Avaliação do efeito do tratamento com alendronato, a longo prazo, sobre a reparação do tecido ósseo em defeitos de calvárias. Estudo em ratas
Carregando...
Data
2016-08-31
Autores
Fióri, Léslie Cristine [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os bifosfonatos têm sido amplamente utilizados no tratamento de doenças relacionadas a distúrbios no metabolismo ósseo. Dentre os bifosfonatos mais potentes, o alendronato (ALD), possui grande potencial em inibir a reabsorção óssea. O objetivo deste estudo foi avaliar, in vivo, o efeito da administração de alendronato, a longo prazo, na reparação óssea em defeitos críticos confeccionados em calvárias de ratas. Para isto, 160 ratas foram distribuídas aleatoriamente em 2 grupos que receberam, semanalmente, injeções subcutâneas de placebo [grupo controle (CTL)] e de alendronato [grupo alendronato (ALD)]. Após 120 dias do início do tratamento, foi confeccionado um defeito crítico na calvária de todos os animais e 10 animais de cada grupo foram sacrificados aos 5, 10, 15, 20, 25, 30, 45 e 60 dias após a confecção do defeito. No dia do sacrifício foram coletadas amostras de urina e sangue e foram removidas as calvárias e os fêmures dos animais. Os fêmures foram avaliados quanto à densidade mineral óssea (BMD). Para a avaliação sistêmica do efeito do medicamento no metabolismo ósseo, foram investigados os níveis urinários de deoxipiridinolina (DPD) e os níveis séricos de propeptídeo amino terminal do procolágeno tipo I (P1NP), de telopeptídeo carboxiterminal do colágeno tipo I (CTX) e de osteocalcina por ensaio imunoenzimático (ELISA). Secções de calvárias submetidas ao processamento para inclusão em parafina foram empregados para: 1) imuno-histoquímica para detecção de RANKL (ligante do receptor de ativação do fator nuclear kß), osteoprotegerina (OPG), Catepsina K, RUNX2 (fator de transcrição 2 relacionado ao Runt), Osteopontina (OPN), iNOS (óxido nítrico sinstase induzível) e Fator de Crescimento Endotelial Vascular (VEGF); 2) coloração com Tricrômico de Goldner para análise da quantidade de tecido mineralizado e de matriz osteóide e fechamento do defeito; 3) avaliação da quantidade de osteoclastos TRAP+ (localização de células positivas para a enzima fosfatase ácida tartarato resistente-positivos) e 4) análise morfológica do tecido ósseo. Foram encontrados maiores valores de BMD para os animais no ALD em relação ao CTL. As concentrações de CTX e DPD foram maiores no grupo ALD, enquanto as concentrações de P1NP e osteocalcina foram menores no grupo ALD. Na análise imuno-histoquímica, verificou-se maiores expressões de catepsina K no ALD até os 15 dias, com inversão a partir dos 20 dias. A OPN apresentou menor imunomarcação nos períodos iniciais no ALD, havendo uma inversão aos 45 e 60 dias. O VEGF apresentou expressão significativamente maior no ALD aos 45 dias. O grupo ALD apresentou menor imunopositividade para o iNOS em todos os períodos, com diferença estatística até os 25 dias, enquanto o RUNX2 apresentou valores significativamente menores no ALD até os 20 dias e aos 60 dias em relação ao CTL. O ALD apresentou menor expressão de RANKL e maior expressão de OPG, em relação ao CTL. Observou-se diminuição da quantidade de células TRAP+ ao longo dos períodos no CTL e ALD. Verificou-se menor quantidade de matriz osteóide no ALD. Aos 45 e 60 dias, no ALD, a distância entre as bordas do defeito foi menor em relação aos períodos iniciais. Na análise morfológica, observou-se um relativo atraso na remodelação óssea no ALD. Os resultados em conjunto, sugerem que o ALD pode tem um efeito prejudicial no processo de reparo de defeitos críticas em calvárias de ratas, a longo prazo.
The bisphosphonates have been widely used for the treatment of diseases related to osseous metabolism disturbances. Among the most powerful bisphosphonates, the alendronate (ALD) presents great potential to inhibit the osseous reabsorption. This aim of this study was to evaluate, in vivo, the effect of the administration of alendronate in the long run, in bone repair in critic defects produced in rats’ calvarias. For so, 160 rats were randomly distributed into 2 groups which received, weekly, placebo subcutaneous injections [control group (CTL)] as well as alendronate injections [alendronate group (ALD)]. After 120 days from the beginning of treatment, a critic defect was produced in all animals and 10 animals from each group were sacrificed at 5, 10, 15, 20, 25, 30, 45 and 60 days after the defect production. On the day of sacrifice, urine and blood samples were collected and the calvarias and femurs were removed. The femurs were evaluated as for their bone mineral density (BMD). For the systemic evaluation of the medicine effect over the bone metabolism, the deoxypyridinoline urinary levels were investigated (DPD) as well as the serum levels of amino-terminal procollagen propetide of type 1 (P1NP), of carboxy-terminal telopeptide of collagen type I (CTX) and of osteocalcin by means of enzyme-linked immunosorbent essay (ELISA). Parts of calvarias undergone processing for inclusion in paraffin were employed to: 1) immuno-histochemistry for detection of RANKL (receptor activator of nuclear factor kß ligand), osteoprotegerin (OPG), Cathepsin K, RUNX2 (transcription factor 2 related to Runt), Osteopontin (OPN), iNOS (inducible nitric oxide synthase) and Vascular Endothelial Growing Factor (VEGF); 2) coloration with Goldner’s Trichrome for analyzing the quantity of mineralized tissue and of osteoid matrix and defect closing; 3) evaluation of quantity of osteoclasts TRAP+ (positive cells localization for the acid phosphatase enzyme positive-resistant tartrate) and 4) morphologic analysis of the osseous tissue. Higher values of BMD were found for the animals in the ALD in relation to CTL. The concentration of CTX and DPD were higher in group ALD, while the concentration of P1NP and osteocalcin were smaller in group ALD. In the immuno-histochemicalanalysis, it was verified higher expressions of cathepsin K in ALD until the 15th day, with inversion starting from the 20th day. The OPN presented smaller immunolabeling in the initial periods in the ALD, and there was an inversion on the 45th and 60th days. The VEGF, presented expression meaningfully higher in the ALD on the 45th day. The group ALD presented lesser immunopositivity for the iNOS in all the periods, with difference estatistical till the 25th day, while the RUNX2 presented values meaningfully smaller in the ALD till the 20th day and 60th day in relation to CTL. The ALD presented smaller expression of RANKL and higher expression of OPG, in relation to CTL. It was observed reduction of quantity of TRAP+ cells along the periods in the CTL and ALD. It was verified smaller quantity of osteoid matrix in the ALD. On the 45th and 60th days, in the ALD, the distance between the borders of the defect was smaller in relation to initial periods. In the morphological analysis, it was observed a relative delay in the bone remodeling in the ALD. The results, as a whole, suggest that the ALD may present a damning effect in the repair processing of critic defects in rats’ calvarias, in the long term.
The bisphosphonates have been widely used for the treatment of diseases related to osseous metabolism disturbances. Among the most powerful bisphosphonates, the alendronate (ALD) presents great potential to inhibit the osseous reabsorption. This aim of this study was to evaluate, in vivo, the effect of the administration of alendronate in the long run, in bone repair in critic defects produced in rats’ calvarias. For so, 160 rats were randomly distributed into 2 groups which received, weekly, placebo subcutaneous injections [control group (CTL)] as well as alendronate injections [alendronate group (ALD)]. After 120 days from the beginning of treatment, a critic defect was produced in all animals and 10 animals from each group were sacrificed at 5, 10, 15, 20, 25, 30, 45 and 60 days after the defect production. On the day of sacrifice, urine and blood samples were collected and the calvarias and femurs were removed. The femurs were evaluated as for their bone mineral density (BMD). For the systemic evaluation of the medicine effect over the bone metabolism, the deoxypyridinoline urinary levels were investigated (DPD) as well as the serum levels of amino-terminal procollagen propetide of type 1 (P1NP), of carboxy-terminal telopeptide of collagen type I (CTX) and of osteocalcin by means of enzyme-linked immunosorbent essay (ELISA). Parts of calvarias undergone processing for inclusion in paraffin were employed to: 1) immuno-histochemistry for detection of RANKL (receptor activator of nuclear factor kß ligand), osteoprotegerin (OPG), Cathepsin K, RUNX2 (transcription factor 2 related to Runt), Osteopontin (OPN), iNOS (inducible nitric oxide synthase) and Vascular Endothelial Growing Factor (VEGF); 2) coloration with Goldner’s Trichrome for analyzing the quantity of mineralized tissue and of osteoid matrix and defect closing; 3) evaluation of quantity of osteoclasts TRAP+ (positive cells localization for the acid phosphatase enzyme positive-resistant tartrate) and 4) morphologic analysis of the osseous tissue. Higher values of BMD were found for the animals in the ALD in relation to CTL. The concentration of CTX and DPD were higher in group ALD, while the concentration of P1NP and osteocalcin were smaller in group ALD. In the immuno-histochemicalanalysis, it was verified higher expressions of cathepsin K in ALD until the 15th day, with inversion starting from the 20th day. The OPN presented smaller immunolabeling in the initial periods in the ALD, and there was an inversion on the 45th and 60th days. The VEGF, presented expression meaningfully higher in the ALD on the 45th day. The group ALD presented lesser immunopositivity for the iNOS in all the periods, with difference estatistical till the 25th day, while the RUNX2 presented values meaningfully smaller in the ALD till the 20th day and 60th day in relation to CTL. The ALD presented smaller expression of RANKL and higher expression of OPG, in relation to CTL. It was observed reduction of quantity of TRAP+ cells along the periods in the CTL and ALD. It was verified smaller quantity of osteoid matrix in the ALD. On the 45th and 60th days, in the ALD, the distance between the borders of the defect was smaller in relation to initial periods. In the morphological analysis, it was observed a relative delay in the bone remodeling in the ALD. The results, as a whole, suggest that the ALD may present a damning effect in the repair processing of critic defects in rats’ calvarias, in the long term.
Descrição
Palavras-chave
Remodelação óssea, Crânio, Alendronato