Modificações do osso subcondral após a terapia com ondas de choque em ratas sob privação estrogênica
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Data
2023-12-18
Autores
Rodrigues, Isabela Capucho [UNIFESP]
Orientadores
Reginato, Rejane Daniele [UNIFESP]
Tipo
Dissertação de mestrado
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Objetivo: Investigar os efeitos da terapia com ondas de choque (ESWT) no osso subcondral de ratas sob privação estrogênica, correlacionando com o grau de osteoartrite. Métodos: Foram utilizadas 30 ratas Wistar adultas (6 meses) ooforectomizadas (OVX) ou Sham-operadas (Sham), que foram divididas em 4 grupos: Sham e OVX: não receberam terapia; Sham+SW e OVX+SW: submetidos à ESWT (única sessão - 500 pulsos - 0,13 mJ/mm2/pulso). A densidade mineral óssea (DMO) foi avaliada no dia da OVX (T0), três meses após a cirurgia (T1) e ao final do experimento (T2), por densitometria óssea. Os animais foram submetidos à eutanásia aos 13 meses de idade e os fêmures foram coletados. Foram realizadas análises hitomorfométicas para avaliação do volume ósseo subcondral, técnica de TRAP (Tartrate Resistant Acid Phosphatase) para quantificação e classificação de osteoclastos, histoquímica de picrosirius para análise das fibras colágenas e imunoistoquímicas para a expressão de TNF-α, caspase-3 clivada, Anexina A1 (AnxA1), Fpr1 e Fpr2. Análises de bioinformática foram realizadas utilizando dados de transcriptomas disponibilizados publicamente. As análises estatísticas foram ANOVA, ANOVA medidas repetidas, Teste T, Teste Exato de Fisher e Correlação de Pearson. Resultados: Diminuição de DMO foi observada nos animais ooforectomizados 3 meses após a cirurgia (T1). Em T2, Sham e Sham+SW apresentaram maior DMO e maior volume ósseo subcondral que OVX, e ambos os grupos apresentaram aumento de DMO ao longo do experimento (T0 vs. T1 e T0 vs. T2). O volume ósseo subcondral e a DMO foram negativamente correlacionados com o grau de osteoartrite, porém foi observada correlação positiva entre esses dois parâmetros. Sham+SW apresentou maior número de osteoclastos e, tanto Sham+SW quanto OVX+SW apresentaram maior superfície de reabsorção que Sham. A maior porcentagem de osteoclastos inativos foi observada em Sham, e houve similaridade na porcentagem de osteoclastos mononucleados e multinucleados entre os grupos. Sham+SW e OVX+SW exibiram maior birrefringência esverdeada. Houve diminuição de osteócitos positivos para TNF-α no osso subcondral no grupo OVX+SW. Além disso, Sham, Sham+SW e OVX+SW apresentaram menos osteócitos positivos para caspase-3, comparados ao OVX. A maior porcentagem de osteócitos positivos para AnxA1 foi observada em OVX+SW, e OVX foi maior do que Sham. Os grupos Sham+SW e OVX apresentaram maior porcentagem de osteócitos positivos para Fpr1 que Sham, e maior quantidade de Fpr1 do que Fpr2. As análises de transcriptomas demonstraram maior expressão de Fpr2 em vértebras de camundongos fêmeas OVX, e maior expressão de Fpr1 no osso acetabular de mulheres com idade de 43 a 80 anos. Conclusão: A ESWT provocou mudanças no osso subcondral compatíveis com o mecanismo de remodelação óssea em ratas OVX. Essas modificações foram mais pronunciadas quando não houve a privação estrogênica, com aumento na quantidade de osteoclastos, na superfície de reabsorção e na deposição de novas fibras colágenas, além da diminuição da morte de osteócitos e do aumento da expressão de Fpr1. No entanto, mesmo na ausência do hormônio, a terapia acelerou a remodelação óssea subcondral, evidenciada pela organização do colágeno, aumento da superfície de reabsorção, diminuindo TNF-α e a morte de osteócitos, aumentando AnxA1 e prevenindo a perda de DMO e volume ósseo subcondral. Além disso, o presente estudo demonstrou que a cirurgia de OVX está relacionada ao aumento de AnxA1 no osso subcondral e que o receptor Fpr1 está presente em maior quantidade neste tecido. Sendo assim, nossos resultados demonstraram o grande potencial da ESWT no tratamento da osteoartrite.
Objective: To investigate the effects of extracorporeal shockwave therapy (ESWT) in the subchondral bone of rats with estrogen deficiency, correlating with osteoarthritis score. Methods: Thirty female Wistar rats (6-month-old) underwent ovariectomy (OVX) or sham-operated. The animals were randomized into four groups: Sham and OVX: without therapy; Sham+SW and OVX+SW: subjected to ESWT (single session – 500 shots – 0.13mJ/mm2/shot). Bone mineral density (BMD) was evaluated with densitometry at OVX (T0), three months after OVX (T1) and at the end of experiment (T2). The rats were euthanized at 13-month-old and femurs were collected. Histomorphometric analysis was used to subchondral bone volume evaluation, TRAP method (Tartrate Resistant Acid Phosphatase) to quantify and classify subchondral bone osteoclasts, histochemistry of picrosirius under polarization to analyze collagen fibers and immunohistochemistry to expression of TNF-α, caspase-3 cleaved, annexin A1 (AnxA1), Fpr1 and Fpr2. Bioinformatic analyses was realized with publicly available transcriptome data. Statistical analyses performed were ANOVA, repeated measures ANOVA, t-test, Fisher’s exact test and Pearson correlation test. Results: A decrease of BMD was observed three months after surgery (T1). At T2, Sham and Sham+SW present higher BMD than OVX, and both groups increase BMD throughout the experiment (T0 vs. T1 and T0 vs. T2). Sham and Sham+SW groups presented higher subchondral bone volume than OVX. A negative correlation was observed between osteoarthritis score and both subchondral bone volume and BMD, while a positive correlation was seen between these two parameters. Sham+SW presented the highest number of osteoclasts, and both Sham+SW and OVX+SW presented higher osteoclast resorption surface than Sham. The highest percentage of inactive osteoclasts was observed in Sham, and was a similarity in the percentage of mononucleated and multinucleated osteoclasts among the groups. Sham+SW and OVX+SW presented higher greenish birefringence. There was a decrease of TNF-α positive osteocytes in subchondral bone in OVX+SW group. Futhermore, Sham, Sham+SW and OVX+SW presented lower percentage of caspase-3 positive osteocytes, compared to OVX. The highest percentage of AnxA1 positive osteocytes was observed in OVX+SW, and OVX was higher than Sham. The groups Sham+SW and OVX presented higher percentage of Fpr1 positive osteocytes than Sham, and higher expression of Fpr1 than Fpr2. Transcriptome analysis showed higher expression of Fpr2 in vertebrae of female OVX mice, and higher expression of Fpr1 in acetabular bone samples of female patients with 43-80 years old. Conclusion: The results showed that the ESWT promoted changes in subchondral bone that were associated with bone remodeling in OVX rats. These modifications were more pronounced without estrogen-deprivation, such as increase of osteoclasts number, resorption surface and collagen fibers deposition, followed by a decrease in osteocytes death and higher Fpr1 expression. However, even in ovariectomized rats, the therapy accelerates subchondral bone remodeling, evidenced by collagen organization, increase in resorption surface, besides decrease TNF-α and osteocytes death, increasing AnxA1 and preventing BMD and subchondral bone loss. In addition, the current study demonstrated that OVX surgery was related to increase AnxA1 in subchondral bone while Fpr1 receptor is the most expressed in this tissue. Therefore, our results pointed the great potential of ESWT in the treatment of osteoarthritis.
Objective: To investigate the effects of extracorporeal shockwave therapy (ESWT) in the subchondral bone of rats with estrogen deficiency, correlating with osteoarthritis score. Methods: Thirty female Wistar rats (6-month-old) underwent ovariectomy (OVX) or sham-operated. The animals were randomized into four groups: Sham and OVX: without therapy; Sham+SW and OVX+SW: subjected to ESWT (single session – 500 shots – 0.13mJ/mm2/shot). Bone mineral density (BMD) was evaluated with densitometry at OVX (T0), three months after OVX (T1) and at the end of experiment (T2). The rats were euthanized at 13-month-old and femurs were collected. Histomorphometric analysis was used to subchondral bone volume evaluation, TRAP method (Tartrate Resistant Acid Phosphatase) to quantify and classify subchondral bone osteoclasts, histochemistry of picrosirius under polarization to analyze collagen fibers and immunohistochemistry to expression of TNF-α, caspase-3 cleaved, annexin A1 (AnxA1), Fpr1 and Fpr2. Bioinformatic analyses was realized with publicly available transcriptome data. Statistical analyses performed were ANOVA, repeated measures ANOVA, t-test, Fisher’s exact test and Pearson correlation test. Results: A decrease of BMD was observed three months after surgery (T1). At T2, Sham and Sham+SW present higher BMD than OVX, and both groups increase BMD throughout the experiment (T0 vs. T1 and T0 vs. T2). Sham and Sham+SW groups presented higher subchondral bone volume than OVX. A negative correlation was observed between osteoarthritis score and both subchondral bone volume and BMD, while a positive correlation was seen between these two parameters. Sham+SW presented the highest number of osteoclasts, and both Sham+SW and OVX+SW presented higher osteoclast resorption surface than Sham. The highest percentage of inactive osteoclasts was observed in Sham, and was a similarity in the percentage of mononucleated and multinucleated osteoclasts among the groups. Sham+SW and OVX+SW presented higher greenish birefringence. There was a decrease of TNF-α positive osteocytes in subchondral bone in OVX+SW group. Futhermore, Sham, Sham+SW and OVX+SW presented lower percentage of caspase-3 positive osteocytes, compared to OVX. The highest percentage of AnxA1 positive osteocytes was observed in OVX+SW, and OVX was higher than Sham. The groups Sham+SW and OVX presented higher percentage of Fpr1 positive osteocytes than Sham, and higher expression of Fpr1 than Fpr2. Transcriptome analysis showed higher expression of Fpr2 in vertebrae of female OVX mice, and higher expression of Fpr1 in acetabular bone samples of female patients with 43-80 years old. Conclusion: The results showed that the ESWT promoted changes in subchondral bone that were associated with bone remodeling in OVX rats. These modifications were more pronounced without estrogen-deprivation, such as increase of osteoclasts number, resorption surface and collagen fibers deposition, followed by a decrease in osteocytes death and higher Fpr1 expression. However, even in ovariectomized rats, the therapy accelerates subchondral bone remodeling, evidenced by collagen organization, increase in resorption surface, besides decrease TNF-α and osteocytes death, increasing AnxA1 and preventing BMD and subchondral bone loss. In addition, the current study demonstrated that OVX surgery was related to increase AnxA1 in subchondral bone while Fpr1 receptor is the most expressed in this tissue. Therefore, our results pointed the great potential of ESWT in the treatment of osteoarthritis.
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Citação
RODRIGUES, Isabela Capucho. Modificações do osso subcondral após a terapia com ondas de choque em ratas sob privação estrogênica. 2023. 118 f. Dissertação de Mestrado (Programa de Pós-Graduação em Biologia Estrutural e Funcional) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo.