Bone mineral: new insights into its chemical composition

Abstract

Some compositional and structural features of mature bone mineral particles remain unclear. They have been described as calcium-deficient and hydroxyl-deficient carbonated hydroxyapatite particles in which a fraction of the PO₄^3- lattice sites are occupied by HPO₄^2- ions. The time has come to revise this description since it has now been proven that the surface of mature bone mineral particles is not in the form of hydroxyapatite but rather in the form of hydrated amorphous calcium phosphate. Using a combination of dedicated solid-state nuclear magnetic resonance techniques, the hydrogen-bearing species present in bone mineral and especially the HPO₄^2- ions were closely scrutinized. We show that these HPO₄^2- ions are concentrated at the surface of bone mineral particles in the so-called amorphous surface layer whose thickness was estimated here to be about 0.8 nm for a 4-nm thick particle. We also show that their molar proportion is much higher than previously estimated since they stand for about half of the overall amount of inorganic phosphate ions that compose bone mineral. As such, the mineral-mineral and mineral-biomolecule interfaces in bone tissue must be driven by metastable hydrated amorphous environments rich in HPO₄^2- ions rather than by stable crystalline environments of hydroxyapatite structure.

Figure 1

Detection of hydrogen-bearing species in bone mineral. ¹H-³¹P cross polarization (CP) based magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (ssNMR) spectra of a dry 2-year-old sheep bone tissue sample. (A) two-dimensional (2D) {¹H}³¹P Heteronuclear Correlation (HetCor) spectrum (contact time, t_CP = 1000 µs). Signal intensity increases from blue to red. (B) ¹H projection of the vertical (F1) dimension of the 2D {¹H}³¹P HetCor spectrum shown in (A). {¹H-³¹P}¹H double CP MAS spectra recorded with the following contact times: (C) t_CP1 = t_CP2 = 1000 µs; and, (D) t_CP1 = t_CP2 = 15000 µs. The total experimental time was the same in each experiment (i.e., 9 hours).

Figure 2

Cross-polarization dynamics of the ¹H resonances from bone mineral. {¹H-³¹P}¹H double cross polarization (CP) magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (ssNMR) dynamics of a dry 2-year-old sheep bone tissue sample. Contact time 1 (t_CP1) was fixed at 1000 µs, while contact time 2 (t_CP2) was varied from 75 µs to 1000 µs. Black dashed lines are guidelines for the eyes.

Figure 3

Determination of ³¹P-¹H internuclear distances within the acidic phosphate species present in bone mineral. (A) {¹H-³¹P}¹H double cross polarization (CP) magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (ssNMR) spectrum of a dry 2-year-old sheep bone tissue sample (blue line) and its corresponding fitting (red dashed line). Contact times 1 (t_CP1) was 1000 µs, while contact time 2 (t_CP2) was 500 µs. (B) Numerical modelling of the evolution of the magnetization of the δ(¹H) = 0 (purple, hydroxyl ions); 5.2 (grey, structural water molecules); 9.8 (blue, acidic phosphate species); and, 14.0 (green, acidic phosphate species) ppm peaks shown in (A). The acidic phosphate species peaks were simulated according to Eq. (1), while the hydroxyl ions and structural water molecules resonances were simulated according to Eq. (3). Numerical modelling according to Eq. (1) of the magnetization evolution of the δ(¹H) = 14.0 (C) and 9.8 (D) ppm peaks attributed to acidic phosphate species; together with the calculations of their respective dipolar constant (D_PH) and P•••H distance (d_PH).

Figure 4

Implication of octacalcium phosphate (OCP) in the non-apatitic environments of bone mineral. (A) Two-dimensional (2D) {¹H}³¹P Heteronuclear Correlation (HetCor) magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (ssNMR) spectrum of a fresh 2-year-old sheep bone tissue sample (contact time, t_CP = 1000 µs). Signal intensity increases from blue to red. (B) One-dimensional (1D) individual ³¹P NMR signal of the H₂O and HPO₄²⁻-containing non-apatitic environments attributed to the amorphous surface layer that coats bone mineral particles (blue line); and 1D individual ³¹P NMR signal of the OH⁻-containing apatitic environments that compose the internal crystalline core of bone mineral particles (orange line). These individual ³¹P NMR signals were generated from the 2D {¹H}³¹P HetCor ssNMR spectrum shown in (A). To do so, the F2 slices taken at the bound water molecules position [from δ(¹H) = 3 to 7 ppm, blue area] and hydroxyl ions position [from δ(¹H) = −2 to 2 ppm, orange area] in F1 have been summed. (C) 1D ³¹P CP MAS ssNMR spectrum (t_CP = 1000 µs) of a synthetic octacalcium phosphate (OCP) sample. P1 to P6 correspond to the six different phosphate groups present in the OCP crystal lattice according to the work of Davies et al.. The red dashed-line marks the most intense resonance in the signal of OCP which is not detected in bone mineral (B).

Figure 5

Quantification of HPO₄²⁻ and CO₃²⁻ ions present in bone mineral. (A) Quantitative ³¹P single pulse (SP) magic angle spinning (MAS) solid-state Nuclear Magnetic Resonance (ssNMR) spectrum of a fresh 2-year-old sheep bone tissue sample (blue line) and its corresponding fitting (red dashed line) with two peaks. Those two peaks, whose lineshape and linewidth were revealed in Fig. 4B, correspond to the PO₄³⁻-containing internal crystalline core in the form of hydroxyapatite (orange peak) and the HPO₄²⁻-containing non-apatitic environments in the form of an amorphous surface layer (purple peak). (B) Fourier Transform-Infrared (FT-IR) spectrum of the ν₂(CO₃) vibration mode for a 2-year-old sheep bone tissue sample (blue line) and its corresponding fitting (red dashed line). Type B CO₃²⁻ ions occupy the PO₄³⁻ sites within the hydroxyapatite’s crystal lattice; type A CO₃²⁻ ions occupy the OH⁻ sites within the hydroxyapatite’s crystal lattice; whereas non-apatitic CO₃²⁻ are present within the amorphous surface layer that coats bone mineral particles.

Figure 6

Chemical and structural model of mature bone mineral particles. Schematic representation of platelet-shaped mature bone mineral particles including dimensions and ionic composition according to the results obtained from our 2-year-old sheep bone tissue sample. They are composed by an internal crystalline core in the form of carbonated hydroxyapatite coated by an amorphous layer in which the HPO₄²⁻ ions are concentrated.