Laser and LED photobiomodulation effects in osteogenic or regular medium on rat calvaria osteoblasts obtained by newly forming bone technique.

The purposes of this study are to evaluate the effects of photobiomodulation (PBM) with laser and LED on rat calvaria osteoblasts (rGO lineage), cultured in osteogenic (OST) or regular (REG) medium, after induction of a quiescent state and to test if PBM is capable of osteogenic induction and if there is a sum of effects when combining OST medium with PBM. Before irradiation, the cells were put in a quiescent state (1% FBS) 24 h, when red (AlGaInP-660 nm) and infrared laser (GaAlAs-808 nm) and LED (637 ± 15 nm) were applied. The groups were as follows: red laser (RL3-5 J/cm2, 3 s and RL5-8.3 J/cm2, 5 s, 1.66 W/cm2); infrared laser (IrL3-5 J/cm2, 3 s and IrL5-8.3 J/cm2, 5 s); LED (LED3-3 s and LED5-5 s, 0.02 J/cm2, 0.885 W/cm2); positive (C+, 10% FBS) and negative control (C-, 1% FBS). For alkaline phosphatase (ALP) and mineralization assays, the cells were cultured in REG (DMEM 10% FBS) and OST medium (DMEM 10% FBS, 50 µg/mL ascorbic acid, 10 mM ß-glycerophosphate). Statistical analysis was performed using ANOVA and Tukey's tests (p < 0.05). RL5 and LED5 increased proliferation, in vitro wound closure, ALP, and mineralization in rGO cells (p < 0.05). PBM with red laser and LED induced mineralization by itself, without osteogenic medium, not observed for infrared laser (p < 0.05). A sum of effects was observed in osteogenic medium and PBM by infrared, red laser, and LED (5 s). Red laser and LED increased proliferation, migration, and secretory phases in rGO cells in a dose-dependent manner. PBM with red laser and LED promotes osteogenic induction by itself. PBM with infrared laser and osteogenic medium potentializes mineralization.

Comparison of the effect of root surface modification with citric acid, EDTA, and aPDT on adhesion and proliferation of human gingival fibroblasts and osteoblasts: an in vitro study.

Root demineralization is used in Periodontics as an adjuvant for mechanical treatment. The aim of this study was to evaluate the effects of root surface modification with mechanic, chemical, and photodynamic treatments on adhesion and proliferation of human gingival fibroblasts and osteoblasts. Root fragments were treated by scaling and root planing (C-control group), EDTA (pH 7), citric acid plus tetracycline (CA-pH 1), and antimicrobial photodynamic therapy (aPDT) with toluidine blue O and red laser (pH 4). Cells were seeded (104 cells/well, 6th passage) on root fragments of each experimental group and cultured for 24, 48, and 72 h. Cells were counted in scanning electron microscopy images by a calibrated examiner. For fibroblasts, the highest number of cells were present at 72-h period (p < 0.05). EDTA group showed a very low number of cells in relation to CA group (p < 0.05). CA and aPDT group presented higher number of cells in all periods, but without differences between other treatment groups (p > 0.05). For osteoblasts, there was a significant increase in cell numbers for aPDT group at 72 h (p < 0.05). In conclusion, aPDT treatment provided a positive stimulus to osteoblast growth, while for fibroblasts, aPDT and CA had a tendency for higher cell growth.

Low level laser therapy modulates viability, alkaline phosphatase and matrix metalloproteinase-2 activities of osteoblasts.

Low level laser therapy (LLLT) has been shown to stimulate bone cell metabolism but their impact on the matrix metalloproteinase (MMP) expression and activity is little explored. This study evaluated the influence of LLLT at two different wavelengths, red and infrared, on MC3T3-E1 preosteoblast viability, alkaline phosphatase (ALP) and MMP-2 and -9 activities. To accomplish this, MC3T3-E1 cells were irradiated with a punctual application of either red (660nm; InGaAIP active medium) or infrared (780nm; GaAlAs active medium) lasers both at a potency of 20mW, energy dose of 0.08 or 0.16J, and energy density of 1.9J/cm2 or 3.8J/cm2, respectively. The control group received no irradiation. Cellular viability, ALP and MMP-2 and -9 activities were assessed by MTT assay, enzymatic activity and zymography, respectively, at 24, 48 and 72h. The treatment of cells with both red and infrared lasers significantly increased the cellular viability compared to the non-irradiated control group at 24 and 48h. The ALP activity was also up modulated in infrared groups at 24 and 72h, depending on the energy densities. In addition, the irradiation with red laser at the energy density of 1.9J/cm2 promoted an enhancement of MMP-2 activity at 48 and 72h. However, no differences were observed for the MMP-9 activity. In conclusion, when used at these specific parameters, LLL modulates both preosteoblast viability and differentiation highlighted by the increased ALP and MMP-2 activities induced by irradiation.

"Aroeira" (Myracrodruon urundeuva) methanol extract: the relationship between chemical compounds and cellular effects.

CONTEXT: "Aroeira" [Myracrodruon urundeuva Allemão (Anacardiaceae)] is a tree whose leaves have been studied for therapeutic purposes in medicine and dentistry. OBJECTIVE: The study chemically identifies the leaf extract of aroeira and determines its effect on human gingival fibroblasts. MATERIALS AND METHODS: An 80% methanol leave extract was obtained by maceration and chemically identified through flow-injection analysis-electrospray ionization-ion trap-tandem mass spectrometry (FIA-ESI-IT-MSn). Cytotoxicity of the aroeira's methanol extract was evaluated in lineage of fibroblasts. Adherent cells were treated with different concentrations of aroeira's methanol extract in the medium: 0.1, 1, 10, 100 and 1000 µg/mL. Control cells were cultivated in the medium only. Analyses were done at 24, 48, 72 and 96 h of culture by neutral red assay; and at 24, 48 and 96 h by crystal violet assay. RESULTS: FIA-ESI-IT-MS analysis determined the presence of compounds, for the first time in the species: quercetin-O-glucuronide and quercetin-O-deoxyhexose-O-glucose in the extract. On one hand, neutral red and crystal violet assay showed a reduction (to 50% up until 100%) of cellular viability of groups of 100 and 1000 µg/mL compared with control at 96 h (p < 0.05). On the other hand, lower concentrations (0.1; 1 and 10 µg/mL) of the extract were similar to that of the control at 96 h (p < 0.05), in general. CONCLUSIONS: In view of the results, we can conclude that the extract of aroeira presents tannins and flavonoids. Furthermore, the extract is capable of modulating the viability of human gingival fibroblasts according to its concentration.

Viability of Human Gingival Fibroblast (FGH) Treated with Ethanolic "Aroeira" Extract (Myracrodruon urundeuva Allemão)

ABSTRACT The aim of this study was to evaluate the effect of ethanolic "aroeira" (Myracrodruon urundeuva) extract on the viability of human gingival fibroblast. For this, fibroblasts (2x103 cells/well) were plated in a 96-well plate and incubated for 24 h; the medium (Eagle's medium modified by Dulbecco - DMEM) supplemented with 10% fetal bovine serum was replaced by DMEM with different ethanolic extract concentration (0, 0.1, 1, 10, 100, and 1000μg / mL). The fibroblast viability was analyzed after 48,72, and 96 h by the neutral red capture test and violet crystal. The "aroeira" extract, at high concentrations (100 and 1000 µg/mL) caused decrease in both cellular viability tests (p<0.05). However, dilutions between 0.1 and 10 µg/mL did not affect the viability of the cells. It was concluded that "aroeira" extract was able to change the gingival fibroblast viability, and this effect was concentration dependent.

Laser and light-emitting diode effects on pre-osteoblast growth and differentiation.

The acceleration of bone regeneration by low-intensity laser irradiation may hold potential benefits in clinical therapy in orthopedics and dentistry. The purpose of this study is to compare the effects of light-emitting diode (LED) and laser on pre-osteoblast MC3T3 proliferation and differentiation. Cells were irradiated with red, infrared, and LED (3 and 5 J/cm(2)). Lasers had a power density of 1 W/cm(2) and irradiation time of 2 and 5 s. LED had a power density of 60 mW/cm(2) and irradiation time of 50 and 83 s. Control group did not receive irradiation. Cell growth was assessed by a colorimetric test (MTT) (24, 48, 72, and 96 h), and cell differentiation was evaluated by alkaline phosphatase (ALP) quantification after growth in osteogenic medium (72 and 96 h and 7 and 14 days). At 24 h, the cell growth was enhanced 3.6 times by LED (5 J/cm(2)), 6.8 times by red laser (3 J/cm(2)), and 10.1 times by red laser (5 J/cm(2)) in relation to control group (p < 0.05). At the other periods, there was no influence of irradiation on cell growth (p > 0.05). The production of ALP was not influenced by irradiation at any period of time (p > 0.05). Low-intensity laser and LED have similar effects on stimulation of cell growth, but no effect on cell differentiation.

Laser phototherapy at high energy densities do not stimulate pre-osteoblast growth and differentiation.

OBJECTIVE: The aim of this study is to evaluate the effects of red and infrared lasers at high energy densities on pre-osteoblast MC3T3 proliferation and differentiation. BACKGROUND DATA: The acceleration of bone regeneration by low intensity laser irradiation may hold potential benefits in clinical therapy in orthopedics and dentistry. MATERIALS AND METHODS: Cells were irradiated with red (660 nm) and infrared (780 nm) lasers (90 and 150 J/cm2, 40 mW). The control group did not receive irradiation. Cell growth was assessed by a colorimetric test (MTT) (24, 48, 72, 96 h) and cell differentiation was evaluated by alkaline phosphatase (ALP) quantification after growth in osteogenic medium (72, 96 h; 7, 14 days). RESULTS: None of the irradiation groups had an enhancement in cell growth (p<0.05). The production of ALP was not influenced by irradiation at any period of time (p>0.05). CONCLUSIONS: The low intensity laser stimulated neither cell growth nor the production of alkaline phosphatase.

Evaluation of tissue reaction to Aroeira (Myracrodruon urundeuva) extracts: a histologic and edemogenic study.

OBJECTIVES: This study evaluated subcutaneous tissue response to Aroeira (Myracrodruon urundeuva) extract employing edemogenic and histological analyses. MATERIAL AND METHODS: Test groups consisted of aqueous and ethanolic Aroeira extracts and saline (control). For edema quantification, 18 rats received an intravenous injection of Evan's Blue. After 30 min, the extracts and saline were injected on the dorsum of the rats, which were then sacrificed after 3 and 6 h. Readings were performed in a spectrophotometer. For subcutaneous implantation, 30 rats received a polyethylene tube containing the extracts on their dorsum and then they were killed after 7 and 28 days. The samples were processed for histological analysis and evaluated with a light microscope. The inflammatory infiltrate was quantified. RESULTS: There were no statistically significant differences between aqueous extract and saline groups in relation to edema quantification in the different periods (p>0.05). Ethanolic solution resulted in more edema independently of the experimental period (p<0.05). Histological analysis showed similar results on the 7-day period for the 3 groups. There was a notable reduction on inflammatory cell number for saline and aqueous extract groups at 28 days. CONCLUSION: The aqueous extract showed biocompatible properties similar to those of saline.

Evaluation of tissue reaction to Aroeira (Myracrodruon urundeuva) extracts: a histologic and edemogenic study

OBJECTIVES: This study evaluated subcutaneous tissue response to Aroeira (Myracrodruon urundeuva) extract employing edemogenic and histological analyses. MATERIAL AND METHODS: Test groups consisted of aqueous and ethanolic Aroeira extracts and saline (control). For edema quantification, 18 rats received an intravenous injection of Evan's Blue. After 30 min, the extracts and saline were injected on the dorsum of the rats, which were then sacrificed after 3 and 6 h. Readings were performed in a spectrophotometer. For subcutaneous implantation, 30 rats received a polyethylene tube containing the extracts on their dorsum and then they were killed after 7 and 28 days. The samples were processed for histological analysis and evaluated with a light microscope. The inflammatory infiltrate was quantified. RESULTS: There were no statistically significant differences between aqueous extract and saline groups in relation to edema quantification in the different periods (p>0.05). Ethanolic solution resulted in more edema independently of the experimental period (p<0.05). Histological analysis showed similar results on the 7-day period for the 3 groups. There was a notable reduction on inflammatory cell number for saline and aqueous extract groups at 28 days. CONCLUSION: The aqueous extract showed biocompatible properties similar to those of saline.

Effect of laser phototherapy on the release of fibroblast growth factors by human gingival fibroblasts.

The effects of laser phototherapy on the release of growth factors by human gingival fibroblasts were studied in vitro. Cells from a primary culture were irradiated twice (6 h interval), with continuous diode laser [gallium-aluminum-arsenium (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP),_660 nm] in punctual and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). Positive [10% fetal bovine serum (FBS)] and negative (1%FBS) controls were not irradiated. Production of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) was quantified by enzyme-linked immunosorbent assay (ELISA). The data were statistically compared by analysis of variance (ANOVA) followed by Tukey's test (P

Terapia com laser em baixa intensidade na cicatrização de feridas: revisão de literatura / Low-intensity laser therapy on wound healing: review of literature

Os lasers em baixa intensidade são utilizados como agentes terapêuticos após o tratamento convencional,mostrando propriedades anti-inflamatórias, analgésicas e de aceleração da cicatrização de feridas; assim, podem propiciar um pós-operatório mais confortável ao paciente, possibilitando a redução do uso de medicamentos. Apesar de grandes controvérsias na literatura, ora mostrando efeitos positivos desta terapia, ora negativos, os estudos com cultura de células têm se mostradopromissores. Tais pesquisas comprovaram que o laser é responsável por aumento da proliferação da atividade celular, aumento da produção de colágeno e da síntese de DNA, modulação da produção dos fatoresde crescimento e redução na produção de prostaglandinas. O objetivo desta revisão de literatura é mostrar oscontrastes existentes entre trabalhos sobre a terapia com laser em baixa intensidade, citando estudos em humanos e em animais e dando ênfase aos estudos com cultura de células. Estes últimos têm mostrado resultadospromissores e auxiliam no entendimento dos mecanismos de ação do laser nos tecidos e na cicatrização de feridas. Como conclusão, é possível afirmar que, apesar de a literatura ser controversa a respeito do assunto, as pesquisas básicas in vitro destacam-se pelos seusresultados promissores. Com base nesses estudos será possível esclarecer os mecanismos de ação do laser nacicatrização de feridas e determinar protocolos-padrão de aplicação in vivo

Efeito da terapia com laser em baixa intensidade (LILT) na expressão de fatores de crescimento da família FGF por fibroblastos gengivais humanos / Low-intensity laser therapy (LILT) effects on fibroblast growth factors expression by human gingival fibroblasts

A terapia com laser em baixa intensidade, conhecida como Low-intensity lasertherapy (LILT), tem sido alvo de pesquisas que buscam esclarecer os mecanismospelos quais o laser age na cicatrização de feridas. Este processo é dependente datransmissão de sinais entre epitélio e conjuntivo, os quais influenciam a proliferaçãoe a migração celular, onde participam fatores de crescimento. O objetivo destapesquisa foi verificar os efeitos da radiação com lasers vermelho e infravermelho embaixa intensidade na produção de fatores de crescimento por fibroblastos in vitro.Foi obtida uma cultura primária de fibroblastos gengivais humanos (linhagem FGH).As células foram cultivadas em placas de 96 poços (5 x 103 células /poço), emestado de quiescência (meio de cultura suplementado com 1% de soro fetal bovino)e irradiadas com lasers de diodo (AlGaAs - 660nm, e AlGaInP - 780nm). O laser emmodo contínuo, foi aplicado em contato e na forma pontual. A potência utilizada foide40mW numa área de 0,042cm2 e com densidades de energia de 2, 3, 4, 5 e6J/cm² com respectivos tempos de aplicação de 2, 3, 4, 5 e 6s. As culturas foramirradiadas duas vezes com 6h de intervalo. Os grupos controle positivo e negativoforam cultivados com 10% e 1% de soro fetal bovino, respectivamente e nãoreceberam irradiação. A produção do fator de crescimento de queratinócitos (KGF) edo fator de crescimento de fibroblastos básico (bFGF) foi analisada e quantificadapor ensaio imunoenzimático (método ELISA) no meio condicionado pelas células. Osdados foram comparados estatisticamente pela análise de variância e teste de.

Low-intensity laser therapy (LILT) has been the subject of researches to understandthe mechanisms by which the laser acts stimulating wound healing. This process isdependent of a signaling transfer between epithelium and connective tissue and itinfluences cellular proliferation and migration. Growth factors are directly involved inthese mechanisms. The aim of this study was to analyze the effects of red andinfrared low-level laser on production of fibroblast growth factors by human gingivalfibroblasts in vitro. The cells were obtained by primary culture (FGH cell line). Theywere grown in 96 wells plates (5x10³ cells / well) in a quiescence environment (1%fetal bovine serum) and then they were irradiated with a diode laser (GaAlAs -660nm, and InGaAlP - 780nm). The continuous laser was applied in contact and in apunctual mode. The power was40mW, the area of irradiation was 0,042cm2, theenergy densities were 2, 3, 4, 5 and 6J/cm² with 2, 3, 4, 5 and 6s of time application,respectively. The cultures were irradiated twice with 6 h-interval. The positive andnegative control groups were grown in 10% and 1% of bovine fetal serum,respectively and didn’t receive any irradiation. The production of keratinocyte growthfactor (KGF) and basic fibroblast growth factor (bFGF) was analyzed and quantifiedby immunoenzymatic test (ELISA) in the conditioned medium. The data werecompared statistically by analysis of variance and Kruskal-Wallis, complemented byTukey’s and Dunn’s tests, respectively (p<0,05). Positive controls produced moreKGF and bFGF than negative controls. The KGF production was similar in all.