Page 109 - Physico-Chemical Niche Conditions for Bone Cells

(unpublished results). Wnt3a was not detectable in MC3T3-E1 pre-osteoblasts at any time point measured, under any condition. LRP5 and LRP6 were quantifiable, but were not affected by PFF at any time point measured. Wnt5a was also quantifiable, and increased by 1 h PFF after 1 h post-incubation, suggesting that bone adaptation regulated by osteoblasts might mainly rely on the Wnt noncanonical pathway, i.e. Wnt5a [2]. Axin, a key scaffolding protein, is involved into the Wnt signaling pathway. In the absence of Wnt prontein, b-catenin interacts directly with Axin [54]. In the presence of Wnt protein, LRP5 and LRP6 co-receptor bind to the Wnt ligand and stimulate the phosphorylation of LRP5 and LRP6 [54]. When Wnt binds to frizzled as well as LRP5/6 the complex targeting b-catenin for destruction is recruited to the cell membrane and inactivated, thereby allowing b-catenin to accumulate and translocate to the nucleus, where it affects the transcription of Wnt target genes by TCF/LEF. One such Wnt target gene is Axin. [54,55]. Mepe plays a multifunctional role in the regulation of mineral homeostasis, cell signaling, and mineralization [56]. Mepe belongs to the small integrin- binding ligands. In this study, 1 h PFF did not affect Mepe gene expression. This suggests that Mepe might be not involved in the PFF-upregulated mineralization in the long-term. Dmp1 is a non-collagenous ECM protein found in dentin and bone, similar as osteopontin and bone sialoprotein, that combines ligand N-linked glycoprotein family, and is part of the small integrin- binding ligand [57]. Collagen type I secreted by early undifferentiated osteoblast-like cells or pre-osteoblasts constitutes 90% of the total organic part of the ECM in mature bone. In this study, 1 h PFF stimulated Dmp1 and Col1⍺1 expression after post-incubation, which was likely as the result of enhanced ALP activity by PFF treatment. Gene upregulation of the osteogenic marker Dmp1 is generally believed to be positively correlated to the differentiation process. Our finding that PFF upregulated Dmp1 expression is in line with the upregulation of osteogenic differentiation of MC3T3-E1 pre-osteoblasts. Therefore, one single bout of mechanical loading by PFF plays a vital role in the upregulation of Dmp1 and Col1⍺1 expression, which is likely responsible for the enhanced cell function or mineralization in the long-term.
Long-term down-stream impact of PFF and post-incubation in osteogenic medium
PFF causes stress of all parts of bone cells including increased membrane stress, and thereby a rapid intracellular calcium response, ion channel activity, and/or modulation of NO production [43,58], which may all affect osteogenic differentiation. During post-incubation, PFF might have affected nutrient uptake from the culture medium containing non-essential amino acids, vitamins, biotin, sodium pyruvate, cyanocobalamin, lipoic acid, nucleosides, and growth factors [59], which could have affected cell behavior, e.g. osteogenic differentiation. On the other hand, soluble factors produced by the cells themselves as a result of PFF treatment
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