The mineralization process
Hydroxyapatite crystals are usually confined to areas where there is a need for high mechanical strength, rigidity, or hardness as in bone, dentine, and enamel. They do not normally occur at all in soft tissues, the only other site of occasional local occurrence being within such epithelial keratins as hard claws and beaks. The restriction of hydroxyapatite crystals to certain kinds of tissue suggests that some mechanism exists for their deposition in tissues where they are functionally useful and nowhere else. The nature of this mechanism is still not completely understood. An idea that persists is that one or more of the organic components of hard tissues is specifically concerned with deposition of minute apatite crystals, and this is sometimes referred to as the organic matrix or nucleator of the crystallites. The term “matrix” is also often used more loosely in a histological context to denote a continuum that fills spaces between discrete entities. Thus, the continuous material, rich in proteoglycans and collagen, that fills the spaces between the cells of cartilage is referred to as the cartilage matrix, despite the fact that the cells have themselves produced this intercellular material rather than been produced by it.
Hydroxyapatite crystals are usually confined to areas where there is a need for high mechanical strength, rigidity or hardness as in bone, dentine and enamel. They do not normally occur at all in ‘soft tissues’, the only other site of occasional local occurrence being within such epithelial keratins as hard claws and beaks.
The restriction of hydroxyapatite crystals to certain kinds of tissue suggests that some mechanism (or mechanisms) exists for their deposition in tissues where they are functionally useful and nowhere else. The nature of this mechanism is still not completely understood. An idea that persists is that one or more of the organic components of hard tissues is specifically concerned with deposition of minute apatite crystals and this is sometimes referred to as the organic matrix or nucleator of the crystallites.
The term ‘matrix’ is also often used more loosely in a histological context to denote a continuum that fills spaces between discrete entities. Thus the continuous material, rich in proteoglycans and collagen, that fills the spaces between the cells of cartilage, is referred to as the ‘cartilage matrix’, despite the fact that the cells have themselves produced this intercellular material rather than been produced by it. This is not in accord with the strict definition of a matrix as ‘the place or medium in which something is bred or developed’, which is applicable to the deposition of inorganic crystallites that occurs during mineralization and suggests that a true hard tissue matrix should: (1) be in existence before whatever is bred, i.e. the crystals, (2) participate in their development and (3) enclose them spatially.
Though the idea of an organic matrix for such tissues as bone and enamel is widely held, the exact manner in which the matrix participates in the development of crystallites still needs to be demonstrated. Other important problems concern why mineralization occurs only at specific sites, and what physicochemical principles govern the equilibrium between hydroxyapatite and its ions in solution.
Difficulties arise, not so much in explaining why mineralization readily and regularly occurs in certain tissues, but rather why other tissues, which resemble them in many ways, do not normally mineralize. Thus it is relatively easy to explain how crystals of a very sparingly soluble substance such as hydroxyapatite can be formed in bone, on the basis of the concentrations of calcium and phosphate ions present in blood; these are sufficiently high to permit small crystals of biological apatite to grow at the expense of ions in solution. It is more difficult to appreciate why, under apparently similar conditions, a tissue such as skin which, like bone, contains collagen as a major constituent, does not mineralize, except rarely after injury. A complete explanation of mineralization should indicate what factors determine where it will occur, what limits its spread and how its timing in different regions of bone is coordinated.
Although it has now been almost completely abandoned, Robison’s alkaline phosphatase theory formulated in 1923, emphasizes some of the essential problems that must be solved by any satisfactory theory of mineralization. Robison noticed that sites of mineralization frequently contain an enzyme operating at high pH values which is capable of hydrolysing organic phosphate esters with the release of inorganic phosphate ions:
He suggested that in mineralization a possible function of this alkaline phosphatase is to raise the local concentration of inorganic phosphate ions and thus to cause the precipitation of calcium phosphate, when its solubility product is exceeded.
A number of objections raised to this theory led to its gradual abandonment. Firstly, it was argued that the concentration of organic phosphate in plasma was too low for this to serve as an effective source of phosphate ions. Robison conceded this point but suggested that a store of organic phosphate was built up at the calcification site by the alkaline phosphatase acting in reverse, before calcification began. A second criticism was that some other sites, such as the kidney, which do not normally calcify, contain considerably higher concentrations of alkaline phosphatase than the calcification sites themselves. Thirdly, it was pointed out that the inorganic phase is more highly organized than can be accounted for by simple precipitation, since the apatite crystallites are regular in size, distribution and orientation. To answer this, Robison proposed a ‘second mechanism’, necessary for smooth deposition of inorganic crystals, which partly anticipated the later epitactic concept (page 456). Finally, it appeared that there is no necessity for the phosphate ion concentration to be raised for deposition of solid calcium phosphate to occur. Slices of cartilage from rachitic rats became mineralized when incubated in solutions containing the same concentration of calcium and phosphate ions as those found in the serum of young normal rats. The same conclusion can be reached on the theoretical grounds discussed in the following section.
When a sparingly soluble salt is placed in water, both anions and cations leave the surface of the solid and dissolve. This process continues until the solution is saturated with respect to the salt; at this point a dynamic equilibrium is established in which the same number of ions are deposited on the surface as leave it in a given time. Once saturation has been reached, there is no net gain or loss of the solid phase. Conversely if the solid salt is placed in an aqueous solution, which is supersaturated with respect to the solid, there will be a net deposition of ions on the surface of the solid until equilibrium is reached at the same solute concentration as before. This concentration will be constant for a given substance at a specified temperature. Furthermore, the product of the ionic concentrations will be constant at a value known as the solubility product. Thus considering the case of the sparingly soluble salt, calcium sulphate, the solubility product can be defined as follows:
Biological apatite, the crystalline phase of bone, dentine and enamel, consists essentially of hydroxyapatite modified by the presence of carbonate, small amounts of magnesium, sodium, and possibly citrate, either within the lattice or adsorbed on the crystal surfaces (page 430). The theoretical possibilities for ionization of hydroxyapatite are complex because more than one calcium ion and more than one phosphate ion can arise per unit of Ca10(PO4)6(OH)2. In addition the pH (which controls hydroxyl ion concentration), the carbonate concentration and the presence of macromolecules all affect the solubility product. The concepts used to describe the solubility characteristics of simple salts are therefore not easily applied to biological apatite. Furthermore, the formula given for hydroxyapatite probably has little meaning in relation to particles of molecular size, since such small aggregates of calcium and phosphate ions are known to be very unstable. Fortunately, experiments on the solubility of hydroxyapatite have shown that the simplest of all possible solubility products, namely , best describes the actual solubility behaviour of this substance. The solubility products of hydroxyapatite itself, biological apatite and calcium hydrogen phosphate at pH 7·4, an ionic strength of 0·16 and 37°C (i.e. under physiological conditions) may therefore be considered as simple products of the calcium ion concentration and the concentration of the monohydrogen phosphate ion , which is the most abundant phosphate ion under these conditions.
The solubility product of biological apatite has been determined by suspending fresh defatted powder from cortical bone in an artificial solution containing inorganic ions at the concentrations present in an ultrafiltrate of normal serum. When equilibrium had been established the solution was removed and replaced by a fresh portion which was equilibrated, removed and analysed. Solutions obtained by repeating this procedure several times were found, after the first few changes, to have the same calcium and phosphate levels and these were used to calculate the solubility product for biological apatite.