Three structural roles for water in bone observed by solid-state NMR

Abstract

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, suggesting a role for this water in mediating mineral-organic matrix interactions. Direct evidence of monohydrogen phosphate in a (1)H NMR spectrum of unmodified bone is presented for the first time. We obtained clear evidence for the presence of hydroxide ion in deproteinated bone by (1)H MAS NMR. A (1)H-(31)P heteronuclear correlation experiment provided unambiguous evidence for hydroxide ion in unmodified bone as well. Hydroxide ion in both unmodified and deproteinated bone mineral was found to participate in hydrogen bonding with neighboring water molecules and ions. In unmodified bone mineral hydroxide ion was found, through a (1)H-(31)P heteronuclear correlation experiment, to be confined to a small portion of the mineral crystal, probably the internal portion.

FIGURE 1

Pulse sequence for ¹H-³¹P frequency-switched Lee-Goldburg Heteronuclear Correlation (FSLG HETCOR) experiment. ¹H magnetization is prepared and allowed to evolve under chemical shift while ¹H-¹H dipolar coupling is suppressed, and then cross-polarized to ³¹P for detection. ³¹P signal with respect to ¹H chemical shift evolution time is Fourier transformed to produce an indirectly detected ¹H spectrum.

FIGURE 2

¹H MAS NMR spectra of synthetic B-type carbonated apatite mineral at (A) 25°C, (B) 125°C, (C) 175°C, and (D) 225°C. Asterisk indicates spinning sideband. The synthetic mineral contains two hydroxide environments and has two water environments at room temperature, one of which resolves into two distinct environments at higher temperature.

FIGURE 3

¹H MAS NMR spectra of deproteinated cortical bone at (A) 25°C, (B) 125°C, (C) 175°C, and (D) 225°C. Deproteinated bone spectra clearly indicate the presence of hydroxide ion and two distinct water environments within the crystallite.

FIGURE 4

(A) ¹H MAS NMR spectrum of partially hydrated cortical bone at room temperature. ¹H dimension projection spectra of (B) hydrated, (C) partially hydrated, and (D) dehydrated cortical bone acquired at room temperature using ¹H-³¹P FSLG HETCOR. Bone samples were dehydrated in ambient air (see Experimental). For the purposes of this work we have defined hydrated bone as bone that exhibits a sharp water peak in its ¹H NMR spectrum and dehydrated bone as bone that has no sharp peaks in its ¹H spectrum and exhibits bulk hydrophobicity. Some water remains in even the dehydrated bone samples; however, enough water has been lost to observe significant changes in the bulk properties of the material. (E) Full 2D ¹H-³¹P FSLG HETCOR spectrum of partially hydrated cortical bone.