Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
Supporting Online Material
Detection of Hydroxyl Ions in Bone Mineral Using
Solid State NMR Spectroscopy
Gyunggoo Cho, Yaotang Wu and Jerome L. Ackerman
Materials and Methods
Calcium hydroxyapatite (HA), Ca10(OH)2(PO4)6, and brushite (BRU), CaHPO4·2H2O,
powders were obtained from Aldrich Chemical (St. Louis, MO, USA), checked with 31P solid state
NMR spectroscopy and x-ray powder diffraction, and used without further preparation to prepare
two synthetic test mixtures. Stoichiometric hydroxyapatite is generally accepted as the most basic
model compound for the mineral phase of bone, and for 31P NMR spectroscopy is a good model for
the unprotonated orthophosphate (PO4–3) ions in bone mineral. Brushite has often been used as a
model for the acid phosphate (HPO4–2) ions in bone mineral. The first mixture prepared, containing
95 weight percent hydroxyapatite and 5 weight percent brushite (95% HA/5% BRU), roughly
simulates the typical proportions of PO4–3 and HPO4–2 ions found in bone mineral. The second
mixture, containing 10 weight percent hydroxyapatite and 90 weight percent brushite (10%
HA/90% BRU), gives a
31
P cross polarization spectrum with comparable maximum peak heights
for both HA and BRU, in order to visualize the BRU spectrum in the presence of HA.
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Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
Animal and human tissue samples were obtained as discarded specimens in procedures
approved by the Massachusetts General Hospital Subcommittee on Research Animal Care and the
Human Research Committee respectively. Bovine cortical bone obtained abattoir, containing all its
organic constituents and water, was frozen in liquid nitrogen, ground in a Spex Industries (Edison,
NJ, USA) cryogenic mill under liquid nitrogen, and stored in a watertight container at ambient
temperature. Rat cortical bone was ground in the cryogenic mill at liquid nitrogen temperature
immediately after the animal was sacrificed, and stored at about –70 °C. Human cortical bone was
obtained from autopsy, and following 1–2 days of storage at refrigerator temperature (about 4 °C),
was similarly ground and stored at about –70 °C. All biological changes, and certainly all relevant
solid state chemical reactions, are stopped by storage at –70 °C, leaving only minor surface drying
of the specimens while in small airtight containers as a possible change in sample composition.
Although there is the possibility that the mineral composition was altered by the slight amount of
autolysis that might have occurred between the time of death and time of freezing, and by small
amounts of drying during storage, these processes are unlikely to have substantially altered the
NMR spectra. As shown in the results (Report Figure 3), drying at room temperature in an open
container produces NMR-visible changes in water content that are only observable over time scales
on the order of one day. Furthermore, essentially the same results with respect to hydroxide ion
content were obtained for specimens stored in closed containers at room temperature for a month,
for 1–2 days in the refrigerator, and frozen immediately following sacrifice.
A specimen of mineral-free bone matrix, was prepared from rat cortical bone by exhaustive
treatment with EDTA. The freshly harvested bone was immersed in 0.5 M EDTA at about pH 8 for
two weeks with constant stirring. The EDTA solution was changed every three days.
31
P NMR
spectroscopy of the resulting demineralized collagenous matrix indicated that the residual mineral
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Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
content, as well as any other phosphorus content, was undetectable under the conditions used in the
subsequent experiments.
The NMR spectra were acquired on a Bruker Instruments (Karlsruhe, Germany and
Billerica, MA, USA) MSL–400 spectrometer equipped with an Oxford Instruments 9.4 T 89 mm
superconducting magnet and a Bruker high range cross polarization/magic angle spinning
(CP/MAS) probe with a 7 mm OD rotor. The Larmor frequencies were 161.98 MHz for 31P and
400.13 MHz for 1H respectively. Ninety-degree pulse lengths for 31P and 1H were 5.0–6.0 µs, and
spinning speeds were 5.0 kHz.
31
P – 1H CP/MAS spectra were obtained with cross polarization
contact times (CT) in the range 0.3–10 ms, and decoupling fields of about 40–50 kHz. Proton
chemical shifts are reported downfield from TMS, and phosphorus chemical shifts are reported
downfield from 85 percent phosphoric acid; shifts are accurate to ±0.2 ppm. Hydroxyapatite was
used as an external secondary chemical shift reference for
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P (3.1 ppm) and 1H (0.2 ppm). The
proton chemical shifts of the hydroxyapatite standard were referenced by moistening the powder
with liquid acetone (proton chemical shift = 2.05 ppm) and spinning at 3 kHz to eliminate
referencing errors from isotropic magnetic susceptibility differences.
Proton and phosphorus one-dimensional MAS spectra were recorded for all samples. Twodimensional 1H–31P HetCor MAS NMR spectra were obtained using a WISE-type (1,2) pulse
sequence with pure phase techniques (3). Because of the relative diluteness of the protons in the
mineral, RF line narrowing techniques were not necessary to achieve the desired selective
polarization transfer in the f1 dimension and adequate spectral resolution in the f2 dimension in the
HetCor measurements. The number of steps for t1 incrementation was 64, with a 20 µs interval (50
kHz f1 dimension spectral width). This yields a limiting digital spectral resolution in the proton
projections of just under 2 ppm, which is adequate for accurately capturing the intrinsic spectral
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Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
linewidths of the hydroxyl resonances of the synthetic hydroxyapatite (about 2.5 ppm) and the bone
mineral (about 4 ppm). The OH– spectral linewidths of both hydroxyapatite and bone are highly
sample dependent. Spectra were processed with the Nuts (Acorn NMR, Livermore, CA, USA)
software package. Two-dimensional time domain data were zero-filled twice in the t1 dimension to
yield 256 points prior to Fourier transformation.
The HetCor NMR signal is the result of a complex and sensitive balance among many
factors: the proton flip angle, the proton T1, the recycle delay, the phosphorus T , and the crosspolarization rate (which further depends on the accuracy of the Hartmann-Hahn matching
condition, and to some extent on the spinning rate because of the relatively small 1H – 31P dipolar
coupling). For these reasons, quantitative HetCor spectroscopy must be carried out with great
attention paid to the use of intensity reference standards with nuclear spin properties comparable to
those of the unknown compounds, and with due account taken of the relaxation times. For
quantitative OH– determination, all materials were dried to constant weight at 60 °C prior to NMR
spectroscopy, and the mineral content of the bone was determined following NMR spectroscopy by
ashing the specimens at 550 °C for 24 hr. Spectral band areas were determined by least squares
fitting in all quantitative measurements for both 1-D spectra and 1-D slices of 2-D spectra; this was
required in order to cleanly resolve the hydroxyl and water signals in the apatites.
One-dimensional proton MAS spectra of dried weighed amounts of CaHPO4, a
stoichiometric reference compound, and a hydroxyapatite standard, were compared, taking care to
insure that the spectrometer gain was constant and that the recycle delay exceeded the longest T1 by
a factor of 5, to yield an OH– content of 93 % of the stoichiometric value on the basis of the
integrated signal intensity for the relevant spectral line. For each of three contact times HetCor
spectra of bone were obtained in two separate acquisitions.
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The OH– signal intensity was
Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
referenced to that of the hydroxyapatite standard, and corrected for the 93% OH– content of the
standard to yield a hydroxyl content. These data are plotted in Figure S1. Error bars represent the
range of the two determinations, and the solid symbols represent the average of the two
determinations. An exponential function with two parameters was least squares fitted to the three
points to yield an intercept at CT = 0 of 21 ± 1 % (fitted value ± standard deviation, correlation
coefficient = 0.99) for the hydroxyl content of this human cortical bone sample. Because the CT
values were chosen to be very short to minimize OH– signal loss so that the most accurate yintercept is obtained, the fitted value of the decay constant is unreliable. At much longer CT
values, the decay curve in Figure S1 would be seen to be an initial fast decaying component
superimposed on a much slower decaying component (see the text of the Report).
To demonstrate how the OH– content of whole bone can easily fail to be detected with
conventional one-dimensional proton MAS NMR spectroscopy, the proton spectrum of whole fresh
human cortical bone (Figure S2a) was compared with the spectrum of hydroxyapatite (Figure S2b)
vertically scaled such that the hydroxyapatite OH– resonance intensity (area under the lineshape) is
equivalent to 20 % of the value it would have if the bone mineral in the bone sample were
stoichiometric in OH– content (Figure S2). This scaling was based on the mineral content of the
bone sample as determined by ashing subsequent to NMR spectroscopy, taking into account the 93
% OH– content of the hydroxyapatite sample. If the hydroxyapatite spectrum is added to that of the
bone, the result is essentially indistinguishable from the original bone spectrum (Figure S2c). Even
if the OH– content of the bone were to be fully 100 % stoichiometric, there is little likelihood that
OH– could be identified in fresh whole bone samples by one-dimensional proton MAS NMR.
References
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Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
1. N. Zumbulyadis, Phys. Rev. B Condens. Matter 33, 6495 (1986).
2. K. Schmidt-Rohr, J. Clauss, H. W. Spiess, Macromolecules 25, 3273 (1992).
3. D. J. States, R. A. Haberkorn, D. J. Ruben, J. Magn. Reson. 48, 286 (1982).
Figures
Figure S1. OH– peak area (converted to OH– ion content by comparison to a hydroxyapatite
standard of known stoichiometry) in HetCor spectroscopy of whole human cortical
bone (stored frozen, cryogenically ground, but otherwise not pretreated) versus cross
polarization contact time CT (symbols). Very short CT values were used to obtain a
reliable exptrapolation to CT = 0, which corresponds to the true OH– content as
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Manuscript 1078470 SOM (11 April 2003): Cho, Wu, Ackerman, Detection of Hydroxyl Ions in Bone Mineral Using Solid State NMR
Spectroscopy
determined by HetCor NMR spectroscopy. The solid curve is a least squares fit to an
exponential function.
Figure S2. Conspicuity of the OH– resonance in one-dimensional proton MAS NMR spectrum of
unpretreated whole human cortical bone. a) Spectrum of hydroxyapatite, vertical scale
adjusted so that OH– resonance area corresponds to 20 % of the of the value
corresponding to fully stoichiometric mineral content in the bone sample. b) Spectrum
of bone sample. c) Sum of the spectra in a and b. This spectrum is indistinguishable
from b; the OH– signal is obscured by the organic and water content of the bone. Onedimensional proton MAS NMR spectroscopy cannot be used to detect OH– in bone
mineral in unpretreated bone.
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