**5. Conclusions**

In conclusion, our results emphasize the role of protein microarchitecture on the mechanical properties of a material. We first illustrated the unique mechanical properties of the male *EC* gular tissue, contrasted with other tissue types, other species of frogs, gender, and rat bladder. The high elongation and moderate UTS demonstrated the tissues' ability to reduce performance trade-o ffs. That is, the tissue was able to elongate while maintaining higher stresses. Importantly, the specific multi-dimensional hierarchical collagen structure allowed the tissue to elongate as well as maintain moderately higher stresses. Together, the two components allow the unique functionality of the gular tissue to inflate and withstand the internal air pressure during calling. The elongation similarities of *EC* gular skin and rat bladder are reinforced by the ultrastructural likeness of the two tissues. Taken together, these findings

demonstrate the potential of the *EC* gular skin as a novel biomimetic example for developing materials for tissue engineering of large deforming tissues, such as the bladder. The characterization of this microarchitecture can provide a simple template for future artificial biomaterial scaffold designs for regenerative medicine applications such as biomimicry in 3D printed bladder tissues.

**Author Contributions:** Conceptualization, J.H. and A.S; methodology, J.H. and A.S.; investigation, J.H., S.S. and S.R., writing—original draft preparation, J.H.; writing—review and editing, S.R. and A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding sources gratefully acknowledged are the Johns Hopkins Greenberg Bladder Cancer award, Johns Hopkins Brady Urological Institute start-up funding, and NIH-NIBIB R21 trailblazer award.

**Acknowledgments:** We greatly thank Chelsea Thomas and Brad Wilson at the Atlanta Botanical Garden for their help in animal procurement and processing. We would also like to thank the Department of Materials Science and Engineering of Johns Hopkins University for allowing us to perform experiments on the MTS Criterion tensile testing equipment.

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