Effects of Repetitive Compressive Loading on the Expression of Autophagy-Related Factors, Autophagy Capacity and Cellular Activity in Human Subchondral Bone Osteoblasts

Bone metabolism is known to change in response to mechanical stress. In osteoarthritis (OA), the subchondral bone as well as the articular cartilage layer is damaged by mechanical stress, which adversely affects the mechanical environment and homeostatic balance of the entire joint, leading to the onset of OA. However, the impact of mechanical stress on osteoblasts in the subchondral bone, especially their defense mechanism against stress, remains unclear. We focused on the mechanical stress response capacity of subchondral bone osteoblasts, which, like chondrocytes in the cartilage layer, are routinely exposed to repetitive mechanical stress. The purpose of this study was to determine whether repetitive mechanical force (at a physiologic level of cyclic compressive loading) affects the expression of factors regulating autophagy, autophagy capacity and cellular activity in subchondral osteoblasts. Three-dimensional cultured tissue was generated from human subchondral bone osteoblasts using a collagen sponge scaffold. Repetitive compressive loading was applied to the 3D cell culture constructs. After mechanical loading on 3D cell–collagen sponge constructs, we comparatively analyzed the expression of autophagy-related factors (ATG5, Beclin-1, Parkin) in human osteoblasts. Levels of osteoblast differentiation activity (alkaline phosphatase) were also analyzed, and transmission electron microscopy (TEM) was used to detect autophagy processes with or without the repetitive compressive loading. In both premature and mature osteoblasts, repetitive compressive loading decreased expression of the autophagy-related factors ATG5, Beclin-1 and Parkin in comparison with the non-loading group, suggesting that repetitive compressive loading may induce downregulation of mitophagy as well as cellular autophagy in subchondral osteoblasts. TEM analysis indicated that, in both premature and mature osteoblasts of the non-loading group, autophagy processes were progressing. In contrast, repetitive loading decreased the number of autophagosomes and autolysosomes in mature and premature osteoblasts. Our results demonstrated that repetitive mechanical loading resulted in a reduction in cellular autophagy and mitophagy in both premature and mature osteoblasts, while also inducing osteoblast differentiation. While mechanical stress promotes osteoblast differentiation, it also alters autophagy, a cellular defense mechanism, suggesting that under conditions of sustained repetitive loading, alterations in the autophagy response—which is essential for maintaining intracellular homeostasis—occur alongside the adaptive bone-forming response, potentially affecting osteoblast function in the long term. Our data demonstrate that reductions in both mitophagy and cellular autophagy in response to repetitive mechanical stress, even at a physiologic level, may lead to accumulation of defective mitochondria and abnormal cellular proteins in the cells, thus reducing osteoblast activity and affecting the homeostasis of subchondral bone tissue, and ultimately contributing to OA progression.