*3.13. Statistical Analysis*

All experiments were performed in triplicate and the results are expressed as mean ± standard deviation. Metabolic activity of MRC-5 cells and shape descriptors showed a non-parametric distribution; therefore Mann-Whitney was used to assess statistically significant differences between results. Difference was considered statistically significant at *p* < 0.05.

#### **4. Conclusions**

The present study showed that collagen type I can be obtained from the skin of codfish with a high degree of purity, representing a valuable strategy for the valorization of a marine by-product. Properties such as molecular weight, amino acid composition and molecular structure were close to those of collagen of mammalian origin. The main difference was in regard to the protein denaturation temperature. Collagen with a low denaturation temperature presents poor gelling properties, which limit its application as a gel-forming agent, causing a processing bottlenecking to achieve cohesive gels at physiological temperatures. However, this could be overcome using chemical crosslinking, such as with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), without significant effects on collagen's biological functions. Besides the biomechanical properties, collagen is also involved in a wide range of biological functions. Collagen can specifically interact with particular receptors at the cell surface, such as integrins, discoidin-domain receptors, and glycoprotein VI [42], thus signaling cell adhesion, differentiation and growth, as well as cell survival. Moreover, heavy metals were undetectable and therefore below the regulated limits. Marine-origin collagen may be used as a safe source of adhesion points and molecular modulators that can be used in combination with biopolymers for the development of biomaterials with promising biomedical application.

**Author Contributions:** Conceptualization and methodology, A.M.C., A.P.M., T.H.S. and R.L.R.; Technical execution, A.M.C.; Discussion of results, A.M.C., A.P.M. and T.H.S.; Writing-Original Draft Preparation, A.M.C.; Writing—Review & Editing, A.P.M. and T.H.S.; Supervision, R.L.R.; Funding Acquisition, R.L.R.

**Funding:** This research was funded by European Research Council grant agreement ERC-2012-ADG 20120216-321266 for the project ComplexiTE.

**Acknowledgments:** The authors acknowledge to Leonardo Aires (Frigoríficos da Erminda, Portugal) for the kind contribution of skin from codfish and Ana M. Gomes and Fátima Silva (Catholic University of Portugal–Porto) for the heavy metals analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.
