Investigation of the Nature of the Protein−Mineral Interface in Bone by Solid-State NMR

Jaeger, Christian; Groom, Nicholas; Bowe, Elizabeth A.; Hörner, A.; Davies, Malcolm; Murray, Rachel C.; Duer, Melinda J.

doi:10.1021/cm050492k

Cited by 94 publications

(151 citation statements)

References 17 publications

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“…23,[44][45][46][47] More recently, 13 C- 31 P correlation experiments have suggested that glycosaminoglycan molecules are located at the interface between the organic and mineral phases of bone and teeth. [48][49][50] To the best of our knowledge, however, solid state NMR has only been used to date to probe the structural environment of the mineral anions: the local environment around the metal cations (Ca 2+ , Mg 2+ or Na + . .…”

Section: àmentioning

confidence: 99%

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy

Laurencin

Wong

Chrzanowski

et al. 2010

Phys. Chem. Chem. Phys.

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110

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International audienceDespite the numerous studies of bone mineral, there are still many questions regarding the exact structure and composition of the mineral phase, and how the mineral crystals become organised with respect to each other and the collagen matrix. Bone mineral is commonly formulated as hydroxyapatite, albeit with numerous substitutions, and has previously been studied by 31P and 1H NMR, which has given considerable insight into the complexity of the mineral structure. However, to date, there has been no report of an NMR investigation of the other major component of bone mineral, calcium, nor of common minority cations like sodium. Here, direct analysis of the local environment of calcium in two biological apatites, equine bone (HB) and bovine tooth (CT), was carried out using both 43Ca solid state NMR and Ca K-edge X-ray absorption spectroscopy, revealing important structural information about the calcium coordination shell. The 43Ca diso in HB and CT is found to correlate with the average Ca–O bond distance measured by Ca K-edge EXAFS, and the 43Ca NMR linewidths show that there is a greater distribution in chemical bonding around calcium in HB and CT, compared to synthetic apatites. In the case of sodium, 23Na MAS NMR, high resolution 3Q-MAS NMR, as well as 23Na{31P} REDOR and 1H{23Na} R3-HMQC correlation experiments give the first direct evidence that some sodium is located inside the apatite phase in HB and CT, but with a greater distribution of environments compared to a synthetic sodium substituted apatite (Na-HA)

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Section: àmentioning

confidence: 99%

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy

Laurencin

Wong

Chrzanowski

et al. 2010

Phys. Chem. Chem. Phys.

Self Cite

110

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“…1D. Strong signals are observed at 182, 169, 76, and 49 ppm; those at 182 and 76 ppm, as well as the inorganic carbonate signal at 169 ppm, had been detected before (17,18), but the strong signal intensity near 49 ppm had not been recognized. We assign the peaks at 182, 76, and 49 ppm to citrate (see structure at top of Fig.…”

mentioning

confidence: 94%

“…Similar to previously published data (17-19), the spectra are dominated by signals of collagen, the fibrous protein rich in glycine (33%), proline, hydroxyproline, and alanine (each 11%) that forms the matrix of the bone nanocomposite. The spectrum of 13 C near 31 P in bovine bone, obtained by 13 Cf 31 Pg rotational echo double resonance (REDOR) NMR (17,20,21), i.e., with 13 C observation and 31 P recoupling pulses, is shown in Fig. 1D.…”

mentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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“…Solid-state nuclear magnetic resonance (NMR) spectroscopy is ideally suited for the characterization of disordered or nanoscale materials and has been shown to provide important structural information in natural dentine [12,13], cartilage [14], and bone minerals [15][16][17][18] and their model compounds [19][20][21][22][23]. The presence of certain moieties known to exist in calcified tissues such as calcium phosphates, hydroxide groups, and water molecules as well as their arrangement and order, can be examined using 31 P and 1 H solid-state NMR.…”

Section: Introductionmentioning

confidence: 99%

Early Stages of Biomineral Formation—A Solid-State NMR Investigation of the Mandibles of Minipigs

et al. 2017

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Solid-state nuclear magnetic resonance (NMR) spectroscopy allows for the identification of inorganic species during the biomineral formation, when crystallite particles visible in direct imaging techniques have not yet been formed. The bone blocks surrounding dental implants in minipigs were dissected after the healing periods of two, four, and eight weeks, and newly formed tissues formed around the implants were investigated ex vivo. Two-dimensional 31 P-1 H heteronuclear correlation (HETCOR) spectroscopy is based on the distance-dependent heteronuclear dipolar coupling between phosphate-and hydrogen-containing species and provides sufficient spectral resolution for the identification of different phosphate minerals. The nature of inorganic species present at different mineralization stages has been determined based on the 31 P chemical shift information. After a healing time of two weeks, pre-stages of mineralization with a rather unstructured distribution of structural motives were found. After four weeks, different structures, which can be described as nanocrystals exhibiting a high surface-to-volume ratio were detected. They grew and, after eight weeks, showed chemical structures similar to those of matured bone. In addition to hydroxyapatite, amorphous calcium phosphate, and octacalcium phosphate, observed in a reference sample of mature bone, signatures of ß-tricalcium phosphate and brushite-like structures were determined at the earlier stages of bone healing.

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Investigation of the Nature of the Protein−Mineral Interface in Bone by Solid-State NMR

Cited by 94 publications

References 17 publications

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy

Probing the calcium and sodium local environment in bones and teeth using multinuclear solid state NMR and X-ray absorption spectroscopy

Strongly bound citrate stabilizes the apatite nanocrystals in bone

Early Stages of Biomineral Formation—A Solid-State NMR Investigation of the Mandibles of Minipigs

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