Time-Resolved Dehydration-Induced Structural Changes in An Intact Bovine Cortical Bone Revealed by Solid-State NMR Spectroscopy

Abstract

Understanding the structure and structural changes of bone, a highly heterogeneous material with a complex hierarchical architecture, continues to be a significant challenge even for high-resolution solid-state NMR spectroscopy. While it is known that dehydration affects mechanical properties of bone by decreasing its strength and toughness, the underlying structural mechanism at atomic-level is unknown. Solid-state NMR spectroscopy, controlled dehydration, and H/D exchange are used for the first time to reveal the structural changes of an intact piece of bovine cortical bone. Proton spectra are used to monitor the dehydration of bone inside the rotor and high-resolution 13 C chemical shift spectra obtained under magic angle spinning are used evaluate the dehydration-induced conformational changes in bone. Experiments reveal the slow denaturation of collagen while the trans-Xaa-Pro conformation in collagen is unchanged due to dehydration. Our results suggest that GAGs in the collagen fiber and mineral interface may chelate with a Ca 2+ ion present on the surface of the mineral through sulfate or carboxylate groups. These results provide insights into the role of water molecules in the bone structure and shed light on the relationship between the structure and mechanics of bone.Supporting Information Available: Assignment and values of 13 C chemical shifts, 1 H NMR spectra, Raman spectra, and water content evaluation of cortical bone. This material is available free of charge via the Internet at

Strongly bound citrate stabilizes the apatite nanocrystals in bone

“…1 A-C shows the 13 C NMR spectra of fish, avian, and bovine bone. Similar to previously published data (17)(18)(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.…”

“…We agree with ref. 17 that the 76-and 182-ppm signals are not from collagen, but the subsequent assignment to sugar residues (18,19) is not tenable for the majority of this signal, because the C─OH carbons in sugars are protonated, not quaternary. The dominant contribution of the nonprotonated carbon of citrate at 76 ppm and the weak signal of anomeric (O─C─O) carbons near 100 ppm, which is a band characteristic of sugar rings (23), indicates that the polysaccharide concentration is only about 1∕4 of that of citrate.…”

Solid-state NMR Study Reveals Collagen I Structural Modifications of Amino Acid Side Chains upon Fibrillogenesis

“…1 and supplemental Fig. S1), and previous work on a collagen-like peptide (8) and native collagen I (9,10,17). No ambiguity was found for carbon resonances except for some C␣ and C␤ for which we observed overlaps in the two-dimensional { 1 H}- 13 C INEPT MAS spectra obtained in native conditions.…”

“…1 H NMR has only shown moderate potential to solve collagen-like peptide structure because of many overlaps of the resonances (8). Moreover, reported 13 C NMR experiments did not display a sufficient spectral resolution to allow a clear distinction among the different amino acid carbon resonances (9,10). NMR studies using specific residue labeling have given valuable information on dynamics (11)(12)(13).…”

“…Torchia et al (14) have compared native and denatured resonances of collagen fragment CB2. More recently, Zhu et al (10) have studied structural modifications of bovine cortical bone induced by dehydration. Even though all of these studies reveal important structural characteristics of collagen, many 13 C chemical shifts were "hidden" due to a poor spectral resolution.…”