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An In Vitro Orbital Flow Model to Study Mechanical Loading Effects on Osteoblasts. | LitMetric

AI Article Synopsis

  • The study investigates the effects of orbital shaker-induced flow on osteoblasts, focusing on how different shaking frequencies impact cell behavior and signaling pathways.
  • Osteoblasts from mice were subjected to varying frequencies (0.7, 1.4, 3.3 Hz), with lower frequencies (0.7 and 1.4 Hz) showing significant increases in alkaline phosphatase (ALP) activity and cell proliferation compared to static controls.
  • Results suggest that lower frequency orbital flow is a useful, low-cost method for examining the molecular mechanisms related to mechanical strain effects in osteoblasts, with specific pathways (mTOR and WNT) being critical for mediating these effects.

Article Abstract

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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11428209PMC
http://dx.doi.org/10.3390/biology13090646DOI Listing

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