Efeitos do ultrassom pulsado de baixa intensidade na prolifera??o e mineraliza??o de pr?-osteoblastos in vitro

• Main Author: Tassinary, Joao Alberto Fioravante
• Other Authors: Oliveira, Jarbas Rodrigues de
• Format: Others
• Language: Portuguese
• Published: Pontif?cia Universidade Cat?lica do Rio Grande do Sul 2015
• Subjects: MEDICINA; OSSOS; CALCIFICA??O FISIOL?GICA; ULTRASSOM; PR?-OSTEOBLASTOS; CIENCIAS DA SAUDE::MEDICINA
• Online Access: http://tede2.pucrs.br/tede2/handle/tede/6328

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS ([e-mail]) 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). No. of bitstreams: 1 475275 - Texto Completo.pdf: 1472800 bytes, checksum: fe0676cb911e95563a60b4837d542763 (MD5) Previous issue date: 2015-08-21 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES === 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-?1, magnesium and osteopontin and osteocalcin mRNA levels, NF-?B1 and p38? 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-?B1 and mTOR complex via p38? and promoted a decrease in TGF-?1 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- ?1 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 terap?utico de alto potencial para o tratamento de doen?as osteometabolicas e fraturas com atraso de consolida??o ?ssea. Entretanto, investiga??es recentes demonstram resultados controversos, sugerindo a necessidade de mais estudos acerca do entendimento das respostas biol?gicas e na padroniza??o dos par?metros das modalidades de tratamento. Sendo assim, o presente estudo teve o objetivo de avaliar o efeito do ultrassom na prolifera??o e mineraliza??o de pre-?steoblastos a partir de bioensaios in vitro. Foram utilizados pr?-osteoblastos da linhagem MC3T3-E1, sendo estas tratadas com ultrassom terap?utico no modo pulsado a 20%, com uma frequ?ncia de 1MHz e intensidade de 0,2 W/cm2. No estudo de prolifera??o celular, avaliamos c?lcio intracelular, o TGF-?1, magn?sio e os n?veis de mRNA de osteopontina e osteocalcina, NF-?B1 e p38?. Al?m disso, utilizamos nifedipina e a rapamicina para investigar rotas de prolifera??o. Inicialmente, os resultados mostraram que o ultrassom aumenta prolifera??o de MC3T3-E1, diminuindo o teor de c?lcio e magn?sio no sobrenadante. Al?m disso, aumenta a concentra??o de c?lcio intracelular, ativa NF-?B1 e o complexo mTOR via p38? e promove uma diminui??o na s?ntese de TGF-?1, que ? um inibidor do crescimento celular. Nos ensaios de mineraliza??o celular avaliamos inicialmente a deposi??o n?dulos de minerais na placa de cultura e a express?o g?nica da osteocalcina por PCR convencional, e, posterirormente a concentra??o no sobrenadante celular de col?geno, fosfato, c?lcio, TGF-? al?m de fosfatase alcalina no lizado de c?lulas. Os resultados mostraram que o ultrassom tem a capacidade de estimular a mineraliza??o pr?-osteoblastica em 192 horas ap?s o tratamento a partir do aumento da express?o osteocalcina, capta??o de c?lcio e fosfato e consequente forma??o de hidroxiapatita.