**Mechanical and Biological Properties of Bio-Inspired Nano-Fibrous Elastic Materials from Collagen**

Nobuhiro Nagai1,2, Ryosuke Kubota2, Ryohei Okahashi2 and Masanobu Munekata2 *1Division of Clinical Cell Therapy, Center for Advanced Medical Research and Development, ART, Tohoku University, Graduate School of Medicine 2Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University Japan* 

### **1. Introduction**

258 Biomaterials – Physics and Chemistry

Flammang, P.; Ribesse, J. & Jangoux, M. (2002) Biomechanics of adhesion in sea cucumber

Hamel, J.-F. & Mercier, A. (2000). Cuvierian tubules in tropical holothurians; usefulness and

Hwang, D.S.; Zeng, H.; Masic, A.; Harrington, M.J.; Israelachvili, J.N. & Waite, J.H. (2010).

Jones, I.; Lindberg, C.; Jakobsson, S.; Hellqvist, A.; Hellman, U.; Borg, B. & Olsson, P.E.

Kamino, K. (2008). Underwater adhesive of marine organisms as the vital link between

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. *Nature,* Vol.227, No.5259, (August), pp. 680-685, ISSN 0028-0836 Lawrence, J.M. (2001). Function of eponymous structures in echinoderms; a review. *Canadian Journal of Zoology,* Vol.79, No.7, (July), pp. 1251-1264, ISSN 0008-4301 Lin, Q.; Gourdon, D.; Sun, C.; Holten-Andersen, N.; Anderson, T.H.; Waite, J.H. &

Mancuso-Nichols, C.A.; Nairn, K.M.; Glattauer, V.; Blackburn, S.I.; Ramshaw, J.A.M. &

Müller, W.E.G.; Zahn, R.K. & Schmid, K. (1972). The adhesive behaviour in Cuvierian

Nakano, M.; Shen, J.R. & Kamino, K. (2007). Self-assembling peptide inspired by a barnacle

VandenSpiegel, D. & Jangoux, M. (1987). Cuvierian tubules of the holothuroid *Holothuria* 

VandenSpiegel, D.; Jangoux, M. & Flammang, P. (2000). Maintaining the line of defence:

Watson, M.R. & Silvester, N.R. (1959). Studies of invertebrate collagen preparations. *Biochemical Journal,* Vol.71, No.3, (March), pp. 578-584, ISSN 0264-6021 Zahn, R.K.; Müller, W.E.G. & Michaelis, M. (1973). Sticking mechanisms in adhesive organs from a *Holothuria. Research in Molecular Biology,* Vol.2, pp. 47-88, ISSN 0340-5400

*Chemistry,* Vol.276, No.21, (May), pp. 17857-17863, ISSN 0021-9258

*America,* Vol.104, No.10, (March), pp. 3782-3786, ISSN 0027-8424

*Cytobiology,* Vol.5, No.3, pp. 335-351, ISSN 0070-2463

*Biology,* Vol.42, No.6, (December), pp. 1107-1115, ISSN 1540-7063

Vol.33, pp. 115-139, ISSN 1023-6244

25850-25858, ISSN 0021-9258

pp. 111-121, ISSN 1436-2228

pp. 97-125, ISSN 0021-8464

pp. 263-275, ISSN 0025-3162

34-49, ISSN 0006-3185

ISSN 1525-7797

Cuvierian tubules (Echinodermata, Holothuroidea). *Integrative and Comparative* 

efficiency as a defence mechanism. *Marine and Freshwater Behaviour and Physiology,* 

Protein- and metal-dependent interactions of a prominent protein in mussel adhesive plaques. *Journal of Biological Chemistry,* Vol.285, No.33, (August), pp.

(2001). Molecular cloning and characterization of spiggin. *Journal of Biological* 

biological science and material science. *Marine Biotechnology,* Vol.10. No.2, (March),

Israelachvili, J.N. (2007). Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3. *Proceeding of the National Academy of Sciences of the United States of* 

Graham, L.D. (2009) Screening microalgal cultures in search of microbial exopolysaccharides with potential as adhesives *Journal of Adhesion,* Vol.85, No.2-3,

tubules of *Holothuria forskåli.* Biochemical and biophysical investigations.

underwater adhesive protein. *Biomacromolecules,* Vol.8, No.6, (June), pp. 1830-1835,

*forskali* (Echinodermata): a morphofunctional study. *Marine Biology,* Vol.96, No.2,

regeneration of Cuvierian tubules in the sea cucumber *Holothuria forskali* (Echinodermata, Holothuroidea). *Biological Bulletin,* Vol.198, No.1, (February), pp. Collagen-based biomaterials have been widely used in medical applications, because of its many advantages, including low antigenicity, abundant availability, biodegradability, and biocompatibility [1]. Collagen represents the major structural protein, accounting for nearly 30% of all vertebrate body protein. The collagen molecule comprises three polypeptide chains (α-chains) which form a unique triple-helical structure (Fig. 1) [2]. Each of the three chains has the repeating structure glycine–X–Y, where X and Y are frequently the imino acids proline and hydroxyproline (Fig. 1b). The collagen molecules self-aggregate through fibrillogenesis into microfibrils forming extracellular matrix (ECM) in the body [3-5]. The fibrils provide the major biomechanical matrix for cell growth (Fig. 1a), allowing the shape of tissues to be defined and maintained. The main application of collagen for biomaterials is as a scaffold for tissue engineering and a carrier for drug delivery [2, 6-9]. Many different forms of collagen biomaterials, such as film [10, 11], gel [12-17], sponge [18-20], micro- /nano-particle [21, 22], and fiber [23], have been fabricated and used in practice. However, most collagen biomaterials become brittle and fail under quite low strains, which limit their application to biomedical engineering fields that need larger mechanical properties, especially elasticity [16].

Recently, we reported a novel crosslinking method of improving the mechanical properties and thermal stability of collagen [24]. The method mimics actual biological events to form collagen matrix in the body; monomeric collagens extruded from cells into extracellular environment initially form microfibrillar aggregates, then lysyl oxidase crosslinking during their assembly to form fibrils (Fig. 1). The *in vitro* crosslinking during collagen fibrillogenesis, namely "bio-inspired crosslinking", creates a crosslinked collagen fibrillar gel with high mechanical properties at certain crosslinking agent concentrations [25, 26]. Fibril formation involves the aggregation and alignment of collagen molecules, and helps increase the collagen's thermal stability. The introduction of crosslinking during fibril formation further increases the thermal stability of collagen. The synergistic effects of crosslinking and fibril formation are found to enable an increase in the thermal stability of

Mechanical and Biological Properties of

especially in vascular tissue engineering.

determined to be 50-100 mM.

agent in this study.

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

Bio-Inspired Nano-Fibrous Elastic Materials from Collagen 261

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,

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

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

Fig. 1. Schematic representation of collagen synthesis process. Procollagen consists of a 300 nm long triple helical domain (comprised of three alpha-chains each of approximately 1000 residues) flanked by a trimeric globular C-propeptide domain and a trimeric N-propeptide domain. Procollagen is secreted from cells and is converted into collagen by the removal of the N- and C-propeptides by proteases. The collagen spontaneously self-assembles into cross-striated fibrils that occur in the extracellular matrix of connective tissues. The fibrils are stabilized by covalent crosslinking, which is initiated by oxidative deamination of specific lysine and hydroxylysine residues in collagen by lysyl oxidase. (a) Scanning electron microscopy image of a human fibroblast adhered on reconstituted salmon collagen fibrillar matrix [17]. (b) Chemical structure of a collagen alpha-chain. (c) Atomic force microscopy image of reconstituted salmon collagen fibril [17]. The repeating broad dark and light zones (where D=67 nm, the characteristic axial periodicity of collagen) can be seen in the fibril.

salmon-derived collagen (SC) [24]. Additionally, heat denaturation of the bio-inspired crosslinked SC gel provides elastic materials, which has the elongation of over 200% at break point [24]. So far, the elasticity of the collagens that had been crosslinked "after" fibrillogenesis is not as high as that crosslinked "during" fibrillogenesis (bio-inspired crosslinking) [25]. This may indicate that the bio-inspired crosslinking confer intrafibrillar

Fig. 1. Schematic representation of collagen synthesis process. Procollagen consists of a 300 nm long triple helical domain (comprised of three alpha-chains each of approximately 1000 residues) flanked by a trimeric globular C-propeptide domain and a trimeric N-propeptide domain. Procollagen is secreted from cells and is converted into collagen by the removal of the N- and C-propeptides by proteases. The collagen spontaneously self-assembles into cross-striated fibrils that occur in the extracellular matrix of connective tissues. The fibrils are stabilized by covalent crosslinking, which is initiated by oxidative deamination of specific lysine and hydroxylysine residues in collagen by lysyl oxidase. (a) Scanning electron microscopy image of a human fibroblast adhered on reconstituted salmon collagen fibrillar matrix [17]. (b) Chemical structure of a collagen alpha-chain. (c) Atomic force microscopy image of reconstituted salmon collagen fibril [17]. The repeating broad dark and light zones (where D=67 nm, the characteristic axial periodicity of collagen) can be seen in the fibril.

salmon-derived collagen (SC) [24]. Additionally, heat denaturation of the bio-inspired crosslinked SC gel provides elastic materials, which has the elongation of over 200% at break point [24]. So far, the elasticity of the collagens that had been crosslinked "after" fibrillogenesis is not as high as that crosslinked "during" fibrillogenesis (bio-inspired crosslinking) [25]. This may indicate that the bio-inspired crosslinking confer intrafibrillar 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.
