The effect of low-level laser therapy (660 nm) on the gene expression involved in tissue repair.

The effect of low-level laser therapy (LLLT) on the healing of skin lesions has been evaluated in many studies; however, the molecular mechanisms involved in the biostimulatory effects resulting from this treatment need to be better understood. The paper aims to analyze the effects of LLLT (660 nm) at doses of 1 and 5 J/cm2 on cell viability and expression of vascular endothelial growth factor (VEGF) and interleukin (IL6) genes in L929 fibroblast cells. The dose-response curve was performed with the GaInAlAs (660 nm) laser-treated cells at energy rates of 1 and 5 J/cm2. Cell viability was quantified at 24, 48, and 72 h after irradiation and the effects of TLBP on the cytoskeleton and endoplasmic reticulum were evaluated by fluorescence microscopy and the RT-qPCR method was used for the analysis of gene expression. It was observed that the 72 h group had a statistically significant increase in cell viability compared to the 48 h group (p < 0.01) and when compared to the 72 h control (p = 0.03). In 72 h, a greater distribution of the cytoskeleton filaments and the more evident endoplasmatic reticulum was verified, indicating an increase in the protein synthesis when compared with the control group. In the expression of the VEGF gene, a significant increase of 1.98 times (p < 0.05) in the number of transcripts was observed; whereas for the IL6 gene, a decrease of the transcripts was 4.05 times (p < 0.05), both occurring within 72 h after irradiation at 5 J/cm2. The LLLT (660 nm) at the dose of 5 J/cm2 should modulate cellular viability, upregulated VEGF, and downregulated IL6 expression of messenger RNA in culture of L929 fibroblast cells.

Assessment of cytoskeleton and endoplasmic reticulum of fibroblast cells subjected to low-level laser therapy and low-intensity pulsed ultrasound.

OBJECTIVE: The aim of the present study was to compare the effect of low-level laser therapy (LLLT) and low-intensity pulsed ultrasound (LIPUS) on the cytoskeleton and endoplasmic reticulum of L929 cells. Thermal and non-thermal physical mechanisms such as LLLT and LIPUS induce clinically significant responses in cells, tissues, and organs. MATERIALS AND METHODS: L929 fibroblast cell cultures were irradiated with LLLT and subjected to LIPUS. Cultures irradiated with the laser (904 nm) were divided into three groups: group I, control (no irradiation); group II, irradiated at 6 J/cm(2); and group III, irradiated at 50 mJ/cm(2). Cultures subjected to ultrasound were divided into five groups: group I, control (no LIPUS); group II, LIPUS at 0.2 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group III, LIPUS at 0.6 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group IV, LIPUS at 0.2 W/cm(2) in pulsed mode at 20% (2:8 duty cycle); and group V, LIPUS at 0.6 W/cm(2) in pulsed mode at 20% (2:8 duty cycle). Each group was irradiated at 24-h intervals, with the following post-treatment incubation times: 24, 48, and 72 h. The effects of LLLT and LIPUS on the cytoskeleton and endoplasmic reticulum was evaluated by the use of fluorescent probes and with fluorescence microscopy analysis. RESULTS: The results following LLLT and LIPUS demonstrate that ultrasound was more effective than laser on fibroblast cell cultures when the endoplasmic reticulum was assessed, whereas there was a better distribution of the filaments of the cytoskeleton in the cells subjected to laser irradiation. CONCLUSION: The study demonstrated that both LLLT and LIPUS promote changes on the cellular level. However, LIPUS was more effective than LLLT at the doses used here, as assessed by fluorescence microscopy, which revealed increased reticulum activity and increased protein synthesis. However, when the organization of actin filaments was assessed, LLLT achieved a better result.

Assessment of fibroblast cells submitted to ultrasonic irradiation.

Physiotherapists consider ultrasound an indispensable tool, which is commonly employed in clinical practice as a treatment aid for musculoskeletal dysfunctions. The aim of our study has been to analyze fibroblast cell structures following low-intensity pulsed ultrasonic irradiation. Fibroblast cell cultures irradiated with ultrasound were analyzed through electron microscopy to determine an ideal irradiation beam that preserved cell morphology and integrity. Analysis by fluorescence microscopy and transmission electron microscopy was used to follow morphological changes of the nucleus and cytoskeleton following different ultrasound irradiation intensities. According to the parameters used in the pulsed irradiation of fibroblast cultures, control over the intensity employed is fundamental to the optimal use of therapeutic ultrasound. Cell cultures submitted to low-intensity pulsed ultrasonic irradiation (0.2-0.6 W/cm2) at 10% (1:9 duty cycle) and 20% (2:8 duty cycle) maintained shape and cellular integrity, with little damage. In the group irradiated with an intensity of 0.8 W/cm2, a loss of adhesion was observed along with an alteration in the morphology of some cells at an intensity of 1.0 W/cm2, which resulted in the presence of cellular fragments and a decrease of adhering cells. In cells irradiated at 2.0 W/cm2, there was a complete loss of adhesion and aggregation of cellular fragments. The present study confirms that biophysical properties of pulsed ultrasound may accelerate proliferation processes in different biological tissues.

Evaluation of low-level laser therapy of osteoblastic cells.

OBJECTIVE: The purpose of the present study was to evaluate the effect of biomodulation on osteoblastic cells using a gallium-aluminium-arsenide diode laser. BACKGROUND DATA: Low-level laser therapy (LLLT) is a non-pharmacological therapeutic resource to which biological tissues respond well, producing such effects as the acceleration of bone formation and bone repair. MATERIALS AND METHODS: Osteoblastic cell cultures (OFCOL II) were irradiated with a gallium-aluminium-arsenide diode laser (GaAlAs lambda = 830 nm; 50 mW; 3 J/cm(2); 600-microm-diameter optical fiber) and divided into two groups: group 1--irradiated cells, and group 2--non-irradiated cells. Irradiation occurred at 24-h intervals for a total of 3 d. After each interval, the cells were marked with Mito Tracker Orange dye to assess the biostimulatory effect on mitochondrial activity and cell proliferation using an MTT assay. RESULTS: Intense grouping of mitochondria in the perinuclear region was observed at 24 h and 48 h following irradiation. Changes from a filamentous to a granular appearance in mitochondrial morphology and mitochondria distributed throughout the cytoplasm were observed 72 h following proliferation. Such changes led to an in vitro proliferation process, as confirmed by the MTT assay. CONCLUSION: LLLT has shown itself capable of altering mitochondrial activity and the population of OFCOL II cells.

Comparison between the effect of low-level laser therapy and low-intensity pulsed ultrasonic irradiation in vitro.

OBJECTIVE: The objective of this study was to compare the effect of low-level laser therapy (LLLT) and low-intensity pulsed ultrasound (LIPUS) on fibroblast cell culture. Several methods, including ultrasound treatment and LLLT, are being used to facilitate tissue repair and healing processes. MATERIALS AND METHODS: L929 fibroblast cell cultures were irradiated with low-level laser energy and LIPUS. Cultures irradiated with ultrasound were divided into five groups: group 1: control (did not receive irradiation); group 2: 0.2 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group 3: 0.6 W/cm(2) in pulsed mode at 10% (1:9 duty cycle); group 4: 0.2 W/cm(2) in pulsed mode at 20% (2:8 duty cycle); and group 5: 0.6 W/cm(2) in pulsed mode at 20% (2:8 duty cycle). Cultures irradiated with laser energy were divided into three groups: group 1: control (did not receive irradiation); group 2: 6 J/cm(2); and group 3: 50 mJ/cm(2). Each group was irradiated at 24-h intervals, with the following incubation periods post-irradiation: 24, 48, and 72 h; after each irradiation cycle the cultures were analyzed using MTT [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide]. RESULTS: Analysis of results after LLLT and LIPUS demonstrated that the effect of laser therapy on fibroblast cell culture was greater than that of LIPUS (p < 0.05). CONCLUSION: Results demonstrated that LLLT significantly increased fibroblastic activity more than LIPUS. Therefore, in the first and second phases of tissue repair, laser treatment may be more effective than ultrasound treatment.