Chitosan-triclosan particles modulate inflammatory signaling in gingival fibroblasts.

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)-1ß. On the other hand, 943 were downregulated in fibroblasts stimulated with IL-1ß plus chitosan-triclosan particles. Fifty-one genes were identified as molecular targets upregulated by IL-1 ß 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-1ß 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.

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.

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-ß1. Cigarette smoke condensate (CSC) was used to evaluate the effects of tobacco smoke on gel contraction. Protein levels of alpha-smooth muscle actin, ß1 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-ß1 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, ß1 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.

Chitosan and platelet-derived growth factor synergistically stimulate cell proliferation in gingival fibroblasts.

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.

The Role of Aphid Abundance, Species Diversity, and Virus Titer in the Spread of Sweetpotato Potyviruses in Louisiana and Mississippi.

Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2) are sweetpotato (Ipomoea batatas) potyviruses nonpersistently transmitted by aphids. Our objective was to determine how aphid abundance, aphid species diversity, and virus titers relate to the spread of SPFMV, SPVG, and SPV2 in Louisiana and Mississippi sweetpotato fields. The most abundant aphid species were Aphis gossypii, Myzus persicae, Rhopalosiphum padi, and Therioaphis trifolii. Aphids were captured during the entire crop cycle but virus infection of sentinel plants occurred mainly during the months of June to August. SPFMV was more commonly detected than SPVG or SPV2 in sentinel plants. Virus titers for SPFMV were higher in samples beginning in late June. Because significant aphid populations were present during April to June when virus titers were low in sweetpotato and there was very little virus infection of sentinel plants, low virus titers may have limited aphid acquisition and transmission opportunities. This is the first study to comprehensively examine aphid transmission of potyviruses in sweetpotato crops in the United States and includes the first report of R. maidis and R. padi as vectors of SPFMV, though they were less efficient than A. gossypii or M. persicae.

Effects of cigarette smoke and nicotine on cell viability, migration and myofibroblastic differentiation.

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 (0.4­500 lg/mL) and the corresponding nicotine concentrations (0.025­32 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.

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.

Triclosan inhibits tumor necrosis factor-alpha-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 stimulates urokinase production through the generation of reactive oxygen species and activation of the mitogen activated protein kinase pathways in human gingival fibroblasts.

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.

Effects of Thiamethoxam-Treated Seed on Mexican Bean Beetle (Coleoptera: Coccinellidae), Nontarget Arthropods, and Crop Performance in Southwestern Virginia Snap Beans.

Thiamethoxam is a neonicotinoid insecticide commonly applied directly to the seeds (seed-treatment) of commercial snap beans, Phaseolus vulgaris L. While previous studies have examined target and nontarget effects of thiamethoxam seed-treatments in snap beans and other crops, to our knowledge, none have been conducted in agroecosystems predominated by the pest Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae). This study examined the effects of thiamethoxam-treated snap beans on E. varivestis, other arthropods, and crop performance in southwestern Virginia. Greenhouse experiments were conducted to evaluate residual toxicity of treated snap beans to E. varivestis and a key predator, Podisus maculiventris (Say) (Hemiptera: Pentatomidae). Treated plants were highly toxic to E. varivestis at 13 d, moderately toxic from 16 to 20 d, and minimally toxic at 24 d. P. maculiventris was unaffected by exposure to treated plants or by feeding on E. varivestis that consumed treated plants. Small plot field experiments in 2014 and 2015 showed no significant effects of thiamethoxam seed-treatments on E. varivestis densities, other arthropods, crop injury, or yield. In 2016, planting was delayed by persistent rain, resulting in early E. varivestis colonization. In this year, thiamethoxam-treated plants had significantly lower densities and feeding injury from E. varivestis, followed by significantly higher yields. Natural enemies were unaffected by seed-treatments in all field experiments. These experiments demonstrated that thiamethoxam seed-treatments provide control of E. varivestis when beetles infest fields within 2 to 3 wk after planting; but otherwise provide negligible advantages. Negative effects from thiamethoxam seed-treatments on nontarget arthropods appear minimal for snap beans in this region.

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-1ß strongly stimulated PGE2 protein levels in gingival fibroblasts, and chitosan inhibited this response at 50 µg/mL. IL-1ß-stimulated PGE2 production was dependent on the JNK pathway, and chitosan strongly inhibited this response. IL-1ß 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.

Hipovitaminosis D en pacientes hospitalizados por trastornos por consumo de sustancias en Unidad de Adicciones del Hospital Clínico Universidad de Chile / Hypovitaminosis D in patients hospitalized for substance use disorders in the Addictions Unit of the Hospital Clínico Universidad de Chile

The pathological consumption of alcohol and other drugs is associated with calcium metabolism disfunction through different pathways. Hypovitaminosis D contributes to acute a chronic neuronal injury in alcohol dependent patients. We do not have national evidence regarding the presence of hypovitaminosis D in addicted patients and there is a lack of information in the literature regarding polysubstance users. In this retrospective study, we evaluate the presence of hypovitaminosis of D in Substance Use Disorder inpatients treated in the Psychiatric Clinic of the University during the months of August to November 2017 and we described their main characteristics. 24 patients were evaluated, 19 of whom presented levels lower than 30 ng/ml of Vitamin D. Of those patients with hypovitaminosis 79% were men and 90% of them consumed alcohol, although in only 26% alcohol was the main substance. The main substance reported by the patients was cocaine (37%), smokable cocaine (32%) and marijuana (5%). Despite the methodological limitations of the study and the high prevalence of Hypovitaminosis D reported in the Chilean population, the results of this study suggest the need for a systematic evaluation of Vitamin D levels in patients hospitalized for addictions to adequately supplement those who require it. (AU)

Diabetes y su impacto en el territorio periodontal / Diabetes and its impact in periodontal tissues

Diabetes y enfermedad periodontal corresponden probablemente al mejor ejemplo de cómo una enfermedad sistémica puede tener un efecto en el territorio periodontal. Si bien esta asociación ha sido extensamente estudiada, muchas de las asociaciones propuestas presentan contradicciones. En la presente revisión de la literatura se analizan los siguientes tópicos relevantes para la práctica clínica en periodoncia e implantología: i) Identificación 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 reparación periodontal y periimplantaria; iv) Efecto del tratamiento periodontal sobre el control metabólico de la diabetes.

Diabetes and periodontal disease correspond to conditions that probably exemplify how a systemic disease may have a strong impact in the periodontium. Although this association has been studied for several years, many of these studies still show contradictory results. The present review analyses the following questions relevant for the clinician in the fields of periodontology: i) Value of the diagnosis of severe periodontitis and its capacity to identify previously un-diagnosed cases of diabetes; ii) Effects of diabetes on periodontal disease; iii) Effects of diabetes on periodontal and peri-implant tissue repair and regeneration and; iv) Effect of periodontal therapy on the metabolic control of diabetes.