The efficacy of low-level 940 nm laser therapy with different energy intensities on bone healing

Abstract The aim of this study was to evaluate the efficacy of low-level 940 nm laser therapy with energy intensities of 5, 10 and 20 J/cm2 on bone healing in an animal model. A total of 48 female adult Wistar rats underwent surgery to create bone defects in the right tibias. Low-level laser therapy (LLLT) was applied immediately after surgery and on post-operative days 2, 4, 6, 8, 10 and 12 in three study groups with energy intensities of 5 J/cm2, 10 J/cm2 and 20 J/cm2 using a 940 nm Gallium-Aluminium-Arsenide (Ga-Al-As) laser, while one control group underwent only the tibia defect surgery. All animals were sacrificed 4 or 8 weeks post-surgery. Fibroblasts, osteoblasts, osteocytes, osteoclasts and newly formed vessels were evaluated by a histological examination. No significant change was observed in the number of osteocytes, osteoblasts, osteoclasts and newly formed vessels at either time period across all laser groups. Although LLLT with the 10 J/cm2 energy density increased fibroblast activity at the 4th week in comparison with the 5 and 20 J/cm2 groups, no significant change was observed between the laser groups and the control group. These results indicate that low-level 940 nm laser with different energy intensities may not have marked effects on the bone healing process in both phases of bone formation.

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro / Avaliação do efeito da riboflavina e luz ultravioleta sobre os ceratócitos in vitro

ABSTRACT Purpose: To culture quiescent human keratocytes and evaluate the effects of ultraviolet light and riboflavin on human corneal keratocytes in vitro. Methods: Keratocytes were obtained from remaining corneoscleral ring donor corneas previously used in corneal transplant surgeries and cultured in DMEM/F12 with 2% FBS until confluence. Characterization of cultured cells was performed by immunofluorescence analysis for anti-cytokeratin-3, anti-Thy-1, anti-α-smooth muscle actin, and anti-lumican. Immunofluorescence was performed before and after treatment of cultured cells with either ultraviolet light or riboflavin. Corneal stromal cells were covered with collagen (200 µL or 500 µL) and 0.1% riboflavin, and then exposed to ultraviolet light at 370 nm for 30 minutes. After 24 hours, cytotoxicity was determined using MTT colorimetric assays, whereas cell viability was assessed using Hoechst 33342 and propidium iodide. Results: Cell cultures achieved confluence in approximately 20 days. Expression of the lumican was high, whereas no expression of CK3, Thy-1, and α-SMA was observed. After crosslinking, MTT colorimetric assays demonstrated a low toxicity rate, whereas Hoechst 33342/propidium iodide staining demonstrated a low rate of apoptosis and necrosis, respectively, in all collagen-treatment groups. Conclusion: Keratocytes can be successfully cultured in vitro and characterized by immunofluorescence using lumican. MTT colorimetric assays, and Hoechst 33342, and propidium iodide staining demonstrated a higher rate of cell death in cells cultured without collagen, indicating collagen protects keratocytes from the cytotoxic effects of ultraviolet light.

RESUMO Objetivo: Avaliar o efeito da aplicação da luz ultravioleta e riboflavina sobre ceratócitos da córnea humana in vitro. Métodos: Os ceratócitos foram obtidos a partir das rimas corneoesclerais remanescentes da trepanação de córneas previamente utilizadas em cirurgias de transplante de córnea e cultivadas em meio DMEM/F12 com 2% de FBS até atingir confluência. As culturas de células foram caracterizadas por imunofluorescência com os anticorpos K3 (marcador de células epiteliais), Thy-1 (marcador de fibroblasto) SMA (marcador de miofibroblasto) e Lumican (marcador de ceratócitos). Imunofluorescência também foi feita após o tratamento. As células do estroma da córnea foram cobertas com colágeno (200 µL e 500 µL) e 0,1% de riboflavina e exposta a luz UVA a 370 nm por 30 minutos. Após 24 horas, citotoxicidade foi determinada por ensaio de MTT e a viabilidade celular foi feita por Hoechst 33342/Iodeto de propideo. Resultados: As culturas de células atingiram confluência em aproximadamente 20 dias. Imunofluorescência apontou alta expressão para o marcador de ceratócitos (Lumican) e expressão negativa par os marcadores de células epiteliais (K3), fibroblasto (Thy-1) e miofibroblasto (α-SMA). Após o cross linking a análise de MTT mostrou baixa taxa de toxicidade e com a coloração de Hoechst 33342/Iodeto de propideo baixa taxa de apoptose e necrose respectivamente em todos os grupos que continham colágeno. Conclusão: As culturas de ceratócitos foram obtidas e caracterizadas por imunofluorescência através do marcador Lumican com sucesso. O ensaio de MTT e a coloração por Hoechst 33342 e iodeto de propídio, apresentaram maior índice de morte celular nos grupos que não continham colágeno, provando que protege as células contra os efeitos da luz UVA.

Influência de diferentes densidades de energia do laser de baixa intensidade em fibroblastos derivados da polpa de dentes decíduos / Influence of low-level laser with different energy densities on pulp fibroblasts from human primary teeth

O objetivo deste trabalho foi comparar os efeitos de diferentes densidades de energia e irradiâncias do Laser de Baixa Intensidade (LBI), variando em função do tempo de irradiação e potência, na viabilidade e proliferação de fibroblastos derivados da polpa de dentes decíduos humanos (HPF). HPF foram cultivados em DMEM e usados entre a 4ª e 8ª passagem. Os grupos foram divididos de acordo com diferentes densidades de energia, variando: Tempo de irradiação - Grupo I Ia (1,2 J/cm2 - 5 mW - 10 s), Ib (2,5 J/cm2 - 5 mW - 20 s), Ic (3,7 J/cm2 - 5 mW - 30 s), Id (5,0 J/cm2 - 5 mW - 40 s), e Ie (6,2 J/cm2 - 5 mW - 50 s); ou potência - Grupo II IIa (1,2 J/cm2 - 5 mW - 10 s), IIb (2,5 J/cm2 - 10 mW - 10 s), IIc (3,7 J/cm2 - 15 mW - 10 s), IId (5,0 J/cm2 - 20 mW - 10 s), e IIe (6,2 J/cm2 - 25 mW - 10 s). Células não irradiadas - cultivadas em condições nutricionais regulares - 10% Soro Fetal Bovino (SFB) (If e IIf) e células não irradiadas - cultivadas em déficit nutricional - 1% SFB (Ig e IIg), foram consideradas controles positivos e negativos, respectivamente. A viabilidade e proliferação celular foram avaliadas, repesctivamente, pelas técnicas MTT e Cristal violeta (CV), nos períodos de 24, 48 e 72 horas após a irradiação. Os dados obtidos foram submetidos à análise estatística por ANOVA 2 critérios, seguido pelo teste de Tukey (P<0,05). No ensaio MTT, os controles negativos, Ig e IIg, apresentaram significativamente menor viabilidade em relação aos correspondentes grupos experimentais: IIa e IIb, 24 horas após a irradiação; Ia, Ib, Ie, If e IIf no período de 48 horas; e Ib-If, assim como, IIa-IIf após 72 horas. Nos diferentes períodos de avaliação do ensaio CV, todos os grupos, exceto Ie, IIe e If, exibiram significativamente maior proliferação em comparação aos respectivos controles negativos. Dentro de um mesmo grupo nos diferentes períodos, os grupos If e IIe apresentaram menor viabilidade durante o período de 24 horas em comparação ao período de 72 horas pelo ensaio MTT. Na avaliação intragrupos, o ensaio CV revelou menor proliferação no período de 24 horas em comparação aos períodos de 48 e 72 horas, independente do grupo avaliado. Os diferentes protocolos de irradiação, grupos I e II, não apresentaram diferença estatisticamente significativa na viabilidade e proliferação celular entre densidades de energia iguais com irradiâncias diferentes durante os períodos avaliados. De acordo com os resultados obtidos, as diferentes densidades de energia e irradiâncias propostas não prejudicaram a viabilidade e proliferação de fibroblastos pulpares de dentes decíduos humanos. A variação do protocolo de irradiação LBI, em função do tempo ou da potência, não interferiram nas respostas celulares após a aplicação da mesma densidade de energia com irradiâncias diferentes.(AU)

The aim of this study was to compare the effects of Low-level laser (LLL) with different energy densities and irradiances, varying according to the irradiation time and power, on cell viability and proliferation of pulp fibloblasts from human primary teeth (HPF). HPF were culture in DMEM and used between 4th and 8th passages. Groups were divided according to different energy densities, varying: Time of irradiation Ia (1.2 J/cm2 - 5 mW - 10 s), Ib (2.5 J/cm2 - 5 mW - 20 s), Ic (3.7 J/cm2 - 5 mW - 30 s), Id (5.0 J/cm2 - 5 mW - 40 s), and Ie (6.2 J/cm2 - 5 mW - 50 s); or output power - Grupo II IIa (1.2 J/cm2 - 5 mW - 10 s), IIb (2.5 J/cm2 - 10 mW - 10 s), IIc (3.7 J/cm2 - 15 mW - 10 s), IId (5.0 J/cm2 - 20 mW - 10 s), e IIe (6.2 J/cm2 - 25 mW - 10 s). Non-irradiated cells - grown in regular nutritional conditions - 10% Fetal Bovine Serum (FSB) (If and IIf) and non-irradiated cells - grown in nutritional deficit - 1% FBS (Ig and IIg) were considered positive and negative controls, respectively. Cell viability and proliferation were respectively assessed through MTT and Crystal violet (CV) assays at 24, 48 and 72h after irradiation. Data were submitted to statistical analysis by ANOVA 2 criteria, followed by Tukey test (P<0.05). In the MTT assay, the negative controls, Ig and IIg, showed significantly lower viability in relation to the corresponding groups: IIa and IIb 24 hours after irradiation; Ia, Ib, Ie, If and IIf at 48 hours period; and Ib-If, as IIa-IIf, after 72 hours. At different periods of evaluation of CV assay, all groups, except Ie, IIe and If, exhibited significantly higher proliferation compared to the respective negative controls. Within the same group at different periods, groups If and IIe showed lower viability during 24 hours compared to 72 hours period by MTT assay. In the intragroup evaluation, CV assay revealed lower proliferation at 24 hours compared to 48 and 72 hours periods, regardless of the evaluated group. Different irradiation protocols, groups I and II, showed no statistically significant differences on cell viability and proliferation among equals energy densities with different irradiances at the evaluated periods. According to these findings, different LLL energy densities and irradiances proposed did not impair viability and proliferation of pulp fibloblasts from human primary teeth. The variation of the LLL irradiation protocol, by the time or power, did not interfere in cellular responses after the application of the same energy density with different irradiances.(AU)

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence

BACKGROUND: Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. RESULTS: Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. CONCLUSION: We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

Effects of radiofrequency on the healing of skin wounds in rats: analysis using digital planigraphy and histological evaluation / Efeitos da radiofrequência na cicatrização de feridas cutâneas em ratos: análise por planigrafia digital e avaliação histológica

Introduction: Given the extensive use of plastic surgery and the search for better aesthetic and functional results, it is necessary to research ways to improve healing and scarring. The objective is to evaluate the effects of three radiofrequency (RF) sessions in healing the skin of mice. Methods: Forty-eight rats were divided into four groups by day of sacrifice and treatment (RF group: RG; control group: CG). Dissection of the excisional wound of 2 cm x 2 cm (4 cm²) was performed and a 6-mm punch was used to hold two excisional wounds 0.6 cm in diameter. After 24 h, radiofrequency was performed using Spectra® directly on the wound in the dorsal region for 7 minutes at 38°C. This was repeated three times on alternate days. For the control group, the radiofrequency protocol was performed with the device switched off. Results: A larger area of the square wound was measured on postoperative day three in RG (RG7: 3.3 cm² ± 0.7 cm² vs. CG7: 2.4 cm² ± 0.4 cm²; p = 0.009). On day 14, the square wound in RG was greater than in CG (RG14: 1.9 cm² ± 0.5 cm² vs. CG14: 1.0 cm² ± 0.3 cm²; p = 0.001). There was a 90% closure of wounds in CG14. In RG14, 60% of the wounds were re-epithelized while 40% remained ulcerated. In CG7, 70% of the remaining wounds were ulcerated and 30% were re-epithelized. In RG7, 8% were re-epithelized and 92% remained ulcerated. Conclusion: Radiofrequency has a negative influence on the healing process, as indicated by mice that received radiofrequency having a persistent ulcerated wound.

Introdução: Tendo em vista o número de cirurgias plásticas e a busca por melhores resultados estético-funcionais fazem-se necessárias pesquisas para encontrar meios para melhorar a cicatrização e as cicatrizes. O objetivo é avaliar os efeitos de três sessões de radiofrequência na cicatrização da pele de ratos. Métodos: Quarenta e oito ratos machos foram divididos em quatro grupos conforme ao grupo que pertenciam e o dia do sacrifício (grupo radiofrequência - GR - e grupo controle - GC). Realizada a dissecação da ferida excisional de 2 cm x 2 cm (4 cm²) e utilizou-se um punch de 6 mm para a realização de duas feridas excisionais de 0,6 cm de diâmetro. Após 24 h, foi realizada a radiofrequência com o equipamento Spectra® na região dorsal, diretamente sobre as feridas por 7 minutos, com temperatura de 38ºC. Repetida por três vezes, em dias alternados. No grupo controle foi realizada com o aparelho desligado. Resultados: Foi encontrada área maior na ferida quadrada, no 3º dia pós-operatório do GR (GR7 3,3 cm² ± 0,7 cm² vs. GC7 2,4 cm² ± 0,4 cm², p = 0,009). No 14º dia a ferida quadrada do GR foi maior do que no GC (GR14 1,9 cm² ± 0,5 cm² vs. GC14 1,0 cm² ± 0,3 cm², p = 0,001). Houve fechamento de 90% das feridas no GC14. No GR14, 60% das feridas foram reepitelizadas enquanto 40% permaneceram ulceradas. No GC7, 70% das feridas de permaneceram ulceradas e 30% foram reepitelizadas. Já no GR7, 8%, foram reepitelizadas e 92% permaneceram ulceradas. Conclusão: A radiofrequência tem influência negativa no processo cicatricial, mostrando que, nos ratos que receberam a radiofrequência, o quadrado permaneceu ulcerado.

Action of AlGaInP laser and high frequency generator in cutaneous wound healing. A comparative study

ABSTRACT PURPOSE: To evaluate in a macroscopic, histological and histomorphometric manner the healing process of cutaneous wounds in mice. METHODS: The sample consisted of 40 male mice and was divided in four groups: 1st group (control, n=10), 2nd group (High Frequency Generator - HF, the maximum amplitude range, 120s, n=10), 3rd group (AlGaInP Laser 660 nm, 30mW power, 5 J/cm2, applying scan mode, 120s, n=10) and 4thgroup (AlGaInP Laser 660 nm, 30 mW power, 8 J/cm2, applying scan mode, n=10). The surgical incision was made with an 8 mm diameter punch perpendicularly to the back of the animal. The statistical analysis was achieved by the statistical test One Way Anova post hoc Tukey Test and significance at p<0.05 in GraphPad Prism program. RESULTS: It was observed that in the acute phase the AlGaInP Laser at 5 J/cm2 provided a greater stimulus to healing, and both lasers were effective in the remodeling phase. CONCLUSION: The AlGaInP lasers from 5 J/cm2 to 8 J/cm2 showed better biomodulatory results in the acute and remodeling phases respectively, however, the HF was less effective than the laser, providing significant benefits only in the acute phase of tissue repair.

Analysis of the healing process of the wounds occurring in rats using lasertherapy in association with hydrocolloid

PURPOSE: To evaluate wound healing in rats by using low-level laser therapy (LLLT) associated with hydrocolloid occlusive dressing. METHODS: Forty male, adult, Wistar rats were used, distributed into four groups: LG (received 2 J/cm² of laser therapy); HG (treated with hydrocolloid); LHG (treated with 2 J/cm² of laser therapy and hydrocolloid); and the CG (treated with 1 mL of 0.9% saline). The wound was evaluated at pre-determined periods 3rd and 7th days, considering the macroscopic and histological parameters (inflammatory cells, capillary neoformation, fibroblasts, collagen formation and reepithelialization).RESULTS: The LG group at seven days showed increased collagen formation, the LHG group at 3 days showed mild collagen formation. The HG group and the CG at 7 days showed complete reepithelialization.CONCLUSION: Low-level laser therapy as well as the hydrocolloid dressing have favored the wound-healing process in rats.

Biomodulation effects of LED and therapeutic ultrasound combined with semipermeable dressing in the repair process of cutaneous lesions in rats

PURPOSE: To compare the biomodulatory effects of LED and ultrasound combined with semipermeable dressing in the repair of cutaneous lesions. METHODS: Eighty-four Wistar rats were submitted to surgical injury (2.5 cm) and divided into four groups (n=21): Group I (control), Group II (LED therapy, LED), Group III (LED therapy + dressing, LED+D) and Group IV (ultrasound + dressing, US+D). At seven, 14 and 21 days, the animals were euthanized, and the specimens of interest removed for histological analysis. RESULTS: Histological and histomorphometric analysis revealed a greater percent wound regression in animals receiving the dressing (group III: 55.97; group IV: 53.06), as well as a greater reduction in the inflammatory infiltrate (group III: 29.14; group IV: 31.71) since day 7. A later effect, with progression of the tissue repair process only after 14 and 21 days, was observed in the LED group intense fibroblast proliferation and greater collagen fiber production and organization were seen in the LED+D and US+D groups compared to the other groups. CONCLUSION: LED combined with a dressing was more effective at accelerating in the repair of cutaneous lesions. .

Effect of reduced exposure times on the cytotoxicity of resin luting cements cured by high-power led

OBJECTIVE: Applications of resin luting agents and high-power light-emitting diodes (LED) light-curing units (LCUs) have increased considerably over the last few years. However, it is not clear whether the effect of reduced exposure time on cytotoxicity of such products have adequate biocompatibility to meet clinical success. This study aimed at assessing the effect of reduced curing time of five resin luting cements (RLCs) polymerized by high-power LED curing unit on the viability of a cell of L-929 fibroblast cells. MATERIAL AND METHODS: Disc-shaped samples were prepared in polytetrafluoroethylene moulds with cylindrical cavities. The samples were irradiated from the top through the ceramic discs and acetate strips using LED LCU for 20 s (50 percent of the manufacturer's recommended exposure time) and 40 s (100 percent exposure time). After curing, the samples were transferred into a culture medium for 24 h. The eluates were obtained and pipetted onto L-929 fibroblast cultures (3x10(4) per well) and incubated for evaluating after 24 h. Measurements were performed by dimethylthiazol diphenyltetrazolium assay. Statistical significance was determined by two-way ANOVA and two independent samples were compared by t-test. RESULTS: Results showed that eluates of most of the materials polymerized for 20 s (except Rely X Unicem and Illusion) reduced to a higher extent cell viability compared to samples of the same materials polymerized for 40 s. Illusion exhibited the least cytotoxicity for 20 s exposure time compared to the control (culture without samples) followed by Rely X Unicem and Rely X ARC (90.81 percent, 88.90 percent, and 83.11 percent, respectively). For Rely X ARC, Duolink and Lute-It 40 s exposure time was better (t=-1.262 p=0,276; t=-9.399 p=0.001; and t=-20.418 p<0.001, respectively). CONCLUSION: The results of this study suggest that reduction of curing time significantly enhances the cytotoxicity of the studied resin cement materials, therefore compromising their clinical performance.

In vitro effect of 470 nm LED (Light Emitting Diode) in keloid fibroblasts / Efeito in vitro do LED (Light Emitting Diode) de 470 nm em fibroblastos de quelóide

Purpose: To quantify keloid fibroblasts after irradiation with 470nm blue LED, in vitro. Methods: Fibroblasts from keloid and adjacent skin have been obtained from 6 patients. Cells have been cultivated and maintained in DMEM culture medium. In Petri dishes, they were irradiated with energy doses of 6J, 12J and 18J. After 24 h, counting was done by the average of the triplicates for each sample. Results: There were no significant differences in the number of irradiated keloid fibroblasts at the studied doses (p=0.261). In adjacent skin fibroblasts, differences were observed (p=0.025) concerning the doses of 18 J and 6 J (p=0.03). Conclusions: There was a reduction in the number of adjacent skin fibroblasts irradiated with 470nm blue LED at the energy dose of 18 J compared to the ones irradiated at the energy dose of 6 J. There were no changes in keloid fibroblasts counting at any of the doses applied, 24 h after irradiation.

Objetivo: Quantificar fibroblastos de quelóide após irradiação com LED azul de 470nm, in vitro. Métodos: Foram obtidos fibroblastos de quelóide e pele adjacente, de seis pacientes. As células foram cultivadas e mantidas em meio de cultura DMEM. Em placas de Petri, receberam irradiação com doses de energia de 6J, 12J e 18J. Após 24 horas a contagem foi feita pela média da triplicata para cada amostra. Resultados: Não houve diferença na quantidade de fibroblastos de quelóide irradiados nas doses estudadas (p=0,261). Observou-se diferença nos fibroblastos de pele adjacente (p=0,025), com relação às doses de 18 J e 6 J (p=0,03). Conclusões: Houve redução dos fibroblastos de pele adjacente irradiados com LED azul de 470 nm na dose de energia de 18 J em relação à dose de 6 J. Não houve alteração na quantidade de fibroblastos de quelóide nas doses aplicadas após 24 horas da irradiação.

Efeitos bioestimulantes do laser de baixa potência no processo de reparo / Biostimulation effects of low-power laser in the repair process

Os lasers de baixa potência promovem efeitos biológicos benéficos, de caráter analgésico, antiinflamatório e cicatrizante, por meio de um fenômeno de bioestimulação. A radiação emitida pelo laser terapêutico afeta os processos metabólicos das células-alvo, produzindo efeitos bioestimulantes que resultam na ocorrência de eventos celulares e vasculares, os quais parecem interferir diretamente no processo de reparo. Este trabalho visa estudar o fenômeno da bioestimulação e destacar os principais efeitos bioestimulantes do laser de baixa potência na reparação tecidual.

The wound healing process has always been an excellent subject for researchers. The use of low-power laser on wounds during the postoperative phase has increased the speed of the healing process. It has been implied that low power radiation affects cellular metabolic processes and promotes beneficial biological effects (analgesic, anti-inflammatory, and healing). Laser biostimulation appears to influence the behavior of the repair process. This paper aims at reviewing the most interesting aspects of the use of low-power laser in the tissue-repair process.

In vitro effect of low intensity laser on the cytotoxicity produced by substances released by bleaching gel

This in vitro study aimed to analyze the effect of different parameters of phototherapy with low intensity laser on the viability of human dental pulp fibroblasts under the effect of substances released by bleaching gel. Cells were seeded into 96 wells plates (1 x 10³ cells/well) and placed in contact with culture medium conditioned by a 35 percent hydrogen peroxide bleaching gel for 40 minutes, simulating the clinical condition of the in-office bleaching treatment. Cells cultured in ideal growth conditions served as positive control group (PC), and the cells grown in conditioned medium and non-irradiated served as negative control group (NC). Cells grown in conditioned medium were submitted to a single irradiation with a diode laser (40 mW, 0.04 cm²) emitting at visible red (660 nm; RL) or near infrared (780 nm; NIR) using punctual technique, in contact mode and energy densities of 4, 6 or 10 J/cm². The cell viability was analyzed through the MTT reduction assay immediately and 24 hours after the irradiation. The data was compared by ANOVA followed by the Tukey's test (p < 0.05). The cell viability increased significantly in 24 hours within each group. The PC presented cell viability significantly higher than NC in both experimental times. Only the NIR/10 J/cm² group presented cell viability similar to that of PC in 24 hours. The phototherapy with low intensity laser in defined parameters is able to compensate the cytotoxic effects of substances released by 35 percent hydrogen peroxide bleaching gel.

Single exposure of human oral mucosa fibroblasts to ultraviolet B radiation reduces proliferation and induces COX-2 expression and activation

The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV) sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX)-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM), primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001). HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05). The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

Induction of chromosomal aberrations in human primary fibroblasts and immortalized cancer cells exposed to extremely-low-frequency electromagnetic fields

Rapidly increasing possibilities of exposure to environmental extremely low-frequency electromagnetic fields [ELF-EMF] have become a topic of worldwide investigation. Epidemiological and laboratory studies suggest that exposure to ELF-EMF may increase cancer risk therefore assessment of chromosomal damage in various cell lines might be of predictive value for future risk estimation. Primary cultures of fibroblasts from human skin biopsy were exposed to continuous extremely low-frequency electromagnetic fields [3, 50 and 60 Hz, sinusoidal, 3h, and 4 mT]. Also immortalized cell lines, SW480, MCF-7 and 1321N1 were exposed to continuous ELF-EMF [50 Hz, sinusoidal, 3 h, 4 mT]. Metaphase plates were prepared according to standard methods and stained in 5% Giemsa solution. Chromosomal aberrations of both chromosome and chromatid types were scored to evaluate the effects of ELF-EMF on primary or established cell lines. Results indicate that by increasing the frequency of ELF-EMF, chromosomal aberrations were increased up to 7-fold above background levels in primary human fibroblast cells. In addition, continuous exposure to a 50 Hz electromagnetic field led to a significant increase in chromosomal aberrations in SW480, MCF-7 and 1321N1 cell lines compared to sham control. Results obtained indicate that ELF-EMF has the potential for induction of chromosomal aberrations in all cell types

Avaliação do uso do laser de baixa intensidade e do Light-Emitting Diode (LED) no comportamento de fibroblastos e na redução da incidência da mucosite bucal em crianças sob tratamento quimioterápico / Evaluation of the use of low-intensity laser and Light-Emitting Diode (LED) on the behavior of fibroblasts and in reducing the incidence of oral mucositis in children under chemotherapy

Avaliou-se o uso do laser de baixa intensidade e do Light-Emitting Diode (LED) no comportamento de fibroblastos e na redução da incidência da mucosite bucal em crianças sob tratamento quimioterápico. Para tanto, o estudo foi executado em dois períodos experimentais distintos. No primeiro período, foi realizada a análise da viabilidade de fibroblastos Balb/c 3T3 cultivados sob déficit nutricional irradiados com laser vermelho (660nm, 40mW), laser infravermelho (780nm, 50mW) e LED vermelho (637 ± 15nm, 40mW) por 4 e 8 segundos através dos ensaios de redução do MTT e captação do vermelho neutro. No segundo período experimental realizou-se ensaio clínico randomizado duplo-cego para avaliar a eficácia do laser vermelho (660nm, 40mW) e do LED vermelho (637 ± 15nm, 40mW) na redução da incidência e da severidade da mucosite bucal e da dor relacionada em crianças portadoras de câncer submetidas a quimioterapia utilizando-se o sistema de graduação da mucosite bucal da Organização Mundial de Saúde e a Escala de Dor de Faces Revisada associada à escala analógica visual. A análise estatística foi realizada utilizando-se o teste não paramétrico de Kruskal-Wallis e analise de variância do modelo linear geral com nível de significância de 5% (p = 0,05). Observou-se com a redução do MTT uma tendência de aumento da proliferação celular relacionado diretamente com o tempo de irradiação, no entanto, não significante estatisticamente. Após 72 horas, os grupos que apresentaram maior proliferação celular foram: grupo irradiado com laser infravermelho, grupo irradiado com LED, grupo irradiado com laser vermelho, grupo controle positivo e grupo controle negativo. Já através da captação do vermelho neutro, após 72 horas o grupo que apresentou maior proliferação celular foi o grupo controle positivo (cultivado sob condições nutricionais ideais) seguido pelo grupo irradiado com laser infravermelho, grupo controle negativo e grupos irradiados com LED e laser vermelho.

The use of low-intensity laser and Light-Emitting Diode (LED) on the behavior of fibroblasts and in reducing the incidence of oral mucositis in children under chemotherapy were evaluated. The study was performed in two separate experiments. In the first moment, the viability of fibroblasts Balb/c 3T3 cultured under nutritional stress irradiated with red laser (660nm, 40mW), infrared laser (780nm, 50mW) and red LED (637 ± 15nm, 40mW) for 4 and 8 seconds was analyzed through the MTT and neutral red assays. The second experiment carried out was a double-blind randomized clinical trial aiming to assess the effectiveness of red laser (660nm, 40mW) and red LED (637 ± 15nm, 40mW) in reducing the incidence and severity of oral mucositis and related pain in children with cancer undergoing chemotherapy using the World Health Organization oral mucositis grading system and the Faces Pain Scale - Revised associated with a visual analogue scale. Statistical analysis was performed using the Kruskal-Wallis nonparametric test and ANOVA with a significance level of 5% (p = 0.05). It was observed with the MTT assay a trend of cell proliferation increase directly related to the irradiation time, however, not statistically significant. After 72 hours, the groups that showed higher cell proliferation were: infrared laser irradiated group, LED irradiated group, red laser irradiated group, positive control group and negative control group. By the neutral red assay, though, after 72 hours the group that showed higher cell proliferation was the positive control (grown under ideal nutritional conditions) followed by the group irradiated with infrared laser, the negative control group and groups irradiated with red laser and LED. By analyzing the results and considering the used parameters and phototherapy protocol, it is plausible to conclude that phototherapy with lowintensity laser and light emitting diode (LED) showed no toxicity at...

Niveles de luz ultravioleta ambiental asociados con apoptosis y necrosis en fibroblstos humanos / Environmental levels of ultraviolet light associated with apoptosis and necrosis in human fibroblstos

La apoptosis involucra una serie de episodios altamente organizados y programados que tienen por objeto mantener la estabilidad genómica eliminando células huésped defectuosas. El propósito de este estudio fue determinar las dosis umbral y los tiempos de exposición a UV-A y UV-B medioambientales suficientes como para producir apoptosis y necrosis en la células normales de una línea celular de fobroblastos humanos. Se midieron dosis de UV-A y UV-B durante un período de seis años con un radiómetro UV de cuatro canales. Se irradiaron los fibroblastos una vez utilizando Simuladores Solar UV Oriel con seis dosis de UV basados en el medio ambiente. Las dosis correspondieron a 0, 11, 19, 23 y 45 minutos de radiación ambiental de UV-A y UV-B al sol del mediodía en Puerto Rico. Se utilizó en método de unión de la anexina-V para diferenciar entre los fibroblastos normales y fibroblastos apoptóticos o necróticos. La dosis umbral de la apoptosis a la necrosis se halló entre 24-28 KJ/m2, correspondiente a los 19 y 23 minutos de exposición ambiental a UV-A y UV-B. Este estudio proporciona los primeros datos que especifican las dosis de umbral medioambientales de UV-A y UV-B en las que los fibroblastos humanos experimentas apoptosis y necrosis. Estos resultados pueden proporcionar valiosos umbrales dosis-respuesta de apoptosis y necrosis para futuros estudios mecanicistas y datos de línea de base para programas de prevención de cáncer de piel.

Efeitos da irradiação com o laser HeNe 632.8nm sobre a cicatrização de feridas em ratos / Los efectos de la aplicación del laser HeNe 632.8nm sobre heridas en ratones / Effects of HeNe 632.8nm laser application on wound healing in rats

O objetivo deste estudo foi investigar os efeitos de laser HeNe 632.8nm sobre o conteúdo de fibroblastos e sua influência no processo cicatrizacional de feridas em ratos. Os animais, após incisão intencional, foram submetidos a irradiação com laser HeNe 632.8nm (4J/cm², única e diária) por períodos de 1, 2, 3, 4, 5, 7, 9, 11, 15, 21 e 29 dias. A avaliação histológica e a contagem de fibroblastos foram realizadas através do programa de captura de imagem Image Pro Plus 5. A irradiação com o laser HeNe 632.8nm promoveu uma redução no conteúdo de fibroblastos na área da ferida irradiada, sugerindo um aumento da síntese de colágeno e na diferenciação de fibroblastos. Os dados indicaram que a irradiação pelo laser HeNe 632.8nm interfere no processo de cicatrização de feridas.

The aim of this study was to investigate the effects of HeNe 632.8nm laser on fibroblast content and its influence on the wound healing process in rats. After a deliberate incision, the animals were subjected to irradiation with HeNe laser 632.8nm (4J/cm², once a day) for periods of 1, 2, 3, 4, 5, 7, 9, 11, 15, 21 and 29 days. The histological analysis and fibroblasts count were analyzed using Image Pro Plus 5 imaging software. HeNe laser irradiation promoted a decrease in the fibroblast content in the irradiated wound area, suggesting an increase in collagen synthesis and fibroblasts differentiation. The data indicated that irradiation with HeNe 632.8nm interferes in the wound healing process.

El objetivo del presente estudio fue investigar los efectos de HeNe 632.8nm láser sobre el contenido de fibroblastos y su influencia en el proceso de cicatrización de heridas en ratones. Los animales tras incisión intencional han sido sometidos a irradiación con laser HeNe 632.8nm (4J/cm., una vez por día) por períodos de 1, 2, 3, 4, 5, 7, 9, 11, 15,21 y 29 días. La evaluación histológica y el número de los fibroblastos fueron analizadas por un programa de captura de imagen Image Pro Plus 5. La irradiación con láser HeNe 632.8nm promovió una disminución en el número de los fibroblastos en el área de la herida, lo que sugiere un aumento en el proceso de síntesis de colágeno y de la diferenciación de fibroblastos. Los datos indicaron que la irradiación por el láser HeNe interfirió en el proceso de cicatrización de la herida.

Hybrid spheroids as a tool for prediction of radiosensitivity in tumor therapy.

Optimization in radiotherapy may be conceivably achieved by individualized treatment regimens. For this, the radiosensitivity of the tumor cells to be treated must be known. A method is presented to show that the effect of radiation on tumor cells in spheroids can be quantitatively evaluated without complicated cell determinations of spheroid composition. This evaluation is based on the dynamics of inactivation of the colony forming ability of whole spheroids composed chiefly of non-transformed diploid fibroblasts and a minority of HeLa "test" cells. Here, spheroids of identical composition, but of different sizes are inactivated proportional to their sizes, thus obviating the need for tedious single cell procedures. The use of spheroids of different sizes permits the deduction of dose-effect relationships, and the innate radiosensitivity of tumors cells. This is a novel method for measuring the radio and chemosensitivity of tumors in primary culture, i.e. cells directly isolated from tumors.

Use of lectins to evaluate the effects of GaAS softlaser on dog tendon

1. Lectins labeled with colloidal gold particles were used for the ultrastructural evaluation of the biological effects of GaAs softlaserirradiation on the healing of dog tendon wounds. 2. Six dogs were submitted to tenotomy and tenorrhaphy on the right and left hind legs. All animals received laser irradiation (4J/cm²) daily for ten days only on the left leg, and the right leg of the same animal was used as control. Biopsies were taken 11, 22 and 40 days after surgery. 3. Laser-irradiated and control tendon tissues were embedded in L.R. White resin and thin section were incubated in the presence of gold-labeled Arachis hypogaea (PNA), Canavalia ensiformes (Con A) and Triticum vulgare (WGA). 4. In general, the control and laser-irradiated tissues presented homogeneous and similar labelling of heterochromatin, rough endoplasmic reticulum and extracellular matrix. 5. We conclude that GaAs softlaser irradiation does not produce significant changes in the glycosylation of healing tendons

Biostimulation by weak laser radiation: a survey

Bioestimulaçäo por radiaçäo laser de baixa intensidade - Um comentário. Relato sobre a situaçäo experimental atual relativa à aplicaçäo médica do laser em bioestimulaçäo