3.8 RT-qPCR
Expression levels of myogenic genes in C2C12 cells cultivated onto CA and CA@A scaffolds were determined using RT-qPCR to assess the progress of these cells during myogenic differentiation. C2C12 cells were cultivated onto both scaffolds in only GM, and MyoD , Myf5,and MyoG expression levels were evaluated after 7 and 14 days of culture.
We found upregulation of the expressions of MyoD(~4 fold change) and MyoG (~1.8 fold change) in the C2C12 cells cultivated onto CA scaffolds after 14 days compared to 7 days in culture; for Myf5 in the same comparison, no effects were seen (Figure 11A). An entirely different gene expression pattern was seen when cells were cultivated onto CA@A scaffolds (Figure 11B): MyoD (~0.03 fold change) and Myf5 (~0.100 fold change) were downregulated, while MyoG expression was upregulated after 14 days compared to 7 days in culture (Figure 11B). According to Chal et al. (2017) [48],MyoD , Myf5, and MyoG are directly linked to the cell differentiation stage in myogenesis, when cells assume a more elongated shape. MyoD and Myf5 should be expressed first, since they are more related to the proliferation stage of these cells, while MyoG is related to the cell fusion stage and is necessary to form multinucleated myotubes [48]. Together, these results suggest that the CA scaffold favors cell proliferation and initiation of the first stages of skeletal muscle cell differentiation, while CA@A blocks the expression of the determination factors (MyoD andMyf5 ) and favors the terminal stages of cell differentiation.