Investigation of action of shear stress, which is produced by Mechanical loading applied to the skeleton, on osteodast formation and bone-resorbing activity

背景：应用于骨骼的机械载荷对骨骼完整性和结构的发展和维持至关重要。由于在太空中长时间固定或失重而导致的机械负荷的降低会降低骨形成率，导致骨丢失。另一方面，机械负荷的增加会导致骨密度的增加。因此，骨组织对机械刺激很敏感。在骨骼上施加机械载荷，产生细胞外基质变形和流体流动，机械刺激分别转化为机械应变和流体剪应力等力学信号。从体外研究中获得的证据表明，嵌于骨陷窝/骨小管系统中的骨细胞以及排列在骨表面的成骨细胞和骨细胞是检测负荷产生的机械刺激的机械传感器。通过这些骨形成细胞，机械刺激被转化为细胞信号因子。然而，正常的骨代谢抗体…更多的骨质疏松症是通过骨形成细胞和骨吸收细胞之间平衡的骨重塑实现的。到目前为止，流体剪应力对成骨细胞的影响已经得到了广泛的研究；然而，对破骨细胞导致骨吸收的作用仍有待阐明。因此，本研究的目的是阐明流体剪应力直接或间接地通过成骨细胞对破骨细胞生成和破骨细胞功能的影响。方法：从小鼠颅骨分离成骨细胞，用单一流动系统加载流体剪应力在这些细胞上。在破骨细胞形成的研究中，我们采用了依赖于M-CSF的骨髓细胞来源的破骨细胞前体细胞的体外培养系统，用于依赖RANKL的破骨细胞形成。结果和讨论：分离的小鼠骨痂来源的成骨细胞在长期培养中能够形成结节和钙化。当成骨细胞受到流体剪切力时，COX-2和RANKL的mRNAs表达早在暴露后2小时就开始增加。此外，RANKL的可溶性诱骗受体OPG的基因表达也受到细胞内流体切应力的影响，但增幅小于RANKL的表达。剪应力作用下的成骨细胞条件培养液可促进低浓度RANKL诱导的破骨细胞的形成，而卸应力作用下的成骨细胞条件培养液不能促进破骨细胞的形成。短期的剪应力负荷足以诱导RANKL基因表达，即使在释放负荷后，这种诱导作用仍然存在。切应力诱导的RANKL表达依赖于ERK的激活，而不依赖于切应力诱导的前列腺素。最后，流体剪应力直接刺激破骨细胞前体细胞在M-CSF和低剂量RANKL处理后形成破骨细胞。综上所述，流体剪应力通过诱导成骨细胞RANKL的表达，直接或间接地刺激破骨细胞的形成。较少

Background : Mechanical loading applied to the skeleton is crucial to the development and maintenance of bone integrity and architecture. A decrease in the mechanical loading due to prolonged immobilization or weightlessness in space reduces the bone formation rate, resulting in bone loss. On the other hand, an increase in mechanical loading causes a gain in bone density. Thus, bone tissue is sensitive to mechanical stimulation. Mechanical loading on bone generates extracellular matrix deformation and fluid flow, and the mechanical stimuli are translated to mechanical signals such as mechanical strain and fluid shear stress, respectively. Evidence obtained from in vitro studies indicates that osteocytes embedded in the lacunae/canaliculi system and osteoblasts and bone cells lining the bone surface are mechanosensors that detect load-derived mechanical stimuli. By these bone-forming cells, the mechanical stimuli are translated into cellular signaling factors. However, normal bone metab … More olism is achieved by bone remodeling on balance between bone-forming cells and bone-resorbing cells. To date, the effect of fluid shear stress on osteoblasts has been extensively investigated ; however, the action on osteoclasts responsible for bone resorption remains to be elucidated. Therefore, the aim of this study is to elucidate the effect of fluid shear stress on osteoclastogenesis and osteoclast function directly or indirectly via osteoblasts.Methods : We isolated osteoblastic cells from mouse calvaria, and loaded fluid shear stress on these cells with a single flow-through system. In the case of study on osteoclast formation, we employed in vitro culture system of M-CSF dependent bone marrow cells-derived osteoclast progenitors for osteoclast formation dependent on RANKL. Results and Discussion : Isolated mouse calvalia-derived osteoblasts were capable of forming nodules and calcifing in a long-term culture. When the osteoblasts were exposed to fluid shear stress, expression of Cox-2 and RANKL mRNAs was increased as early as 2h after the start of exposure. In addition, gene expression of OPG, a soluble decoy receptor of RANKL, was also increased by the fluid shear stress in the cells ; however, the increase was less than that of RANKL expression. Conditioned medium obtained from the culture of shear stress-loading osteoblasts increased osteoclast formation induced by low concentration of RANKL, whereas the conditioned medium from the stress-unloading osteoblasts did not stimulated the formation. Short-term shear stress loading was sufficient for induction of RANKL gene expression, and the induction maintained even after freeing the loading. The shear stress-induced RANKL expression was dependent on the activation of Erk, but independent of prostaglangin induced by the shear stress. Finally, fluid shear stress directly stimulated the osteoclast formation from osteoclast precursors that were treated with M-CSF and low doses of RANKL. Taken together, fluid shear stress stimulates osteoclastogenesis directly or indirectly via inducing RANKL expression in osteoblasts. Less