**1. Introduction**

For millions of years, animals have developed many unique organs for the sole purpose of attracting a suitable mate. These organs take advantage of phenomena such as color [1], scent [2], sound [3], and visual size [4]. Many of these phenomena are exemplified and exploited by many male frogs (*anuran*) to attract a suitable female [5]. Most commonly known is the sound of a frog and the iconic inflation of its gular skin (vocal sac). The characteristic inflation of the gular skin serves as a visual stimulus during mating season and facilitates the energetic mechanical effectiveness of air movement during calls [5]. Investigating the extraordinary inflating action of the gular skin can uncover novel mechanisms behind their ability to stretch and guide the development of artificial compliant biomaterials for regenerative medicine.

Most families in the order *anuran* have inflatable gular skin, such as *Hylidae*, *Eleutherodactylidae*, and *Leptopelis;* however, the size, shape, and color can vary significantly between species [6–8]. Commonly associated with frogs is the single inflated gular skin located under the floor of the gular; however, some species can have two external vocal sacs or no external vocal sac at all [5]. The vocal sac is imperative to the success of the male frog in attracting its female counterpart. *Anuran* vocal sacs play the same role in enhancing the calling effectiveness of the male frog to penetrate their often heavily forested habitats. The calling process begins with the frog filling its lungs with air and subsequently passing it over its vocal cords to produce a call. To reduce the time required to inhale after every call, the vocal sac stores and pushes air back into the lungs, effectively removing the need to inhale [5]. The vocal sac is able to increase the energetic mechanical efficiency of this process because of its elastic property. It stores the energy while the muscles push the air into the sac and release the energy to push the air back into the lungs, much like a helical spring or rubber balloon. The evolution of this elastic organ allows frogs to be very efficient in their calling and produce thousands of calls per night [5]. Doing so enables females to pinpoint the male's location for mating.

In this study, we focused on the male *Eleutherodactylidae Coqui* (*EC*) for its single inflatable gular skin. The highly stretchable nature of the gular skin in frogs is reminiscent of organs in several other species, such as the body of *Lagocephalus Gloveri* [9] and *Diodon Holocanthus* (pufferfish) [10], *Nerodia Sipedon* (snake) gular [11], the gular skin of *Fregata Magnificens* (frigate bird) [12], and the urinary bladder [13]. These organs possess the material phenomena known as elasticity and compliance that allow them to expand to such grea<sup>t</sup> volumes. Compliant materials can stretch easily under low forces, as opposed to stiff materials, which require significant force to deform the material slightly. These mechanical features originate from the composition of structural proteins and their architectures.

Elastin is a protein often associated with higher elasticity and compliance in tissues [14,15]. Several groups have incorporated elastin to develop highly elastic biomaterials, with the caveat that most methods include crosslinkers that are inherently cytotoxic (i.e., glutaraldehyde, hexamethylene diisocyanate) [16]. Other strategies used either peptide materials [17–19] or micro/nanofiber reinforced materials [20,21] to enhance elasticity and compliance; however, they are still unable to match the high extensibility of the urinary bladder. The inability of these materials to recapitulate the biomechanics of the bladder warrants further investigation into other mechanisms of elasticity and compliance, such as tissue ultrastructure. Using scanning electron microscopy (SEM), Murakumo et al. identified a unique helical architecture of collagen type III within the bladder, suggesting a role of collagen architecture in its biomechanics [13]. Although some groups have incorporated micro/nanofibers [20,21] into the material, the fibers are deposited in its fully elongated state and simply act to increase tensile strength instead of compliance. While it is possible to electrospin intertwined nanofibers [22], the authors of the study did not apply their technique in scaffold construction. We believe that the configuration or microarchitecture of collagen in tissues likely plays a significant role. Thus, the characterization of collagen microarchitectures in several compliant tissues in nature could reveal alternative ultrastructures that may be translated into biomaterial design.

Here, we characterize the commonly observed biomechanics and biocomposition of the gular skin, shown in Figure 1A. To fully understand the mechanism behind the highly inflatable gular skin, we performed experiments to characterize the tensile strength, tissue morphology, ultrastructure, and collagen/elastin content. We further compared the male EC gular skin tissue with frogs without a visually inflatable gular skin, namely *XL*, *XM*, female *EC*, and the male rat urinary bladder. In addition, leg tissues (Figure 1B) were dissected from the frogs and underwent the same analyses to identify the key features that allow the *EC*'s gular skin to be functionally unique. We performed TEM, tensile tests, histology, and biochemical assays. We demonstrate that the gular skin of *EC* can achieve comparable elongation to the rat bladder and is more compliant than *Xenopus Laevis* (*XL*) and *Xenopus Muelleri* (*XM*).

**Figure 1.** (**A**) A male *Hyperolius Cinnamomeoventris,* with inflated gular skin tissue to resonate its mating call. Reproduced from [5]. (**B**) Gross visual of tissue dissection areas. Shown here is the Xenopus Laevis specimen.

Furthermore, we identified a unique and more sophisticated collagen ultrastructure in *EC* gular skin, different from the helical structure observed in the urinary bladder. *EC* gular skin has a combination of several structures, such as layering, crimping, and twisting. The significance of these features is further emphasized by the statistically insignificant amount of elastin in most of the test samples. We believe that biomimicry of the collagen microstructure present in the *EC*'s gular skin may provide researchers with an alternative solution to reconstruct a more mechanically significant scaffold for regenerative medicine.
