RESUMO
The present study aimed to evaluate the effect of photobiomodulation therapy (PBM) on different stages of osteogenesis in vitro. For this, osteoblastic-like cells (Saos-2 cell lineage) were irradiated in two different periods: during the Proliferation phase (PP; from the second to the fourth day) and during the Differentiation phase (DP; from the seventh to the ninth day). The energy density used in the study was 1.5 J/ cm2. The following parameters were evaluated: 1) quantification of collagen type 1 (COL 1), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2); 2) quantification of alkaline phosphatase (ALP) activity; and 3) quantification of extracellular matrix (ECM) mineralization. Non-irradiated cultures were used as controls. The data were analyzed using the Student's t-test or one-way ANOVA, considering a significance level of 5%. The results indicated that COL 1 and BMP-2 quantification was higher in Saos-2 irradiated during the DP in relation to the control group at day 10 (p < 0.05). No differences were observed for other comparisons at this time point (p > 0.05). OPN expression was greater in PP compared with the other experimental groups at day 10 (p < 0.05). Irradiation did not affect ALP activity in Saos-2 regardless of the exposure phase and the time point evaluated (p > 0.05). At day 14, ECM mineralization was higher in Saos-2 cultures irradiated during the DP in relation to the PP (p < 0.05). In conclusion, the results suggested that the effects of PBM on osteoblastic cells may be influenced by the stage of cell differentiation.
Assuntos
Fosfatase Alcalina , Proteína Morfogenética Óssea 2 , Diferenciação Celular , Proliferação de Células , Colágeno Tipo I , Terapia com Luz de Baixa Intensidade , Osteoblastos , Osteogênese , Osteopontina , Osteogênese/efeitos da radiação , Humanos , Proteína Morfogenética Óssea 2/metabolismo , Fosfatase Alcalina/metabolismo , Osteopontina/metabolismo , Diferenciação Celular/efeitos da radiação , Colágeno Tipo I/metabolismo , Osteoblastos/efeitos da radiação , Osteoblastos/citologia , Osteoblastos/metabolismo , Proliferação de Células/efeitos da radiação , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiaçãoRESUMO
The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.
Assuntos
Embrião não Mamífero/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Matriz Extracelular/efeitos da radiação , Palaemonidae/efeitos da radiação , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/genética , Matriz Extracelular/genética , Água Doce/química , Palaemonidae/genética , Palaemonidae/crescimento & desenvolvimentoRESUMO
The present study aims to assess the influence of Aluminum-Gallium-Indium-Phosphide laser (AlGaInP laser, λâ¯=â¯660â¯nm), whether or not in association with the application of a membrane of bacterial cellulose (Nexfill™), during recovery from induced second-degree burns at the dorsum of Wistar rats. (Rattus norvegicus, Wistar). Forty-eight animals have been distributed into four groups: Control (burns remained untreated), Group I (laser-treated), Group II (treated with Nexfill), and Group III (laser + Nexfill™). In addition to a morphological analysis, immunohistochemical analysis has been performed for type I collagen, type III collagen, fibronectin, and laminin. The Fisher's Test was used to assess differences among groups (pâ¯<â¯0,05). A larger amount of collagen type III was observed in Control, Group II and Group III when compared with Group I (pâ¯<â¯0,05). Group I and Group III have shown a greater collagen deposition when compared with Group II (pâ¯<â¯0,05), but the amount of collagen was similar in Group I, Group III, and Control. Group III has shown larger fibronectin amounts in comparison with Group II (pâ¯<â¯0,05). As regards laminin, Group I has shown a predominant discontinuity pattern on the basal lamina in comparison with Control, Group II, and Group III (pâ¯<â¯0,05). It is concluded that in this current study the laser when used alone (Group I) hasn't influenced collagen deposition neither has it acted on fiber pattern (fibril and/or reticular). Moreover, laser application hasn't accelerated the repair of wounds caused by inflicted second-degree burns.
Assuntos
Queimaduras/terapia , Celulose , Matriz Extracelular/efeitos da radiação , Lasers , Cicatrização/efeitos dos fármacos , Cicatrização/efeitos da radiação , Animais , Bactérias/metabolismo , Celulose/farmacologia , Celulose/uso terapêutico , Colágeno/metabolismo , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/metabolismo , Imuno-Histoquímica , Laminina/metabolismo , Masculino , Membranas Artificiais , Ratos , Ratos WistarRESUMO
INTRODUCTION: Teeth are often included in the radiation field during head and neck radiotherapy, and recent clinical evidence suggests that dental pulp is negatively affected by the direct effects of radiation, leading to impaired sensitivity of the dental pulp. Therefore, this study aimed to investigate the direct effects of radiation on the microvasculature, innervation, and extracellular matrix of the dental pulp of patients who have undergone head and neck radiotherapy. METHODS: Twenty-three samples of dental pulp from patients who finished head and neck radiotherapy were analyzed. Samples were histologically processed and stained with hematoxylin-eosin for morphologic evaluation of the microvasculature, innervation, and extracellular matrix. Subsequently, immunohistochemical analysis of proteins related to vascularization (CD34 and smooth muscle actin), innervation (S-100, NCAM/CD56, and neurofilament), and extracellular matrix (vimentin) of the dental pulp was performed. RESULTS: The morphologic study identified preservation of the microvasculature, nerve bundles, and components of the extracellular matrix in all studied samples. The immunohistochemical analysis confirmed the morphologic findings and showed a normal pattern of expression for the studied proteins in all samples. CONCLUSIONS: Direct effects of radiotherapy are not able to generate morphologic changes in the microvasculature, innervation, and extracellular matrix components of the dental pulp in head and neck cancer patients.
Assuntos
Polpa Dentária/efeitos da radiação , Neoplasias de Cabeça e Pescoço/radioterapia , Actinas/análise , Adolescente , Adulto , Antígenos CD34/análise , Antígeno CD56/análise , Corantes , Cárie Dentária/etiologia , Esmalte Dentário/efeitos da radiação , Polpa Dentária/irrigação sanguínea , Polpa Dentária/citologia , Polpa Dentária/inervação , Dentina/efeitos da radiação , Matriz Extracelular/efeitos da radiação , Feminino , Fibroblastos/efeitos da radiação , Corantes Fluorescentes , Humanos , Filamentos Intermediários/química , Masculino , Microvasos/efeitos da radiação , Pessoa de Meia-Idade , Fibras Nervosas/efeitos da radiação , Moléculas de Adesão de Célula Nervosa/análise , Dosagem Radioterapêutica , Proteínas S100/análise , Vimentina/análiseRESUMO
Osteoarthritis (OA) resulting from injury or disease is associated with increased levels of several matrix metalloproteinases (MMPs), which degrade all components of the complex extracellular matrix in the cartilage. The objective of this study is to investigate the effect of low-level laser therapy (LLLT) on papain-induced joint damage in rats by histopathology and analysis of metalloproteinase 2 and 9 production. Sixty male Wistar rats were randomly distributed into four groups of 15 animals: (1) non-injury negative control, (2) injury positive control, (3) treated with LLLT at 50 mW, and (4) treated with LLLT at 100 mW. OA was induced in animals using papain (4 % solution) followed by treatment with LLLT. After 7, 14, and 21 days, the animals were euthanized. The articular lavage was collected and centrifuged; then, the supernatant was stored prior to protein analysis by western blot. The material was stained with hematoxylin and eosin for histopathological analysis, and Picrosirius Red was used to estimate the percentage of collagen fibers. To determine normal distribution, ANOVA and Tukey's post hoc test were used for comparison between and within each group at each time period. All data are expressed as mean and standard deviation values, with the null hypothesis considered as p < 0.05. Both laser groups (50 and 100 mW) were effective in tissue repair, decreasing collagen type III expression and increasing type I expression in all experimental periods; however, LLLT at 50 mW reduced metalloproteinase 9 more than at 100 mW in 21 days. LLLT at 50 mW was more efficient in the modulation of matrix MMPs and tissue repair.
Assuntos
Cartilagem Articular/efeitos da radiação , Lasers Semicondutores/uso terapêutico , Terapia com Luz de Baixa Intensidade , Cicatrização/efeitos da radiação , Animais , Western Blotting , Cartilagem Articular/lesões , Cartilagem Articular/fisiopatologia , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Papaína , Ratos , Ratos WistarRESUMO
Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and (7)Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.
Assuntos
Apoptose/fisiologia , Terapia por Captura de Nêutron de Boro/métodos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Transferência Linear de Energia/fisiologia , Melanoma Experimental/radioterapia , Análise de Variância , Apoptose/efeitos da radiação , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Colágeno/metabolismo , Primers do DNA/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Citometria de Fluxo , Proteínas de Choque Térmico HSP47/metabolismo , Humanos , Imuno-Histoquímica , Transferência Linear de Energia/efeitos da radiação , Melanócitos , Microscopia Eletrônica de Transmissão , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator de Necrose Tumoral/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Boron Neutron Capture Therapy (BNCT) involves the selective accumulation of boron carriers in tumor tissue followed by irradiation with a thermal or epithermal neutron beam. This therapy is therefore a cellular irradiation suited to treat tumors that have infiltrated into healthy tissues. BNCT has been used clinically to treat patients with cutaneous melanomas which have a high mortality. Human normal melanocytes and melanoma cells were treated with BNCT at different boronophenylalanine concentrations for signaling pathways analysis. BNCT induced few morphological alterations in normal melanocytes, with a negligible increase in free radical production. Melanoma cells treated with BNCT showed significant extracellular matrix (ECM) changes and a significant cyclin D1 decrease, suggesting cell death by necrosis and apoptosis and cell cycle arrest, respectively. BNCT also induced a significant increase in cleaved caspase-3 and a decrease in the mitochondrial electrical potential with selectivity for melanoma cells. Normal melanocytes had no significant differences due to BNCT treatment, confirming the data from the literature regarding the selectivity of BNCT. The results from this study suggest that some signaling pathways are involved in human melanoma treatment by BNCT, such as cell cycle arrest, ECM changes and intrinsic apoptosis.
Assuntos
Terapia por Captura de Nêutron de Boro/efeitos adversos , Melanócitos/efeitos da radiação , Melanoma/radioterapia , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/efeitos da radiação , Radicais Livres/metabolismo , Humanos , Masculino , Melanócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos da radiação , Necrose/induzido quimicamenteRESUMO
OBJECTIVE: Patients who have had pelvic radiotherapy as part of their cancer therapy may develop subsequent urinary bladder effects such as hyperactive bladder, incontinence, and dysuria. Therefore, the goal of this study was to evaluate whether glutamine supplementation could prevent collagen expression damage in healthy urinary bladder caused by radiotherapy. METHODS: Fifteen adult Wistar rats were separated into a control group that received food and water ad libitum (C group), an irradiated group that received a single pelvic radiation dose of 1164 cGy (I group), and an irradiated group supplemented with l-glutamine every day during the entire experimental period (0.65 g/kg of body weight; I+G group). All animals were sacrificed 15 d after irradiation. The extracellular matrix and muscle were quantified by a morphometric method. Picro Sirius Red was used to visualize the different collagen types. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to determine collagen type I and III expressions. RESULTS: The extracellular matrix (C group 36.84±4.37, I group 31.64±5.00, I+G group 35.53±2.60, P=0.0001), muscle (C group 36.43±6.15, I group 29.39±7.08, I+G group 31.38±3.14, P=0.0001), and gene expressions of collagen type I (C group 1.067±0.31, I group 0.579±0.17, I+G group 1.816±0.66, P=0.0009) and type III (C group 0.99±0.28, I group 0.54±0.13, I+G group 1.07±0.28, P=0.0080) were decreased in the I group. Apart from muscle, glutamine supplementation prevented these alterations. Immunohistochemistry and Picro Sirius Red showed similar results. CONCLUSION: Supplementation with l-glutamine seems to prevent bladder wall damage in relation to extracellular matrix volumetric density and collagen expression. These results suggest that glutamine supplementation could be efficient in protecting healthy tissues from the adverse effects of radiotherapy.
Assuntos
Colágeno/metabolismo , Suplementos Nutricionais , Glutamina/uso terapêutico , Músculo Liso , Lesões por Radiação/prevenção & controle , Doenças da Bexiga Urinária/prevenção & controle , Bexiga Urinária/efeitos dos fármacos , Animais , Colágeno/genética , Colágeno/efeitos da radiação , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/efeitos da radiação , Colágeno Tipo III/sangue , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo III/efeitos da radiação , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos da radiação , Expressão Gênica/efeitos dos fármacos , Glutamina/farmacologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Músculo Liso/efeitos da radiação , Neoplasias/radioterapia , Lesões por Radiação/etiologia , Lesões por Radiação/metabolismo , Ratos , Ratos Wistar , Valores de Referência , Bexiga Urinária/metabolismo , Bexiga Urinária/efeitos da radiação , Doenças da Bexiga Urinária/etiologia , Doenças da Bexiga Urinária/metabolismoRESUMO
OBJECTIVE: Studies on the influence of low-power red laser on the repair of dental structures are very scarce. This study investigated the effects of the laser therapy on the ultrastructure of the dentine-pulp interface after conservative class I cavity preparation. DESIGN: Two female volunteers with 8 premolars indicated for extraction for orthodontic reasons were recruited. Class I cavities were prepared and the teeth were randomly divided into two groups. The first group received treatment with a GaA1As laser, lambda=660nm, power of 30mW and energy dose of 2J/cm(2), directly and perpendicularly into the cavity in a single visit. After the irradiation, the cavities were filled with composite resin. The second group received the same treatment, except by the laser therapy. RESULTS: Twenty-eight days post-preparation, the teeth were extracted and processed for transmission electron microscopy analysis. Two sound teeth, without cavity preparation, were also studied. The irradiated group presented odontoblast process in higher contact with the extracellular matrix and the collagen fibrils appeared more aggregated and organised than those of control group. These results were also observed in the healthy teeth. CONCLUSION: These findings suggest that laser irradiation accelerates the recovery of the dental structures involved in the cavity preparation at the predentine region.
Assuntos
Preparo da Cavidade Dentária , Polpa Dentária/efeitos da radiação , Dentina/efeitos da radiação , Lasers Semicondutores/uso terapêutico , Terapia com Luz de Baixa Intensidade , Adulto , Colágeno/efeitos da radiação , Colágeno/ultraestrutura , Resinas Compostas , Preparo da Cavidade Dentária/classificação , Polpa Dentária/ultraestrutura , Restauração Dentária Permanente , Dentina/ultraestrutura , Matriz Extracelular/efeitos da radiação , Matriz Extracelular/ultraestrutura , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Odontoblastos/efeitos da radiação , Odontoblastos/ultraestrutura , Doses de RadiaçãoRESUMO
1. Lectins labeled with colloidal gold particles were used for the ultrastructural evaluation of the biological effects of GaAs softlaser irradiation 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 (4 J/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 sections 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.
Assuntos
Lasers , Lectinas , Tendões/efeitos da radiação , Animais , Colágeno/efeitos da radiação , Colágeno/ultraestrutura , Cães , Matriz Extracelular/efeitos da radiação , Matriz Extracelular/ultraestrutura , Feminino , Fibroblastos/efeitos da radiação , Fibroblastos/ultraestrutura , Masculino , Microscopia Eletrônica , Tendões/cirurgia , Tendões/ultraestrutura , Fatores de Tempo , Cicatrização/efeitos da radiaçãoRESUMO
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