R. Arancibia

Effects of Chitosan Particles in Periodontal Pathogens and Gingival Fibroblasts

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.

Tumor Necrosis Factor-α Inhibits Transforming Growth Factor-β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

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.

Involvement of MT1-MMP and TIMP-2 in human periodontal disease

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.

c-Jun N terminal kinase modulates NOX-4 derived ROS production and myofibroblasts differentiation in human breast stromal cells.

BackgroundHard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-β1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts.MethodsWe used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-β1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-β1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation.ResultsTGF-β1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-β1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-β1-dependent expression of CTGF, NOX4 and α-SMA.ConclusionsHuman mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.

Triclosan inhibits tumor necrosis factor-α-stimulated urokinase production in human gingival fibroblasts

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.

Cigarette smoke condensate inhibits collagen gel contraction and prostaglandin E2 production in human gingival fibroblasts.

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.

Methylglyoxal and methylglyoxal-modified collagen as inducers of cellular injury in gingival connective tissue cells

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.

Diabetes y su impacto en el territorio periodontal

Resumen Diabetes y enfermedad periodontal corresponden probablemente al mejor ejemplo de como una enfermedad sistemica puede tener un efecto en el territorio periodontal. Si bien esta asociacion ha sido extensamente estudiada, muchas de las asociaciones propuestas presentan contradicciones. En la presente revision de la literatura se analizan los siguientes topicos relevantes para la practica clinica en periodoncia e implantologia: i) Identificacion de enfermedad periodontal severa y su capacidad para diagnosticar casos de diabetes; ii) Efectos de la diabetes sobre la enfermedad periodontal; iii) Efectos de la diabetes sobre la reparacion periodontal y periimplantaria; iv) Efecto del tratamiento periodontal sobre el control metabolico de la diabetes.

Empleo de Agentes Farmacológicos en Dentífricos como Moduladores de la Infección e Inflamación Periodontal

Resumen La enfermedad periodontal causa importantes efectos deletereos en la denticion humana, provocando la degradacion del tejido de soporte periodontal y eventualmente la perdida de la denticion. Por otro lado, existen evidencias que sugieren que esta infeccion puede comprometer la salud general del paciente. La prevencion de esta enfermedad puede tener entonces importantes repercusiones en la salud publica y las herramientas para prevenir o controlar esta patologia ameritan la implementacion de medidas basadas en el autocuidado y la prevencion por parte del propio paciente. Es en este sentido que el desarrollo de productos que faciliten el control de placa bacteriana y reduzcan los niveles de inflamacion en el tejido periodontal podria tener efectos beneficiosos en la salud oral de nuestra comunidad. En la presente revision se analiza el papel de agentes farmacologicos utilizados en dentifricos que pueden tener un efecto positivo en el control de la gingivitis y enfermedad periodontal.