Avaliação do reparo ósseo na interface osso/implante em ratas com deficiência de estrógeno tratadas com fitoestrógeno genisteína. Análise microtomográfica, molecular por PCR e imunoistoquímica.
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Data
2018-01-29
Autores
Batista, Fábio Roberto de Souza [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente projeto teve como proposta avaliar o processo de reparo ósseo na interface osso/implante em ratas submetidas à ovariectomia para a indução da deficiência de estrógeno e tratadas com fitoestrógeno genisteína (1mg/dia) por gavagem. Para isso, 30 ratas (Rattus novergicus albinus, Wistar), com peso médio de 200 gramas foram divididas em 3 grupos conforme a análise a que foram submetidas e o tratamento medicamentoso, sendo 10 ratas para o grupo SHAM SAL = Ratas submetidas à cirurgia fictícia, sem tratamento medicamentoso (gavagem com solução salina 0.9%); 10 ratas para o grupo OVX SAL = Ratas submetidas à ovariectomia bilateral, sem tratamento medicamentoso (gavagem com solução salina 0.9%); 10 ratas para o grupo OVX GEN = Ratas submetidas à ovariectomia bilateral, tratadas com fitoestrógeno genisteína (gavagem com 1mg/dia de genisteína). Foram utilizados 60 implantes, cada animal recebeu 2 implantes, sendo 1 em cada metáfise tibial. A eutanásia dos animais foi realizada após 60 dias da instalação dos implantes, por meio de sobredosagem anestésica. Para os grupo calcificados: nas análises por microtomografia computadorizada foram utilizadas as tíbias do lado direito de 6 ratas e para a análise da expressão gênica por PCR em tempo real, as 6 tíbias do lado esquerdo das mesmas ratas. Para a análise dos grupos descalcificados: 4 ratas foram submetidas à eutanásia na análise de imunoistoquímica osteoprotegerina – (OPG-SC21038), ativador do receptor nuclear kappa B ligante – (RANKL-SC 7627), osteocalcina – (OC-SC18319) e fosfatase ácida resistente ao ácido tartárico (TRAP-SC30832) e fosfatase alcalina (ALP -SC23430). Todos os dados quantitativos foram submetidos ao teste de homocedasticidade e após a determinação do teste mais adequado, o nível de significância foi estabelecido em 0<0,05. A análise morfométrica do grupo OVX GEN mostrou maior percentual de volume ósseo, menor separação entre as trabéculas e menor porosidade. As análises molecular por PCR e Imunohistoquímica evidenciam uma maior expressão para OPG e RANKL (remodelação óssea), ALP e OC (mineralização) de cada um desses marcadores, o que sugere que a genisteína atua como um modulador seletivo natural dos receptores de estrogenio favorecendo a expressão de proteínas importantes para a modulação da atividade osteoblástica no metabolismo do tecido ósseo. Assim, os resultados deste estudo confirmaram que a terapia com genisteína mostra-se capaz de melhorar a reparação óssea na interface osso / implante sob condições de osteopenia/osteoporose.
The present project had as proposal to evaluate the process of bone repair in the bone / implant interface in rats submitted to ovariectomy for induction of estrogen deficiency and treated with phytoestrogen genistein (1mg / day) by gavage. For this, 30 rats (Rattus novergicus albinus, Wistar), with an average weight of 200 grams were divided into 3 groups according to an analysis and the drug treatment, being 10 rats for the group SHAM SAL = Rats submitted to the fictional surgery , without drug treatment (gavage with 0.9% saline solution); 10 rats for the OVX group SAL = Rats submitted to bilateral ovariectomy without drug treatment (gavage with 0.9% saline solution); 10 rats for the OVX group GEN = Rats subjected to bilateral ovariectomy, treated with phytoestrogen genistein (gavage with 1mg / day of genistein). 60 implants were installed, with 2 implants in each animal. The euthanasia of the animals was performed 60 days after implant’s installation, by anesthetic overdose. The calcified groups analysis with computerized microtomography was peformed in right tibiae of 6 rats and the gene expression analysis by PCR was performed in left tíbia of the same animals. For the decalcified analysis through immunolabeling experiments, 4 rats were submited to eutanásia in order to evaluate the presence of Osteoprotegerin (OPG – SC21038), activator of nuclear kappa B ligand (RANKL – SC7627), Osteocalcin (OC – SC 18319), and acid resistant phosphatase tartaric acid (TRAP – SC 30832) and alcaline phosphatase (ALP SC 23430). All the quantitative data were submitted to the normality curve to determine the most adequate test. The significance level of p<0.05 was considered for all tests. A morphometric analysis of the OVX GEN group showed higher percentage of bone volume, less separation between trabeculae and lower porosity. As molecular analyzes by PCR and Immunohistochemistry evidenced a greater expression for OPG and RANKL (bone remodeling), ALP and OC (mineralization) of each the markers, which suggests that genistein acts as a natural selective modulator of estrogen receptors favoring expression of proteins important for a modulation of osteoblastic activity in the metabolism of bone tissue. Thus, the results of this study confirm that genistein therapy is shown to improve bone repair at the bone / implant interface under conditions of osteopenia / osteoporosis.
The present project had as proposal to evaluate the process of bone repair in the bone / implant interface in rats submitted to ovariectomy for induction of estrogen deficiency and treated with phytoestrogen genistein (1mg / day) by gavage. For this, 30 rats (Rattus novergicus albinus, Wistar), with an average weight of 200 grams were divided into 3 groups according to an analysis and the drug treatment, being 10 rats for the group SHAM SAL = Rats submitted to the fictional surgery , without drug treatment (gavage with 0.9% saline solution); 10 rats for the OVX group SAL = Rats submitted to bilateral ovariectomy without drug treatment (gavage with 0.9% saline solution); 10 rats for the OVX group GEN = Rats subjected to bilateral ovariectomy, treated with phytoestrogen genistein (gavage with 1mg / day of genistein). 60 implants were installed, with 2 implants in each animal. The euthanasia of the animals was performed 60 days after implant’s installation, by anesthetic overdose. The calcified groups analysis with computerized microtomography was peformed in right tibiae of 6 rats and the gene expression analysis by PCR was performed in left tíbia of the same animals. For the decalcified analysis through immunolabeling experiments, 4 rats were submited to eutanásia in order to evaluate the presence of Osteoprotegerin (OPG – SC21038), activator of nuclear kappa B ligand (RANKL – SC7627), Osteocalcin (OC – SC 18319), and acid resistant phosphatase tartaric acid (TRAP – SC 30832) and alcaline phosphatase (ALP SC 23430). All the quantitative data were submitted to the normality curve to determine the most adequate test. The significance level of p<0.05 was considered for all tests. A morphometric analysis of the OVX GEN group showed higher percentage of bone volume, less separation between trabeculae and lower porosity. As molecular analyzes by PCR and Immunohistochemistry evidenced a greater expression for OPG and RANKL (bone remodeling), ALP and OC (mineralization) of each the markers, which suggests that genistein acts as a natural selective modulator of estrogen receptors favoring expression of proteins important for a modulation of osteoblastic activity in the metabolism of bone tissue. Thus, the results of this study confirm that genistein therapy is shown to improve bone repair at the bone / implant interface under conditions of osteopenia / osteoporosis.
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Palavras-chave
Osteoporose, Interface osso-implante, Osseointegração, Fitoestrógenos, Genisteína, Osteoporosis, Bone-implant interface, Osseointegration, Phytoestrogens, Genistein