Boning up on NMR

Solid-state NMR spectroscopy can analyse intact bone and could lead to atomic-level explorations of how disease and aging affect bone. It could show, for instance, how age-related water loss leads to structural changes.

Peizhi Zhu, Jiadi Xu, Nadder Sahar, Michael Morris, David Kohn, and Ayyalusamy Ramamoorthy of the Department of Chemistry, at the University of Michigan, in Ann Arbor, recognised just how much of a challenge bone represents analytically speaking.

"If people think of bone at all - and they usually don't, until they have a fracture - they think of it as an inert material," Ramamoorthy says. "But like everything else, bone is also made up of molecules whose behaviour is reflected in its structure, toughness and mechanical strength, making bone really exciting in terms of its chemistry." Unpicking the chemical secrets of bone, however, requires powerful tools.

According to Ramamoorthy, magic-angle spinning solid-state NMR spectroscopy is just such a tool as it allows chemists to obtain structural information with nanoscopic resolution without physical damage to the bone and with no chemical modifications. Previous investigations have resorted to pulverized bone samples, but the U-M team was able to analyze an intact sample of cow bone, shaped to fit the NMR probe.

By applying controlled dehydration, and hydrogen-deuterium exchange to the bone, the team was able, for the first time, to obtain information about structural changes. They explain that proton NMR spectra were used to monitor the dehydration of the bone inside the rotor, and then high-resolution carbon-13 NMR under MAS was used evaluate any dehydration-induced conformational changes in the bone. "The 13C spectrum of a fresh cortical bone exhibits well-resolved spectral lines and is dominated by resonances from collagen," the team says. "The spectra of a dehydrated cortical bone and a H/D-exchanged cortical bone are similar and exhibit much broader spectral lines than the fresh cortical bone," the team adds.

"The experiments revealed the slow denaturation of collagen due to dehydration while the trans-Xaa-Pro conformation in collagen remained unchanged," the team says. "Our results suggest that glycosaminoglycans in the collagen fibre and mineral interface may chelate with a calcium 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." Water after all constitutes 20 percent of healthy, young bone by volume and interstitial water molecules help stabilize both collagen and mineral content through enthalpic stabilization and hydrogen bonding.

Such findings are important not only to those hoping to mimic the properties of bones for materials science applications but because the water content of bone tissue decreases with age. Such changes affect both collagen and mineral content, reducing bone's strength and toughness. "We were able to see dynamical structural changes with the main protein, collagen," Ramamoorthy explains. "Its characteristic triple helix structure was not completely damaged, but its mobility was altered, in addition to a disorder in the structure."

The details of the results also suggest that using higher magnetic fields and sophisticated radio frequency pulse sequences under ultrafast MAS might also provide even better atomic-level structural data about intact bone structure that is precluded by the cryogenic milling of bone samples.

The research could pave the way for more detailed studies of how bone behaves under different conditions. "We'd like to look at how bone changes at the atomic level, as a function of aging," Ramamoorthy adds, "and to make comparisons between diseased and healthy bone." Such studies may provide insights into the susceptibility of bone to fracture, especially in people with bone disorders, such as osteoporosis, a leading cause of debilitating and potential lethal fractures in the elderly.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.