Strongly bound citrate stabilizes the apatite nanocrystals in bone

Abstract

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...

Компьютерные Исследования Наноструктур Гидроксиапатита, Их Особенности И Свойства

“…Citrates have the same influence on the shapes of HAP, which can bound and stabilize the HAP structures by various adhesion features on different surfaces, as it was shown by AFM technique (Fig. 16,a) [58,59]. It was confirmed by molecular modeling [31,32], where interactions of citrate molecules with HAP surface was studied (see Fig.…”

Citrate bridges between mineral platelets in bone

“…The existence of OCP-dicarboxylic acid and OCP-tricarboxylic acid double salts (the nomenclature double salt is used here in line with how these materials have been referred to in the literature previously) was first shown in 1983 (17)(18)(19), with a structural formula of Ca 16 (20). Subsequently, these double salt compounds were shown to have crystallographic structures similar to the parent OCP structure (Fig.…”

“…Its presence in bone is an accepted fact, but until 2010, there was no attempt to rationalize its presence within a structural model of bone mineral. In 2010, the association of citrate with bone mineral was shown by NMR, and it was proposed that citrate was bound to the surface of bone mineral particles (16). Given the very close spacing of the mineral platelets in bone [below the resolution of even the highest resolution transmission electron microscopy (TEM) studies], there is the distinct possibility that, in fact, citrate binds between the surfaces of mineral platelets, helping perhaps to establish the continuum of platelet structures of which bone mineral is observed to consist.…”

Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footprinting

“…LC-MS analysis indicated a substantial and gradual increase in citrate and cis-aconitate production throughout osteogenic induction ( peaking at day 15) in comparison with standard medium control. Interestingly, citric acid has been described as essential for bone organic nanostructure 47 and bone tissue engineering in bone marrow-derived rat MSCs. 48 This suggests that the change in citrate levels may be linked to osteogenic treatment.…”