Alterações morfológicas e imunoistoquímicas da cartilagem articular de ratas submetidas à terapia com ondas de choque na presença e ausência do hormônio estrogênio
Data
2021-02-24
Autores
Rodrigues, Isabela Capucho 
Orientadores
Reginato, Rejane Daniele
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A osteoartrite (OA) é uma doença multifatorial que afeta as articulações e é caracterizada pela degradação da cartilagem articular. A privação estrogênica na pós-
menopausa é considerada um importante fator de risco para o desenvolvimento da OA. A terapia com ondas de choque (SW) estimula o reparo tecidual em diversas doenças musculoesqueléticas, porém pouco se sabe sobre seus efeitos na cartilagem articular. Objetivo: Investigar os efeitos da terapia com SW nos condrócitos e na matriz da cartilagem articular de ratas na presença e na ausência do hormônio estrogênio. Métodos: Trinta ratas Wistar com 6 meses de idade foram submetidas ao procedimento cirúrgico de ooforectomia (OVX) ou cirurgia simulada e divididas em: Sham: cirurgia simulada e sem terapia; Sham+SW: cirurgia simulada e submetidas à terapia com SW; OVX: ooforectomizadas e sem terapia; OVX+SW: ooforectomizadas e submetidas à terapia com SW. A terapia com SW foi aplicada em uma única sessão (500 pulsos a 0,13mJ/mm²/pulso) nos joelhos dos animais, seis meses após a OVX. A massa total, densidade (DMO) e conteúdo (CMO) mineral ósseo foram avaliados por densitometria no dia da OVX, três meses após a cirurgia e ao final do experimento. Os animais foram eutanasiados com 13 meses de idade e os fêmures com suas respectivas cartilagens articulares foram coletados. As alterações na cartilagem foram analisadas por histomorfometria para quantificação de condrócitos isolados, clones, condrócitos totais e espessura da cartilagem articular. Condroitim sulfato e ácido hialurônico foram avaliados por eletroforese em gel de agarose e ELISA, respectivamente. Imunoistoquímicas foram realizadas para avaliar condrócitos em processo de morte (caspase-3), colágeno tipo II e fator de necrose tumoral alfa (TNF-α). Cortes evidenciados por Safranina O e Fast Green foram analisados para avaliar a degradação de proteoglicanos e para classificação do grau de OA segundo a Osteoarthritis Research Society International (OARSI). Resultados: O grupo Sham+SW exibiu classificação OARSI de 0,5, alta intensidade de coloração de Safranina O, menor número de clones de condrócitos, a maior concentração de ácido hialurônico, maior imuno marcação de colágeno II em relação a OVX+SW e menos TNF-α comparado a Sham, além de aumento de DMO. O grupo OVX+SW apresentou grau OARSI entre 1,0-1,5, aumento do número de clones em relação a Sham+SW, aumento de massa e diminuição de DMO. Os grupos submetidos a terapia com SW apresentaram uma menor quantidade de condrócitos positivos para caspase-3 em relação ao grupo OVX. O maior grau de degradação da cartilagem (2,0) foi observado no OVX, além de uma redução da DMO. Sham apresentou grau OARSI de 0,5-1,0, alta intensidade de coloração de Safranina O, a menor quantidade de condrócitos positivos para caspase-3 e aumento de CMO. Não foram observadas diferenças na espessura da cartilagem articular e no conteúdo de condroitim sulfato. Conclusão: A terapia com ondas de choque foi efetiva em reduzir os efeitos deletérios da privação estrogênica na cartilagem articular. Entretanto, os efeitos foram mais pronunciados quando a terapia foi aplicada na presença do hormônio, melhorando a qualidade da matriz cartilaginosa.
Introduction: Osteoarthritis (OA) is a multifactorial joint disorder characterized by progressive destruction of articular cartilage. The estrogen deprivation in post-menopause is considered an important risk factor for OA development. The extracorporeal shockwave (SW) therapy promotes tissue repair in several musculoskeletal disorders, however the action of this therapy in articular cartilage has not been completely elucidated. Objective: To investigate the effects of SW therapy in chondrocytes and extracellular matrix of the articular cartilage, in the presence and absence of estrogen hormone. Methods: Thirty 6-month-old female Wistar rats underwent ovariectomy (OVX) or sham-operated and were randomized into: Sham: sham- operated without therapy; Sham+SW: sham-operated and subjected to SW therapy; OVX: ovariectomized and without SW therapy; OVX+SW: ovariectomized and subjected to SW therapy. Animals were exposed to one SW session (500 shots delivered at 0.13mJ/mm2) in the knees, six months after OVX. Total mass, bone mineral density (BMD) and bone mineral content (BMC) were evaluated with densitometry at OVX, three months after OVX, and at the end of experiment. The rats were euthanized with 13-months-old, and the femurs were collected without damaging the cartilage surface. Changes in cartilage were assessed using histomorphometry, for quantification of chondrocytes, clusters, total of chondrocytes and articular cartilage thickness. Chondroitin sulfate and hyaluronic acid were analyzed by agarose gel electrophoresis and ELISA, respectively. Immunohistochemistry were realized to evaluate chondrocyte death (caspase-3), type II collagen and tumor necrosis factor-α (TNF-α). Safranin O-Fast Green sections were evaluated for proteoglycans degradation and the osteoarthritis grade were analyzed according Osteoarthritis Research Society International (OARSI). Results: Sham+SW group exhibited grater intensity of Safranin O, 0,5 OARSI grade, smaller number of clusters, the highest hialuronic acid concentration, greater positive immunostaining of type II collagen regarding to OVX+SW, and lower TNF-α compared to Sham, in addition improved BMD. OVX+SW presented 1,0-1,5 OARSI grade, increased number of clusters compered to Sham+SW, enhanced total mass, and decreased BMD. Both groups subjected to SW therapy presented smaller number of caspase-3 positive chondrocytes in comparison to OVX. The grade of cartilage degradation observed in OVX group was 2,0, besides reduced BMD. Sham exhibited grater intensity of Safranin O, 0,5-1,0 OARSI degree, the lowest caspase-3 and increased BMC. There were no differences in the articular cartilage thickness and concentration of CS. Conclusions: Extracorporeal shockwave therapy was effective in reducing the deleterious effects of estrogen-deprivation in the articular cartilage. However, the benefits were more pronounced when therapy was applied in the presence of hormone, improving cartilage matrix quality.
Introduction: Osteoarthritis (OA) is a multifactorial joint disorder characterized by progressive destruction of articular cartilage. The estrogen deprivation in post-menopause is considered an important risk factor for OA development. The extracorporeal shockwave (SW) therapy promotes tissue repair in several musculoskeletal disorders, however the action of this therapy in articular cartilage has not been completely elucidated. Objective: To investigate the effects of SW therapy in chondrocytes and extracellular matrix of the articular cartilage, in the presence and absence of estrogen hormone. Methods: Thirty 6-month-old female Wistar rats underwent ovariectomy (OVX) or sham-operated and were randomized into: Sham: sham- operated without therapy; Sham+SW: sham-operated and subjected to SW therapy; OVX: ovariectomized and without SW therapy; OVX+SW: ovariectomized and subjected to SW therapy. Animals were exposed to one SW session (500 shots delivered at 0.13mJ/mm2) in the knees, six months after OVX. Total mass, bone mineral density (BMD) and bone mineral content (BMC) were evaluated with densitometry at OVX, three months after OVX, and at the end of experiment. The rats were euthanized with 13-months-old, and the femurs were collected without damaging the cartilage surface. Changes in cartilage were assessed using histomorphometry, for quantification of chondrocytes, clusters, total of chondrocytes and articular cartilage thickness. Chondroitin sulfate and hyaluronic acid were analyzed by agarose gel electrophoresis and ELISA, respectively. Immunohistochemistry were realized to evaluate chondrocyte death (caspase-3), type II collagen and tumor necrosis factor-α (TNF-α). Safranin O-Fast Green sections were evaluated for proteoglycans degradation and the osteoarthritis grade were analyzed according Osteoarthritis Research Society International (OARSI). Results: Sham+SW group exhibited grater intensity of Safranin O, 0,5 OARSI grade, smaller number of clusters, the highest hialuronic acid concentration, greater positive immunostaining of type II collagen regarding to OVX+SW, and lower TNF-α compared to Sham, in addition improved BMD. OVX+SW presented 1,0-1,5 OARSI grade, increased number of clusters compered to Sham+SW, enhanced total mass, and decreased BMD. Both groups subjected to SW therapy presented smaller number of caspase-3 positive chondrocytes in comparison to OVX. The grade of cartilage degradation observed in OVX group was 2,0, besides reduced BMD. Sham exhibited grater intensity of Safranin O, 0,5-1,0 OARSI degree, the lowest caspase-3 and increased BMC. There were no differences in the articular cartilage thickness and concentration of CS. Conclusions: Extracorporeal shockwave therapy was effective in reducing the deleterious effects of estrogen-deprivation in the articular cartilage. However, the benefits were more pronounced when therapy was applied in the presence of hormone, improving cartilage matrix quality.
Descrição
Citação
RODRIGUES, I. C. Alterações morfológicas e imunoistoquímicas da cartilagem articular de ratas submetidas à terapia com ondas de choque na presença e ausência do hormônio estrogênio. 2021. 71 f. Trabalho de Conclusão de Curso (Graduação em Ciências Biológicas) - Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema.