**The Specific Molecular Composition and Structural Arrangement of** *Eleutherodactylus Coqui* **Gular Skin Tissue Provide Its High Mechanical Compliance**

#### **Justin Hui 1, Shivang Sharma 2, Sarah Rajani 1 and Anirudha Singh 2,3,\***


3 Department of Urology, The James Buchanan Brady Urological Institute, TheJohnsHopkinsSchoolofMedicine,Baltimore,MD21287,USA

**\*** Correspondence: asingh29@jhu.edu

Received: 29 June 2020; Accepted: 3 August 2020; Published: 5 August 2020

**Abstract:** A male *Eleutherodactylus Coqui* (*EC*, a frog) expands and contracts its gular skin to a grea<sup>t</sup> extent during mating calls, displaying its extraordinarily compliant organ. There are striking similarities between frog gular skin and the human bladder as both organs expand and contract significantly. While the high extensibility of the urinary bladder is attributed to the unique helical ultrastructure of collagen type III, the mechanism behind the gular skin of *EC* is unknown. We therefore aim to understand the structure–property relationship of gular skin tissues of *EC*. Our findings demonstrate that the male EC gular tissue can elongate up to 400%, with an ultimate tensile strength (UTS) of 1.7 MPa. Species without vocal sacs, *Xenopus Laevis* (*XL*) and *Xenopus Muelleri* (*XM*), elongate only up to 80% and 350% with UTS~6.3 MPa and ~4.5 MPa, respectively. Transmission electron microscopy (TEM) and histological staining further show that *EC* tissues' collagen fibers exhibit a layer-by-layer arrangemen<sup>t</sup> with an uninterrupted, knot-free, and continuous structure. The collagen bundles alternate between a circular and longitudinal shape, suggesting an out-of-plane zig-zag structure, which likely provides the tissue with greater extensibility. In contrast, control species contain a nearly linear collagen structure interrupted by thicker muscle bundles and mucous glands. Meanwhile, in the rat bladder, the collagen is arranged in a helical structure. The bladder-like high extensibility of *EC* gular skin tissue arises despite it having eight-fold lesser elastin and five times more collagen than the rat bladder. To our knowledge, this is the first study to report the structural and molecular mechanisms behind the high compliance of *EC* gular skin. We believe that these findings can lead us to develop more compliant biomaterials for applications in regenerative medicine.

**Keywords:** collagen; elastin; bladder; compliance; microarchitecture; biomimicry
