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1.
J Orthop Res ; 42(3): 628-637, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37804213

RESUMEN

Chondrocytes are mechanosensitive cells able to sense and respond to external mechanical stimuli through the process of mechanotransduction. Previous studies have demonstrated that mechanical stimulation causes mitochondrial deformation leading to mitochondrial reactive oxygen species (ROS) release in a dose-dependent manner. For this reason, we focused on elucidating the role of mitochondrial ROS as anabolic signaling molecules in chondrocyte mechanotransduction. Chondrocyte-seeded agarose gels were subjected to mechanical stimuli and the effect on matrix synthesis, ROS production, and mitogen-activated protein kinases (MAPK) signaling was evaluated. Through the use of ROS-specific staining, superoxide anion was the primary ROS released in response to mechanical stimuli. The anabolic effect of mechanical stimulation was abolished in the presence of electron transport chain inhibitors (complexes I, III, and V) and superoxide anion scavengers. Subsequent studies were centered on the involvement of MAPK pathways (ERK1/2, p38, and JNK) in the mechanotransduction cascade. While disruption of the ERK1/2 pathway had no apparent effect, the anabolic effect of mechanical stimulation was abolished in the presence of p38 and JNK pathway inhibitors. This suggest the involvement of apoptosis stimulating kinase 1 (ASK1), an upstream redox-sensitive MAP3K shared by both the JNK and p38 pathways. Future experiments will focus on the involvement of the thioredoxin-ASK1 complex which disassociates in the presence of oxidative stress, allowing ASK1 to phosphorylate several MAP2Ks. Overall, these findings indicate superoxide anion as the primary ROS released in response to mechanical stimuli and that the resulting anabolic effect on chondrogenic matrix biosynthesis arises from the ROS-dependent activation of the p38 and JNK MAPKs.


Asunto(s)
Anabolizantes , Condrocitos , Especies Reactivas de Oxígeno/metabolismo , Condrocitos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacología , Superóxidos , Anabolizantes/farmacología , Mecanotransducción Celular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/farmacología , Apoptosis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/farmacología
2.
Biomech Model Mechanobiol ; 21(2): 605-614, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-35091857

RESUMEN

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0-50 mM LiCl for 24 h, with different pre-culture durations (0 to 4 days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50 mM LiCl compared to other concentrations (P < 0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50 mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P < 0.1) and proteoglycan (1.5-fold, P < 0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.


Asunto(s)
Cartílago Articular , Condrocitos , Cartílago , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/fisiología , Cilios/fisiología , Cloruro de Litio/metabolismo , Cloruro de Litio/farmacología , Mecanotransducción Celular/fisiología , Ingeniería de Tejidos/métodos
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