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1.
Braz Oral Res ; 34: e013, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074213

RESUMO

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


Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Lectinas de Plantas/farmacologia , Prostaglandina D2/análogos & derivados , Fator de Crescimento Transformador beta1/metabolismo , Análise de Variância , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Gengiva/citologia , Humanos , Prostaglandina D2/farmacologia , Valores de Referência , Estatísticas não Paramétricas , Fatores de Tempo , Fator de Crescimento Transformador beta1/efeitos dos fármacos
2.
Arch Oral Biol ; 110: 104602, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31734544

RESUMO

OBJECTIVE: Oxidative stress, which is defined as an imbalance between pro-oxidant and antioxidant systems, has been implicated in the development and/or progression of several inflammatory diseases, including periodontal disease. The reactive oxygen species (ROS) are the primary inducers of oxidative stress. In the induction of cytoprotective enzymes, the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling in antioxidant systems takes a main role. Notably, 10-oxo-trans-11-octadecenoic acid (KetoC), known as a bioactive metabolite generated by intestinal microorganisms, has been reported to have beneficial effects on several biological responses. Therefore, we investigated the antioxidant effect of KetoC on gingival epithelial cells (GECs) in this present study. METHODS: An SV40-T antigen-transformed human gingival epithelial cell line (Epi4) was used for experiments. The alteration of anti-oxidative stress related genes was analyzed by qPCR. The cellular ROS levels were evaluated by flow cytometry. To explore its molecular mechanisms, ARE promotor activity was analyzed by luciferase assay; the involvement of mitogen-activated protein kinase (MAPK) and G protein-coupled receptor 120 (GPR120) were evaluated by Western blotting and luciferase assay, respectively. RESULTS: KetoC significantly increased the expression of antioxidant-related genes in GECs. The level of ROS was significantly inhibited by the pretreatment of KetoC. Extracellular signal-regulated kinase (ERK) phosphorylation by KetoC promoted both the nuclear translocation of Nrf2 and its binding to the ARE in GECs. Further, GPR120 regulated the activation of KetoC induced-Nrf2-ARE signaling. CONCLUSION: KetoC exerts a protective function against the oxidative stress in GECs through GPR120-dependent ERK-Nrf2-ARE signaling.


Assuntos
Elementos de Resposta Antioxidante , Gengiva , Ácidos Linoleicos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Antioxidantes , Células Epiteliais , Gengiva/citologia , Gengiva/metabolismo , Humanos , Ácidos Linoleicos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio
3.
Bioelectrochemistry ; 131: 107386, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706116

RESUMO

The biocompatibility of human gingival fibroblasts (HGF) was evaluated in different concentrations of poly(vinyl alcohol) and sodium alginate (PVA/SA) nanofibres (3.5 wt% 4 wt% and 5 wt%). The PVA/SA nanofibres were deposited on the surface of an electrode microchip by using the electrospinning technique. Electrochemical impedance spectroscopy (EIS) was applied to measure the dielectric properties of each system. In order to provide a detailed analysis as well as a right physical interpretation of the EIS results, the data was fitted with an electric equivalent circuit based on the EIS and the microscopic assessments. The results registered three different time constants (TCs) of the PVA/SA scaffold which indicated different layers at different depths of the scaffold. The TCs changed their dielectric properties depending on the PVA/SA concentration. The 4 wt% system showed the highest biocompatibility properties, given that its resistance and electrochemical capacitance show the formation of a mature-stage cell interaction of HGF. The EIS data offers an exhaustive analysis of the biological activity of the cell response in real time to determine its biocompatibility features. Fluorescence analysis demonstrated a heterogeneous growth of the HGF on the PVA/SA scaffold surface.


Assuntos
Materiais Biocompatíveis , Espectroscopia Dielétrica/métodos , Gengiva/metabolismo , Tecidos Suporte , Fibroblastos/metabolismo , Gengiva/citologia , Humanos
4.
J Biol Regul Homeost Agents ; 33(6): 1715-1723, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31797649

RESUMO

Ascorbic acid (AS), also known as vitamin C or ascorbate, is an essential dietary nutrient which plays a vital role in biological processes through various different mechanisms, in particular for the biosynthesis of collagen. The aim of the study was to establish the possibility of enhancing the osteogenic differentiation potential by manipulating the cellular micro-environment, through AS supplementation in human gingival mesenchymal stem cells (hGMSCs) at different concentrations, such as 60 and 90 µg/mL, for three weeks. Human GMSCs are considered a stem cell population, easily obtainable and displaying a remarkable immunotherapeutic potential and regenerative repair expression. Osteogenic differentiation level induced from AS was assayed by histochemical characterization, using light microscopy through Alizarin red S staining. The transcript levels of Collagen 1A1 (COL1A1), runtrelated transcription factor 2 (RUNX2), bone morphogenetic protein 2/4 (BMP2/4), osteopontin (OPN) and osteonectin (SPARC) were determined by quantitative RT-PCR. Protein expression of COL1A1, RUNX2, BMP2/4, OPN, SPARC were studied through Western blotting and confocal laser scanning microscopy (CLSM). Our results demonstrate that AS supports osteogenic differentiation in stem cells from gingiva niche as shown by osteogenic marker upregulation and by de novo production of calcium phosphate deposits as revealed by Alizarin red S staining. In summary, the results of the current study provide evidence that hGMSCs undergo osteogenic differentiation with AS treatment, for that reason AS could be a promising candidate for the prevention and healing of bone-related diseases.


Assuntos
Ácido Ascórbico/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Células Cultivadas , Gengiva/citologia , Humanos
5.
Int. j. morphol ; 37(4): 1229-1233, Dec. 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1040117

RESUMO

SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.


RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.


Assuntos
Humanos , Células do Tecido Conjuntivo , Técnicas de Cultura de Células/métodos , Bioengenharia/métodos , Gengiva/citologia , Biologia Celular , Fibroblastos
6.
Adv Exp Med Biol ; 1197: 55-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31732934

RESUMO

Gingival epithelium plays a pivotal role in protecting the underlying periodontium from the microbial colonization found in the gingival sulcus. Having an appropriate phenotype displayed by gingival epithelial cells is a critical host component required for protection against bacterial invasion into gingival tissues. In the present study, gingival epithelial homeostasis associated with the CXCL-8/IL-8 chemokine response was investigated in vitro to determine the mechanisms that gingival epithelial cells utilize for sensing gram-positive and gram-negative microorganisms. The findings of this study have demonstrated, by using Fusobacterium nucleatum, a heterogeneity of gingival epithelial cell response by Toll-like receptor (TLR) 2, a lipoprotein sensor. Notably, however, lipopolysaccharide (LPS), a major virulence factor of gram-negative bacteria, is not recognized by gingival epithelial cells unless the LPS is internalized into the cells. Activation of TLR4 in gingival epithelial cells occurs in the endosome, an intracellular event that requires a vesicular acidification to turn on TLR4 signaling, indicating their stringency for fine-tuning a local LPS response. This study has identified a unique LPS sensing mechanism of the oral epithelium to overcome a periodontal infection associated with LPS derived from gram-negative microbes that arises during dysbiosis.


Assuntos
Gengiva , Lipopolissacarídeos , Periodontite , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Gengiva/citologia , Gengiva/imunologia , Gengiva/microbiologia , Humanos , Interleucina-8/imunologia , Lipopolissacarídeos/metabolismo , Periodontite/imunologia , Periodontite/microbiologia
7.
Drug Des Devel Ther ; 13: 3291-3306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571831

RESUMO

Objectives: This study was performed to evaluate the effects of muscone on the proliferation, migration and differentiation of human gingival mesenchymal stem cells (GMSCs) and to explore the relevant mechanisms. Materials and methods: We performed studies to determine the effects and mechanisms of muscone on GMSC proliferation, migration and differentiation. We conducted CCK-8, colony formation, transwell chamber, scratch wound, alkaline phosphatase (ALP) staining and activity, and alizarin red and oil red O staining assays, as well as real-time quantitative polymerase chain reaction (qRT-PCR), to ascertain the effects of muscone on GMSC proliferation, migration and differentiation in vitro. The mechanism by which muscone influences the osteogenic and adipogenic differentiation of GMSCs was elucidated by qRT-PCR and Western blotting. Results: We found that muscone significantly promoted GMSC proliferation, chemotaxis, wound healing and fat droplet formation and inhibited ALP activity and mineral deposition. Notably, we observed that the Wnt/ß-catenin pathway was closely related to the ability of muscone to inhibit the osteogenic differentiation and promote the adipogenic differentiation of GMSCs. The effect of muscone on the multidirectional differentiation capacity of GMSCs was significantly reversed by the agonist lithium chloride through the Wnt/ß-catenin signaling pathway. Conclusion: Muscone effectively increased the proliferation and migration, promoted the adipogenic differentiation and inhibited the osteogenic differentiation of GMSCs by inhibiting the Wnt/ß-catenin signaling pathway. These results may provide a theoretical basis for the application of GMSCs and muscone in tissue engineering and regenerative medicine.


Assuntos
Adipogenia/efeitos dos fármacos , Cicloparafinas/farmacologia , Gengiva/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Adipogenia/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Humanos , Cloreto de Lítio/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
8.
Molecules ; 24(19)2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31574951

RESUMO

Shikonin, an active ingredient of Lithospermum erythrorhizon, exerts anti-inflammatory and antibacterial effects, and promotes wound healing. We investigated whether shikonin stimulated gingival tissue wound healing in human gingival fibroblasts (hGF). In addition, we evaluated the effects of shikonin on the mitogen-activated protein kinase (MAPK) signaling pathway, which has an important role in wound healing. hGF were subjected to primary culture using gingiva collected from patients. The cells were exposed to/treated with Shikonin at concentrations ranging from 0.01 to 100 µM. The optimal concentration was determined by cell proliferation and migration assays. Type I collagen and fibronectin synthesis, the gene expression of vascular endothelial growth factor (VEGF) and FN, and the phosphorylation of Extracellular signal-regulated kinase (ERK) 1/2 were investigated. Identical experiments were performed in the presence of PD98059 our data suggest, a specific ERK 1/2 inhibitor. Shikonin significantly promoted hGF proliferation and migration. Shikonin (1 µM) was chosen as the optimal concentration. Shikonin promoted type I collagen and FN synthesis, increased VEGF and FN expression, and induced ERK 1/2 phosphorylation. These changes were partially suppressed by PD98059. In conclusion, Shikonin promoted the proliferation, migration, type I collagen and FN synthesis, and expression of VEGF and FN via ERK 1/2 signaling pathway in hGFs. Therefore, shikonin may promote periodontal tissue wound healing.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Fibroblastos/metabolismo , Gengiva/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Naftoquinonas/farmacologia , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Lactato Desidrogenases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacos
9.
Nat Commun ; 10(1): 4496, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582750

RESUMO

Solitary chemosensory cells (SCCs) are epithelial sentinels that utilize bitter Tas2r receptors and coupled taste transduction elements to detect pathogenic bacterial metabolites, triggering host defenses to control the infection. Here we report that SCCs are present in mouse gingival junctional epithelium, where they express several Tas2rs and the taste signaling components α-gustducin (Gnat3), TrpM5, and Plcß2. Gnat3-/- mice have altered commensal oral microbiota and accelerated naturally occurring alveolar bone loss. In ligature-induced periodontitis, knockout of taste signaling molecules or genetic absence of gingival SCCs (gSCCs) increases the bacterial load, reduces bacterial diversity, and renders the microbiota more pathogenic, leading to greater alveolar bone loss. Topical treatment with bitter denatonium to activate gSCCs upregulates the expression of antimicrobial peptides and ameliorates ligature-induced periodontitis in wild-type but not in Gnat3-/- mice. We conclude that gSCCs may provide a promising target for treating periodontitis by harnessing innate immunity to regulate the oral microbiome.


Assuntos
Células Quimiorreceptoras/imunologia , Gengiva/imunologia , Imunidade Inata , Microbiota/imunologia , Periodontite/imunologia , Animais , Células Quimiorreceptoras/metabolismo , Modelos Animais de Doenças , Feminino , Gengiva/citologia , Gengiva/microbiologia , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Periodontite/microbiologia , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/imunologia , Canais de Cátion TRPM/metabolismo
10.
Molecules ; 24(20)2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31619000

RESUMO

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.


Assuntos
Catecóis/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada/genética , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/genética , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Gengiva/citologia , Produtos Finais de Glicação Avançada/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Arch Oral Biol ; 107: 104514, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31394382

RESUMO

OBJECTIVE: To investigate the effect of adenosine triphosphate (ATP) on inflammasome activation by Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) stimulation and the anti-inflammatory eff ;ect of doxycycline (Dox) in human gingival fibroblasts (HGFs). DESIGN: The optimal concentration of P. gingivalis-LPS (1.0 µg/mL) for cellular viability was determined by observing cell morphology and measuring the amount of formazan and the expression of pro-caspase-1. The expression of genes and proteins related to the NAcht Leucine-rich repeat Protein 3 (NLRP3) inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, and the inflammatory factor interleukin-1ß (IL-1ß) and its activated forms were measured. RESULTS: The NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by stimulation with P. gingivalis-LPS or ATP. However, a combination of P. gingivalis-LPS and ATP significantly enhanced inflammasome activation and IL-1ß production at the gene and protein levels as measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Furthermore, doxycycline addition markedly inhibited inflammasome activation and IL-1ß production induced by a combination of P. gingivalis-LPS and ATP. CONCLUSIONS: LPS, ATP, and doxycycline play critical roles in regulating host immune responses. This evidence provides guidance for the application of tetracycline drugs for the clinical treatment of periodontal disease.


Assuntos
Doxiciclina/farmacologia , Gengiva/citologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Porphyromonas gingivalis , Trifosfato de Adenosina/farmacologia , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia
12.
Acta Med Okayama ; 73(4): 315-323, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31439954

RESUMO

The periodontal pathogen Porphyromonas gingivalis shows colonial pigmentation on blood agar and produces gingipains (Kgp, RgpA, and RgpB), cysteine proteases involved in an organism's virulence and pigmentation. We showed previously that deletion of the PGN_0300 gene abolished the pigmentation activity and reduced the proteolytic activity of gingipains. The role of the PGN_0297 gene, which consists of an operon with the PGN_0300 gene, is unclear. Herein we examined the effect of PGN_0297 gene deletion on the pigmentation and proteolytic activities and transcriptional levels of gingipains. A PGN_0297 gene deletion mutant (ΔPGN_0297) did not exhibit the pigmentation. The proteolytic activity of the gingipains was decreased in the culture supernatant and on the cell surface of ΔPGN_0297. The mutant ΔPGN_0297 failed to attenuate Akt phosphorylation at Thr308 and Ser473, but both phosphorylations were attenuated in the wild-type and its complementation strain. The deletion of PGN_0297 gene did not substantially affect the transcriptional levels of the gingipain genes kgp, rgpA, and rgpB. Taken together, these results indicate that PGN_0297 is closely involved in the secretion and maturation of gingipains.


Assuntos
Proteínas de Bactérias/metabolismo , Porphyromonas gingivalis/genética , Proteínas de Bactérias/genética , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Gengiva/citologia , Humanos , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Pigmentos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo
13.
Am J Dent ; 32(3): 152-156, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31295398

RESUMO

PURPOSE: To investigate the effect of silver diamine fluoride (SDF) and fluoride varnish (FV) on human gingival fibroblasts (HGF) and bacteria. METHODS: HGF cell viability was assessed after exposure to various dilutions of SDF or FV. Hydroxyapatite (HA) discs treated with SDF, FV, or saline were rinsed in artificial saliva for 84 days. HGF were exposed to treated discs and viability assessed fluorescently. Oral bacteria were exposed to treated discs and survival quantified. RESULTS: At 0.01%, SDF was almost 100% cytotoxic to HGF. SDF and FV treated HA discs, induced near-complete cell death after 24 hours of contact. After rinsing FV discs for 21 days, cell survival exceeded 95%. SDF treated discs were toxic to HGF and bacteria after 9 weeks of rinsing. CLINICAL SIGNIFICANCE: SDF and FV can induce cell death. FV lost its cytotoxicity within 3 weeks, while SDF remained cytotoxic even after 9 weeks of rinsing. This research confirms that SDF has long lasting antimicrobial effects at very low concentrations although it does raise concerns regarding cytotoxicity. However, HGF cells are exposed to other cytotoxic substances in dentistry with little, if any, long-term effects.


Assuntos
Fluoretos Tópicos , Compostos de Amônio Quaternário , Compostos de Prata , Fluoretos , Fluoretos Tópicos/toxicidade , Gengiva/citologia , Gengiva/efeitos dos fármacos , Humanos , Compostos de Amônio Quaternário/toxicidade , Compostos de Prata/toxicidade
14.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269731

RESUMO

Bone tissue regeneration strategies require approaches that provide an osteogenic and angiogenic microenvironment able to drive the bone growth. Recently, the development of 3D printing biomaterials, including poly(lactide) (3D-PLA), enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as extracellular vesicles (EVs) has been achieving promising results. In this study, in vitro results showed an increased expression of osteogenic and angiogenic markers, as RUNX2, VEGFA, OPN and COL1A1 in the living construct 3D-PLA/human Gingival MSCs (hGMSCs)/EVs. Considering that EVs carry and transfer proteins, mRNA and microRNA into target cells, we evaluated miR-2861 and miR-210 expression related to osteoangiogenesis commitment. Histological examination of rats implanted with 3D-PLA/hGMSCs/EVs evidenced the activation of bone regeneration and of the vascularization process, confirmed also by MicroCT. In synthesis, an upregulation of miR-2861 and -210 other than RUNX2, VEGFA, OPN and COL1A1 was evident in cells cultured in the presence of the biomaterial and EVs. Then, these results evidenced that EVs may enhance bone regeneration in calvaria defects, in association with an enhanced vascularization offering a novel regulatory system in the osteoangiogenesis evolution. The application of new strategies to improve biomaterial engraftment is of great interest in the regenerative medicine and can represent a way to promote bone regeneration.


Assuntos
Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Poliésteres/química , Tecidos Suporte/química , Animais , Células Cultivadas , Vesículas Extracelulares/genética , Vesículas Extracelulares/transplante , Gengiva/citologia , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Osteogênese , Impressão Tridimensional , Ratos Wistar , Regulação para Cima
15.
Saudi Med J ; 40(7): 657-668, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31287125

RESUMO

OBJECTIVES: To investigate the use of leukocyte-platelet rich fibrin on suppressing the porphyromonas gingivalis (PG-LPS)-induced secretion of proinflammatory cytokines. Methods:This quantitative experimental study was conducted at the School and Hospital of Stomatology, Jilin University, Changchun, China, between September 2017 and January 2019. A modified technique was used to obtain human gingival fibroblast cells (HGFCs). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Cell Counting Kit-8 tests were established to determine the proliferation rate. Human gingival fibroblast cells were treated by PG-LPS at different periods and the isolated mRNA was subjected to reverse transcription polymerase chain reaction and real time quantitative polymerase chain reaction. The release of platelet-derived growth factor and transforming-growth factor-ß1 at various time intervals was observed. Results: We successfully established a modified technique for the production of HGFCs culture. One µg/mL PG-LPS was the recommended concentration to inhibit fibroblast proliferation. The expression of the pro-inflammatory cytokines messenger ribnucleic acid was notably raised at 3 and 6 hours post-PG-LPS treatment. The cumulative release of growth factors peaked during the first 24 hours and the production continued for 10 days. However, the fibroblast expression of cytokines was significantly suppressed after treatment with leucocyte- and platelet-rich fibrin (L-PRF). Conclusion: This study provided a novel way of obtaining HGFCs and greater understanding of the clinical impacts through the assessment of the anti-inflammatory properties of L-PRF in vitro.


Assuntos
Citocinas/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fibrina Rica em Plaquetas , Porphyromonas gingivalis/metabolismo , Estomatite , Animais , Técnicas de Cultura de Células , Proliferação de Células , Citocinas/genética , Citocinas/imunologia , Fibroblastos/imunologia , Gengiva/citologia , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase em Tempo Real
16.
Arch Oral Biol ; 105: 81-87, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31288145

RESUMO

AIMS: We investigated the effect of a specific inhibitor of the receptor for advanced glycation (FPS-ZM1) against lipopolysaccharide (LPS)-induced increase in expressions of high mobility group protein B1 (HMGB1) and interleukin-6 (IL-6) in human gingival fibroblasts (HGFs). Furthermore, we explored the potential molecular mechanisms and assessed the involvement of the NF-κB pathway in mediating the changes in the expressions of HMGB1 and IL-6 expression in response to LPS and FPS-ZM1. METHODS: HGFs were cultured with enzymatic digestion-tissue explants method. The proliferation of LPS-stimulated HGFs pretreated with FPS-ZM1 at 24, 48, and 72 h was determined by cell counting kit 8 assay. The expressions of HMGB1 and IL-6 were measured using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Western blot analysis was used to assess the expressions of receptor for advanced glycation end products (RAGE) and NF-κB. RESULTS: LPS enhanced the protein expression of RAGE in HGFs. At the same time, LPS stimulated mRNA and protein expressions of HMGB1 and IL-6 in HGFs. However, pretreatment with FPS-ZM1 attenuated these effects. Pretreatment with FPS-ZM1 (250, 500 nM) significantly inhibited the LPS-induced NF-κB activity. CONCLUSION: FPS-ZM1 down-regulated the LPS-induced HMGB1 and IL-6 expression in HGFs through blocking NF-κB activation. FPS-ZM1 is a promising therapeutic agent for inflammatory diseases caused by oral bacteria.


Assuntos
Proteína HMGB1/metabolismo , Interleucina-6/metabolismo , NF-kappa B , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Transdução de Sinais , Benzamidas/farmacologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Humanos , Lipopolissacarídeos
17.
Int J Pharm ; 569: 118564, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31352049

RESUMO

Control of infection and inflammation is crucial for the success of periodontal treatment. In this study, in-situ forming implants (ISFI) loaded with chlorhexidine dihydrochloride (CHX) and ibuprofen (IBU) were developed and tested to optimize periodontal treatment outcomes. Release profiles were promising. Exposure to 1.5% and 5.3% CHX-IBU loaded ISFI's release media decreased significantly the P. gingivalis growth up to 20-fold and 35-fold, respectively, after 48 h (p < 0.05). The metabolic activity assay of gingival epithelial cells (EC) demonstrated 1.5% CHX-IBU-loaded ISFI to be non-toxic, therefore, it was selected for further experimentation. Furthermore, significant down-regulation of TNF-α release (34% at 6 h and 43% at 24 h, p < 0.05) in P. gingivalis lipopolysaccharide (Pg-LPS) stimulated EC exposed to 1.5% CHX-IBU ISFI release medium was demonstrated by ELISA. In vivo, 1.5% CHX-IBU ISFI was injected into the periodontal pocket in an experimental periodontitis mouse model and the reduction in inflammation and improvement in periodontal wound healing was evaluated through inflammatory cell scoring and histomorphometry at 7- and 15-days post-treatment. The results indicate that CHX-IBU loaded ISFI could be efficient as adjuvant to periodontal therapy for the control of infection and inflammation. Moreover, other (e.g., pro-regenerative) drugs could be incorporated into ISFI to further improve periodontal treatment outcomes.


Assuntos
Anti-Infecciosos Locais/administração & dosagem , Anti-Inflamatórios não Esteroides/administração & dosagem , Clorexidina/administração & dosagem , Ibuprofeno/administração & dosagem , Periodontite/tratamento farmacológico , Animais , Anti-Infecciosos Locais/química , Anti-Inflamatórios não Esteroides/química , Linhagem Celular , Clorexidina/química , Implantes de Medicamento , Liberação Controlada de Fármacos , Células Epiteliais/efeitos dos fármacos , Gengiva/citologia , Humanos , Ibuprofeno/química , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Estudo de Prova de Conceito , Cicatrização/efeitos dos fármacos
18.
Toxicol In Vitro ; 60: 252-260, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31195088

RESUMO

Universal adhesives are the most important innovation in restorative dentistry. They are composed of different monomers, solvents and fillers. The potential cytotoxic effect of these materials is an important scientific aspect in recent literature. The aim of this study was to determine, using different in vitro techniques, the cytotoxicity evaluation of seven universal enamel-dental adhesives on human gingival fibroblasts. For this purpose, seven universal dental enamel adhesives have been evaluated by in vitro cytotoxicity tests using direct contact tests (an unpolymerized and a polymerized method) and an indirect contact test: preparation of extracts. The polymerized method showed a cytotoxicity range from 36% (G-PremioBond, GPB) to 79% (FuturaBond M+, FB). With the unpolymerized direct methods the range was from 4% (Prime&Bond Active, PBA) to 40% (Ibond Universal, IB) for undiluted adhesives; generally passing to the major dilutions the test showed a strong inhibitory activity by all the adhesives. Whereas with the indirect method by diluting the extracts of all dental adhesives the cell viability increased. The data obtained from the work has shown a lower cytotoxic effect of Optibond Solo Plus (OB) and Adhesive Universal (AU) with more reliable results with the extracts technique. The choice of reliable in vitro cytotoxic technique could represent, in dental practice, an important aid for clinical procedures in the use of adhesive systems.


Assuntos
Cimentos Dentários/toxicidade , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Metacrilatos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos
19.
Bull Exp Biol Med ; 167(1): 47-49, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31177448

RESUMO

We studied the influence of synthetic indolicidin analogues on the development of acute periodontitis. The corrective effect was found in indolicidin analogues Nos. 7 and 8; it manifested in a decrease in the edema of gingival epithelium and lamina propria, a decrease in the relative area of inflammatory infiltrates, and a significant increase in the relative area of normal connective tissue. These changes were revealed as soon as on day 14 and were most pronounced in 21 days after the removal of the ligature. Indolicidin analogues Nos. 7 and 8 demonstrated similar effectiveness on the model of acute periodontitis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Periodontite/tratamento farmacológico , Animais , Peptídeos Catiônicos Antimicrobianos/química , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Membrana Mucosa/efeitos dos fármacos , Membrana Mucosa/metabolismo , Ratos Wistar
20.
Int J Biol Macromol ; 136: 823-830, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228504

RESUMO

Chitosan as a biopolymer is an attractive vehicle for biomedical applications due to its unique characteristics. In order to improve chitosan's physicochemical features, chemical modification has been carried out to make it more suitable for such approaches. The aim of this study was to prepare and evaluate thiolated chitosan-lauric acid as a new chitosan derivative for biomedical use. Lauric acid was introduced to chitosan via stable amide bond between carboxylic acid group of fatty acid and the amine in the chitosan and thiolation was carried out using thioglycolic acid. Resulted polymers were characterized by FTIR, 1H NMR and TGA. Moreover, cell viability assessment of new derivative was performed using MTT method. FTIR and 1H NMR results showed that both substitution reactions were successfully completed. Furthermore, new synthesized polymer had no significant cytotoxicity against normal gingiva human cells (HGF1-PI 1).These findings confirm that this new derivative can be introduced as a suitable polymer for biomedical purposes such as mucosal drug delivery.


Assuntos
Quitosana/síntese química , Quitosana/toxicidade , Citotoxinas/síntese química , Citotoxinas/toxicidade , Ácidos Láuricos/química , Compostos de Sulfidrila/química , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Quitosana/química , Citotoxinas/química , Relação Dose-Resposta a Droga , Gengiva/citologia , Humanos , Interações Hidrofóbicas e Hidrofílicas
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