Detection of Hydroxyl Ions in Bone Mineral by Solid-State NMR Spectroscopy

Cho, Gyunggoo; Wu, Yaotang; Ackerman, Jerome L.

doi:10.1126/science.1078470

Cited by 261 publications

(287 citation statements)

References 20 publications

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“…For example, Xu et al [8] have suggested that in hydroxyapatite the hydroxide ions move under pressures above 3 GPa. There is less hydroxide in bone than in hydroxyapatite [25]. Nonetheless, movement of any hydroxide would influence the larger phosphate, some of which is adjacent to the column of remaining hydroxide, more than the smaller carbonate.…”

Section: Discussionmentioning

confidence: 99%

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

et al. 2005

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Abstract. While the biomechanical properties of bone are reasonably well understood at many levels of structural hierarchy, surprisingly little is known about the response of bone to loading at the ultrastructural and crystal lattice levels. In this study, our aim was to examine the response (i.e., rate of change of the vibrational frequency of mineral and matrix bands as a function of applied pressure) of murine cortical bone subjected to hydrostatic compression. We determined the relative response during loading and unloading of mineral vs. matrix, and within the mineral, phosphate vs. carbonate, as well as proteinated vs. deproteinated bone. For all mineral species, shifts to higher wave numbers were observed as pressure increased. However, the change in vibrational frequency with pressure for the more rigid carbonate was less than for phosphate, and caused primarily by movement of ions within the unit cell. Deformation of phosphate on the other hand, results from both ionic movement as well as distortion. Changes in vibrational frequencies of organic species with pressure are greater than for mineral species, and are consistent with changes in protein secondary structures such as alterations in interfibril cross-links and helix pitch. Changes in vibrational frequency with pressure are similar between loading and unloading, implying reversibility, as a result of the inability to permanently move water out of the lattice. The use of high pressure Raman microspectroscopy enables a deeper understanding of the response of tissue to mechanical stress and demonstrates that individual mineral and matrix constituents respond differently to pressure.Key words: Raman spectroscopy -Bone -Biomechanics -Diamond anvil cell -High pressureThe chemical composition and crystal structure of bone play an essential role in its biological and structural functions [1,2]. Bone tissue can be described as a composite of an organic matrix reinforced with an inorganic mineral phase. The organic matrix is about 90% type I collagen fibrils locally oriented predominantly parallel to each other (in long bone) that provide a supporting matrix upon which the mineral crystals grow. The mineral fraction of bone is a highly impure carbonated apatite situated between collagen fibril cross-links and fibril ends. Both the organic and mineral components and the interactions between the two contribute to boneÕs mechanical properties, including strength, toughness and elasticity. It is not clear though how mineral crystallites deform in response to mechanical loading nor how the matrix deforms. This is particularly true at the atomic level. To better understand deformation it is useful to extract atomic-level compositional information about the changes in inorganic and organic phases that occur when bone undergoes mechanical loading.Raman spectroscopy is a powerful technique for relating bone mechanical properties with bone ultrastructural and crystal lattice changes [3]. Raman spectroscopy provides bone vibrational spectra and, like infrared spectros...

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Section: Discussionmentioning

confidence: 99%

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

et al. 2005

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“…Citrate in bone is fundamentally different from carbonate, fluoride, sodium, magnesium, hydroxide (29), calcium, or phosphate ions, in that it is too large to be incorporated into the apatite crystal lattice. Therefore, bound citrate must remain an interfacial component that affects properties of the bone nanocomposite more profoundly than do simple substitutions of carbonate for phosphate or of sodium for calcium.…”

Section: Resultsmentioning

confidence: 99%

Strongly bound citrate stabilizes the apatite nanocrystals in bone

Rawal

Schmidt‐Rohr

2010

Proc. Natl. Acad. Sci. U.S.A.

472

495

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Nanocrystals of apatitic calcium phosphate impart the organicinorganic nanocomposite in bone with favorable mechanical properties. So far, the factors preventing crystal growth beyond the favorable thickness of ca. 3 nm have not been identified. Here we show that the apatite surfaces are studded with strongly bound citrate molecules, whose signals have been identified unambiguously by multinuclear magnetic resonance (NMR) analysis. NMR reveals that bound citrate accounts for 5.5 wt% of the organic matter in bone and covers apatite at a density of about 1 molecule per ð2 nmÞ 2 , with its three carboxylate groups at distances of 0.3 to 0.45 nm from the apatite surface. Bound citrate is highly conserved, being found in fish, avian, and mammalian bone, which indicates its critical role in interfering with crystal thickening and stabilizing the apatite nanocrystals in bone.T he load-bearing material in bone is a fascinating organicinorganic nanocomposite whose stiffness is provided by thin nanocrystals of carbonated apatite, a calcium phosphate, imbedded in an organic matrix consisting mostly of collagen, a fibrous protein (1-5). The small (ca. 3-nm) thickness of the apatite nanocrystals is favorable for mechanical properties, likely preventing crack propagation (6). While the size and shape of the nanocrystals have been studied extensively (4, 5), the mechanism stabilizing them at a thickness corresponding to only about four unit cells has not been elucidated. A better understanding of the factors controlling the nanocrystals in bone is desirable for prevention and treatment of bone diseases such as osteoporosis, which causes millions of fractures each year (7), and for more efficient synthesis of biomimetic nanocomposites (8, 9). In vitro experiments have shown that carboxylate-rich proteins such as osteocalcin and osteopontin (7) can affect hydroxyapatite crystal formation and growth (10, 11). These observations might suggest that such proteins limit nanocrystal thickening (12); however, these proteins are not sufficiently abundant in vivo to bind to all the nanocrystal surfaces at high enough area concentration; possibly, they control the length of the nanocrystals (7).Here we show instead that the surfaces of the apatite crystals in bone are studded with strongly bound citrate molecules, at a density of ca. 1∕ð2 nmÞ 2 , using advanced solid-state nuclear magnetic resonance (NMR) as a unique tool for probing buried interfaces. Citrate is quite abundant in bone (ca. 1 wt%, or 5 wt% of the organic components) (13,14). Before 1975, citrate in bone was studied by simple wet-chemical methods and thought to regulate bone demineralization (14). However, citrate is no longer even mentioned in most of the prominent literature on the bone nanocomposite published during the last thirty years (1)(2)(3)(4)(5)(15)(16)(17)(18). We now highlight the importance of citrate in bone by demonstrating that it is not a dissolved calcium-solubilizing agent but a strongly bound, integral part of the nanocomposite. Structurally, citrate s...

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“…The environment and structure of the citrate anion in OCPcitrate are both readily probed by 13 C Bloch decay (BD) and cross-polarization (CP) NMR spectra of the material (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…We investigated the possibility of dynamic disorder by cooling a sample of OCP-citrate to 200 K and rerecording 13 C CP magic angle spinning NMR spectra. Cooling the sample to 200 K does not change the relative intensities of the CH 2 (1) and CH 2 (2) signals (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…One suggestion has been that this tightly bound water occupies hydroxyl sites in the core apatitic structure (6). Although hydroxyl groups have been found to be present in bone mineral at only ∼20% of the concentration expected for hydroxyapatite (12,13), it is difficult to see how such a model could incorporate sufficient water molecules while retaining an apatitic structure-certainly, no such synthetic material has been made. Another fact to be rationalized is that NMR shows that the hydrogen phosphate groups in bone mineral, proposed to be in highly hydrated, labile surface layers, are, in fact, relatively immobile: the 31 P chemical shift anisotropies of these groups are comparable with those measured for mineral brushite, for instance (14), in which the hydrogen phosphate groups are static; if the hydrogen phosphate groups in bone mineral are highly mobile, their effective 31 P chemical shift anisotropy should be significantly smaller than that for static hydrogen phosphate groups.…”

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Citrate bridges between mineral platelets in bone

Davies

Müller

Wong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

256

246

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We provide evidence that citrate anions bridge between mineral platelets in bone and hypothesize that their presence acts to maintain separate platelets with disordered regions between them rather than gradual transformations into larger, more ordered blocks of mineral. To assess this hypothesis, we take as a model for a citrate bridging between layers of calcium phosphate mineral a double salt octacalcium phosphate citrate (OCPcitrate). We use a combination of multinuclear solid-state NMR spectroscopy, powder X-ray diffraction, and first principles electronic structure calculations to propose a quantitative structure for this material, in which citrate anions reside in a hydrated layer, bridging between apatitic layers. To assess the relevance of such a structure in native bone mineral, we present for the first time, to our knowledge, 17 O NMR data on bone and compare them with 17 O NMR data for OCP-citrate and other calcium phosphate minerals relevant to bone. The proposed structural model that we deduce from this work for bone mineral is a layered structure with thin apatitic platelets sandwiched between OCP-citrate-like hydrated layers. Such a structure can explain a number of known structural features of bone mineral: the thin, plate-like morphology of mature bone mineral crystals, the presence of significant quantities of strongly bound water molecules, and the relatively high concentration of hydrogen phosphate as well as the maintenance of a disordered region between mineral platelets.NMR crystallography | biomineralization B one is a complex organic-inorganic composite material (1), in which calcium phosphate nanoparticles are held within a primarily collagen protein matrix. The mineral component is a poorly crystalline phase, closely related to hydroxyapatite. The currently accepted model of bone mineral is ∼50-to 150-nmthick stacks of very closely packed apatitic platelets, each of order 2.5-4 nm in thickness (1-4), arranged so that their large (100) faces are parallel to each other and their c axes are strongly ordered (parallel to collagen fibrils) (5). NMR studies show that, in addition to the largely ordered but nonstoichiometric apatitic phase, there is a substantial, highly hydrated, disordered phase containing up to 55% of the bone mineral phosphatic ions (6, 7) but in the form of hydrogen phosphate or phosphate strongly hydrogen-bonded to water rather than apatitic orthophosphate (8). This phase has been assigned as a surface phase, but whether the surface in question is that of individual mineral platelets or the surface of the overall structure formed by a stack of such platelets is not yet clear. There is, however, significant experimental evidence that is not explained by this model as it stands. First, there has never been any observation of an isolated mineral platelet in mature bone, even in preparations in which there have been attempts to disperse the mineral structures (9). This feature suggests that the mineral platelets are not independent structures-indeed, their ordered aggregati...

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Detection of Hydroxyl Ions in Bone Mineral by Solid-State NMR Spectroscopy

Cited by 261 publications

References 20 publications

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

Strongly bound citrate stabilizes the apatite nanocrystals in bone

Citrate bridges between mineral platelets in bone

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