1.1. Background
1.1.1 Collagen Basics
The most abundant protein in mammals, collagen, exists within the extracellular matrix (ECM) providing structure and support to connective tissue. Collagen plays an important role in the formation of tissue and organs (Abraham et al., 2007). It can be found in tendons, ligaments, skin, bone, teeth, cartilage, blood vessels, nerves and organ capsules. Collagen is surface active, biodegradable and excellently biocompatible, even when retrieved from animal resources, making it a candidate material for various biomedical applications (Lee, 2001). Collagen accounts for approximately 20 - 30% of the total protein in a human body (Harkness, 1961; Shoulders, 2009). Currently, over 28 distinct collagen types have been discovered, with various properties, and from various body regions (Shoulders, 2009; Gelse, 2003; Kühn, 1986; Sherman, 2015). Complexity and structural diversity, splice variants, presence of non-helical domains, assembly and function are factors considered in collagen type classification (Gelse, 2003). The general groups of collagen include: fibrillar collagens, FACIT (Fibril Associated Collagens with Interrupted Triple Helices) and FACIT-like collagens, beaded filament collagens, basement membrane collagens, short chain collagens, transmembrane collagens, and others with unique functions (Gelse, 2003; Sherman, 2015). The most common group in the human body, making up nearly 90% of the total collagen, is the fibrillar collagens, consisting of Types I, II, III, V and XI (Gelse, 2003; Hulmes, 2008; Ottani, 2002). The most common form in mammal tissue is type I (Hulmes, 2008). Collagens, type I in particular, are most commonly used in research due to their ability to form highly oriented, structural hierarchies of fibrils and their prevalence in human tissue (Gelse, 2003).
This article focuses on type I collagen only. In an evaluation and rating free fashion it reviews the diverse methods demonstrated to fabricate artificial type I oriented collagen in 1D, 2D and 3D architectures, mimicking natural structures and the achieved orientation, as well as orientation distribution and/or order. In the following type I collagen will often be referred to as just collagen.
1.1.2. Collagen Hierarchy
Type I collagen molecules consist of two α1 chains and one α2 chain that form a right-handed triple helix around an axis (Figure1a and b) (Gelse, 2003, Sherman, 2015; Ottani, 2002; Kadler, 1996; Chattopadhyay, 2014). These three polypeptide chains consist of nearly one thousand residues whereby glycine occurs as every third residue (Gelse, 2003; Ottani, 2002; Kadler, 1996; Chattopadhyay, 2014). The “collagenous” structure of amino acids is commonly expressed as a repeating triplet of Gly-X-Y, where X and Y can be any amino acid; however, it has been reported that the most frequently occurring amino acids in the triplet are proline and hydroxyproline (Kadler, 1996; Chattopadhyay, 2014; Ramshaw, 1998). At the beginning of the in-vivo synthesis (Figure1b), the protein exists as procollagen, with a triple helix and two globular terminals. The terminals are cleaved by specific proteases, which leave a triple helix with some terminal non-helical portions (Figure1 c and d). The assembly is now called tropocollagen (molecule). It has a diameter of ~1.6 nm and a length of ~300 nm, and can be viewed as a nanoscopic rod. It can undergo spontaneous fibrillogenesis (Ottani, 2002; Olsen, 1963) via an entropy-driven self-assembly process of the tropocollagen into fibrils (Ottani, 2002; Kadler, 1996).
The type I collagen assembly to form fibrillar collagen is unique because it possesses a staggered, quasi-hexagonal arrangement (Vuorio, 1990). The tropocollagen, staggered by multiples of 68 nm, is called a D-period (Vuorio, 1990). Within one D-period, there are five molecules in cross-section: each molecule staggered by one D-period (Figure1e) (Sherman, 2015; Ottani, 2002; Olsen, 1963). Within each D-period, there is a gap of ~36 nm, and an overlapping region of 31 nm between adjacent molecules (Figure1e) (Vuorio, 1990). Following the self-assembly into the D-period, the tropocollagen becomes cross-linked in-vivo by an enzyme. This enzyme undergoes a reaction with the amine side chains of lysine and hydroxylysine and converts the residues into aldehydes (Smith, 1968; Pinnell, 1968; Kruger, 2013). After a hydration reaction the two collagen molecules are cross-linked via peptide bonds. These collagen micro-fibrils further self-assemble into fibrils, and finally into larger macroscopic units called fibers (Figure1f).
Figure 1. Type I collagen consists of a) two identical α1(I) and one α2(I) peptide chains; b) self-assembly to form procollagen; c) in vivo procollagen peptidase removes the loose termini creating, d) type I tropocollagen. Tropocollagen undergoes a second self-assembly process, e) forming collagen fibrils with a D-period, and f) by yet another self-assembly process, collagen fibrils form a collagen fiber (Kruger, 2013). This work is licensed under Creative Commons Attribution 3.0 Unported).
1.1.3. Mechanical Properties
The packing of fibrils in a fiber may vary from one tissue to another and influences the mechanical properties. The internal structure of the collagen fibrils and fibers is highly ordered, thus has highly anisotropic physical properties. One distinct property set is the mechanical characteristics, which are used by nature in connective tissue. Numerous studies on mechanical properties of collagen have been performed: both experimentally and theoretically (Fratzl, 2008). Collagen exhibits strength and elasticity that vary with respect to orientation when a constant force is applied. The Young’s modulus for collagen fibrils has been measured along the fibrils and values from 0.2 MPa to 12 GPa were found depending on the source of the fibrils (e.g. rat tail, tendon, fish scales, bone, animal, human, etc), their dehydration state, the degree of cross-linking and their multiple hierarchical levels (molecular, fibril, fiber, tissue) (Sherman, 2015; Wenger, 2007; Hamed, 2010). The Young’s modulus in the direction perpendicular to the fibrils was not measured satisfactorily yet, due to experimental uncertainties and the interference between the mechanical measurement tools as well as the internal structure of the fibrils (Sherman, 2015; Annaidh, 2012). Indirect measurements have been performed with tissue, e.g. with human skin, selecting samples oriented perpendicular and parallel to the Langer lines (Annaidh, 2012). Langer lines correspond to the natural orientation of collagen fibers in the dermis. A difference in the elastic moduli of the two directions of roughly 50% (a relatively large error) was found (Annaidh, 2012).
All collagen-based tissues exhibit non-linear visco-elastic properties, reflected by a “J”-shaped stress - strain curve: collagen exhibits greater compliance at low stresses than at higher stresses. This enhances the energy absorption capacity at low-stress levels. A greater compliance at the onset of loading in combination with some viscous damping are the main properties of collagen reducing the susceptibility to damage (Fratzl, 2008).