Mohan Subburaman, Surisetty Ritika, Kesavan Chandrasekhar
Musculoskeletal Disease Center, VA Loma Linda Healthcare System, Loma Linda, CA 92357, USA.
Department of Medicine, Loma Linda University, Loma Linda, CA 92354, USA.
Biology (Basel). 2024 Aug 23;13(9):646. doi: 10.3390/biology13090646.
Flow induced by an orbital shaker is known to produce shear stress and oscillatory flow, but the utility of this model for studying mechanical loading effects in osteoblasts is not well defined. To test this, osteoblasts derived from the long bones of adult male C57BL/6J mice were plated on 6-well plates and subjected to orbital shaking at various frequencies (0.7, 1.4, and 3.3 Hz) for 30 and 60 min in serum-free differentiation media. The shear stress on cells produced by 0.7, 1.4, and 3.3 Hz shaking frequencies were 1.6, 4.5, and 11.8 dynes/cm, respectively. ALP activity measured 72 h after shaking (orbital flow) showed a significant increase at 0.7 and 1.4 Hz, but not at 3.3 Hz, compared to static controls. Orbital flow-induced mechanical stress also significantly increased (25%) osteoblast proliferation at a 0.7 Hz flow compared to static controls. Additionally, expression levels of bone formation markers , , , and were significantly increased (1.5- to 3-fold, < 0.05) in cells subjected to a 0.7 Hz flow compared to non-loaded control cells. We also evaluated the effect of orbital flow on key signaling pathways (mTOR, JNK, and WNT) known to mediate mechanical strain effects on osteoblasts. We found that blocking mTOR and WNT signaling with inhibitors significantly reduced (20-30%) orbital flow-induced ALP activity compared to cells treated using a vehicle. In contrast, inhibition of JNK signaling did not affect flow-induced osteoblast differentiation. In conclusion, our findings show that the flow produced by an orbital shaker at a lower frequency is an appropriate inexpensive model for studying the molecular pathways mediating mechanical strain effects on primary cultures of osteoblasts in vitro.
已知轨道振荡器产生的流动会产生剪切应力和振荡流，但该模型在研究成骨细胞机械负荷效应方面的实用性尚未明确界定。为了对此进行测试，将源自成年雄性C57BL/6J小鼠长骨的成骨细胞接种于6孔板中，并在无血清分化培养基中于不同频率（0.7、1.4和3.3赫兹）下进行30分钟和60分钟的轨道振荡。0.7、1.4和3.3赫兹振荡频率对细胞产生的剪切应力分别为1.6、4.5和11.8达因/平方厘米。与静态对照相比，振荡（轨道流）72小时后测得的碱性磷酸酶（ALP）活性在0.7和1.4赫兹时显著增加，但在3.3赫兹时未增加。与静态对照相比，0.7赫兹流的轨道流诱导的机械应力也显著增加（25%）成骨细胞增殖。此外，与未加载的对照细胞相比，在0.7赫兹流作用下的细胞中骨形成标志物、、和的表达水平显著增加（1.5至3倍，<0.05）。我们还评估了轨道流对已知介导成骨细胞机械应变效应的关键信号通路（mTOR、JNK和WNT）的影响。我们发现，与使用载体处理的细胞相比，用抑制剂阻断mTOR和WNT信号可显著降低（20 - 30%）轨道流诱导的ALP活性。相比之下，抑制JNK信号并不影响流诱导的成骨细胞分化。总之，我们的研究结果表明，较低频率的轨道振荡器产生的流动是一种合适的、廉价的模型，用于研究体外介导原代培养成骨细胞机械应变效应的分子途径。
