Self-assembly of mineralized collagen composites

Self-assembly of mineralized collagen composites

(Parte 1 de 8)

Self-assembly of mineralized collagen composites

Fu-Zhai Cui*, Yan Li, Jun Ge

Advanced Materials Laboratory, Department of Materials Science & Engineering, Tsinghua University, Beijing 100084, China


This paper presents a review of the current understanding of the structure, self-assembly mechanisms, and properties of mineralized collagen fibril composites in connective tissues, such as in lamellar bones, woven bones, zebrafish skeletal bone, and ivory. Recent work involving biomimetic synthesis of new materials with the structure of mineralized collagen is described. The focus in the paper is mainly on materials containing type I collagen, with mineralization by Ca–P crystals although some other systems are also described. Investigation and simulation of naturally occurring fibril structures can offer some new ideas in the design and fabrication of new functional materials, for applications such as bone grafts or for use as scaffolds in tissue engineering and biomimetic engineering materials. The development of bone grafts based on the mineralization of selfassembled collagen fibrils in vivo and in vitro is an active area of research. This kind of bone graft composite has already shown great promise and success in clinical applications, on account of its compositional and structural similarity to autologous bone. It is suggested that future work in this should focus on both basic theoretical aspects as well as the development of applications. In particular issues including control of morphology, incorporation of foreign ions, interaction with biomolecules, and the assembly of organic and inorganic phases are all still not well understood. In the area of applications, the design of composite materials with a hierarchical structure closer to that of natural hard tissues, and the synthesis of bone grafts and tooth regenerative materials, as well as biomimetic functional materials, are areas currently being examined by many research groups. # 2007 Elsevier B.V. All rights reserved.

Keywords: Self-assembly; Biomaterials; Collagen; Calcium phosphate

1. Introduction2
2. Hierarchical assembly of mineralized collagen in natural tissue3
2.1. Collagen and model of self-assembled fibrils3
2.2. Organization of mineralized collagen fibrils4
2.2.1. Organization in lamellar bones4
2.2.2. Organization in woven bones6
2.2.3. Organization in zebrafish skeletal bone6
2.2.4. Organization in ivory9
2.2.5. Size and shape of crystals in the mineralized collagen fibrils9

Contents Materials Science and Engineering R 57 (2007) 1–27

* Corresponding author. Tel.: +86 10 6277 2850; fax: +86 10 6277 2850. E-mail address: (F.-Z. Cui).

0927-796X/$–see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mser.2007.04.001

3.1. Nucleation sites for collagen fibrils and the initial stage of collagen mineralization10
3.2. Assembly of nano-fibrils of mineralized collagen12
3.3. Assembly and mineralization of peptide-amphiphilic nanofibers14
4. Applications of mineralized collagen composites in bone regeneration16
4.1. Synthesis and properties of nano-HA/collagen composites16
4.1.1. Direct blended nano-HA/collagen composites16
4.1.2. SBF immersion technology17
4.1.3. Co-precipitation self-assembly method18
4.2. Applications of nano-HA/collagen composites19
4.2.1. Gels and powders as a filler for orthopaedic surgery19
4.2.2. Surface coatings for implant materials19
4.2.3. Three-dimensional scaffolds for bone tissue engineering20
4.3. Related improvements of mineralized collagen composites20
4.3.1. Optimization of controllable ionic substitution in nano-HA crystals20
4.3.2. Improved mechanical properties for implantation at load-wearing sites21
nano-micro synthesis methods2
4.4. Animal models and clinical applications2
4.5. Other applications of self-assembled collagen fibrils23
4.5.1. Combination with cartilage regeneration23
4.5.2. Combination with vascular tissue regeneration23
4.5.3. Combination with neural regeneration23

3. Biomimetic fabrication with self-assembled collagen mineralization . . .... .... .... .... .... ..... .... .. 10 4.3.3. Development of hierarchical biomimetic technology by combination with other advanced

1. Introduction

In this paper we review the current understanding of structure, formation and properties of mineralized collagen fibril composites in connective tissues, as well as recent work involving biomimetic synthesis of new materials with the structure of mineralized collagen. The focus in the paper is mainly on type I collagen, of which at least twenty collagens have so far been discovered. By mineralized collagen we refer mostly to calcium phosphate based crystals, which in bone are found to consist primarily of calcium and phosphate ions, with traces of magnesium, carbonate, hydroxyl, chloride, fluoride, and citrate ions [1]. Mineralized collagen is one kind of material that can be produced by self-assembly at ambient temperatures. In this paper we have used the concept of self-assembling defined by Whitesides and Grzybowski [2], i.e., self-assembling is the autonomous organization of components into patterns or structures without human intervention. It is considered that self-assembling processes are common throughout nature and technology.

Connective tissues are among the most advanced structural composite materials known to be made of macromolecular building blocks [3–5]. A wide range of tissues, each possessing very different properties are successfully synthesized in natural environments with only the same basic macromolecular design [6]. Nevertheless, these tissues in many instances show some common features—they are assembled in numerous assembly ways that allow control of the formation of varying hierarchical structures, from the nanometer scale to the macroscale [7]. The concept of hierarchical assembly has been recognized and emphasized by more and more scientists over the last decades, as exemplified by the investigations of numerous biocomposite systems. The hierarchical levels of organization with highly specific interconnectivity and with unique architectures are designed to give the required spectrum of properties for each oriented composite system. Based on these lessons in biology, the laws for the formation of complex composite systems for functional macromolecular assemblies have been probed [8,9].I n addition to gaining knowledge of the fundamental mechanisms for assembly of such materials, the ability to build architectures as a direct consequences of the precision in assembly would certainly open the gate to some new areas of materials science. Examples could be the design and construction of inorganic materials with specified atomic structure, size, shape, crystal orientation, and number of defects and the integration of these architectures into

F.-Z. Cui et al./Materials Science and Engineering R 57 (2007) 1–272 functioning devices for anticipated electrical, optical, magnetic, and chemical outputs [4,10–16]. Only with an appreciation and understanding of the unique structure–property relationships in such biosystems can such proposals be achieved.

Collagens comprise a family of extracellular matrix molecules responsible for the integrity and mechanical properties of both soft and hard connective tissue [17], including cornea, skin, tendon, cartilage, and bone [18–21]. Almost all of the connective tissues with collagen fibrils as the basic building blocks have remarkably similar chemistry at the macromolecular and fibrillar levels of structure. However, differentiation in the hierarchical structure takes place as these fibrils are arranged in the specific architecture required from the construction of special tissues each with unique functions, which is generally considered as the functions of other non-collagen molecules.

In this review, we focus principally on the self-assembly of mineralized collagen composites in hard connective tissues and on the relative involvement of mimetic insoluble organic structures in controlled mineralization (referred to as organic matrix-mediated mineralization). In most cases of such mineralization where collagen fibrils are involved, the matrix is a polymeric framework that consists of a complex assembly of macromolecules. Previous studies have revealed that natural bone is a representative example with a typical hierarchically ordered organization. Bone tissues are mainly constructed from nano-sized hydroxyapatite (HA) crystals and a collagen framework in which the crystals form, resulting in a highly complex but ordered mineral–organic composite material. This composite itself is organized into layers or lamellae, each with the thickness of a few microns, and in turn these are arranged into higher order structures in a variety of ways depending on the specific bone type [20]. Therefore we begin this paper with a short review of the model for self-assembled collagen fibrils, and then discuss the organization of mineralized collagen fibrils in natural hard tissue. We also describe the response of the organization during collagen assembly to gene mutations by reference to the zebrafish skeleton system. Some systems specifically designed to simulate the collagen mineralization are then also reviewed. Finally, we finish by giving a description of work on biomemitic fabrication using self-assembled collagen mineralization and review the current applications of such materials.

2. Hierarchical assembly of mineralized collagen in natural tissue 2.1. Collagen and model of self-assembled fibrils

Collagens represent an important family of proteins in the vertebrate body. At least 20 distinct human collagens are known, the most abundant being found in fibrils with an axial 67 nm periodicity. In addition, collagen-like domains are crucial to ligand binding and self-association in host-defence proteins, such as mannose-binding proteins [2]. Mutations in collagen are the cause of various connective tissue diseases, including osteogenesis imperfecta and hereditary aortic aneurysm [23–25]. Additionally, the results of the altered assembly of collagen fibrils have also been implicated in the etiology of these diseases listed above that put collagen in the context of protein assembly and aggregation diseases [26–28]. Hereafter, as used in this paper collagen will always refer, unless stated otherwise, to type I collagen.

The biological synthesis, secretion, and assembly into definitive extracellular structures of collagen can be described briefly as follows [29]. Firstly, the individual polypeptide chains, called pre-procollagen chains, are synthesized on membrane-bound ribosomes. These chains have three major domains: the a-chain, the amino-terminal peptide, and the carboxy-terminal peptide. In the cisternae of the rough endoplasmic reticulum, three pro-a chains associate, and a procollagen molecule with a triple helix structure is formed. During this process, the prepeptide sequences are removed, praline and lysine residues become hydroxylated, the propeptides are glycosylated, and disulfide bondings are formed. After all these post-translational modifications take place, individual procollagen molecules are transported to the Golgi complex, packaged into granules, and secreted into the extracellular space. In the case of type I collagen, the extension peptides at both ends of the molecule must be cleaved before the fibers can be assembled. After removal of the extension propeptides, several collagen molecules associate in a quarter-staggered manner to form collagen fibrils, and in turn, collagen fibers and bundles are formed.

The common elements in the structure of collagen fibrils begin at the molecular level with similarities in the distinctive amino sequences—this in fact is probably the most outstanding characteristic of these molecules. Additionally, the similarities lay the groundwork for the development of a variety of tissues that share common chemical and physical properties [6]. Collagens all contain the amino acid hydroxyproline (Hyp). Along with proline and glycine, these three amino acids account for more than fifty percent of the total amino acid content [18]. Addition

F.-Z. Cui et al./Materials Science and Engineering R 57 (2007) 1–27 3 of other amino acids differentiates between the different collagen types. It is now accepted that amino acids play a major role in determining the three-dimensional assembly conformations. The representative hallmark of collagen molecules is the multiple repetition of Gly-X-Y sequences. This feature is dictated by the unique triple helical conformation, built of three polypeptide chains, and is a widespread structural element occurring in collagens. The basic three-dimensional structure of the collagen triple-helix was first inferred from fiber diffraction studies on collagen in tendon [30,31]. Each of the three chains in the molecule forms a left-handed polyproline-I-type helix, which has exactly three residues per turn. The chains are arranged in parallel, staggered by one residue relative to each other, and supercoiled along a common axis to form a right-handed triple helix of 290 nm in length. In contrast to righthanded a-helices, left-handed polyproline-I-helices occur relatively rarely as structural elements in proteins, with the striking exception of collagens. The fiber diffraction performed on tendon fibers has revealed a clear right-handed supercoiling of the three individual chains to form a triple helix, resulting in an increase to 3.3 residues per turn and to a reduction of the axial repeat distance to 0.286 nm per residue.

A functionally important structural feature of the collagen triple helix is the orientation of the side chains. Side chains of residues in X- and Y-positions point out of the helix, and are freely accessible for binding interactions, which play an important role in fibril formation through intermolecular interactions between oppositely charged residues and through hydrophobic interactions between residues of different molecules [32]. The interactions between the collagen molecules result in the characteristic quarter repeat of 67 nm and in a complex cross-striation banding, which results in agglomeration of the collagen triple helices into microfibrils, forming the supramolecular structures [3–35]. The most widely accepted model for packing of collagen molecules is that five triple helices align longitudinally with an overlap of approximately a quarter of the molecular length to form a microfibril [34]. Considering that the diameter of each collagen molecule is 1.5 nm [36], the diameter of the collagen fibrils in the five-stranded packing model should be approximate 3.6 nm. This has been verified by transmission electron microscope (TEM) observations of selfassembled collagen fibrils. This so-called quarter stagger, combined with the gap between successive macromolecules, is responsible for the typical 67 nm periodical cross-striation patterns as observed by TEM, atomic force microscope (AFM), and X-ray diffraction (XRD) investigations [21,34,37]. The microfibrils are then assembled into collage fibrils that may vary in thickness from 35 to 500 nm. These are further combined, oriented and laid up to form ordered structures with particular morphologies for tissues. The overwhelming consideration in the arrangement of collagen fibrils to form connective tissues is the resulting tissue function. This will be illustrated and discussed in the following sections. Recently, the formation of synthetic collagen triple helices as long or even longer than natural collagen has been reported [38], although applications for these materials are yet to be developed.

2.2. Organization of mineralized collagen fibrils

2.2.1. Organization in lamellar bones

Mineralized collagen fibrils are the basis for various connective tissues such as bone and cartilage. The structure and organization of mineralized collagen in such mineralized tissues are both fundamentally important for many biochemical, physicochemical, and biomechanical events that determine the normal function of these tissues. Bone refers to a family of materials that are constructed by mineralized collagen fibrils with complex hierarchically assembled structures [39], of which lamellar bone is the most abundant type in many mammals, including humans. Previous studies have demonstrated that bone tissues are primarily adapted to the variety of mechanical functions that they fulfill.

(Parte 1 de 8)