Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.     

Morphological and molecular analysis of idiopathic gingival fibromatosis: a case report

AIM: We analyse a case of idiopathic gingival overgrowth using morphological and molecular methods. As this overgrowth involves collagen accumulation in the gingival connective tissue, we measured the collagen turnover to clarify the pathogenic mechanisms potentially involved. MATERIALS AND METHODS: The patient was a 29-year-old Italian woman with enlargement of the gingivae throughout the entire mandible and maxilla. Morphological analyses were carried out on haematoxylin-eosin and Sirius red-stained paraffin-embedded gingival sections. mRNA levels of collagen type I and III, matrix metalloproteinase (MMP)-1, transforming growth factor-beta1 and lysyl hydroxylase (LH)2b were determined by RT-PCR on cultured gingival fibroblasts and compared with healthy control fibroblasts. Interstitial collagen and MMP-1 content in the supernatants were assessed, respectively, by dot blot and SDS zymography. RESULTS AND CONCLUSIONS: In Sirius red-stained sections of the patient's overgrown gingivae, interstitial collagen content was 29% higher than controls. Her gingival fibroblasts had higher collagen type I, MMP-1 and LH2b gene expression and unmodified interstitial collagen, type I protein levels in the supernatants. These findings would seem to suggest that in this case collagen accumulation in the gingival connective tissue was not associated with increased synthesis and decreased degradation.     

水蛭素对牙龈成纤维细胞碱性成纤维细胞生长因子和转化生长因子-β1表达的影响

目的 观察水蛭素对人牙龈成纤维细胞（HGFs）碱性成纤维细胞生长因子（bFGF）及转化生长因子-β1（TGF-β1）表达变化的规律,探讨水蛭素影响牙龈改建的可能作用机制。方法 体外培养并鉴定HGFs,利用不同浓度的水蛭素分别作用于正常（对照组）和受长期机械外力作用后增生的HGFs（实验组）,通过实时定量聚合酶链反应法和免疫细胞化学法检测TGF-β1及bFGF的表达。结果 未加入水蛭素时,受长期机械外力作用后,实验组促进HGFs增殖胶原合成的TGF-β1表达升高,而抑制胶原合成的bFGF表达降低（P<0.05）。加入水蛭素干预增生的HGFs后,可正向调节bFGF表达,而负向调节TGF-β1的表达（P<0.05）。结论 外力作用干扰了HGFs胶原合成与降解之间的平衡,水蛭素可能通过调节这一平衡而促进牙龈改建过程。     

Effects of interleukin-1alpha on the production and release of basic fibroblast growth factor in cultured nifedipine-reactive gingival fibroblasts

The effect of interleukin-1alpha (IL-1alpha) on the production of basic fibroblast growth factor (bFGF) in human gingival fibroblasts originated from a nifedipine-reactive patient was investigated. Ca(2+)-mobilizing agents, thapsigargin and bradykinin, were also tested to determine whether they affected the production and release of bFGF. The release of bFGF from IL-1alpha-pretreated cells in relation to the transient increase in intracellular Ca(2+)([Ca(2+)]i) and extracellular Ca(2+)levels was also investigated. IL-1alpha and thapsigargin yielded significantly higher bFGF production, and also enhanced bFGF mRNA expression. IL-1alpha-pretreated cells showed significantly greater release of bFGF under the present experimental conditions. Levels of released bFGF were significantly higher in cells pretreated with IL-1alpha, followed by bradykinin and thapsigargin in the presence of extracellular Ca(2+). The transient mobilization of intracellular Ca(2+) accelerated the release of bFGF in IL-1alpha-pretreated cells, but not in untreated cells. Thus, IL-1alpha increases bFGF production in nifedipine-reactive gingival fibroblasts and also influences the release of bFGF in the IL-1alpha-pretreated cells.     

Elevation of collagen type I in fibroblast-keratinocyte cocultures emphasizes the decisive role of fibroblasts in the manifestation of the phenotype of cyclosporin A-induced gingival overgrowth

Background and Objective: Collagen type I elevation in cyclosporin A‐induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo‐like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth‐derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. Material and methods: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. Results: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. Conclusion: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth‐phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth‐derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A‐induced effects under more in vivo‐like conditions.     

High IL-6 synthesis in cultured fibroblasts isolated from radicular cysts

OBJECTIVE: Inflammatory cytokines have been reported to be related with inflammation and expansion of jaw cysts. In this study, to examine the relationship between radicular cysts and inflammatory cytokines, it was found that there was notable unique evidence on cytokine synthesis from fibroblasts isolated from radicular cysts. METHODS: The expression of such cytokines, namely, interleukin-1beta, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and granulocyte-macrophage colony-stimulating (GM-CSF) mRNA, in nine radicular cysts was examined and compared with that detected in six specimens of healthy gingival mucosa. Furthermore, separating all fibroblasts from their respective radicular cysts, healthy gingival mucosa, and healthy periodontal ligaments, these fibroblast groups were cultured without stimulators and a supernatant for each was obtained to analyse IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma by ELISA. RESULTS: Differences between radicular cysts and healthy gingival mucosa were not clearly shown by the expression of cytokine mRNA. Analysing inflammatory cytokine synthesis in fibroblast groups from these three kinds of tissues, surprisingly, the levels of IL-6 mRNA and protein were recognised to be higher in fibroblasts of radicular cysts than in those of control tissues by ELISA and a real-time RT-PCR. Significant differences in the cultured supernatants of these fibroblast groups were not recognised in the release of IL-1beta, IL-8, TNF-alpha, and IFN-gamma by ELISA. CONCLUSIONS: From these results, it was suggested that fibroblasts inducing IL-6 production might play important roles in the expansion of radicular cysts. It is considered that fibroblasts around radicular cysts may lead to high IL-6 synthesis over time in chronic inflammation.     

Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.     

不同来源人牙龈成纤维细胞体外降解胶原能力研究

目的 :体外培养条件下比较健康人及侵袭性牙周炎患者的牙龈成纤维细胞对鼠I型胶原降解的影响和差异。方法 :在 6孔板底制备胶原膜 ,取固定数量的对数生长期健康人及侵袭性牙周炎患者的牙龈成纤维细胞接种于胶原膜中央 ,分别于孵育 2 4、48、72h后消化弃去细胞 ,考马斯亮兰液染色胶原膜 ,显微镜下测定降解区和非降解区的光密度来比较不同时间段各组细胞的胶原降解量。结果 :健康人和侵袭性牙周炎各 3例标本来源细胞的结果基本一致 ,表现为随时间的延长 ,胶原降解量相应增加 ;而各时间段病变组的胶原降解量较健康组明显增加(P <0 .0 5 )。结论 :健康人和侵袭性牙周炎的牙龈成纤维细胞对鼠I型胶原均有降解作用并呈现出时间效应 ,而且病变组的作用更强。     

Direct interaction between gingival fibroblasts and lymphoid cells induces inflammatory cytokine mRNA expression in gingival fibroblasts

In inflamed periodontal lesions, dense infiltration of lymphocytes is usually observed in the extravascular periodontal connective tissue, adjacent to gingival fibroblasts. Our previous study revealed that activated lymphocytes can adhesively interact with gingival fibroblasts in vitro. In the present study, we investigated whether gingival fibroblasts are activated through direct interaction with lymphoid cells by monitoring the expression of inflammatory cytokine mRNA in human gingival fibroblasts (HGF). Co-culture with various human lymphoid cells in vitro resulted in a marked increase in the expression of IL-1alpha, IL-1beta, and IL-6 mRNA by the HGF. In addition, expression of the mRNA of the IL-1beta-converting enzyme (ICE), which is essential to produce the mature form of IL-1beta, was constitutively observed in the HGF, suggesting that mature IL-1beta is produced by these cells. When HGF were cultured with the culture supernatant of the lymphoid cells, the increase in the inflammatory cytokine mRNA expression was not observed. Similarly, when HGF and lymphoid cells were cultured in the same well but separated by a membrane which prevented direct contact between the cells, no increase in inflammatory cytokine mRNA expression was observed. These results strongly indicate that direct interaction between these heterotypic cell types transduces activation signals into HGF that induce an increase in inflammatory cytokine mRNA expression. Furthermore, IL-1beta mRNA expression in the HGF was synergistically increased when HGF directly interacted with lymphoid cells in the presence of exogeneous IL-1beta. The present study demonstrates that direct interaction between HGF and lymphoid cells stimulates HGF to increase inflammatory cytokine mRNA expression, and raises the possibility that heterotypic cell-cell interaction may facilitate local inflammatory reactions.     

Regulation of fibroblast-induced collagen gel contraction by interleukin-1β

Fibroblasts incorporated within collagen gels induce a cell-mediated contraction of the gel to form a three-dimensional, tissue-like structure by a mechanism thought to mimic wound contraction in vivo . In this study a gel contraction model was used to investigate the ability of fibroblasts derived from adult gingiva, adult skin and fetal skin to organise a collagen matrix. In addition the effects of interleukin-1β (IL-1β) on the contraction process was also investigated. Over the concentration range 5-50 U/ml, IL-1β induced a statistically significant inhibition of gel contraction in all fibroblast cell types ( P <0.05), although fetal fibroblasts appeared least responsive and gingival fibroblasts most responsive to the inhibitory effects of this cytokine. Comparison of gel contraction by the different fibroblast strains indicated that fetal and gingival fibroblasts shared similar contraction kinetics. For the adult skin fibroblasts, three of five strains studied showed significantly diminished levels of gel contraction compared to fetal and gingival cells. This apparent difference in fibroblast phenotype may, at least in part, explain the fetal-like wound healing pattern seen in the oral mucosa.     

Increased expression of collagen prolyl 4-hydroxylases in Chinese patients with hereditary gingival fibromatosis

OBJECTIVES: Hereditary gingival fibromatosis (HGF) is characterized by excess accumulation of interstitial collagen. However, until now, there has been controversy about the mechanism of collagen accumulation in HGF gingivae. The present study aimed to clarify the pathogenic mechanisms potentially involved. DESIGN: Gingival fibroblasts from three Chinese HGF patients and three healthy subjects were cultured. Cell proliferation was assessed by MTT assay. The mRNA levels of type I collagen, MMP-1, MMP-3, TIMP-1, prolyl 4-hydroxylase (P4H)alpha(I), alpha(II), alpha(III) and P4Hbeta were analyzed in gingival fibroblasts by RT-PCR. The protein production of type I collagen and P4H was examined respectively by ELISA and Western blot. RESULTS: In culture, HGF gingival fibroblasts showed similar growth characteristics to fibroblasts isolated from control gingivae. The mRNA and protein levels of type I collagen and P4Halpha in HGF fibroblasts were higher than those in controls. There were no detected differences in mRNA expression levels of MMP-1, MMP-3, TIMP-1, P4Halpha(II), alpha(III) and P4Hbeta between HGF and control fibroblasts. CONCLUSIONS: These data suggest that increased collagen post-translational modification by P4H may be one mechanism by which increased collagen accumulation occurs in some forms of HGF.     

Cyclosporin-A increases type I procollagen production and mRNA level in human gingival fibroblasts in vitro

In order to study the pathogenesis of gingival overgrowth induced by the immunosuppressive drug cyclosporine-A (CyA), we investigated its effect on 3H thymidine incorporation and on collagen production and mRNA levels in fibroblast cultures obtained from normal human gingiva. At concentrations of 100, 500 and 1000 ng/ml, CyA did not modify thymidine incorporation after 24 and 72 h of incubation. However, after 24 h it significantly increased the level of 3H proline-containing proteins in the medium. In addition, CyA increased alpha-procollagen chains by up to three times. This CyA-induced change was related to a rise in the level of type I procollagen. The CyA effect on fibroblasts was markedly reduced by cycloheximide, an inhibitor of protein synthesis, and it correlated well with an increase of type I procollagen mRNA. Overall, our data indicate a direct stimulatory action of CyA on collagen synthesis, but not on DNA synthesis, in human gingival fibroblasts.     

Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor

Background: Transforming growth factor‐β1 (TGF‐β1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF‐β1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF‐β1–mediated myofibroblast transformation. Methods: To study the inhibitory effect of Smad7 on TGF‐β1/CTGF‐mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α‐SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme‐linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGF's role on myofibroblast transformation was examined by enzyme‐linked immunosorbent assay and small interference RNA. Results: TGF‐β1 induced the expression of α‐SMA and CTGF, and small interference RNA–mediating CTGF silencing prevented fibroblast‐myofibroblast switch induced by TGF‐β1. In Smad7‐overexpressing fibroblasts, ablation of TGF‐β1–induced Smad2 phosphorylation marked decreased α‐SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α‐SMA and phospho‐Smad2 and significant reduction on CTGF and type I collagen production. Conclusions: CTGF is critical for TGF‐β1–induced gingival fibroblast‐myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.     

The Effect of α‐Tocopherol and Selenium on Human Gingival Fibroblasts and Periodontal Ligament Fibroblasts In Vitro

Background: The aim of the present study is to evaluate the effect of α‐tocopherol and selenium on gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) in terms of proliferation, basic fibroblast growth factor (bFGF) release, collagen type I synthesis, and wound healing. Methods: Primary cultures of human GFs and PDLFs were isolated. Four test groups and a control group free of medication was formed. In group E, 60 μM α‐tocopherol was used, and in groups ES1, ES2, and ES3, the combination of 60 μM α‐tocopherol with 5 × 10^?9 M, 10 × 10^?9 M, and 50 × 10^?9 M selenium was used, respectively. Viability, proliferation, bFGF, and collagen type I synthesis from both cell types were evaluated at 24, 48, and 72 hours, and healing was compared on a new wound‐healing model at 12, 24, 36, 48, and 72 hours. Results: α‐Tocopherol alone significantly increased the healing rate of PDLFs at 12 hours and increased bFGF and collagen type I release from GFs and PDLFs at 24, 48, and 72 hours. The α‐tocopherol/selenium combination significantly enhanced the proliferation rate of both cells at 48 hours, decreased the proliferation of PDLFs at 72 hours, and increased the healing rate of GFs at 12 hours and PDLFs at 12 and 48 hours. bFGF and collagen type I synthesis was also increased in both cell types at 24, 48, and 72 hours by α‐tocopherol/selenium combination. Conclusion: α‐Tocopherol and α‐tocopherol/selenium combination is able to accelerate the proliferation rate and wound‐healing process and increase the synthesis of bFGF and collagen type I from both GFs and PDLFs.     

Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival eipthelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular martix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibitied enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular martix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.     

Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to bacterial challenge

ObjectiveTo investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge.DesignmRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot.ResultsHuman gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts.ConclusionOur data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production.