# Chicken Feather Keratin Peptides for the Control of Keratinocyte Migration
## 3 Results and Discussion
### Cell Metabolic Activity and Migration
As can be seen in Figure 2A,B, the presence of the 20% peptide fraction decreased the metabolic activity and migration rate of the keratinocytes, although it did not reach statistical significance. On the other hand, fractions 40 and 80% do not seem to have any effect on either mechanism. As these fractions did not induce significant changes in the keratinocyte proliferation and migration rate, an additional fractionation was performed, where peptides were separated according to their molecular weight, ranging from 9090 to 401 Da for the 20% fraction (P188D 20%) and from 8082 to 382 Da for the 40% fraction (P188D 40%) (Table 1). Once again, the keratinocyte proliferation and migration rate were determined in contact with these new peptide fractions. In this analysis, the results obtained were very distinct (Figure 3) from the previous one (fraction 20%, 40% and 80%). Significant changes were observed in the metabolic activity of cells in the presence of peptides, particularly in the presence of 40% fraction \begin{table}
\begin{tabular}{c c c} \hline \hline
**Peptide Fraction.** & **Chromatographic Peak** & **Molecular Weight (Da)** \\ \hline P1 & P9090 \\ P188D 20\% & P2 & 1361 \\ P3 & 401 \\ \hline P1 & P1 & 8082 \\ P2 & 4699 \\ P3 & 2473 \\ P4 & 1199 \\ P5 & 382 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Peptide fractions and respective molecular weight. Figure 1: Keratinocyte migration in absence (**A**) and presence (**B**) of P188D peptide over time. 1—before contact, 2—2 h, 3—6 h, 4—24 h, respectively. Scale bar—200 mm. Figure 2: Keratinocyte proliferation (**A**) and migration rate (**B**) (in comparison to the control) in the presence of peptide fractions (45 g/mL). peptides. Analysing Figure 3A, it is clear that fractions P188D 20% P1, P188D 40% P1 seem to induce a higher metabolic activity of these cells; in contrast, P188D 40% P3 induced a decrease in the metabolic activity. Even though the results showed that sub-fraction P1 from 20% fraction peptide induced the highest metabolic activity in comparison to P2 or P3 from the same fraction, these differences did not reach statistical significance. On the other hand, significant changes were observed for the peptides from the 40% fraction, particularly P188D 40% P1, P3 and P5. While P1 significantly increased the metabolic activity, P3 decreased it. All the peptide sub-fractions induced an increase in the keratinocyte migration rate (Figure 3B), with significant differences. P188D 20%, P2 and P3 are significantly different, with the latter presenting a higher migration rate in comparison to the former. A similar  
Figure 3: Keratinocyte metabolic activity (**A**) and keratinocyte migration rate (**B**) (in comparison to the control) in the presence of peptide fraction. Same symbol shows significant differences between groups (_p_ < 0.05). *. + statistical significant between the groups. result was observed for P188D 40% P2, wherein P188D 20% P2 and P188D 40% P2 are significantly different, with the latter inducing a higher migration rate. P188D 40 % P3 also significantly affects migration in comparison with P188D 20% P1 and P188D 20 % P3. It seems that the presence of these peptides has a more significant effect on the migratory ability of these cells than on their metabolic activity. In this case, the use of fractioned peptides is very important for skin reepithelial and therefore for the treatment of chronic wounds. It is important to mention that resazurin assay is based on the capacity of living cells to transform resazurin to resorufin [29]. This ability is correlated with cell viability and cell number/proliferation. The basis for this understanding is that the higher the conversion of the dye, the higher the cell number. Therefore, taking into consideration this fact, it is possible to interpret the results observed, as the peptides fraction has a higher influence on cellular migration than on proliferation.