**4. Mechanical properties of e-BC gel**

The mechanical properties of the e-BC gel were evaluated by tensile tests. The original BC gel rarely had elasticity and stretchability similar to the usual collagen materials. However, the e-BC gel showed rubberlike elasticity and high stretchability. Figure 7 shows the stress– strain curves to the breaking point obtained in the strain rate of 0.1 mm/s (n = 5). Salmonderived elastic collagen gels (e-SC gel) were used as controls [24]. The mean values ± standard deviation (SD) of elongation at the break of the e-BC gel and e-SC gel were 201 ± 47% and 260 ± 59%, respectively (Fig. 7c). At the early stage of loading, stress was almost linearly increased depending on the strain. Above a strain of ca. 100%, an increase in strain hardening was observed. There was no significant difference in elongation between the two e-gels. According to the report by Koide and Daito [31], collagen films reinforced by traditional cross-linking reagents, glutaraldehyde and tannic acid, showed only small elongation at the breaking point (6.6% and 12.4%, respectively). Weadock et al. showed small ultimate strains (approximately 40% and 30%) of collagen fibers cross-linked by UV irradiation and dehydrothermal treatment, respectively [36]. Even a purified skin with an intact fibrous collagen network gives elongation at a breaking point of 125% [36]. Recently, it was reported that a chemically cross-linked collagen-elastin-glycosaminoglycan scaffold, which are the contents analogous to the actual tissue/organs, demonstrated good stretchability (150% strain) [37]. To the best of our knowledge, this is the first report of a material from bovine collagen with elongation at a breaking point over 200%. Although the mechanism of the high stretchability was not well understood, the denaturation of the

The collagen fibrils were observed by high-resolution scanning electron microscopy (SEM). The preparation of the specimen was performed according to a previous report [16, 24]. Figure 6 shows the well-developed networks of nano-fibrils on the BC gel and e-BC gel. The width of the fibrils on the BC-gel was in the range of 50–100 nm (Fig. 6a). However, a wider (width; >200 nm) and winding fibril-like structure was observed on the e-BC gel (Fig. 6b), indicating that the fibril structure of collagen was deformed through the heat treatment. The wide and winding fibril-like structure of the e-BC gel should be directly derived from the collagen nano-fibrils of the original BC gel through swelling of the fibrils by comparison of

The mechanical properties of the e-BC gel were evaluated by tensile tests. The original BC gel rarely had elasticity and stretchability similar to the usual collagen materials. However, the e-BC gel showed rubberlike elasticity and high stretchability. Figure 7 shows the stress– strain curves to the breaking point obtained in the strain rate of 0.1 mm/s (n = 5). Salmonderived elastic collagen gels (e-SC gel) were used as controls [24]. The mean values ± standard deviation (SD) of elongation at the break of the e-BC gel and e-SC gel were 201 ± 47% and 260 ± 59%, respectively (Fig. 7c). At the early stage of loading, stress was almost linearly increased depending on the strain. Above a strain of ca. 100%, an increase in strain hardening was observed. There was no significant difference in elongation between the two e-gels. According to the report by Koide and Daito [31], collagen films reinforced by traditional cross-linking reagents, glutaraldehyde and tannic acid, showed only small elongation at the breaking point (6.6% and 12.4%, respectively). Weadock et al. showed small ultimate strains (approximately 40% and 30%) of collagen fibers cross-linked by UV irradiation and dehydrothermal treatment, respectively [36]. Even a purified skin with an intact fibrous collagen network gives elongation at a breaking point of 125% [36]. Recently, it was reported that a chemically cross-linked collagen-elastin-glycosaminoglycan scaffold, which are the contents analogous to the actual tissue/organs, demonstrated good stretchability (150% strain) [37]. To the best of our knowledge, this is the first report of a material from bovine collagen with elongation at a breaking point over 200%. Although the mechanism of the high stretchability was not well understood, the denaturation of the

both surface structures.

Fig. 6. SEM images of BC gels (a) and e-BC gels (c). Bars: 1 μm.

**4. Mechanical properties of e-BC gel** 

collagen molecules probably plays an important role in the elongation, i.e., the bend structure of denatured collagen fibrils observed in Fig. 6b is considered to provide its rubber-like stretchability.

Fig. 7. Stress–strain curves generated from tensile testing of e-BC gels (a) and e-SC gels (b). The specimen (5 × 3 × 12 mm) was gripped to achieve a gauge length of 8 mm and stretched in the strain rate of 1.25%/s. (c) Elongation at the break of the e-BC gel and e-SC gel. (d) Ultimate strength at the break of the e-BC gel and e-SC gel. Values are mean ± SD (n=5).

The mean values ± SD of ultimate strength at the break of the e-BC gel and e-SC gel were 4.1 ± 2.6 kPa and 9.0 ± 4.8 kPa, respectively (Fig. 7d). The strength of the e-BC gels was lower than that of e-SC gels, although there was no significant difference between the two. Collagen exhibits a limited mechanical resistance so that collagen requires an additional skeletal material such as inorganic materials [38]. Bio-inspired crosslinking can provide a collagen scaffold with high mechanical strength as well as stretchability. It is useful without the addition of any other material. Because a collagen solution is a precursor for fabrication of various collagen forms, bio-inspired crosslinking would be a useful fabrication process of widely various collagen biomaterials.
