Irsalan Asif

Abstract

Type 1 Collagen is essential in the formation of bone and mineralization. This is due to its high tensile strength and the structural scaffold it forms for the mineral hydroxyapatite. The Biochemical structure of Type 1 Collagen allows it to form strong and tight alpha triple helical structures and its assembly into fibres gives Type 1 Collagen its strength. The role of repetitive amino acids such as Glycine gives Collagen its tight triple helical structure. The role of Collagen and its longitudinal alignment in the bone matrix allows bone mineral to settle within the fibres. The bone mineral is mainly hydroxyapatite, which makes up the inorganic matrix, adding to the strength and toughness of the tissue. Along with ground substance, Collagen fibrils form a structure similar to reinforced concrete. The high abundance of Collagen in nature has led to intensive study of Collagen as a biomaterial.

Introduction

Collagen is the most common protein in the animal world totalling 25% of total body protein, and its high abundance underlines the necessity of collagenous protein in the body. Collagen (type I) has the critical role in forming the scaffold for the bones in the human body, building up an extracellular framework for multicellular organisms. However, there are 36 different types of Collagen with slightly different properties, for example Type III Collagen is more flexible than type I Collagen and type V Collagen is a heteropolymer. This allows mixing of fibres to vary the elastic modulus (1). Construction of Collagen is essential to bone function as minor deformities in the arrangement can lead to serious consequences in the case of Osteogenesis imperfecta.

Biochemical composition of Collagen

The linear sequence of amino acids that make Collagen polypeptides (primary structure) are predominantly Glycine (33 %) whilst Proline, and hydroxyproline makes up 21% of the principal structure (2). Glycine at every third residue forms a Gly - X – Y sequence, where X and Y are more likely Proline and hydroxyproline. This aids the structure of Collagen as Glycine compromises of just a hydrogen atom as its side chain, consequently allowing chains to pack closely, fitting in

restricted spaces (figure 1). Proline stabilises the

FIGURE 1 Collagen triple helix illustrating role of Glycine in packing chains. (3)

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right handed helical conformation and is essential because it causes ‘kinks’ in Collagen chains due to the ring structure (secondary structure) (3). However, not all the Collagen molecules are identical to one another as in Type I Collagen only two chains are identical known as an alpha 1 chain, and one is non-identical known as an alpha 2 chain(2).

Assembly of collagen molecules to create fibres (figure 2)

The primary structure of Collagen fibres are built by ribosomes from their constituent amino acids and enter the rough endoplasmic reticulum (RER) labelled as pro alpha chains. The addition of propeptide molecules, to N and C terminals prevents binding of Collagen fibers and guides the chains through the cell during assembly (3). While in the RER selected Proline and lysine are hydroxylated, and Glycosylation occurs on various hydroxylysine amino acids by attaching a carbohydrate group (2). The pro alpha chains leave the RER and hydrogen bond with other pro alpha chains to create the procollagen triple helix (secondary structure). The Propeptides are cleaved to form tropocollagen molecules, and are secreted into the extracellular space where they self-assemble to form fibrils (3). The strands are held furthermore by cross links (tertiary structure), between Lysine on adjacent tropocollagen molecules catalysed by lysyl oxidase (3).The cross linking and the staggering of the chains are ‘like bricks in a wall’ providing strength and stability (4). Aggregation of the fibrils to form fibers occurs on the outside of the cell (Quaternary structure) (Figure 3).

FIGURE 3 Cross links formed by Collagen molecules (3).

FIGURE 2 Formation of Collagen fibre in Osteoblasts (3)

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Role of Collagen type I in bone formation

Type I Collagen makes up 25-30 % of organic material in bone and is the cellular equivalent of steel rebar whilst summing up to 90% of the organic matrix (Osteoid)(4,5). Collagen provides the scaffold and the tensile structure of the bone preventing snapping and fracturing, whilst providing an area where the hydroxyapatite (bone mineral) can be inserted. The protein is synthesised and secreted by the Osteoblasts into the Extracellular matrix and the longitudinal staggered arrangement leads to gaps in the structure. These regions are known as gap regions and overlap regions, which are situated periodically where the gap regions occupy 60% and overlap regions 40% of the axial periods (figure 4). The parallel arrangement ensures the mineral crystals

form stacks rather than orientated randomly in the cross section of the fibril. This also achieves a higher packing density, and therefore improved mechanical strength and stiffness (6). Organisation of collagen fibres and the size of the gap regions limits the crystal size of hydroxyapatite, as larger crystals between fibres can cause Osteogenesis imperfecta (7)

How bone mineral and Collagen assemble to form bone

The bone mineral constitutes 69% of the bone matrix and bone mineral is

predominantly hydroxyapatite, which is a form of calcium phosphate. However,

the hydroxyapatite crystals in the body are much smaller than geologic

hydroxyapatite crystals therefore allowing support to mineral metabolism.

Collagen fibrils are first embedded into ground substance like steel rebar in

reinforced concrete (4). Following the embedment, Hydroxyapatite is deposited

in gap regions between fibers, and then secreted in the ground substance,

leading to super-saturation and precipitation of the crystals (figures 5 and 6) (4)

FIGURE 4 Diagram of staggered orientation of collagen fibrils (9)

FIGURE 6 3D arrangement of Collagen fibrils and Hydroxyapatite (12).

FIGURE 5 Collagen fibril interaction with ground substance (11)

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(6)(4). Cemented to the fibrils are seed crystals, secreted by Osteoblasts, which

are nucleation sites allowing heterogeneous nucleation. Extracellular vesicles aid

in this by acting as micro environments for the hydroxyapatite to form (8).

Macromolecules such as bone sialoprotein attach to the crystals regulating their

size and shape. The presence of hydroxyapatite in the tissue increases the

tensile strength of the fibrils by two fold and the elastic modulus by tenfold (9).

This results in a highly organised collagen-hydroxyapatite matrix which can

perform the mechanical function of the skeleton. Some scientists believe,

however, the participation of Collagen in mineralisation is not a priority. Most of

the mineral contained in the interfibrillar space associates with non- collagenous

protein and not Collagen itself (5).These molecules termed as crystal ‘ghosts’

because they resemble crystals, but the nature of these crystals is yet to be

defined. Therefore, Research still has to be done to find a definitive answer.

Conclusion

Collagen is responsible for the structural integrity of the human body but also

defines the shape of the tissue. It forms an important component of bones

providing strength which prevents easy breakage. This is because of the tight

aggregation of fibrils leading to closely packed tropocollagen molecules.

Collagen has been studied intensely in the field of biomaterials due to its

abundance in nature and ease of modification. Collamend® made by Bard is

used for soft tissue repair as Collagen is a major protein in most connective

tissue (10). It is also widely used as therapeutic applications such as Botox and

the Collagen used is harvested from the Achilles tendon. Collagen has been

recently looked into as a scaffold for Cartilage because of its structural role in the

body (10). Collagen is a wide ranging and valuable protein which is paramount to

bone strength and as a scaffold in the body.

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