Dec 15, 2013 - Florida College of Dentistry; 2Department of. Periodontology and Oral Biology, University of. Florida College of Dentistry; 3Department of.
Dental Traumatology 2014; 30: 362–367; doi: 10.1111/edt.12096
Propolis decreases lipopolysaccharideinduced inflammatory mediators in pulp cells and osteoclasts Kathleen G. Neiva1, Dana L. Catalfamo2, Shannon Holliday3, Shannon M. Wallet2, Roberta Pileggi1 1 Department of Endododntics, University of Florida College of Dentistry; 2Department of Periodontology and Oral Biology, University of Florida College of Dentistry; 3Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL, USA
Key words: propolis; inflammation; pulp cells; osteoclasts; intracanal medicament Correspondence to: Kathleen G. Neiva, Department of Endodontics, University of Florida College of Dentistry, 1395 Center Drive, Room D10-41, PO Box 100436, Gainesville, FL 32610-0436, USA Tel.: (352) 273 5435 Fax: (352) 273 5446 e-mail: kneiva@dental.ufl.edu Accepted 15 December, 2013
Abstract – Background: Intracanal medicaments are used to disinfect the root canal system, reduce interappointment pain and inflammation, and prevent resorption. Bacterial components such as lipopolysaccharide (LPS) are implicated in the development of pulpal and periapical inflammation and inducing osteoclastogenesis. Propolis is a natural, non-toxic substance collected from bee’s wax that has been used for many years in folk medicine. Propolis has been demonstrated to have antibacterial and anti-inflammatory properties. Our previous studies have shown that propolis inhibits osteoclast maturation. However, the effect of propolis on the inflammatory response of pulp cells and osteoclasts has not been explored. Aim: The purpose of this study was to evaluate whether propolis alters the inflammatory response of three endodontically relevant cell lines: mouse odontoblast-like cells (MDPC23), macrophages (RAW264.7), and osteoclasts. Material and methods: Cells were exposed to 0–20 ug ml 1 LPS to induce an inflammatory response, in the presence of propolis or vehicle control. Culture supernatants were collected after 6 and 24 h, and expression of multiple soluble mediators was determined using Luminexâ multiplex technology. Results: Propolis was effective in reducing secretion of the LPS-induced inflammatory cyto/chemokines: IL-1a, IL-6, IL-12(p70), IL-15, G-CSF, TNF-a, MIP-1a, MCP-1, and IP-10. Conclusion: Our results demonstrate that propolis suppresses the LPSinduced inflammatory response of key cells within the root canal system.
An intracanal medicament may be necessary during root canal therapy in order to eliminate bacteria (1), to reduce pain and inflammation in pulp and periapical tissues in cases of pulpectomy (2), and to prevent inflammatory and replacement resorption in cases of trauma (3). Biocompatibility and stability are essential properties for all intracanal medicaments. Propolis is a natural, non-toxic substance collected from bee’s wax that has been used for many years in folk medicine. It contains amino acids, flavonoids, terpenes, and cinnamic acid derivatives (4, 5). Propolis has been demonstrated to have antibacterial properties along with anti-inflammatory effects (6, 7). Notably, propolis’ significant antibacterial activity against oral pathogens has been demonstrated, including Enterococcus faecalis, Staphylococcus aureus, Candida albicans, Streptococcus sanguis, Streptococcus mutans, Streptococcus sobrinus, Streptococcus cricetus, Actinomyces naeslundii, Actinomyces viscosus, Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella denticola (6, 8–10). Interestingly, one study has demonstrated that propolis was more effective than calcium hydroxide and as effective as a triantibiotic mixture in inhibiting the growth of E. faecalis (11). 362
In vitro experiments have shown that propolis exhorts its anti-inflammatory properties by inhibiting the cyclo-oxygenase pathway and eicosanoid synthesis (7, 12, 13). Similarly, we have observed that propolis can decrease lipopolysaccharide (LPS)-stimulated iNOS activity (unpublished data). LPS is the major component of the outer membrane in Gram-negative bacteria and is largely responsible for the pathogenicity of these organisms (14, 15). Gram-negative bacteria are prevalent in deep caries and pulpitis (16–18). These bacteria play a significant role in the development of clinical symptoms through the production of pro-inflammatory cytokines and chemokines such as tumor necrosis factor-(TNF)a, interleukin (IL)-1a (19, 20), IL-1b (21), IL-6 (22), IL-8 (23), and vascular endothelial growth factor (VEGF) (24). Although propolis has only been investigated in dental research to a minimal extent, we have observed its biocompatibility with the oral tissues, including the pulp and periodontium (25, 26). In addition, studies have observed that propolis promotes regeneration of dental pulp, enhancement of pulp healing, and greater dentinal bridge formation (27, 28). Finally, we have demonstrated that propolis is also able to inhibit osteoclast maturation, suggesting it could also contribute to © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Propolis decreases inflammatory mediators preventing replacement resorption (29). However, the effect of propolis on osteoclasts and pulpal cell inflammatory responses has not been explored. Therefore, the purpose of this study was to evaluate whether propolis decreases the LPS-induced responsiveness of three endodontically relevant cell lines: murine-derived odontoblast-like cells (MDPC-23), macrophages (RAW264.7), and RAW264.7-derived osteoclasts. Material and methods
363
murine soluble RANK-L (rmsRANK-L) (Peprotech, Rocky Hill, NJ, USA) and allowed to culture for 6 days with complete media refreshed every 3 days to stimulate osteoclast differentiation. After that, odontoblast-like cells, macrophages, and osteoclasts were exposed to 0– 100 ng ml 1 of non-pure Escherichia coli LPS (SigmaAldrich, St. Louis, MO, USA) for 1 h. Cells were then treated with 1:100 dilution of propolis or vehicle control (ethanol) for an additional 6 or 24 h. Supernatants were collected, and cyto/chemokine expression was analyzed using multiplex technology.
Propolis
A single source of propolis from the Baccharis dracunculifolia (the most important botanical source of Southeastern Brazilian propolis) was used throughout the study. It was obtained from the same Brazilian bee species (Africanized Apis mellifera), dissolved, purified, and prepared at 80% concentration using 0.4% ethanol solution as vehicle. Cell culture
Mouse odontoblast-like cells (MDPC-23) and macrophages (RAW264.7) (gift from T. Botero, University of Michigan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Mediatech, Manassas, VA, USA), 1% L-glutamine (Thermo Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin/ amphotericin B (Fisher, Waltham, MA, USA). Cells were cultured in T-75 flasks (Fisher) at 80% confluency. Cells were then seeded at a concentration of 4 9 104 in 24-well plates (Fisher). Alternatively, RAW macrophages were supplemented with 50 ng ml 1 recombinant
Tartrate-resistant acid phosphate (Trap) staining
After 6 days of RANKL stimulation, cells plated on glass coverslips were fixed with 2% paraformaldehyde/ PBS (Fisher). Cells were washed with PBS and permeabilized in 0.5% Triton X-100/PBS (Fisher). Cells were washed and probed for leukocyte acid phosphatase (TRAP) (1:1:1:2:4 Fast Garnet GBC Base Solution: Sodium Nitrite Solution:Naphthol AS-BI Phosphate Solution:Tartrate Solution:Acetate Solution) (SigmaAldrich) after which cells were washed and mounted on glass slides with MOWIOL 4-88 solution (Calbiochem). TRAP-positive cells (stained in purple) were observed using light microscopy at 209 and 409 magnification in order to confirm osteoclast differentiation. Soluble mediator analysis
Cytokines and chemokines from supernatants were detected and quantified using a mouse 22-cyto/ chemokine multiplex (Millipore) according to the manufacturer’s instructions. Briefly, supernatant and antibody-coated beads were allowed to incubate over-
(a)
(b)
(c)
(d)
Fig. 1. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in odontoblast-like cells. MDPC-23 were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 24 h, supernatants were collected and cyto/ chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent expression of (a) IL1a, (b) MIP-1a, (c), IL-12(p70), and (d) IL-15. Asterisks represent statistically significant difference (P < 0.05). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
364
Neiva et al.
night at 4°C in a 96-well primed plate. Following three washes, reactivity was probed with biotinylated detection antibodies and streptavidin (SAV)–phycoerythrin (PE). All incubations occurred while gently shaking in the dark. Following three washes, beads were resuspended in sheath fluid and reactivity acquired using a Luminex 200 with XPONENT software (Millipore, Billerica, MA, USA). MILLIPLEXâ ANALYST software (Millipore), 5-parameter logistics, and a standard curve were used to determine pg ml 1 concentrations. Outcome measures were normalized to cell number per well. Statistical analysis
One-way ANOVA with Dunn’s correction was used to analyze and determine statistical significance (P < 0.05). Results
Pilot studies were conducted to determine the ideal concentration and timing of E. coli LPS stimulation to induce inflammatory mediators in all cell types. Here, a 20 ng ml 1 concentration of LPS induced significant
(a)
(b)
(c)
(d)
(e)
(f)
upregulation of soluble mediators at 24 h poststimulation for odontoblast-like cells, while upregulation of these mediators was observed at both 6 and 24 h for macrophages and osteoclasts (data not shown). Bacterial LPS induced upregulation of several cyto/ chemokines in odontoblast-like cells at 24 h. In addition, treatment with propolis alone or vehicle control had no effect on the baseline expression of inflammatory cytokines measured (Fig. 1a–d). However, treatment with propolis significantly reduced the LPSinduced expression of interleukin (IL)-1a (Fig. 1a), MIP-1a (Fig. 1b), IL-12(p70) (Fig. 1c), and IL-15 (Fig. 1d) in odontoblast-like cells. Lipopolysaccharide stimulation upregulated the expression of multiple cyto/chemokines by RAW264.7 macrophages, and propolis was again able to inhibit this LPS-induced expression (Fig. 2a–f). Specifically, 6 h post-treatment, MIP-1a (Fig. 2a), G-CSF (Fig. 2b), tumor necrosis factor (TNF)-a (Fig. 2c), and IL-6 (Fig. 1d) were significantly inhibited by treatment with propolis as compared to LPS treatment alone. Similarly, 24 h following propolis administration, monocyte chemoattractant protein (MCP)-1 (Fig. 2e) expression was significantly reduced. The expression level of IL-6
Fig. 2. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in macrophages. RAW264.7 macrophages were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 6 or 24 h, supernatants were collected and cyto/chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent the expression of (a) MIP-1a, (b) G-CSF, (c) TNF-a, and (d) IL-6 at 6 h after stimulation; (e) MCP-1 and (f) IL-6 at 24 h after stimulation. Asterisks represent statistically significant difference (P < 0.05). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Propolis decreases inflammatory mediators was higher at 24 h than at 6 h, and propolis continued to inhibit the LPS-induced IL-6 (Fig. 2f) expression. As seen in odontoblast-like cells, treatment with propolis or vehicle control alone did not change baseline expression level of cyto/chemokines (Fig. 2a–f). Tartrate-resistant acid phosphate staining was used to confirm that macrophages differentiated into osteoclasts (Fig. 3a,b). TRAP-positive cells showed mononuclear (Fig. 3a) and multinucleated osteoclasts (Fig. 3b). Lastly, LPS also induced the expression of inflammatory cyto/chemokines by osteoclasts (Fig. 3c–f), which was again reduced by propolis treatment. Propolis treatment or vehicle alone had no effect on expression of inflammatory cyto/chemokines (Fig. 3c–f). Six hours following propolis treatment, MIP-1a (Fig. 3c) expression was significant lower when compared to LPS treatment alone. Similarly, the expression of IL-6 (Fig. 3d), IP-10 (Fig. 3e), and MCP-1 (Fig. 3f) was significantly reduced after 24 h of propolis treatment. Discussion
The identification of an intracanal medicament which can not only control infection but also minimize inflammation would be beneficial for more effective healing and success of root canal therapies. Here, we demonstrated that bacterial LPS induces a panel of
(a)
Fig. 3. Propolis reduced lipopolysaccharide (LPS)-induced expression of cyto/chemokines in osteoclasts. (a, b) Tartrate-resistant acid phosphate-positive cells. RAW264.7 macrophages were supplemented with 50 ng ml 1 rmsRANK-L and cultured for 6 days to stimulate osteoclast differentiation. Representative images of (a) mononuclear and (b) multinucleated osteoclasts. (c–f) RAW264.7-derived osteoclasts were exposed to 0 or 20 ng ml 1 LPS for 1 h. Cells were then treated with propolis or vehicle control. After 6 or 24 h, supernatants were collected and cyto/chemokine expression was analyzed using a Luminexâ multiplex assay. Graphs represent expression of (c) MIP-1a at 6 h after stimulation, and (d) IL-6, (e) IP-10, and (f) MCP-1 at 24 h after stimulation. Asterisks represent statistically significant difference (P < 0.05).
inflammatory cytokines and/or chemokines in odontoblast-like cells, macrophages, and osteoclasts. Several studies (19–24) have shown that LPS induces expression of inflammatory mediators in cells associated with endodontic lesions. Here, we demonstrated that propolis decreased this LPS-induced cyto/chemokine expression in odontoblast-like cells, macrophages, and osteoclasts. Concentrated natural ethanol extracts have been shown to have anti-inflammatory effects (30, 31). In this study, we used ethanol to dilute propolis and as vehicle control. Propolis stock solution was diluted before use, and the final concentration of ethanol was not enough to cause cytotoxic or anti-inflammatory effect on the cells (data not shown). Thus, the reduction in LPS-induced inflammatory mediators is attributed to the effect of propolis itself and not of ethanol. The cell types tested here, odontoblast-like cells, macrophages, and osteoclasts, are particularly relevant in endodontics. Odontoblasts are the first pulp cells to encounter external stimulation, playing an important role in innate immunity and inflammatory events in response to cariogenic bacteria (32). Odontoblasts sense pathogens by Toll-like receptors (TLRs) and produce cyto/chemokines upon cell stimulation with microbial by-products (33). Macrophages are the most predominant immune cells of the dental pulp (34, 35) and are activated in the early stages of pulpitis. They
(b)
(c)
(d)
(e)
(f)
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
365
366
Neiva et al.
have three major functions: antigen presentation, phagocytosis, and immunomodulation through the production of various cyto/chemokines and growth factors. Activated macrophages produce TNF-a, IL-1, IL-12, IL-10, chemokines, and short-lived lipid mediators such as platelet-activating factor (PAF), prostaglandins, and leukotrienes to orchestrate a local inflammation (36). Studies have shown significant increase in TNF-a and IL-1 in irreversible pulpitis (37, 38). Similarly, while osteoclasts are involved in maintenance of bone homeostasis, they are also involved in tooth resorption following traumatic injury or irritation of the pulp. Osteoclast precursors are recruited and activated in the presence of inflammatory mediators which can result in inflammatory resorption. Root resorption is the main challenge to clinicians when dealing with traumatized teeth (39–41). Studies have attempted to find a medicament that would ameliorate this problem, and to date, calcium hydroxide is the treatment of choice. Several studies have demonstrated its efficacy as antimicrobial agent (42–47). However, the anti-inflammatory and antiresorptive properties of calcium hydroxide are not evident. Here, we demonstrated that LPS induces the expression of pro-inflammatory cytokines (IL-1a, IL-12), chemokines (MIP-1a), and growth factors (IL-15) by odontoblast-like cells, and that this response can be inhibited by propolis. Similarly, we demonstrated that propolis can inhibit LPS-induced expression of proinflammatory cytokines (TNF-a and IL-6), chemokines (MIP-1a and MCP-1), and growth factors (G-CSF) from macrophages as well as from osteoclasts [IL-6 (cytokine), MIP-1a, MCP-1, and IP-10 (chemokines)]. While IL-1a, IL-12, and IL-15 support the survival, activation, and proliferation of adaptive immune cells (T cells and B cells), G-CSF supports the differentiation and proliferation of innate immune cell populations (neutrophils and macrophages). On the other hand, MIP-1a and MCP-1 are best known for their chemotactic and pro-inflammatory (i.e. induction of cytokines) properties, while IL-6 and TNF-a are more pleiotropic in nature, affecting chemoattractants, induction of cytokine expression, and angiogenic properties. Thus inhibition of these molecules would significantly dampen pulpal and periapical inflammation and enhance the endodontic wound healing processes. Indeed, inhibition of these cytokines has provided potent therapeutic effects in the treatment of multiple inflammatory diseases (48, 49). Here, we demonstrated that propolis, which has successfully been used in the medical field as an antiinflammatory and antimicrobial agent (50–52), had similar efficacy in suppressing the induction of cyto/ chemokines in key cells within the root canal system. Acknowledgement
The authors thank Dr. Tatiana Botero (University of Michigan) for the cells used in this study. Conflict of interest
The authors deny any conflicts of interest.
References 1. Vera J, Siqueira JF Jr, Ricucci D, Loghin S, Fernandez N, Flores B et al. One- versus two-visit endodontic treatment of teeth with apical periodontitis: a histobacteriologic study. J Endod 2012;38:1040–52. 2. Ramos IF, Biz MT, Paulino N, Scremin A, Della Bona A, Barletta FB 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. Trope M, Yesilsoy C, Koren L, Moshonov J, Friedman S. Effect of different endodontic treatment protocols on periodontal repair and root resorption of replanted dog teeth. J Endod 1992;18:492–6. 4. Khalil ML. Biological activity of bee propolis in health and disease. Asian Pac J Cancer Prev 2006;7:22–31. 5. Sforcin JM. Propolis and the immune system: a review. J Ethnopharmacol 2007;113:1–14. 6. Kayaoglu G, Omurlu H, Akca G, Gurel M, Gencay O, Sorkun K et al. Antibacterial activity of propolis versus conventional endodontic disinfectants against Enterococcus faecalis in infected dentinal tubules. J Endod 2011;37:376–81. 7. Paulino N, Teixeira C, Martins R, Scremin A, Dirsch VM, Vollmar AM et al. Evaluation of the analgesic and anti-inflammatory effects of a Brazilian green propolis. Planta Med 2006;72:899–906. 8. Koo H, Gomes BP, Rosalen PL, Ambrosano GM, Park YK, Cury JA. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch Oral Biol 2000;45:141–8. 9. Arslan S, Ozbilge H, Kaya EG, Er O. In vitro antimicrobial activity of propolis, BioPure MTAD, sodium hypochlorite, and chlorhexidine on Enterococcus faecalis and Candida albicans. Saudi Med J 2011;32:479–83. 10. Oncag O, Cogulu D, Uzel A, Sorkun K. Efficacy of propolis as an intracanal medicament against Enterococcus faecalis. Gen Dent 2006;54:319–22. 11. Madhubala MM, Srinivasan N, Ahamed S. Comparative evaluation of propolis and triantibiotic mixture as an intracanal medicament against Enterococcus faecalis. J Endod 2011;37:1287–9. 12. Ribeiro LR, Mantovani MS, Ribeiro DA, Salvadori DM. Brazilian natural dietary components (annatto, propolis and mushrooms) protecting against mutation and cancer. Hum Exp Toxicol 2006;25:267–72. 13. Ozturk F, Kurt E, Inan UU, Emiroglu L, Ilker SS. The effects of acetylcholine and propolis extract on corneal epithelial wound healing in rats. Cornea 1999;18:466–71. 14. Cohen J. The immunopathogenesis of sepsis. Nature 2002;420:885–91. 15. Rietschel ET, Brade H. Bacterial endotoxins. Sci Am 1992;267:54–61. 16. Hoshino E. Predominant obligate anaerobes in human carious dentin. J Dent Res 1985;64:1195–8. 17. Massey WL, Romberg DM, Hunter N, Hume WR. The association of carious dentin microflora with tissue changes in human pulpitis. Oral Microbiol Immunol 1993;8:30–5. 18. Martin FE, Nadkarni MA, Jacques NA, Hunter N. Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis. J Clin Microbiol 2002;40:1698–704. 19. Tani-Ishii N, Wang CY, Stashenko P. Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure. Oral Microbiol Immunol 1995;10:213–9. 20. Hong CY, Lin SK, Kok SH, Cheng SJ, Lee MS, Wang TM et al. The role of lipopolysaccharide in infectious bone resorption of periapical lesion. J Oral Pathol Med 2004;33:162–9. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Propolis decreases inflammatory mediators 21. Hosoya S, Matsushima K. Stimulation of interleukin-1 beta production of human dental pulp cells by Porphyromonas endodontalis lipopolysaccharide. J Endod 1997;23:39–42. 22. Tokuda M, Sakuta T, Fushuku A, Torii M, Nagaoka S. Regulation of interleukin-6 expression in human dental pulp cell cultures stimulated with Prevotella intermedia lipopolysaccharide. J Endod 2001;27:273–7. 23. Nagaoka S, Tokuda M, Sakuta T, Taketoshi Y, Tamura M, Takada H et al. Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide. J Endod 1996;22:9–12. 24. Botero TM, Mantellini MG, Song W, Hanks CT, Nor JE. Effect of lipopolysaccharides on vascular endothelial growth factor expression in mouse pulp cells and macrophages. Eur J Oral Sci 2003;111:228–34. 25. Martin MP, Pileggi R. A quantitative analysis of propolis: a promising new storage media following avulsion. Dent Traumatol 2004;20:85–9. 26. Gjertsen AW, Stothz KA, Neiva KG, Pileggi R. Effect of propolis on proliferation and apoptosis of periodontal ligament fibroblasts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:843–8. 27. Scheller S, Ilewicz L, Luciak M, Skrobidurska D, Stojko A, Matuga W. Biological properties and clinical application of propolis. IX. Experimental observation on the influence of ethanol extract of propolis (EEP) on dental pulp regeneration. Arzneimittelforschung 1978;28:289–91. 28. Bretz WA, Chiego DJ Jr, Marcucci MC, Cunha I, Custodio A, Schneider LG. Preliminary report on the effects of propolis on wound healing in the dental pulp. Z Naturforsch [C] 1998;53:1045–8. 29. Pileggi R, Antony K, Johnson K, Zuo J, Shannon Holliday L. Propolis inhibits osteoclast maturation. Dent Traumatol 2009;25:584–8. 30. Tao JY, Zhao L, Huang ZJ, Zhang XY, Zhang SL, Zhang QG et al. Anti-inflammatory effects of ethanol extract from Kummerowia striata (Thunb.) Schindl on lps-stimulated RAW 264.7 cell. Inflammation 2008;31:154–66. 31. Daniela L, Alla P, Maurelli R, Elena D, Giovanna P, Vladimir K et al. Anti-inflammatory effects of concentrated ethanol extracts of Edelweiss (Leontopodium alpinum Cass.) callus cultures towards human keratinocytes and endothelial cells. Mediators Inflamm 2012;2012:498373. 32. Hahn CL, Liewehr FR. Innate immune responses of the dental pulp to caries. J Endod 2007;33:643–51. 33. Farges JC, Keller JF, Carrouel F, Durand SH, Romeas A, Bleicher F et al. Odontoblasts in the dental pulp immune response. J Exp Zool B Mol Dev Evol 2009;312B:425–36. 34. Bergenholtz G, Nagaoka S, Jontell M. Class II antigen expressing cells in experimentally induced pulpitis. Int Endod J 1991;24:8–14.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
367
35. Jontell M, Okiji T, Dahlgren U, Bergenholtz G. Immune defense mechanisms of the dental pulp. Crit Rev Oral Biol Med 1998;9:179–200. 36. Hahn CL, Liewehr FR. Update on the adaptive immune responses of the dental pulp. J Endod 2007;33:773–81. 37. D’Souza R, Brown LR, Newland JR, Levy BM, Lachman LB. Detection and characterization of interleukin-1 in human dental pulps. Arch Oral Biol 1989;34:307–13. 38. Pezelj-Ribaric S, Anic I, Brekalo I, Miletic I, Hasan M, Simunovic-Soskic M. Detection of tumor necrosis factor alpha in normal and inflamed human dental pulps. Arch Med Res 2002;33:482–4. 39. Tronstad L. Root resorption–etiology, terminology and clinical manifestations. Endod Dent Traumatol 1988;4:241–52. 40. Andreasen JO, Kristerson L. The effect of extra-alveolar root filling with calcium hydroxide on periodontal healing after replantation of permanent incisors in monkeys. J Endod 1981;7:349–54. 41. Dumsha T, Hovland EJ. Evaluation of long-term calcium hydroxide treatment in avulsed teeth–an in vivo study. Int Endod J 1995;28:7–11. 42. Spangberg LSWHM. Rationale and efficacy of root canal medicaments and root filling materials with emphasis on treatment outcome. Endod Topics 2002;2:35–58. 43. Law A, Messer H. An evidence-based analysis of the antibacterial effectiveness of intracanal medicaments. J Endod 2004;30:689–94. 44. Kawashima N, Wadachi R, Suda H, Yeng T, Parashos P. Root canal medicaments. Int Dent J 2009;59:5–11. 45. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J 1999;32:361–9. 46. Evanov C, Liewehr F, Buxton TB, Joyce AP. Antibacterial efficacy of calcium hydroxide and chlorhexidine gluconate irrigants at 37°C and 46°C. J Endod 2004;30:653–7. 47. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93–8. 48. Schett G. Effects of inflammatory and anti-inflammatory cytokines on the bone. Eur J Clin Invest 2011;41:1361–6. 49. Braun T, Schett G. Pathways for bone loss in inflammatory disease. Curr Osteoporos Rep 2012;10:101–8. 50. Kuropatnicki AK, Szliszka E, Krol W. Historical aspects of propolis research in modern times. Evid Based Complement Alternat Med 2013;2013:964149. 51. Watanabe MA, Amarante MK, Conti BJ, Sforcin JM. Cytotoxic constituents of propolis inducing anticancer effects: a review. J Pharm Pharmacol 2011;63:1378–86. 52. Daleprane JB, Abdalla DS. Emerging roles of propolis: antioxidant, cardioprotective, and antiangiogenic actions. Evid Based Complement Alternat Med 2013;2013:175135.
