Page 107 - Physico-Chemical Niche Conditions for Bone Cells

morphogenesis need to be better understood, but we are just starting to uncover the complexity of interactions between fluid flow and cells. More research is needed to fully understand the immediate physiological response of pre-osteoblasts to physical loads as a result of PFF resulting in long-term bone adaptation to mechanical loading.
Initial down-stream impact of PFF on NO production and cell morphology
In this study, one hour PFF stimulated NO production. NO has biphasic effects on bone cells in vitro. High NO concentrations (>30 μM) inhibit cell proliferation, differentiation, and survival, whereas low NO concentrations derived from sodium nitroprusside (SNP, 1 μM ) have opposite effects [36–38]. Low concentrations of NO derived from SNP (100 μM) induce early mineralization via activation of ALP in rat bone marrow MSCs [39]. In addition, mechanical loading-upregulated NO production stimulates osteogenic differentiation of MSCs after 7, 14, and 21 days, e.g. increased ALP activity, collagen synthesis, and mineralization in vitro [40]. NO also significantly contributes to the activation of Fgf2 expression during angiogenesis [41], which is prerequisite for new bone formation. In our study, the initial down-stream impact of PFF-upregulated NO production from 10 nM to 150 nM, which concentration, albeit low, is still higher than the NO produced by the static control cells. PFF-upregulated NO production might have stimulated Fgf2 expression in the short-term, which could have contributed to the increased ECM production in the long-term. On the other hand, during post-incubation, NO induced by PFF could lead to production of another critical soluble factor. Our previous work showed that NO production was increased in a single MC3T3-E1 pre-osteoblast from 0-90 min post-incubation after 1 min mechanical stimulation [42]. Future study should measure NO soluble factor in the culture medium after post-incubation.
Mechanical loading stimulates expression of cyclooxygenase 2 (COX2), a key enzyme for PGE2 production in bone cells [43]. COX2 is involved in the reorganization of the F-actin stress fibers [43]. Such an F-actin stress fiber reorganization might provide an explanation of our finding that mechanical loading by PFF affected F-actin fluorescence intensity in bone cells. Our data are in agreement with findings by others showing that mechanical loading affects the reorganization of actin filaments, e.g. prominent F-actin stress fibers [13]. These cytoskeletal changes might allow nuclear genomic adaptation to fluid shear stress by adjusting cellular morphology in the most force-efficient shape. On the other hand, in the time frame measured we did not find a shift in cell orientation relative to the direction of the flow.
The attachment of pseudopodia to the ECM is achieved by several types of special adhesions, e.g. hemidesmosomes, podosomes, fibrillar adhesions, invadopodia, focal complexes, and focal adhesions [44]. As an active organelle, pseudopodia not only participate in cell migration [45], but also sense instantaneous changes in the environment, which is
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