## 3 Results
### Effect of Optimized Mechanical Fluid Shear Stress on Myogenic Differentiation  
Immunofluorescence was used to detect myogenic markers and examine whether FSS promotes myogenic differentiation (MD). After 24 h, a slight reduction in the number of MyoD-positive cells was observed compared to the static culture condition, but this was not statistically significant (Figure 2a). However, at 72 h, the number of myogenin-positive cells showed a significant increase under the PFSS condition compared to both static (\(p<0.001\)) and CFSS (\(p<0.01\)) conditions (Figure 2b-f). To confirm our data, we evaluated the expression of the myogenic markers up to day 7 post-induction. The data analysis revealed upregulated MyD expression for cells cultivated under static, CFSS, and PFSS conditions (\(p<0.001\), \(p<0.05\), and \(p<0.01\)) compared to non-myogenic induced cells, respectively (Figure 2g). The data showed an upregulated myogenic expression for all experimental groups at 72 h (\(p<0.05\)) and day 7 (\(p<0.001\)) compared to 3 h post-induction. The data analysis indicates a significant upregulation of myogenin for those cells under static, CFSS, and PFSS conditions compared to the non-induced cells at day 7 post-induction (Figure 2h). Additionally, the data showed an upregulated MHC7 expression for all differentiated groups at 72 h and day 7 regardless of FSS (\(p<0.001\)) compared to early induction at 3 h. Furthermore, there was an enhanced MHC7 expression for all treated groups at day 7 (\(p<0.001\)) compared to non-induced cells in the control medium (Figure 2i). Similarly, an upregulated MHC1 expression was detected for all differentiated groups regardless of FSS at day 7 (\(p<0.05\)) compared to early induction at 3 h (Figure 2j). To examine whether FSS influences protein synthesis, the expression level of mTOR was quantified. The data analysis demonstrated an upregulation of mTOR in cells under both static and PFSS conditions at day 7 (\(p<0.05\)) compared to matched groups after 3 h induction (Figure 2k). Figure 1: Effect of optimized mechanical fluid shear stress on the cell viability. (**a**–i) Phalloidin staining (green) of C2C12 cultivated under both cyclic fluid shear stress (CFSS) and periodic fluid shear stress (PFSS) in a growth medium after 3 h, 24 h, and 3 d. Cells cultivated in parallel under static condition served as a negative control. Morphologic alterations combined with numerous cellular processes under CFSS (arrow) were observed. (**j**) Semi-quantitative analysis of cell viability (\(n=6\)) at 570 nm absorbance using MTT assay. (**k**) Semi-quantitative analysis of cell count (\(n=6\)) at 565 nm absorbance using the sulforhodamine B assay (SRB). (**l**–o) Colony formation unit assay for C2C12 cultivated under CFSS, PFSS and static conditions at various seeding densities: 1 \(\times\) 10\({}^{2}\), 5 \(\times\) 10\({}^{2}\), and 1 \(\times\) 10\({}^{3}\) cells per flask in a growth medium for 7 days (\(n=6\)). (**l**) Average colony size (\(\upmu\)m\({}^{2}\)) at 1 \(\times\) 10\({}^{3}\) cell density. (**m**) Number of colonies at 1 \(\times\) 10\({}^{2}\) cell density. (**n**) Number of colonies at 1 \(\times\) 10\({}^{2}\) cell density. at \(5\times 10^{2}\) cell density. (**o**) Number of colonies at \(1\times 10^{3}\) cell density. Data are presented as means \(\pm\) SEM. * = \(p\) \(<\) 0.05, ** = \(p\) \(<\) 0.01, *** = \(p\) \(<\) 0.001. DAPI was used as a nuclear stain (blue). Scale bar = \(10\)\(\mu\)M.  
Figure 2: Effect of optimized mechanical fluid shear stress on the myogenic differentiation capacity of C2C12 under static, CFSS, and PFSS conditions at 3 h, 72 h and 7 days. Non-induced cells cultivated in basal medium served as a negative control. (**a**) Percentage of MyoD-positive cells after 24 h. (**b–e**) Immunofluorescence shows myogenin-positive cells (red) after 72 h under static, CFSS and PFSS conditions. Cells processed in parallel without adding a primary antibody served as a negative  
control ((**(b)**), NC).