- Email: [email protected]
Effects of Enamel Matrix Derivative on the Viability, Cytokine Secretion, and Phagocytic Activity of Human Monocytes
Basic Research—Biology
Effects of Enamel Matrix Derivative on the Viability, Cytokine Secretion, and Phagocytic Activity of Human Monocytes Sedigheh Khedmat, DDS, MSc,* Jamshid Hadjati, PhD,† Azita Iravani, DDS,* and Maryam Nourizadeh, PhD† Abstract Introduction: There is some controversy about the effect of enamel matrix derivative (EMD) on inflammation and resorption. The aim of this study was to investigate the effect of EMD on the inflammatory response of monocytes and their phagocytic activity in vitro. Methods: Human monocytes were incubated in complete medium (CM) and exposed to 50, 100, and 200 mg/mL EMD for different time points (12, 24, 48, and72 hours). Untreated monocytes were considered as controls. Cellular viability was evaluated through a 3-(4, 5 dimethylthiazol-2-yl) 2, 5-diphenyl-2 tetrazolium bromide assay. For cytokine measurements, the cells were treated simultaneously with 50, 100, or 200 mg/mL EMD and 10 mg/mL Escherichia coli lipopolysaccharide. Cell-free supernatants were collected after 12, 24, 48, and 72 hours of incubation. Tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) concentrations were measured by an enzyme-linked immunosorbent assay kit. Phagocytic activity of the cells was assayed using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Results: The viability of cells exposed to 50, 100, and 200 mg/mL EMD for 12, 24, 48, and 72 hours were similar to the controls. There was no significant differences in the production of TNF-a and IL-1b among samples with various concentrations (50, 100, and 200 mg/mL) of EMD and control (EMD = 0) at 12, 24, 48, and 72 hours. Phagocytic activity of monocytic cells increased significantly after 72 hours compared with 12 hours. Conclusions: Based on the results of this study, EMD does not promote releasing of the two studied proinflammatory and resorbing cytokines, TNF-a and IL-1b. By increasing the phagocytic activity of monocytic cells, EMD might accelerate wound healing. (J Endod 2010;36:1000–1003)
E
Key Words
Isolation of Monocytes Peripheral blood mononuclear cells were separated from fresh buffy coats (Tehran Blood Transfusion Center, Tehran, Iran) containing human white blood cells. The
Emdogain, inflammation, interleukin-1b, phagocytic activity
namel matrix derivative (EMD) contains amelogenin and other enamel matrix proteins. Several researches have confirmed the efficiency of EMD in promoting the formation of acellular cementum as well as strengthening the periodontal ligament and accelerating tissue regeneration (1–4). Moreover, EMD has been suggested as a pulp tissue regenerative agent in vital pulp therapy (5, 6). In the Sabbarini et al study (5), EMD, as a pulpotomy agent, showed better radiographic results compared with formocresol. Min et al (6) suggested that EMD as a pulp capping agent may facilitate the formation of hard tissue on the exposed pulp. In some clinical studies, the application of EMD on the root surfaces and into the socket of traumatized teeth has eliminated inflammatory root resorption and prevented ankylosis of these teeth (3, 4). In the study by Sato et al (7), LPS-stimulated monocytes exposed to EMD exhibited a significant decrease in proinflammatory tumor necrosis factor-a (TNF-a) production. Also, prostaglandin E-2– mediated inhibition of TNF-a production was enhanced in the presence of EMD. In contrast, St George et al (8) showed that external inflammatory root resorption occurred after the application of EMD into infrabony defects. On the other hand, two clinical studies showed that EMD had no significant effect on systemic secretion of interleukin-1b (IL-1b) (9, 10). TNF-a and IL-1b are two important proinflammatory cytokines mainly produced by monocytes/macrophages. These cytokines are strong stimulators of osteoclastic or odontoclastic activity in the resorption process (11). Considering the controversy about the effect of EMD on inflammation and resorption, these two cytokines were chosen to be studied. The aim of this study was to investigate the viability of human monocytes exposed to different concentrations of EMD, its influence on the release of two monocytic inflammatory cytokines (TNF-a and IL-1 b), and to assay the phagocytic function of monocytic cells exposed to EMD at various time points.
Materials and Methods Preparation of Emdogain Emdogain gel (Straumann, Basel, Switzerland) was dissolved in 0.1% acetic acid (pH = 5.7) to provide a soluble form of EMD (30 mg/mL). Then, it was diluted to working strength dilutions (50, 100, and 200 mg/mL) in complete medium (CM) containing RPMI1640 (Gibco; Life Technologies, Carlsbad, CA) supplemented with 2 mmol/L L-glutamine, 100 mg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (Gibco).
From the *Department of Endodontics and Dental Research Center, Faculty of Dentistry and †Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Supported by Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant no. 132/6570) Address requests for reprints to Dr Sedigheh Khedmat, Department of Endodontics, Faculty of Dentistry, Tehran University of Medical Sciences, Ghods Avenue, Enghelab Street, Tehran, Iran. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.02.032
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Khedmat et al.
JOE — Volume 36, Number 6, June 2010
Basic Research—Biology buffy coats were obtained from the healthy volunteers without any known viral or chronic infectious diseases. Briefly, blood samples was layered over Ficoll Histoprep (BAG Healthcare, Lich, Germany) 1.077 and centrifuged at 3,000 rpm for 20 minutes at 22 C .The mononuclear cell interface was removed and washed twice with prewarmed RPMI to omit ficoll and platelets. The remaining peripheral blood mononuclear cells were cultured into 96-well microplates (106 cells/well) in CM for 2 hours. Then, microplates were washed three times to remove nonadherent mononuclear cells such as T and B lymphocytes. The monocytes were isolated by their adherent capacity. Therefore, the experiment was performed on monocytes, which adhered to the microplates.
Viability Assay The viability of monocytes was assayed using 3-(4, 5 dimethylthiazol-2-yl) 2, 5-diphenyl-2 tetrazolium bromide (MTT) (Sigma, St. Louis, MO) (5 mg/mL) according to the manufacturer’s instructions. One hundred thousand monocytes were seeded in 96-well microplates containing 200 mL CM and exposed to 50, 100, and 200 mg/mL EMD. Some wells without EMD treatment were considered as controls. The experiment was performed in triplicate, and cells were incubated at 37 C in 5% CO2/95% O2 for 12, 24, 48, and 72 hours. Four hours before completing the incubation time, 20 mL MTT was added to the each well. The microplates were centrifuged at 2,000 rpm for 5 minutes, and then the upper medium was discarded. This assay is based on the cellular conversion of MTT into a blue formazan product that can be read by placing 96-microplates into spectrophotometer. Crystalline formazan deposits were dissolved 1 hour after the addition of 100 mL acidic alcohol isopropanol to each well. Optical density at 570 nm absorbance is directly related to the number of viable cells in culture.
Quenching solution was added to the samples in order to suppress the fluorescence of adhering bacteria not being phagocytosed. All tubes were vortexed immediately after the addition of the quenching solution and washed twice with washing solution. Each tube received 2 mL lysing solution afterwards and was vortexed and incubated for 20 minutes at room temperature. The samples were centrifuged, and the supernatant was discarded .After the final washing, 200 mL DNA staining solution was added to each tube. The tubes were vortexed and incubated for 10 minutes in iced water. The samples were analyzed within 30 minutes by a flowcytometer (Partec).
Statistical Evaluation The results were analyzed using two-way analysis of variance, Tukey HSD, and Tamhane post hoc tests. A p value of <0.05 was considered significant.
Results Viability Analysis The viability of cells exposed to 50, 100, and 200 mg/mL EMD for 12, 24, and 72 hours were similar to the controls. A partial nonsignificant increase in cell viability was observed after a 72-hour incubation time with 200 mg/mL concentration of EMD (Fig. 1A).
TNF-a and IL-1b Detection Assay Human monocytes were cultured in 96-well microplates at a density of 105 cell/well in triplicate. Then, cells were treated simultaneously with 50, 100, or 200 mg/mL EMD and 10 mg/mL Escherichia coli LPS. Cultures without LPS did not produce sufficient TNF-a or IL1b to be detected by enzyme-linked immunosorbent assay (ELISA). Therefore, monocytes treated with 10 mg/mL LPS without EMD were considered as the control. Cell-free supernatants were collected for cytokine measurement after 12, 24, 48, and 72 hours of incubation. TNF-a and IL-1b concentrations were measured by an ELISA kit (U-Cytech Biosciences, Utrecht, The Netherlands) according to the manufacturer’s recommendations. The detection limit of these two cytokines in the ELISA system used in this study was 5 pg/mL. THP-1 Phagocytosis Assay In order to get the appropriate amounts of cells, human acute monocytic leukemia cell line (THP-1 cells) was cultured for 10 days. Then cells were incubated in 6-well plates containing CM for 6, 12, and 72 hours. Three wells were considered for EMD treatment and the remaining wells as controls. Phagocytic activity of the cells was assayed using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, 5 105 cells were used per test. Then, 20 mL precooled FITC-labeled E. coli bacteria was added to the cells in 5-mL flowcytometery tubes. All tubes were vortexed and incubated for 2 hours at 37 C. The control tubes were remained on ice. Phagocytosis was then terminated by removing all samples simultaneously and placing them in iced water. JOE — Volume 36, Number 6, June 2010
Figure 1. The effect of different concentrations of EMD (0, 50, 100, and 200 mg/mL) on (A) viability and the release of (B) TNF-a and (C) IL-1b of human monocytes.
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Basic Research—Biology Effect of EMD on TNF-a and IL-1b Production by Monocytes TNF-a and IL-1b production from the samples treated with EMD (no LPS) and control (EMD = 0) were below the limit of ELISA detection. After the stimulation of monocytes with 10 mg/mL LPS, TNF-a and IL-1b production was detectable. TNF-a and IL-1b production among samples with different concentrations (50,100, and 200 mg/mL) of EMD and control after 12, 24, 48, and 72 hours of incubation were not significantly different (Fig. 1B and C). Effect of EMD on Phagocytosis of THP 1 Cells By flowcytometric analysis, it was shown that the phagocytic activity of THP-1 cells in 72-hour cultures in the presence of EMD was higher than 12-hour cultures (p = 0.045) (Fig. 2).
Discussion The aim of this study was a further investigation about the effect of EMD on the inflammatory response of monocytes. Based on our knowledge, this is the first study about the phagocytic activity of human monocytic cells in the presence of EMD. The MTT assay was used in order to test the safe concentrations of EMD for monocytic viability, and 50, 100, and 200 ug/mL concentrations were revealed to be nontoxic. According to the results of this study, the viability of monocytes in the presence of 50, 100, and 200 mg/mL EMD were not different. In the Davenport et al study (12), the viability of periodontal ligament fibroblasts decreased with increasing concentrations (25, 50, 75, and100 mg/mL) of EMD. The largest reduction in the viability of the fibroblasts was detected at 100 mg/mL. The difference between the Davenport study and ours might be related to the different types cells examined. It has been shown that the response of mesenchymal cells to EMD is dependent on the origin of the cells and the stage of maturation (13–15). In the Pischon et al study (14), although 100 mg/mL of EMD had no effect on osteoblasts, it increased DNA synthesis of PDL cells significantly. TNF-a is a proinflammatory cytokine that is mainly produced in response to gram-negative bacteria. The major cellular source of this cytokine is mononuclear phagocytes (16). TNF-a can stimulate fibroblast apoptosis, increase vascular permeability, and induce the production of other cytokines and chemokines (17). TNF-a also stimulates the differentiation and activation of osteoclasts (18). In the present study, EMD and LPS were added to the cultures at the same time, and they had no significant effect on TNF-a production from monocytes in all of the tested time points. This finding was in
Figure 2. The percentage of phagocytic cells in the presence (200 mg/mL) and absence (0 mg/mL) of EMD. *Statistically significant compared with 12 hours (p < 0.05).
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agreement with Myhre et al (19), who showed that EMD administration together with LPS to whole-blood cell cultures does not significantly decrease TNF-a production. Interestingly, in the study of Myhre et al, when cells were exposed to EMD before stimulation with LPS, they produced significantly less TNF-a in comparison to simultaneous administration. This in vitro study showed that EMD had no significant effect on IL-1b production. This result was consistent with two clinical studies that showed that EMD had no significant effect on the systemic secretion of IL-1b (9, 10). This cytokine is the most active cytokine that has the capacity to activate osteoclastic bone resorption, and its bone resorbing capacity is 1,000-fold that of TNF-a (20). TNF-a and IL-1b are two proinflammatory cytokines that are involved in the dissolution of hard tissues by the stimulation and proliferation of clastic cells (21). These cytokines are also strong stimulators of osteoclastic or odontoclastic activity in the resorption process. In the current study, EMD did not promote TNF-a and IL-1b secretion, so it does not appear to have any negative effect on inflammation and resorption. This study showed that EMD significantly increases phagocytic activity of human monocytes after 72 hours compared with 12 hours. This finding indicates that EMD might accelerate wound healing through clearing of the inflammation site from the foreign bodies. However, this study was a preliminary investigation about the effect of EMD on phagocytosis, and more investigations are needed to confirm this concept. Furthermore, it would be interesting to assess phagocytic activity in time points between 12 and 72 hours in future studies.
Conclusions According to the results of this study, EMD did not promote TNF-a and IL-1b secretion; therefore, it does not appear to have any negative effect on inflammation and resorption. By increasing the phagocytic activity of monocytic cells, EMD might also accelerate wound healing through clearing of the inflammation site from the foreign bodies.
Acknowledgment The authors would like to thank Dr. Katayoon Bidad for reviewing this article.
References 1. Slavkin HC, Bringasp Jr, Bessem C, et al. Hertwig’s epithelial root sheath differentiation and initial cementum and bone formation during long-term organ culture of mouse mandibular first molars using serumless, chemically-defined medium. J Periodontal Res 1989;24:28–40. 2. Hammarstrom L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997;24:658–68. 3. Filippi A, Pohl Y, Von Arx T. Treatment of replacement resorption with Emdogainpreliminary results after 10 months. Dent Traumatol 2001;17:134–8. 4. Iqbal MK, Bamaas N. Effect of enamel matrix derivative (EMDOGAIN) upon periodontal healing after replantation of permanent incisors in beagle dogs. Dent Traumatol 2001;17:36–45. 5. Sabbarini J, Mohamed A, Wahba N, et al. Comparison of enamel matrix derivative versus formocresol as pulpotomy agents in the primary dentition. J Endod 2008; 34:284–7. 6. Min KS, Yang SH, Kim EC. The combined effect of mineral trioxide aggregate and enamel matrix derivative on odontoblastic differentiation in human dental pulp cells. J Endod 2009;35:847–50. 7. Sato S, Kitagawa M, Sakamoto K, et al. Enamel matrix derivative exhibits antiinflammatory properties in monocytes. J Periodontol 2008;79:535–40. 8. St Geroge G, Darbar U, Thomas G. Inflammatory external root resorption following surgical treatment for intra-bony defects: a report of two cases involving Emdogain and a review of the literature. J Clin Periodontol 2006;33:449–54.
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Basic Research—Biology 9. Giannopoulou C, Andersen E, Brochut P, et al. Enamel matrix derivative and systemic antibiotics as adjuncts to non surgical periodontal treatment :biologic response. J Periodontol 2006;77:707–13. 10. Gundersen RY, Ruud TE, Jorgensen PF, et al. Systemic administration of enamel matrix derivative to lipopolysacchride challenged pigs;effects on the inflammatory response. Surg Infect (Larchmt) 2008;9:161–9. 11. Stashenko P, Teles R, D’Souza R. Periapical inflammatory responses and their modulation. Crit Rev Oral Biol Med1998;9:498–521. 12. Davenport DR, Mailhot JM, Wataha JC, et al. Effects of enamel matrix protein application of the viability, proliferation and attachment of human periodontal ligament fibroblasts to diseased root surfaces in vitro. J Clin Periodontol 2003;30:125–31. 13. Schwartz Z, Carnes DL Jr, Pulliam R, et al. porcine fetal enamel matrix dervative stimulates proliferation but not differentiation of osteoblast- like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOST cells. J Periodontol 2000;71:1287–96. 14. Pischon N, Zimmermann B, Bernimoulin JP, et al. Effects of an enamel matrix derivative on human osteoblasts and PDL cells grown in organoid cultures. OralSurg Oral Med Oral PatholOral Radiol Endod 2006;102:551–7.
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15. Dean DD, Lohmann CH, Sylvia VL, et al. Effect of porcine fetal enamel matrix derivative on chondrocyte proliferation, differentiation, and local factor production is dependent on cell maturation state. Cells Tissues Organs 2002;171:117–27. 16. Ingle JI, Bakland LK, Baumgartner JC. Endodontics. 6th ed. Hamilton: BC Decker Inc.; 2008:356–358. 17. Graves DT, Oskoui M, Volenjikova S, et al. Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment and osteoclast formation in response to P. gingivalis infection. J Dent Res 2001;80:1875–9. 18. Azuma Y, Kaji K, Katogi R, et al. Tumor necrosis factor-alpha inducse differentiation of and bone resorption by osteoclasts. J Biol Chem 2000;275:4858–64. 19. Myhre AE, Lyngstadaas SP, Dahle MK, et al. Anti inflammatory properties of enamel matrix derivative in human blood. J Periodont Res 2006;41:208–13. 20. Stashenko P, Dewhirst FE, Peros WJ, et al. Synergistic interactions between interleukin 1, tumor necrosis factor, and lymphotoxin in bone resorption. J Immunol 1987;138:1464–8. 21. Okamura T, Shimokawa H, Takagi Y, et al. Detection of collagenase mRNA in odontoclasts of bovine root resorbing tissue by in situ hybridization. Calcif Tissue Int 1993;52:325–30.
Effects of Enamel Matrix Derivative on Human Monocytes
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Effects of Enamel Matrix Derivative on the Viability, Cytokine Secretion, and Phagocytic Activity of Human Monocytes Sedigheh Khedmat, DDS, MSc,* Jamshid Hadjati, PhD,† Azita Iravani, DDS,* and Maryam Nourizadeh, PhD† Abstract Introduction: There is some controversy about the effect of enamel matrix derivative (EMD) on inflammation and resorption. The aim of this study was to investigate the effect of EMD on the inflammatory response of monocytes and their phagocytic activity in vitro. Methods: Human monocytes were incubated in complete medium (CM) and exposed to 50, 100, and 200 mg/mL EMD for different time points (12, 24, 48, and72 hours). Untreated monocytes were considered as controls. Cellular viability was evaluated through a 3-(4, 5 dimethylthiazol-2-yl) 2, 5-diphenyl-2 tetrazolium bromide assay. For cytokine measurements, the cells were treated simultaneously with 50, 100, or 200 mg/mL EMD and 10 mg/mL Escherichia coli lipopolysaccharide. Cell-free supernatants were collected after 12, 24, 48, and 72 hours of incubation. Tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) concentrations were measured by an enzyme-linked immunosorbent assay kit. Phagocytic activity of the cells was assayed using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Results: The viability of cells exposed to 50, 100, and 200 mg/mL EMD for 12, 24, 48, and 72 hours were similar to the controls. There was no significant differences in the production of TNF-a and IL-1b among samples with various concentrations (50, 100, and 200 mg/mL) of EMD and control (EMD = 0) at 12, 24, 48, and 72 hours. Phagocytic activity of monocytic cells increased significantly after 72 hours compared with 12 hours. Conclusions: Based on the results of this study, EMD does not promote releasing of the two studied proinflammatory and resorbing cytokines, TNF-a and IL-1b. By increasing the phagocytic activity of monocytic cells, EMD might accelerate wound healing. (J Endod 2010;36:1000–1003)
E
Key Words
Isolation of Monocytes Peripheral blood mononuclear cells were separated from fresh buffy coats (Tehran Blood Transfusion Center, Tehran, Iran) containing human white blood cells. The
Emdogain, inflammation, interleukin-1b, phagocytic activity
namel matrix derivative (EMD) contains amelogenin and other enamel matrix proteins. Several researches have confirmed the efficiency of EMD in promoting the formation of acellular cementum as well as strengthening the periodontal ligament and accelerating tissue regeneration (1–4). Moreover, EMD has been suggested as a pulp tissue regenerative agent in vital pulp therapy (5, 6). In the Sabbarini et al study (5), EMD, as a pulpotomy agent, showed better radiographic results compared with formocresol. Min et al (6) suggested that EMD as a pulp capping agent may facilitate the formation of hard tissue on the exposed pulp. In some clinical studies, the application of EMD on the root surfaces and into the socket of traumatized teeth has eliminated inflammatory root resorption and prevented ankylosis of these teeth (3, 4). In the study by Sato et al (7), LPS-stimulated monocytes exposed to EMD exhibited a significant decrease in proinflammatory tumor necrosis factor-a (TNF-a) production. Also, prostaglandin E-2– mediated inhibition of TNF-a production was enhanced in the presence of EMD. In contrast, St George et al (8) showed that external inflammatory root resorption occurred after the application of EMD into infrabony defects. On the other hand, two clinical studies showed that EMD had no significant effect on systemic secretion of interleukin-1b (IL-1b) (9, 10). TNF-a and IL-1b are two important proinflammatory cytokines mainly produced by monocytes/macrophages. These cytokines are strong stimulators of osteoclastic or odontoclastic activity in the resorption process (11). Considering the controversy about the effect of EMD on inflammation and resorption, these two cytokines were chosen to be studied. The aim of this study was to investigate the viability of human monocytes exposed to different concentrations of EMD, its influence on the release of two monocytic inflammatory cytokines (TNF-a and IL-1 b), and to assay the phagocytic function of monocytic cells exposed to EMD at various time points.
Materials and Methods Preparation of Emdogain Emdogain gel (Straumann, Basel, Switzerland) was dissolved in 0.1% acetic acid (pH = 5.7) to provide a soluble form of EMD (30 mg/mL). Then, it was diluted to working strength dilutions (50, 100, and 200 mg/mL) in complete medium (CM) containing RPMI1640 (Gibco; Life Technologies, Carlsbad, CA) supplemented with 2 mmol/L L-glutamine, 100 mg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum (Gibco).
From the *Department of Endodontics and Dental Research Center, Faculty of Dentistry and †Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Supported by Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant no. 132/6570) Address requests for reprints to Dr Sedigheh Khedmat, Department of Endodontics, Faculty of Dentistry, Tehran University of Medical Sciences, Ghods Avenue, Enghelab Street, Tehran, Iran. E-mail address: [email protected]. 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.02.032
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Khedmat et al.
JOE — Volume 36, Number 6, June 2010
Basic Research—Biology buffy coats were obtained from the healthy volunteers without any known viral or chronic infectious diseases. Briefly, blood samples was layered over Ficoll Histoprep (BAG Healthcare, Lich, Germany) 1.077 and centrifuged at 3,000 rpm for 20 minutes at 22 C .The mononuclear cell interface was removed and washed twice with prewarmed RPMI to omit ficoll and platelets. The remaining peripheral blood mononuclear cells were cultured into 96-well microplates (106 cells/well) in CM for 2 hours. Then, microplates were washed three times to remove nonadherent mononuclear cells such as T and B lymphocytes. The monocytes were isolated by their adherent capacity. Therefore, the experiment was performed on monocytes, which adhered to the microplates.
Viability Assay The viability of monocytes was assayed using 3-(4, 5 dimethylthiazol-2-yl) 2, 5-diphenyl-2 tetrazolium bromide (MTT) (Sigma, St. Louis, MO) (5 mg/mL) according to the manufacturer’s instructions. One hundred thousand monocytes were seeded in 96-well microplates containing 200 mL CM and exposed to 50, 100, and 200 mg/mL EMD. Some wells without EMD treatment were considered as controls. The experiment was performed in triplicate, and cells were incubated at 37 C in 5% CO2/95% O2 for 12, 24, 48, and 72 hours. Four hours before completing the incubation time, 20 mL MTT was added to the each well. The microplates were centrifuged at 2,000 rpm for 5 minutes, and then the upper medium was discarded. This assay is based on the cellular conversion of MTT into a blue formazan product that can be read by placing 96-microplates into spectrophotometer. Crystalline formazan deposits were dissolved 1 hour after the addition of 100 mL acidic alcohol isopropanol to each well. Optical density at 570 nm absorbance is directly related to the number of viable cells in culture.
Quenching solution was added to the samples in order to suppress the fluorescence of adhering bacteria not being phagocytosed. All tubes were vortexed immediately after the addition of the quenching solution and washed twice with washing solution. Each tube received 2 mL lysing solution afterwards and was vortexed and incubated for 20 minutes at room temperature. The samples were centrifuged, and the supernatant was discarded .After the final washing, 200 mL DNA staining solution was added to each tube. The tubes were vortexed and incubated for 10 minutes in iced water. The samples were analyzed within 30 minutes by a flowcytometer (Partec).
Statistical Evaluation The results were analyzed using two-way analysis of variance, Tukey HSD, and Tamhane post hoc tests. A p value of <0.05 was considered significant.
Results Viability Analysis The viability of cells exposed to 50, 100, and 200 mg/mL EMD for 12, 24, and 72 hours were similar to the controls. A partial nonsignificant increase in cell viability was observed after a 72-hour incubation time with 200 mg/mL concentration of EMD (Fig. 1A).
TNF-a and IL-1b Detection Assay Human monocytes were cultured in 96-well microplates at a density of 105 cell/well in triplicate. Then, cells were treated simultaneously with 50, 100, or 200 mg/mL EMD and 10 mg/mL Escherichia coli LPS. Cultures without LPS did not produce sufficient TNF-a or IL1b to be detected by enzyme-linked immunosorbent assay (ELISA). Therefore, monocytes treated with 10 mg/mL LPS without EMD were considered as the control. Cell-free supernatants were collected for cytokine measurement after 12, 24, 48, and 72 hours of incubation. TNF-a and IL-1b concentrations were measured by an ELISA kit (U-Cytech Biosciences, Utrecht, The Netherlands) according to the manufacturer’s recommendations. The detection limit of these two cytokines in the ELISA system used in this study was 5 pg/mL. THP-1 Phagocytosis Assay In order to get the appropriate amounts of cells, human acute monocytic leukemia cell line (THP-1 cells) was cultured for 10 days. Then cells were incubated in 6-well plates containing CM for 6, 12, and 72 hours. Three wells were considered for EMD treatment and the remaining wells as controls. Phagocytic activity of the cells was assayed using the PHAGOTEST kit (Glycotope Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Briefly, 5 105 cells were used per test. Then, 20 mL precooled FITC-labeled E. coli bacteria was added to the cells in 5-mL flowcytometery tubes. All tubes were vortexed and incubated for 2 hours at 37 C. The control tubes were remained on ice. Phagocytosis was then terminated by removing all samples simultaneously and placing them in iced water. JOE — Volume 36, Number 6, June 2010
Figure 1. The effect of different concentrations of EMD (0, 50, 100, and 200 mg/mL) on (A) viability and the release of (B) TNF-a and (C) IL-1b of human monocytes.
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Basic Research—Biology Effect of EMD on TNF-a and IL-1b Production by Monocytes TNF-a and IL-1b production from the samples treated with EMD (no LPS) and control (EMD = 0) were below the limit of ELISA detection. After the stimulation of monocytes with 10 mg/mL LPS, TNF-a and IL-1b production was detectable. TNF-a and IL-1b production among samples with different concentrations (50,100, and 200 mg/mL) of EMD and control after 12, 24, 48, and 72 hours of incubation were not significantly different (Fig. 1B and C). Effect of EMD on Phagocytosis of THP 1 Cells By flowcytometric analysis, it was shown that the phagocytic activity of THP-1 cells in 72-hour cultures in the presence of EMD was higher than 12-hour cultures (p = 0.045) (Fig. 2).
Discussion The aim of this study was a further investigation about the effect of EMD on the inflammatory response of monocytes. Based on our knowledge, this is the first study about the phagocytic activity of human monocytic cells in the presence of EMD. The MTT assay was used in order to test the safe concentrations of EMD for monocytic viability, and 50, 100, and 200 ug/mL concentrations were revealed to be nontoxic. According to the results of this study, the viability of monocytes in the presence of 50, 100, and 200 mg/mL EMD were not different. In the Davenport et al study (12), the viability of periodontal ligament fibroblasts decreased with increasing concentrations (25, 50, 75, and100 mg/mL) of EMD. The largest reduction in the viability of the fibroblasts was detected at 100 mg/mL. The difference between the Davenport study and ours might be related to the different types cells examined. It has been shown that the response of mesenchymal cells to EMD is dependent on the origin of the cells and the stage of maturation (13–15). In the Pischon et al study (14), although 100 mg/mL of EMD had no effect on osteoblasts, it increased DNA synthesis of PDL cells significantly. TNF-a is a proinflammatory cytokine that is mainly produced in response to gram-negative bacteria. The major cellular source of this cytokine is mononuclear phagocytes (16). TNF-a can stimulate fibroblast apoptosis, increase vascular permeability, and induce the production of other cytokines and chemokines (17). TNF-a also stimulates the differentiation and activation of osteoclasts (18). In the present study, EMD and LPS were added to the cultures at the same time, and they had no significant effect on TNF-a production from monocytes in all of the tested time points. This finding was in
Figure 2. The percentage of phagocytic cells in the presence (200 mg/mL) and absence (0 mg/mL) of EMD. *Statistically significant compared with 12 hours (p < 0.05).
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agreement with Myhre et al (19), who showed that EMD administration together with LPS to whole-blood cell cultures does not significantly decrease TNF-a production. Interestingly, in the study of Myhre et al, when cells were exposed to EMD before stimulation with LPS, they produced significantly less TNF-a in comparison to simultaneous administration. This in vitro study showed that EMD had no significant effect on IL-1b production. This result was consistent with two clinical studies that showed that EMD had no significant effect on the systemic secretion of IL-1b (9, 10). This cytokine is the most active cytokine that has the capacity to activate osteoclastic bone resorption, and its bone resorbing capacity is 1,000-fold that of TNF-a (20). TNF-a and IL-1b are two proinflammatory cytokines that are involved in the dissolution of hard tissues by the stimulation and proliferation of clastic cells (21). These cytokines are also strong stimulators of osteoclastic or odontoclastic activity in the resorption process. In the current study, EMD did not promote TNF-a and IL-1b secretion, so it does not appear to have any negative effect on inflammation and resorption. This study showed that EMD significantly increases phagocytic activity of human monocytes after 72 hours compared with 12 hours. This finding indicates that EMD might accelerate wound healing through clearing of the inflammation site from the foreign bodies. However, this study was a preliminary investigation about the effect of EMD on phagocytosis, and more investigations are needed to confirm this concept. Furthermore, it would be interesting to assess phagocytic activity in time points between 12 and 72 hours in future studies.
Conclusions According to the results of this study, EMD did not promote TNF-a and IL-1b secretion; therefore, it does not appear to have any negative effect on inflammation and resorption. By increasing the phagocytic activity of monocytic cells, EMD might also accelerate wound healing through clearing of the inflammation site from the foreign bodies.
Acknowledgment The authors would like to thank Dr. Katayoon Bidad for reviewing this article.
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