Melvin J. Glimcher

The carbonate environment in bone mineral: a resolution-enhanced Fourier Transform Infrared Spectroscopy Study.

SummaryThe environment of carbonate ions in bones of different species (rat, rabbit, chicken, cow, human) was investigated by Fourier Transform Infrared Spectroscopy (FTIR) associated with a self-deconvolution technique. The carbonate bands in thev2 CO32− domain show three components which were identified by using synthetic standards and different properties of the apatitic structure (ionic affinity for crystallographic locations, ionic exchange). The major component at 871 cm−1 is due to carbonate ions located in PO43− sites (type B carbonate). A band at 878 cm−1 was exclusively assigned to carbonate ions substituting for OH− ions in the apatitic structure (type A carbonate). A band at 866 cm−1 not previously observed was shown to correspond to a labile carbonate environment. The intensity ratio of type A to type B carbonate appears remarkably constant in all bone samples. The 866 cm−1 carbonate band varies in its relative intensity in different species.

Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging

SummaryThe environment of CO32− ions in the bone mineral of chickens of different ages and in bone fractions of different density have been investigated by resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopy. Three carbonate bands appear in thev2 CO3 domain at 878, 871, and 866 cm−1, which may be assigned to three different locations of the ion in the mineral: in monovalent anionic sites of the apatitic structure (878 cm−1), in trivalent anionic sites (871 cm−1), and in unstable location (866 cm−1) probably in perturbed regions of the crystals. The distribution of the carbonate ions among these locations was estimated by comparing the intensities of the corresponding FTIR spectral bands. The intensity ratio of the 878 and 871 cm−1 bands remains remarkably constant in whole bone as well as in the fractions obtained by density centrifugation. On the contrary, the intensity ratio of the 866 cm−1 to the 871 cm−1 band was found to vary directly and decreased with the age of the animal. In bone of the same age, the relative content of the unstable carbonate ion was found to be highest in the most abundant density centrifugation fraction. A resolution factor of the CO32− band (CO3 RF) was calculated from the FTIR spectra which was shown to be very sensitive to the degree of crystallinity of the mineral. The crystallinity was found to improve rapidly with the age of the animal. The CO3 RF in the bone samples obtained by density centrifugation from bone of the same animal was found to be essentially constant. This indicates a fairly homogeneous, crystalline state of the mineral phase. A comparison of the maturation characteristics of synthetic carbonated apatites with bone mineral indicates that a simple, passive, physicochemical maturation process cannot explain the changes observed in the mineral phase of whole bone tissue or in the density centrifugation fractions of bone during aging and maturation.

Regulation of Adult Bone Mass by the Zinc Finger Adapter Protein Schnurri-3

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.

A resolution-enhanced Fourier transform infrared spectroscopic study of the environment of the CO3(2-) ion in the mineral phase of enamel during its formation and maturation.

SummaryA resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopic study of the CO32− ion in pig enamel of increasing age and maturity has demonstrated the existence of four different, main carbonate locations. The major CO32− site arises as a result of the substitution of CO32− ions in the positions occupied by PO43− ions in the apatitic lattice. In addition, two minor locations have been identified in positions in which the CO32− ions substitute for OH− ions. The fourth carbonate group appears to be in an unstable location. Its concentration has been found to decrease with aging and maturation, during which there is a progressive increase in the amount of mineral deposited in the enamel. The distribution of the carbonate ions in the different apatitic sites varies randomly during the formation of the mineral phase in enamel and during its maturation. Although these changes have been shown to be related to changes in the composition of the mineral phase, a comparison of the parameters assessing the degree of crystallinity of the mineral phase from υ2CO32− and υ4PO32− infrared absorption data reveals a significant discrepancy related to the nonhomogeneous partition of the CO32− ion in the mineral phase. After maximum mineralization is reached, the composition of the mature mineral phase is decidedly different than that of the initial mineral deposited; the changes affect principally the concentrations of Ca2+, OH−, and HPO42− ions, but not the CO32− ions.

Nuclear Magnetic Resonance Spin‐Spin Relaxation of the Crystals of Bone, Dental Enamel, and Synthetic Hydroxyapatites

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short‐range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus‐31 solid state nuclear magnetic resonance (NMR) spin‐spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei.31P magnetization prepared by a 90° pulse or by proton‐phosphorus cross‐polarization (CP) yielded different second moments and experienced different degrees of proton spin‐spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4−2) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4−3) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4−2 groups is higher in the younger, less mature biological crystals.

The presence of γ-carboxyglutamic acid in the proteins associated with ectopic calcification

Abstract γ-Carboxyglutamic acid (Gla) is identified in the proteins associated with several types of ectopic calcifications in which hydroxyapatite is the major mineral component. These included the calcified skin and subcutaneous plaques from a patient with dermatomyositis, the calcium containing material extruded from the skin of a patient with scleroderma, and the calcified, atheromatous plaques from aorta. Alkaline hydrolysis of biopsy material from these and from normal tissue revealed the presence of Gla only in the plaque specimens. Since a γ-carboxyglutamic acid-containing protein is normally present in bone and absent in unmineralized tissues, the presence of Gla in soft tissue calcifications is a potentially significant finding, especially in view of its known calcium and phospholipid binding properties.

Poorly crystalline apatites: evolution and maturation in vitro and in vivo

Poorly crystalline apatites (PCA) are the major mineral component of mineralized tissues in vertebrates. Their physical-chemical properties are, however, not very well known due to their relative instability and the difficulties to characterize nanocrystalline compounds. Several studies using spectroscopic techniques (Fourier transform infrared [FTIR]; 31P nuclear magnetic resonance [NMR]) have demonstrated the existence, both in precipitated and biological PCA, of labile non-apatitic environments of the mineral ions. These environments are involved in the high surface reactivity and evolution ability of PCA and they are believed to form a hydrated layer at the surface of the nanocrystals in aqueous media. The extent of the hydrated layer may vary considerably depending on the conditions of precipitation and maturation time. As PCA age, the decrease of the non-apatitic environments proportion is associated with a decrease of intracrystalline disorder and an increase of stable apatitic domains. For synthetic and biological apatites, the carbonation rate of the mineral and the uptake of essential or toxic trace elements can be related to the maturation processes. The mineral ions of the hydrated layer can be easily and reversibly substituted by other ions which can either be included in the growing stable apatite lattice during maturation or remain in the hydrated layer. In addition, the non-apatitic environments seem to be involved in the binding of soluble non-collagenic proteins. This phenomenon could be related to calcium phosphate formation; we showed that, at an albumin concentration close to that in human serum, this protein has an inhibitory effect on octacalcium phosphate crystallization on collagen in vitro.

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.

Evidence of hydroxyl-ion deficiency in bone apatites: an inelastic neutron-scattering study

The novelty of very large neutron-scattering intensity from the nuclear-spin incoherence in hydrogen has permitted the determination of atomic motion of hydrogen in synthetic hydroxyapatite and in deproteinated isolated apatite crystals of bovine and rat bone without the interference of vibrational modes from other structural units. From an inelastic neutron-scattering experiment, we found no sharp excitations characteristic of the vibrational mode and stretch vibrations of OH ions around 80 and 450 meV (645 and 3630 cm(-1)), respectively, in the isolated, deproteinated crystals of bone apatites; such salient features were clearly seen in micron- and nanometer-size crystals of pure hydroxyapatite powders. Thus, the data provide additional definitive evidence for the lack of OH(-) ions in the crystals of bone apatite. Weak features at 160-180 and 376 meV, which are clearly observed in the apatite crystals of rat bone and possibly in adult mature bovine bone, but to a much lesser degree, but not in the synthetic hydroxyapatite, are assigned to the deformation and stretch modes of OH ions belonging to HPO(4)-like species.

The Nature of the Mineral Phase in Bone: Biological and Clinical Implications

The subjects that will be presented in this chapter are: (1) The nature of the mineral phase in bone; that is, the chemical composition and crystal structure of the solid calcium-phosphate (Ca-P) mineral phase in bone, and the changes that occur in the mineral phase per se with time and maturation; (2) the relationship of these maturational changes which occur with time to the biological, physiological, and mechanical functions of the crystals and to Ca and P mineral metabolism; and (3) the ultrastructural location of the mineral phase and its relationship to the basic underlying physical chemical mechanism responsible for the initiation of calcification.