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The calcified cartilage of the epiphyseal growth plate of young calves has been studied by x-ray diffraction, Fourier transform infrared spectroscopy, magic angle 31P nuclear magnetic resonance spectroscopy, and chemical composition. The powdered tissue was separated by density centrifugation as a function of mineral content and thus qualitatively of the age of the calcium-phosphorus mineral phase. The individual density centrifugation fractions were examined separately. X-ray diffraction of the samples, especially of the lowest density fractions, revealed very poorly crystalline apatite. Fourier transform infrared spectroscopy and 31P nuclear magnetic resonance spectroscopy revealed the presence of significant amounts of nonapatitic phosphate ions. The concentration of such nonapatitic phosphates increases during the early stages of mineralization but then decreases as the mineral content steadily rises until full mineralization is achieved. The total concentration of carbonate ions was found to be much lower in calcified cartilage than in bone from the same organ (scapula). The carbonate ions are located in both A sites (OH−) and B sites (PO43-), with a distribution similar to that found in bone mineral. However, discrepancies between infrared resolution factors of phosphate and carbonate bands are consistent with a heterogeneous distribution of carbonate ions in poorly organized domains of the solid phase of calcium phosphate. These initial studies permit one to characterize the calcium phosphate mineral phase as a very poorly crystalline, immature calcium phosphate apatite, rich in labile nonapatitic phosphate ions, with a low concentration of carbonate ions compared with bone mineral of the same animal, indeed from the bone of the same organ (scapula).

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.

The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy. The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached.

Bone mineral: update on chemical composition and structure

The density of the organic matrix of bone substance is a critical parameter necessary to clinically evaluate and distinguish structural and metabolic pathological conditions such as osteomalacia in adults and rickets in growing children. Water and fat suppressed proton projection MRI (WASPI) was developed as a noninvasive means to obtain this information. In this study, a density calibration phantom was developed to convert WASPI intensity to true bone matrix density. The phantom contained a specifically designed poly(ethylene oxide)/poly(methyl methacrylate) blend, whose MRI properties (T1, T2 and resonance linewidth) were similar to those of solid bone matrix (collagen, tightly bound water, other immobile molecules), minimizing the need to correct for differences in T1 and/or T2 relaxation between the phantom and the subject. Cortical and trabecular porcine bone specimens were imaged using WASPI with the calibration phantom in the field of view as a stable intensity reference. Gravimetric and amino acid analyses were carried out on the same specimens after WASPI and the chemical results were found to be highly correlated (r2 = 0.98 and 0.95 respectively) to the WASPI intensity. By this procedure the WASPI intensity can be used to obtain the true bone matrix mass density in g cm−3.

New insights into the bone collagen fibril superstructure have been obtained by novel small-angle X-ray scattering analysis. The analysis was carried out on the small-angle equidistant meridional reflections resulting from the periodic structure of collagen fibrils in their axial direction. Conventional two-dimensional analysis is difficult because of the large discrepancy of longitudinal and lateral length scales for individual fibrils, as well as their preferred orientation. The new approach represents an unapproximated analysis of the equidistant meridional reflections, which takes the exact separation of preferred orientation and fibril size effects into account. The analytical results (e.g.axial period, fibril diameteretc.) agree well with the parameters obtained from transmission electron microscopy.

Bone sialoprotein (BSP) is one of the major non-collagenous glycosylated phosphoproteins of the extracellular matrix in bone. In vitro studies suggest that BSP may play important roles in the initiation and/or growth of calcium-phosphate crystals. To investigate the potential role of BSP in more complex in vivo environments, we implanted purified bovine BSP with type-I collagen as a carrier into surgically created rat calvarial defects and thoracic subcutaneous pouches. The responses to the implants were assessed by histochemistry, immunohistochemistry, in situ hybridization, quantitative real-time PCR, and biochemical analyses. BSP-collagen, but not collagen alone, elicited mineral deposition in the matrix of proliferating cells near the dura at days 4-5 followed by osteoblast differentiation and synthesis of new bone in the mid-portion of the calvarial defects. In contrast, implantation of BSP-collagen into subcutaneous pouches did not induce calcification or osteogenesis over the same experimental period. We explored the underlying mechanisms for the site-specific responses to BSP-collagen implants and found that higher levels of calcium content and alkaline phosphatase activity at the cranial site at days 2-5 were associated with the BSP-mediated calcification. We also found that BSP stimulated osteoblast differentiation through up-regulation of cbfa1 and osterix, key transcription factors of osteoblast differentiation, which occurred in the calvarial defects but not in the subcutaneous tissue. These results demonstrate that BSP stimulates calcification and osteogenesis in a site-specific manner, and that local environment and the specificities of responding cells may play critical roles in the function of BSP in vivo.

Calcium phosphate is the dominant solid mineral phase within the skeletal and dental tissues of vertebrates. This chapter concentrates on the structure and composition of the solid calcium inorganic orthophosphate (Ca–Pi) phase in bone and the mechanisms that are thought to induce the onset of this mineralization process as an example of biological mineralization in general. It is important to recognize that the Ca–Pi mineral phase is deposited in a living tissue and is a substance that is continuously being synthesized, resorbed and replaced by the action of living cells. Therefore, the composition, structure and other properties of the solid Ca–Pi mineral phase will change in space and time, depending on the general body metabolism and the local cellular functions in specific regions of bone. Similar considerations arise in studies of the mineralized tissues of invertebrates, where the crystals consist of various lattice arrangements of CaCO3 (calcite, aragonite, vaterite) deposited in hierarchical arrangement with the constituents of the ordered organic matrices. Furthermore, the nature of the Ca–Pi phase in bone is significantly different from synthetic, highly crystallized and geological hydroxylapatites, which is reflected in the physical, structural properties and physiological functions of the biological apatites. This chapter is an attempt to summarize at least some of the historical background leading to the more recent research over the past several decades. The focus here is on investigations at the molecular and nano-scales, now possible both theoretically and experimentally, which have been applied to determine the “nature” of the solid Ca–Pi mineral phase of bone and other calcified vertebrate tissues from the inception of mineralization to the changes that occur during crystal maturation (“crystal aging”) and the “normal” aging of the animal. The topics addressed below include an introduction to the basic concepts, relevant terminology and cellular events involved in bone …

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDS(P)EENGVFK), 51-86 (FYPELKRFAVQSSS(P)DS(P)S(P)EENGNGDS(P)S(P)EEEEEEEETS(P)), 151-165 (EDES(P)DEEEEEEEEEE), and 295-305 (GRGYDS(P)YDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.

Simultaneous small- and wide-angle X-ray diffraction of in vitro calcified highly ordered decalcified shad fish bone collagen have identified the calcium phosphate (Ca-P) crystals, formed as poorly crystalline apatite, and their highly ordered spatial, axial distribution with respect to the supramolecular packing of collagen fibrils. The extent of in vitro calcification was significantly diminished when the supramolecular collagen packing was disrupted. These findings are similar to both electron microscopic and small angle X-ray scattering (SAXS) studies of native shad and other species of fish bone and other animal species and of in vitro experiments of the calcification of purified and reconstituted native type collagen fibrils. The results emphasize the important role of the supramolecular packing of collagen fibrils in the heterogeneous nucleation of apatite crystals initiating calcification. The exquisite spatial relationships of the inorganic crystal and the supramolecular packing of native collagen fibrils also form a two-phase composite substance, providing distinct mechanical properties and other physiological functions to bone substance and tissue.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

Water- and fat-suppressed projection MR imaging (WASPI) utilizes the large difference between the proton Ts of the solid organic matrix and the fluid constituents of bone to suppress the fluid signals while preserving solid matrix signals. The solid constituents include collagen and some molecularly immobile water and exhibit very short T. The fluid constituents include mobile water and fat, with long T. In WASPI, chemical shift selective low-power π/2 pulses excite mobile water and fat magnetization which is subsequently dephased by gradient pulses, while the magnetization of collagen and immobile water remains mostly in the z-direction. Additional selective π pulses in alternate scans further cancel the residual water and fat magnetization. Following water and fat suppression, the matrix signal is excited by a short hard pulse and the free induction decay acquired in the presence of a gradient in a 3D projection method. WASPI was implemented on a 4.7 T MR imaging system and tested on phantoms and bone specimens, enabling excellent visualization of bone matrix. The bone matrix signal per unit volume of bovine trabecular specimens was measured by this MR technique and compared with that determined by chemical analysis. This method could be used in combination with bone mineral density measurement by solid state 31P projection MRI to determine the degree of bone mineralization. Magn Reson Med 50:59–68, 2003. © 2003 Wiley-Liss, Inc.

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 the 31P 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 degree 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 inorganic phase of bone is comprised primarily of very small mineralites. The size and shape of these mineralites play fundamental roles in maintaining ionic homeostasis and in the biomechanical function of bone. Using atomic force microscopy, we have obtained direct three-dimensional visual evidence of the size and shape of native protein-free mineralites isolated from mature bovine bone. Approximately 98% of the mineralites are less than 2 nm thick displaying a plate-like habit. Distributions of both thickness and width show single peaks. The distribution of lengths may be multimodal with distinct peaks separated by approximately 6 nm. Application of our results is expected to be of use in the design of novel orthopaedic biomaterials. In addition, they provide more accurate inputs to molecular-scale models aimed at predicting the physiological and mechanical behavior of bone.

Characterization of the very early calcium phosphate (CaP) crystals deposited in bone or in osteoblast cell cultures has been hampered by the overwhelming presence of organic matrix components and cells that obscure spectral analyses. We have overcome this problem using isolated protein-free crystals and have obtained new data including 31P nuclear magnetic resonance (NMR) spectra for the first time from mineral crystals deposited during osteoblast calcification in culture. Crystals were isolated from cultures at two time points: (a) at first calcium accumulation (day 8-10) and (b) after 60 days of culture, to assess maturational changes. The analyses show that the chemical composition overall and short range order of the early and mature crystals are characteristic of the apatite crystals found in young embryonic chick bone in vivo. No mineral phase other than apatite was detected by any of the methods used. 31P NMR spectroscopy identified the HPO4 groups as those present in bone apatite. Similar to bone apatites, no OH groups were detected by Fourier transform infrared (FTIR) spectroscopy. The temporal maturational changes in composition and structure of the mineral phase were difficult to assess because of the continuous deposition of crystals throughout culturing. The pathway of the maturational changes observed were similar to those occurring in chick bone in vivo and synthetic apatite crystals in vitro although to a much smaller extent.

Surface modification of biomaterials to improve biocompatibility without changing their bulk properties is desired for many clinical applications and has become an emerging technology in biomaterial research and industry. In the present study, a simple method of coating the solid surfaces of metals, organic tissue matrices, glasses, inorganic ceramics as well as organic polymers with a thin film of low-crystalline apatite crystals (LCA) was developed. Acidic solution containing calcium and phosphate ions was neutralized with alkaline solution to form calcium phosphate precipitates at low temperature. Precipitates of solid calcium phosphate particles were, then, removed by filtration. Concentration of free ions in the filtered ion solution which were not involved in the formation of calcium phosphate precipitate was high enough to induce the heterogeneous nucleation on the solid surfaces at low temperature. Thin layers of calcium phosphate crystals were formed on the surfaces of metals, glasses, inorganic ceramics, organic polymers including hydrophobic ones, and biological tissue matrices with this solution. The thin layer of crystals consisted of poorly crystalline calcium phosphate apatite crystals which contain high amount of labile ions like bone crystals and did not dissolve in the physiologic solutions. Various cells attached to this crystal layer and proliferated well.

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.

Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus–type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-γ production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.

Multinuclear three-dimensional solid-state MRI of bone, tooth, and synthetic calcium phosphates is demonstrated in vitro and in vivo with a projection reconstruction technique based on acquisition of free induction decays in the presence of fixed amplitude magnetic field gradients. Phosphorus-31 solid-state MRI provides direct images of the calcium phosphate constituents of bone substance and is a quantitative measurement of the true volumetric bone mineral density of the bone. Proton solid-state MRI shows the density of bone matrix including its organic constituents, which consist principally of collagen. These solid-state MRI methods promise to yield a biological picture of bone richer in information concerning the bone composition and short range-crystalline order than the fluid-state images provided by conventional proton MRI or the density images produced by radiologic imaging techniques. Three-dimensional solid-state projection reconstruction MRI should be readily adaptable to both human clinical use and nonmedical applications for a variety of solids in materials science.

Synchrotron small-angle X-ray scattering (SAXS) was conducted on native intramuscular shad/herring bone samples. Two-dimensional SAXS patterns were quantitatively analyzed with special consideration for preferred orientation effects, leading to new insights into the three-dimensional superstructure of mineralized collagen fibrils in shad/herring bone.

International audience; The adsorptive properties of synthetic calcium phosphates analogous to bone mineral were examined with respect to cisplatin and risedronate, two biological active drugs; the uptake and release experiments were carried out under various conditions in order to understand the basic mechanism of interaction. The effect of temperature and solution composition were highlighted and discussed. The adsorption results obtained for the therapeutic agents demonstrated that, depending on the conditions investigated (nature of the sorbent, concentration range, ionic composition, temperature. . . ), the shape of the isotherms is of Freundlich or Langmuir type. The adsorption is described as an ion-exchange process in dilute solutions, while the interaction appears to be reactive for concentrated solutions (dissolution of mineral ions from the apatite substrate and formation of soluble calcium complex and/or precipitation of calcium salts involving sorbate molecules). The information gained on the surface reactivity of calcium phosphate were exploited to associate an antibiotic to calcium phosphate cements for drug delivery applications. The specimens were obtained by combination of calcium phosphate and calcium carbonate powders upon mixing with water. The physicochemical properties of the paste were altered by the drug loading method (in the liquid or solid phase). Thus, a dose-dependent effect was noticed for the paste setting time, hardening and the release process.

Apatite crystals of the calcified zone of the subarticular cartilaginous growth plates of the long bones of young growing chickens and calves were isolated by low temperature reaction with hydrazine and plasma ashing and examined by electron microscopy, electron diffraction and microprobe analysis, and computer-generated deconvolution of the spectra obtained by Fourier transform infrared spectroscopy. The crystal habit was that of wide, very thin, relatively long rectangular plates, which tended to form small clusters of crystals, possibly because reaction with hydrazine alone did not remove all of the organic matrix constituents. Further reaction with low power plasma ashing released more of the isolated crystals although to a lesser extent than was possible with bone. Stereograms of the small clusters showed that many of the crystals in the small isolated aggregates of crystals were bent and/or curved. Together with the resultant overlap of individual adjacent crystals, they also produced images of sharp, very dense lines, reminiscent of the electron-dense needle or rod-like appearances frequently observed by transmission electron microscopy of thin sections of calcified cartilage and thought to represent the habit of the apatite crystals. No true rod or needle-like crystals were observed in the isolated crystals. Although the overall general apatitic structure of the apatite crystals was similar to that of the apatitic crystals of bone, the individual crystals were significantly larger than those of bone from the same specimen, and there were small but significant differences in the concentrations of acid phosphate and carbonate groups and in their short range order.

The CD44 family of surface receptors regulates adhesion, movement, and activation of normal and neoplastic cells. The cytokine osteopontin (Eta-1), which regulates similar cellular functions, was found to be a protein ligand of CD44. Osteopontin induces cellular chemotaxis but not homotypic aggregation, whereas the inverse is true for the interaction between CD44 and a carbohydrate ligand, hyaluronate. The different responses of cells after CD44 ligation by either osteopontin or hyaluronate may account for the independent effects of CD44 on cell migration and growth. This mechanism may also be exploited by tumor cells to promote metastasis formation.

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.

Activating transcription factor-2 (ATF-2) is a basic region leucine zipper protein whose DNA target sequence is the widely distributed cAMP response element (CRE). We report here that mice carrying a germline mutation in ATF-2 demonstrated unique actions of ATF-2 not duplicated by other ATF/CREB family members. Mutant mice had decreased postnatal viability and growth, with a defect in endochondral ossification at epiphyseal plates similar to human hypochondroplasia. The animals had ataxic gait, hyperactivity and decreased hearing. In the brain, there were reduced numbers of cerebellar Purkinje cells, atrophic vestibular sense organs and enlarged ventricles. Unlike CREB alpha/delta-deficient mice whose main defect is in long-term potentiation, the widespread abnormalities in ATF-2 mutant mice demonstrate its absolute requirement for skeletal and central nervous system development, and for maximal induction of select genes with CRE sites, such as E-selectin.

Calcium phosphate crystals deposited in the organic matrix synthesized by chick bone osteoblasts in culture were studied by x-ray and electron diffraction, Fourier transform infrared spectroscopy and chemical composition. The amounts of mineral phase deposited with time and the extent of calcification (% of mineral phase in the tissue) were also determined as a function of time, as were the nature of the changes in the short range order of the crystals. The amount of mineral deposited and the extent of calcification increased with time; the tissue not only contained more crystals of apatite, but the extent of calcification also increased with time as it does in vivo. After 30 days of culture the extent of calcification in the cell culture matrix was similar to that in late chick embryonic and early postnatal chick tibiae. The nature of the CO3 and HPD4 environments were similar to those found in vivo although the concentrations of these ions and the changes in their concentrations with time appeared to develop more slowly in cell culture than they do in vivo. However, the general overall pathway of maturation was similar in cell culture to that observed in vivo.

Chicken bone powder, sequentially extracted with 4M guanidine HCl (NG), 0.3M HCl (H), 0.5 NaCl at neutral pH (NS), and 4M guanidine HCl pH 7.4 (G), preferentially solubilized osteocalcin (OC), osteopontin (OPN), alpha 2 HS-glycoprotein, bone sialoprotein (BSP), and osteonectin (ON). These studies provide the first amino acid sequences for a non-mammalian form of ON and both N-terminal and internal amino acid sequences obtained for ON demonstrated that the avian form of this protein is more than 80% conserved for all the peptides sequenced in this study. A novel approximately 60 kDa protein was identified with a unique N-terminal amino acid sequence and several internal amino acid sequences. Amino acid composition of this protein indicates that it is similar to other acidic glycosylated phosphoprotein of bone, and it is phosphorylated principally on serine residues, although a small amount of phosphorylated threonine residues were also detected. Solubility characteristics of this protein differed from those of ON, OPN and Decorin, and, it was not recognized by polyclonal antibodies to ON, BSP, OPN or by a monoclonal antibody to Decorin. This protein was not present in chicken serum and no comparable sequences were found in the GenBank or EMBL protein sequence database. These studies provide the first sequence for a non-mammalian form of ON and identify a novel acidic bone protein.

The detailed chemical composition and microstructure of freshly deposited bone mineral, and how these properties change with maturation of the mineral, have been studied intensively and still remain controversial. For example, current analytical technology is inadequate for the unambiguous characterization of the monohydrogen phosphate ions in bone mineral. Using a differential cross polarization/magic angle spinning solid state nuclear magnetic resonance spectroscopy technique, we suppress the dominant orthophosphate (PO4-3) signal to reveal the spectra of the minor phosphate constituents. This method depends upon differences in the cross polarization time constants for phosphorus-31 nuclei in protonated and non-protonated phosphate ions. It is now possible for the first time to directly measure both the proportion of acid phosphate (HPO4-2) as well as the parameters which characterize its isotropic and anisotropic chemical shift. In bone from three species at several developmental stages, we have found a single type of acid phosphate species, identical in all of the specimens examined. The phosphorus-31 isotropic chemical shift of this acid phosphate group in bone mineral corresponds precisely with that of acid phosphate in octacalcium phosphate, and not with that of brushite. In contrast, the bone acid phosphate anisotropic chemical shift parameters are close to those of brushite, and differ significantly from those of octacalcium phosphate. The orthophosphate resonances of bone mineral, synthetic hydroxyapatite and synthetic octacalcium phosphate share identical chemical isotropic shifts, and similar chemical shift anisotropies. The implication of these results is that the intimate structure of the acid phosphate group in bone mineral is unique, and that none of the common synthetic calcium phosphates accounts well for all of the observed solid state phosphorus-31 NMR properties of bone mineral.

The spatial and temporal distribution of transcripts for the TRE/CRE-binding basic region-leucine zipper protein hXBP-1 was determined by in situ hybridization. Analysis of embryos from day 10.5 to 18.5 pc revealed high level expression of hXBP-1 RNA in two developing organ systems: (1) in bone and cartilage cells of the developing skeleton and toothbuds, and (2) in exocrine glands including the pancreas and the submandibular and salivary glands. High level expression was also found in whisker follicles and in selected cells in brown adipose tissue. In the developing skeleton, hXBP-1 RNA was expressed starting on day 11.5 pc in osteoblasts of newly formed intramembranous bone. Thereafter, hXBP-1 was expressed in both osteoblasts and preosteoblasts in bone formed directly by intramembranous formation as well as in bone formed during endochondral ossification. The most intese signal was observed in preosteoblasts and osteoblasts of newly forming bone. At day 11.5 pc low level hXBP-1 expression was also observed in matrix secreting chondroblasts of bones which are formed initially of cartilage, at the stage where they consist entirely of cartilage, at the stage where they consist entirely of cartilage. Signal was also present in matrix producing chondroblasts of the mature zone of the growth region during endochondral ossification although at significantly lower level than in osteoblasts. hXBP-1 is thus the first transcription factor described, to our knowledge, whose level of expression is modulated during the osteoblast developmental sequence in vivo. The pattern of expression of hXBP-1 in the developing skeleton was found to be very similar to that of the genes encoding the tissue inhibitor of metalloproteinase and alkaline phosphatase throughout development. These observations suggest that hXBP-1 may play a role in regulating the expression of tissue specific genes (TIMP, osteonectin, osteopontin, osteocalcin) expressed in osteoblasts. It is intriguing that the promoter regions of several such genes contain potential hXBP-1 binding sites. © 1993 Wiley-Liss, Inc.

To understand the role of post-translational modifications on the structure and function of osteopontin, a secreted glycosylated phosphoprotein, we expressed mouse osteopontin in E. coli as a fusion protein with glutathione-S-transferase (GST). The purified fusion protein was cleaved by factor Xa generating GST (26 kDa) and recombinant osteopontin (60 kDa). The fusion protein was phosphorylated in vitro by cytosolic, microsomal, and casein kinase II fractions from mouse kidney homogenates. The fusion protein and recombinant osteopontin were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The suitability of the fusion and recombinant proteins as model substrates for the study of the function(s) and post-translational modifications of osteopontin is discussed.

Affinity chromatography of “Enamelin Extracts” of developing bovine molar enamel on CNBr activated Sepharose 4B to which polyclonal antibodies of whole bovine serum and fetuin were cross-linked, revealed that at most, only 1–2% of the proteins in the extracts were not bound to the columns. The approximately 98% or more of the proteins in such extracts were bound to the resin and were eluted in the position of the serum proteins and fetuin. The small amount of protein which was not bound to the affinity column and which was eluted very early, was subjected to SDS-PAGE and immunostained with polyclonal antibodies to two non-amelogenin proteins isolated and identified previously (∼26kDa and 22kDa). The antibody to the ∼50kDa protein immunostained strongly with only one protein band of ∼26kDa. The antibody to the ∼22kDa band reacted strongly with one protein band of ∼22kDa, weakly with an ∼100kDa and very weakly with several lower molecular weight bands, suggesting either aggregation and degredation of the 22...

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Purpose: To demonstrate water- and fat-suppressed proton projection MRI (WASPI) in a clinical scanner to visualize the solid bone matrix in animal and human subjects. Materials and Methods: Pig bone specimens and polymer pellets were used to optimize the WASPI method in terms of soft-tissue suppression, image resolution, signal-to-noise ratio, and scan time on a 3T MRI scanner. The ankles of healthy 2–3-month-old live Yorkshire pigs were scanned with the optimized method. The method was also applied to the wrists of six healthy adult human volunteers to demonstrate the feasibility of the WASPI method in human subjects. A transmit/receive coil built with proton-free materials was utilized to produce a strong B1 field. A fast transmit/receive switch was developed to reduce the long receiver dead time that would otherwise obscure the signals. Results: Clear 3D WASPI images of pig ankles and human wrists, showing only the solid bone matrix and other tissues with high solid content (eg, tendons), with a spatial resolution of 2.0 mm in all three dimensions were obtained in as briefly as 12 minutes. Conclusion: WASPI of the solid matrix of bone in humans and animals in vivo is feasible. J. Magn. Reson. Imaging 2010;31:954–963. ©2010 Wiley-Liss, Inc.

One-dimensional 31P nuclear magnetic resonance images (projections) of synthetic calcium hydroxyapatite, Ca10(OH)2(PO4)6, have been obtained for samples on the order of 0.5 to 1.0 cm in linear extent at 7.4 T magnetic field strength. Because of the solid state nature of these samples, short 31P spin-spin relaxation times under 1 ms occur, necessitating echo times of 1 ms and phase-encoding magnetic field gradient pulses shorter than 500 microseconds. Optimal projection quality and shortest acquisition times result from pulsed gradient phase-encoding of the spatial dimension, using a compensating gradient pulse to cancel the distorting effects of gradient waveform transients. The exceedingly long 31P spin-lattice relaxation times could lead to potentially intolerable image acquisition times; these have been reduced with a flipback pulse technique. In addition to holding great potential as a novel research tool in the study of biomineralization of those organisms containing calcium phosphate solid phases, these methods should be of general utility in the multinuclear imaging of a wide variety of solids of interest in biophysics and materials science.

Despite the widespread use of bisphosphonates, its effects on normal bone microarchitecture of the proximal femur are still poorly studied. The purpose of this study was to determine the effects of long-term high-dose treatment of alendronate on microstructure and bone mineral density of cancellous, cortical compact and subchondral compact bone of the femoral head and neck region in normal adult male rabbits. Thirty-two adult, male rabbits were randomized into and were treated with either alendronate or placebo for 6 and 12 months. Micro-QCT measurements were taken in the (1) trabecular region, (2) cortical region of the femoral neck and (3) the subchondral region of the femoral head. In the trabecular region of the femoral head, alendronate treatment significantly increased vBMD at 6 and 12 months (+21.0%, p

A resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopic study of the CO3(2-) ion in pig enamel of increasing age and maturity has demonstrated the existence of four different, main carbonate locations. The major CO3(2-) site arises as a result of the substitution of CO3(2-) ions in the positions occupied by PO4(3-) ions in the apatitic lattice. In addition, two minor locations have been identified in positions in which the CO3(2-) 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 upsilon 2CO3(2-) and upsilon 4PO4(3-) infrared absorption data reveals a significant discrepancy related to the nonhomogeneous partition of the CO3(2-) 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 HPO4(2-) ions, but not the CO3(2-) ions.

Localization of a ∼66 kD glycosylated phosphoprotein during morphogenesis of the embryonic chick tibia has been accomplished using immunohistochemistry. Although initial expression of the tibial osteoblast phenotype is detected as early as stage 28.5, with the deposition of osteoid matrix beginning at stage 30, little or no immunoreactivity against the ∼66 kD glycosylated phosphoprotein is observed in pre-osteoblasts, osteoblasts, osteocytes, or in the uncalcified osteoid matrix during the early events of tibia development. Immunoreactivity was first observed at stage 32 when mineralization of the osteoid matrix is initiated. At this and all later stages, the phosphoprotein is located almost exclusively in the extracellular matrix at the mineralization front with essentially no detectable staining in the adjacent unmineralized osteoid matrix. Similarly, no cellular staining is observed when even the lightly mineralized extracellular matrix is strongly immunoreactive. Only scant immunostaining is present over the heavily mineralized regions, although demineralization of these areas with EDTA exposes a low intensity, punctate staining pattern. Additionally, cryosections of developing calvaria stained with this antiserum only display reactivity in regions of bone matrix undergoing mineralization. These localization studies support the hypothesis that this phosphoprotein is intimately associated with the process of bone matrix mineralization in the developing chick long bone.

The density of the organic matrix of bone substance is a critical parameter necessary to clinically evaluate and distinguish structural and metabolic pathological conditions such as osteomalacia in adults and rickets in growing children. Water- and fat-suppressed proton projection MRI (WASPI) was developed as a noninvasive means to obtain this information. In this study, a density calibration phantom was developed to convert WASPI intensity to true bone matrix density. The phantom contained a specifically designed poly(ethylene oxide)/poly(methyl methacrylate) (PEO/PMMA) blend, whose MRI properties (T(1), T(2), and resonance linewidth) were similar to those of solid bone matrix (collagen, tightly bound water, and other immobile molecules), minimizing the need to correct for differences in T(1) and/or T(2) relaxation between the phantom and the subject. Cortical and trabecular porcine bone specimens were imaged using WASPI with the calibration phantom in the field of view (FOV) as a stable intensity reference. Gravimetric and amino acid analyses were carried out on the same specimens after WASPI, and the chemical results were found to be highly correlated (r(2) = 0.98 and 0.95, respectively) to the WASPI intensity. By this procedure the WASPI intensity can be used to obtain the true bone matrix mass density in g cm(-3).

To assess possible differences between the mineral phases of cortical and cancellous bone, the structure and composition of isolated bovine mineral crystals from young (1-3 months) and old (4-5 years) postnatal bovine animals were analyzed by a variety of complementary techniques: chemical analyses, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and (31)P solid-state magic angle spinning nuclear magnetic resonance spectroscopy (NMR). This combination of methods represents the most complete physicochemical characterization of cancellous and cortical bone mineral completed thus far. Spectra obtained from XRD, FTIR, and (31)P NMR all confirmed that the mineral was calcium phosphate in the form of carbonated apatite; however, a crystal maturation process was evident between the young and old and between cancellous and cortical mineral crystals. Two-way analyses of variance showed larger increases of crystal size and Ca/P ratio for the cortical vs. cancellous bone of 1-3 month than the 4-5 year animals. The Ca/(P + CO(3)) remained nearly constant within a given bone type and in both bone types at 4-5 years. The carbonate and phosphate FTIR band ratios revealed a decrease of labile ions with age and in cortical, relative to cancellous, bone. Overall, the same aging or maturation trends were observed for young vs. old and cancellous vs. cortical. Based on the larger proportion of newly formed bone in cancellous bone relative to cortical bone, the major differences between the cancellous and cortical mineral crystals must be ascribed to differences in average age of the crystals.

Osteopontin is a noncollagenous, phosphorylated extracellular glycoprotein, expressed in mineralized and nonmineralized tissues, organs and body fluids. The protein contains an RGD tripeptide cell-binding motif, and is subjected to a variety of posttranslational modifications that play important roles in its multiple biological functions, such as bone remodeling and inhibition of pathological calcification. In this study, we have expressed bovine osteopontin in a prokaryotic system and identified the seven amino acid residues phosphorylated in vitro by CKII.

Embryonic chicken osteoblasts cultured over a 30 day period were used as a model system for studying the expression of bone phosphoproteins during cellular differentiation and the possible role of these proteins in extracellular matrix mineralization. Accumulation of total phosphoprotein in the cultures, as determined by O-phosphoserine (Ser-P) and O-phosphothreonine (Thr-P) amino acid analysis, revealed a greater than 10-fold increase over the 30 day period. Total phosphoprotein synthesis, as assessed by (32P)-, (3H)-Ser-P, and (14C)-Thr-P protein labeling, showed the highest levels concurrent with initial mineral deposition within the matrix. The major phosphoprotein present in chicken bones and synthesized by the cultured osteoblasts had a molecular weight of approximately 66 kDa. This 66 kDa bone phosphoprotein (66 kDa BPP) was purified to homogeneity and was used for antibody production. Application of this antibody in Western blot analysis revealed that 66 kDa BPP was present only in protein extracts of mineralizing cultured osteoblasts and was absent in cultures of non-mineralizing chondrocytes, myoblasts, and tendon fibroblasts. The 66 kDa BPP in vitro accumulated continuously in the extracellular matrix in a manner that paralleled both phosphoprotein synthesis and total phospho-amino acid production. A comparison of the results obtained in vitro to those from developing embryonic tibiae in vivo demonstrated a similar qualitative and temporal expression of phosphoprotein and a continual accumulation of 66 kDa BPP in the matrix with advancing mineralization and developmental age. Ultrastructural immunocytochemistry using the 66 kDa BPP antibody and the protein A-gold technique revealed specific immunolabeling over electron-dense regions of mineralization in the cultures that appeared identical to the distribution of labeling observed in vivo (McKee et al.: Connect. Tissue Res., 21:21-29, 1989; Anat. Rec., 228:77-92, 1990). These results demonstrate that this major 66 kDa BPP was expressed concurrently with other differentiated osteoblast functions and suggests that it may play a role in the initiation or regulation of mineralization.

Biochemical analyses and immunocytochemistry were used to examine the developmental appearance of a major approximately 66 kDa bone phosphoprotein (66 kDa BPP) in the mid-diaphyseal region of embryonic and post-natal chicken tibiae in vivo. Total protein and O-phosphoserine (Ser-P) and O-phosphothreonine (Thr-P) content of 8-, 12-, and 18-day embryonic, and 4-wk post-natal chicken tibiae were determined by amino acid analysis. Similar bone samples were carried through a wide variety of tissue-processing regimes including different protocols for fixation, decalcification, dehydration, and embedding prior to electron microscopy. For immunocytochemistry, tissue sections were incubated with a polyclonal antibody raised in rabbits against 66 kDa BPP, and the antigen was revealed by the high-resolution protein A-gold technique. Amino acid analysis, Western blotting, and immunocytochemistry all showed the presence and increasing concentration of bone phosphoprotein with advancing developmental age. Immunogold labeling was observed over osteoblasts and mineral deposits throughout the bone with the most intense reaction occurring at the mineralization front in embryonic tibiae. Electron probe X-ray microanalysis confirmed the association of 66 kDa BPP with mineral. The levels of phosphoprotein in the tissue were directly correlated with increasing degrees of mineralization. These observations are consistent with previous proposals suggesting that phosphoproteins may play a significant role in the calcification of bone matrix.

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with polyclonal and monoclonal antibodies to albumin. Two-dimensional separation of the components in the enamelin extract by isoelectric focusing and SDS/PAGE reveal that the major approximately 67-kDa component and almost all of the minor Coomassie-staining protein components of approximately 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The approximately 67-kDa band eluted after SDS/PAGE, as well as the major approximately 67-kDa spots eluted after two-dimensional separation, were found to have N-terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70-80% of the total protein content of the enamelin extract and was essentially the only protein in the approximately 67-kDa component. The serum proteins alpha-2 HS glycoprotein, gamma-globulin and fetuin, and the proline-rich salivary protein termed P-B were also identified in the enamelin extract. The serum proteins and the salivary protein account for greater than 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non-serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N-terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true 'enamelins' synthesized by ameloblasts. One additional protein had the first five N-terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N-terminally blocked on attempted sequencing. None of the components in the neutral soluble low-ionic-strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti-albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain non-amelogenin proteins. On the basis of the data presented, studies employing antibodies to the so-called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed.

In order to understand the mechanism of the post-translational processing of bone phosphoproteins in embryonic bone, periosteal bone strips isolated from 12-day-embryonic-chick tibiae were cultured and the bone proteins labelled with Na2H32PO4. Of the total radiolabelled proteins recovered from the medium and bone extracts in the absence of SDS (‘medium’, ‘EDTA extract’ and ‘EDTA/guanidinium chloride extract’), nearly 80% of the radioactivity was found in the EDTA extract. The three major radiolabelled phosphoproteins in the EDTA extract of apparent Mr 68,000, 63,000 and 58,000 reacted with polyclonal as well as monoclonal antibodies raised against ‘32-kDa’ and ‘150-kDa’ bone phosphoproteins which were derived from 14-week-old chicken. Therefore these phosphorylated embryonic proteins are identified as chicken bone phosphoproteins. Judging from their common N-terminal sequences, differences in the patterns obtained by labelling them with several radioisotopes, and slightly different amino acid compositions, these components seem to have been derived from the same original protein by sequential proteolytic cleavage and other processing such as glycosylation and phosphorylation.

In order to investigate the possible existence in biological and poorly crystalline synthetic apatites of local atomic organizations different from that of apatite, resolution-enhanced, Fourier transform infrared spectroscopy studies were carried out on chicken bone, pig enamel, and poorly crystalline synthetic apatites containing carbonate and HPO4 2− groups. The spectra obtained were compared to those of synthetic well crystallized apatites (stoichiometric hydroxyapatite, HPO4 2−-containing apatite, type B carbonate apatite) and nonapatitic calcium phosphates which have been suggested as precursors of the apatitic phase [octacalcium phosphate (OCP), brushite, and β tricalcium phosphate and whitlockite]. The spectra of bone and enamel, as well as poorly crystalline, synthetic apatite in thev 4 PO4 domain, exhibit, in addition to the three apatitic bands, three absorption bands that were shown to be independent of the organic matrix. Two low-wave number bands at 520–530 and 540–550 cm−1 are assigned to HPO4 2−. Reference to known calcium phosphates shows that bands in this domain also exist in HPO4 2−-containing apatite, brushite, and OCP. However, the lack of specific absorption bands prevents a clear identification of these HPO4 2− environments. The third absorption band (610–615 cm−1) is not related to HPO4 2− or OH− ions. It appears to be due to a labile PO4 3− environment which could not be identified with any phosphate environment existing in our reference samples, and thus seems specific of poorly crystalline apatites. Correlation of the variations in band intensities show that 610–615 cm−1 band is related to an absorption band at 560 cm−1 superimposed on an apatite band. All the nonapatitic phosphate environments were shown to decrease during aging of enamel, bone, and synthetic apatites. Moreover, EDTA etching show that the labile PO4 3− environment exhibited a heterogeneous distribution in the insoluble precipitate.

The vibrational circular dichroism (VCD) spectra of metal-free phosvitin are presented as a function of pH and analyzed both qualitatively and by using a factor analysis approach referenced to a protein data set. The qualitative pattern of both the IR and VCD changes is consistent with a coil-to-sheet transition occurring as pH is progressively decreased to values lower than 3. A similar transition was seen in commercial preparation of phosvitin which still contained metal ions, but there the transition was more gradual and occurred at somewhat different pH values. Such a gradual change is also evident in the solution phase absorption band profile but is made clearer using Fourier deconvolution. Based on VCD results, the low pH transition appears to occur with two distinct manifestations of the beta-sheet form. However, at the lowest pH values the sample may precipitate. These two forms are not distinguishable with Fourier transform infrared alone and may be due to a twist of the beta-sheet form or to aggregation.

Carbonate ions which replace phosphates—type B—and hydroxide ions—type A—in bone and synthetic apatites were studied by 13C and 1H solid state NMR spectroscopy. Heated apatites and dipolar suppression experiments have been employed to assign the spectra lines. Two overlapping peaks around 170 ppm which behaved differently on heating were assigned to type B carbontes. Pure type A carbonate gave a sharp signal at 166.5 ppm, and we were able to identify this signal in type AB apatite and in a presumably pure type B carbonate apatite. Amorphous calcium phosphate carbonates gave a broad gaussian peak of about 3 ppm at full width half-maximum with a center of gravity at about 168 ppm. The same resonance line, but with lower intensity, was also observed for apatites containing type A and B carbonates. Magic angle spinning 1H NMR spectra of these apatites showed resolved OH− and H2O signals but were inconclusive in identifying specific structural and adsorbed H2O groups. Implications of the NMR data to the crystal structure of carbonate apatites is discussed.