Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro

Detalhes bibliográficos
Autor(a) principal: Tassinary, Joao Alberto Fioravante
Data de Publicação: 2015
Tipo de documento: Tese
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da PUC_RS
Texto Completo: http://tede2.pucrs.br/tede2/handle/tede/6328
Resumo: Low-intensity pulsed ultrasound (LIPUS) has been proposed as a high potential therapeutic technique for the treatment of metabolic bone diseases and fractures with delayed healing. However, recent investigations have shown controversial results, suggesting the need for more studies on the understanding of biological responses and the standardization of methods and parameters. For this reason, the present study aimed to evaluate the effect of ultrasound on the proliferation and mineralization of osteoblasts using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were used and treated with therapeutic pulsed ultrasound, 20%, frequency of 1MHz and 0,2W/cm2 of intensity. In the study of cellular proliferation, intracellular calcium, TGF-β1, magnesium and osteopontin and osteocalcin mRNA levels, NF-κB1 and p38α were evaluated. In addition, nifedipine and rapamycin were used to investigate the proliferation pathways. Initially, the results showed an increase in proliferation of MC3T3-E1 and a decrease in calcium and magnesium content in supernatant with LIPUS exposure. In addition, LIPUS increased calcium deposition, activated NF-κB1 and mTOR complex via p38α and promoted a decrease in TGF-β1 synthesis, which is an inhibitor of cell growth. On cell mineralization assays, we evaluated mineral nodules deposition and expression of osteocalcin mRNA, collagen concentrations on culture supernatant, phosphate, calcium, TGF- β1 and ALP on cell lysates. The results showed that US stimulates the mineralization of preosteoblasts 192h after treatment by stimulating osteocalcin mRNA expression, calcium and phosphate uptake amd consequent formation of HA. Later, in different experimental conditions, the results showed that LIPUS had the ability to stimulate pre-osteoblastic mineralization with decreased concentration of collagen, calcium and phosphate in the cell supernatant 192 hours after treatment. It also changed the alkaline phosphatase concentration, as well as the osteocalcin gene expression.
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spelling Oliveira, Jarbas Rodrigues de140.906.430-15http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781448U1Melo, Denizar Silvahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703281J6008.692.650-08http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4468329Z7Tassinary, Joao Alberto Fioravante2015-09-28T12:04:15Z2015-08-21http://tede2.pucrs.br/tede2/handle/tede/6328Low-intensity pulsed ultrasound (LIPUS) has been proposed as a high potential therapeutic technique for the treatment of metabolic bone diseases and fractures with delayed healing. However, recent investigations have shown controversial results, suggesting the need for more studies on the understanding of biological responses and the standardization of methods and parameters. For this reason, the present study aimed to evaluate the effect of ultrasound on the proliferation and mineralization of osteoblasts using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were used and treated with therapeutic pulsed ultrasound, 20%, frequency of 1MHz and 0,2W/cm2 of intensity. In the study of cellular proliferation, intracellular calcium, TGF-β1, magnesium and osteopontin and osteocalcin mRNA levels, NF-κB1 and p38α were evaluated. In addition, nifedipine and rapamycin were used to investigate the proliferation pathways. Initially, the results showed an increase in proliferation of MC3T3-E1 and a decrease in calcium and magnesium content in supernatant with LIPUS exposure. In addition, LIPUS increased calcium deposition, activated NF-κB1 and mTOR complex via p38α and promoted a decrease in TGF-β1 synthesis, which is an inhibitor of cell growth. On cell mineralization assays, we evaluated mineral nodules deposition and expression of osteocalcin mRNA, collagen concentrations on culture supernatant, phosphate, calcium, TGF- β1 and ALP on cell lysates. The results showed that US stimulates the mineralization of preosteoblasts 192h after treatment by stimulating osteocalcin mRNA expression, calcium and phosphate uptake amd consequent formation of HA. Later, in different experimental conditions, the results showed that LIPUS had the ability to stimulate pre-osteoblastic mineralization with decreased concentration of collagen, calcium and phosphate in the cell supernatant 192 hours after treatment. It also changed the alkaline phosphatase concentration, as well as the osteocalcin gene expression.O ultrassom pulsado de baixa intensidade surge como recurso terapêutico de alto potencial para o tratamento de doenças osteometabolicas e fraturas com atraso de consolidação óssea. Entretanto, investigações recentes demonstram resultados controversos, sugerindo a necessidade de mais estudos acerca do entendimento das respostas biológicas e na padronização dos parâmetros das modalidades de tratamento. Sendo assim, o presente estudo teve o objetivo de avaliar o efeito do ultrassom na proliferação e mineralização de pre-ósteoblastos a partir de bioensaios in vitro. Foram utilizados pré-osteoblastos da linhagem MC3T3-E1, sendo estas tratadas com ultrassom terapêutico no modo pulsado a 20%, com uma frequência de 1MHz e intensidade de 0,2 W/cm2. No estudo de proliferação celular, avaliamos cálcio intracelular, o TGF-β1, magnésio e os níveis de mRNA de osteopontina e osteocalcina, NF-κB1 e p38α. Além disso, utilizamos nifedipina e a rapamicina para investigar rotas de proliferação. Inicialmente, os resultados mostraram que o ultrassom aumenta proliferação de MC3T3-E1, diminuindo o teor de cálcio e magnésio no sobrenadante. Além disso, aumenta a concentração de cálcio intracelular, ativa NF-κB1 e o complexo mTOR via p38α e promove uma diminuição na síntese de TGF-β1, que é um inibidor do crescimento celular. Nos ensaios de mineralização celular avaliamos inicialmente a deposição nódulos de minerais na placa de cultura e a expressão gênica da osteocalcina por PCR convencional, e, posterirormente a concentração no sobrenadante celular de colágeno, fosfato, cálcio, TGF-β além de fosfatase alcalina no lizado de células. Os resultados mostraram que o ultrassom tem a capacidade de estimular a mineralização pré-osteoblastica em 192 horas após o tratamento a partir do aumento da expressão osteocalcina, captação de cálcio e fosfato e consequente formação de hidroxiapatita.Submitted by Setor de Tratamento da Informação - BC/PUCRS (tede2@pucrs.br) on 2015-09-28T12:04:15Z No. of bitstreams: 1 475275 - Texto Completo.pdf: 1472800 bytes, checksum: fe0676cb911e95563a60b4837d542763 (MD5)Made available in DSpace on 2015-09-28T12:04:15Z (GMT). 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dc.title.por.fl_str_mv Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
title Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
spellingShingle Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
Tassinary, Joao Alberto Fioravante
MEDICINA
OSSOS
CALCIFICAÇÃO FISIOLÓGICA
ULTRASSOM
PRÉ-OSTEOBLASTOS
CIENCIAS DA SAUDE::MEDICINA
title_short Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
title_full Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
title_fullStr Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
title_full_unstemmed Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
title_sort Efeitos do ultrassom pulsado de baixa intensidade na proliferação e mineralização de pré-osteoblastos in vitro
author Tassinary, Joao Alberto Fioravante
author_facet Tassinary, Joao Alberto Fioravante
author_role author
dc.contributor.advisor1.fl_str_mv Oliveira, Jarbas Rodrigues de
dc.contributor.advisor1ID.fl_str_mv 140.906.430-15
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781448U1
dc.contributor.advisor-co1.fl_str_mv Melo, Denizar Silva
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4703281J6
dc.contributor.authorID.fl_str_mv 008.692.650-08
dc.contributor.authorLattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4468329Z7
dc.contributor.author.fl_str_mv Tassinary, Joao Alberto Fioravante
contributor_str_mv Oliveira, Jarbas Rodrigues de
Melo, Denizar Silva
dc.subject.por.fl_str_mv MEDICINA
OSSOS
CALCIFICAÇÃO FISIOLÓGICA
ULTRASSOM
PRÉ-OSTEOBLASTOS
topic MEDICINA
OSSOS
CALCIFICAÇÃO FISIOLÓGICA
ULTRASSOM
PRÉ-OSTEOBLASTOS
CIENCIAS DA SAUDE::MEDICINA
dc.subject.cnpq.fl_str_mv CIENCIAS DA SAUDE::MEDICINA
description Low-intensity pulsed ultrasound (LIPUS) has been proposed as a high potential therapeutic technique for the treatment of metabolic bone diseases and fractures with delayed healing. However, recent investigations have shown controversial results, suggesting the need for more studies on the understanding of biological responses and the standardization of methods and parameters. For this reason, the present study aimed to evaluate the effect of ultrasound on the proliferation and mineralization of osteoblasts using in vitro bioassays. Pre-osteoblastic MC3T3-E1 cells were used and treated with therapeutic pulsed ultrasound, 20%, frequency of 1MHz and 0,2W/cm2 of intensity. In the study of cellular proliferation, intracellular calcium, TGF-β1, magnesium and osteopontin and osteocalcin mRNA levels, NF-κB1 and p38α were evaluated. In addition, nifedipine and rapamycin were used to investigate the proliferation pathways. Initially, the results showed an increase in proliferation of MC3T3-E1 and a decrease in calcium and magnesium content in supernatant with LIPUS exposure. In addition, LIPUS increased calcium deposition, activated NF-κB1 and mTOR complex via p38α and promoted a decrease in TGF-β1 synthesis, which is an inhibitor of cell growth. On cell mineralization assays, we evaluated mineral nodules deposition and expression of osteocalcin mRNA, collagen concentrations on culture supernatant, phosphate, calcium, TGF- β1 and ALP on cell lysates. The results showed that US stimulates the mineralization of preosteoblasts 192h after treatment by stimulating osteocalcin mRNA expression, calcium and phosphate uptake amd consequent formation of HA. Later, in different experimental conditions, the results showed that LIPUS had the ability to stimulate pre-osteoblastic mineralization with decreased concentration of collagen, calcium and phosphate in the cell supernatant 192 hours after treatment. It also changed the alkaline phosphatase concentration, as well as the osteocalcin gene expression.
publishDate 2015
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