Chapter 11. Mechanisms of mineralization
Matrix vesicle-mediated mineralization 135
Heterogenous nucleation 136
Mineralization of circumpulpal dentine 136
Initial mineralization of root dentine 136
Mineralization of bone 136
Mineralization of enamel 137
Mineralization of biological tissue is both strictly regulated and unique to each mineralized tissue. For each tissue, the mineral crystal in question is hydroxyapatite. The mechanisms of mineralization of bone and dentine share close similarities whereby an unmineralized matrix, osteoid or predentine, is calcified by a combination of matrix vesicle-mediated mineralization and heterogenous mineralization. Enamel is mineralized purely by heterogenous mineralization. However, for all three tissues, a number of tissue-specific proteins and proteoglycans tightly regulate mineralization by inhibiting mineralization processes or by guiding mineral deposition. Such proteins, and the cells that synthesize them, are therefore crucial in controlling the rate of mineralization, allowing ordered growth of the mineral crystals and preventing premature crystal fusion.
• know the mechanisms relating to matrix vesicle-mediated mineralization and understand the concept of heterogenous nucleation (epitaxy) as related to mineralization of bone and dentine organic matrix
• understand the similarities with and differences from the mineralization of enamel
• understand the role of bone- and dentine-specific proteins in inhibiting or promoting mineralization.
When considering the basic concepts of mineralization of bone and dentine it becomes clear that mineralization is a controversial subject. Very close similarities exist in the mineralization of bone and dentine, whereas the mineralization of enamel is unique. To understand mineralization we have to answer several important questions:
• What initiates mineralization?
• Where does the mineral come from?
• What controls the rate of mineralization?
• Where are mineral crystals deposited?
Although the area is still subject to much debate, it is clear that the matrix-producing cells (the osteoblasts, odontoblasts and ameloblasts) are essential and key elements in initiating and regulating mineralization of the various hard tissues. They:
• produce the organic matrix that becomes mineralized
• control the transport of calcium ions into the matrix
• determine the presence and distribution of specific matrix components (proteoglycans and glycoproteins) that regulate the process.
The basic mechanisms for mineralization are matrix vesicle-mediated mineralization and heterogenous mineralization or epitaxy.
Matrix vesicle-mediated mineralization
Hydroxyapatite cannot be precipitated directly, as it is too complex and requires a high degree of saturation of calcium and phosphate. To obtain hydroxyapatite, we must first precipitate a less stable, simpler calcium phosphate called brushite and then transform this into hydroxyapatite. However, there is not enough calcium or phosphate in serum to precipitate brushite so that, to form hydroxyapatite mineral where none existed before, there is a need for a mechanism to raise calcium and phosphate concentrations within a localized environment. This first mineral in mantle dentine, early bone and calcified cartilage is provided by matrix vesicles. These vesicles provide a controlled micro-environment to concentrate calcium and phosphate, in the presence of a variety of enzymes (including alkaline phosphatase). Mineral crystals develop within the vesicles and eventually burst out and become associated with the organic collagenous matrix of predentine or osteoid. These vesicles ‘bud off’ from the odontoblast process membrane in dentine formation, forming membrane-bound organelles of approximately 30–200 nm in size. As matrix vesicles are the only crystalline structures present in the first-formed dentine (mantle dentine) and bone, they have been credited with having an initiating role in mineralization.
Mineral crystal formation involves two stages:
• Firstly, nucleation or the formation of small embryo crystals
• Secondly, crystal growth, which is characterized by the deposition of calcium and phosphate onto the crystal surface.
In heterogenous nucleation, crystal growth is induced by the provision of a second, solid phase on which a crystal lattice may be formed. Crystal growth is better promoted on crystalline material having similar lattice spacings, a concept known as epitaxy. In the context of mineralizing tissues, the prime candidate is the organic matrix. This method requires the ‘seeding’ of small quantities of hydroxyapatite crystals into the matrix and allowing it to grow at the expense of the calcium and phosphate surrounding it.
Mineralization of circumpulpal dentine
Investigations into the mineralization of circumpulpal dentine have suggested that odontoblasts actively transport calcium ions to the mineralization site via active transport through the cell. This intracellular route actively controls the level of calcium in the mineralizing area and maintains calcium concentrations that are not in equilibrium with other body fluids. Deposition of calcium occurs on to a template formed by type I collagen, and mineral crystal deposition occurs at the gap zone within the collagen fibrils. A pool of chondroitin sulphate (CS), decorin and biglycan is secreted at the mineralization front and is transported intracellularly along the odontoblast process. Although general levels of proteoglycans at the mineralization site and in mineralized dentine are lower than those found in predentine, the CS-rich proteoglycans are distributed in mineralized connective tissues interfibrally and associated with the gap zones of the collagen fibrils. It is this CS proteoglycan pool that is associated with guiding mineral deposition, as the CS may be involved in transport of calcium and phosphate to gap zones in the collagen.
Another important glycoprotein in directing mineralization is dentine phosphoprotein (DPP). This dentine-specific protein is secreted at the mineralizing front and is highly phosphorylated and highly acidic, thus having a high affinity for calcium and hydroxyapatite surfaces. Changes in the conformation of the protein allow it to bind increasing numbers of calcium ions. In high concentrations, it inhibits crystal formation and so, by controlling the release and level of DPP, the odontoblast controls the initiation of mineralization and rate of deposition.
Several other proteins have been shown in vitro to be associated with mineralization. The classical bone-associated glycoproteins are also found within the dentine. Osteopontin is a phosphorylated protein capable of promoting mineralization, and osteonectin has been shown to inhibit growth of hydroxyapatite crystals whilst promoting the binding of calcium and phosphate to collagen.
Other factors may also play a role in limiting or controlling mineralization. Collagen is found in many other soft connective tissues that are not mineralized. Although specific glycoproteins and proteoglycans have been shown to have differing roles in promoting and inhibiting mineralization and crystal growth (including the presence of dermatan sulphate proteoglycans in predentine), other factors are also important in regulating mineralization. Pyrophosphate is found in soft tissues and body fluids and actively inhibits mineralization. In hard tissues, pyrophosphatase can be identified and degrades any pyrophosphate present, allowing mineralization to occur.
Why two mechanisms?
Matrix vesicles are required for the mineralization of mantle dentine, not only as there is the need to produce mineral where none existed before, but also because the matrix formed is secreted from newly differentiating odontoblasts, with some contribution from the adjacent dental papilla cells. Not all the matrix components are present and some nucleation sites are not expressed. There is also the need to form mineral that can be seeded onto/>