E c J L C E S a b c d e a A R R 1 A A K E R P C C 1 e p g l ( k ( ( s h 1 Photodiagnosis and Photodynamic Therapy 13 (2016) 97–100 Contents lists available at ScienceDirect Photodiagnosis and Photodynamic Therapy jou rn al homepage: www.elsev ier .com/ locate /pdpdt valuation of photodynamic therapy on fibroblast viability and ytokine production oão Eduardo Gomes-Filhoa, Gustavo Sivieri-Araujoa,∗, Carla Renata Sipertb, udmilla Mota da Silva Santosa, Índia Olinta de Azevedo Queiroza, hristine Men Martinsa, Nayara Kívilla do Carmo Maiaa, Luciano Tavares Angelo Cintraa, loi Dezan-Juniora, Vanderlei Salvador Bagnatoc, Antônio Hernandes Chaves-Netod, andra Helena Penha de Oliveirae Department of Restorative Dentistry, Discipline of Endodontics, Araç atuba School of Dentistry, São Paulo State University, Araç atuba, SP, Brazil Department of Restorative Dentistry, Discipline of Endodontics, Dental School, University of São Paulo, São Paulo, SP, Brazil Optics Group, Physics Institute of São Carlos, University of São Paulo, São Carlos, SP, Brazil Department of Basic Science, Discipline of Biochemistry, Araç atuba School of Dentistry, São Paulo State University, Araç atuba, SP, Brazil Department of Basic Science, Discipline of Pharmacology, Araç atuba School of Dentistry, São Paulo State University, Araç atuba, SP, Brazil r t i c l e i n f o rticle history: eceived 12 September 2015 eceived in revised form 9 December 2015 ccepted 14 January 2016 vailable online 18 January 2016 eywords: ndodontic treatment oot canal irrigants hotodynamic therapy a b s t r a c t Background: The aim of this study was to evaluate the effects of photodynamic therapy with curcumin (PDT) comparatively to 5% sodium hypochlorite (NaOCl) and saline solution on cell viability and cytokine (IL-1� and IL-6) production by mouse fibroblasts. Methods: Sixty seconds of pre-irradiation time with curcumin 500 mg/L and Led wavelength (�) 480 nm, 72 J cm2, for 300 s was used for PDT. Solutions were diluted in culture medium DMEM (1 × 104 cells) and placed into 24-well cell culture plates with mouse fibroblasts L-929. Culture medium was used as control. After 6, 24 and 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) was used to evaluate the cell viability and the supernatant was collected for cytokine evaluation using enzyme-linked immunosorbent assay (ELISA). The results were statistically analyzed by ANOVA and BonFerroni correction (p < 0.05) for MTT and Kruskal–Wallis test and Dunn (p < 0.05) for ELISA. ytotoxicity ytokine Results: PDT and saline solution presented low cytotoxic effect similar to the control group (p > 0.05) while 5% NaOCl was more cytotoxic than PDT (p < 0.05) in all periods of time. All materials similarly expressed IL-1� and IL-6 regardless to the experimental period (p < 0.05). Conclusions: PDT with curcumin was not cytotoxic to L929 fibroblasts differently from 5% NaOCl. In all groups occurred similar expression of IL-1� and IL-6. © 2016 Elsevier B.V. All rights reserved. . Introduction Root canal cleaning and shaping are essential to reduce and/or liminate the population of micro-organisms (MO) and their toxic roducts (endotoxins and apical biofilm) present in endodontic ∗ Corresponding author. Fax: +55 18 3636 3253. E-mail addresses: joao@foa.unesp.br (J.E. Gomes-Filho), ustavosivieri@uol.com.br (G. Sivieri-Araujo), carlasipert@gmail.com (C.R. Sipert), udmillasantos@yahoo.com.br (L.M. da Silva Santos), indiaodonto@gmail.com Í.O. de Azevedo Queiroz), christinemen@hotmail.com (C. Men Martins), ivilla@hotmail.com (N.K. do Carmo Maia), lucianocintra@foa.unesp.br L.T.A. Cintra), dezan@foa.unesp.br (E. Dezan-Junior), vander@ifsc.usp.br V.S. Bagnato), antoniohernandes@foa.unesp.br (A.H. Chaves-Neto), hpoliv@foa.unesp.br (S.H.P. de Oliveira). ttp://dx.doi.org/10.1016/j.pdpdt.2016.01.007 572-1000/© 2016 Elsevier B.V. All rights reserved. infection [1]. The use of irrigating solution that assist cleaning during endodontic treatment is essential to maximize the decon- tamination by its chemical action [2]. Sodium hypochlorite (NaOCl) is the most employed irriga- tion solution primarily for its antimicrobial action [2,3]. However, despite the scientific-technical progress, authors show the persis- tence of MO in root canal system post-treatment [1,2,4]. Therefore, new therapeutic strategies must be investigated to potentiate the combat of endodontic infections. Recently new methods as photodynamic therapy (PDT) are used on treatments to promote disinfection in periodontitis, den- tal caries, endodontic diseases, among other dental specialties [5–7]. Photodynamic therapy uses specific wavelength light Laser (light amplification by stimulated emission of radiation) or Led dx.doi.org/10.1016/j.pdpdt.2016.01.007 http://www.sciencedirect.com/science/journal/15721000 http://www.elsevier.com/locate/pdpdt http://crossmark.crossref.org/dialog/?doi=10.1016/j.pdpdt.2016.01.007&domain=pdf mailto:joao@foa.unesp.br mailto:gustavosivieri@uol.com.br mailto:carlasipert@gmail.com mailto:ludmillasantos@yahoo.com.br mailto:indiaodonto@gmail.com mailto:christinemen@hotmail.com mailto:kivilla@hotmail.com mailto:lucianocintra@foa.unesp.br mailto:dezan@foa.unesp.br mailto:vander@ifsc.usp.br mailto:antoniohernandes@foa.unesp.br mailto:shpoliv@foa.unesp.br dx.doi.org/10.1016/j.pdpdt.2016.01.007 9 nd Photodynamic Therapy 13 (2016) 97–100 ( p v t m t b a p i m i d a e i b s d o i s t c v 2 2 b m U t a b c 2 T c i m b ( i P I B d c w 2 c a 2 w 8 J.E. Gomes-Filho et al. / Photodiagnosis a light emitting diode) that activates the photosensitizer (PS) and roduces highly reactive specie of oxygen (singlet oxygen) inacti- ating the target cell [8,9] and assists antimicrobial action without he risk to promote microbial resistance [10]. Several kinds of PS ay be associated with Laser or Led: hematoporphyrin deriva- ives, porphyrin, phenothiazine (toluidine blue and methylene lue), chlorophyll, fluorones (rhodamine B), cyanine phytotherapic gents, phthalocyanines and curcumin [5,11]. Curcumin, a com- ound from Curcuma longa L., the drug has a variety of applications, ncluding treatment of liver diseases, wounds, inflamed joints and icrobial effects [6,12,13]. In vitro studies with porphyrin and methylene blue PS [14] and n vivo studies with chlorophyll and methylene blue PS [14–17] emonstrated PDT as a new antimicrobial therapeutic modality iming to increase the disinfection of root canal system during ndodontic treatment. It was also evidenced with toluidine blue PS n PDT against Enterococcus faecalis once PS fixes in the cell mem- rane and reaches the peak of absorption leading to generation of inglet oxygen, which destroys cellular wall and leads to bacterial eath [18]. However, Frota et al. [19], did not show total disinfection f root canals PDT against E. faecalis with curcumin PS. Although PDT has been already employed for tumor treatment n oncology [20] its cytotoxicity effect is not completely under- tood, especially when using curcumin as a photosensitizer. Thus, he aim of this study was to determine the effects of PDT with cur- umin comparatively to 5% NaOCl and saline solution on fibroblasts iability and on IL-1� and IL-6 cytokine releasing. . Materials and methods .1. Fibroblasts culture L-929 mouse lineage fibroblasts were maintained in culture ottles with Dulbecco’s modified Eagle’s medium (DMEM) supple- ented with 10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD, SA), streptomycin (50 g/mL), and 1% antibiotic/antimycotic cock- ail (300 units/mL penicillin, 300 �g/mL streptomycin, 5 �g/mL mphotericin B and 200 �g/mL of glutamin) (GIBCO BRL, Gaithers- urg, MD, USA). The cultures were maintained under standard cell ulture conditions (37 ◦C, 100% humidity, 5% CO2) [21,22]. .2. Group division and photodynamic therapy It was used 50 �L of each solution diluted in 450 �L of DMEM. he solutions were 20 times more concentrated to get the desired oncentration after dilution. A total volume of 500 �L was inserted n each well. The groups were distributed as follow: G1-Culture edium (control); G2-5% NaOCl; G3-Saline solution; G4-PDT. The PDT was performed with curcumin at 500 mg/L [1,7- is-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] PDTPharma, Cravinhos, SP, Brazil), in the period of 60 s of pre- rradiation as recommended by Paschoal et al., 2013 [6]. Then the S was activated with blue Led for � 480 nm, 72 J cm2, (Physics nstitute of São Carlos, University of São Paulo, São Carlos, SP, razil) for 300 s [6]. During the pre-irradiation of PS and also uring the activation of the LED, the lighting of the laminar flow hamber was turned off and the ambient light in the laboratory as reduced to avoid PS degradation. .3. Cytotoxicity testing L929 fibroblasts were seeded into the 24-well plates (1 × 104 ell/well). The cells were incubated for 24 h in a humidified air tmosphere of 5% CO2 at 37 ◦C. The solutions were tested for 6 h, 4 h and 48 h. Tree wells were used for each solutions, and wells ith DMEM were used as the control. Viable cells were stained with Fig. 1. Viability of fibroblasts in the presence of PDT and irrigating solutions, the periods 6 h, 24 h and 48 h. *(p < 0.05) indicates significant statistical difference. formazan dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra- zolium bromide) (MTT) (Sigma Chemical Co., St. Louis, MO) that was dissolved in phosphate- buffered saline at 5 mg/mL and fil- tered in order to sterilize and remove a small amount of insoluble residue. At the experimental times, stock MTT solution (40 �L per 360 �L medium) was added to all wells of an assay, and plates were incubated at 37 ◦C for 3 h. The medium was then removed by the inversion of the plate and 200 mL of isopropilic alcohol was added to the wells and mixed during 20 minutes in order to dis- solve the dark blue crystals. The blue solution was transferred to a 96-well plate, and the absorbance was read in the microplate reader using 570 nm wavelength [23]. The treatments were clas- sified according to the cytotoxic effect of: 0—non-cytotoxic (less inhibition than 25%); 1—slightly cytotoxic (inhibition between 25% and 50%); 2—moderately cytotoxic (inhibition between 50% and 75%) and 3—strongly cytotoxic (greater than 75% inhibition). Assays were performed in triplicate. 2.4. Enzyme-linked immunosorbent assay For cytokine assay, supernatant samples were used in the exper- iment originated from cytotoxicity to analyze the production of cytokines (IL-1� and IL-6) by fibroblasts, according to Gomes-Filho et al. [21,22]. After the incubation periods, supernatants were col- lected and analyzed to detect were measured using Peprotech Murine IL-1� and IL-6 mini ELISA development kits (Manufacturer, Rocky Hill, NJ, USA), according to the manufacturer’s instructions. This assay also revealed the presence and levels of IL-1� and IL- 6 (pg/ml). Wells with no added GT solution or irrigant, but with cell culture served as a control. The measurements were made in triplicate. 2.5. Statistical analysis The results were statistically analyzed by ANOVA test with Bon- Ferroni correction (p < 0.05) for MTT and Kruskal–Wallis test and Dunn’s (p < 0.05) for ELISA. 3. Results MTT assay in the periods of 6 h, 24 h and 48 h is shown in Fig. 1. It was revealed that control, saline solution, and PDT did inhibit less than 25% the cell viability. The same result was not seen in the 5% NaOCl group that was greater than 75% inhibition and was the most cytotoxic (p < 0.05). The mean concentrations of IL-1� for the different groups are shown in Fig. 2a. All groups were able to induce similar IL-1� pro- duction observed by ELISA assay (p < 0.05). The expression of IL-6 are shown in Fig. 2b. ELISA assay revealed that the mean concentrations of IL-6 was similar among the groups (p > 0.05), although 5% NaOCl had been highly cytotoxic. J.E. Gomes-Filho et al. / Photodiagnosis and Ph Fig. 2. Mean levels of IL-1� (a) and IL-6 (b) were raised when the cells were grown i s 4 d ( e z t s e t c [ N d [ b s b a i T t l O p [ p s c o a a i i 10.1016/j.pdpdt.2010.03.004. n the presence of the solutions, the periods 6 h, 24 h and 48 h. *(p < 0.05) indicates ignificant statistical difference. . Discussion The cell viability was determined in this study by 3-(4,5- imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay MTT) based on the ability of mitochondrial dehydrogenase nzymes in living cells to convert the yellow water-soluble tetra- olium salt MTT into dark blue formazan crystals, a simple method hat showed advantages as rapidness and precision [21,22] and the ubstrate used in the assay does not interfere with measurement nd [23]. Complementary, ELISA assay was used for the measured he cytokines secreted. In the present study, it was observed that 5% NaOCl was highly ytoxic observed by the low cell viability similarly to previous study 24]. These results can be explain due the chemical properties of aOCl as ability to dissolve organic tissue [25,26] and its toxicity ue to the recruitment of expressive number of inflammatory cells 27]. The concentration of curcumin PS associated with 72 J cm2 of lue Led was biocompatible agreement with a previous in vivo tudy [28]. The present study employed a blue Led � 480 nm, ecause curcumin PS has an absorption peak ranging between 300 nd 500 nm, according with Paschoal et al. [6]. Saline solution and PDT were capable to maintain the cell viabil- ty similar to that observed for control during all periods evaluated. hese results are similar to previous studies that have showed hat PDT did not affect host cell viability from gingival fibrob- asts, osteoblasts, and human periodontal ligament cells [22–31]. n the other hand, disagree with others that have showed that PDT romoting reduction in fibroblast, macrophage and human head 32,33]. The differences can be due to concentrations, treatment rotocol, or cell types. The synthesis of cytokines is very complex, and their expres- ion and effects are governed by many factors that include other ells and mediators [21,22]. During inflammatory process, numer- us pro- and anti-inflammatory cytokines are secreted such as IL-1 nd IL-6 [21]. IL-1 is a cytokine that mediates the bone resorption fter synthesis by macrophages; it is an important mediator of the nflammatory response, involved in a variety of cellular activities, ncluding cell proliferation, differentiation, and apoptosis [34,35]. otodynamic Therapy 13 (2016) 97–100 99 On the other hand, IL-6 is a cytokine that mediates the host response to injury and infection, and it is secreted during the inflammatory process after tumor necrosis factor-alpha (TNF-�) and IL-1 secre- tion, consequently IL-6 inhibits the secretion of TNF-� and IL-1 [36,37]. All irrigants investigated were able to express similarly Il- 6 and IL-1� cytokines, even 5% NaOCl that was highly cytotoxic agreeing to previous studies that showed that NaOCl even being toxic can express cytokines [38]. It is important to consider that a small amount of cells remained after exposure to 5% NaOCL infer- ring that proportionally the cytokine release after its contact had been expressively higher than the other solutions converging to its high cytotoxicity. Previous studies showed that PDT has immunomodulatory activity and was capable to modulate host response reducing the capacity to stimulate the proliferation of T cells [39,40]. In the present study, PDT stimulated the production of IL-1� and IL-6 by fibroblasts agreeing to previous study that showed PDT induc- ing IL-1� and IL-6 secretion [41]. By the other hand, disagree to studies that showed that PDT decreased cytokines production [42] or promoted the inactivation of key proinflamatory markers [43]. The differences can be due to the photosensitizer, treatment pro- tocol, or cell types. In the present study, it was used the previously described drug and light parameters [6]. After PDT, molecular bio- logical mechanisms lead to biostimulation (increased proliferation rate), repair of the damage leading to rescue of the cells, autophagy, apoptosis and necrosis. The different modes of cellular responses depend mainly on the PDT-protocol, photosensitizer localization, cellular damage protection and available intracellular energy [44]. This fact may also contribute to tissue repair and suggests the use of PDT in Endodontics. 5. Conclusion According to the methodology employed, photodynamic ther- apy with curcumin was not cytotoxic and did not inhibit the L-929 fibroblasts viability and release of cytokines IL-1� and IL-6. Conflict of interest The authors declare that they have no conflict of interest. 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dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1016/j.pdpdt.2008.02.001 dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1007/s10103-015-1749-y dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 dx.doi.org/10.1016/j.pdpdt.2014.10.011 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dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.1902/jop.2009.090214 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 dx.doi.org/10.2174/092986712799034842 Evaluation of photodynamic therapy on fibroblast viability and cytokine production 1 Introduction 2 Materials and methods 2.1 Fibroblasts culture 2.2 Group division and photodynamic therapy 2.3 Cytotoxicity testing 2.4 Enzyme-linked immunosorbent assay 2.5 Statistical analysis 3 Results 4 Discussion 5 Conclusion Conflict of interest Acknowledgements References