Page 108 - Physico-Chemical Niche Conditions for Bone Cells

PFF affects pre-osteoblast behavior
considered as a sensor of basal material morphology [46]. Cells loaded by fluid shear stress experience a larger overturning effect, while strain deriving from the substrate mainly affects cell-substrate attachment [43]. We found that PFF treatment did not modulate pseudopodia length.
The current study showed that PFF was capable of changing F-actin fluorescence intensity. This might be explained by F-actin stress fiber reorganization when the cytomembrane was subjected to fluid shear stress. In addition, we found that although PFF caused a change in the internal structure of the cells, it did not alter the orientation of the cells.
Short-term down-stream impact of PFF on cell metabolic activity, ALP activity, and gene expression
In our study, PFF did not affect cell metabolic activity. However, ALP activity was affected by 1 h post-incubation after PFF treatment. Mechanical loading enhances ALP activity and ALP mRNA of human bone marrow mesenchymal stem cells [47]. However, the loading-induced difference is 40% higher in ALP activity than that in ALP mRNA [47]. Therefore, in our study, we did not measure ALP gene expression. Runx2 is a crucial transcription factor associated with bone cell differentiation. In the cell cycle exit and entry, Runx2 has a vital cell proliferation regulatory role in osteoblasts [48]. We found that PFF increased Runx2 mRNA expression at 3 h post-incubation. This increase might be due to the effects of amino acids, vitamins, lipoic acid, and growth factors in the culture medium taken up by osteoblasts after 1 h PFF treatment at that specific time point (3 h post-incubation). There is an excellent correlation between Ki67 expression and cell growth or proliferation [49]. In the present study, 1 h PFF did not influence Ki67 gene expression, which indicates that PFF may not affect cell proliferation. This result is not agreement with the findings of Wiesner et al [49]. Osteocalcin (Ocn) is secreted solely by osteoblasts, and plays an important role in the regulation of bone metabolism [50]. Ocn is involved in calcium ion homeostasis and bone mineralization, similar to Fgf2. Fibroblast growth factors (Fgfs) affect osteoblast gene expression in a biphasic fashion, depending on the stage of osteoblast maturation [51,52]. Fgf2 can activate osteoblast proliferation and Ocn production in immature pre-osteoblasts [53]. We found no significant difference in Ocn expression in static control and PFF-treated cells without post-incubation, although we might have expected an upregulation since mineralization in the long-term was affected by PFF. Both Ocn and Fgf2 expression were not affected by 1 h PFF treatment without post-incubation. However, Ocn and Fgf2 expression were affected by PFF after 3 h post-incubation, which might be the result of factors present in the culture medium at the right time. This indicates that bone cells are indeed able to respond to PFF with expression of key factors that are involved in bone formation, e.g. Wnt signaling molecules, which regulate bone adaptation. In our study, we performed additional PCR analysis for Wnt3a, Wnt5a, LRP5, and LRP6
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