Effect of ScLL and 15d-PGJ₂ on viability and cytokine release in LPS-stimulated fibroblasts: an in vitro study

Abstract

This study evaluated the effect of a cyclopentenone-type PG, 15-Deoxy-^Δ12,14-PG J2 (15d-PGJ₂), and lectin (ScLL) on the viability of human gingival fibroblasts (HGFs), and on IL-6 and TGFβ-1 release by these fibroblasts, stimulated with lipopolysaccharide (LPS). HGFs were stimulated with LPS 10 μg/ml and treated with 15d-PGJ₂ 1 and 2 μg/ml, and ScLL 2 and 5 μg/ml, for 1 and 3h, and then evaluated for viability by MTT assay. Supernatant was collected to detect IL-6 and TGFβ-1 release, by ELISA. Positive control was cells kept in Dulbecco’s Modified Eagle’s Medium, and negative control was those kept in LPS. Data were analyzed by ANOVA and Dunnett’s test (α = 0.05). No significant difference was found in viability among experimental groups at 1h (p > 0.05). Percentage of ScLL 5 µg/ml viable cells was similar to that of positive control at evaluated periods (p > 0.05), whereas the other groups had lower levels than the positive control (p < 0.05). IL-6 release was statistically higher for ScLL 5 μg/ml and 15d-PGJ₂ 2 µg/ml at 1h, compared with the other treated groups and positive control (p < 0.05). No significant differences were found among the groups at 3h (p > 0.05), except for ScLL 2 µg/ml and 15d-PGJ₂ 1 µg/ml, which showed lower IL-6 release compared with that of negative control (p < 0.05). No significant difference was found among the groups for TGFβ-1 release (p > 0.05). Results indicated that ScLL 5 μg/ml did not interfere in viability, and ScLL 2 µg/ml and 15d-PGJ₂ 1 µg/ml demonstrated reduced IL-6 release. Tested substances had no effect on TGFβ-1 release.

Cell Survival; Fibroblasts; Lectins

Introduction

The ideal procedure for avulsed teeth is immediate replantation, but this is not possible in most cases.¹ For this reason, the duration of the extra-alveolar dry time of the tooth, its storage medium, its replantation time and the survival of PDL cells are crucial factors determining the prognosis.² A more favorable prognosis after replantation must take these required factors into consideration. It is believed that storage media that can preserve PDL cell viability is more important than the extra-alveolar period.² However, under clinical conditions, it has been observed that patients do not know that the storage media is important, thus leading to delayed replantation.³ In this situation, dry storage of avulsed teeth causes death of PDL cells, and the presence of necrotic PDL remnants can trigger the development of root resorption.¹ The application of several therapies before replantation has been proposed to prevent or delay root resorption⁴ in cases of late replantation, and thus increase the chance of avulsed tooth survival, including 2% acidulated phosphate fluoride on the root surface. However, this substance does not have active principles that could modulate immune inflammatory reactions triggering root resorption.⁵ In this respect, a new and interesting challenge would be to investigate substances having the potential to modulate the inflammatory response, and act as a possible therapy to replace acid root treatments, in cases of late replanted teeth.

Some substances tested to treat the root surface before delayed tooth replantation have efficacy as osteoporotic therapeutic agents that inhibit osteoclast activity and modulate immune inflammatory reactions.⁶ , ⁷ , ⁸ These substances include Emdogain, alendronate, odanacatib⁶ and propolis.⁷ , ⁸ In recent years, compounds of natural origin, obtained from plants such as curcumin and piperine, have been tested to formulate anti-inflammatory and anti-osteoclastogenic drugs having the potential to treat and prevent replacement resorption of replanted teeth.⁹ In this respect, a recent study tested the effect of a plant lectin on stimulated gingival fibroblasts, with promising results¹⁰ for future use as a possible therapy for delayed replanted teeth. Lectins are carbohydrate-binding proteins involved in various biological process, including cell-cell recognition, proliferation, migration, adhesion and biochemical responses.¹¹ The lectin isolated from the Euphorbiaceae Synadenium carinatum latex (ScLL) plant exhibits immunoregulatory properties.¹² This ability of ScLL to modulate inflammatory activity has been demonstrated in models of chronic inflammation, such as leishmaniose,¹³ asthma¹² and neosporosis.¹⁴

Another component with immunomodulatory capability is 15-Deoxy-^12,14-PG J₂ (15d-PGJ₂), a cyclopentenone-type PG that has demonstrated promising results in different experimental models in vitro ¹⁵ and in vivo. ¹⁵ , ¹⁶ 15d-PGJ₂ displays a wide spectrum of physiological activities, and is one of the terminal products of the cyclooxygenase (COX)-2 pathway.¹⁶ It differs from other prostaglandins in several aspects, especially chemically and biologically, mainly due to its anti-tumor¹⁵ and anti-inflammatory effects.¹⁶ 15d-PGJ₂ was previously evaluated in a periodontitis mouse model, in which it exhibited immunomodulatory effects, by decreasing bone resorption and inflammatory responses.¹⁶ However, more in-depth studies are of interest to the dental field, especially to evaluate the effect of 15d-PGJ₂ on other cells of the oral cavity, and compare this agent with other potentially immunomodulating substances.

The purpose of this study was to investigate the effect of 15d-PGJ₂ and ScLL on gingival fibroblasts stimulated with Porphyromonas gingivalis lipopolysaccharides (LPS) regarding viability and IL-6 and TGFβ-1 release. The hypotheses were that a) cells stimulated with LPS and treated with ScLL or 15d-PGJ₂ would demonstrate cell viability and TGFβ-1 release results similar to those of the positive control group; and b) IL-6 levels would be lower in the treated groups than in the negative control group.

Methodology

Cell culture

Immortalized human gingival fibroblasts (HFGs) (Cell Bank of Rio de Janeiro, Rio de Janeiro, RJ, Brazil) were cultured in T-50 culture flasks containing Dulbecco’s Modified Eagle’s Medium (DMEM) (Vitrocell Embriolife, Campinas, Brazil) and supplemented with 10% fetal bovine serum (FBS) (Vitrocell Embriolife), in a humidified incubator with 5% CO₂ and 95% O₂ at 37^oC, until confluence. HFGs were trypsinized, and seeded in 96-well culture plates (Coastar Corp., Cambridge, USA) with 10% DMEM at a cell density of 2x10⁴ cells/well. After 24 h, the medium was gently removed from each well and replaced by P. gingivalis LPS 10 µg/ml (Invivogen, San Diego, USA) in 10% DMEM for 24 h. Then, the medium was removed and treated with ScLL (2 µg/ml, 5 µg/ml) (Herbarium of the Federal University of Uberlandia, UFU), and 15d-PGJ₂ (1 µg/ml, 2 µg/ml) (Sigma-Aldrich, St. Louis, USA) in 10% DMEM for 1 and 3 h. The positive control group was maintained in 10% DMEM (not LPS-stimulated), and the negative control group was stimulated with LPS. After the incubation period, the cells were prepared for cell viability analysis and cytokine assay.

Cell viability assay

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay. After removing the medium, the cells were rinsed with a phosphate-buffer solution (PBS), and an MTT solution (5 mg/ml) was added to the cells, which were incubated at 37^oC for 4 h to allow development of formazan crystals. The supernatant of each well was replaced with the same volume of dimethyl sulfoxide (Sigma-Aldrich) to dissolve the crystals. Optical density was measured at 570 nm using a microplate reader (Biochrom, Cambridge, UK, England). Cell viability evaluation was performed according to the absorbance level, and was expressed as a percentage of viable cells.

Cytokine assay

IL-6 and TGFβ-1 levels were analyzed after 24 h of cell exposure to ScLL and 15d-PGJ₂. Cytokine levels were measured in culture supernatants using a sandwich Elisa kit for IL-6 (PeproTech, Rocky Hill, USA) and TGFβ-1 (Sigma-Aldrich), according to the manufacturer’s instructions. Absorbance was recorded using a microplate reader (Biochrom), at 405 nm wavelength for IL-6, and 450 nm for TGFβ-1. The results obtained for absorbance were interpolated into a standard curve, using Microplate Manager 4.0 software (Bio-rad, Hercules, USA).

Statistical analysis

MTT, IL-6 and TGFβ-1 data were subjected to analysis of normality using Shapiro-Wilk and Levene’s tests. The MTT data were evaluated with log transformation followed by one-way analysis of variance (ANOVA) and Tukey’s test to compare the experimental groups (p < 0.05). Data for IL-6 and TGFβ-1 were analyzed using one-way ANOVA followed by Tukey’s test. Dunnett’s test was performed to compare the control with the experimental groups. The statistical analyses were performed using SigmaPlot 12.0 software (Systat Software, San Jose, USA), with statistical significance set at α = 0.05.

Results

Cell viability

The results for cell viability evaluated by MTT are shown in Figure 1 . No significant difference was found among the experimental groups at 1 h (p > 0.05). However, ScLL 5 µg/ml presented higher cell viability than 15d-PGJ₂ 1 µg/ml and 2 µg/ml, at 3 h (p < 0.001). The percentage of viable ScLL 5 µg/ml cells was similar to that of the positive control group (DMEM) in both periods evaluated (p > 0.05). All the other groups had lower levels of cell viability than the positive control, at 1 and 3 h (p < 0.05). The experimental groups were similar to the negative control (LPS) in both periods evaluated (p > 0.05), but not to the positive control (p < 0.05).

Figure 1
Effect of 15d-PGJ2 and ScLL on cell viability of HGFs at 1 h and 3 h by MTT assay. Statistical significance was determined using one-way ANOVA and Dunnett’s test. #p<0.05 compared among the experimental groups.

*p < 0.05 compared with positive control (DMEM). **p < 0.05 compared with negative control (LPS).

Release of IL-6 and TGFβ-1

The results for IL-6 and TGFβ-1 release are shown in Figures 2 and 3 , respectively. The IL-6 level was statistically higher for the ScLL 5 μg/ml-treated and the 15d-PGJ₂ 2 µg/ml-treated groups at 1 h, compared with the other treated groups and the positive control group (p < 0.05). Similar values were found for the negative control and the treated groups at 1 h, except for 15d-PGJ₂ 1 µg/ml, which showed lower IL-6 release (p = 0.04). No significant differences were found among the groups at 3 h (p > 0.05), except for ScLL 2 µg/ml and 15d-PGJ₂ 1 µg/ml, which showed lower IL-6 release than the negative control group (p < 0.05).

Figure 2
Release of IL-6 in HGFs examined by Elisa at 1 and 3 h. Statistical significance was determined using one-way ANOVA and Dunnett’s test. #p<0.05 compared among the experimental groups.

*p < 0.05 compared with positive control (DMEM). **p < 0.05 compared with negative control (LPS).

Figure 3
Release of TGFβ-1 in HGFs examined by Elisa at 1 and 3 h. Statistical significance was determined using one-way ANOVA and Dunnett’s test.

Regarding TGFβ-1 release, no significant difference was found among the groups in either evaluated period (p > 0.05).

Discussion

The first hypothesis was rejected, because cell viability analysis revealed lower levels of HFG viability in the treated groups than the positive control group, even though TGF-β release presented values similar to this group. The second hypothesis was accepted, because IL-6 release was lower in the treated groups than the negative control group. HFG is a immortalized cell line obtained from gingival tissue. It is important to note that the use of immortalized cell cultures performed in preliminary studies assessing drugs and medicines was recommended in the literature.⁹ , ¹⁰ Moreover, established cell lines have enhanced reproducibility of results, an unlimited life span, and a higher rate of cell multiplication than primary culture cells.¹⁷ There are many cell types in the oral environment, which may react differently to the test products, a possibility which should be carefully considered. Although the phenotypic and genotypic characteristics of gingival fibroblasts differ from those of periodontal fibroblasts,¹⁸ immortalized PDL cells are not available for this protocol. This is why the HGF lineage was selected for this preliminary study.

In the present study, the cells were stimulated with LPS in order to mimic situations that occur clinically. Studies have shown that gingival fibroblasts express Toll-like receptors and CD14 in response to LPS.¹⁷ It is known that LPS induces cell stress, associated with the activation of mitogen-activated protein kinase (MAPK), after an inflammatory response.¹⁹ MAPK mediates the intracellular signaling related to cellular activities, such as apoptosis, cell differentiation and cell survival.²⁰ Studies using fibroblasts activated with LPS in drug tests are recommended in this area.¹⁰

Considering that LPS could interfere in cell viability, the current research evaluated its presence by MTT assay. Tetrazolium salts have become widely used in cell biology assays, because of their ability to convert yellow MTT tetrazolium salt into dark blue formazan crystals, by the mitochondrial activity of living cells. Compared with other viability assays, the MTT assay is simple, fast, precise, sensitive and reproducible.²¹ In the current study, the highest viability levels were found for the positive control group. This result can be attributed to the absence of LPS, which may trigger the cell death mechanism.²² The fact that ScLL 5 μg/ml showed viability similar to the positive control group suggests that this ScLL concentration may act at any point of the apoptotic pathway, blocking the molecular events in the apoptotic process, thus maintaining viability levels similar to basal cell parameters. However, it should be emphasized that, in the present study, the LPS percentage remained above 80%, despite its reduced cell viability.

Analyses of IL-6 and TGFβ-1 release were performed based on the belief that LPS could induce fibroblasts to display some sort of pro-inflammatory reaction, and that treatment with ScLL and 15d-PGJ₂ could have a regulatory effect on this parameter. IL-6 is produced by human fibroblasts when stimulated by bacteria and their products, and plays a key role in regulating the immune response.²³ Furthermore, IL-6 is involved in a broad spectrum of biological activities, plays a role in cellular defense, and acts on several target cells. When this cytokine acts on bone, it affects mainly osteoclastogenesis and bone resorption.²³ Taking into consideration the anti-inflammatory role of the tested substances, it was expected that lower levels of IL-6 would be found in the treated groups than in the negative control group, in the current study. However, only ScLL 2 µg/ml and the 15d-PGJ₂ 1 µg/ml showed a significant reduction in IL-6 release. This result may indicate that these substances were able to modulate the release of this inflammatory cytokine in stimulated fibroblasts, in a concentration-dependent manner.

Another relevant factor evaluated was release of the TGFβ cytokine, which plays an important role in promoting fibroblast differentiation at inflammation and repair sites.²⁴ Curiously, no differences were found in the TGFβ-1 levels in the different groups of the current study. Possibly, the 24 h time period established for detection of TGFβ-1 levels in the present study was not enough to release this cytokine in gingival fibroblasts. Furthermore, the amount of LPS used to stimulate HGF cells may not have been enough to induce TGFβ-1 release during this time. It has been demonstrated that TGF-β production in PDL cells versus gingival fibroblasts differs, and that this difference could be attributed to the highest concentration of P. gingivalis LPS used to stimulate this production.²⁵ Although it was not evaluated in the present study, joint production of TGFβ-1 and IL-6 may induce release of the Th17 cell profile,²⁶ which can result in tissue inflammation, since they have been found to occur in chronic periodontal inflammation.²⁷ Regarding the beneficial results of ScLL, this study was unable to define the effect of this substance on prolonged extra-alveolar periods, since only the periods of 1 and 3 h were evaluated. Therefore, more studies evaluating other cytokines, other cellular profiles and different experimental periods should be performed to further advance the present findings.

Despite the limitations of the present study, the results presented here suggest that lectin ScLL and 15d-PGJ₂ may be promising in dentistry, specifically in lower concentrations. This reflects their positive effect on the parameters evaluated, such as cell viability and IL-6 and TGFβ-1 release. However, it should borne in mind that 15d-PGJ₂ is a commercial product sold in small aliquots, thus representing a higher cost and greater difficulty in handling, in comparison with ScLL. This is why preference should be given to ScLL in future research to investigate biological models of greater complexity.

It is important emphasize that ScLL is a protein with significant immunoregulatory activity, like that of leukocyte trafficking and inhibition of TH2 cytokine production;¹² however, it has no nutritional potential. Thus, the main objective of this lectin is to control immune inflammatory reactions, which trigger root resorption. Application of this substance as a product for avulsed teeth should be associated with a storage media that has the nutritional ability to combine its modulatory property with maintenance of cell viability.

Therefore, more research using different in vitro and in vivo models is required to evaluate the potential of ScLL as an immunomodulator and anti-resorption agent for avulsion therapy. Moreover, future research should evaluate the use of immunomodulator agents together with storage media of nutritional capability for use as a product for avulsed teeth.

Conclusion

Based on the results of the present study, it can be concluded that ScLL 5 µg/ml did not interfere in cell viability. In relation to the parameter of IL-6 release, ScLL 2 µg/ml and 15d-PGJ₂ 1 µg/ml demonstrated reduced levels of this inflammatory cytokine. None of the concentrations tested had any effect on TGFβ-1 release.

Acknowledgements

This study was supported by the Research Support Foundation of the State of Minas Gerais (FAPEMIG APQ 02145-14), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (CNPq 474505/2013-8).

References

• ¹
  Trope M. Avulsion of permanent teeth: theory to practice. Dent Traumatol. 2011 Aug;27(4):281-94. https://doi.org/10.1111/j.1600-9657.2011.01003.x
  » https://doi.org/10.1111/j.1600-9657.2011.01003.x
• ²
  Gopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D. Comparison of coconut water, propolis, HBSS, and milk on PDL cell survival. J Endod. 2008 May;34(5):587-9. https://doi.org/10.1016/j.joen.2008.01.018
  » https://doi.org/10.1016/j.joen.2008.01.018
• ³
  Andersson L, Bodin I. Avulsed human teeth replanted within 15 minutes: a long-term clinical follow-up study. Endod Dent Traumatol. 1990 Feb;6(1):37-42. https://doi.org/10.1111/j.1600-9657.1990.tb00385.x
  » https://doi.org/10.1111/j.1600-9657.1990.tb00385.x
• ⁴
  Gulinelli JL, Panzarini SR, Fattah CM, Poi WR, Sonoda CK, Negri MR, et al. Effect of root surface treatment with propolis and fluoride in delayed tooth replantation in rats. Dent Traumatol. 2008 Dec;24(6):651-7. https://doi.org/10.1111/j.1600-9657.2008.00667.x
  » https://doi.org/10.1111/j.1600-9657.2008.00667.x
• ⁵
  Roskamp L, Westphalen VP, Carneiro E, Fariniuk LF, Silva Neto UX. The positive influence of atopy on the prognosis of avulsed and replanted teeth despite differences in post-trauma management. J Endod. 2011 Apr;37(4):463-5. https://doi.org/10.1016/j.joen.2010.12.013
  » https://doi.org/10.1016/j.joen.2010.12.013
• ⁶
  Yang W, Ko H, Kim H, Kim M. The effect of cathepsin K inhibitor on osteoclastic activity compared to alendronate and enamel matrix protein. Dent Traumatol. 2015 Jun;31(3):202-8. https://doi.org/10.1111/edt.12152
  » https://doi.org/10.1111/edt.12152
• ⁷
  Yuan XJ, Wang YY, Shi BQ, Zhao YM. Effect of propolis on preserving human periodontal ligament cells and regulating pro-inflammatory cytokines. Dent Traumatol. 2018 May;34(4):245-53. https://doi.org/10.1111/edt.12411
  » https://doi.org/10.1111/edt.12411
• ⁸
  Wimolsantirungsri N, Makeudom A, Louwakul P, Sastraruji T, Chailertvanitkul P, Supanchart C, et al. Inhibitory effect of Thai propolis on human osteoclastogenesis. Dent Traumatol. 2018 Apr;34(4):237-44. https://doi.org/10.1111/edt.12401
  » https://doi.org/10.1111/edt.12401
• ⁹
  Martins CA, Leyhausen G, Volk J, Geurtsen W. Curcumin in Combination with Piperine Suppresses Osteoclastogenesis In Vitro. J Endod. 2015 Oct;41(10):1638-45. https://doi.org/10.1016/j.joen.2015.05.009
  » https://doi.org/10.1016/j.joen.2015.05.009
• ¹⁰
  Reis MV, Moura CC, Silva MV, Souza MA, Soares PB, Soares CJ. Effect of lectin (ScLL) on fibroblasts stimulated with LPS - an in vitro study. Braz Oral Res. 2016 Dec;30(1):e140. https://doi.org/10.1590/1807-3107bor-2016.vol30.0140
  » https://doi.org/10.1590/1807-3107bor-2016.vol30.0140
• ¹¹
  Souza MA, Carvalho FC, Ruas LP, Ricci-Azevedo R, Roque-Barreira MC. The immunomodulatory effect of plant lectins: a review with emphasis on ArtinM properties. Glycoconj J. 2013 Oct;30(7):641-57. https://doi.org/10.1007/s10719-012-9464-4
  » https://doi.org/10.1007/s10719-012-9464-4
• ¹²
  Rogerio AP, Cardoso CR, Fontanari C, Souza MA, Afonso-Cardoso SR, Silva EV, et al. Anti-asthmatic potential of a D-galactose-binding lectin from Synadenium carinatum latex. Glycobiology. 2007 Aug;17(8):795-804. https://doi.org/10.1093/glycob/cwm053
  » https://doi.org/10.1093/glycob/cwm053
• ¹³
  Afonso-Cardoso SR, Silva CV, Ferreira MS, Souza MA. Effect of the Synadenium carinatum latex (ScLL) on Leishmania (Leishmania) amazonesis infection in murine macrophages. Exp Parasitol. 2011 May; 128(1):61-7. https://doi.org/10.1016/j.exppara.2011.02.006
  » https://doi.org/10.1016/j.exppara.2011.02.006
• ¹⁴
  Cardoso MR, Mota CM, Ribeiro DP, Noleto PG, Andrade WB, Souza MA, et al. Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis. Vet Res (Faisalabad). 2012 Oct;43(1):76. https://doi.org/10.1186/1297-9716-43-76
  » https://doi.org/10.1186/1297-9716-43-76
• ¹⁵
  Nakamura M, Tsumura H, Satoh T, Matsumoto K, Maruyama H, Majima M, et al. Tumor apoptosis in prostate cancer by PGD(2) and its metabolite 15d-PGJ(2) in murine model. Biomed Pharmacother. 2013 Feb;67(1):66-71. https://doi.org/10.1016/j.biopha.2012.10.012
  » https://doi.org/10.1016/j.biopha.2012.10.012
• ¹⁶
  Napimoga MH, da Silva CA, Carregaro V, Farnesi-de-Assunção TS, Duarte PM, de Melo NF, et al. Exogenous administration of 15d-PGJ2-loaded nanocapsules inhibits bone resorption in a mouse periodontitis model. J Immunol. 2012 Jul;189(2):1043-52. https://doi.org/10.4049/jimmunol.1200730
  » https://doi.org/10.4049/jimmunol.1200730
• ¹⁷
  Wang PL, Oido-Mori M, Fujii T, Kowashi Y, Kikuchi M, Suetsugu Y, et al. Heterogeneous expression of Toll-like receptor 4 and downregulation of Toll-like receptor 4 expression on human gingival fibroblasts by Porphyromonas gingivalis lipopolysaccharide. Biochem Biophys Res Commun. 2001 Nov;288(4):863-7. https://doi.org/10.1006/bbrc.2001.5842
  » https://doi.org/10.1006/bbrc.2001.5842
• ¹⁸
  Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodontal Res. 2010 Apr;45(2):262-70. https://doi.org/10.1111/j.1600-0765.2009.01229.x
  » https://doi.org/10.1111/j.1600-0765.2009.01229.x
• ¹⁹
  Seo T, Cha S, Kim TI, Lee JS, Woo KM. Porphyromonas gingivalis-derived lipopolysaccharide-mediated activation of MAPK signaling regulates inflammatory response and differentiation in human periodontal ligament fibroblasts. J Microbiol. 2012 Apr;50(2):311-9. https://doi.org/10.1007/s12275-012-2146-x
  » https://doi.org/10.1007/s12275-012-2146-x
• ²⁰
  Lim W, Choi H, Kim J, Kim S, Jeon S, Zheng H, et al. Anti-inflammatory effect of 635 nm irradiations on in vitro direct/indirect irradiation model. J Oral Pathol Med. 2015 Feb;44(2):94-102. https://doi.org/10.1111/jop.12204
  » https://doi.org/10.1111/jop.12204
• ²¹
  Sepet E, Pinar A, Ilhan B, Ulukapi I, Bilir A, Tuna S. Cytotoxic effects of calcium hydroxide and mineral trioxide aggregate on 3T3 fibroblast cell line in vitro. Quintessence Int. 2009 Sep;40(8):e55-61.
• ²²
  Urnowey S, Ansai T, Bitko V, Nakayama K, Takehara T, Barik S. Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling. BMC Microbiol. 2006 Mar;6(1):26. https://doi.org/10.1186/1471-2180-6-26
  » https://doi.org/10.1186/1471-2180-6-26
• ²³
  Akdis M, Burgler S, Crameri R, EIwegger T, Fujita H, Gomez E, et al. Interleukins, from 1 to 37, and interferon-γ: receptors, functions, and roles in diseases. J Allergy Clin Immunol. 2011 Mar;127(3):701-21.e1-70. https://doi.org/10.1016/j.jaci.2010.11.050
  » https://doi.org/10.1016/j.jaci.2010.11.050
• ²⁴
  Rahimi F, Hsu K, Endoh Y, Geczy CL. FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8. FEBS J. 2005 Jun;272(11):2811-27. https://doi.org/10.1111/j.1742-4658.2005.04703.x
  » https://doi.org/10.1111/j.1742-4658.2005.04703.x
• ²⁵
  Morandini AC, Sipert CR, Ramos-Junior ES, Brozoski DT, Santos CF. Periodontal ligament and gingival fibroblasts participate in the production of TGF-β, interleukin (IL)-8 and IL-10. Braz Oral Res. 2011 Mar-Apr;25(2):157-62. https://doi.org/10.1590/S1806-83242011000200010
  » https://doi.org/10.1590/S1806-83242011000200010
• ²⁶
  Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009;27(1):485-517. https://doi.org/10.1146/annurev.immunol.021908.132710
  » https://doi.org/10.1146/annurev.immunol.021908.132710
• ²⁷
  Cardoso CR, Garlet GP, Crippa GE, Rosa AL, Júnior WM, Rossi MA, et al. Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease. Oral Microbiol Immunol. 2009 Feb;24(1):1-6. https://doi.org/10.1111/j.1399-302X.2008.00463.x
  » https://doi.org/10.1111/j.1399-302X.2008.00463.x

Publication Dates

• Publication in this collection
  17 Feb 2020
• Date of issue
  2020

History

• Received
  15 July 2019
• Accepted
  1 Nov 2019
• Reviewed
  29 Nov 2019