Strongly bound citrate stabilizes the apatite nanocrystals in bone

Abstract

Nanocrystals of apatitic calcium phosphate impart the organic-inorganic 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 (2 nm)(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.

Fig. 1.

¹³C NMR spectra of bone, of organic residues at the interface with apatite, and of ¹³C-labeled citrate in bone. (A) Fish bone; (B) avian bone; and (C) bovine bone. (D) Spectra of ¹³C near ³¹P in bovine bone. Thin line: ¹³C{³¹P} REDOR difference (ΔS) spectrum (total measuring time: 19 d). Thick line: Same data but with the spectrum S scaled up by 1.1 to match the 43-ppm peak of Gly in S₀ and thus remove signal of abundant interior collagen residues. (E) Thin line: Spectrum of bovine bone with uniformly ¹³C-labeled bound citrate, introduced after (partial) removal of native citrate by treatment with hot dilute acid. Thick line: Same data after subtraction of the collagen background, isolating the signals of bound citrate. (F) Spectrum of calcium citrate, with minimal line broadening (thin line) and broadened (thick line) to match that in (D) and (E). (G) Same as (F) after 40 μs dipolar dephasing, which selects signals of carbons with weak C─H dipolar couplings, i.e., nonprotonated C and mobile CH_n. (H) Spectrum of bovine bone after 40 μs dipolar dephasing. Citrate signals are highlighted by red arrows.

Fig. 2.

The 76-ppm ¹³C NMR signal in bone is from an immobile (nonprotonated) C─OH group. (A) Wideline ¹H spectra associated with 76-ppm ¹³C signals in bone (solid red line) and sodium citrate dihydrate (dashed orange line), from ¹H-¹³C wideline-separation NMR. Both show no significant motional narrowing. (B) Corresponding ¹H spectra of NCH (protein backbone, dashed line) and CH₃ groups. Only the latter, which undergo rotational jumps, exhibit motional narrowing.

Fig. 3.

Determination of distances of citrate carbons from bone apatite, measured by ¹³C{³¹P} REDOR NMR. The measured dephasing S/S₀ is plotted along with simulated curves for distances z_C-P of 0.3, 0.35, 0.4, 0.45, and 0.5 nm of ¹³C from the first ³¹P layer at the interface (see inset schematic and Fig. S1). (A) Filled black circles: carboxylate resonance at 181 ppm in native bovine bone; open circles: sum of terminal groups in ¹³C-labeled citrate absorbed into bovine bone. Triangles pointing right and left: Dephasing of 178- and 180-ppm ¹³C citrate signals, respectively. (B) Data for the quaternary C-OH of citrate in bone (filled black squares), as well as quaternary C─OH (Cq, open squares), CH₂ (open blue triangles) and the central COO^- (open purple circles) in ¹³C-labeled citrates absorbed into bovine bone. Error margins were determined from the signal-to-noise ratios in the REDOR spectra. The dashed curve is for a ³¹C-³¹P spin pair (two-bond distance of 0.24 nm) as in phosphocitrate.

Fig. 4.

Distances between apatite-bound citrate molecules, probed by CODEX ¹³C NMR with ¹³C spin exchange during the time t_m. Data points (red circles) for citrate with terminal ¹³COO groups on purified bone mineral are compared with fit curves for seven different citrate distributions (Fig. S2). Data points for 2.5 times less citrate, with little intermolecular spin exchange, are shown for reference as open triangles. Inset: One of the best-fit distributions of ¹³C spins (black dots), with some citrate molecules identified by ellipses.

Fig. 5.

Schematic of apatite-bound citrate (with oxygen of the carboxylates in red) interacting with Ca²⁺ on two surfaces of high morphological importance of an idealized bone apatite nanocrystal, at a realistic citrate surface density of ca. 1/(2 nm)². Calcium ions are blue filled circles on top and front surfaces, P is green (omitted on the top surfaces), OH^- ions are pink dots, while phosphate oxygen is omitted for clarity. The hexagonal crystal structure projected along the c-axis (with greater depth of atoms indicated by lighter shading) shown in front reveals various layers of phosphate and calcium ions.