Page 105 - Physico-Chemical Niche Conditions for Bone Cells

DISCUSSION
This study aimed to map any rapid (seconds, minutes) changes of apex height and fluid dynamics over an osteoblast after a single bout of mechanical loading, and investigate whether osteoblasts produce (a mix of) signaling factors in the intermediate-term (hours) that affect osteogenic differentiation in the long-term (days). Computational FE analysis revealed that fluid dynamics (fluid velocity, fluid pressure, and fluid shear stress) on an adherent pre- osteoblast inside a parallel-plate flow chamber immediately changed as a result of PFF. This data was in line with the movement of the cell in vertical direction during loading by PFF. We also found that PFF considerably affected NO production, slightly affected actin stress fibers, but did not affect cell orientation, or the length of pseudopodia after 1 h PFF. In the short-term, PFF did not alter cell metabolic activity, but enhanced ALP activity and osteogenic gene expression, i.e. it caused a significant increase in Runx2, Ocn, Fgf2, Dmp1, and Col1⍺1 expression. In the long-term, PFF did not affect ALP and collagen protein production, but changed matrix mineralization, dependent on the post-incubation time that the cells had experienced without mechanical loading and refreshment of medium. These results indicate that a single bout of mechanical loading by PFF acutely affected signaling molecule gene expression. The observation that PFF treatment followed by post-incubation in culture medium up to 3 h enhanced the differentiation of the cells in the long-term (i.e. at 28 days) indicates that the PFF-induced release of soluble factors had long lasting effects on osteoblast differentiation.
We treated the cells with PFF of 6.5 Pa/s peak shear stress rate, 1.0 Pa amplitude, and 1 Hz frequency. PFF of 6.5 Pa/s peak shear stress rate was used to treat the cells, since we have found earlier that the response of MC3T3-E1 pre-osteoblasts is linearly dependent on the rate of fluid shear stress, which depends on the amplitude and frequency of stress [20,21]. The fluid shear stress amplitudes and frequencies in bone have been determined by a combination of experiments and computer models, where the peak fluid shear stress around mouse osteocytes in situ has been estimated to range up to 5 Pa [22]. That this range of fluid shear stress is enough to stimulate bone cells was confirmed by in vitro studies [1,23]. In the current study, we have tested the effect of shear stress of a single magnitude within the physiological range, but not shear stresses resembling disuse or overuse, since this will cause bone cell apoptosis and cell death [24]. Alterations in osteoblast cytoskeletal structure in response to shear stress occur within minutes [22,25]. Therefore, we have chosen 1 h PFF as an end point for our investigations. Previously, we have shown that post-incubation might affect the behavior of MLO-Y4 osteocytes and MC3T3-E1 pre-osteoblasts [26]. Little is known whether post-incubation affects pre-osteoblast function after mechanical loading.
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