## 3 Results and Discussion
### Carboxymethylcellulose Characterization
#### 3.1.4 Pro-Collagen I \(\alpha\) I Production  
Collagen, the most abundant protein found in humans, and the primary structural component of the dermis, is responsible for conferring strength and support to human skin. Alterations in collagen play an integral role in the ageing process. Type I collagen, the most abundant dermal collagen, is composed of two types of pro-collagen: pro-collagen I \(\alpha\) I  
Figure 3: Cytotoxicity of CMCs towards HaCaT (**a**) and HDFa (**b**) cells. The dotted line represents the 30% cytotoxicity limit as defined by the ISO 10993-5:2009 standard. and \(\alpha\) 2 chains. Thus, by measuring the protein levels of pro-collagen I \(\alpha\) I it is possible to estimate alterations in the levels of collagen type I [47, 48], which may be a critical step toward the development of any possible cosmetic ingredient. With regards to the effects of CMC upon pro-collagen, I \(\alpha\) I production, the first step was the quantification of the protein content of the cellular extracts obtained. As can be seen from Figure 4, CMC2 and CMC4 significantly (\(p\) < 0.05) reduced the total protein content recovered from HDFa cells. However, considering the lack of impact on the cellular metabolism previously observed, it is most likely that the reductions here observed are a result of protein adsorption by the CMC fibers, as previous works described that anionic polymers, such as CMC, are capable of adsorbing biomolecules [49, 50, 51]. As to why only two of the four CMC samples assayed displayed this behavior, the most likely explanation is the crystallinity index. Considering that both samples had a higher crystallinity index and that, as previously shown, higher crystallinity indexes are usually associated with higher degrees of interaction with biological molecules, it is only natural that these samples will have higher protein adsorption properties [52, 53]. When analyzing the results regarding the pro-collagen I \(\alpha\) I production (Figure 5), one can see that CMC was only capable of modulating intracellular production (Figure 5a) and not extracellular (Figure 5b). Figure 4: Total protein content of HDFa cells in the presence of CMC. (**a**) Total protein content; (**b**) percentage of protein production relative to the basal level (control). Different letters represent the statistically significant differences (\(p\) < 0.05) found between conditions. Figure 5: Pro-collagen I \(\alpha\) I relative percentage of production in the presence of different CMC samples relative to the basal level (control). (**a**) Intracellular; (**b**) extracellular. Different letters represent the statistically significant differences (\(p\) < 0.05) found between conditions. This discrepancy may be a result of the addition of CMC to the culture media which may alter the reaction kinetics of collagen assembly and increase the deposition of collagen in the extracellular matrix, a phenomenon previously described by [PERSON] and [PERSON] [54]. A similar explanation can be a mechanism similar to the steric hindrance of fibril formation reported by [PERSON] and [PERSON] [55] for an alginate hydrogel, in which pro-collagen was observed in the pericellular space, but was not present in the extracellular matrix due to basically a lack of space for lateral fibril growth. Additionally, authors [PERSON] and [PERSON] [56] and [PERSON] and [PERSON] [57] have previously described cationic collagen as being adsorbed to CMC through interactions with its -COOH group. It is likely that the lack of modulation observed in the extracellular setting was a consequence of this interaction and not a lack of activity of CMC samples. This hypothesis has been recently confirmed by [PERSON] and [PERSON] [58], who showed through docking analysis that CMC interacted with the Lys, Thr, and Pro amino acids present in collagen through hydrogen bonds and van der Waals and that the affinity for said interaction was high as it had a low energy.