Figure 12. Relative expression levels of MyoD ,Myf5, and MyoG in C2C12 cells cultured in GM for 7 days and in DM for an additional 7 days onto cellulose acetate nanofibers (A) and cellulose acetate nanofibers with annatto extract (B).
Minor differences in expression were observed when cells were cultivated onto CA and CA@A nanofibers for 7 days in GM, followed by 7 days in DM (Figure 12). Under these conditions, while the CA scaffold induced upregulation of expression of all the tested transcripts includingMyf5 (Figure 12A), CA@A’s positive effect on MyoGexpression was no longer visible but its negative effect on expression of the determination factors (MyoD and Myf5 ) remained (Figure 12B).
Therefore, these results corroborate the hypothesis that the CA scaffold with annatto in impairs the differentiation process, indicating that annatto extract does not synergize with the cell differentiation phenotype for C2C12 cells. However, the MTT assay results suggesting that annatto can improve C2C12 cell viability/proliferation indicate that this natural additive is useful when progenitor proliferation is required (for example, when establishing a construct for cultured meat production). A plausible hypothesis for the negative effect of annatto on myogenesis is that it can pair synergistically with other cell types, which is confirmed by research by Capella et al. (2016) [50], who found that annatto extract assisted in the process of healing open wounds when connective tissue was analyzed.