Modificações do tecido ósseo decorrentes da administração intermitente do PTH (1-34), ao longo de 30 dias, em camundongos ooforectomizados
Data
2023-04-10
Autores
Pereira, Nathalia Mestre 
Orientadores
Reginato, Rejane Daniele
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A matriz óssea é um ambiente enriquecido principalmente por colágeno, proteínas não colágenas, glicosaminoglicanos, proteoglicanos e componentes inorgânicos, que se organizam e fornecem às células ósseas um ambiente para manter a homeostase óssea. Desequilíbrios na remodelação óssea podem ocasionar patologias, como a osteoporose, um problema de saúde pública mundial, que atinge cerca de 200 milhões de mulheres no mundo todo. Um dos tratamentos anabólicos utilizados para a osteoporose e na iminência de fraturas é o PTH (1-34), um análogo sintético do PTH humano, que quando administrado de forma intermitente possui efeito anabólico no tecido ósseo, culminando no aumento de massa óssea. No entanto, faltam estudos sobre as modificações que ocorrem no tecido ósseo ao longo do tratamento. Objetivo: Investigar as modificações do tecido ósseo ao longo do tratamento (0, 7, 14, 21 e 30 dias) com PTH (1-34) administrado intermitentemente em camundongos fêmeas ooforectomizados. Materiais e métodos: 70 camundongos fêmeas com 3 meses de idade foram ooforectomizadas, e após 3 meses divididos e tratados com: 5µg/kg/dia de PTH (1-34) (grupos PTH) ou 5µg/kg/dia de solução salina (grupos controle). O tratamento foi realizado diariamente durante 0, 7, 14, 21 e 30 dias (d). A densidade mineral óssea (DMO) foi avaliada por densitometria óssea e após, os animais foram eutanásiados. Os fêmures foram destinados ás análises histomorfométricas (volume ósseo trabecular-BV/TV e espessura do osso cortical-Ct.Th), análises biofísicas (densidade óssea, porcentagem de material orgânico, porcentagem de material mineral e porcentagem de água), histoquímica do TRAP (quantificação dos osteoclastos TRAP-positivos, classificação quanto ao tamanho, atividade, superfície de reabsorção e número de osteoclastos multinucleados), histoquímica do picrosirius red (avaliação da birrefringência das fibras colágenas) e eletroforese em gel de agarose e ELISA para quantificar condroitim sulfato (CS) e ácido hialurônico (AH), respectivamente. As tíbias foram destinadas ao ensaio colorimétrico para quantificação de cálcio e fósforo. Resultados: A DMO permaneceu inalterada durante o tratamento, no entanto, houve aumento do BV/TV já aos 7d de tratamento, seguido de uma perda óssea aos 14d. Aos 21d houve um aumento no BV/TV culminando na recuperação da microarquitetura óssea aos 30d de tratamento. A menor Ct.Th foi observada com 21d de tratamento. O osso trabecular respondeu primeiro a ação anabólica do PTH, comparado ao osso cortical, pois a perda ocorreu em torno de 14d, enquanto na região cortical essa perda ocorreu aos 21d de tratamento. As análises biofísicas demonstraram um aumento da densidade óssea, material orgânico e material mineral com 7d sustentado até 14d de tratamento e uma redução após esse período, inversamente, a porcentagem de água foi reduzida até 14d de tratamento e aumentada após esse período. O número de osteoclastos TRAP-positivos aumentou no osso trabecular em 21d com posterior diminuição em 30d, além disso, com 7d e 30d de tratamento os osteoclastos tiveram uma superfície de reabsorção menor em comparação com seus respetivos controles, indicando menor reabsorção óssea. No osso cortical não houve diferença no número de osteoclastos, no entanto, o número de osteoclastos multinucleados foi maior com 21d e menor com 7d de tratamento. Não foi observada diferença entre os grupos quanto ao tamanho e atividade dos osteoclastos. Em relação ao colágeno, a menor birrefringência avermelhada e esverdeada, no osso trabecular, foi observada aos 7d de tratamento. Após esse período os grupos tratados exibiram aumento de ambas birrefringências, sendo que a birrefringência avermelhada foi maior aos 21d de tratamento, comparado ao seu controle. Não foram observadas diferenças de birrefringências na região cortical. A maior concentração de CS e AH foi observada aos 21d de tratamento. Os resultados da porção inorgânica da matriz mostraram que a concentração de cálcio, mas não de fósforo, é aumentada em 7, 21 e 30d de tratamento, sendo que aos 14d de tratamento foi observada a menor concentração de cálcio. Conclusões: Os resultados mostraram que o efeito anabólico do PTH (1-34) é precedido por perda óssea, diminuindo o volume ósseo e aumentando a superfície de reabsorção dos osteoclastos, sendo que o osso trabecular reponde primeiro, que o osso cortical, ao tratamento com PTH (1-34). Durante o tratamento com PTH (1-34), ocorrem alterações no perfil dos componentes da matriz óssea, como colágeno e condroitim sulfato, que desencadeiam mecanismos para a perda óssea e posterior recuperação do tecido.
Introduction: The bone matrix is an environment enriched mainly by collagen, non-collagenous proteins, glycosaminoglycans, proteoglycans and inorganic components, which organize themselves and provide bone cells with an environment to maintain bone homeostasis. Imbalances in bone remodeling can lead to pathologies such as osteoporosis, a global public health problem that affects approximately 200 million women worldwide. One of the most used anabolic treatments for osteoporosis and imminent fractures is PTH (1-34), a synthetic analogue of human PTH, which administered intermittently has an anabolic effect on bone tissue, culminating in an increase in bone mass. However, there is a lack of studies about changes that occur in bone tissue during treatment. Objective: To investigate changes in bone tissue during treatment (0, 7, 14, 21 and 30 days) with PTH (1-34) administered intermittently in ovariectomized female mice. Materials and methods: Seventy 3-month-old female mice were ovariectomized, after 3 months divided and treated with: 5µg/kg/day of PTH (1-34) (PTH groups) or 5µg/kg/day of saline solution (control groups). Treatment was carried out daily for 0, 7, 14, 21 and 30 days (d). Bone mineral density (BMD) was evaluated by bone densitometry and afterwards, the animals were euthanized. The femurs were destined to histomorphometric analyzes (cancellous bone volume-BV/TV and cortical bone thickness-Ct.Th), biophysical analyzes (bone density, percentage of organic material, percentage of mineral material and percentage of water), histochemistry of TRAP (quantification of TRAP-positive osteoclasts, classification according to size, activity, resorption surface and number of multinucleated osteoclasts), histochemistry of picrosirius red (assessment of birefringence of collagen fibers) and agarose gel electrophoresis and ELISA to quantify chondroitin sulfate (CS) and hyaluronic acid (HA), respectively. The tibias were destined to the colorimetric assay for the quantification of calcium and phosphorus. Results: BMD remained unchanged during treatment, however, there was an increase in BV/TV already at 7d of treatment, followed by bone loss at 14d. At 21d there was an increase in BV/TV culminating in the recovery of bone microarchitecture at 30d of treatment. The lowest Ct.Th was observed with 21d of treatment. Cancellous bone responded first to the anabolic action of PTH, compared to cortical bone, as the loss occurred around 14d, while in the cortical region this loss occurred at 21d of treatment. Biophysical analyzes demonstrated an increase in bone density, organic material and mineral material sustained 7d up to 14d of treatment, after this period was observed a reduction, conversely, the percentage of water was reduced up to 14d of treatment and increased after this period. The number of TRAP-positive osteoclasts increased in the cancellous bone in 21d with a subsequent decrease in 30d, in addition, with 7d and 30d of treatment the osteoclasts had a smaller resorption surface compared to their respective controls, indicationg a reduction of bone resorption. In the cortical bone there was no difference in the number of osteoclasts, however, the number of multinucleated osteoclasts was higher with 21d and lower with 7d of treatment. No difference was observed among groups regarding the size and activity of osteoclasts. Regarding collagen, the lowest reddish and greenish birefringence in cancellous bone was observed at 7d of treatment. After this period, the treated groups showed an increase in both birefringence, and the reddish birefringence was higher at 21d of treatment, compared to the control group. No differences in birefringence were observed in the cortical region. The highest concentration of CS and AH was observed at 21d of treatment. The results of the inorganic portion of the matrix showed that the concentration of calcium, but not of phosphorus, is increased in 7, 21 and 30d of treatment, and at 14d of treatment the lowest concentration of calcium was observed. Conclusions: The results showed that the anabolic effect of PTH (1-34) is preceded by bone loss, decreasing bone volume and increasing the surface area for osteoclast reabsorption; and cancellous bone responds first then cortical bone to PTH (1-34). During treatment with PTH (1-34), changes occur in the profile of bone matrix components, such as collagen and chondroitin sulfate, which trigger mechanisms for bone loss and subsequent tissue recovery.
Introduction: The bone matrix is an environment enriched mainly by collagen, non-collagenous proteins, glycosaminoglycans, proteoglycans and inorganic components, which organize themselves and provide bone cells with an environment to maintain bone homeostasis. Imbalances in bone remodeling can lead to pathologies such as osteoporosis, a global public health problem that affects approximately 200 million women worldwide. One of the most used anabolic treatments for osteoporosis and imminent fractures is PTH (1-34), a synthetic analogue of human PTH, which administered intermittently has an anabolic effect on bone tissue, culminating in an increase in bone mass. However, there is a lack of studies about changes that occur in bone tissue during treatment. Objective: To investigate changes in bone tissue during treatment (0, 7, 14, 21 and 30 days) with PTH (1-34) administered intermittently in ovariectomized female mice. Materials and methods: Seventy 3-month-old female mice were ovariectomized, after 3 months divided and treated with: 5µg/kg/day of PTH (1-34) (PTH groups) or 5µg/kg/day of saline solution (control groups). Treatment was carried out daily for 0, 7, 14, 21 and 30 days (d). Bone mineral density (BMD) was evaluated by bone densitometry and afterwards, the animals were euthanized. The femurs were destined to histomorphometric analyzes (cancellous bone volume-BV/TV and cortical bone thickness-Ct.Th), biophysical analyzes (bone density, percentage of organic material, percentage of mineral material and percentage of water), histochemistry of TRAP (quantification of TRAP-positive osteoclasts, classification according to size, activity, resorption surface and number of multinucleated osteoclasts), histochemistry of picrosirius red (assessment of birefringence of collagen fibers) and agarose gel electrophoresis and ELISA to quantify chondroitin sulfate (CS) and hyaluronic acid (HA), respectively. The tibias were destined to the colorimetric assay for the quantification of calcium and phosphorus. Results: BMD remained unchanged during treatment, however, there was an increase in BV/TV already at 7d of treatment, followed by bone loss at 14d. At 21d there was an increase in BV/TV culminating in the recovery of bone microarchitecture at 30d of treatment. The lowest Ct.Th was observed with 21d of treatment. Cancellous bone responded first to the anabolic action of PTH, compared to cortical bone, as the loss occurred around 14d, while in the cortical region this loss occurred at 21d of treatment. Biophysical analyzes demonstrated an increase in bone density, organic material and mineral material sustained 7d up to 14d of treatment, after this period was observed a reduction, conversely, the percentage of water was reduced up to 14d of treatment and increased after this period. The number of TRAP-positive osteoclasts increased in the cancellous bone in 21d with a subsequent decrease in 30d, in addition, with 7d and 30d of treatment the osteoclasts had a smaller resorption surface compared to their respective controls, indicationg a reduction of bone resorption. In the cortical bone there was no difference in the number of osteoclasts, however, the number of multinucleated osteoclasts was higher with 21d and lower with 7d of treatment. No difference was observed among groups regarding the size and activity of osteoclasts. Regarding collagen, the lowest reddish and greenish birefringence in cancellous bone was observed at 7d of treatment. After this period, the treated groups showed an increase in both birefringence, and the reddish birefringence was higher at 21d of treatment, compared to the control group. No differences in birefringence were observed in the cortical region. The highest concentration of CS and AH was observed at 21d of treatment. The results of the inorganic portion of the matrix showed that the concentration of calcium, but not of phosphorus, is increased in 7, 21 and 30d of treatment, and at 14d of treatment the lowest concentration of calcium was observed. Conclusions: The results showed that the anabolic effect of PTH (1-34) is preceded by bone loss, decreasing bone volume and increasing the surface area for osteoclast reabsorption; and cancellous bone responds first then cortical bone to PTH (1-34). During treatment with PTH (1-34), changes occur in the profile of bone matrix components, such as collagen and chondroitin sulfate, which trigger mechanisms for bone loss and subsequent tissue recovery.
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Citação
PEREIRA, Nathalia Mestre. Modificações do tecido ósseo decorrentes da administração intermitente de PTH (1-34), ao longo de 30 dias, em camundongos ovariectomizados. 2023 81 f. Dissertação (Mestrado em Biologia Estrutural e Funcional) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2023.