**3. Fibril formation of BC**

264 Biomaterials – Physics and Chemistry

(Fig. 3a, b). The rate of fibril formation decreased with increasing EDC concentration, which indicates EDC exhibited an inhibitory effect on collagen fibril formation. These inhibitory effects were probably the results of the rapid reaction of EDC to monomeric collagens upon mixing of the EDC containing buffer and acidic collagen solution, by which the ability to form fibrils was reduced or lost through random and nonfibrous aggregation of monomeric collagens. Further increased EDC concentration above 90 mM completely suppressed fibril formation irrespective of NaCl concentrations. Based on these results, it appeared that a buffer that would enable a faster fibril formation rate would be desirable. According to a

previous report, EDC is sufficiently stable and active under such conditions [35].

Fig. 4. Degree of crosslinking in the collagen. Values are mean ± SD (n=4).

concentrations of 70 mM and 100 mM, respectively.

The degree of crosslinking was determined as the decrease in the free amino group content of the collagen molecules [19, 26]. The free amino group content was measured spectrophotometrically after the reaction of the free amino groups with 2,4,6-trinitrobenzensulfonic acid and was expressed as the decrease in the ratio of the free amino group content of the crosslinked sample to that of the uncrosslinked sample. An increase in the degree of crosslinking with increase in the EDC concentration was observed (Fig. 4). The degree of crosslinking was slight lower at NaCl concentration of 100 mM compared to the NaCl concentration of 50 mM. This may be attributed to lower ability of fibril formation observed at NaCl concentration of 50 mM. Fig 2 shows that the plateau level in turbidity at 50 mM NaCl was lower than that at 100 mM NaCl. Lower fibril formation may result in an increase of nonfibrous aggregates, which can lead to high degree of crosslinking due to increased sites of crosslinking in monomeric collagens. The synergistic effects of crosslinking and fibril formation were thought to be complete at EDC and NaCl To produce an elastic material from bio-inspired crosslinked BC gel (e-BC gel), heat denaturation process is needed. By heat treatment, the cross-linked collagen fibrils shrink, maintaining the cross-linkage among the collagen molecules and fibrils through the denaturation of triple-helical collagen molecules to the random-coil form [24]. At the same time, uncross-linked collagen molecules and fibrils may be lost through dissolution to water. In fact, the original BC gels crosslinked with the EDC concentrations of 30-70 mM showed drastic shrinkage (Fig. 5) and rubber-like elasticity after heat treatment at 60ºC for 5 min. The BC gels crosslinked with EDC concentrations of 0-10 mM dissolved away after heat treatment due to incomplete crosslinking.

Fig. 5. Appearances of BC gels (a, b) and e-BC gels (c, d). The values in the graphs indicate the final EDC concentrations (mM) in the gels.

Mechanical and Biological Properties of

widely various collagen biomaterials.

**5. Biological properties of e-BC gel** 

rubber-like stretchability.

Bio-Inspired Nano-Fibrous Elastic Materials from Collagen 267

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

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

To investigate the potential of the e-BC gel for use as a cell culture scaffold, we measured the growth rates of human umbilical vein endothelial cells (HUVECs) cultured on the e-BC gel.

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 both surface structures.

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