Efeitos da terapia com ondas de choque isolada ou associada à vibração mecânica de baixa frequência e intensidade na cartilagem articular de ratas sob privação estrogênica
Data
2021-04-20
Autores
Mierzwa, Aline Gomes Hidalgo 
Orientadores
Reginato, Rejane Daniele
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Investigar os efeitos da terapia com ondas de choque (ESWT) isolada ou associada à vibração mecânica (MV) na cartilagem articular de ratas sob privação estrogênica. Materiais e métodos: Sessenta e duas ratas Wistar fêmeas, com 6 meses de idade, foram submetidas ao procedimento cirúrgico de ooforectomia (OVX) ou ooforectomia simulada (Sham). Após três meses foram divididas em: Sham: não receberam terapia; Sham+MV: submetidas à MV; Sham+SW: submetidas à ESWT; Sham+MV+SW: submetidas à MV e à ESWT; OVX: não receberam terapia; OVX+MV: submetidas à MV; OVX+SW: submetidas à ESWT; OVX+MV+SW: submetidas à MV e à ESWT. A MV de baixa frequência e intensidade (60Hz-0,6g por 30min/dia, 5 dias/semana) foi realizada durante três meses. A ESWT (500 pulsos a 0,13mJ/mm²) foi aplicada em única sessão nos joelhos dos animais, seis meses após os procedimentos cirúrgicos. A densidade mineral óssea (DMO) e a composição corporal foram avaliadas no dia da OVX, após três meses e ao final do experimento por densitometria óssea. Os animais foram submetidos à eutanásia aos 13 meses de idade e os fêmures distais com suas respectivas cartilagens foram processadas e destinadas às análises histomorfométricas (quantificação de condrócitos e clones de condrócitos), imunoistoquímicas para avaliar condrócitos em processo de morte (caspase-3 clivada), fator de necrose tumoral alfa (TNF-α) e colágeno tipo II, e histoquímica de Picro-Sirius para avaliar fibras colágenas. Cortes evidenciados por Safranina O e Fast Green foram submetidos a classificação do grau de degeneração da cartilagem segundo a Osteoarthritis Research Society International (OARSI). Condroitim sulfato e ácido hialurônico foram avaliados por eletroforese em gel de agarose e ELISA, respectivamente. Resultados: O maior grau de degeneração da cartilagem (2,0) e redução de DMO foi observado no grupo OVX. A ESWT aplicada de forma isolada em ratas ooforectomizadas (OVX+SW), atenuou a degradação da matriz, aumentou o número de clones e preservou a viabilidade dos condrócitos, diminuindo o grau OARSI para 1,0-1,5. Nos animais sem privação estrogênica (Sham+SW) a ESWT reduziu o número de clones e a expressão de caspase-3 e TNF-α, elevou a concentração de ácido hialurônico e a expressão de colágeno II, culminando na menor classificação OARSI (0,5), além de aumentar a DMO. A associação da ESWT com a MV (OVX+MV+SW) atenuou a degradação da cartilagem, reduzindo a expressão de TNF-α e caspase-3, levando a diminuição do grau OARSI (1,5) comparado ao grupo OVX. A MV isolada (OVX+MV), reduziu os clones de condrócitos, aumentou a expressão de colágeno tipo II e a deposição de novas fibras colágenas, além de elevar a concentração de condroitim sulfato, contribuindo na redução da classificação OARSI (1,0-1,5). Ao grupo Sham foi atribuído a classificação entre 0,5 e 1,0 devido a preservação da matriz, menor expressão de caspase-3 e aumento de clones de condrócitos. Conclusões: A ESWT atenua os efeitos deletérios da privação estrogênica, no entanto, na presença do hormônio estrogênio os efeitos benéficos na matriz cartilaginosa e nos condrócitos são mais evidentes. Quando associada à MV reduz a expressão de TNF-α e aumenta a viabilidade dos condrócitos, e quando aplicada de forma isolada, a MV modifica o perfil dos componentes da matriz cartilaginosa em ratas sob privação estrogênica. Esses resultados sugerem que as terapias com ondas de choque e vibração mecânica demonstram potencial clínico como tratamento auxiliar para a osteoartrite.
Objective: To investigate the effects of extracorporeal shockwave therapy (ESWT) isolated or associated with mechanical vibration (MV) in the articular cartilage of estrogen-deficient rats. Material and methods: Sixty-two 6-month-old female Wistar rats were bilaterally ovariectomized (OVX) or sham-operated (Sham). After three months the animals were assigned into Sham: without therapy; Sham+MV: subjected to MV; Sham+SW: subjected to ESWT; Sham+MV+SW: subjected to MV and ESWT; OVX: without therapy; OVX+MV: subjected to MV; OVX+SW: subjected to ESWT; OVX+MV+SW: subjected to MV and ESWT. The low-intensity and high-frequency MV (0,6g-60Hz, 30min/day, 5 days/week) was performed for three months. A single session of ESWT (500 shots delivered at 0.13mJ/mm2) was applied in the animals’ knees six months after surgical procedure. Bone mineral density (BMD) and body composition were evaluated at the day of OVX, three months after surgery, and at the end of experiment by densitometry. The animals were euthanized with 13-month-old, and the distal femurs with their respective cartilages were processed and subjected to the following analysis: histomorphometry (quantification of chondrocytes and clusters), immunohistochemistry to evaluate chondrocyte death (cleaved caspase-3), tumor necrosis factor-α (TNF-α) and type II collagen; and Picro-Sirius to evaluate collagen fibers. Sections stained with Safranin O-Fast Green were evaluated according to the Osteoarthritis Research Society International (OARSI) for articular cartilage degeneration. Chondroitin sulfate and hyaluronic acid were analyzed by agarose gel electrophoresis and ELISA, respectively. Results: The highest degree of cartilage degradation (2.0) and reduction of BMD was observed in the OVX group. ESWT applied alone in ovariectomized rats (OVX+SW) attenuated matrix degradation, increased the number of clusters and preserved the chondrocytes viability, decreasing OARSI grade to 1.0-1.5. In animals without estrogen deprivation (Sham+SW), ESWT reduced chondrocyte clusters, caspase-3 and TNF-α expression, increased hialuronic acid concentration and expression of type II collagen, culminating in the lowest OARSI classification (0.5), besides to increase BMD. The association of ESWT with MV (OVX+MV+SW) attenuated the cartilage degeneration, reducing the expression of TNF-α and caspase-3, leading to a slight decrease of OARSI grade (1.5) compared to the OVX group. MV applied alone (OVX+MV), reduced the chondrocyte clusters, increased type II collagen expression and the deposition of new collagen fibers, in addition elevated the chondroitin sulfate concentration, contributing to reduce the OARSI classification (1.0-1.5). Sections from Sham group were classified between 0.5 and 1.0, due to the preservation of cartilage matrix, lower expression of caspase-3 and increased number of chondrocyte clusters. Conclusions: ESWT attenuates the deleterious effects of estrogen deprivation, however, in the presence of hormone the beneficial effects on the cartilage matrix and chondrocytes are more evident. The ESWT associated with MV reduces the expression of TNF-α and increases the chondrocytes viability, and MV applied alone modifies the profile of cartilage matrix components in estrogen-deficient rats. These results suggest that ESWT and MV therapies demonstrate clinical potential as an adjunct treatment for osteoarthritis.
Objective: To investigate the effects of extracorporeal shockwave therapy (ESWT) isolated or associated with mechanical vibration (MV) in the articular cartilage of estrogen-deficient rats. Material and methods: Sixty-two 6-month-old female Wistar rats were bilaterally ovariectomized (OVX) or sham-operated (Sham). After three months the animals were assigned into Sham: without therapy; Sham+MV: subjected to MV; Sham+SW: subjected to ESWT; Sham+MV+SW: subjected to MV and ESWT; OVX: without therapy; OVX+MV: subjected to MV; OVX+SW: subjected to ESWT; OVX+MV+SW: subjected to MV and ESWT. The low-intensity and high-frequency MV (0,6g-60Hz, 30min/day, 5 days/week) was performed for three months. A single session of ESWT (500 shots delivered at 0.13mJ/mm2) was applied in the animals’ knees six months after surgical procedure. Bone mineral density (BMD) and body composition were evaluated at the day of OVX, three months after surgery, and at the end of experiment by densitometry. The animals were euthanized with 13-month-old, and the distal femurs with their respective cartilages were processed and subjected to the following analysis: histomorphometry (quantification of chondrocytes and clusters), immunohistochemistry to evaluate chondrocyte death (cleaved caspase-3), tumor necrosis factor-α (TNF-α) and type II collagen; and Picro-Sirius to evaluate collagen fibers. Sections stained with Safranin O-Fast Green were evaluated according to the Osteoarthritis Research Society International (OARSI) for articular cartilage degeneration. Chondroitin sulfate and hyaluronic acid were analyzed by agarose gel electrophoresis and ELISA, respectively. Results: The highest degree of cartilage degradation (2.0) and reduction of BMD was observed in the OVX group. ESWT applied alone in ovariectomized rats (OVX+SW) attenuated matrix degradation, increased the number of clusters and preserved the chondrocytes viability, decreasing OARSI grade to 1.0-1.5. In animals without estrogen deprivation (Sham+SW), ESWT reduced chondrocyte clusters, caspase-3 and TNF-α expression, increased hialuronic acid concentration and expression of type II collagen, culminating in the lowest OARSI classification (0.5), besides to increase BMD. The association of ESWT with MV (OVX+MV+SW) attenuated the cartilage degeneration, reducing the expression of TNF-α and caspase-3, leading to a slight decrease of OARSI grade (1.5) compared to the OVX group. MV applied alone (OVX+MV), reduced the chondrocyte clusters, increased type II collagen expression and the deposition of new collagen fibers, in addition elevated the chondroitin sulfate concentration, contributing to reduce the OARSI classification (1.0-1.5). Sections from Sham group were classified between 0.5 and 1.0, due to the preservation of cartilage matrix, lower expression of caspase-3 and increased number of chondrocyte clusters. Conclusions: ESWT attenuates the deleterious effects of estrogen deprivation, however, in the presence of hormone the beneficial effects on the cartilage matrix and chondrocytes are more evident. The ESWT associated with MV reduces the expression of TNF-α and increases the chondrocytes viability, and MV applied alone modifies the profile of cartilage matrix components in estrogen-deficient rats. These results suggest that ESWT and MV therapies demonstrate clinical potential as an adjunct treatment for osteoarthritis.
Descrição
Citação
MIERZWA, A G H. Efeitos da terapia com ondas de choque isolada ou associada à vibração mecânica de baixa frequência e intensidade na cartilagem articular de ratas sob privação estrogênica. São Paulo, 2021. 162 p. Tese (Doutorado em Biologia Estrutural e Funcional) - Universidade Federal de São Paulo. São Paulo, 2021.