Klaus Schmidt-Rohr, a chemist at the US Department of Energy’s Ames Laboratory, used solid-state nuclear magnetic resonance spectroscopy to examine the role citrate plays in bone composition, work that may help scientists better understand and treat or prevent bone diseases such as osteoporosis. Credit: U.S. Dept. of Energy’s Ames Laboratory |
Bone
is one of nature’s surprising “building materials.” Pound-for-pound
it’s stronger than steel, tough yet resilient. Scientists at the U.S.
Department of Energy’s Ames Laboratory have identified the composition
that gives bone its outstanding properties and the important role
citrate plays, work that may help science better understand and treat or
prevent bone diseases such as osteoporosis.
Using
nuclear magnetic resonance (NMR) spectroscopy, Ames Laboratory
scientist and Iowa State University chemistry professor Klaus
Schmidt-Rohr and his colleagues studied bone, an organic-inorganic
nanocomposite whose stiffness is provided by thin nanocrystals of
carbonated apatite, a calcium phosphate, imbedded in an organic matrix
of mostly collagen, a fibrous protein.
By
understanding the nanostructure of naturally occurring materials,
researchers may be able to develop new light-weight, high-strength
materials that will require less energy to manufacture and that could
make the products in which they are used more energy efficient.
“The
organic, collagen matrix is what makes bones tough,” Schmidt-Rohr said,
“while the inorganic apatite nanocrystals provide the stiffness. And
the small thickness – about 3 nanometers – of these nanocrystals appears
to provide favorable mechanical properties, primarily in prevention of
crack propagation.”
While
bone structure has been studied extensively, how these apatite
nanocrystals form and what prevents them from growing thicker was a
mystery. Some research pointed to sugars being involved, but that
didn’t match with the NMR spectra that Schmidt-Rohr was seeing.
“We
can see all the peaks clearly,” he says of a spectral graph which shows
the points at which specific components in bone samples resonate;
these specific signatures are the key to NMR technology, “even those at
the organic-inorganic interface, where the organic material’s signal
strength is relatively weak.”
After
studying bone structure over a five-year period, it was actually
serendipitous that Schmidt-Rohr came across a signature that appeared to
match what he was seeing.
“We
had gotten some crystalline collagen samples to study,” he said, “and
it turned out that the supplier, Sigma-Aldrich, had used citrate to
dissolve the collagen. And the citrate signature in the collagen samples
matched the signature we were seeing in bone.”
According
to Schmidt-Rohr, the role of citrate in bone had been studied up until
about 1975, but since that time, no mention was made in any of the newer
literature on bone. So in essence, his research team had to rediscover
it.
This diagram shows the effect of citrate concentration on the size of hydroxyapatite crystals fabricated with self-assembling block copolymer templates. Just as it does with actual bone structure, as the concentration of citrate increases, the thickness of the nanocrystals decreases and the thinner nanocrystals appear to make the bone more resistant to stress cracking. Credit: U.S. Dept. of Energy’s Ames Laboratory |
The
case for citrate was made most convincingly when graduate research
assistant Yanyan Hu was able to extract citrate from cow bone and
replace it with carbon 13 (C13) -enriched citrate, resulting in a
30-fold enhancement of the NMR signals of the bone sample. The peaks
matched exactly, confirming the presence of citrate on the surface where
the apatite nanocrystals had formed.
Schmidt-Rohr
further hypothesized that, since citrate is too large to be
incorporated into the apatite crystal lattice, it must be bound to the
nanocrystals’ surface where it stabilizes the nanocrystals’ size by
preventing their further growth. The findings were published in the Dec. 28, 2010 issue of the Proceedings of the National Academy of Sciences.
“Based
on the old literature, we looked at the citrate levels in a variety of
types of bone and found that herring spine had the highest citrate
concentration – about 13 percent by weight,” Schmidt-Rohr said. “So it
should hold that the citrate signal for herring spine should be three
times higher than for cow bone, and indeed it was.”
In
further studies, the group found that higher concentration of citrate,
the thinner the apatite nanocrystals in bone. This was further confirmed
on bone-mimetic nanocomposites in a collaboration with Ames Lab faculty
scientists Surya Mallapragada and Muffit Akinc, using a polymer
template with various concentrations of citrate to synthesize apatite
nanocrystals. At higher concentrations, the nanocrystals that formed
were thinner and should therefore be more resistant to crack
propagation. This work was published in the April 12 issue of Chemistry of Materials.
“At
this point, we feel that citrate probably also has a role in the
biomineralization of the apatite,” Schmidt-Rohr said. “It’s also been
noted in the literature that as an organism ages, the nanocrystal
thickness increases and the citrate concentration goes down,”
Schmidt-Rohr said, “and there’s also support from clinical studies that
citrate is good for bones,” adding that one of the leading supplements
for bone strength contains calcium citrate.
“While
calcium loss is a major symptom in osteoporosis, the decline of citrate
concentration may also contribute to bone brittleness,” he said.
