## 4 Discussion
In contrast, the enhanced osteogenic differentiation under CFSS as previously reported for MSCs in equines [21] suggests that either cell type or the differentiation fate might play a role in cell response to the FSS induction. In agreement with our data, a study revealed that intermittent FSS was able to promote osteogenic differentiation more effectively compared to CFSS, which enables a recovery period and enhances the sensitivity to mechanical stimulation [63]. Morphometric evaluation of myotubes, indicative of cell differentiation capacity, showed a reduction in myonuclear fusion and myotube formation under PFSS compared to CFSS. The data suggest improved cell differentiation under CFSS; however, more shear stress should be applied to achieve significant differentiation induction. In agreement with our data, another study demonstrated that an FSS of 16 mPa effectively induced myogenic differentiation in C2C12 cells characterized by the formation of long myotubes alignedwith the direction of flow together with an elongation of the cell nuclei. In contrast, an FSS of 3 mPa was less able to trigger myogenic differentiation parameters [24]. In the present work, neither an orientation of the myotubes nor an elongation of the cell nucleus could be detected, possibly due to the different values of the FSS, as well as the different forms of the applied FSS in terms of a laminar flow compared to an oscillating FSS in the present study. A similar study using pulsating FSS reported no differences in MyoD and myogenin expression in primary murine myoblasts compared to static conditions [58]. Like mechanical FSS, oxygen concentration plays an important role in the performance of stem cells. It is well established that oxygen concentration in the muscle is lower than the atmospheric O\({}_{2}\), suggesting that the standard in vitro cultivation might provide inaccurate parameters about the in vivo stem cell response. The present results demonstrated an improved cell viability up to 72 h under 3% O\({}_{2}\) compared to 21% O\({}_{2}\), which was confirmed by a parallel increase in cell count. Interestingly, however, a reduction in the cell count after 72 h was combined with no changes in cell viability under hypoxia. The data suggest that hypoxia promoted cell viability even with a low cell number. Similar results have previously been reported by our group for up to 7 days of cultivation under hypoxia [40]. Consistent with these results, hypoxic conditions enhance the proliferation of primary satellite cells isolated from mice, rats, bovine, and humans [14; 37; 38; 71]. The data suggest that hypoxia activates cell cyclic progression, intracellular regulators, and signaling pathways that promote cell viability. In agreement with this, another study revealed that a 3% oxygen concentration activates G1/S cyclins and cyclin-dependent kinases, while it inhibits cell cycle inhibitor p27 and phosphorylation of Akt, which induces enhanced cell proliferation [37]. In the same line as previous results, an increase in the number of colonies was detected under hypoxia. Similar reports have concluded that the combined high number of colonies under hypoxia condition was a result of increased metabolic activity and the high doubling capacity [37; 40]. Our data exhibited a reduction in the number of MyoD-positive cells after 24 h and myogenin-positive cells after 72 h under hypoxia. Moreover, MyoD was upregulated at 72 h under normoxia while myogenin expression was upregulated at day 7 for both experimental groups, regardless of the effect of hypoxia. Taken altogether, these data suggest that hypoxia impairs the myogenic precursor's commitment to the myogenic direction. In the same line, another study demonstrated inhibition of myogenic differentiation at day 3 under 0.5% O\({}_{2}\); in contrast, an irreversible impairment cell differentiation was detected after day 6 [72]. In addition, hypoxic conditions promote the quiescence of primary mouse myoblasts through the upregulation of Pax7 and the downregulation of MyoD and myogenin [45].