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Mineralized tissues are biological tissues that incorporate minerals into soft matrices. Typically these tissues form a protective shield or structural support. Bone, mollusc shells, deep sea sponge Euplectella species, radiolarians, diatoms, antler bone, tendon, cartilage, tooth enamel and dentin are some examples of mineralized tissues.
These tissues have been finely tuned to enhance their mechanical capabilities over millions of years of evolution. Thus, mineralized tissues have been the subject of many studies since there is a lot to learn from nature as seen from the growing field of biomimetics. The remarkable structural organization and engineering properties makes these tissues desirable candidates for duplication by artificial means. Mineralized tissues inspire miniaturization, adaptability and multifunctionality. While natural materials are made up of a limited number of components, a larger variety of material chemistries can be used to simulate the same properties in engineering applications. However, the success of biomimetics lies in fully grasping the performance and mechanics of these biological hard tissues before swapping the natural components with artificial materials for engineering design.
Mineralized tissues combine stiffness, low weight, strength and toughness due to the presence of minerals (the inorganic part) in soft protein networks and tissues (the organic part). There are approximately 60 different minerals generated through biological processes, but the most common ones are calcium carbonate found in mollusk shells and hydroxyapatite present in teeth and bones. Although one might think that the mineral content of these tissues can make them fragile, studies have shown that mineralized tissues are 1,000 to 10,000 times tougher than the minerals they contain. The secret to this underlying strength is in the organized layering of the tissue. Due to this layering, loads and stresses are transferred throughout several length-scales, from macro to micro to nano, which results in the dissipation of energy within the arrangement. These scales or hierarchical structures are therefore able to distribute damage and resist cracking. Two types of biological tissues have been the target of extensive investigation, namely nacre from mollusk shells and bone, which are both high performance natural composites. Many mechanical and imaging techniques such as nanoindentation and atomic force microscopy are used to characterize these tissues. Although the degree of efficiency of biological hard tissues are yet unmatched by any man-made ceramic composites, some promising new techniques to synthesize them are currently under development. Not all mineralized tissues develop through normal physiologic processes and are beneficial to the organism. For example, kidney stones contain mineralized tissues that are developed through pathologic processes. Hence, biomineralization is an important process to understand how these diseases occur.
The evolution of mineralized tissues has been puzzling for more than a century. It has been hypothesized that the first mechanism of mammalian tissue mineralization began either in the oral skeleton of conodont or the dermal skeleton of early agnathans. The dermal skeleton is just surface dentin and basal bone, which is sometimes overlaid by enameloid. It is thought that the dermal skeleton eventually became scales, which are homologous to teeth. Teeth were first seen in chondrichthyans and were made from all three components of the dermal skeleton, namely dentin, basal bone and enameloid. The mineralization mechanism of mammalian tissue was later elaborated in actinopterygians and sarcopterygians during bony fish evolution. It is expected that genetic analysis of agnathans will provide more insight into the evolution of mineralized tissues and clarify evidence from early fossil records.
Hierarchical structures are distinct features seen throughout different length scales. To understand how the hierarchical structure of mineralized tissues contributes to their remarkable properties, those for nacre and bone are described below. Hierarchical structures are characteristic of biology and are seen in all structural materials in biology such as bone and nacre from seashells
Nacre has several hierarchical structural levels.
Some mollusk shells protect themselves from predators by using a two layered system, one of which is nacre. Nacre constitutes the inner layer while the other, outer, layer is made from calcite. The latter is hard and thus prevents any penetration through the shell, but is subject to brittle failure. On the other hand, nacre is softer and can uphold inelastic deformations, which makes it tougher than the hard outer shell. The mineral found in nacre is aragonite, CaCO3, and it occupies 95% vol. Interestingly, nacre is 3000 times tougher than aragonite and this has to do with the other component in nacre, the one that takes up 5% vol., which is the softer organic biopolymers. Furthermore, the nacreous layer also contains some strands of weaker material called growth lines that can deflect cracks.
The Microscale can be imagined by a three-dimensional brick and mortar wall. The bricks would be 0.5 μm thick layers of microscopic aragonite polygonal tablets approximately 5-8 μm in diameter. What holds the bricks together are the mortars and in the case of nacre, it is the 20-30 nm organic material that plays this role. Even though these tablets are usually illustrated as flat sheets, different microscopy techniques have shown that they are wavy in nature with amplitudes as large as half of the tablet’s thickness. This waviness plays an important role in the fracture of nacre as it will progressively lock the tablets when they are pulled apart and induce hardening.
The 30 nm thick interface between the tablets that connects them together and the aragonite grains detected by scanning electron microscopy from which the tablets themselves are made of together represent another structural level. The organic material “gluing” the tablets together is made of proteins and chitin.
To summarize, on the macroscale, the shell, its two layers (nacre and calcite), and weaker strands inside nacre represent three hierarchical structures. On the microscale, the stacked tablet layers and the wavy interface between them are two other hierarchical structures. Lastly, on the nanoscale, the connecting organic material between the tablets as well as the grains from which they are made of is the final sixth hierarchical structure in nacre.
Like nacre and the other mineralized tissues, bone has a hierarchical structure that is also formed by the self-assembly of smaller components. The mineral in bone (known as bone mineral) is hydroxyapatite with a lot of carbonate ions, while the organic portion is made mostly of collagen and some other proteins. The hierarchical structural of bone spans across to a three tiered hierarchy of the collagen molecule itself. Different sources report different numbers of hierarchical level in bone, which is a complex biological material. The types of mechanisms that operate at different structural length scales are yet to be properly defined. Five hierarchical structures of bone are presented below.
Compact bone and spongy bone are on a scale of several millimetres to 1 or more centimetres.
There are two hierarchical structures on the microscale. The first, at a scale of 100 μm to 1 mm, is inside the compact bone where cylindrical units called osteons and small struts can be distinguished. The second hierarchical structure, the ultrastructure, at a scale of 5 to 10 μm, is the actual structure of the osteons and small struts.
There are also two hierarchical structures on the nanoscale. The first being the structure inside the ultrastructure that are fibrils and extrafibrillar space, at a scale of several hundred nanometres. The second are the elementary components of mineralized tissues at a scale of tens of nanometres. The components are the mineral crystals of hydroxyapatite, cylindrical collagen molecules, organic molecules such as lipids and proteins, and finally water. The hierarchical structure common to all mineralized tissues is the key to their mechanical performance.
The mineral is the inorganic component of mineralized tissues. This constituent is what makes the tissues harder and stiffer. Hydroxyapatite, calcium carbonate, silica, calcium oxalate, whitlockite, and monosodium urate are examples of minerals found in biological tissues. In mollusc shells, these minerals are carried to the site of mineralization in vesicles within specialized cells. Although they are in an amorphous mineral phase while inside the vesicles, the mineral destabilizes as it passes out of the cell and crystallizes. In bone, studies have shown that calcium phosphate nucleates within the hole area of the collagen fibrils and then grows in these zones until it occupies the maximum space.
The organic part of mineralized tissues is made of proteins. In bone for example, the organic layer is the protein collagen. The degree of mineral in mineralized tissues varies and the organic component occupies a smaller volume as tissue hardness increases. However, without this organic portion, the biological material would be brittle and break easily. Hence, the organic component of mineralized tissues increases their toughness. Moreover, many proteins are regulators in the mineralization process. They act in the nucleation or inhibition of hydroxyapatite formation. For example, the organic component in nacre is known to restrict the growth of aragonite. Some of the regulatory proteins in mineralized tissues are osteonectin, osteopontin, osteocalcin, bone sialoprotein and dentin phosphophoryn. In nacre, the organic component is porous, which allows the formation of mineral bridges responsible for the growth and order of the nacreous tablets.
Formation of minerals
Understanding the formation of biological tissues is inevitable in order to properly reconstruct them artificially. Even if questions remain in some aspects and the mechanism of mineralization of many mineralized tissues need yet to be determined, there are some ideas about those of mollusc shell, bone and sea urchin.
The main structural elements involved in the mollusk shell formation process are: a hydrophobic silk gel, aspartic acid rich protein, and a chitin support. The silk gel is part of the protein portion and is mainly composed of glycine and alanine. It is not an ordered structure. The acidic proteins play a role in the configuration of the sheets. The chitin is highly ordered and is the framework of the matrix. The main elements of the overall are:
- The silk gel fills the matrix to be mineralized before the mineralization takes place.
- The highly ordered chitin determines the orientation of the crystals.
- The components of the matrix are spatially distinguishable.
- Amorphous calcium carbonate is the first form of the mineral.
- Once nucleation begins on the matrix, the calcium carbonate turns into crystals.
- While crystals grow, some of the acidic proteins get trapped within them.
In bone, mineralization starts from a heterogeneous solution having calcium and phosphate ions. The mineral nucleates, inside the hole area of the collagen fibrils, as thin layers of calcium phosphate, which then grow to occupy the maximum space available there. The mechanisms of mineral deposition within the organic portion of the bone are still under investigation. Three possible suggestions are that nucleation is either due to the precipitation of calcium phosphate solution, caused by the removal of biological inhibitors or occurs because of the interaction of calcium-binding proteins.
Sea urchin embryo
The sea urchin embryo has been used extensively in developmental biology studies. The larvae form a sophisticated endoskeleton that is made of two spicules. Each of the spicules is a single crystal of mineral calcite. The latter is a result of the transformation of amorphous CaCO3 to a more stable form. Therefore, there are two mineral phases in larval spicule formation.