seelze, Ger-many), penicillin G 100 mg/mL (Sigma, Seelze, Ger-many), streptomycin 100 µg/mL (Sigma, Seelze, Germany) and 1% Fungizone (Sigma, Seelze, Ger-many) and incubated at 37°C in humidified 95% air and 5% CO2 for 3 weeks.14 The medium was re-JODDD, Vol. 8, No. 3 Summer 2014 132     Jahromi et al. freshed every 3 days until the cells reached 80% con-fluency.  In the third passage the cells were plated at 1×105 cell/mL per well onto 96-well plates. Then the cells maintained at 37°C and 5% CO2 in a humidified in-cubator (Memmert, Frankfurt, Germany).15 Then 10 µL of 1 mg/mL propolis and calcium hydroxide (Merck, Frankfurter, Germany) was added to each well and incubated for 24 hours again. After expo-sure to the solutions, the cells were washed twice in PBS to remove dead cells.16 To determine cell viabil-ity 100 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma, Seelze, Germany) solution (5 mg/mL) was added to each well of the plate and further incubated at 37°C for 4 hours. To dissolve the formazan precipitate the me-dium in each well was changed to 100 µL dimethyl sulfoxide (DMSO) (Sigma, Seelze, Germany) and mixed thoroughly. After 10 minutes at room tem-perature to ensure that all crystals were dissolved, optical densities of each plate were read with ELISA reader (Biorad, Mannheim, Germany) at a wave-length of 550 nm.17 Cells exposed to cell culture me-dium served as a control for cell viability. All the experiments were conducted in six replicates per group and statistical analysis was performed by t-test. In addition, the following grading system was used for qualitative comparison of the results: in 90% cell viability, the material was considered bio-compatible; in 60-90% cell viability, it was consid-ered mild toxic; in 30-59% moderately toxic, and in less than 30% of cell viability it was considered se-verely toxic.18Results The percentage of cell viability was calculated as the relative absorbance of sample versus control wells as follows: % cell viability = (mean optical density of experimental well/mean optical density of control well) ×100. In this context, treatment of dental pulp fibroblasts with 1 mg/mL of propolis and calcium hydroxide resulted in 75.20% and 11.34% cell vi-ability, respectively. Comparison of the cell viability showed that at concentration of 1 mg/mL, cells sub-jected to propolis significantly were more viable and calcium hydroxide was extremely toxic (P 0.05) (Table 1). Thus, the cytotoxicity of calcium hydrox-ide and propolis in these concentrations was consid-ered severe and mild. Discussion There have been considerable discussions about finding ideal intracanal medicaments; however, to justify their application, antibacterial activity must exceed cytotoxicity. Antibacterial agents that are cytotoxic and have enough potential to eliminate bacteria may damage periapical tissues.19 Trevino et al20 in a study concluded that irrigants and intracanal medicaments greatly affect the survivability of cells within the root canal environment; therefore, evalua-tion of cytotoxicity of these agents beside antibacte-rial  activity seems to be  necessary. Calcium hy-droxide has been introduced as an effective intraca-nal medicament because of its alkaline PH and anti-bacterial effect. However, because of high PH, cal-cium hydroxide is potentially toxic and tends to dis-solve soft tissues.3 Additionally, evidence indicates that a number of microorganisms such as E. fae-calis21, Candida species22 and Actinomyces radici-dentis23,24 are resistant to calcium hydroxide. Due to these disadvantages, the new and natural materials with minimum side effects and high antibacterial activity have attracted the attention of endodontists in endodontic therapy. One of these materials is pro-polis. Since calcium hydroxide is toxic and biofla-vonoids in propolis are antibacterial and anti-inflammatory, and due to the low content (0.5%) of alcohol in propolis extraction, this study intended to examine the effect of propolis on the fibroblasts of dental pulp as the first step in its possible use as an alternative intracanal medication. Considering the fact that the effect of propolis varies in different geographical regions and seasons of the year, spring-extracted propolis of Iran was used. To quantify vi-able cells, MTT assay was used. MTT is a simple, fast and reliable method for cytotoxicity assay. The methyl-tetrazolium ring is cleared to formazan by mitochondrial dehydrogenases in viable cells; for-mazan is blue in color and can be measured with a spectrophotometer.25 The amount of formazan pro-duced is directly proportional to the total viable cell number.25 In a study by Al-Shaher et al,3 calcium hydroxide was approximately 10 times more cyto-toxic than propolis. These results are similar to our findings. Our comparison of the toxicity of propolis to calcium hydroxide showed that at concentration of 1 mg/mL of calcium hydroxide, which is the most commonly used concentration for cytotoxicity assay in similar studies,1,26 resulted in less cell viability than propolis. The cause of more toxicity of calcium Table 1. Statistical values of the optical density of 1 mg/mL of propolis and Ca(OH)2Group N Mean Std. Deviation P value Ca(OH)2 6 0.35500 0.037084 <0.001 Propolis 6 2.35400 0.117849  Control 6 3.1300 0.199198  JODDD, Vol. 8, No. 3 Summer 2014 Iranian Propolis vs. Calcium Hydroxide Cytotoxicity    133 hydroxide may be attributed to high pH and hy-droxyl ions adjacent to cells, resulting in cell necro-sis and apoptosis.27 Anti-inflammatory properties might be the causes of lower toxicity of propolis, perhaps through suppression of immune cells, activa-tion of macrophage-derived nitric oxide and cytokine production, neutrophil activation and also synthesis of collagene.28 According to the results of this study and because of antimicrobial properties and ability to enhance immune response3 and lower cytotoxicity, propolis can be considered an intracanal medica-ment. However, further studies should be performed in order to investigate other properties of this sub-stance.  Limitations of this study were difficulty of finding open-apex third molars with minimal trauma and a simple way out, difficulty of ethanol extract of pro-polis and achieving appropriate levels of EEP, which is time-consuming and increases the cost of the study. Conclusions Calcium hydroxide significantly reduced fibroblasts viability more than Iranian propolis. Therefore, per-haps after evaluating other properties, Iranian propo-lis might be considered an intracanal medicament. References 1. Ruparel NB, Teixeria FB, Ferraz CC, Diogenes A. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372-5. 2. Santos Ramos I, Tillmann Biz M, Paulino N, Scremin A, Della Bona A, Barletta F, et al. Histopathological analysis of corticosteroid-antibiotic preparation and propolis paste formulation as intracanal medication after pulpectomy: an in vivo study. J Appl Oral Sci 2012;20:50-6. 3. Al-shaher A, Wallace J, Agarwal S, Bretz W, Baugh D. Ef-fect of propolis on human Fibroblast from the pulp & perio-dontal ligament. J Endod 2004;30:359-61. 4. Wang J, Hume WR. Diffusion of hydrogen ion and hydroxyl ion from various sources through dentine. Int Endod J 1988;21:17-26. 5. Tronstad L, Andreasen Jo, Hasselgren G, kristerson L. PH changes in dental tissues after root canal filling with calcium hydroxide. J Endod 1981;7:17-21. 6. Bankova VS, Popova SS, Marekov NL. A study of flavon-oids of propolis. J Nat Prodn 1983;46:471-4. 7. Wade C, Friedrich JA. Propolis Power Plus: The Health-Promoting Properties Of The Amazing Beehive Energizer. New Canaan, CT: keats; 1996. 8. Awawdeh L, AL-Beitawi M, Hammad M. Effectiveness of propolis & calcium hydroxide as a short- term intracanal medicament against Enterococcus faecalis: a laboratory study. Aust Endod J 2009;35:52-8. 9. Ahangari Z, Eslami G, Koosedghi H, Ayatolahi A. Com-parative study of antibacterial activity of propolis and Ca(OH)2 against lactobacillus, entrococusfeacalis, pepto-streptococus and Candida albicans. JIDA 2009;21:50-6. 10. Victorino FR, Bramante CM, Zapata RO, Casaroto AR, Gar-cia RB, Moraes IG, et al. Removal efficiency of propolis paste dressing from the root canal. J Appl Oral Sci 2010;18:621-4. 11. Martin MP, Pileggi R. A quantitative analysis of Propolis: a promising new storage media following avulsion. Dent Traumatol 2004;20:85-9. 12. Boyce, Steven T, Warden, Glenn D, Holder, Ian Alan. Cyto-toxicity testing of topical antimicrobial agents on human ke-rationcytes and fibroblasts, for cultured skin grafts. J Burn Care Rehabil 1995;16(2 Pt 1): 97-103. 13. Atari M, Barajas M. Isolation of pluripotent stem cells from human third molar dental pulp. Histol Histopathol 2011;26:1057-70. 14. Park SH, Hsiao GY, Huang GT. Role of substance P and calcitonin gene-related peptide in the regulation of inter-leukin-8 and monocyte chemotactic protein-1 expression in human dental pulp. Int Endod J 2004;37:185-92. 15. Martin Trope. Regenerative potential of dental pulp. J Endod 2008;34(7 Suppl):S13-7. 16. Chueh LH, Huang GT-J. Immature teeth with periradicular periodontitis or abscess undergoing apexogenesis: a para-digm shift. J Endod 2006; 32:1205-13. 17. Loushine BA, Bryan TE, Looney SW, Gillen BM, Loushine RJ, Weller RN, et al. Setting properties and cytotoxicity evaluation of a premixed bioceramic root canal sealer. J En-dod 2011;37: 673-7. 18. Ebadi R. beekeeping, 1st ed. Isfahan: Isfahan University of technology publications; 1990. P. 330-6. 19. Messer HH and Feigal RJ. A comparison of the antibacterial and cytotoxic effect parachlorophenol. J Dent Res 1985;64:818-21.  20. Trevino EG, Patwardhan AN, Henry MA. Effect of irrigants on the survival of human stem cells of the apical papilla in platelet–rich plasma scaffold in human root tips. J Endod 2011;37:1109-15. 21. Orstavik D, Haapasalo M. Disinfection by endodontic ir-rigants & dressings of experimentally infected dentinal tu-bules. Dent Traumatol 1990;6:142-9. 22. Waltimo T, Siren E, Orstavik D, Haapasalo M. Susceptibil-ity of oral candida species to calcium hydroxide in vitro. Int Endod J 1999;32:96-101. 23. Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch  J. Bac-teria isolated after unsuccessful  endodontic treatment in a North American population. Oral Surg Oral Med Oral Pa-thol Oral Radiol Endod 2001;91:579-86. 24. Kalfas S, Figdor D, sundqvist G. A new bacterial species associated with failed endodontic treatment: identification & description of Actinomyce sradicidentis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:208-14. 25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity as-says. J Immunol, Methods 1983;65:55-63. 26. Abdul Al,WallaceJ,Agarwal S,Bretz W& BaughD. Effect of propolis on human Fibroblast from the pulp & periodontal ligament. J Endod 2004; 30:359-61 27. Silva EJ, Herrera DR, Almedia JF, Ferraz CC, Gomes BP, Zaia AA. Evaluation of cytotoxicity and upregulation of ge-latinases in fibroblast cells by three root repair materials. Int Endod J 2012;45:815-20. 28. Sabir A, Tabbu CR, Agustiono P, Sosroseno W. Histological analysis of rat dental pulp tissue capped with propolis. J Oral Scien 2005;47:135-8.  JODDD, Vol. 8, No. 3 Summer 2014 

Cytotoxicity Evaluation of Iranian Propolis and Calcium

Hydroxide on Dental Pulp Fibroblasts

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Cytotoxicity Evaluation of Iranian Propolis and Calcium Hydroxide on Dental Pulp Fibroblasts

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