ReviewCollagen for bone tissue regeneration
Introduction
In mammals, collagen is the most abundant protein, constituting more than one-third by weight of body protein tissue [1]. Around 28 types of collagen [2] have so far been identified and, among these, type I collagen is the most prevalent type found in the extracellular matrix (ECM), especially in tissues such as tendon and bone [2], [3]. The ECM plays an important role in the morphogenesis and cellular metabolism of new tissues, conferring mechanical and biochemical properties [2]. Collagen has potential as a biomaterial for bone tissue engineering due to its abundance, biocompatibility, high porosity, facility for combination with other materials, easy processing, hydrophilicity, low antigenicity, absorbability in the body, etc. [4], [5].
Collagen protein has a complex hierarchical conformation divided in four structures: primary structure (amino acid triplet), secondary structure (the α-helix), tertiary structure (triple helix) and quaternary structure (fibrils) [2].
Collagen protein is recognized by the characteristic domain of proline-rich Gly-X-Y polypeptide (Fig. 1) with two unique features: (i) Gly is found every third residue with the strict repeating –(Gly-X-Y-)n– tripeptide sequence along the entire length of the ∼1000 amino acid chain. However, a single substitution of a Gly with an Ala residue has been found in the crystal structure of a triple-helical molecule after 10 repeating Pro-Hyp-Gly units [6]. (ii) A high proportion of residues (∼20%) in the tripeptide sequences is frequently comprised of proline (X) and hydroxyproline (Y). Hydroxyproline is not commonly found in other proteins, while in collagen it constitutes more than 50% of the total amino acid content [7], [8].
The α-chains are formed by repetitions of the tripeptide –(Gly-X-Y-)n– and are linked to each other, building the characteristic triple helix of type I, II and III collagen [9]. The non-helical domains are at the end of the α-chains, where the C-terminus is involved in the initiation of triple-helix formation and the N-terminus is involved in the regulation of primary fibril diameters. The short non-helical telopeptides of collagen are linked by covalent cross-links which form between the collagen molecules and/or between collagen and other molecules present in the ECM [2], [10].
The triple helix, especially in collagen type I, is usually formed as a heterotrimer of two identical α1(I)- and α1(II)-chains and one α2(I)-chain with about 1000 amino acids, and is approximately 300 nm in length (L) and 1.5 nm in diameter [9], [11]. The three α-chains form a left-handed, rod-like helix, where the glycine residues are located around a central axis, while larger amino acids belonging to the X and Y residues (usually proline and hydroxyproline) occupy outer positions [9] (Fig. 1). The α-chains are linked to each other by hydrogen bonds through the single interstrand N–H(Gly)…O = C(X) as well as Cα–H(Gly/Y)…O = C(X/Gly), which are the major stabilizing interactions of the α-triple helical and β-sheet protein structures [8], [12], [13]. Some studies of collagen molecule assembling have hypothesized that the C-terminal (COOH-terminal propeptide) globular domains of the α2(I)-chain in the collagen type I play a crucial role in the initiation of the intermolecular assembly, chain association and stable collagen heterotrimer formation [14], [15], [16].
Collagen molecules are able to self-assemble into a supramolecular form via a quarter-stagger package pattern of five triple-helical collagen molecules highly oriented with D-periodic banding spaces, where D is ∼67 nm (Fig. 2) [11], [17]. The telopeptides, composed of non-helical regions about 20 amino acid residues in length, play an important role in the fibrillogenesis, contributing to the stabilization of the mature collagen molecules by cross-link formation [18]. In fact, collagen cross-links are divided into two types: enzymatic cross-links, mediated by lysine hydroxylase and lysyl oxidase; and non-enzymatic cross-links, commonly called glycation or oxidation induced Advanced glycation end products (AGE) cross-links [19]. Fig. 2 shows an example of enzymatic cross-linking mediated by lysyl oxidase. The two chemical forms of 3-hydroxypyridinum cross-linking, namely hydroxyl lysyl pyridinoline (HP) and lysyl pyridoline (LP) cross-links, are formed between the amine side group present in the lysine and hydroxy lysine residues in collagen telopeptides, which are converted into aldehydes by the lysyl oxidase enzyme, and the specific active binding sites present in neighboring triple helices [10], [11].
Various non-collagen proteins and bound water fill the space between cells and fibers of the connective tissue defining the features of the tissue. These macromolecules can be grouped into two main classes: glycosaminoglycans (GAGs) and glycoproteins [20]. Proteoglycans are complex molecules that resemble the shape of a brush used to clean test tubes and comprise around 80 GAG chains bound covalently (with the exception of hyaluronic acid) to the central core of a protein. A large number of anionic charges, such as carboxyl and sulfate groups, are present in the GAGs and interact electrostatically with water molecules, regulating the hydration of the connective tissue, and with ECM proteins, such as collagen, forming an interlocked supramolecular matrix [20], [21].
Historically, the industrial uses of collagen in the form of leather and gelatin are widespread, including photographic gelatin, cosmetics, food and pharmaceutical applications, enzyme production, etc. [22]. Collagen, as a fibrous protein, is the principal component of connective tissues in mammals. The fibrillar collagens are insoluble in their native structure but can be solubilized in aqueous solution if they are denatured to soluble procollagens [23]. The denaturation of collagen is an irreversible kinetic process [24] and it may be obtained by thermal treatment: once the helix–coil transition temperature (e.g. ∼37 °C for bovine collagen) is exceeded, collagen is converted into a randomly coiled gelatin [25]. Other methods to produce gelatin include acid or alkaline chemical treatments [22].
For the past decade, collagen has been among the most widely used biomaterials for biomedical applications, due to its excellent biological features and physicochemical properties [26]. Collagen may be easily modified by reaction of its functional groups, introducing cross-links or grafting biological molecules to create a wide variety of materials with tailored mechanical or biological properties [5], [27], [28]. The main drawbacks of collagen include the high costs of manufacturing (due to the time-consuming and complex procedures required for isolation and purification), careful selection of processing conditions to avoid denaturation, and high swelling in vivo, due to collagen hydrophilicity [21], [22].
In recent years, demand for the development of innovative products aimed at the replacement, correction and improvement of poorly functioning tissues in humans or animals has increased. Collagen can be easily modified into different physical forms such as powder/particles, fibers/tubing, gel/solution, films/membranes, sponges, blends (with other polymers) and composites (with ceramics). Collagen has found a wide variety of applications in the field of medicine including: sutures, hemostatic agents, tissue replacement and regeneration (bone, cartilage, skin, blood vessels, trachea, esophagus, etc.), cosmetic surgery (lips, skin), dental composites, skin regeneration templates, membrane oxygenators, contraceptives (barrier method), biodegradable matrices, protective wrapping of nerves, implants, corneal bandage, contact lens, drug delivery, etc. [22], [25], [28].
In particular, among the various collagen types, type I collagen is the most abundant component of the ECM and may be used as scaffolding material, promoting cell migration, wound healing and tissue regeneration. As the bone ECM is very rich in type I collagen, it has found important applications in bone tissue engineering where a collagen-based scaffold provides the innate biological information required for cell adhesion, proliferation and orientation, and promotes the chemostatic response [29].
Section snippets
Bone tissue engineering
In the human body, bone belongs to a family of tissues with a complex structure organized hierarchically. Bone is composed of calcium phosphate (69–80 wt.%, mainly hydroxyapatite), collagen (17–20 wt.%) and other components (water, proteins, etc.) [30]. Natural bones are a complex assembly of parallel type I collagen nanofibrils and HA crystals precipitated on their surface [31].
Two types of cells play an important role in the formation of bone: osteoblasts (bone-forming) and osteoclasts
Conclusions and future remarks
Collagen is a fibrous protein comprising the natural ECM of tissues, from which it can be extracted by a variety of techniques. A proteolytic treatment of animal tissues in acidic environment (e.g. using pepsin) is the most widely used collagen extraction procedure: it cleaves collagen cross-links as well as telopeptides, making collagen non-immunogenic. As native collagen contains amino acidic sequences (GFOGER, RGD, etc.) for cell bio-recognition, it has been widely used as a material for the
Acknowledgments
The authors acknowledge support for this work provided by CRT-Progetto Lagrange for A.M.F.’s grant.
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