Biomedical Engineering Reference
In-Depth Information
crosslinking as well as interfibrillar one, allowing for homogenous crosslinking sites and
larger mechanical properties. Therefore, the bio-inspired crosslinking may provide more
wide range of applications of collagen biomaterials. However, the bio-inspired crosslinking
has not yet been applied to bovine collagen (BC), which is widely used for medical
applications for past decades, because the bio-inspired crosslinking has been developed for
thermal stabilization of “fish collagens” with low-denaturation temperature to develop
marine-derived collagen biomaterials [13-17, 24-26]. In this study, we studied the
crosslinking condition to create bio-inspired crosslinked BC gel and prepared the elastic
material from the BC gel (e-BC gel) by heat treatment. The mechanical properties (tensile
strength and elongation rate) and biological properties (biodegradability, cell culture, and
blood compatibility) of the e-BC gel were evaluated. Herein, we report the fabrication of the
bio-inspired elastic material from BC and demonstrate its applicability for biomaterials,
especially in vascular tissue engineering.
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.
