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The properties of bone mineral change with age and maturation. Several investigators have suggested the presence of an initial or "precursor" calcium phosphate phase to help explain these differences. We have used solid state 31P magic angle sample spinning (MASS) nuclear magnetic resonance (NMR) and X-ray radial distribution function (RDF) analyses to characterize 11- and 17-day-old embryonic chick bone and fractions obtained from them by density fractionation. Density fractionation provides samples of bone containing Ca-P solid-phase deposits even younger and more homogeneous with respect to the age of mineral than the calcium phosphate (Ca-P) deposits in the whole bone samples. The analytical techniques yield no evidence for any distinct phase other than the poorly crystalline hydroxyapatite phase characteristic of mature bone mineral. In particular, there is no detectable crystalline brushite [DCPD, CaHPO4 2H2O less than 1%] or amorphous calcium phosphate (less than 8-10%) in the most recently formed bone mineral. A sizeable portion of the phosphate groups exist as HPO4(2-) in a brushite (DCPD)-like configuration. These acid phosphate moieties are apparently incorporated into the apatitic lattice. The most likely site for the brushite-like configuration is probably on the surface of the crystals.

We have reexamined the question of whether brushite (CaHPO4 X 2H2O) occurs in embryonic bone. On the basis of the experiments reported here, and a reexamination of data previously obtained in this laboratory, we have concluded that crystalline brushite as a separate phase identifiable by X-ray diffraction does not occur in embryonic chick or bovine bone. Crystalline brushite previously identified in this laboratory in the less-mineralized fractions of embryonic chick bone was apparently formed artifactually during the fractionation process, possibly from preexisting domains of HPO4(-2) groups in a noncrystalline brushite-like configuration.

The crystallinity of bone mineral at different stages of maturation has been measured by quantitative X-ray diffraction methods. Crystallinity measurements were made on tibial mid-diaphyses from 17-day embryonic chicks, newly-formed periosteal bone from embryonic chicks, and density-fractionated bone from post-hatch chickens from 5 weeks to 2 years of age. For a given animal age and degree of mineralization, crystallinity increases with animal age, indicating that changes in bone mineral occur even after mineralization is complete or nearly complete.

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.

X-ray diffraction radial distribution function analysis was used to determine if a significant amount of an amorphous solid phase of calcium phosphate exists in bone, and if so, whether the amount varies as a function of age and maturation. Unfractionated cortical bone from embryonic and posthatch chicks of various ages and a low-density fraction of embryonic bone were studied. No evidence was found for the presence of an amorphous solid phase of calcium phosphate in any of the samples studied, including the recently deposited bone mineral of the low density fraction of embryonic bone. As little as 12.5% of synthetic amorphous calcium phosphate (ACP) added to bone was readily detected by the radial distribution function technique used. The results clearly indicate that the concept that ACP is the initial solid mineral phase deposited in bone, and the major mineral constituent of young bone is no longer tenable. The concept does not provide an accurate description of the nature of the initial bone mineral deposited, or the changes that occur with maturation, nor can it account for the compositional and X-ray diffraction changes that the mineral component undergoes during maturation and aging.