**3. Discussion**

To understand the fundamental mechanisms behind the iconic inflatable gular tissue seen in select frog species, in the present study, we analyzed multiple frog tissues. Among all tested frog tissues, *EC* tissue displayed lower secant moduli in uniaxial tensile tests, more sophisticated collagen microarchitectures, and higher elastin to collagen ratios. These mechanical characteristics allow the tissue to meet the functional requirements of the gular tissue to move air between the vocal sac and the lungs mechanically. The low UTS (1263 ± 134 kPa) of the male *EC* gular tissue reflects the easily extensible tissue, allowing the frog to inflate its gular skin without excessive force. Interestingly, the gular tissue in all species had a higher UTS when compared to its leg tissue. This is likely indicative of the need for the tissue to withstand repeated stress from fast air movement. Furthermore, gular tissue from male *EC* had the highest average elongation of nearly 400% compared to the other species. In contrast to expectations, the gular tissue of *XM* was able to elongate, on average, 350% ± 9% of its original length, surpassing the female *EC* but not achieving the same elongation of the male *EC*. On the other hand, *XL* elongated substantially less, with only an average of 104% ± 17%. The leg tissues of all species had lower peak stress and strain values compared to their gular tissue counterparts. In all species, leg skin tissue displayed half the peak stress of the gular skin except for male *EC*. The male *EC* gular skin had a UTS of 1263 kPa while the leg skin had 193 kPa. This trend shows that the gular skin is a mechanically more durable material than that of the leg. These UTS values also occurred at smaller strain values than those of the gular skin. Male *EC* gular skins' UTS occurred at 187%, compared to its leg tissue at 83%, as shown in Table 1. The high elongation and moderate UTS of *EC* gular tissue reflect its role as a mechanically dynamic tissue. Its frequent expansion and loading require it to sustain higher stress while still elongating, much like the rat urinary bladder. Gular tissue of the male *EC* and *XM* had comparable elongations to the male rat bladder (~412%). The rat bladder also had UTS averaging ~3000 kPa, higher than tissue from *EC* but lower than *XM* and *XL*. The bladder can withstand higher stresses without compensating for elongation, which is essential to retain urine pressure at higher volumes. We believe that the high variability in the data, as indicated by Table 1, is due to the limited number of EC frogs and inherent biological- and age-related variations.

TEM and histological staining illustrated the relationship between microarchitecture and the mechanical characteristics of the tissues. The typical crimp pattern observed in H&E and trichrome stains were also found in other elastic tissues [9]. Evident in Figure 3 is the lack of large muscle bundles in *EC* tissues. Therefore, the large muscle bundles likely contribute to the tissues' higher UTS. Furthermore, the more crimped structure found in the *EC* suggests a greater elongation when stretched. The nearly linear collagen structure found in *Xenopus* tissues (Figures 3 and 4) has clear linear collagen bundles, which restrict the tissues' ability to elongate. In combination with the stress vs. strain behaviors, the crimped collagen structure is critical to the extensibility of the tissue. However, collagen fibers from the skin of the leg were less compact, which explains the lower stress values and lower strain at failure.

Further investigations with high magnification TEM show a sophisticated collagen microstructure in the male *EC* gular tissue. Firstly, it displayed increased folding of the crimp structure, with two folds of crimp angles of 80◦ and 70◦, compared to the female, with a single fold of crimp angle of 77◦. The compact collagen allows the tissue to stretch with less force and to a higher degree. Secondly, the collagen bundles can be observed to alternate between a circular and longitudinal shape, suggesting an out-of-plane zig-zag structure. This structure likely provides the tissue with greater extensibility because the collagen bundles have additional degrees of freedom to realign in space.

Additionally, each collagen strand twists about its central axis, allowing it to unwind during elongation for higher extension (Figure 5). Lastly, the layering structure throughout the cross-section of the tissue has an alternating round and longitudinal shape, indicating a mesh-like structure. This technique is documented to aid in dissipated energy during loading [23]. Taken altogether, the gular tissue of the male *EC* has a compact three-dimensional structure that allows collagen bundles to extend significantly. Its female counterpart also displayed these features but to a lesser extent. The crimp structure was less compact, indicated by the larger curvature seen in Figure 5B. The unique features found in *EC* were not present in the *Xenopus* frogs but, instead, many locations were identified to be potential areas of stress concentration and elongation inhibition. Figure 5C,D show apparent discontinuities in the overall collagen structure in the *Xenopus* gular tissues. The discontinuities show clear breaks where the collagen aligned parallel to the tissue is impeded by collagen aligned perpendicularly. These areas are a likely cause of the higher secant modulus as they are the site of stress concentration during loading. Architecturally, *EC* tissue is unique because of its continuous hierarchical collagen structure. We believe that the multi-dimensional crimp structure allows for easy elongation of the tissue in all directions, which is key to mate calling. The di fference between male and female gular tissue lies in the compactness of the structure, where a compact crimp allows greater elongation under lower forces. This property is also known as the "crimp angle" and is often used to describe the collagen structure in human tendons [24–26]. As mentioned in previous studies, the crimped collagen structure was determined to act as a recoiling system during muscle relaxation. This structure in the gular tissue likely serves a similar purpose in recoiling the tissue post-inflation and working in tandem with elastin.

The lamina propria in the rat bladder consisted of collagen interspersed between muscle bundles (Figures 3 and 4). This feature is in clear contrast to the frog tissues, where *EC* tissues consisted of no muscle, and *XM*/*XL* had muscle bundles set apart from a collagen layer. Both TEM and histology showed that collagen in the rat bladder was less compact and less regularly structured compared to frog tissues.

As expected, the biochemical analysis showed a higher concentration of elastin in the *EC* tissues than in *Xenopus* tissues (see Figure 7). A high elastin content is one of the major contributing factors to elastic tissues and plays a key role in elongation. Male and female *EC* gular tissue had 58 ± 17 and 64 ± 10 μg/mg of wet tissue, respectively, whereas *XL* and *XM* only had 22 ± 5 and 42 ± 12 μg/mg of wet tissue, respectively. The higher concentration of elastin present in *EC* gular tissue sets it apart, bio-compositionally, from *XL* and *XM*. The biocomposition translates to a higher elongation, evident in the mechanical characteristics shown in Figure 2. Furthermore, male and female *EC* gular tissues have similar elastin concentrations, but the male *EC* has larger elongation. This emphasizes the benefits of the collagen ultrastructure for the mechanics of the tissue.

Although very dissimilar under histology stains, TEM was able to uncover similar collagen ultrastructure with the rat bladder. As shown in Figure 6A,B, the rat bladder and *EC* gular skin share ultrastructural features such as out-of-plane zig-zag and crimp structures. Additionally, *EC* gular skin and rat bladder had similar average elongations, of 398% and 412%, respectively. This clearly shows the importance of the collagen ultrastructure on the elongation of the tissue. Furthermore, rat bladder contained 485 ± 122 μg elastin/mg of wet tissue, eight-fold higher than *EC* gular skin. The significantly higher amount of elastin in the rat bladder did not translate to higher elongation. However, an abundance of elastin quickly recoils collagen bundles during contraction. [13] These results show that this unique collagen ultrastructure or high amounts of elastin can lead to higher elongation of a material. Further investigations are required in order to elucidate the individual impact of high elastin content and various collagen ultrastructures (i.e., crimp or helical) on compliance.

Tensile tests on these tissues show the much higher UTS of the gular tissue compared to that of the leg. This is counter-intuitive because of the gular tissue's lower collagen content; however, the increase in mechanical strength can be attributed to the hierarchical microstructure seen in the male *EC*. Shown in a previous study [27], the microstructure of the material has a significant role in the mechanical behavior of the material. Evidently shown here is that reducing performance compromises is one of the key reasons behind intricate microarchitectures. The layered structure of the collagen provides the tissue with much of its strength [27–30], while the waviness enhances tissue elongation. This hierarchical structure provides a balance between tensile strength and compliance, two mechanical properties that are not often positively correlated. As shown in a previous study [13], elastin is only regionally present in the bladder, and the helical shape of collagen provides much of the bladder's compliance. While collagen type I provides the bladder with its strength, collagen type III and elastin provide its compliance [13,31–35]. The unique conformation of collagen type III and elastin provide recoverable deformation due to hydrophobic interactions. This conformation may not be required to achieve the same biomechanical action, as gular skin did not contain coiled collagen or a high elastin content. The compliance of male *EC* tissue is a result of its tissue-specific collagen ultrastructure, thus reinforcing the importance of material architecture in bulk material properties. The functional similarities between the gular tissue and the bladder prove that the same fundamental mechanisms can be used in biomaterial design for bladder reconstruction.

Taken altogether, we believe that the collagen architecture shown here in EC gular skin tissue can be relevant in tissue engineering of large-deforming compliant tissues. The unique orthogonal arrangemen<sup>t</sup> of collagen layers can also be potentially considered for corneal tissue engineering [36]. Future work will involve recapitulating this structure using 3D bioprinting techniques to develop reinforced hydrogels [37–39]. Additionally, the area of decellularized tissues in tissue engineering has generated considerable attention in recent works [40,41]. Decellularized EC gular tissues have the potential to act as a temporary functional graft, although additional experiments will need to be conducted to validate its potential.
