Mechanical loading up-regulates early remodeling signals from osteocytes subjected to physical damage

In the mineralized bone matrix, mechanical loading causes micrometer-sized cracks. These cracks trigger targeted remodeling along the micro-crack. Physical damage to osteocytes was shown to be involved in the initiation of this remodeling process. However, the role of subsequent mechanical loading osteocyte response to physical damage is unclear. In this study, we have designed and developed an in vitro cell model to study the impact of mechanical loading on osteocytes with physical damage. Specifically, a system was developed to create sub-cellular physical damage on MLO-Y4 osteocytes in vitro. This model re-created the spatial distribution of non-viable cells and VEGF expression around microdamage as reported in vivo. Using this system, the short term (24h) effects of fluid shear stress in regulation of osteocyte response to physical damage were investigated. We have observed that the mechanical stimuli had an additive effect in terms of COX-2, VEGF mRNA expressions, as well as PGE2, VEGF concentrations in the media. Interestingly, other inflammatory signals such as IL-6 and TNF-α did not change with these stimuli, at this time point. Moreover, fluid shear also had a modulating effect in regulation of osteoclast differentiation by osteocyte with physical damage. These results show that (1) subcellular physical damage upregulates remodeling signals in osteocytes at early time point, (2) mechanical loading substantially upregulates these signals for remodeling in osteocytes with physical damage.