*3.10. Statistical Analysis*

All the experiments were conducted three times and the data were presented as mean with standard error based on descriptive statistics. One way ANOVA (*p* < 0.05) and post hoc test were adapted using the statistical software SPSS version 16.0 (SPSS, Chicago, IL, USA).

### **4. Conclusions**

In this study, two types of collagens, PSC and ASC were isolated and purified from blue shark skin, which contained three distinct chains, 2 alpha, and beta chains. The amino acid composition and the FTIR secondary structure of PSC varied with ASC due to pepsin hydrolysis. Among two collagens, PSC had high thermal stability, which might be due to high imino acid content. Most importantly, the obtained collagens promoted osteoblast cell growth and upregulated collagen synthesis in bone cells, which is a most desirable property of biomaterials for the treatment of a bone disorder. Proliferation of differentiated osteogenic cells by blue shark skin collagen may be achieved through activation of Runx2 dependent FAK/JNK signaling pathway. Overall, the blue shark skin collagens with good biochemical and osteogenic properties may be considered as a potential drug in biomedical application. Conversely, the in vivo evaluation of blue shark skin collagen and its molecular interaction with bone cells needs to be addressed by further research in order to understand the actual mechanism of the action.

**Author Contributions:** J.E. and J.W.L. carried out the laboratory works, compiled data, and wrote the manuscript. Y.H., S.W., J.E.M.S.d.V., S.Y.L. and J.M.R. revised the manuscript and provided valuable advice on interpretation of data. W.W. and B.B. designed and directed the study, inferred the data, and revised the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China, project grant number 81750110548, the National High Technology Research and Development Program of China, project grant number 2011AA09070109), and the Plan of Innovation Action in Shanghai project grant number 15410722500 and 17490742500.

**Acknowledgments:** The authors gratefully acknowledge the funding support received from the National Natural Science Foundation of China (Project Grant No. 81750110548), an International Young Scientist Research Fellowship, the National High Technology Research and Development Program of China (Project Grant No. 2011AA09070109), and the Plan of Innovation Action in Shanghai (Project Grant No. 15410722500 and 17490742500). The authors also thank Shanghai Ocean University, Shanghai, China for providing the necessary facilities to undertake this work.

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