# An In Vitro Orbital Flow Model to Study Mechanical Loading Effects on Osteoblasts

## TL;DR

This study shows that orbital shaking at low frequencies can stimulate bone-forming cells in a cost-effective lab model.

## Contribution

The study introduces an inexpensive in vitro model using orbital shaking to investigate mechanical strain effects on osteoblasts.

## Key findings

- Orbital shaking at 0.7 Hz increases osteoblast proliferation and differentiation marker expression.
- Blocking mTOR and WNT signaling reduces flow-induced osteoblast differentiation.
- Lower frequency orbital shaking (0.7 Hz) is more effective than higher frequencies for stimulating osteoblasts.

## Abstract

Models to study the effects of exercise-induced benefits on bone-forming osteoblasts are limited and expensive. Although an orbital shaker model is simple and inexpensive, the impact of the flow produced by an orbital shaker on osteoblasts is not well defined, and this was evaluated in this study using an in vitro model. The findings of this study show that the flow produced by orbital shaking at physiological levels stimulates anabolic effects on osteoblasts, as evident from an increase in cell number and expression levels of osteoblast differentiation markers and mechanosensitive genes. Therefore, an orbital shaker at lower frequency provides an inexpensive and appropriate model to study mechanical strain effects on osteoblasts in vitro.

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/cm2, 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 Osf2, Hif1a, Vegf, and Cox2 were significantly increased (1.5- to 3-fold, p < 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.