Yaotang Wu

Density of organic matrix of native mineralized bone measured by water- and fat-suppressed proton projection MRI.

Water‐ and fat‐suppressed projection MR imaging (WASPI) utilizes the large difference between the proton T u20092* s 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 u20092* . The fluid constituents include mobile water and fat, with long T u20092* . 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.

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.

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.

Phosphate Ions in Bone: Identification of a Calcium–Organic Phosphate Complex by 31P Solid-State NMR Spectroscopy at Early Stages of Mineralization

Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO4−3 phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO4−2 component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

A Comparison of the Physical and Chemical Differences Between Cancellous and Cortical Bovine Bone Mineral at Two Ages

To assess possible differences between the mineral phases of cortical and cancellous bone, the structure and composition of isolated bovine mineral crystals from young (1–3xa0months) and old (4–5xa0years) postnatal bovine animals were analyzed by a variety of complementary techniques: chemical analyses, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and 31P 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 31P 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 1–3xa0month than the 4–5xa0year animals. The Ca/(Pxa0+xa0CO3) remained nearly constant within a given bone type and in both bone types at 4–5xa0years. 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.

EVALUATION OF BONE MINERAL DENSITY USING THREE-DIMENSIONAL SOLID STATE PHOSPHORUS-31 NMR PROJECTION IMAGING

Abstract. A solid state magnetic resonance imaging technique is used to measure true three-dimensional mineral density of synthetic hydroxyapatite phantoms and specimens of bone ex vivo. The phosphorus-31 free induction decay at 2.0 T magnetic field strength is sampled following application of a short, hard radiofrequency excitation pulse in the presence of a fixed amplitude magnetic field gradient. Multiple gradient directions covering the unit sphere are used in an efficient spherical polar to Cartesian interpolation and Fourier transform projection reconstruction scheme to image the three-dimensional distribution of phosphorus within the specimen. Using 3–6 Gauss/cm magnetic field gradients, a spatial resolution of 0.2 cm over a field of view of 10 cm is achieved in an imaging time of 20–35 minutes. Comparison of solid state magnetic resonance imaging with dual energy X-ray absorptiometry (DXA), gravimetric analysis, and chemical analysis of calcium and phosphorus demonstrates good quantitative accuracy. Direct measurement of bone mineral by solid state magnetic resonance opens up the possibility of imaging variations in mineral composition as well as density. Advantages of the solid state magnetic resonance technique include avoidance of ionizing radiation; direct measurement of a constituent of the mineral without reliance on assumptions about, or models of, tissue composition; the absence of shielding, beam hardening, or multiple scattering artifacts; and its three-dimensional character. Disadvantages include longer measurement times and lower spatial resolution than DXA and computed tomography, and the inability to scan large areas of the body in a single measurement, although spatial resolution is sufficient to resolve cortical from trabecular bone for the purpose of measuring bone mineral density.

Water- and fat-suppressed proton projection MRI (WASPI) of rat femur bone.

Investigators often study rats by μCT to investigate the pathogenesis and treatment of skeletal disorders in humans. However, μCT measurements provide information only on bone mineral content and not the solid matrix. CT scans are often carried out on cancellous bone, which contains a significant volume of marrow cells, stroma, water, and fat, and thus the apparent bone mineral density (BMD) does not reflect the mineral density within the matrix, where the mineral crystals are localized. Water‐ and fat‐suppressed solid‐state proton projection imaging (WASPI) was utilized in this study to image the solid matrix content (collagen, tightly bound water, and other immobile molecules) of rat femur specimens, and meet the challenges of small sample size and demanding submillimeter resolution. A method is introduced to recover the central region of k‐space, which is always lost in the receiver dead time when free induction decays (FIDs) are acquired. With this approach, points near the k‐space origin are sampled under a small number of radial projections at reduced gradient strength. The typical scan time for the current WASPI experiments was 2 hr. Proton solid‐matrix images of rat femurs with 0.4‐mm resolution and 12‐mm field of view (FOV) were obtained. This method provides a noninvasive means of studying bone matrix in small animals. Magn Reson Med 57:554–567, 2007.

Structure, Composition, and Maturation of Newly Deposited Calcium-Phosphate Crystals in Chicken Osteoblast Cell Cultures

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 including31P 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.

Quantitative bone matrix density measurement by water- and fat-suppressed proton projection MRI (WASPI) with polymer calibration phantoms

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 (T1, T2, 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 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 (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 (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. Magn Reson Med 60:1433–1443, 2008.

Quantitative 31P NMR spectroscopy and 1H MRI measurements of bone mineral and matrix density differentiate metabolic bone diseases in rat models

In this study, bone mineral density (BMD) of normal (CON), ovariectomized (OVX), and partially nephrectomized (NFR) rats was measured by (31)P NMR spectroscopy; bone matrix density was measured by (1)H water- and fat-suppressed projection imaging (WASPI); and the extent of bone mineralization (EBM) was obtained by the ratio of BMD/bone matrix density. The capability of these MR methods to distinguish the bone composition of the CON, OVX, and NFR groups was evaluated against chemical analysis (gravimetry). For cortical bone specimens, BMD of the CON and OVX groups was not significantly different; BMD of the NFR group was 22.1% (by (31)P NMR) and 17.5% (by gravimetry) lower than CON. For trabecular bone specimens, BMD of the OVX group was 40.5% (by (31)P NMR) and 24.6% (by gravimetry) lower than CON; BMD of the NFR group was 26.8% (by (31)P NMR) and 21.5% (by gravimetry) lower than CON. No significant change of cortical bone matrix density between CON and OVX was observed by WASPI or gravimetry; NFR cortical bone matrix density was 10.3% (by WASPI) and 13.9% (by gravimetry) lower than CON. OVX trabecular bone matrix density was 38.0% (by WASPI) and 30.8% (by gravimetry) lower than CON, while no significant change in NFR trabecular bone matrix density was observed by either method. The EBMs of OVX cortical and trabecular specimens were slightly higher than CON but not significantly different from CON. Importantly, EBMs of NFR cortical and trabecular specimens were 12.4% and 26.3% lower than CON by (31)P NMR/WASPI, respectively, and 4.0% and 11.9% lower by gravimetry. Histopathology showed evidence of osteoporosis in the OVX group and severe secondary hyperparathyroidism (renal osteodystrophy) in the NFR group. These results demonstrate that the combined (31)P NMR/WASPI method is capable of discerning the difference in EBM between animals with osteoporosis and those with impaired bone mineralization.