Laurence C. Bonar

Neutron diffraction studies of collagen in fully mineralized bone.

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.

Recent studies of bone mineral: Is the amorphous calcium phosphate theory valid?

Abstract Recent studies on bone mineral are reviewed, with particular emphasis on the nature of the initial mineral deposited and the changes occuring during further mineralization and maturation. It is shown that amorphous calcium phosphate, which has been proposed as the precursor of hydroxyapatite in bone mineral, cannot be detected in significant quantity in the earliest bone mineral formed, and furthermore that there is evidence that no progressive decrease in amount of any amorphous phase occurs with bone maturation. We conclude in general that the X-ray difraction pattern and non-stoichiometric composition of bone mineral, and the changes in these characteristic observed with age and maturation cannot be explained by the presence of a second phase in progressively decreasing proportion whether it be amorphous calcium phosphate, octacalcium phosphate, brushite or some other distinct phase. The structural and compositional changes observed with age and maturation in bone mineral are consistent with a general increase in the degree of crystal perfection of a calcium phosphate phase best described as a poorly crystalline hydroxyapatite, although the exact details of the stuctural and compositional modification taking place with maturation must await further research. The role of brushite, which has been proposed as the earliest solid calcium phosphate phase deposited in at least one species, is reviewed.

X-ray diffraction studies of the crystallinity of bone mineral in newly synthesized and density fractionated bone

SummaryThe crystallinity of bone mineral at different stages of maturation has been measured by quantitative X-ray diffraction methods. Crystallinity measurements were made on tibial middiaphyses from 17-day embryonic chicks, newlyformed 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.

Comparative studies of the organic matrices of invertebrate mineralized tissues

The organic matrices of the mineralized tissues of four phyla of invertebrates, representing eight species, has been investigated by means of electron microscopy, X-ray diffraction, and amino acid analyses. The echinoid plates of Strongylocentrotus and Lytechinus, which contain CaCO3 as the major inorganic crystal phase, contained collagen as the principal organic constituent. The amino acid compositions of the proteins of the mineralized tissues of the phyla Porifera and Mollusca varied very markedly from species to species, but all were characterized by their relatively high concentrations of aspartic acid, glycine, and serine, and by the fact that a large proportion of the total amino acid residues was represented by as few as three, and at the most, five, different amino acids. In this respect, they resemble the protein of embryonic and mature enamel proteins and certain of the silk fibroins. Native type collagen fibrils were observed by electron microscopy in the matrices of the phyla Porifera and Mollusca, although they constitute only a very minor structural component of these matrices.

Failure to detect an amorphous calcium-phosphate solid phase in bone mineral: a radial distribution function study

SummaryX-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 acount for the compositional and X-ray diffraction changes that the mineral component undergoes during maturation and aging.

A study of dense mineralized tissue by neutron diffraction

Abstract Neutron diffraction studies of mineralized tissue show a close relationship between the wet state equatorial diffraction spacing and wet tissue density expressable as a second-order polynomial. The molecular fractional shrinkage when the tissue is dried shows a straight line dependence on wet tissue density with a correlation of 0.98. Since the dry state equatorial diffraction spacing is much less than for the corresponding wet state, even in fully mineralized bone, the collagen molecules must be displaced through a mineral-free volume while drying. The mineral can only be located within the available volume of the dried tissue whether intra- or extrafibrillar. The dimension of the dry state equatorial spacing for each of the tissues examined is close to that of dried tendon collagen. It appears unlikely that hydroxyapatite crystallites can be accommodated radially between collagen molecules in bone if the packing is like that of dried tail tendon collagen. The only mineral within the fibrils must be in the intermolecular gaps. It is estimated on the basis of the volume of the axial intermolecular gaps and the minimum extrafibrillar volume that the intrafibrillar mineral can be no more than 20% of the total mineral and may be less than 10%.

The molecular structure of the protein matrix of bovine dental enamel.

The protein matrix of bovine dental enamel, as well as various layers from the enamel-forming organ, remaining after decalcification with ethylenediamine-tetra-acetic acid, have been isolated from mid-embryonic and early post-fetal incisor teeth. It has been possible to separate enamel matrix proper from the enamel organ. The enamel matrix proper gave a cross- β X-ray diffraction pattern. Furthermore, X-ray diffraction patterns of the enamel organ also indicated the presence of a cross- β component. These findings are discussed in relation to current concepts of the role of ordered organic matrices in biological mineralization.

Characterization of very young mineral phases of bone by solid state 31Phosphorus magic angle sample spinning nuclear magnetic resonance and X-ray diffraction

SummaryThe 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 crystal-line hydroxyapatite phase characteristic of mature bone mineral. In particular, there is no detectable crystalline brushite [DCPD, CaHPO4 2H2O< 1%] or amorphous calcium phosphate (< 8–10%) in the most recently formed bone mineral. A sizeable portion of the phosphate groups exist as HPO42− 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.

Thermal denaturation of mineralized and demineralized bone collagens

Polarization microscopy, X-ray diffraction, and electron microscopy have revealed an extremely intimate relationship between the highly ordered and “crystalline” organic matrix of collagen and the inorganic crystals in bone and dentine (2). In bone it has been shown that a major portion of the mineral phase is deposited within “holes” or compartments formed in the collagen fibrils as a result of the specific way in which macromolecules are aggregated in native type fibrils (2). One of the fundamental properties of collagen is the thermal denaturation and shortening observed when the collagen is heated above a well-defined shrinkage temperature. We have examined, by wide- and low-angle X-ray diffraction, the role of the mineral phase in stabilizing both the short-range (helical) and long-range (the packing of collagen macromolecules in the characteristic staggered arrangement of native collagen fibrils) structure of the collagen against thermal denaturation.

The molecular structure of the neutral-soluble proteins of embryonic bovine enamel in the solid state

Cross- β X-ray diffraction patterns have been obtained from fibers prepared from the cold, neutral-soluble protein fraction of decalcified, bovine, embryonic enamel matrix. Similar cross- β patterns have previously been reported from the intact organic matrix (8). The structural implications of the unusually high proline content of these proteins are discussed.