RESUMEN
Pulsed electromagnetic field (PEMF) stimulation has been widely applied clinically to promote bone healing; however, its detailed mechanism of action, particularly in endochondral ossification, remains elusive, and long-term stimulation is required for its satisfactory effect. The aim of this study was to investigate the involvement of the mammalian target of rapamycin (mTOR) pathway in chondrocyte differentiation and proliferation using a mouse prechondroblast cell line (ATDC5), and establish an efficient PEMF stimulation strategy for endochondral ossification. The changes in cell differentiation (gene expression levels of aggrecan, type II collagen, and type X collagen) and proliferation (cellular uptake of bromodeoxyuridine [BrdU]) in ATDC5 cells in the presence or absence of rapamycin, an mTOR inhibitor, was measured. The effects of continuous and intermittent PEMF stimulation on changes in cell differentiation and proliferation were compared. Rapamycin significantly suppressed the induction of cell differentiation markers and the cell proliferation activity. Furthermore, only intermittent PEMF stimulation continuously activated the mTOR pathway in ATDC5 cells, significantly promoting cell proliferation. These results demonstrate the involvement of the mTOR pathway in chondrocyte differentiation and proliferation and suggest that intermittent PEMF stimulation could be effective as a stimulus for endochondral ossification during fracture healing process, thereby reducing stimulation time.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Condrocitos , Campos Electromagnéticos , Osteogénesis , Serina-Treonina Quinasas TOR , Animales , Ratones , Osteogénesis/efectos de la radiación , Condrocitos/citología , Condrocitos/metabolismo , Condrocitos/fisiología , Línea Celular , Serina-Treonina Quinasas TOR/metabolismo , Sirolimus/farmacología , Cartílago/metabolismo , Cartílago/citología , Cartílago/fisiología , Transducción de Señal , Regulación de la Expresión Génica/efectos de la radiaciónRESUMEN
Pulsed electromagnetic fields (PEMFs) have been shown to be a noninvasive physical stimulant for bone fracture healing. However, PEMF stimulation requires a relatively long period of time and its mechanism of action has not yet been fully clarified. Recently, the mammalian target of rapamycin (mTOR) pathway has been shown to be involved in bone formation. This study aimed to investigate the effects of PEMFs on osteoblastic MC3T3-E1 cells by examining various cellular responses including changes in the mTOR pathway. Continuous PEMF stimulation induced a transient phosphorylation of the mTOR pathway, whereas intermittent PEMF stimulation (1 cycle of 10 min stimulation followed by 20 min of stimulation pause) revitalized the reduced phosphorylation. Moreover, PEMF stimulation stimulated cell proliferation (bromodeoxyuridine incorporation) rather than differentiation (alkaline phosphatase activity), with a more notable effect in the intermittently stimulated cells. These results suggest that intermittent PEMF stimulation may be effective in promoting bone fracture healing by accelerating cell proliferation, and in shortening stimulation time. Bioelectromagnetics. 2019;40:412-421. © 2019 Bioelectromagnetics Society.
Asunto(s)
Campos Electromagnéticos , Osteoblastos/citología , Serina-Treonina Quinasas TOR/metabolismo , Fosfatasa Alcalina/metabolismo , Bromodesoxiuridina/metabolismo , Línea Celular , Proliferación Celular , Estimulación Eléctrica , Humanos , Fosforilación , Transducción de SeñalRESUMEN
Duchenne muscular dystrophy is a lethal X-linked disease with no effective treatment. Progressive muscle degeneration, increased macrophage infiltration, and ectopic calcification are characteristic features of the mdx mouse, a murine model of Duchenne muscular dystrophy. Because dietary phosphorus/phosphate consumption is increasing and adverse effects of phosphate overloading have been reported in several disease conditions, we examined the effects of dietary phosphorus intake in mdx mice phenotypes. On weaning, control and mdx mice were fed diets containing 0.7, 1.0, or 2.0 g phosphorus per 100 g until they were 90 days old. Dystrophic phenotypes were evaluated in cryosections of quadriceps and tibialis anterior muscles, and maximal forces and voluntary activity were measured. Ectopic calcification was analyzed by electron microscopy to determine the cells initially responsible for calcium deposition in skeletal muscle. Dietary phosphorus overload dramatically exacerbated the dystrophic phenotypes of mdx mice by increasing inflammation associated with infiltration of M1 macrophages. In contrast, minimal muscle necrosis and inflammation were observed in exercised mdx mice fed a low-phosphorus diet, suggesting potential beneficial therapeutic effects of lowering dietary phosphorus intake on disease progression. To our knowledge, this is the first report showing that dietary phosphorus intake directly affects muscle pathological characteristics of mdx mice. Dietary phosphorus overloading promoted dystrophic disease progression in mdx mice, whereas restricting dietary phosphorus intake improved muscle pathological characteristics and function.