**2. The bio-inspired crosslinking conditions for BC**

Acid-soluble collagen molecules self-assemble and form fibrils under physiological conditions. The pH, NaCl concentraton, and temperature are important factors to provide a successful reconstituted collagen fibrillar gel. First, we evaluated the effect of NaCl concentrations on fibril formation of BC at constant pH of 7.4 and temperature of 37ºC. The fibril formation of BC was monitored by a turbidity change observed at 310 nm [26, 27]. Figure 2 shows that a rapid rise in turbidity was observed in the mixture of BC solution and 30 mM Na-phosphate buffer at NaCl concentration from 50mM to 100 mM. Then, the rise in turbidity increase was gradually decreased at over 140 mM NaCl. The fibril formation rate of collagen is known to be reduced by addition of salts [4], which appears to reduce electrostatic interaction among collagen molecules. The bio-inspired crosslinking needs active fibrillogenesis during crosslinking (see below) [17, 26]. Therefore, the optimum range of NaCl concentration for BC fibril formation was determined to be 50-100 mM.

Cross-linking generally reinforces the biomaterials composed of collagen fibrils for further improvement of mechanical properties. Various techniques for stabilizing collagen have been developed and reported. These techniques are divided into chemical treatments and physical treatments. Glutaraldehyde is one of the most widely used chemical agents [28, 29]; it is known, however, that there are side effects to its use in cross-linking [30], for example, cytotoxicity, enhancement of calcification, and a mild inflammatory response compared with using other reagents. The water soluble condensign agent 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) has been reported to be significantly less cytotoxic than glutaraldehyde because EDC reagents do not remain in the linkage and are simply washed away during the cross-linking process [28, 29]. On the other hand, physical cross-linking methods such as UV irradiation [31, 32] and dehydrothermal treatment [33, 34] do not introduce any additional chemical units. These methods may therefore be more biocompatible than chemical treatments. However, the mechanical properties of materials cross-linked by physical treatments are lower than those cross-linked by chemical treatments. Therefore, EDC was used for a crosslinking agent in this study.

Mechanical and Biological Properties of

the gels.

Bio-Inspired Nano-Fibrous Elastic Materials from Collagen 263

Fig. 3. Effect of EDC concentrations on fibril formation curve in the presence of 50 mM NaCl (a) and 100 mM NaCl (b). The values in the graphs indicate the final EDC concentrations in

The turbidity changes in BC solution mixed with Na-phosphate buffer including various EDC concentration at 50 mM or 100 mM NaCl were monitored using a spectrophotometer

Fig. 2. Effect of NaCl concentration on fibril formation curve. The values in the graphs indicate the final NaCl concentrations in the gels.

Fig. 2. Effect of NaCl concentration on fibril formation curve. The values in the graphs

indicate the final NaCl concentrations in the gels.

Fig. 3. Effect of EDC concentrations on fibril formation curve in the presence of 50 mM NaCl (a) and 100 mM NaCl (b). The values in the graphs indicate the final EDC concentrations in the gels.

The turbidity changes in BC solution mixed with Na-phosphate buffer including various EDC concentration at 50 mM or 100 mM NaCl were monitored using a spectrophotometer

Mechanical and Biological Properties of

treatment due to incomplete crosslinking.

**3. Fibril formation of BC** 

Bio-Inspired Nano-Fibrous Elastic Materials from Collagen 265

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

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.

(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).

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 concentrations of 70 mM and 100 mM, respectively.
