RESUMEN
O objetivo do presente trabalho foi avaliar o efeito de diferentes densidades de energia do Laser de Baixa Intensidade na viabilidade e proliferação celular de fibroblastos derivados da polpa de dentes decíduos humanos e na expressão de RNAm para DMP- 1, DSPP, VEGF e FGF-2. Amostras de fibroblastos pulpares da polpa de dentes decíduos humanos foram obtidas de um Biorrepositório. Foram utilizadas células entre a 4ª e a 7ª passagem, irradiadas com Laser de Baixa Intensidade (InGaAlP) de acordo com os seguintes grupos experimentais: Grupo 1: 1,2 J/cm2 - 05 mW - 10s; Grupo 2: 2,5 J/cm2 - 05 mW - 20s; Grupo 3: 3,7 J/cm2 - 05 mW - 30s; Grupo 4: 5,0 J/cm2 - 05 mW - 40s; Grupo 5: 6,2 J/cm2 - 05 mW - 50s; Grupo 6: 2,5 J/cm2 - 10 mW - 10s; Grupo 7: 3,7 J/cm2 - 15 mW - 10s; Grupo 8: 5,0 J/cm2 - 20 mW - 10s; Grupo 9: 6,2 J/cm2 - 25 mW - 10s; Controle Negativo: DMEM 1% SFB não irradiado; Controle Positivo: DMEM 10% SFB não irradiado. As técnicas utilizadas para as análises de viabilidade e proliferação celular foram MTT e CV. A técnica utilizada para avaliação da expressão de RNAm para os alvos DMP-1, DSPP, VEGF e FGF-2 foi RT-PCR. Os resultados foram analisados pelo método ANOVA a dois critérios, seguido pelo teste de Tukey (p<0,05). Para o teste MTT, na comparação intragrupos observou-se que houve diferença estatisticamente significativa entre os períodos 6h, 12h e 24h, diminuindo a viabilidade com o passar do tempo, exceto para o Grupo 1. Na comparação intergrupos, o MTT mostrou menor viabilidade para o controle negativo em comparação com os outros grupos (p<0,05), exceto com grupo 5 (5mW/50 seg). Observou-se que os grupos com maiores potências (10mW, 15mW, 20mW e 25mW), menores tempos de aplicação (10 segundos) e densidades de energia entre 2,5 J/cm2 e 6,2 J/cm2, apresentaram estatisticamente maior viabilidade que o grupo com menor potência (5mW), maior tempo de aplicação (50 segundos) e densidade de energia de 6,2 J/cm2. Para o teste CV não houve diferença intragrupos, mas houve diferença intergrupos entre os controles positivo e negativo. Para a expressão de RNAm por RTPCR, os fatores de crescimento VEGF e FGF-2 foram expressos em grande quantidade no primeiro período experimental, enquanto que DMP-1 e DSPP não foram expressos de maneira significativa. De acordo com os resultados obtidos, frente as diferentes densidades de energia, sugere-se que a terapia a laser de baixa intensidade manteve os fibroblastos viáveis e aumentou a expressão de RNAm para VEGF e FGF-2.(AU)
This study aimed to evaluate the effect of different energy densities of Low Level Laser (LLL) on cell viability and proliferation of fibroblasts from the pulp of human primary teeth (DHPF) and on the RNAm expression of DMP-1, DSPP, VEGF and FGF-2. DHPF were obtained from a biorepository and used at passages 4th to 7th. The cells were irradiated with LLL (InGaAlP) according to the following experimental groups: Group 1: 1.2 J/cm2 - 05 mW - 10s; Group 2: 2.5 J/cm2 - 05 mW - 20s; Group 3: 3.7 J/cm2 - 05 mW - 30s; Group 4: 5.0 J/cm2 - 05 mW - 40s; Group 5: 6.2 J/cm2 - 05 mW - 50s; Group 6: 2.5 J/cm2 - 10 mW - 10s; Group 7: 3,7 J/cm2 - 15 mW - 10s; Group 8: 5.0 J/cm2 - 20 mW - 10s; Group 9: 6.2 J/cm2 - 25 mW - 10s; Negative Control: DMEM 1% SFB not irradiated; Positive Control: DMEM 10% SFB not irradiated. The techniques used to evaluate the cell viability/proliferation were MTT and Crystal Violet (CV) assays. RT-PCR was used to verify the RNAm expression of DMP-1, DSPP, VEGF, and FGF-2. Two-way ANOVA, followed by Tukey test (p<0.05) was used to analyze the results. In the intragroup comparison, MTT assay revealed statistically significant differences among the periods of 6h, 12h, and 24h, with viability reduction as time went by, except for Group 1. In the intergroup comparison, the MTT assay showed that the negative control had statistically lower viability than that of the other groups (p<0.05), except for Group 5 (5mW/50 s). The groups with higher powers (10mW, 15mW, 20mW, and 25mW), shortest application periods (10 s), and energy densities between 2.5 J/cm2 and 6.2 J/cm2 exhibited statistically higher viability than that of the group with small power (5mW), longer application period (50 s), and energy density of 6.2 J/cm2 . CV assay did not show intergroup statistically differences. In the intragroup comparison, CV assay revealed statistically significant differences between positive and negative controls (p<0.05). RT-PCR revealed increased RNAm expression of the growth factors VEGF and FGF-2 at the first experimental period, while DMP-1 and DSPP was not significant. Based on the results and different energy densities used, LLL maintained DHPF viability and increased the RNAm expression of VEGF and FGF-2.(AU)
Asunto(s)
Humanos , Pulpa Dental/citología , Proteínas de la Matriz Extracelular/análisis , Factor 2 de Crecimiento de Fibroblastos/análisis , Fibroblastos/efectos de la radiación , Terapia por Luz de Baja Intensidad , Fosfoproteínas/análisis , Sialoglicoproteínas/análisis , Factor A de Crecimiento Endotelial Vascular/análisis , Análisis de Varianza , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Proteínas de la Matriz Extracelular/efectos de la radiación , Factor 2 de Crecimiento de Fibroblastos/efectos de la radiación , Fosfoproteínas/efectos de la radiación , Dosis de Radiación , Sialoglicoproteínas/efectos de la radiación , Factores de Tiempo , Diente Primario/citología , Factor A de Crecimiento Endotelial Vascular/efectos de la radiaciónRESUMEN
OBJECTIVE: It was the aim of the present study to evaluate whether the laser irradiation of osteoblasts could enhance the release of growth factors including basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and receptor of IGF-I (IGFBP3). BACKGROUND DATA: Low-level laser therapy (LLLT) has been shown to have biostimulatory effects on various cell types by enhancing production of some cytokines and growth factors. MATERIALS AND METHODS: Human mesenchymal stem cells (MSCs) were seeded in osteogenic medium and differentiated into osteoblasts. Three groups were formed: in the first group (single dose group), osteoblasts were irradiated with laser (685 nm, 25 mW, 14.3 mW/cm(2), 140 sec, 2 J/cm(2)) for one time; and in the second group, energy at the same dose was applied for 2 consecutive days (double dose group). The third group was not irradiated with laser and served as the control group. Proliferation, viability, bFGF, IGF-I, and IGFBP3 levels were compared between groups. RESULTS: Both of the irradiated groups revealed higher proliferation, viability, bFGF, IGF-I, and IGFBP3 expressions than did the nonirradiated control group. There was increase in bFGF and IGF-I expressions and decrease in IGFBP3 in the double dose group compared to single dose group. CONCLUSIONS: The results of the present study indicate that LLLT increases the proliferation of osteoblast cells and stimulates the release of bFGF, IGF-I, and IGFBP3 from these cells. The biostimulatory effect of LLLT may be related to the enhanced production of the growth factors.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/efectos de la radiación , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/efectos de la radiación , Factor I del Crecimiento Similar a la Insulina/efectos de la radiación , Terapia por Luz de Baja Intensidad/métodos , Osteoblastos/efectos de la radiación , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Células Madre Mesenquimatosas/fisiología , Células Madre Mesenquimatosas/efectos de la radiación , Osteoblastos/fisiología , Dosis de Radiación , Muestreo , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Low-power laser irradiation has a stimulating effect on cells and tissues. Although the carbon dioxide (CO(2)) laser is a hard surgical laser, we have attempted to use it at low energy density to achieve biological alterations. To investigate the effects of CO(2) laser irradiation on BSP gene transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were increased at 12 h after irradiation with the CO(2) laser (2 W, 20 s). Transient transfection assays using various sizes of the rat BSP gene promoter linked to the luciferase reporter gene showed that CO(2) laser irradiation induced luciferase activity of a -116 to +60 BSP promoter construct (pLUC3) at 12 h in the cells. Transcriptional stimulation by CO(2) laser irradiation was abrogated in the pLUC3 construct containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that CO(2) laser irradiation increased the binding of nuclear protein to FRE. These studies demonstrate that CO(2) laser irradiation increases BSP transcription via FRE in the rat BSP gene promoter.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Sialoproteína de Unión a Integrina/genética , Láseres de Gas , Terapia por Luz de Baja Intensidad , Osteoblastos/metabolismo , Transcripción Genética/efectos de la radiación , Animales , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/efectos de la radiación , Sialoproteína de Unión a Integrina/biosíntesis , Luciferasas , Regiones Promotoras Genéticas/efectos de la radiación , Ratas , Elementos de Respuesta/efectos de la radiaciónRESUMEN
OBJECTIVE: The purpose of this study was to investigate the effects of low power laser on basic fibroblast growth factors (bFGF) expression in periodontal tissue during tooth movement. METHODS: 18 white rabbits were randomly divided into 6 groups with 3 rabbits in each group, including groups of 1, 3, 5, 7, 14 and 21 days. Under an anesthesia condition by 2% pentobarbital sodium, the stainless coil springs were fixed between the first maxillary molar and the incisor producing the force of 80 g. The right side of maxilla was considered as the experimental group under the irradiation of low power laser with the left side as the control groups. The expression of bFGF was investigated half-quantitatively through immunohistochemical analysis. RESULTS: The expression of bFGF in periodontal tissue with irradiation of low power laser was higher than the control side. There were significant differences among the 5, 7, and 14 day groups. In the tension area of the experimental side, the expression of bFGF in the osteoblastic surface of alveolar bone was characteristically greater than that of the control side. CONCLUSION: The laser of low power promotes the expression of bFGF in the periodontal tissue and alveolar bone remodeling.