Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
Department of Mechanical and Industrial Engineering, University of Toronto, Toronto, Canada.
J Orthop Res. 2019 Aug;37(8):1681-1689. doi: 10.1002/jor.24302. Epub 2019 Apr 24.
Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1681-1689, 2019.
成骨细胞对骨形成和骨吸收的机械调节是维持骨健康的关键。尽管广泛的体外研究使用成熟的 MLO-Y4 细胞模型探索了成骨细胞的机械生物学，但该细胞系分泌的硬骨素量低，使其不足以研究机械加载下成骨细胞和破骨细胞之间的串扰。在这里，我们研究了表达硬骨素的 OCY454 成骨细胞模型在发挥这一作用中的潜力。通过测量对振荡液流反应的蛋白质分泌、总三磷酸腺苷 (ATP) 含量和细胞内钙的变化来测试完全分化的 OCY454 细胞的机械敏感性。观察到硬骨素表达和总 ATP 含量的增加。然而，检测到非常低水平的核因子κ-B 配体受体激活剂，并且钙反应存在很大的不一致性。然后使用 OCY454 细胞的条件培养基 (CM) 培养成骨细胞和破骨细胞前体。用碱性磷酸酶 (ALP) 和茜素红 S 染色定量成骨细胞活性，用抗酒石酸酸性磷酸酶 (TRAP) 染色定量破骨细胞分化。我们证明，机械刺激的 OCY454 细胞释放的可溶性因子增加了成骨细胞的 ALP 活性 (p<0.05) 和钙沉积 (p<0.05)。当用来自流动刺激的 OCY454 细胞的 CM 处理破骨细胞前体时，也显著减少了大尺寸 TRAP 阳性破骨细胞 (p<0.05)。这项研究的结果表明，OCY454 细胞对机械加载的反应是通过释放关键因子（如硬骨素）来调节下游骨细胞，从而证明其作为体外成骨细胞机械生物学研究的新型细胞模型的潜力。2019 年 Orthopaedic Research Society. 由 Wiley Periodicals, Inc. 出版。J Orthop Res 37:1681-1689，2019。
