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Background and Objective: Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing., Methods: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and β1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-β1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant., Results: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-β1, and β1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and β1 integrin activation. FOXO1 and TGF-β1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed., Conclusion: The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background and Objectives: An important goal of periodontal therapy is the modulation of the inflammatory response. To this end, several pharmacological agents have been evaluated. Triclosan corresponds to an antibacterial and anti-inflammatory agent currently used in periodontal therapy. Chitosan is a natural polymer that may act as a drug delivery agent and exerts antibacterial and anti-inflammatory activities. Therefore, an association between both molecules might be useful to prevent inflammation and tissue destruction in periodontal tissues., Material and Methods: In the present study, we have generated chitosan-triclosan particles and evaluated their morphology, charge, biocompatibility and gene expression analysis in human gingival fibroblasts., Results: The chitosan-triclosan particles size and Z potential were 129 ± 47 nm and 51 ± 17 mV respectively. Human gingival fibroblast viability was not affected by chitosan-triclosan. A total of 1533 genes were upregulated by interleukin (IL)-1β. On the other hand, 943 were downregulated in fibroblasts stimulated with IL-1β plus chitosan-triclosan particles. Fifty-one genes were identified as molecular targets upregulated by IL-1 β and downregulated by the chitosan-triclosan particles. The gene ontology analysis revealed that these genes were enriched in categories related to biological processes, molecular function and cellular components. Furthermore, using real-time reverse transcription-polymerase chain reaction beta-actin, fibronectin, interleukin-6 and IL-1b genes were confirmed as targets upregulated by IL-1β and downregulated by chitosan-triclosan particles., Conclusion: Our results show that chitosan-triclosan particles are able to modulate the inflammatory response in gingival fibroblasts. This effect might be useful in the prevention and/or treatment of inflammation in periodontal diseases., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aging is a gradual biological process characterized by a decrease in cell and organism functions. Gingival wound healing is one of the impaired processes found in old rats. Here, we studied the in vivo wound healing process using a gingival repair rat model and an in vitro model using human gingival fibroblast for cellular responses associated to wound healing. To do that, we evaluated cell proliferation of both epithelial and connective tissue cells in gingival wounds and found decreased of Ki67 nuclear staining in old rats when compared to their young counterparts. We next evaluated cellular responses of primary gingival fibroblast obtained from young subjects in the presence human blood serum of individuals of different ages. Eighteen to sixty five years old masculine donors were classified into 3 groups: "young" from 18 to 22 years old, "middle-aged" from 30 to 48 years old and "aged" over 50 years old. Cell proliferation, measured through immunofluorescence for Ki67 and flow cytometry for DNA content, was decreased when middle-aged and aged serum was added to gingival fibroblast compared to young serum. Myofibroblastic differentiation, measured through alpha-smooth muscle actin (α-SMA), was stimulated with young but not middle-aged or aged serum both the protein levels and incorporation of α-SMA into actin stress fibers. High levels of PDGF, VEGF, IL-6R were detected in blood serum from young subjects when compared to middle-aged and aged donors. In addition, the pro-inflammatory cytokines MCP-1 and TNF were increased in the serum of aged donors. In old rat wound there is an increased of staining for TNF compared to young wound. Moreover, healthy gingiva (non injury) shows less staining compared to a wound site, suggesting a role in wound healing. Moreover, serum from middle-aged and aged donors was able to stimulate cellular senescence in young cells as determined by the expression of senescence associated beta-galactosidase and histone H2A.X phosphorylated at Ser139. Moreover, we detected an increased frequency of γ-H2A.X-positive cells in aged rat gingival tissues. The present study suggests that serum factors present in middle-aged and aged individuals may be responsible, at least in part, for the altered responses observed during wound healing in aging.

Background: Glucose-derived metabolites may alter the structure and biologic properties of important proteins in periodontium, such as collagens. As a consequence, it is possible that collagen-binding cells may change their phenotypic traits. Although the glucose-derived product methylglyoxal (MGO) has been detected in periodontal lesions, the precise effect of collagen glycation on gingival connective tissue biology is not fully understood. The present study evaluates whether collagen glycation by MGO may affect phenotypic properties and remodeling capacity of human gingival fibroblasts (HGFs)., Methods: Primary cultures of HGFs were grown on Type I collagen matrices previously treated with MGO. Cell cultures were tested for cell viability, apoptosis, α-smooth muscle actin (SMA), fibronectin (FN) production, and collagen remodeling. Mechanical properties and morphology of MGO-treated collagen gels were evaluated using rheometry and atomic force microscopy. Statistical analysis was performed by Kruskal-Wallis and Mann-Whitney U tests., Results: MGO-treated collagen did not affect cell viability or apoptosis. In addition, MGO did not induce significant changes in morphology or mechanical properties of the collagen matrix. However, MGO-treated collagen stimulated an increase in the myofibroblast marker α-SMA, production and assembly of FN, and contraction of collagen matrices. Moreover, use of a triple-helical peptide that reconstitutes the collagen-binding domain for integrins GFOGER reverted the assembly of FN induced by MGO-treated collagen., Conclusions: The present study shows that collagen glycation by MGO stimulates differentiation of myofibroblasts and production and assembly of FN. These responses may alter the homeostatic balance and wound-healing response of gingival connective tissues affected by diabetes mellitus or aging.

Background and Objectives: Methylglyoxal is a toxic product derived from glucose metabolism that plays a role in inflammation, diabetes and aging. In addition, the periodontal pathogen Tannerella forsythensis may also generate this compound. However, the effects of methylglyoxal on gingival cells are still poorly understood. In the present study, we have explored whether methylglyoxal or methylglyoxal-treated collagen may modulate cell viability, death and proliferation in gingival connective tissue cells. In addition, we have searched for inflammatory mediators secreted by cells upon exposure to these conditions., Material and Methods: Primary cultures of human gingival fibroblasts were stimulated with soluble methylglyoxal or cultured over a collagen matrix glycated by this agent. Cell viability was evaluated through the MTS assay. Cell death was assessed through DAPI nuclear staining, annexin V and propidium iodide assays. Cell proliferation was evaluated through double immunofluorescence for DAPI and Ki67. Protein levels of matrix metalloproteinases and cytokines were assessed through antibody arrays, enzyme-linked immunosorbent assay, real-time reverse transcription polymerase chain reaction and immunofluorescence. Statistical analysis was performed using the Kruskall-Wallis and Mann-Whitney tests., Results: Soluble methylglyoxal, but not culture of gingival fibroblasts over a methylglyoxal-modified collagen matrix, induced a reduction on cell viability. Moreover, soluble methylglyoxal induced apoptotic cell death as indicated by DAPI nuclear staining, annexin V and propidium iodide assays. Neither soluble methylglyoxal, nor methylglyoxal-modified collagen modified cell proliferation. Using an antibody array, enzyme-linked immunosorbent assay and immunofluorescence assays, we determined that both, soluble methylglyoxal and methylglyoxal-modified collagen stimulated an increase in tissue inhibitor of metalloproteinase (TIMP)-1 protein levels., Conclusions: Soluble methylglyoxal is a highly cytotoxic compound that induces cell death through apoptosis in gingival fibroblasts. TIMP-1 is induced in these cells upon direct exposure to methylglyoxal or after culture of gingival fibroblasts over methylglyoxal-treated collagen. As TIMP-1 has been implicated in cell survival and matrix remodeling, we propose that increased TIMP-1 protein levels may be part of a protective response of gingival connective tissue cells upon exposure to methylglyoxal or after the interaction with the collagen matrix that has been modified by this agent., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Granulation tissue remodeling and myofibroblastic differentiation are critically important events during wound healing. Tobacco smoking has a detrimental effect in gingival tissue repair. However, studies evaluating the effects of cigarette smoke on these events are lacking., Material and Methods: We used gingival fibroblasts cultured within free-floating and restrained collagen gels to simulate the initial and final steps of the granulation tissue phase during tissue repair. Collagen gel contraction was stimulated with serum or transforming growth factor-β1. Cigarette smoke condensate (CSC) was used to evaluate the effects of tobacco smoke on gel contraction. Protein levels of alpha-smooth muscle actin, β1 integrin, matrix metalloproteinase-3 and connective tissue growth factor were evaluated through Western blot. Prostaglandin E(2) (PGE(2)) levels were determined through ELISA. Actin organization was evaluated through confocal microscopy., Results: CSC reduced collagen gel contraction induced by serum and transforming growth factor-β1 in restrained collagen gels. CSC also altered the development of actin stress fibers in fibroblasts cultured within restrained collagen gels. PGE(2) levels were strongly diminished by CSC in three-dimensional cell cultures. However, other proteins involved in granulation tissue remodeling and myofibroblastic differentiation such as alpha-smooth muscle actin, β1 integrin, matrix metalloproteinase-3 and connective tissue growth factor, were unmodified by CSC., Conclusions: CSC may alter the capacity of gingival fibroblasts to remodel and contract a collagen matrix. Inhibition of PGE(2) production and alterations of actin stress fibers in these cells may impair proper tissue maturation during wound healing in smokers., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Gingival wound healing comprises a series of sequential responses that allow the closure of breaches in the masticatory mucosa. This process is of critical importance to prevent the invasion of microbes or other agents into tissues, avoiding the establishment of a chronic infection. Wound healing may also play an important role during cell and tissue reaction to long-term injury, as it may occur during inflammatory responses and cancer. Recent experimental data have shown that gingival wound healing is severely affected by the aging process. These defects may alter distinct phases of the wound-healing process, including epithelial migration, granulation tissue formation, and tissue remodeling. The cellular and molecular defects that may explain these deficiencies include several biological responses such as an increased inflammatory response, altered integrin signaling, reduced growth factor activity, decreased cell proliferation, diminished angiogenesis, reduced collagen synthesis, augmented collagen remodeling, and deterioration of the proliferative and differentiation potential of stem cells. In this review, we explore the cellular and molecular basis of these defects and their possible clinical implications., (© International & American Associations for Dental Research 2014.)

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process., (© International & American Associations for Dental Research.)

Background and Objective: Chitosan is a naturally derived polymer that may be applied in periodontal therapy for tissue-reconstruction purposes. Previous studies have shown that chitosan may stimulate tissue healing. However, reports exploring the cellular responses stimulated by chitosan are lacking. In the present study we analyzed whether chitosan may promote cell proliferation in primary cultures of human gingival fibroblasts., Material and Methods: Chitosan particles were generated, and their size, zeta potential and morphology were characterized using transmission and scanning electron microscopy and zetasizer analysis. The biocompatibility of chitosan particles was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay and by detecting the release of lactate dehydrogenase into the cell-culture medium. The total number of cells was estimated by staining with crystal violet followed by measurement of the absorbance at 560 nm on a microplate reader. Cell proliferation was studied by detecting proliferating cell nuclear antigen protein levels, immunofluorescence for Ki67 and incorporation of 5'-bromo-2'-deoxyuridine., Results: The sizes of the chitosan particles generated were in the micrometer and nanometer ranges. Cell viability was increased in the presence of chitosan. Moreover, the combination of chitosan and platelet-derived growth factor (PDGF-BB) potently stimulated cell viability, cell proliferation and activation of the ERK1/2 pathway involved in cell proliferation., Conclusions: The present study shows that chitosan is well tolerated by gingival fibroblasts and is able to stimulate cell proliferation through the ERK1/2 signaling pathway. A synergistic response between chitosan and growth factors (such as PDGF-BB) may stimulate cell proliferation in gingival fibroblasts exposed to this biomaterial., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chitosan is a naturally derived polymer with antimicrobial and anti-inflammatory properties. However, studies evaluating the role of chitosan in the control of periodontal pathogens and the responses of fibroblasts to inflammatory stimuli are lacking. In the present study, we analyzed whether chitosan particles may inhibit the growth of periodontal pathogens and modulate the inflammatory response in human gingival fibroblasts. Chitosan particles were generated through ionic gelation. They inhibited the growth of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans at 5 mg/mL. Conversely, IL-1β strongly stimulated PGE2 protein levels in gingival fibroblasts, and chitosan inhibited this response at 50 µg/mL. IL-1β-stimulated PGE2 production was dependent on the JNK pathway, and chitosan strongly inhibited this response. IL-1β stimulated NF-κB activation, another signaling pathway involved in PGE2 production. However, chitosan particles were unable to modify NF-κB signaling. The present study shows that chitosan exerts a predominantly anti-inflammatory activity by modulating PGE2 levels through the JNK pathway, which may be useful in the prevention or treatment of periodontal inflammation.

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) modulate myofibroblastic differentiation and the production of ECM components., Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF-β1 and TNF-α. Protein levels of α-smooth muscle actin (α-SMA), type I collagen, heat shock protein-47 (HSP-47), fibronectin (FN), ED-A-FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α-SMA and actin fibers were identified by immunofluorescence. TGF-β1, TNF-α, and α-SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF-β1 activity was evaluated using a plasminogen activator inhibitor-1 (PAI-1) reporter transfected in HGFs., Results: TGF-β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α-SMA and ED-A-FN. Moreover, TGF-β1 induced the production of type I collagen, HSP-47, FN, and periostin. Costimulation with TNF-α and TGF-β1 significantly reduced the expression of all the above-mentioned proteins. TNF-α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI-1 reporter., Conclusions: TNF-α inhibits several cell responses induced by TGF-β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.

Background and Objective: Several studies have analysed the role of nicotine as a prominent agent affecting wound repair in smokers. However, tobacco smoke contains several components that may alter gingival wound healing. The present study aimed to analyse the roles of cigarette smoke condensate (CSC) and nicotine on cell viability, cell migration/invasion and myofibroblastic differentiation using primary cultures of human gingival fibroblasts., Material and Methods: To compare the effects of CSC and nicotine, gingival fibroblasts were stimulated with CSC (0.4–500 lg/mL) and the corresponding nicotine concentrations (0.025–32 lg/mL) present in research cigarettes (1R3F). Cell viability was evaluated through the MTS assay. Cell migration and invasion were assessed through scratch wound assays, collagen nested matrices and trans well migration. a-Smooth muscle actin production was evaluated by western blotting., Results: Cigarette smoke condensate at 50 lg/mL induced a moderate increase in cell viability, whereas the corresponding nicotine concentration (3.2 lg/mL) did not produce this response. Cigarette smoke condensate at 250 lg/mL, but not nicotine at 16 lg/mL (the corresponding nicotine concentration), induced cell death. Both nicotine and CSC stimulated cell migration (50 lg/mL CSC; 3.2 lg/mL nicotine). At 150 lg/mL, CSC inhibited cell migration; however, the corresponding concentration of nicotine (9.6 lg/mL), did not have this effect. Although both nicotine and CSC inhibited a-smooth muscle actin production, only the latter induced a statistically significant effect on this response., Conclusion: Cigarette smoke condensate may stimulate cell survival and migration at low concentrations and inhibit these cell responses at higher levels of exposure. Moreover, CSC may interfere in myofibroblastic differentiation.These results show that cigarette smoke, but not nicotine, may significantly alter cell viability, cell migration and myofibroblastic differentiation in gingival mesenchymal cells.

Objectives: Although platelet-rich plasma (PRP) has been proposed as a therapeutic tool to enhance wound repair, the cellular and molecular mechanisms stimulated by this agent are still not completely understood. The present study was designed to characterize the effects of PRP and platelet-poor plasma (PPP) supernatants on cell responses involved in gingival tissue repair., Methods: We studied the response of human gingival fibroblasts (HGF) to PRP and PPP fractions on: matrix contraction, cell migration, myofibroblastic differentiation, production of matrix components and proteolytic enzymes. PRP and PPP were obtained from donors using a commercial kit. Matrix contraction was evaluated by means of collagen lattices in the presence of matrix metalloproteinase (MMP) and actin polymerization inhibitors. The production of matrix molecules and proteinases was assessed through Western-blot. RhoA activity was evaluated through a pull-down assay. Actin distribution and focal adhesions were assessed through immunofluorescence. Transforming growth factor-beta (TGF-β) was quantified through ELISA., Results: Both PRP and PPP stimulated human gingival fibroblasts-populated collagen gel contraction and Ilomastat and cytochalasin D inhibited this response. PRP and PPP also stimulated MT1-MMP and TIMP-2 production, RhoA activation and actin cytoskeleton remodeling, cell migration/invasion and myofibroblastic differentiation. TGF-β1 was found at significantly higher concentrations in PRP than in PPP., Conclusions: Both PRP and PPP promote wound tissue remodeling and contraction through the stimulation of actin remodeling, the activity of MMPs, promotion of cell migration, and myofibroblastic differentiation. The similar biological responses induced by PRP and PPP suggest that both platelet-derived fractions may exert a positive effect on gingival repair., (© 2011 John Wiley & Sons A/S.)

Background and Objective: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability., Material and Methods: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound-healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active β1 integrin. Rac activation was evaluated by means of a pull-down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann-Whitney U-test., Results: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin-treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of β1 integrin activation. During cell spreading, simvastatin diminished Rac activation., Conclusion: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing., (© 2011 John Wiley & Sons A/S.)

Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type-1 matrix metalloproteinase (MT1-MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined., Methods: MT1-MMP and TIMP-2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis-affected human subjects. MT1-MMP and TIMP-2 expression were assessed through Western-blot of tissue homogenates. The main cell types involved in MT1-MMP and TIMP-2 production were evaluated by means of immunohistochemistry., Results: Both MT1-MMP and TIMP-2 were significantly increased in periodontitis-affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1-MMP and its inhibitor TIMP-2 was altered in periodontitis-affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1-MMP in fibroblasts and macrophages in gingival tissues. MT1-MMP was detected in cells in close association with the gingival collagen matrix. TIMP-2 expression was identified in fibroblasts, macrophages and epithelial cells., Conclusions: Our observations show an increased expression of MT1-MMP and TIMP-2 in periodontitis-affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.

Background and Objectives: Destruction of the supporting periodontal tissues is mediated by the action of several proteolytic enzymes. Urokinase is a serine protease that plays a key role in connective tissue destruction through conversion of plasminogen into plasmin. The present study was conducted to evaluate the effect of triclosan on the production and activity of urokinase in cultured gingival fibroblasts., Material and Methods: Urokinase production was studied in primary cultures of human gingival fibroblasts stimulated with tumor necrosis factor-alpha. Urokinase activity and production were evaluated using casein zymography and western blotting, respectively. Urokinase mRNA expression was evaluated using the reverse transcription-polymerase chain reaction. Triclosan was used to interfere with this stimulatory effect. The roles of different cell-signaling cascades involved in urokinase production were assessed through western blotting and immunofluorescence using several cell-signaling inhibitors., Results: Tumor necrosis factor-alpha was found to be a strong stimulus for urokinase production and triclosan was able to inhibit this response at the protein and mRNA levels. Triclosan was also able to inhibit conversion of plasminogen into plasmin. Tumor necrosis factor-alpha-stimulated urokinase production was shown to be dependent on the nuclear factor-kappaB and c-Jun N-terminal kinase signaling pathways. Triclosan inhibited c-Jun N-terminal kinase phosphorylation and c-Jun production., Conclusions: Within the limits of this study, these results show that triclosan may inhibit urokinase production and plasminogen activation in gingival fibroblasts through modulation of the c-Jun N-terminal kinase signaling pathway.

Background and Objective: Tobacco smoking is a significant risk factor for periodontal disease. It has been suggested that smoking may alter connective tissue remodeling in the periodontium. In the present study, we investigated whether cigarette smoke condensate modulates the production of the serine protease urokinase in human gingival fibroblasts., Material and Methods: Primary cultures of human gingival fibroblasts were stimulated with cigarette smoke condensate. Urokinase production was evaluated through casein zymography and western blotting. Plasmin activation was assessed by means of a radial diffusion assay. The roles of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and reactive oxygen species in cigarette smoke condensate-stimulated urokinase production were studied using distinct selective inhibitors (SP600125, PD98059, N-acetyl cysteine). Reactive oxygen species production was determined using a fluorometric assay. Activation of ERK and JNK pathways were evaluated using western blots., Results: In gingival fibroblasts, cigarette smoke condensate potently stimulated urokinase production and plasmin activation. Cigarette smoke condensate-stimulated urokinase production was dependent on the activity of ERK/JNK pathways and was inhibited by the reactive oxygen species scavenger, N-acetyl cysteine. Cigarette smoke condensate strongly stimulated ERK and JNK phosphorylation and the generation of reactive oxygen species., Conclusion: Cigarette smoke condensate stimulates urokinase production and plasmin activation in gingival fibroblasts. Moreover, cigarette smoke condensate-stimulated urokinase production depends on both the activation of ERK/JNK pathways and on the generation of intracellular reactive oxygen species. These results show that cigarette smoke may alter connective tissue remodeling by inducing production of the urokinase-type plasminogen activator through specific signaling pathways.

Background and Objectives: Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts., Material and Methods: Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction., Results: Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha)., Conclusions: The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

Background: Platelet-rich plasma is a blood-derived fraction that contains a high concentration of platelets and growth factors. It was proposed that the use of this platelet concentrate stimulates tissue repair. However, little is known about the biologic response of gingival fibroblasts to platelet's derived growth factors. In the present study, we evaluated whether platelet-rich plasma modulated cell adhesion, cell migration, and myofibroblastic differentiation in primary cultures of human gingival fibroblasts., Methods: We studied the response of primary cultures of gingival fibroblasts to thrombin-activated platelet-rich plasma fractions. Cell adhesion was evaluated through a colorimetric assay. Cell spreading, actin cytoskeleton remodeling, and focal adhesion distribution were assessed through light and immunofluorescence microscopy. Cell migration was analyzed using a bicameral cell culture system. Smooth muscle actin production was studied through Western blotting., Results: Exposure of gingival fibroblasts to platelet-rich plasma stimulated adhesion and spreading of cells on fibronectin matrices, the development of actin-enriched cellular extensions, and formation of focal adhesions. Platelet-rich plasma also promoted cell migration and invasion through a reconstituted basement membrane matrix. Differentiation into the myofibroblastic phenotype, assessed through the production of smooth muscle actin, was also stimulated by platelet-rich plasma preparations., Conclusion: Platelet-rich plasma may modulate several cell responses potentially involved in wound healing such as cell adhesion, cell migration, and myofibroblastic differentiation.

Transforming Growth Factor-beta1 (TGF-beta1) plays a key role in connective tissue remodeling and inflammation. Under pathological conditions, like periodontal disease, fibroblasts may display an altered response to this growth factor. To investigate this question, we have studied whether TGF-beta1 may differentially regulate the expression of urokinase at the protein level in primary cultures of fibroblasts derived from healthy gingiva, granulation tissue from gingival wounds, and chronic periodontal disease. We observed that TGF-beta1 may repress urokinase expression in healthy gingival fibroblasts and promote its production in granulation-tissue fibroblasts. A significant correlation was found between expression of the myofibroblast marker alpha-smooth-muscle actin and stimulation of urokinase production by TGF-beta1. Immunostaining of gingival wounds showed that myofibroblasts were involved in urokinase production. TGF-beta1-stimulated urokinase expression was blocked after inhibition of the c-jun-NH2 terminal kinase signaling pathway. We propose that stimulation of urokinase production by TGF-beta1 is involved in the responses of activated fibroblasts to tissue injury.

Background: Regulation of the extracellular matrix turnover is a crucial process in wound healing and the progress of periodontal disease. It has been proposed that urokinase-type plasminogen activator (uPA), under the control of growth factors or cytokines, provides the proteolytic potential to the accomplishment of these cellular events. Epidermal growth factor (EGF) is one of the growth factors that has been shown to be active in uPA regulation., Methods: In this study, we have assessed the effect of EGF on uPA expression in primary cultures of human gingival fibroblasts. We also studied the signaling pathways involved in this process and the role of the dietary phytoestrogens curcumin and genistein as potential modulators of this response., Results: Human gingival fibroblasts expressed a basal uPA activity, which was inhibited by genistein, but not by curcumin. After treatment with 10 ng/ml EGF, uPA production was strongly stimulated. Exposure to genistein and curcumin inhibited EGF-stimulated urokinase production, although only genistein showed a statistically significant inhibitory response. Using more specific inhibitors, we found that the mitogen-activated extracellular kinase and c-Jun N-terminal kinase (JNK) inhibitors PD98059 and SP600125 also blocked the EGF-dependent stimulatory effect. On the other hand, SB203580, inhibitor of the p38 member of mitogen-activated protein kinase family, did not alter this response. In accordance to these findings, EGF stimulated a potent activation of JNK and a mild activation of extracellular signal-regulated kinases 1/2. Finally, EGF stimulated the phosphorylation of its receptor and tyrphostin (AG1478), curcumin and genistein were able to inhibit this stimulatory effect., Conclusions: These results indicate that EGF constitutes a strong stimuli on uPA expression in human gingival fibroblasts. Our data also shows that EGF-stimulated uPA production involves the activation of the extracellular signal-regulated kinases 1/2 and JNK signaling pathways and might be modulated by the natural phytoestrogens curcumin and genistein., ((c)Blackwell Munksgaard 2004)

Background and Objective: As the periodontal lesion develops, the junctional epithelium migrates apically in conjunction with the dissolution of the most coronal Sharpey's fibers. Because matrix metalloproteinase-9 (MMP-9) has been identified in migrating epithelial cells and invading tumors, we propose that this enzyme is produced by gingival keratinocytes in advanced periodontal lesions., Methods: To test this idea, biopsies of inflamed gingival tissues were obtained from patients with advanced periodontitis. Healthy gingival tissue samples were utilized as controls. The presence and activity of MMP-9 was evaluated by combining indirect immunofluorescence of gingival tissue samples and gelatin zymography of gingival epithelium separated from connective tissue., Results and Conclusions: The staining pattern showed the presence of MMP-9 in junctional and pocket gingival epithelial cells, polymorphonuclear neutrophils (PMNs) and as a scattered deposit along connective tissues of periodontitis-affected gingival tissues. Gelatin zymography permitted the identification of pro-MMP-9 in surcular/pocket epithelium derived from inflamed gingival tissues. Lower levels of MMP-9 were detected in epithelium not exposed to inflammation. These observations suggest a role for MMP-9 in gingival epithelial response to periodontal infection.