RESUMEN
BACKGROUND: Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. OBJECTIVE: To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. METHODOLOGY: HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). RESULTS: After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. CONCLUSION: The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.
Asunto(s)
Pulpitis , Humanos , Pulpitis/metabolismo , FN-kappa B , Pulpa Dental , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Escherichia coli/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Células CultivadasRESUMEN
Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.
RESUMEN
The aim of this study was to assess the effects of IL-6 and IL-8 cytokines on human gingival fibroblasts (HGF) cultured in a 3-D model and the possible photobiomodulation (PBM) of such effects by low-level laser therapy. In complete culture medium (DMEM), HGF from a healthy patient were seeded in a type I collagen matrix inserted into 24-well plates. After 5 days of incubation, the cytokines were added or not to serum-free DMEM, which was applied to the cell-enriched matrices. Then, PBM was performed: three consecutive irradiations using LaserTable diode device (780 nm, 0.025 W) at 0.5 J/cm2 were delivered or not to the cells. Twenty-four hours after the last irradiation, cell viability and morphology, gene expression, and synthesis of inflammatory cytokines and growth factors were assessed. The histological evaluation demonstrated that, for all groups, matrices presented homogeneous distribution of cells with elongated morphology. However, numerous cytokine-exposed cells were rounded. IL-6 and IL-8 decreased cell viability, synthesis of VEGF, and gene expression of collagen type I. PBM enhanced cell density in the matrices and stimulated VEGF expression, even after IL-6 challenge. Reduced TNF-α synthesis occurred in those cells subjected to PBM. In conclusion, PBM can penetrate collagen matrix and stimulate HGF, highlighting the relevance of this research model for further phototherapy studies and in vitro biomodulation of the healing process.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citocinas/metabolismo , Fibroblastos/patología , Fibroblastos/efectos de la radiación , Encía/patología , Inflamación/patología , Terapia por Luz de Baja Intensidad , Modelos Biológicos , Supervivencia Celular/efectos de la radiación , Colágeno Tipo I/metabolismo , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Interleucina-1beta/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
OBJECTIVES: To assess the biological, antimicrobial, and mechanical effects of the treatment of deep dentin with simvastatin (SV) before application of a glass-ionomer cement (GIC). MATERIALS AND METHODS: Dentin discs were adapted to artificial pulp chambers and SV (2.5 or 1.0 mg/mL) was applied to the occlusal surface, either previously conditioned or not with EDTA (±EDTA). The extracts (culture medium + SV that diffused through dentin) was obtained and then applied to cultured odontoblast-like MDPC-23 cells. Cell viability, alkaline phosphatase (ALP) activity, and mineralization nodule (MN) deposition were evaluated. Untreated discs were used as control. The antibacterial activity of SV (2.5 or 1.0 mg/mL) against Streptococcus mutans and Lactobacillus acidophilus, as well as the bond strength of GIC to dentin in the presence of SV 2.5 mg/mL (±EDTA) were also assessed. The data were analyzed by ANOVA/Tukey tests (α = 5%). RESULTS: EDTA + SV 2.5 mg/mL significantly enhanced the ALP activity and MN deposition in comparison with the control, without changing in the cell viability (p < 0.05). The association EDTA + SV 2.5 mg/mL + GIC determined the highest ALP and MN values (p < 0.05). SV presented intense antimicrobial activity, and the EDTA dentin conditioning followed by SV application increased bond strength values compared with SV treatment alone (p < 0.05). CONCLUSION: SV presents antimicrobial activity and diffuses across conditioned dentin to biostimulate odontoblast-like pulp cells. CLINICAL SIGNIFICANCE: The use of SV as adjuvant agent for indirect pulp capping may biostimulate pulp cells thus preserving vitality and function of the pulp-dentin complex.
Asunto(s)
Recubrimiento de la Cavidad Dental , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Simvastatina , Dentina/efectos de los fármacos , Dentina/microbiología , Cementos de Ionómero Vítreo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Odontoblastos , Simvastatina/uso terapéuticoRESUMEN
The restoration of dentine-pulp complex remains a challenge for dentists; nonetheless, it has been poorly addressed. An ideal system should modulate the host response, as well as enable the recruitment, proliferation and differentiation of relevant progenitor cells. Herein was proposed a photocrosslinkable hydrogel system based on hyaluronic acid (HA) and platelet lysate (PL). PL is a cocktail of growth factors (GFs) and cytokines involved in wound healing orchestration, obtained by the cryogenic processing of platelet concentrates, and was expected to provide the HA hydrogels specific biochemical cues to enhance pulp cells' recruitment, proliferation and differentiation. Stable HA hydrogels incorporating PL (HAPL) were prepared after photocrosslinking of methacrylated HA (Met-HA) previously dissolved in PL, triggered by the Ultra Violet activated photoinitiator Irgacure 2959. Both the HAPL and plain HA hydrogels were shown to be able to recruit cells from a cell monolayer of human dental pulp stem cells (hDPSCs) isolated from permanent teeth. The hDPCs were also seeded directly over the hydrogels (5 × 104 cells/hydrogel) and cultured in osteogenic conditions. Cell metabolism and DNA quantification were higher, in all time-points, for PL supplemented hydrogels (p < 0,05). Alkaline phosphatase (ALPL) activity and calcium quantification peaks were observed for the HAPL group at 21 days (p < 0,05). The gene expression for ALPL and COLIA1 was up-regulated at 21 days to HAPL, compared with HA group (p < 0,05). Within the same time point, the gene expression for RUNX2 did not differ between the groups. Overall, data demonstrated that the HA hydrogels incorporating PL increased the cellular metabolism and stimulate the mineralized matrix deposition by hDPSCs, providing clear evidence of the potential of the proposed system for the repair of damaged pulp/dentin tissue and endodontics regeneration.
Asunto(s)
Plaquetas/citología , Ácido Hialurónico/química , Hidrogeles/química , Células Madre/citología , Fosfatasa Alcalina/metabolismo , Calcio/química , Diferenciación Celular , Proliferación Celular , Quimiotaxis , Reactivos de Enlaces Cruzados/química , Pulpa Dental/citología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Osteogénesis , Fotoquímica , Regeneración , Ingeniería de Tejidos , Diente/citologíaRESUMEN
Reepithelialization and wound closure are the desired outcome for several ulcerative conditions. Such resolution reduces the possibility of wound contamination and maintenance of the injury and improves the reestablishment of tissue morphology and functions. Investigators are seeking adjuvant therapies that can accelerate wound healing and are developing new strategies for clinical applications. This study compared the effects of epidermal growth factor (EGF) application and low-level laser therapy (LLLT) on cultured epithelial cells. Cells were seeded in 24-well plates. After a 24-h incubation, the epithelial cells were either treated with EGF (100 µM in serum-free DMEM for 72 h) or subjected to LLLT (780 nm, 25 mW, 0.5, 1.5, and 3 J/cm2) by three applications every 24 h. Seventy-two hours after cells were treated with EGF or LLLT, cell migration, viability, proliferation, and collagen synthesis were assessed. Cells treated with EGF showed increased cell viability, proliferation, and collagen synthesis compared with those cells that received no treatment. LLLT enhanced cell migration; however, no significant effects of laser irradiation on other cell functions were observed. Comparison of both therapies demonstrated that EGF and LLLT enhanced specific epithelial cell activities related to wound healing.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Terapia por Luz de Baja Intensidad , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Colágeno/biosíntesis , Células Epiteliales/efectos de los fármacos , HumanosRESUMEN
This study assessed the effects of photobiomodulation (PBM) to cells previously exposed to lipopolysaccharides (LPS). Human gingival fibroblasts (HGF) and epithelial cells (HaCaT) were seeded in wells of 24-well plates containing complete culture medium (DMEM). After 24 h, the DMEM was replaced by serum-free DMEM, and cells were exposed to LPS of Escherichia coli (E. coli) (10 µg mL-1 ) for 24, 48, and 72 h. The cells were subjected to specific parameters of phototherapy (PT) (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J cm-2 ). Cell proliferation (alamarBlue® ), viability (Trypan Blue) and synthesis of CCL2 (ELISA) were evaluated. Data were statistically analyzed by the Kruskal-Wallis and Mann-Whitney test (α = 5%). Proliferation and viability of both cell lines decreased after LPS treatment at 48 and 72 h. Enhanced synthesis of CCL2 by gingival fibroblasts occurred at 24 h, while epithelial cells increased synthesis of this chemokine at 48 and 72 h. PBM enhanced cell proliferation and viability in a time-dependent manner for both cell lines exposed or not to LPS, while synthesis of CCL2 by cells exposed to PT decreased over time. PBM caused biomodulatory effects on gingival fibroblasts and epithelial cells previously treated with LPS. These effects may decrease tissue inflammatory response and accelerate wound healing of oral mucosal tissue.
Asunto(s)
Encía/efectos de los fármacos , Lipopolisacáridos/farmacología , Procesos Fotoquímicos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Medio de Cultivo Libre de Suero , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Escherichia coli/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Encía/citología , Encía/metabolismo , HumanosRESUMEN
Several in vitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy-induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low-level laser therapy (LLLT). Oral keratinocytes were seeded in the ex vivo-produced oral mucosal equivalent (EVPOME) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780 nm, 25 mW) at 0.5, 1.5 and 3 J cm-2 . After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF) as well as epidermal (hEGF) growth factors. In general, analysis of these data shows that the three-dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model.
Asunto(s)
Dermis Acelular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Encía/citología , Queratinocitos/efectos de la radiación , Terapia por Luz de Baja Intensidad , Dermis Acelular/veterinaria , Análisis de Varianza , Animales , Técnicas de Cultivo de Célula , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Factor 7 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/efectos de la radiación , Humanos , Queratinocitos/citología , PorcinosRESUMEN
This study evaluated the effects of low-level laser therapy (LLLT) and epidermal growth factor (EGF) on fibroblasts obtained from young and elderly individuals. Gingival fibroblasts from young (Y) and elderly (E) individuals were seeded in wells of 24-well plates with Dulbecco's modified Eagle's medium (DMEM) containing 10 % of fetal bovine serum (FBS). After 24 h, the cells were irradiated (LASERTable-InGaAsP-780 ± 3 nm, 25 mW, 3 J/cm2) or exposed to EGF (100 µM). After 72 h, cells were evaluated for viability, migration, collagen and vascular endothelial growth factor (VEGF) synthesis, and gene expression of growth factors. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 5 %). Y and E fibroblasts irradiated with laser or exposed to EGF showed increased viability and collagen synthesis. Enhanced cell migration was observed for Y fibroblasts after both treatments, whereas only the LLLT stimulated migration of E cells. VEGF synthesis was higher for Y and E cells exposed to EGF, while this synthesis was reduced when E fibroblasts were irradiated. Increased gene expression of VEGF was observed only for Y and E fibroblasts treated with LLLT. Regardless of a patient's age, the LLLT and EGF applications can biostimulate gingival fibroblast functions involved in tissue repair.
Asunto(s)
Fibroblastos/citología , Fibroblastos/efectos de la radiación , Encía/citología , Terapia por Luz de Baja Intensidad , Factor A de Crecimiento Endotelial Vascular/farmacología , Adolescente , Adulto , Anciano , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Colágeno/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Terapia por Láser , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adulto JovenRESUMEN
Introdução: A candidose oral é uma infecção fúngica que se manifesta frequentemente em pacientes imunocomprometidos ou naqueles que fazem uso de prótese dental removível, associada a hábitos de higiene deficitários. Antifúngicos sintéticos, a exemplo do fluconazol, são utilizados no tratamento desta infecção; entretanto algumas cepas apresentam resistência a estes fármacos. Objetivo: Este estudo avaliou o efeito antifúngico dos óleos essenciais de Persea americana (abacate), Cinnamomumzeylanicum (canela folha), Cinnamomumcassia (canela casca) e Cymbopogonwinterianus (citronela), frente à Candidaglabrata. Material e Métodos: O screening da atividade antifúngica dos óleos foi determinado por difusão em meio sólido, utilizando um inóculofúngico ajustado em 1 × 106 UFC/mL. A Concentração Inibitória Mínima (CIM) foi determinada pela técnica da microdiluição. Os óleos essenciais foram avaliados em concentrações entre 1000 µg/mL e 7,81 µg/mL, enquanto que os fármacos Fluconazol e Nistatina foram avaliados nas concentrações que entre 64 µg/mL e 0,5 µg/mL. Resultados: Os halos de inibição mensurados variaram entre 8,2 e 9,2 mm de diâmetro, respectivamente para C. winterianuse C. cassia.Os óleos essenciais de C. winterianuse C. zeylanicumapresentaram CIM de 125 µg/mL, enquanto a CIM de C. cassiafoi 62,5 µg/mL. A CIM dos fármacos utilizados como controle foram estabelecidasem 16 µg/mL(fluconazol) e 2,0 µg/mL(nistatina). O óleo essencial de P. americana não apresentou atividade antifúngica nas concentrações avaliadas. Conclusão: Conclui-se que os óleos essenciais de canela (casca e folha) e citronela apresentaram atividade antifúngica frente a cepa de C. glabrataresistente a fluconazol. (AU)
Introduction: Oral candidiasis is a fungal infection diagnosed mainly in patients with immunosuppression or in denture wearers with deficient hygiene habits. Synthetic antifungal agents, such as fluconazole, have been used to treat this infection, but some strains are resistant to these drugs. Objective: This study aimed to evaluate the antifungal effect of the essential oils from Persia americana (avocado), Cinnamomum zeylanicum (cinnamon - leaf), Cinnamomum cassia (cinnamon - bark) and Cymbopogon winterianus (citronella) against Candida glabrata. Materials and Methods: The essential oils were screened for their antifungal activity using the solid medium diffusion method, with fungal inoculum adjusted to 1 x 106 CFU/mL. The Minimum Inhibitory Concentration (MIC) was determined by the microdilution technique. The essential oils were evaluated at concentrations ranging between 1,000 µg/mL and 7.81 µg/mL, while fluconazole and nystatin were evaluated at concentrations between 64 µg/mL and 0.5 µg/ mL. Results: The zones of inhibition measured varied between 8.2 and 9.2 mm diameter for C. winterianus and C. cassia, respectively. The essential oils from C. winterianusand and C. zeylanicum had MIC of 125 µg/ml, while C. cassia essential oil had MIC of 62.5 µg/ml. The drugs used as controls showed MIC values of 16 µg/mL (fluconazole) and 2.0 µg/mL (nystatin). P. americana essential oil showed no antifungal activity at the concentrations evaluated. Conclusion: We conclude that the essential oils from cinnamon (bark and leaf) and citronella showed antifungal activity against fluconazole-resistant C. glabrata. (AU)
Asunto(s)
Humanos , Candida , Fitoterapia , Productos con Acción AntimicrobianaRESUMEN
Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.
Asunto(s)
Odontoblastos/metabolismo , Fototerapia , Células Cultivadas , HumanosRESUMEN
Abstract Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.
Resumo Fototerapia tem sido indicada como um tratamento adjuvante para o reparo de tecidos, incluindo o tecido pulpar. Entretanto, não há parâmetros de irradiação definidos, o que representa um grande desafio para o uso clínico da fototerapia. O objetivo deste estudo foi avaliar o efeito da fototerapia com LED vermelho em células MDPC-23 com fenótipo odontoblastóide, usando vários parâmetros. As células foram semeadas (104 células/cm2), incubadas por 12 h em DMEM completo e então o meio de cultura foi trocado por DMEM com 0,5% SFB. Após 12 h de incubação, as irradiações foram realizadas (630±10 nm) usando um dispositivo com densidade de potência de 20 ou 40 mW/cm2 e dose de energia de 2 J/cm2. As células foram irradiadas 1 ou 3 vezes, com intervalos de 1 min. Células não irradiadas serviram como controle. Foram avaliadas a viabilidade (ensaio de MTT), dosagem de proteína total (método de Lowry) e número de células viáveis (ensaio de Trypan blue). Os dados (n=12 por grupo) foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (p=0,05). Uma única irradiação com 20 ou 40 mW/cm2 aumentou a viabilidade celular, a qual foi negativamente afetada após 3 irradiações. Células irradiadas apenas uma vez com 20 mW/cm2 produziram mais proteínas comparadas com aquelas irradiadas com 40 mW/cm2. Redução no número de células viáveis ocorreu apenas após 3 irradiações com 40 mw/cm2. Em conclusão, o LED vermelho foi capaz de biomodular a atividade metabólica de células MDPC-23. A melhor bioestimulação celular foi obtida quando uma única irradiação com dose de energia de 2 J/cm2 e densidade de potência de 20 mW/cm2 foi administrada às células pulpares.
Asunto(s)
Humanos , Odontoblastos/metabolismo , Fototerapia , Células CultivadasRESUMEN
BACKGROUND AND OBJECTIVES: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines. METHODS: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05). RESULTS: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6. CONCLUSION: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Citocinas/metabolismo , Fibroblastos/efectos de la radiación , Láseres de Semiconductores/uso terapéutico , Terapia por Luz de Baja Intensidad/métodos , Mucosa Bucal/efectos de la radiación , Estomatitis/radioterapia , Cicatrización de Heridas/efectos de la radiación , Adulto , Biomarcadores/metabolismo , Movimiento Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Fibroblastos/fisiología , Regulación de la Expresión Génica/efectos de la radiación , Encía/fisiología , Encía/efectos de la radiación , Humanos , Mucosa Bucal/fisiología , Estomatitis/genética , Estomatitis/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
Besides extensive data about the effects of low-level laser therapy (LLLT) on different cell types, so far, these results were obtained from monolayer cell culture models, which have limitations in terms of cell morphology and phenotype expression. Therefore, for better in vitro evaluation of the effects of LLLT, this study was performed with a 3D cell culture model, where gingival fibroblasts were seeded in collagen matrix. Cells isolated from a healthy patient were seeded in wells of 24-well plates with culture medium (DMEM) supplemented with 10 % fetal bovine serum and collagen type I solution. After 5 days, a serum-free DMEM was added to the matrices with cells that were subjected or not to three consecutive irradiations of LLLT by means of the LaserTABLE diode device (780 nm, 25 mW) at 0.5, 1.5, and 3 J/cm(2). Twenty-four hours after the last irradiation, cell viability and morphology as well as gene expression of growth factors were assessed. Histological evaluation of matrices demonstrated uniform distribution and morphology of gingival fibroblasts within the collagen matrix. LLLT at 3 J/cm(2) increased gingival fibroblast viability. Enhanced gene expression of hCOL-I and hEGF was observed for 0.5 J/cm(2), while no significant changes were detected for the other irradiation densities tested. In conclusion, LLLT promoted biostimulation of gingival fibroblasts seeded in a 3D cell culture model, demonstrating that this model can be applied for phototherapy studies and that LLLT could penetrate the collagen matrix to increase cell functions related to tissue repair.
Asunto(s)
Fibroblastos/efectos de la radiación , Encía/citología , Terapia por Luz de Baja Intensidad/métodos , Técnicas de Cultivo de Célula , Supervivencia Celular , HumanosRESUMEN
INTRODUCTION: The discovery of new antimicrobials derived from plants could aid in the management of biofilm-associated infections, including denture-induced stomatitis (DS). DS is an oral infection caused by Candida biofilms on the surfaces of poorly cleansed dentures. Effective treatment of DS requires the use of an appropriate denture cleanser and preferably one that exhibits antimicrobial properties. OBJECTIVE: This study aimed to evaluate the anti-Candida and anti-biofilm efficacy of two essential plant oils from Cymbopogon winterianus (citronella) and Cinnamon cassia (cinnamon). MATERIALS AND METHODS: Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs) were determined by broth microdilution, whilst anti-biofilm activity was measured against mature (cultured for 72 h) biofilms on acrylic surfaces. Candida cell viability was assessed immediately (0 h) after treatment (T0) and 48 h after biofilm re-growth (T48). Biofilm structure was determined using Scanning Electron Microscopy (SEM) at T0 and T48. RESULTS: The respective MICs of cinnamon and citronella oils were 65 and 250 µg/ml and these were also the MFC values. For anti-biofilm efficacy, both oils significantly (p < 0.05) reduced the number of viable micro-organisms and accumulation of biofilms at T0. However, at T48, there was no difference between treated and untreated biofilms. CONCLUSIONS: It is concluded that citronella and cinnamon essential oils have potential for daily anti-candidal denture cleansing.
Asunto(s)
Antifúngicos/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Cinnamomum , Cymbopogon , Aceites Volátiles/farmacología , Aceites de Plantas/farmacología , Acroleína/análogos & derivados , Acroleína/farmacología , Resinas Acrílicas/química , Monoterpenos Acíclicos , Aldehídos/farmacología , Película Dental/microbiología , Limpiadores de Dentadura , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica de Rastreo , Monoterpenos/farmacología , Fitoterapia/métodos , Distribución Aleatoria , Propiedades de Superficie , Factores de TiempoRESUMEN
To evaluate the effect of irradiation with light-emitting diode (LED; 455 nm) on the viability and synthesis of dentin matrix proteins by odontoblast-like cells, MDPC-23 cells were cultivated (10(4) cells/cm(2)) in 24-well culture plates. After 12 h incubation in Dulbecco's modified Eagle's medium (DMEM), the cells were submitted to nutritional restriction by means of reducing the concentration of fetal bovine serum (FBS) for an additional 12 h. Cells were irradiated one single time with one of the following energy densities (EDs): 0.5, 2, 4, 10, or 15 J/cm(2) and irradiance fixed at 20 mW/cm(2). Non-irradiated cells served as control. After 72 h, cells were evaluated with regard to viability (methylthiazol tetrazolium technique (MTT)), mineralization nodule (MN) formation, total protein (TP) production, alkaline phosphatase activity (ALP), and collagen synthesis (Sircol), n = 8. The data were submitted to Kruskal-Wallis and Mann-Whitney tests (p > 0.05). There was no statistical difference between the viability of cells irradiated or not (control), for all the EDs. However, an increase in TP was observed for all the EDs when compared with the control group. A reduced ALP activity was seen in all irradiated groups, except for the ED of 0.5 J/cm(2), which did not differ from the control. There was no difference between the irradiated groups and control regarding collagen synthesis, with the exception of the ED of 10 J/cm(2), which inhibited this cell function. Significant reduction in MN occurred only for the EDs of 0.5 and 2 J/cm(2). The single irradiation with blue LED (455 nm), irradiance of 20 mW/cm(2), and energy densities ranging from 0.5 to 15 J/cm(2) exerted no effective biostimulatory capacity on odontoblast-like cells.
Asunto(s)
Odontoblastos/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de la radiación , Colágeno/biosíntesis , Láseres de Semiconductores , Odontoblastos/efectos de la radiación , Ratas , Calcificación de DientesRESUMEN
Blue light emitting diodes (LEDs) are frequently used in dentistry for light activation of resin-based materials; however, their photobiostimulatory effects have not yet been fully investigated. This study aimed to investigate the effect of blue LED (455 nm) on the metabolism of odontoblast-like cells MDPC-23. Energy doses of 2 and 4 J/cm(2) were used at 20 mW/cm(2) fixed power density. MDPC-23 cells were seeded at 10,000 cells/cm(2) density in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal bovine serum (FBS). After 12 h, the culture medium was replaced with new DMEM supplemented with 0.5 % of FBS, and the cells were incubated for further 12 h. After that, single irradiation was performed to the culture, under selected parameters. Cell viability evaluations (Alamar Blue Assay, n = 12), number of viable cells (Trypan Blue Assay, n = 12), morphological analysis by scanning electron microscopy (SEM, n = 2), gene expression (n = 6) of alkaline phosphatase (Alp), collagen (Col-1a1), and dental matrix protein (Dmp-1) (quantitative polymerase chain reaction (qPCR)) were performed 72 h after irradiation. Data were analyzed by Kruskal-Wallis, ANOVA, and Tukey tests (p < 0.05). Direct light application at 4 J/cm(2) energy dose had no negative effects on cell viability, while irradiation with 2 J/cm(2) reduced cell metabolism. None of doses affected the number of viable cells compared with the control group. The two energy doses downregulated the expression of Alp; however, expression of Col-1a1 and Dmp-1 had no alteration. Cells presented change in the cytoskeleton only when irradiated with 2 J/cm(2). In conclusion, the blue LED (455 nm) irradiation, under the evaluated parameters, had no biostimulatory effects on MDPC-23 cells.
Asunto(s)
Luz , Odontoblastos/metabolismo , Odontoblastos/efectos de la radiación , Semiconductores , Fosfatasa Alcalina/metabolismo , Animales , Bovinos , Línea Celular , Supervivencia Celular/efectos de la radiación , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Odontoblastos/citologíaRESUMEN
Despite several reports regarding tissue regeneration, including pulp repair induced by different light sources, only limited data have been reported concerning the effects of light-emitting diodes (LED) on stem cells from human exfoliated deciduous teeth (SHEDs). The aim of this study was to evaluate the effects of different energy densities of infrared LED on the cell viability, number of cells and mineralized tissue production by SHEDs. SHEDs were obtained from near-exfoliation primary teeth (n=3), seeded in plain DMEM (104 cells/cm2), and irradiated by a LED prototype (LEDTable 850 nm, 40 mW/cm2) delivering 0 (control), 2, 4, 8, 15 or 30 J/cm2 (n=9). Cell viability (MTT assay), cell proliferation (trypan blue assay), and mineralized nodule (MN) formation (alizarin red stain) were assessed 12 and 72 h post-irradiation. Data were subjected to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Cells irradiated with 2 or 4 J/cm2 exhibited higher metabolism at 72 h, and all energy densities provided increase in cell proliferation after 12 h. Regarding MN formation, the best results were observed at 72 h after SHED irradiation with 8 and 15 J/cm2. It was concluded that the cell viability, cell number and MN formation by pulp cells are enhanced after exposure to infrared LED irradiation. Overall, the greatest SHED biostimulation was obtained with 4 and 8 J/cm2.
.Apesar de diversos estudos envolvendo regeneração tecidual, incluindo o reparo pulpar induzido por diferentes fontes de luz, dados limitados têm sido reportados a respeito dos efeitos da irradiação com diodos emissores de luz (LED) sobre células-tronco de dentes decíduos esfoliados (SHEDs). O objetivo do presente estudo foi avaliar os efeitos de diferentes doses de energia (DE) do LED infravermelho sobre a viabilidade celular, número de células viáveis e produção de nódulos mineralizados (NM) por SHEDs. As células foram obtidas a partir de dentes decíduos próximos ao período de esfoliação (n=3), semeadas em DMEM completo (104 células/cm2) e irradiadas utilizando um protótipo de LED (LEDTable 850 nm, 40 mW/cm2) com as doses de 0 (controle), 2, 4, 8, 15 ou 30 J/cm2 (n=9). A viabilidade celular (MTT), o número de células viáveis (trypan blue assay) e a formação de NM (alizarin red stain) foram realizados 12 e 72 h após a irradiação. Os dados foram avaliados utilizando os testes Kruskal-Wallis e Mann-Whitney (α=0,05). As células irradiadas com 2 ou 4 J/cm2 exibiram uma maior viabilidade em 72 h, e todas as DE aumentaram o número de células viáveis após 12 h. Para a formação de NM, os melhores resultados foram observados 72 h após a irradição das SHEDs, com as doses de 8 e 15 J/cm2. Concluiu-se que a viabilidade celular, o número de células e a formação de NM por células pulpares são aumentados após exposição ao LED infravermelho. De um modo geral, a melhor bioestimulação celular (SHEDs) foi obtida com 4 e 8 J/cm2.
.Asunto(s)
Humanos , Rayos Infrarrojos , Células Madre/efectos de los fármacos , Diente/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Diente/citologíaRESUMEN
OBJECTIVES: The aim of this study was to evaluate the effects of infrared LED (850nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED). METHODS: Near-exfoliation primary teeth were extracted (n=3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3×10(4) cells/cm(2)) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4J/cm(2) (n=8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). RESULTS: Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4J/cm(2) also increased gene expression of COL I and DMP-1. CONCLUSIONS: In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4J/cm(2). CLINICAL SIGNIFICANCE: Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing.
Asunto(s)
Pulpa Dental/citología , Proteínas de la Matriz Extracelular/efectos de la radiación , Fototerapia/métodos , Células Madre/efectos de la radiación , Diente Primario/citología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/efectos de la radiación , Técnicas de Cultivo de Célula , Células Cultivadas , Colágeno Tipo I/análisis , Colágeno Tipo I/efectos de la radiación , Medios de Cultivo , Proteínas de la Matriz Extracelular/análisis , Humanos , Rayos Infrarrojos , Osteogénesis/efectos de la radiación , Fosfoproteínas/análisis , Fosfoproteínas/efectos de la radiación , Proteínas/análisis , Proteínas/efectos de la radiación , Dosis de Radiación , Sialoglicoproteínas/análisis , Sialoglicoproteínas/efectos de la radiación , Exfoliación Dental , Regulación hacia ArribaRESUMEN
PURPOSE: Clinical studies have shown that low-level laser therapy (LLLT) can improve local tissue healing of bisphosphonate-induced osteonecrosis of the jaw. However, the effects of laser irradiation on bisphosphonate-treated osteoblasts have not been completely elucidated. METHODS: Human osteoblasts were cultured in plain culture medium (DMEM). After 48 h, plain DMEM was replaced by DMEM with no fetal bovine serum, for a 24-h incubation followed by addition of zoledronic acid (5 µM) for additional 48 h. Cells were subjected to LLLT (InGaAsP; 780 ± 3 nm; 0.025 W) at 0.5, 1.5, 3, 5, and 7 J/cm(2), three times every 24 h. Cell viability, total protein production, alkaline phosphatase activity (ALP), mineral nodule formation, gene expression of collagen type I and ALP, and cell morphology were evaluated. RESULTS: LLLT at 0.5 J/cm(2) increased cell viability of cultured osteoblasts. ALP activity and gene expression, in addition to mineral nodule formation and Col-I gene expression, were not increased by LLLT. LLLT applied to ZA-treated cells increased Col-I expression at 0.5, 1.5, and 3 J/cm(2) but did not improve any other cell activity assessed. CONCLUSION: LLLT showed limited effects on bisphosphonate-treated osteoblasts.