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.

Thymus vulgaris L. extract has antimicrobial and anti-inflammatory effects in the absence of cytotoxicity and genotoxicity.

OBJECTIVES: This study evaluated the biological effects of the T. vulgaris L. extract., such as antimicrobial activity on planktonic cultures and mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory activity and genotoxicity. METHODS: Monomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed by C. albicans with each bacterium were formed for 48h and exposed for 5min to the plant extract. Murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7) and cervical carcinoma cells (HeLa) were also exposed to the plant extract for 5min and the cell viability were analyzed by MTT, neutral red (NR) and crystal violet (CV) assays. Interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) produced by RAW 264.7 was quantified by ELISA, after 24h exposure to the plant extract, both in the absence and presence of lipopolysaccharide (LPS) from Escherichia coli. Genotoxicity of the plant extract was evaluated by micronucleus formation (MN) in 1000 cells. The results were analyzed by T-Test or ANOVA and Tukey's Test (P≤0.05). RESULTS: All biofilms showed significant reductions in CFU/mL (colony-forming units per milliliter). Cell viability was above 50% for all cell lines. Anti-inflammatory effect on the synthesis of IL-1ß and TNF-α was observed. The MN was similar or lower than the control group in all cells. CONCLUSIONS: T. vulgaris L. extract was effective against all biofilms, promoted high cell viability, anti-inflammatory effect and presented no genotoxicity.

Plant extracts: initial screening, identification of bioactive compounds and effect against Candida albicans biofilms.

AIM: This study screened plants from Brazilian Pantanal for Candida albicans antibiofilm activity. MATERIAL & METHODS: Sixty extracts were obtained from ten plants using different extraction methods. Antifungal activity was assessed. Effects on biofilm inhibition and disruption and cytotoxicity were also evaluated. The most active extract was chemically characterized. RESULTS: Buchenavia tomentosa ethanolic extract showed noticeable antifungal activity and was selected for biofilm experiments. Subinhibitory concentration of extract inhibited fungal adhesion. Maximum killing reached 90% of C. albicans cells in suspension and 65% of cells in biofilms. The active extract was noncytotoxic. Chemical characterization showed the presence of phenols. Ellagic and gallic acids showed activity on C. albicans. CONCLUSION: B. tomentosa extract and its isolated compound, ellagic acid, presented antibiofilm activity and low toxicity.

Wound healing properties and mucilage content of Pereskia aculeata from different substrates

Physiologic growth parameters Wound healing Pereskia aculeata Mill., Cactaceae, is a cactus with high mucilage production, well-known for its nutritional properties. Folk use consists on skin injuries, and mucilage is probably involved in the wound healing activity. This work studied some aspects of its cultivation, specifically regarding soil (substrate), to correlate the effects of nutritional content to mucilage production and to the wound-healing property. Plants were grown under five different soil treatment (sand, crude soil, sand and soil, sand and cattle manure, soil and cattle manure), and after eight months extracts were prepared by turbo-extraction to obtain a crude hydroethanolic extract. We evaluated the effects of these extracts on swelling index, cytotoxicity, and in vitro wound healing property. The results show that the substrate used in cultivation may interfere with mucilage production, but not with cytotoxicity and wound healing, this shows the safety of its use, despite the soil treatment received along the various biomes where P. aculeata is cultivated. Furthermore, morphological studies demonstrated the beneficial effect of the mucilage-containing extract on the fibroblast cell culture, corroborating its folk use for wound healing.

Effect of photodynamic therapy supplemented with quercetin in HEp-2 cells.

Photodynamic therapy (PDT) is a technique that can be used as a complementary therapy in cancer treatment combined with other therapeutic modalities. Quercetin (QCT) is known to be effective in the treatment of cancer, by reducing the cell viability of different cancer cell lines. This study aimed to evaluate the influence of different concentrations of QCT in PDT on the viability, mitochondrial membrane potential and induction of apoptosis/necrosis in the human larynx carcinoma cells (HEp-2). The HEp-2 cells were treated with aluminum phthalocyanine tetrasulfonate (AlPcS4) and QCT and subsequently irradiated with a diode laser light (685 nm, 35 mW, 4.5 J/cm(2)). The results demonstrated that treatment of HEp-2 cells with high concentrations of QCT (at least 50 µM) reduced cell viability. This response was enhanced in cells subjected to PDT supplemented with high concentrations of QCT. In addition, was observed decrease in the mitochondrial membrane potential and characteristics of late apoptosis and/or initial necrosis process. QCT at concentrations from 50 µM improves PDT-induced cytotoxicity by significantly reducing cell viability by apoptosis and/or necrosis, and mitochondrial membrane potential of Hep-2 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.

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.

Surface morphology of sound deciduous tooth enamel after application of a photo-absorbing cream and infrared low-level laser irradiation: an in vitro scanning electron microscopy study.

OBJECTIVES: The purpose of this descriptive scanning electron microscopic study was to characterize surface alterations in deciduous tooth enamel after in vitro infrared diode laser irradiation, using a photo-absorbing agent alone and also combined with fluoride, before and after laser irradiation. BACKGROUND DATA: Previous investigations have demonstrated increased enamel caries resistance after laser irradiation. METHODS: Seven extracted or exfoliated primary molar teeth underwent soft tissue débridement and fluoride-free prophylaxis. Buccal surfaces were determined to be caries free by macroscopic examination. Sample groups were divided into: (1) control (no treatment); (2) infrared diode laser irradiation (lambda = 810 nm, 68 nm, 60 mW/mm(2), 30 W) using the photo-absorbing agent alone (IRDL + PA; 500 J/cm(2)); and (3) infrared diode laser irradiation using a photo-absorbing agent combined with 2% fluoride (IRDL + PFA; 500 J/cm(2)). Buccal surfaces were evaluated following standard scanning electron microscopy preparation techniques. Control samples of enamel surfaces were relatively smooth but presented occasional enamel prism ends. There were no areas with cavitations or surface defects. RESULTS: After the IRDL + PA treatment, irradiated surfaces became rough and mildly to moderately irregular with scarce enamel cavitations and without exposure of enamel prism ends. The surfaces had adherent granules and only occasional fine cracks and porosities in surface coatings were noted. After the IRDL + PFA treatment, there was a homogenous confluent surface that masked typical enamel surface markings. The surfaces had well-defined globules resulting from the IRDL + PFA treatment, that were not seen after IRDL + PA treatment. CONCLUSIONS: Treatment of deciduous tooth enamel with infrared diode laser irradiation using a photo-absorbing agent and a photo-absorbing agent combined with 2% fluoride created surface coatings that may act as reservoirs for mineral phases during cariogenic activity on enamel, and also provide a certain degree of protection against cariogenic challenge.

Photodynamic therapy with a new photosensitizing agent.

OBJECTIVE: The aim of this study was to investigate the cytotoxicity of octal-bromide zinc phthalocyanine (ZnPcBr(8)) before and after irradiation with a low-power laser (AsGaAl) and analyze the effects of photodynamic therapy (PDT) on the nucleus of L929 cells. BACKGROUND DATA: One of the most recent and promising applications of phthalocyanine in medicine is in the detection and cure of tumors. We studied the ZnPcBr(8) in agreement with the development of new photosensitizing agents for curing tumors. METHODS: L929 cells were cultivated at standard conditions, incubated with ZnPcBr(8) for 1 h at different concentrations, irradiated with a semiconductor laser, and incubated in MEM medium for 1, 12, or 24 h. Cells were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) technique and fluorescence microscopy. RESULTS: The results demonstrated that ZnPcBr(8) at 1 microM was the most effective concentration for PDT, with a decrease of 63% after 1 h, 99% after 12 h, and 100% after 24 h in relation to the control group. The fluorescence microscopy results showed that ZnPcBr(8) was localized in the perinuclear region when analyzed 1 h after incubation. Nucleus staining with DAPI made it possible to observe that nuclear fragmentation occurred 24 h after PDT, cytoplasm retraction at 1, 12, and 24 h after PDT, and vacuoles along the cytoplasm at 12 and 24 h after PDT. CONCLUSION: According to the results obtained in this study, L929 cell death caused by PDT with ZnPcBr(8) possesses characteristics of apoptosis mediated by the mitochondria, due to the decrease in cells viability, the subcellular localization, and the photodamage found.

Laser light prevents apoptosis in Cho K-1 cell line.

OBJECTIVE: The present study investigated the effects of low-level laser therapy (LLLT) on the mitochondria, nucleus, and cytoskeleton of CHO K-1 cells by the use of specific fluorescent probes. BACKGROUND DATA: The use of LLLT has been recommended by several authors for acceleration of the healing process. The literature on the effects of LLLT in this process is highly contradictory because of difficulties in identifying its effects on cells. MATERIALS AND METHODS: CHO K-1 cells were cultivated using MEM containing 5% FBS and were irradiated or not with a semiconductor laser (lambda = 830 nm; phi approximately 0.8 mm; 10 mW; 2 J/cm2). The cells were incubated with specific fluorescent probes--0.1 microM for 30 min with 5,5', 6,6'-tetrachloro-1, 1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) for the mitochondria; 5 mM for 5 min of 4',6'-diamidino, 2'-phenylindole (DAPI)for the nucleus, and 0.1 M of 1:100 PHEM of rhodamine-phalloidin during 1 h for the cytoskeleton--and were analyzed by epifluorescence. RESULTS: Positive biomodulatory effects were observed on irradiated cells compared to their controls as seen on JC-1, DAPI, and rhodamine-phalloidin labeling. Irradiated cells showed an increased level of cellular division, as evidenced by analyzing the intermediary filaments of the cytoskeleton and the chromosomes. Another important observation was that cells maintained under the condition of nutritional deficiency had both membrane and genetic material that was more preserved in comparison to the controls, in which the presence of an apoptotic nucleus could be observed in some cells. CONCLUSION: The results of the present study demonstrate that LLLT, in addition to providing positive biomodulation, acts in the re-establishment of cellular homeostasis when the cells are maintained under the condition of nutritional stress; it also prevents apoptosis in CHO K-1 cells.