## 4 Discussion
These data suggest that the cell type, level, and type of FSS are critical parameters for inducing mechanical FSS. In contrast to unidirectional FSS, a study found that oscillating FSS resulted in delayed or no reorganization of actin filaments in osteoblasts or osteocytes [56]. The data showed that mechanical FSS promoted cell viability and cell number up to 24 h under both CFSS and PFSS. Although the cell colony size was decreased under both mechanical FSS protocols, the colony distribution as indicated by the colony number was increased only under PFSS. The results suggest that the applied stress enhances cell viability; however, the resting period of 1 h as shown in PFSS was sufficient to facilitate colony distribution. Such performance improves the stemness as well as the regenerative ability of stem cells. In contrast, the area of colonies tended to be smaller in both FSS culture conditions to minimize the area exposed to FSS and establish balanced adhesion. The results demonstrate that the duration of FSS applied has a central influence on the function of myoblasts. Previous studies have shown that intermittent mechanical stimulation allows cells to recover and establish adhesion by modulating their cytoskeleton [63]. Consistent with our data, previous studies showed improved cell proliferation under FSS compared to the static culture condition. One possible interpretation is that FSS may enhance stem cell division. It has been observed that pulsed FSS activates cell cycle progression through the upregulation of proliferation gene expression and the activation of signaling pathways including ERK \(\
icefrac{{1}}{{2}}\), p38, MAPK, and cyclin-dependent kinase 4 expression in primary satellite cells [58]. A similar study showed an enhanced cell proliferation in C2C12 under dynamic FSS culture conditions [24]. This enhancement of cell viability and cell number has previously been documented in human and equine MSCs under oscillating or pulsating FSS by our group and others [21; 64]. The potential for colony formation, and thus the ability for self-renewal, is considered to be a characteristic feature of stem cells [65]. Consistent with our data, a study demonstrated an increase in MSCs migration capacity under very low FSS (\(\sim\)0.001 Pa) comparable to the interstitial fluid flow in vivo [66], which could interpret the increased colony count under PFSS as presented in our data. The effect of FSS on MSC migration was relatively dependent on the strength of the applied FSS. Similar to our data, meanwhile, a low FSS of 0.2 Pa promoted cell migration, whereas a higher FSS of \(>\)2 Pa initiated an inhibitory effect on cell migration [67]. In the same line, the pulsating FSS increased the expression of stem cell markers, including SOX2 and Nanog, indicating enhanced stemness properties under mechanical stress [68]. The data showed that myogenic differentiation upregulated MyoD expression in all experimental groups, regardless of the effect of FSS. Although FSS promotes cell viability and colony number, it might delay cells' commitment to their myogenic fate. In the same line, it was reported that FSS increased the expression of the cyclic cell division in primary myoblasts and enhanced the expression of C-Fos is a subunit of the transcription factor activator protein 1, which inhibits the transcription of MyoD by binding to its promoter, thus maintaining cell proliferation and blocking the cell differentiation [58; 69]. Consistent with our results, a study investigating the influence of mechanical stretch on proliferating myoblasts demonstrated a reduced expression of MyoD under mechanical stimulation [70]. Interestingly, the present data showed an increased number of myogenic-positive cells at 72 h only under PFSS conditions, as well as an upregulated mTOR expression in the cells under static and PFSS conditions. The data suggest that under PFSS, the resting period was sufficient to establish cell adhesion and overcome the effect of mechanical FSS by upregulating mTOR and MHC expression.