- Email: [email protected]
Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines
archives of oral biology 58 (2013) 75–81
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines Roy Petel a,d, Moti Moskovitz a,d,*, Nili Tickotsky b, Amal Halabi c, Judith Goldstein c, Yael Houri-Haddad c a
Department of Pediatric Dentistry, Hadassah School of Dental Medicine, Israel Private Practice, Hadera, Israel c Department of Prosthodontics, Hadassah School of Dental Medicine, Israel b
article info
abstract
Article history:
Objective: The present study investigated the effect of the Iodoform-containing root canal
Accepted 18 June 2012
filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion.
Keywords:
Design: The effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line
Root canal treatment
and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha
Primary teeth
(TNFa) from macrophages was examined. Cell vitality was evaluated using a colourimetric
Macrophage cell
XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tet-
Epithelial cell
razolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-
Cyst-like lesion
linked immunosorbent assay (ELISA).
Endoflas F.S.
Results: Direct exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by 70% compared to control media (P < 0.05). Media dilution from 1/16 to 1/1024 demonstrated a proliferative effect, increasing cell viability by about 60% compared to media without Iodoform-containing root canal filling material. Conclusions: Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Root canal treatment in primary teeth is indicated once the teeth are with carious pulp exposures and the radicular pulp exhibits clinical signs of hyperaemia, or evidence of necrosis of the radicular pulp.1 The characteristics of an ideal root filling material have been described in the literature.2,3 Available root canal filling
materials for primary teeth include: zinc oxide-eugenol (ZOE)4,5; Kri paste ((Pharmachemie AG, Zurich, Switzerland)2,6; Maisto paste (Inodon, Porto Alegre, Brazil)7; Vitapex (New Dental Chemical Products Co. Ltd., Tokyo, Japan)8; and Endoflas F.S. (Sanlor & Cia. S. en C.S., Colombia). Except for ZOE, all contain iodoform, and all have demonstrated good clinical results.2,7–11 Endoflas F.S. is composed of a powder component of tri-iodomethane and iodine dibutilorthocresol (40.6%), zinc oxide (56.5%), calcium hydroxide (1.07%) and
* Corresponding author at: Department of Pediatric Dentistry, Hadassah School of Dental Medicine, P.O. Box 12272, Jerusalem 91120, Israel. Tel.: +972 2 776122; fax: +972 2 6431747. E-mail address: [email protected] (M. Moskovitz). d The authors contributed equally to the study. 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2012.06.014
76
archives of oral biology 58 (2013) 75–81
barium sulphate (1.63%), with a liquid consisting of eugenol and paramonochlorophenol.3 Moskovitz et al. found that eight (3.3%) of 242 primary molars that were treated by root canal using Endoflas F.S. presented either a new radiolucent defect or enlargement of an existing periapical radiolucency.12 It was suspected that the radiolucent defect detected in four of these teeth, one of which was confirmed by histological examination as a radicular cyst, resulted from low-grade irritation to the periapical tissue and the follicle of the permanent successor due to one of the components of the root filling material. A number of case reports suggest an association between root canal treatment and periapical lesions in primary dentition.11,13–17 The aim of the present study was to test the hypothesis that iodoform-containing root canal filling material – Endoflas F.S. – may affect cultured macrophages and epithelial cell viability and cytokine secretion after activation with heat-killed Porphyromonas gingivalis (P. gingivalis), an anaerobic Gram-negative bacterium.
2.
Materials and methods
2.1.
Preparation of Endoflas F.S.
as described previously.18 The fraction of viable cells in the Endoflas F.S.-exposed groups was normalised according to 100% cell viability of groups without Endoflas F.S. (control).
2.4.
The secreted TNFa was quantified using the two-site enzymelinked immunosorbent assay (ELISA). This method is based on commercially available antibody pairs (Pharmingen, San Diego, CA, USA) as described previously.19 The range of detection for each specific cytokine was 25–2000 pg ml 1.
2.5.
2.2.
Cell cultures (macrophages and epithelial cells)
A RAW 264.7 macrophage cell line and an RKO epithelial cell line were cultured separately on Petri dishes in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% penicillin/streptomycin and 1% glutamine. Before the assay, the cells were cultured at a density of 60,000 cells in 200 ml media per well in 96-well tissue culture plates (Nunc) (the doubling time of RKO is 18 2.1 h and for RAW cells it is 12.5 2 h). Twenty-four hours after plating, the cells were activated by 10 ml of heat-killed P. gingivalis 33277 ATCC (109 colony forming unit (CFU) ml 1) and exposed to the Endoflas F.S. layered on the special plastic inserts (direct contact). Following 24 h of incubation at 37 8C in a humidified atmosphere of 5% CO2, the plates were analysed for cell viability and levels of TNFa secreted by macrophages. In another set of experiments, the Endoflas F.S. layered on the special inserts was inserted into a 96-well plate with media for 24 h. The macrophage and RKO epithelial cells cultured in separate 96-well tissue culture plates were exposed to several descending dilutions (1/1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/ 256, 1/512 and 1/1024) of the Endoflas F.S.-conditioned media (indirect contact). Every experiment was repeated in eight wells and the average of three repeated experiments was calculated.
2.3.
Heat-killed bacterium P. gingivalis
P. gingivalis, strain ATCC 33277, was grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2 and 10% CO2. After incubation at 37 8C for 2–3 days, the bacteria were inoculated into peptone yeast extract and incubated for 3–4 days under the same conditions. To obtain heatkilled bacteria, the bacteria were washed three times with PBS and then exposed to 80 8C for 10 min as described previously.19
2.6. Endoflas F.S. (Sanlor & Cia. S. en C.S., Colombia) was prepared according to the manufacturer’s instructions and coated in a 1-mm2 layer weighting 15 5 mg per sample on special plastic inserts. The latter were sterilised under UV radiation for 24 h and placed in 96-well tissue culture plates.
Cytokine analysis
Statistical analysis
Data analysis was performed using SigmaStat statistical software (Jandel Scientific, San Rafael, CA, USA). One-way repeated measure of analysis of variance (RM ANOVA) was used to assess a dose-dependent effect of Endoflas F.S. When significance was established, intergroup differences were tested for significance using the Student’s t-test with the Bonferroni correction for multiple testing. The significance of the differences between two treatment groups was evaluated using the t-test. The level of significance was set at P < 0.05. All the results are presented as the mean standard error.
3.
Results
3.1. The effect of direct and indirect contact of Endoflas F.S. on the viability and TNFa secretion of RAW 264.7 macrophages (Fig. 1) For all tested conditions, exposure to Endoflas F.S. decreased the viability of macrophages by 70% compared to the controlmedia group, P < 0.05 (Fig. 1(a)). (Mitochondrial activity was normalised to the control.) The challenge with heat-killed P. gingivalis alone did not significantly affect the viability of the cells. For all conditions, including direct and indirect contact, Endoflas F.S. demonstrated a similar effect on cell viability and on the reduction of P.gingivalis-induced TNFa secretion to almost undetectable levels (P < 0.05) (Fig. 1(b)).
3.2. The dose-dependent effect of Endoflas F.S. on macrophage viability and TNFa secretion
Cell vitality
The vitality of the cells was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay
High concentrations of Endoflas F.S.-conditioned media (dilutions of 1/1, 1/2, 1/4 and 1/8) reduced cell viability by 80% (Fig. 2(a)). However, low concentrations (dilutions of 1/ 16, 1/32, 1/64, 1/128, 1/512 and 1/1024) induced a proliferative
archives of oral biology 58 (2013) 75–81
77
Fig. 1 – The effect of Endoflas F.S. on the mitochondrial activity of the RAW macrophage cell line (A) and its TNFa secretion (B). Mitochondrial activity was normalysed to the control without any stimulation. Macrophages, both unstimulated and stimulated (with heat killed P. gingivalis, with and without Endoflas F.S.) were cultured for 24 h. Cell viability was tested using the XTT test (A); secreted TNFa levels were analysed by ELISA (B). In the group ‘‘media of Endoflas F.S. + P. gingivalis’’, Endoflas F.S. was inserted to the media without cells for 24 h, and then transferred to cells stimulated with P. gingivalis. The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
effect, with cell viability increasing by about 60% compared to the control-media group (Fig. 2(a)). In the presence of P. gingivalis challenge, high concentrations of Endoflas F.S. (low dilutions 1/1, 1/2, 1/4 and 1/8) still induced a cytotoxic effect,
reducing the viability of macrophages to 20%. Endoflas F.S. at 1/16 dilution reduced cell viability to 80% (Fig. 2(b)). The proliferative effect of Endoflas F.S. at low concentration (high dilutions of 1/32, 1/64, 1/128, 1/512 and 1/1024) was less
Fig. 2 – The dose dependent effect of Endoflas F.S. on the mitochondrial activity of TNFa secretion in the RAW macrophage cell line. Mitochondrial activity was normalysed to the control without any stimulation. The macrophages were stimulated by decreasing dilutions of Endoflas media in the presence of heat killed P. gingivalis (B) or without P. gingivalis (A). The macrophages were cultured with the Endoflas media for 24 h and their viability was tested using the XTT test. In the presence of heat killed P. gingivalis. The cells were cultured for 24 h and the secreted TNFa levels were analysed by ELISA (C). The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
78
archives of oral biology 58 (2013) 75–81
Epithelial cell viability as dose dependent of
ANo P. gingivalis
Endoflas dilutions
% cell viability
250 200
*
*
* v
1:16
1:32
150
*
*
*
*
100
*
* *
50 0
media 1:1
1:2
1:4
1:8
1:64 1:128 1:256 1:512
Endoflas dilutions BWith P. gingivalis % cell viability
250 200
*
150
*
100 50
*
* *
1:1
1:2
0 media
1:4
1:8
1:16
1:32
1:64
1:128 1:256 1:512
Endoflas dilutions Fig. 3 – The dose dependent effect of Endoflas on the mitochondrial activity of the RKO epithelial cell line. Mitochondrial activity was normalysed to the control without any stimulation. The epithelial cells were stimulated by decreasing dilutions of Endoflas F.S. media in the presence of heat killed P. gingivalis (B) or without P. gingivalis (A). The epithelial cells were cultured with the Endoflas F.S. media for 24 h and their viability tested using the XTT test. The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
pronounced in the presence of P. gingivalis than in its absence, with cell viability differing significantly from the control only for the 1/256 dilution (P < 0.05). As expected, macrophages challenged with heat-killed P. gingivalis were capable of secreting high levels of TNFa as seen in the media control group (Fig. 2(c)). However, the addition of Endoflas F.S.-conditioned media in various concentrations affected TNFa secretion. For undiluted and high concentration (low dilutions 1/2, 1/4 and 1/8) of Endoflas F.S., levels of TNFa reached very low values – hundreds of pg ml 1 compared to 27,000 pg ml 1 in the media group (Fig. 2(c)). At dilution 1/16, this effect was reduced, and from dilution 1/32 and higher levels of TNFa did not differ significantly from those in the media group (Fig. 2(c)).
3.3. The dose-dependent effect of Endoflas on epithelial cell viability Without bacterial challenge, high concentration (low dilutions) of Endoflas F.S. (1/2 and 1/4) reduced cell viability by 60% compared to samples without Endoflas F.S. (Fig. 3(a)).
From a dilution of 1/8 and higher, the effect was proliferative rather than cytotoxic. The XTT test showed a significant increase in cell viability, indicative of an increase in cell number of about 60% compared to control-media cells (Fig. 3(a)). With P. gingivalis challenge, high Endoflas F.S. concentrations induced a more pronounced cytotoxic effect than without the bacterial challenge. Cell viability was reduced to 30% compared to the control-media group (Fig. 3(b)). For Endoflas F.S. at dilutions of 1/8 to 1/256, a mild cytotoxic effect was observed (Fig. 3(b)). Only the highest tested dilution of 1/ 512 increased cell viability compared to the control-media group, resulting in a proliferative effect of 20%.
4.
Discussion
To the best of our knowledge, this is the first study examining the effect of diluted media of iodoform-containing root canal filing material on macrophage and epithelial cell lines. Macrophages were chosen as cell targets because of their
archives of oral biology 58 (2013) 75–81
major role in the inflammatory process as they are vital for appropriate healing and also function in destructive inflammatory processes. Epithelial cells were selected because radicular cysts arise from the proliferation of epithelial remnants of Malassez in response to stimulation of chronic inflammatory processes in the apical region of the necrotic tooth.20 In the present study, both direct and indirect exposure of the RAW 264.7 macrophage and RKO epithelial cell lines to Endoflas F.S. or its highly concentrated media, diluted up to 1/ 8, resulted in cell death of about 70%. This supports the study of Brisen˜o and Willershausen in which Endoflas F.S. demonstrated significantly higher cytotoxicity to human gingival fibroblasts than did other calcium hydroxide-based root canal sealers.21 The present study’s results indicate that high concentrations of Endoflas F.S. and its conditioned media reduced the secretion of P. gingivalis-induced TNFa to almost undetectable levels. P. gingivalis is one of the anaerobic bacteria that exist in anaerobic oral niches including periapical sites and periodontal pockets. The activation of the host cells by such bacteria was represented in this study by the stimulation of the cell lines with heat-killed P. gingivalis. According to the results, the proliferative effect of Endoflas F.S.-conditioned media was less prominent with the heat-killed P. gingivalis challenge. This may be due to a toxic effect of bacterial particles on cell survival and proliferation, as suppression of mitochondrial activity, suggesting cytotoxicity, may preclude the ability of macrophages to secrete any cytokines. The effect of Endoflas F.S.-conditioned media on the RAW 264.7 macrophage and RKO epithelial cell lines was dose dependent. Highly concentrated media with dilutions of Endoflas F.S. up to 1/8 demonstrated a cytotoxic effect on both cell lines, while media with dilutions from 1/32 to 1/1024 induced cell proliferation. The observation of increased cell proliferation is based on the XTT results, an assay correlated to cell viability. XTT results could also be a reflection of the high mitochondrial activity as an outcome of cells’ stress condition. XTT significant increase in cell viability requires further investigation. Changes of mitochondrial activity by the Endoflas F.S. were probably not mediated by the material itself, but by degradation products released to the surrounding cell-culture medium. It is probable that these degradation products caused changes in both mitochondrial activity and cytokine secretion. This unexpected in vitro effect may be used to explore the clinical reports of cysts forming near deciduous teeth that have been treated with Endoflas F.S. In such teeth, exposed Endoflas F.S. may induce low-grade irritation. During primary tooth shedding and eruption of a successor permanent tooth, the bud of the latter is in close proximity to the resorbed roots of the primary tooth. Cells involved in root resorption, including multinucleated cells, may thus come in contact with media containing remnants of Endoflas F.S. The same occurs with epithelial cells derived from the dental sac of the erupting permanent tooth. In both cell lines, the possible proliferative effect of Endoflas F.S.conditioned media may promote enlargement of the dental sac of the permanent tooth bud.During periapical healing, macrophages secrete a number of inflammatory mediators,
79
including cytokines, that perpetuate inflammation or direct healing.22 Cytokines such as interleukin 6 (IL6), ILb or TNFa mediate periapical inflammation, bone resorption23,24 and inhibition of bone repair that are features of the formation of periapical lesion.25,26 Cytokine production is also stimulated by bacterial lipopolysaccharides that are commonly present as part of the pulpal infection preceding the root canal treatment.27–29 Degradation of primary tooth root canal sealing materials discharges chemicals or particles that may alter the function of monocytes or macrophages attempting phagocytosis.30,31 Thus, the effect of endodontic sealers on monocytic function is an important consideration; aberrant, suppressed or unnecessary inflammatory responses delay periapical healing.32,33 The growth of radicular cysts is accompanied by local bone destruction, tissue osteolysis and accumulation of different cells in the cystic epithelium. During this dynamic inflammatory process, pro-inflammatory cytokines such as TNF-a are actively secreted and accumulated in the cystic fluid,34 and their concentrations are significantly higher in radicular cyst fluids.35,36 TNF-a can act as a pro-inflammatory cytokine and stimulate bone resorption.37 Takahashi et al. found that epithelial cells undergo proliferation in human dental periapical lesions.38 The present study demonstrated the effect of Endoflas F.S. components on both macrophages and epithelial cells. One of the study limitations and a major concern in material biocompatibility is the applicability of in vitro models of biological activity to clinical performance. While evaluating mitochondrial function, cytokine secretion and epithelial proliferation in response to Endoflas F.S. degradation products in a relatively static manner, the present model could not account for the root apex dynamic environment and circulatory removal of degradation products, or the interactions among cell types at the periapical region. The complexities of inflammatory responses cannot be adequately assessed by a single in vitro test and the full impact of this specific endodontic sealer on inflammation deserves further investigation.
4.1.
Clinical implication
The relationship between root canal treatment in primary molars and the appearance of cysts is not fully understood. Clinicians should be aware that chemical components of materials used in root canal treatments of primary teeth may cause long-term local irritation to follicles of permanent successors, thus stimulating formation of cyst-like radiolucent defects.12,39 Further research is needed to elucidate the specific mechanisms involved in this process and to identify the responsible agents.
5.
Conclusions
Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation.
80
archives of oral biology 58 (2013) 75–81
Funding Department funds.
Competing interests The authors deny any conflicts of interest.
Ethical approval Not applicable.
references
1. American Academy of Pediatric Dentistry: Guideline on pulp therapy for primary and young permanent teeth, Adopted 1991 Revised 1998, 2001, 2004, 2009 Pediatric dentistry http:// www.aapd.org/media/Policies_Guidelines/G_Pulp.pdf; 2009. 2. Holan G, Fuks AB. A comparison of pulpectomies using ZOE and KRI in primary molars: a retrospective study. Pediatric Dentistry 1993;15:403–7. 3. Moskovitz M, Sammara E, Holan G. Success rate of root canal treatment in primary molars. Journal of Dentistry 2005;33:41–7. 4. Dummett Jr CO, Kopel HM. Pediatric endodontics. In: Ingle J, editor. Endodontics. Philadelphia: Lea and Febiger; 2002. p. 861–902. 5. Erausquin J, Muruzabal M. Root canal fillings with zinc oxide eugenol cement in the rat molar. Oral Surgery Oral Medicine and Oral Pathology 1967;24:547–58. 6. Garcia-Godoy F. Evaluation of an iodoform paste in root canal therapy for infected primary teeth. ASDC Journal of Dentistry for Children 1987;54:30–4. 7. Mass E, Zilberman LU. Endodontic treatment of infected primary teeth, using Maisto’s paste. ASDC Journal of Dentistry for Children 1989;56:117–20. 8. Nurko C, Gracia-Godoy F. Evaluation of a calcium hydroxide/ iodoform paste (Vitapex) in root canal therapy for primary teeth. The Journal of Clinical Pediatric Dentistry 1999;23:289–94. 9. Fuks AB, Eidelman E, Pauker N. Root fillings with Endoflas in primary teeth: a retrospective study. Journal of Clinical Pediatric Dentistry 2002;27:41–6. 10. Rifkin A. A simple effective, safe technique for the root canal treatment of abscessed primary teeth. ASDC Journal of Dentistry for Children 1980;47:435–41. 11. Rifkin A. The root canal treatment of abscessed primary teeth – a three to four year follow-up. ASDC Journal of Dentistry for Children 1982;49:428–31. 12. Moskovitz M, Yahav D, Tickotsky N, et al. Long-term follow up of root canal treated primary molars. International Journal of Paediatric Dentistry 2010;20:207–13. 13. Savage NW, Adkins KS, Weir AV, et al. A histological study of cystic lesion following pulp therapy in deciduous molars. Journal of Oral Pathology 1986;15:209–12. 14. Bhat SS, Vidhya M, Sargod S. Radicular cyst associated with endodontically treated teeth. Journal of Indian Society of Pedodontic and Preventive Dentistry 2003;21:139–41. 15. Takiguchi M, Fujiwara T, Sobue S, et al. Radicular cyst associated with a primary molar following pulp therapy: a case report. International Journal of Paediatric Dentistry 2001;11:452–5.
16. Mass E, Kaplan I, Hirshberg A. A clinical and histopathological study of radicular cysts associated with primary molars. Journal of Oral Pathology and Medicine 1995;24:458–61. 17. Hill FJ. Cystic lesions associated with deciduous teeth. Proceedings of the British Paedodontic Society 1978;8:9–12. 18. Beyth N, Houri-Haddad Y, Baraness-Hadar L, et al. Surface antimicrobial activity and biocompatibility of incorporated polyethylenimine nanoparticles. Biomaterials 2008;29: 4157–63. 19. Polak D, Wilensky A, Shapira L, et al. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response. Journal of Clinical Periodontology 2009;36: 406–10. 20. Regezi JA, Sciubba JJ, Jordan RCK. Cysts of the Jaws and Neck. 4th ed. USA: Saunders; 2003. 21. Brisen˜o BM, Willershausen B. Root canal sealer cytotoxicity with human gingival fibroblasts: III. Calcium hydroxidebased sealers. Journal of Endodontics 1992;18:110–3. 22. Sjogren U, Ohlin A, Sundqvist G, Lerner UH. Guttapercha stimulated mouse macrophages release factors that activate the bone resorptive system of mouse calvarial bone. European Journal of Oral Sciences 1998;106:872–81. 23. Metzger Z, Nitzan D, Pitaru S, Brosh T, Teicher S. The effect of bacterial endotoxin on the early tensile strength of healing surgical wounds. Journal of Endodontics 2002;28: 30–3. 24. Mosser DM. The many faces of macrophage activation. Journal of Leukocyte Biology 2003;73:209–12. 25. 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. Archives of Medical Research 2002;33:482–4. 26. Zehnder M, Delaleu N, Du Y, Bickel M. Cytokine gene expression – part of host defence in pulpitis. Cytokine 2003;22:84–8. 27. 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. Journal of Oral Pathology and Medicine 2004;33:162–9. 28. Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B. Bacterially induced bone destruction: mechanisms and misconceptions. Infection and Immunity 1996;64:2371–80. 29. Stashenko P. Role of immune cytokines in the pathogenesis of periapical lesions. Endodontics and Dental Traumatology 1990;6:89–96. 30. Nair PN, Sjogren U, Schumacher E, Sundqvist G. Radicular cyst affecting a root-filled human tooth: a long-term posttreatment follow-up. International Endodontic Journal 1993;26:225–33. 31. Nair PN, Sjogren U, Krey G, Sundqvist G. Therapyresistant foreign body giant cell granuloma at the periapex of a root-filled human tooth. Journal of Endodontics 1990;16:589–95. 32. Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dental Materials 1996;12: 186–93. 33. Bernath M, Szabo J. Tissue reaction initiated by different sealers. International Endodontic Journal 2003;36:256–61. 34. Jurisic V, Colic S, Jurisic M. The inflammatory radicular cysts have higher concentration of TNFa in comparison to odontogenic keratocysts (odontogenic tumour). Acta Medica (Hradec Kralove) 2007;50:233–8. 35. Colic´ S, Jurisic´ M, Jurisic´ V. Pathophysiological mechanism of the developing radicular cyst of the jaw. Article in Serbian. Acta chirurgica Iugoslavica 2008;55:87–92.
archives of oral biology 58 (2013) 75–81
36. Muglali M, Komerik N, Bulut E, Yarim GF, Celebi N, Sumer M. Cytokine and chemokine levels in radicular and residual cyst fluids. Journal of Oral Pathology and Medicine 2008;37: 185–9. 37. Bando Y, Henderson B, Meghji S, Poole S, Harris M. Immunocytochemical localization of inflammatory cytokines and vascular adhesion receptors in radicular cysts. Journal of Oral Pathology and Medicine 1993;22:221–7.
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38. Takahashi K, MacDonald D, Murayama Y, Kinane D. Cell synthesis, proliferation and apoptosis in human dental periapical lesions analyzed by in situ hybridization and immunohistochemistry. Oral Diseases 1999;5:313–20. 39. Grundy GE, Adkins KF, Savage NW. Cysts associated with deciduous molars following pulp therapy. Australian Dental Journal 1984;29:249–56.
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
Cytotoxicity and proliferative effects of Iodoform-containing root canal-filling material on RAW 264.7 macrophage and RKO epithelial cell lines Roy Petel a,d, Moti Moskovitz a,d,*, Nili Tickotsky b, Amal Halabi c, Judith Goldstein c, Yael Houri-Haddad c a
Department of Pediatric Dentistry, Hadassah School of Dental Medicine, Israel Private Practice, Hadera, Israel c Department of Prosthodontics, Hadassah School of Dental Medicine, Israel b
article info
abstract
Article history:
Objective: The present study investigated the effect of the Iodoform-containing root canal
Accepted 18 June 2012
filling material on the viability of cultured macrophages and epithelial cells, and on cytokine secretion.
Keywords:
Design: The effect of Endoflas F.S. on the proliferation of a RAW 264.7 macrophage cell line
Root canal treatment
and on a RKO epithelial cell line, and on the production of tumour necrosis factor alpha
Primary teeth
(TNFa) from macrophages was examined. Cell vitality was evaluated using a colourimetric
Macrophage cell
XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tet-
Epithelial cell
razolium inner salt) assay. The presence of cytokines was determined by two-site enzyme-
Cyst-like lesion
linked immunosorbent assay (ELISA).
Endoflas F.S.
Results: Direct exposure of Endoflas F.S. and its media, up to a dilution of 1/8, decreased the viability of macrophages and epithelial cells by 70% compared to control media (P < 0.05). Media dilution from 1/16 to 1/1024 demonstrated a proliferative effect, increasing cell viability by about 60% compared to media without Iodoform-containing root canal filling material. Conclusions: Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Root canal treatment in primary teeth is indicated once the teeth are with carious pulp exposures and the radicular pulp exhibits clinical signs of hyperaemia, or evidence of necrosis of the radicular pulp.1 The characteristics of an ideal root filling material have been described in the literature.2,3 Available root canal filling
materials for primary teeth include: zinc oxide-eugenol (ZOE)4,5; Kri paste ((Pharmachemie AG, Zurich, Switzerland)2,6; Maisto paste (Inodon, Porto Alegre, Brazil)7; Vitapex (New Dental Chemical Products Co. Ltd., Tokyo, Japan)8; and Endoflas F.S. (Sanlor & Cia. S. en C.S., Colombia). Except for ZOE, all contain iodoform, and all have demonstrated good clinical results.2,7–11 Endoflas F.S. is composed of a powder component of tri-iodomethane and iodine dibutilorthocresol (40.6%), zinc oxide (56.5%), calcium hydroxide (1.07%) and
* Corresponding author at: Department of Pediatric Dentistry, Hadassah School of Dental Medicine, P.O. Box 12272, Jerusalem 91120, Israel. Tel.: +972 2 776122; fax: +972 2 6431747. E-mail address: [email protected] (M. Moskovitz). d The authors contributed equally to the study. 0003–9969/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2012.06.014
76
archives of oral biology 58 (2013) 75–81
barium sulphate (1.63%), with a liquid consisting of eugenol and paramonochlorophenol.3 Moskovitz et al. found that eight (3.3%) of 242 primary molars that were treated by root canal using Endoflas F.S. presented either a new radiolucent defect or enlargement of an existing periapical radiolucency.12 It was suspected that the radiolucent defect detected in four of these teeth, one of which was confirmed by histological examination as a radicular cyst, resulted from low-grade irritation to the periapical tissue and the follicle of the permanent successor due to one of the components of the root filling material. A number of case reports suggest an association between root canal treatment and periapical lesions in primary dentition.11,13–17 The aim of the present study was to test the hypothesis that iodoform-containing root canal filling material – Endoflas F.S. – may affect cultured macrophages and epithelial cell viability and cytokine secretion after activation with heat-killed Porphyromonas gingivalis (P. gingivalis), an anaerobic Gram-negative bacterium.
2.
Materials and methods
2.1.
Preparation of Endoflas F.S.
as described previously.18 The fraction of viable cells in the Endoflas F.S.-exposed groups was normalised according to 100% cell viability of groups without Endoflas F.S. (control).
2.4.
The secreted TNFa was quantified using the two-site enzymelinked immunosorbent assay (ELISA). This method is based on commercially available antibody pairs (Pharmingen, San Diego, CA, USA) as described previously.19 The range of detection for each specific cytokine was 25–2000 pg ml 1.
2.5.
2.2.
Cell cultures (macrophages and epithelial cells)
A RAW 264.7 macrophage cell line and an RKO epithelial cell line were cultured separately on Petri dishes in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% penicillin/streptomycin and 1% glutamine. Before the assay, the cells were cultured at a density of 60,000 cells in 200 ml media per well in 96-well tissue culture plates (Nunc) (the doubling time of RKO is 18 2.1 h and for RAW cells it is 12.5 2 h). Twenty-four hours after plating, the cells were activated by 10 ml of heat-killed P. gingivalis 33277 ATCC (109 colony forming unit (CFU) ml 1) and exposed to the Endoflas F.S. layered on the special plastic inserts (direct contact). Following 24 h of incubation at 37 8C in a humidified atmosphere of 5% CO2, the plates were analysed for cell viability and levels of TNFa secreted by macrophages. In another set of experiments, the Endoflas F.S. layered on the special inserts was inserted into a 96-well plate with media for 24 h. The macrophage and RKO epithelial cells cultured in separate 96-well tissue culture plates were exposed to several descending dilutions (1/1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/ 256, 1/512 and 1/1024) of the Endoflas F.S.-conditioned media (indirect contact). Every experiment was repeated in eight wells and the average of three repeated experiments was calculated.
2.3.
Heat-killed bacterium P. gingivalis
P. gingivalis, strain ATCC 33277, was grown on blood agar plates in an anaerobic chamber with 85% N2, 5% H2 and 10% CO2. After incubation at 37 8C for 2–3 days, the bacteria were inoculated into peptone yeast extract and incubated for 3–4 days under the same conditions. To obtain heatkilled bacteria, the bacteria were washed three times with PBS and then exposed to 80 8C for 10 min as described previously.19
2.6. Endoflas F.S. (Sanlor & Cia. S. en C.S., Colombia) was prepared according to the manufacturer’s instructions and coated in a 1-mm2 layer weighting 15 5 mg per sample on special plastic inserts. The latter were sterilised under UV radiation for 24 h and placed in 96-well tissue culture plates.
Cytokine analysis
Statistical analysis
Data analysis was performed using SigmaStat statistical software (Jandel Scientific, San Rafael, CA, USA). One-way repeated measure of analysis of variance (RM ANOVA) was used to assess a dose-dependent effect of Endoflas F.S. When significance was established, intergroup differences were tested for significance using the Student’s t-test with the Bonferroni correction for multiple testing. The significance of the differences between two treatment groups was evaluated using the t-test. The level of significance was set at P < 0.05. All the results are presented as the mean standard error.
3.
Results
3.1. The effect of direct and indirect contact of Endoflas F.S. on the viability and TNFa secretion of RAW 264.7 macrophages (Fig. 1) For all tested conditions, exposure to Endoflas F.S. decreased the viability of macrophages by 70% compared to the controlmedia group, P < 0.05 (Fig. 1(a)). (Mitochondrial activity was normalised to the control.) The challenge with heat-killed P. gingivalis alone did not significantly affect the viability of the cells. For all conditions, including direct and indirect contact, Endoflas F.S. demonstrated a similar effect on cell viability and on the reduction of P.gingivalis-induced TNFa secretion to almost undetectable levels (P < 0.05) (Fig. 1(b)).
3.2. The dose-dependent effect of Endoflas F.S. on macrophage viability and TNFa secretion
Cell vitality
The vitality of the cells was evaluated using a colourimetric XTT (sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) assay
High concentrations of Endoflas F.S.-conditioned media (dilutions of 1/1, 1/2, 1/4 and 1/8) reduced cell viability by 80% (Fig. 2(a)). However, low concentrations (dilutions of 1/ 16, 1/32, 1/64, 1/128, 1/512 and 1/1024) induced a proliferative
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Fig. 1 – The effect of Endoflas F.S. on the mitochondrial activity of the RAW macrophage cell line (A) and its TNFa secretion (B). Mitochondrial activity was normalysed to the control without any stimulation. Macrophages, both unstimulated and stimulated (with heat killed P. gingivalis, with and without Endoflas F.S.) were cultured for 24 h. Cell viability was tested using the XTT test (A); secreted TNFa levels were analysed by ELISA (B). In the group ‘‘media of Endoflas F.S. + P. gingivalis’’, Endoflas F.S. was inserted to the media without cells for 24 h, and then transferred to cells stimulated with P. gingivalis. The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
effect, with cell viability increasing by about 60% compared to the control-media group (Fig. 2(a)). In the presence of P. gingivalis challenge, high concentrations of Endoflas F.S. (low dilutions 1/1, 1/2, 1/4 and 1/8) still induced a cytotoxic effect,
reducing the viability of macrophages to 20%. Endoflas F.S. at 1/16 dilution reduced cell viability to 80% (Fig. 2(b)). The proliferative effect of Endoflas F.S. at low concentration (high dilutions of 1/32, 1/64, 1/128, 1/512 and 1/1024) was less
Fig. 2 – The dose dependent effect of Endoflas F.S. on the mitochondrial activity of TNFa secretion in the RAW macrophage cell line. Mitochondrial activity was normalysed to the control without any stimulation. The macrophages were stimulated by decreasing dilutions of Endoflas media in the presence of heat killed P. gingivalis (B) or without P. gingivalis (A). The macrophages were cultured with the Endoflas media for 24 h and their viability was tested using the XTT test. In the presence of heat killed P. gingivalis. The cells were cultured for 24 h and the secreted TNFa levels were analysed by ELISA (C). The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
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Epithelial cell viability as dose dependent of
ANo P. gingivalis
Endoflas dilutions
% cell viability
250 200
*
*
* v
1:16
1:32
150
*
*
*
*
100
*
* *
50 0
media 1:1
1:2
1:4
1:8
1:64 1:128 1:256 1:512
Endoflas dilutions BWith P. gingivalis % cell viability
250 200
*
150
*
100 50
*
* *
1:1
1:2
0 media
1:4
1:8
1:16
1:32
1:64
1:128 1:256 1:512
Endoflas dilutions Fig. 3 – The dose dependent effect of Endoflas on the mitochondrial activity of the RKO epithelial cell line. Mitochondrial activity was normalysed to the control without any stimulation. The epithelial cells were stimulated by decreasing dilutions of Endoflas F.S. media in the presence of heat killed P. gingivalis (B) or without P. gingivalis (A). The epithelial cells were cultured with the Endoflas F.S. media for 24 h and their viability tested using the XTT test. The results are expressed as the mean + ser. Statistically significant differences between the control (media) and the different groups are indicated by asterisks (P < 0.05) n = 8 wells.
pronounced in the presence of P. gingivalis than in its absence, with cell viability differing significantly from the control only for the 1/256 dilution (P < 0.05). As expected, macrophages challenged with heat-killed P. gingivalis were capable of secreting high levels of TNFa as seen in the media control group (Fig. 2(c)). However, the addition of Endoflas F.S.-conditioned media in various concentrations affected TNFa secretion. For undiluted and high concentration (low dilutions 1/2, 1/4 and 1/8) of Endoflas F.S., levels of TNFa reached very low values – hundreds of pg ml 1 compared to 27,000 pg ml 1 in the media group (Fig. 2(c)). At dilution 1/16, this effect was reduced, and from dilution 1/32 and higher levels of TNFa did not differ significantly from those in the media group (Fig. 2(c)).
3.3. The dose-dependent effect of Endoflas on epithelial cell viability Without bacterial challenge, high concentration (low dilutions) of Endoflas F.S. (1/2 and 1/4) reduced cell viability by 60% compared to samples without Endoflas F.S. (Fig. 3(a)).
From a dilution of 1/8 and higher, the effect was proliferative rather than cytotoxic. The XTT test showed a significant increase in cell viability, indicative of an increase in cell number of about 60% compared to control-media cells (Fig. 3(a)). With P. gingivalis challenge, high Endoflas F.S. concentrations induced a more pronounced cytotoxic effect than without the bacterial challenge. Cell viability was reduced to 30% compared to the control-media group (Fig. 3(b)). For Endoflas F.S. at dilutions of 1/8 to 1/256, a mild cytotoxic effect was observed (Fig. 3(b)). Only the highest tested dilution of 1/ 512 increased cell viability compared to the control-media group, resulting in a proliferative effect of 20%.
4.
Discussion
To the best of our knowledge, this is the first study examining the effect of diluted media of iodoform-containing root canal filing material on macrophage and epithelial cell lines. Macrophages were chosen as cell targets because of their
archives of oral biology 58 (2013) 75–81
major role in the inflammatory process as they are vital for appropriate healing and also function in destructive inflammatory processes. Epithelial cells were selected because radicular cysts arise from the proliferation of epithelial remnants of Malassez in response to stimulation of chronic inflammatory processes in the apical region of the necrotic tooth.20 In the present study, both direct and indirect exposure of the RAW 264.7 macrophage and RKO epithelial cell lines to Endoflas F.S. or its highly concentrated media, diluted up to 1/ 8, resulted in cell death of about 70%. This supports the study of Brisen˜o and Willershausen in which Endoflas F.S. demonstrated significantly higher cytotoxicity to human gingival fibroblasts than did other calcium hydroxide-based root canal sealers.21 The present study’s results indicate that high concentrations of Endoflas F.S. and its conditioned media reduced the secretion of P. gingivalis-induced TNFa to almost undetectable levels. P. gingivalis is one of the anaerobic bacteria that exist in anaerobic oral niches including periapical sites and periodontal pockets. The activation of the host cells by such bacteria was represented in this study by the stimulation of the cell lines with heat-killed P. gingivalis. According to the results, the proliferative effect of Endoflas F.S.-conditioned media was less prominent with the heat-killed P. gingivalis challenge. This may be due to a toxic effect of bacterial particles on cell survival and proliferation, as suppression of mitochondrial activity, suggesting cytotoxicity, may preclude the ability of macrophages to secrete any cytokines. The effect of Endoflas F.S.-conditioned media on the RAW 264.7 macrophage and RKO epithelial cell lines was dose dependent. Highly concentrated media with dilutions of Endoflas F.S. up to 1/8 demonstrated a cytotoxic effect on both cell lines, while media with dilutions from 1/32 to 1/1024 induced cell proliferation. The observation of increased cell proliferation is based on the XTT results, an assay correlated to cell viability. XTT results could also be a reflection of the high mitochondrial activity as an outcome of cells’ stress condition. XTT significant increase in cell viability requires further investigation. Changes of mitochondrial activity by the Endoflas F.S. were probably not mediated by the material itself, but by degradation products released to the surrounding cell-culture medium. It is probable that these degradation products caused changes in both mitochondrial activity and cytokine secretion. This unexpected in vitro effect may be used to explore the clinical reports of cysts forming near deciduous teeth that have been treated with Endoflas F.S. In such teeth, exposed Endoflas F.S. may induce low-grade irritation. During primary tooth shedding and eruption of a successor permanent tooth, the bud of the latter is in close proximity to the resorbed roots of the primary tooth. Cells involved in root resorption, including multinucleated cells, may thus come in contact with media containing remnants of Endoflas F.S. The same occurs with epithelial cells derived from the dental sac of the erupting permanent tooth. In both cell lines, the possible proliferative effect of Endoflas F.S.conditioned media may promote enlargement of the dental sac of the permanent tooth bud.During periapical healing, macrophages secrete a number of inflammatory mediators,
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including cytokines, that perpetuate inflammation or direct healing.22 Cytokines such as interleukin 6 (IL6), ILb or TNFa mediate periapical inflammation, bone resorption23,24 and inhibition of bone repair that are features of the formation of periapical lesion.25,26 Cytokine production is also stimulated by bacterial lipopolysaccharides that are commonly present as part of the pulpal infection preceding the root canal treatment.27–29 Degradation of primary tooth root canal sealing materials discharges chemicals or particles that may alter the function of monocytes or macrophages attempting phagocytosis.30,31 Thus, the effect of endodontic sealers on monocytic function is an important consideration; aberrant, suppressed or unnecessary inflammatory responses delay periapical healing.32,33 The growth of radicular cysts is accompanied by local bone destruction, tissue osteolysis and accumulation of different cells in the cystic epithelium. During this dynamic inflammatory process, pro-inflammatory cytokines such as TNF-a are actively secreted and accumulated in the cystic fluid,34 and their concentrations are significantly higher in radicular cyst fluids.35,36 TNF-a can act as a pro-inflammatory cytokine and stimulate bone resorption.37 Takahashi et al. found that epithelial cells undergo proliferation in human dental periapical lesions.38 The present study demonstrated the effect of Endoflas F.S. components on both macrophages and epithelial cells. One of the study limitations and a major concern in material biocompatibility is the applicability of in vitro models of biological activity to clinical performance. While evaluating mitochondrial function, cytokine secretion and epithelial proliferation in response to Endoflas F.S. degradation products in a relatively static manner, the present model could not account for the root apex dynamic environment and circulatory removal of degradation products, or the interactions among cell types at the periapical region. The complexities of inflammatory responses cannot be adequately assessed by a single in vitro test and the full impact of this specific endodontic sealer on inflammation deserves further investigation.
4.1.
Clinical implication
The relationship between root canal treatment in primary molars and the appearance of cysts is not fully understood. Clinicians should be aware that chemical components of materials used in root canal treatments of primary teeth may cause long-term local irritation to follicles of permanent successors, thus stimulating formation of cyst-like radiolucent defects.12,39 Further research is needed to elucidate the specific mechanisms involved in this process and to identify the responsible agents.
5.
Conclusions
Direct and indirect exposure to high concentrations of iodoform-containing root canal filling material showed a cytotoxic effect on macrophages and epithelial cells, while low concentrations induced cell proliferation.
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Funding Department funds.
Competing interests The authors deny any conflicts of interest.
Ethical approval Not applicable.
references
1. American Academy of Pediatric Dentistry: Guideline on pulp therapy for primary and young permanent teeth, Adopted 1991 Revised 1998, 2001, 2004, 2009 Pediatric dentistry http:// www.aapd.org/media/Policies_Guidelines/G_Pulp.pdf; 2009. 2. Holan G, Fuks AB. A comparison of pulpectomies using ZOE and KRI in primary molars: a retrospective study. Pediatric Dentistry 1993;15:403–7. 3. Moskovitz M, Sammara E, Holan G. Success rate of root canal treatment in primary molars. Journal of Dentistry 2005;33:41–7. 4. Dummett Jr CO, Kopel HM. Pediatric endodontics. In: Ingle J, editor. Endodontics. Philadelphia: Lea and Febiger; 2002. p. 861–902. 5. Erausquin J, Muruzabal M. Root canal fillings with zinc oxide eugenol cement in the rat molar. Oral Surgery Oral Medicine and Oral Pathology 1967;24:547–58. 6. Garcia-Godoy F. Evaluation of an iodoform paste in root canal therapy for infected primary teeth. ASDC Journal of Dentistry for Children 1987;54:30–4. 7. Mass E, Zilberman LU. Endodontic treatment of infected primary teeth, using Maisto’s paste. ASDC Journal of Dentistry for Children 1989;56:117–20. 8. Nurko C, Gracia-Godoy F. Evaluation of a calcium hydroxide/ iodoform paste (Vitapex) in root canal therapy for primary teeth. The Journal of Clinical Pediatric Dentistry 1999;23:289–94. 9. Fuks AB, Eidelman E, Pauker N. Root fillings with Endoflas in primary teeth: a retrospective study. Journal of Clinical Pediatric Dentistry 2002;27:41–6. 10. Rifkin A. A simple effective, safe technique for the root canal treatment of abscessed primary teeth. ASDC Journal of Dentistry for Children 1980;47:435–41. 11. Rifkin A. The root canal treatment of abscessed primary teeth – a three to four year follow-up. ASDC Journal of Dentistry for Children 1982;49:428–31. 12. Moskovitz M, Yahav D, Tickotsky N, et al. Long-term follow up of root canal treated primary molars. International Journal of Paediatric Dentistry 2010;20:207–13. 13. Savage NW, Adkins KS, Weir AV, et al. A histological study of cystic lesion following pulp therapy in deciduous molars. Journal of Oral Pathology 1986;15:209–12. 14. Bhat SS, Vidhya M, Sargod S. Radicular cyst associated with endodontically treated teeth. Journal of Indian Society of Pedodontic and Preventive Dentistry 2003;21:139–41. 15. Takiguchi M, Fujiwara T, Sobue S, et al. Radicular cyst associated with a primary molar following pulp therapy: a case report. International Journal of Paediatric Dentistry 2001;11:452–5.
16. Mass E, Kaplan I, Hirshberg A. A clinical and histopathological study of radicular cysts associated with primary molars. Journal of Oral Pathology and Medicine 1995;24:458–61. 17. Hill FJ. Cystic lesions associated with deciduous teeth. Proceedings of the British Paedodontic Society 1978;8:9–12. 18. Beyth N, Houri-Haddad Y, Baraness-Hadar L, et al. Surface antimicrobial activity and biocompatibility of incorporated polyethylenimine nanoparticles. Biomaterials 2008;29: 4157–63. 19. Polak D, Wilensky A, Shapira L, et al. Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response. Journal of Clinical Periodontology 2009;36: 406–10. 20. Regezi JA, Sciubba JJ, Jordan RCK. Cysts of the Jaws and Neck. 4th ed. USA: Saunders; 2003. 21. Brisen˜o BM, Willershausen B. Root canal sealer cytotoxicity with human gingival fibroblasts: III. Calcium hydroxidebased sealers. Journal of Endodontics 1992;18:110–3. 22. Sjogren U, Ohlin A, Sundqvist G, Lerner UH. Guttapercha stimulated mouse macrophages release factors that activate the bone resorptive system of mouse calvarial bone. European Journal of Oral Sciences 1998;106:872–81. 23. Metzger Z, Nitzan D, Pitaru S, Brosh T, Teicher S. The effect of bacterial endotoxin on the early tensile strength of healing surgical wounds. Journal of Endodontics 2002;28: 30–3. 24. Mosser DM. The many faces of macrophage activation. Journal of Leukocyte Biology 2003;73:209–12. 25. 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. Archives of Medical Research 2002;33:482–4. 26. Zehnder M, Delaleu N, Du Y, Bickel M. Cytokine gene expression – part of host defence in pulpitis. Cytokine 2003;22:84–8. 27. 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. Journal of Oral Pathology and Medicine 2004;33:162–9. 28. Nair SP, Meghji S, Wilson M, Reddi K, White P, Henderson B. Bacterially induced bone destruction: mechanisms and misconceptions. Infection and Immunity 1996;64:2371–80. 29. Stashenko P. Role of immune cytokines in the pathogenesis of periapical lesions. Endodontics and Dental Traumatology 1990;6:89–96. 30. Nair PN, Sjogren U, Schumacher E, Sundqvist G. Radicular cyst affecting a root-filled human tooth: a long-term posttreatment follow-up. International Endodontic Journal 1993;26:225–33. 31. Nair PN, Sjogren U, Krey G, Sundqvist G. Therapyresistant foreign body giant cell granuloma at the periapex of a root-filled human tooth. Journal of Endodontics 1990;16:589–95. 32. Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dental Materials 1996;12: 186–93. 33. Bernath M, Szabo J. Tissue reaction initiated by different sealers. International Endodontic Journal 2003;36:256–61. 34. Jurisic V, Colic S, Jurisic M. The inflammatory radicular cysts have higher concentration of TNFa in comparison to odontogenic keratocysts (odontogenic tumour). Acta Medica (Hradec Kralove) 2007;50:233–8. 35. Colic´ S, Jurisic´ M, Jurisic´ V. Pathophysiological mechanism of the developing radicular cyst of the jaw. Article in Serbian. Acta chirurgica Iugoslavica 2008;55:87–92.
archives of oral biology 58 (2013) 75–81
36. Muglali M, Komerik N, Bulut E, Yarim GF, Celebi N, Sumer M. Cytokine and chemokine levels in radicular and residual cyst fluids. Journal of Oral Pathology and Medicine 2008;37: 185–9. 37. Bando Y, Henderson B, Meghji S, Poole S, Harris M. Immunocytochemical localization of inflammatory cytokines and vascular adhesion receptors in radicular cysts. Journal of Oral Pathology and Medicine 1993;22:221–7.
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38. Takahashi K, MacDonald D, Murayama Y, Kinane D. Cell synthesis, proliferation and apoptosis in human dental periapical lesions analyzed by in situ hybridization and immunohistochemistry. Oral Diseases 1999;5:313–20. 39. Grundy GE, Adkins KF, Savage NW. Cysts associated with deciduous molars following pulp therapy. Australian Dental Journal 1984;29:249–56.