Effect of Low-Level Laser Therapy (660 nm) on Angiogenesis in Wound Healing: A Immunohistochemical Study in a Rodent Model

The aim of the present investigation was to evaluate the angiogenesis on dorsal cutaneous wounds in a rodent model treated with λ660 nm laser light. New vessel formation is a multistep process involving vessel sprouting, endothelial cell migration, proliferation and tube formation. Although several in vivo studies have shown that laser phototherapy influences tissue repair, a fully understanding of angiogenesis mechanisms are not yet known. Twenty-four young adult male Wistar rats weighing between 200 and 250 g were used. Under general anesthesia, one excisional wound was created on the dorsum of each animal and they were randomly distributed into two groups: one control and one treated with laser (λ660 nm, 16 mW, 10 J/cm2). Each group was subdivided into three subgroups according to the animal death timing (2, 4 and 6 days). Laser irradiation started immediately after surgery and was repeated every other day during the experiment and marked with Sirius Red, specific for collagen, and immunomarked with anti-TGF-β and anti-von Willebrand factor. Marked sections underwent histological analysis by light microscopy and the mean area of the wound of each animal was calculated and analyzed by ANOVA and Tukey's test (α=0.05). Although at some death periods, collagen expression and number of blood vessels on irradiated animals were higher than in the control ones, no significant differences were found at any time in relation to TGF-β expression (p>0.05). It was concluded that laser treatment (λ660 nm) contributed to increase angiogenesis.

O objetivo do trabalho foi avaliar a angiogênese em feridas cutâneas no dorso de ratos tratados com o laser de λ660 nm. A neovascularização é um processo que envolve o aparecimento vascular, a migração das células endoteliais, a proliferação e a formação tubular. Embora diversos estudos in vivo demonstrem que a fototerapia laser influencia no reparo tecidual, uma compreensão completa dos mecanismos da angiogênese ainda não é conhecida. Foram utilizados 24 ratos Wistar novos, machos e adultos pesando entre 200 e 250 g. Uma ferida excisional foi criada no dorso de cada animal sob anestesia geral e os animais foram distribuídos aleatoriamente em dois grupos: G0 (controle) e G1 (laser λ660 nm, 16 mW, 10 J/cm2). Cada grupo foi subdividido em três subgrupos de acordo com o sincronismo da morte dos animais (2, 4 e 6 dias). A irradiação laser foi iniciada imediatamente após a cirurgia, sendo repetida diariamente durante a experiência, avaliada por meio de vermelho de Sirius, específico para o colágeno e avaliação imunológica com anti-TGF-β e o Fator anti-von Willebrand. As seções marcadas foram submetidas à análise histológica no foto-microscópio, onde a área média de cada subgrupo foi calculada e analisada usando o teste ANOVA e de Tukey (α=0,05). Os valores dos animais irradiados foram maiores em certos períodos da morte, na expressão do colágeno e no número de vasos em comparação com os grupos controles. Nenhuma diferença significativa foi encontrada na expressão do TGF-β entre os grupos nos períodos. Conclui-se que o tratamento com laser λ660 nm contribuiu para o aumento da angiogênese.

Evaluation of two protocols for low-level laser application in patients submitted to orthodontic treatment

INTRODUCTION: Different low-level laser (LLL) irradiation protocols have been tested to accelerate orthodontic tooth movement (OTM). Nevertheless, divergent results have been obtained. It was suggested that the stimulatory action of low level laser irradiation occurs during the proliferation and differentiation stages of bone cellular precursors, but not during later stages. OBJECTIVE: The purpose of this study was to determine the effect of two protocols of LLL irradiation on experimental tooth movement: One with daily irradiations and another with irradiations during the early stages. METHODS: Thirty-six rats were divided into control groups (CG1, CG2, CG3) and irradiated groups (IrG1, IrG2, IrG3) according to the presence of: experimental tooth movement, laser irradiation, type of laser irradiation protocol and date of euthanasia (3th or 8th day of experiment). At the end of experimental periods, a quantitative evaluation of the amount of OTM was made and the reactions of the periodontium were analyzed by describing cellular and tissue reactions and by counting blood vessels. RESULTS: The amount of OTM revealed no significant differences between groups in the same experimental period (p < 0.05). Qualitative analysis revealed the strongest resorption activity in irradiated groups after seven days, especially when using the daily irradiation protocol. There was a higher number of blood vessels in irradiated animals than in animals without orthodontic devices and without laser irradiation (p < 0.05). CONCLUSION: Moreover, angiogenesis was verified in some of the irradiated groups. The irradiation protocols tested were not able to accelerate OTM and root resorption was observed while they were applied.

INTRODUÇÃO: diferentes protocolos de irradiação por laser de baixa potência (LBP) têm sido testados para potencializar o movimento ortodôntico; entretanto, há resultados divergentes. Foi sugerido que seu efeito bioestimulador ocorre nas fases de proliferação e diferenciação celular, não agindo em estágios tardios. OBJETIVO: avaliar o efeito de dois protocolos de irradiação do LBP na movimentação ortodôntica: um com irradiações diárias e outro em que irradiações foram realizadas apenas nos períodos iniciais. MÉTODOS: trinta e seis ratos Wistar foram divididos em grupos controles (GC1, GC2 e GC3) e irradiados (GIr1, GIr2 e GIr3), de acordo com a presença de dispositivo ortodôntico, a presença de irradiação, o tipo de protocolo de irradiação e a data de eutanásia (3º ou 8º dia de experimento). Ao final dos períodos experimentais, foram realizadas mensurações da movimentação dentária, análise qualitativa das reações celulares e teciduais do periodonto e contagem de vasos sanguíneos no ligamento periodontal. RESULTADOS: a quantidade de movimentação não diferiu entre os grupos num mesmo tempo experimental (p < 0,05). A análise qualitativa revelou maior atividade absortiva nos grupos irradiados ao final de 7 dias, especialmente quando as irradiações foram diárias. Nos grupos irradiados diariamente, a contagem de vasos foi aumentada em relação aos animais isentos de dispositivo ortodôntico e de aplicações de LBP (p < 0,05). CONCLUSÃO: apesar de verificada angiogênese em certos grupos irradiados, os protocolos de irradiação testados não foram capazes de acelerar a movimentação dentária, e foi possível verificarem-se absorções radiculares.

Low-level laser on femoral growth plate in rats / Laser terapêutico de baixa potência na placa de crescimento de ratos

PURPOSE: To determine the influence of low-level laser therapy on femoral growth plate in rats. METHODS: Thirty male Wistar rats aged 40 days were divided into two groups, G1 and G2. In G1 the area of the distal growth plate of the right femur was irradiated at one point using GaAlAs laser 830 nm wavelength, output power of 40 mW, at an energy density of 10 J/cm². The irradiation was performed daily for a maximum of 21 days. The same procedure was done in G2, but the probe was turned off. Five animals in each group were euthanized on days 7, 14 and 21 and submitted to histomorphometric analysis. RESULTS: In both groups the growth plate was radiographically visible at all moments from both craniocaudal and mediolateral views. On the 21st day percentage of femoral longitudinal length was higher in G2 than G1 compared to basal value while hypertrophic zone chondrocyte numbers were higher in G1 than G2. Calcified cartilage zone was greater in G1 than in G2 at all evaluation moments. Angiogenesis was higher in G1 than in G2 at 14th and 21st days. CONCLUSION: The low-level laser therapy negatively influenced the distal femoral growth plate.

OBJETIVO: Determinar a influência do Laser Terapêutico de Baixa Potência sobre a placa de crescimento de ratos. MÉTODOS: Trinta ratos Wistar machos com 40 dias de idade foram divididos em dois grupos, G1 e G2. O grupo G1 foi submetido à irradiação com laser GaAlAs 830 nm, potência de saída de 40 mW, e densidade de energia de 10 J/cm2. A irradiação foi aplicada diariamente por um período máximo de 21 dias. O mesmo procedimento foi realizado no grupo G2, com a probe desativada. Cinco animais em cada grupo foram sacrificados nos dias 7, 14 e 21 e submetidas à análise histomorfométrica. RESULTADOS: Em ambos os grupos, o disco fisário esteve radiograficamente visível em todos os momentos nas incidências craniocaudal e médio-lateral. No 21º dia a porcentagem de comprimento longitudinal do fêmur foi maior em G1 que em G2 em relação ao valor basal, e o número de condrócitos da zona hipertrófica foi maior em G1 que em G2. A zona de cartilagem calcificada estava maior em G1 em relação a G2 em todos os momentos de avaliação. A angiogênese foi maior em G1 que em G2 nos 14º e 21º dias. CONCLUSÃO: A terapia com laser terapêutico de baixa potência influenciou negativamente o disco fisário distal do fêmur de ratos.

Histological and radiographic evaluation of the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2) in a scaffold of inorganic bone and after stimulation with low-power laser light.

Objective: The present study histologically and radiologically evaluates the muscle tissue of rats after implantation of bone morphogenic protein (rhBMP-2) in a natural inorganic bone mineral scaffold from a bull calf femur and irradiation with low-power light laser. Materials and Methods: The right and left hind limbs of 16 rats were shaved and an incision was made in the muscle on the face corresponding to the median portion of the tibia, into which rhBMP-2 in a scaffold of inorganic bone was implanted. Two groups of limbs were formed: control (G1) and laser irradiation (G2). G2 received diode laser light applied in the direction of the implant, at a dose of 8 J/cm2 for three minutes. On the 7th, 21st, 40th and 112th days after implantation, hind limbs of 4 animals were radiographed and their implants removed together with the surrounding tissue for study under the microscope. The histological results were graded as 0=absence, 1=slight presence, 2=representative and 3=very representative, with regard to the following events: formation of osteoid structure, acute inflammation, chronic inflammation, fibrin deposition, neovascularization, foreign-body granuloma and fibrosis. Results: There were no statistically significant differences in these events at each evaluation times, between the two groups (P>0.05; Mann-Whitney test). Nevertheless, it could be concluded that the natural inorganic bone matrix with rhBMP-2, from the femur of a bull calf, is a biocompatible combination. Conclusions: Under these conditions, the inductive capacity of rhBMP-2 for cell differentiation was inhibited. There was a slight acceleration in tissue healing in the group that received irradiation with low-power laser light.