Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells / Laser de baixa intensidade induz à proliferação in vitro de células-tronco mesenquimais

Objective : To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells. Methods : Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm2). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4’-6-diamidino-2-phenylindole) at 72 hours. Results : Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm2, especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm2, but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups. Conclusion : Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering. .

Objetivo : Avaliar o efeito da terapia com laser de baixa intensidade sobre a proliferação e as possíveis alterações morfológicas nucleares em células-tronco mesenquimais de camundongos. Métodos : Células-tronco mesenquimais derivadas da medula óssea e do tecido adiposo foram submetidas a duas aplicações (T0 e T48 horas) de laser de baixa intensidade (660nm; doses de 0,5 e 1,0J/cm2). O ensaio de azul de tripan foi utilizado para a avaliação da viabilidade celular, e curvas de crescimento foram usadas para avaliar a proliferação das células em zero, 24, 48, e 72 horas. Alterações nucleares foram avaliadas por coloração com DAPI (4-6-diamidino-2-fenilindolo) em 72 horas. Resultados : As células-tronco mesenquimais derivadas da medula óssea responderam a terapia com laser de forma dose-dependente. Um maior crescimento celular foi observado quando as células foram irradiadas com dose de 1,0J/cm2, especialmente depois de 24 horas (p<0,01). As células-tronco mesenquimais derivadas do tecido adiposo responderam melhor à dose de 1,0J/cm2, com maior proliferação após 48 (p<0,05) e 72 horas (p<0,01). Nem alterações nucleares nem a mudança significativa na viabilidade celular foi detectada nos grupos estudados. Conclusão : Laser de baixa intensidade estimulou a proliferação de células-tronco mesenquimais sem causar alterações nucleares. A bioestimulação de células-tronco mesenquimais por laserterapia pode ser uma ferramenta importante para a terapia regenerativa e a engenharia tecidual. .

Efeito do laser de baixa potência sobre células tronco de polpa de dentes decíduos esfoliados humanos (SHED) / Effect of low level laser on stem cells from human exfoliated deciduous teeth (SHED)

Avaliou-se a proliferação das células tronco da polpa de dentes decíduos esfoliados humanos (SHED) após aplicação única do laser de baixa potência. Foi realizada a análise da viabilidade das SHED cultivadas sob déficit nutricional e em condições ideais após irradiação com o laser de baixa potência vermelho de Indio Gálio Alumínio e Fósforo - InGaAlP (660nm, 40mW e 10J/cm2) e infravermelho (780nm, 40mW e 10J/cm2) durante 4 e 8 segundos, nos períodos de 24, 48 e 72 horas através dos ensaios de redução do MTT e do ensaio colorimétrico de Busatti e Gomes. Para análise estatística utilizou-se o teste ANOVA complementado pelo teste de Tukey com nível de significância de 5% (p< 0,05). Observou-se tanto com o MTT quanto com o ensaio colorimétrico de Busatti e Gomes uma tendência de aumento da proliferação celular diretamente relacionada à dose do LBP, estatisticamente significante nos períodos de 24, 48 e 72 horas. Ao analisar os resultados e considerando os parâmetros utilizados e o protocolo de LBP, pode-se concluir que o LBP promoveu a proliferação das SHED tanto a 660 nm quanto a 780nm, pode influenciar a viabilidade e a proliferação das SHED nas doses e comprimentos de onda utilizados e os ensaios do MTT e colorimétrico de Busati e Gomes demonstraram dentro de suas limitações ser eficientes para determinar a viabilidade e proliferação das SHED.

It was evaluated the proliferation of stem cells from human exfoliated deciduous teeth (SHED) after a single application of low power laser. The viability of SHED grown under ideal conditions and under nutritional deficit after irradiation with red laser (660/780nm, 10J/cm2 and 40mW) during periods of 4 and 8 seconds was analyzed through the MTT reduction assays and rapid colorimetric assay of Busatti and Gomes. Statistical analysis was performed using the ANOVA and Tukey´s multiple comparisons test with a significance level of 5% (p < 0.05). It was observed with the MTT assay and Busatti and Gomes assay a trend of cell proliferation increase directly releated to the irradiation dose, statistically significant. After 24, 48 and 72 hours, all the groups showed higher cell proliferation when compared to control. Analyzing the results and considering the used parameters and LBP protocol, it can be concluded that LBP promoted the proliferation of SHED both 660nm and 780nm according to the dosage and wavelengths used, and MTT assay and colorimetric Busatti and Gomes demonstrated within their limitations to be effective in determining the viability and proliferation of SHED.

Efeito do laser de baixa potência sobre células tronco de polpa de dentes decíduos esfoliados humanos (SHED) / Effect of low level laser on stem cells from human exfoliated deciduous teeth (SHED)

Avaliou-se a proliferação das células tronco da polpa de dentes decíduos esfoliados humanos (SHED) após aplicação única do laser de baixa potência. Foi realizada a análise da viabilidade das SHED cultivadas sob déficit nutricional e em condições ideais após irradiação com o laser de baixa potência vermelho de Indio Gálio Alumínio e Fósforo - InGaAlP (660nm, 40mW e 10J/cm2) e infravermelho (780nm, 40mW e 10J/cm2) durante 4 e 8 segundos, nos períodos de 24, 48 e 72 horas através dos ensaios de redução do MTT e do ensaio colorimétrico de Busatti e Gomes. Para análise estatística utilizou-se o teste ANOVA complementado pelo teste de Tukey com nível de significância de 5% (p< 0,05). Observou-se tanto com o MTT quanto com o ensaio colorimétrico de Busatti e Gomes uma tendência de aumento da proliferação celular diretamente relacionada à dose do LBP, estatisticamente significante nos períodos de 24, 48 e 72 horas. Ao analisar os resultados e considerando os parâmetros utilizados e o protocolo de LBP, pode-se concluir que o LBP promoveu a proliferação das SHED tanto a 660 nm quanto a 780nm, pode influenciar a viabilidade e a proliferação das SHED nas doses e comprimentos de onda utilizados e os ensaios do MTT e colorimétrico de Busati e Gomes demonstraram dentro de suas limitações ser eficientes para determinar a viabilidade e proliferação das SHED...

It was evaluated the proliferation of stem cells from human exfoliated deciduous teeth (SHED) after a single application of low power laser. The viability of SHED grown under ideal conditions and under nutritional deficit after irradiation with red laser (660/780nm, 10J/cm2 and 40mW) during periods of 4 and 8 seconds was analyzed through the MTT reduction assays and rapid colorimetric assay of Busatti and Gomes. Statistical analysis was performed using the ANOVA and Tukey´s multiple comparisons test with a significance level of 5% (p < 0.05). It was observed with the MTT assay and Busatti and Gomes assay a trend of cell proliferation increase directly releated to the irradiation dose, statistically significant. After 24, 48 and 72 hours, all the groups showed higher cell proliferation when compared to control. Analyzing the results and considering the used parameters and LBP protocol, it can be concluded that LBP promoted the proliferation of SHED both 660nm and 780nm according to the dosage and wavelengths used, and MTT assay and colorimetric Busatti and Gomes demonstrated within their limitations to be effective in determining the viability and proliferation of SHED...

Single-fraction gamma[-60]Co radiation induces apoptosis in cultured rat C6 cells

Radiotherapy is frequently applied in the treatment of malignant gliomas, but it is unclear if radiotherapy exerts its effects via induction of apoptosis. The present study was designed to determine whether a single-fraction gamma-60Co radiation can induce apoptosis. In vitro cytological controlled study performed at a military medical university from October 2006 to June 2008. C6 cells were treated with a single fraction of gamma-60Co radiation at various doses [0, 4, 16, and 64 Gy]. The 3-[4,5]-dimethylthiazol-2]-2,5-diphenyl tetrazolium bromide [MTT] assay, apoptosis assays using Annexin V-fluorescein isothiocyanate /propidium iodide or Hoechst 33258 staining, and the cell cycle assay were performed, and the expression of p53 and p21 proteins was evaluated. The C6 cell numbers in the 16 Gy and 64 Gy groups were much lower than in the control group at 48, 96, and 144 hours after irradiation. The irradiated cells underwent apoptosis in a dose-dependent manner. Irradiation also impacted cell cycle progression, arresting cells in the G1 phase. The p53 protein expression was shown in both the nucleus and the cytoplasm of irradiated cells, whereas p53 was only expressed in the nucleus of control [untreated] cells. The p21 protein was expressed in irradiated cells but not in control cells. Single-fraction gamma-60Co radiation inhibited C6 cell growth by inducing apoptosis and G1 arrest, which correlated with the up-regulation of the p53-p21 pathway. The extent of apoptosis and G1 arrest was positively correlated with the dose of radiation. Better understanding of apoptosis induced by radiation therapy will help design optimal dosing schedules for radiation therapy, especially in combination with chemotherapy

Effects of low-level laser therapy on human osteoblastic cells grown on titanium

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) by using gallium aluminum arsenide (GaAlAs) diode laser on human osteoblastic cells grown on titanium (Ti). Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured on Ti discs for up to 17 days. Cells were exposed to LLLT at 3 J/cm2 (wavelength of 780 nm) at days 3 and 7 and non-irradiated cultures were used as control. LLLT treatment did not influence culture growth, ALP activity, and mineralized matrix formation. Analysis of cultures by epifluorescence microscopy revealed an area without cells in LLLT treated cultures, which was repopulated latter with proliferative and less differentiated cells. Gene expression of ALP, OC, BSP, and BMP-7 was higher in LLLT treated cultures, while Runx2, OPN, and OPG were lower. These results indicate that LLLT modulates cell responses in a complex way stimulating osteoblastic differentiation, which suggests possible benefits on implant osseointegration despite a transient deleterious effect immediately after laser irradiation.

Este estudo teve como objetivo investigar o efeito do laser diodo de gálio-alumínio-arsênio (GaAlAs) em células osteoblásticas humanas cultivadas sobre discos de Ti. Para tanto, células osteoblásticas foram obtidas por digestão enzimática de osso alveolar humano e cultivadas sobre discos de Ti por 17 dias. As células foram submetidas à irradiação no 3º e 7º dias na dose de 3 J/cm2 e comprimento de onda de 780 nm e células não irradiadas foram usadas como controle. A irradiação não alterou a proliferação celular, atividade de ALP e formação de matriz mineralizada. Microscopia por epifluorescência indicou que após 24 h da aplicação do laser, as culturas irradiadas apresentaram áreas sem células, que mais tarde foram repovoadas por células em fase de proliferação e menos diferenciadas. O laser aumentou a expressão gênica relativa da ALP, OC, BSP e BMP-7 e reduziu a de RUNX2, OPN e OPG. Os resultados indicam que a terapia com laser modula de forma complexa as respostas celulares, estimulando a diferenciação osteoblástica. Assim, é possível sugerir possíveis benefícios do laser na osseointegração de implantes de Ti apesar do efeito deletério às células imediatamente após a irradiação.