Thermal stability and structure of cancellous bone mineral
from the femoral head of patients with osteoarthritis or
L D Mkukuma, C T Imrie, J M S Skakle, D W L Hukins, R M Aspden
............................................................... ............................................................... .
See end of article for
Professor R M Aspden,
University of Aberdeen,
Institute of Medical
Aberdeen AB25 2ZD,
Scotland, UK; r.aspden@
Accepted 1 June 2004
Ann Rheum Dis 2005;64:222–225. doi: 10.1136/ard.2004.021329
Background: Cancellous bone from patients with osteoarthritis (OA) has been reported to be
undermineralised and that from patients with osteoporosis (OP) is more liable to fracture. Changes in
the mineral component might be implicated in these processes.
Objectives: To investigate the thermal stability and the mineral structure of cancellous bone from femoral
heads of patients with either OA or OP.
Methods: Powdered bone was prepared from femoral heads of patients with either OA or OP and a
control group. Composition and thermal stability were determined using a thermogravimetric analyser
coupled to a mass spectrometer. Unit cell dimensions and the crystallite size of the mineral were measured
using x ray diffraction.
Results: Thermal stability of the bone matrix, or of the mineral phase alone, was little altered by disease,
though OA bone contained less mineral than OP or control bone. In all three groups, x ray diffraction
showed that the mineral unit cell dimensions and crystallite sizes were the same. The mean carbonate
content in the mineral from all three groups was between 7.2 and 7.6% and is suggested to be located in
both the A site (that is, substituting for hydroxyl groups), and the B site (that is, substituting for phosphate
Conclusions: These results confirm that there is a lower mass fraction of mineral in OA bone, and indicate
that the nature of the mineral is not a factor in either disease process.
and material properties of the bone matrix appear to be little
different from people of the same age without OP.1–4
However, there may be changes in the material properties
that have previously been overlooked5and there are reports of
alterations in collagen crosslinking that could alter the
Although osteoarthritis (OA) is traditionally believed to be
a disease of articular cartilage, there is increasing interest in
the changes occurring in the bone, including the suggestion
that these are part of a disorder of the whole joint7or even
the whole musculoskeletal system,8rather than secondary to
cartilage degeneration. Subchondral bone sclerosis is one of
the clinical signs of OA and appears to result from changes in
trabecular orientation, thickness, and number,9–11and an
expansion of the subchondral bone plate.12 13In addition to
this proliferation of bone, there is mounting evidence for
changes in the bone matrix,2–4 14–16even at sites remote from
the affected joints.7 17
Previous studies have shown that the cancellous bone from
all sites over the femoral head and neck of patients with OA
is hypomineralised.2Consequently, it has a reduced material
density, confirming earlier studies using a density fractiona-
tion method,14and the stiffness increases more slowly with
apparent density than does normal or OP bone.2Scanning
electron microscopy indicates a more porous texture, similar
to that found in woven bone, and greater heterogeneity in
mineralisation.16The basis of these material changes is
The study reported here investigates further the nature of
these changes by investigating the extent of mineralisation
andthe mineral structure
n osteoporosis (OP) an exaggerated loss of mainly
cancellous bone results in bone fragility and an increased
risk of fracture following minimal trauma. The mechanical
ofthe bone matrix.
Thermogravimetric analysis (TGA) linked to mass spectro-
metry (MS) was used to investigate the thermal decomposi-
tion of the matrix and hence its mineral content and the
carbonate content of the mineral. In this combined TGA-MS
technique, a sample is heated in a stream of gas, its mass is
determined as a function of temperature, and the thermal
decomposition products are monitored by mass spectrometry.
TGA has been used previously to investigate synthetic
apatites18–21and mineral extracted from bovine bone.22The
lattice dimensions in the mineral crystals and the crystallite
size were measured by x ray diffraction (XRD), which is a
standard method for determining these properties in syn-
thetic and natural apatites.23–26The aims of this study were to
confirm the mineralisation deficit in OA cancellous bone of
the hip and determine whether there were any concomitant
changes in the mineral phase.
MATERIALS AND METHODS
Source of material
Femoral heads were obtained from patients undergoing a hip
replacement for either a fractured femoral neck attributed to
OP (18 patients) or for primary OA of the hip (27 patients).
Control bone tissue was obtained from the distal femur from
above knee amputations (seven patients), because femoral
heads from non-diseased patients proved impossible to
obtain. Local ethics committee approval was obtained to
use tissue removed during the normal course of surgery and
consent was obtained from patients for this purpose. The
median ages and the oldest and youngest in each group are
shown in table 1 for the various analyses.
Abbreviations: CHA, carbonate hydroxyapatite; MS, mass
spectrometry; OA, osteoarthritis; OP, osteoporosis; TGA,
thermogravimetric analysis; XRD, x ray diffraction
Femoral heads were wrapped in sterile gauze soaked in
phosphate buffered saline, vacuum sealed into plastic bags
and stored frozen at 220˚C until required. A transverse slice
of bone, about 10 mm thick, was taken from the distal femur
of patients undergoing an above the knee amputation and
stored in the same way.
To prepare samples for analysis, stored bones were
removed from the freezer and thawed at room temperature.
One core of cancellous bone, 9 mm in diameter and about
10 mm long, was prepared from the superior aspect of each
femoral head as described previously2and left to air dry. They
were then powdered using a freezer mill (Model 6750; Glen
Creston Ltd, Middlesex, UK) by pre-cooling in liquid nitrogen
for 6 minutes followed by milling for 1 minute at 10 impacts
per second. The resulting powder was put through a 63 mm
mesh sieve (BS410/1986; Endocotts Ltd, London, UK) in
order to produce a uniform particle size. Powdered samples
were stored in tightly sealed glass containers at 220˚C until
x ray diffraction
Unit cell dimensions and crystallite size were measured from
the prepared bone powder using XRD. A diffraction pattern
was recorded from each sample of bone powder using a Stoe
Stadi/P diffractometer (Stoe & Cie GmbH, Darmstadt,
Germany) in transmission mode, using Cu Ka1 radiation
(wavelength 0.15406 nm). The sample was prepared on a flat
plate and rotated about the axis of the beam throughout the
period of irradiation to reduce any effects of ‘‘graininess’’ in
the sample. Crystallite size was determined using the
Scherrer equation27from the full width at half maximum of
the 002 scattering peak and the a and c unit cell dimensions
from the positions of the 20 strongest scattering peaks.
Fitting of the patterns was performed using Stoe FIT software
with a squared Lorentzian function. Peak positions were
calibrated with a silicon standard.
A sample of each powder (mass 10.0 mg) was heated in a
thermogravimetric analyser (model TGA/SDTA851; Mettler-
Toledo, Schwerzenbach, Switzerland) linked to a mass
spectrometer (Balzers ThermoStar; Balzers Instruments,
Liechtenstein). Samples were enclosed in a 70 mL alumina
crucible. They were heated at a rate of 10˚C/min up to a
maximum of 1500˚C, in a stream of dry air delivered from a
regulated cylinder (BOC Gases; Guildford, Surrey, UK). MS
was used to monitor for species with relative molecular
masses of 18 (water) and 44 (CO2). A sample of CuSO4.5H2O
(BDH Laboratory Supplies, Poole, UK) was heated between
every two bone samples, and the presence of the expected
water peaks was used to ensure the capillary leading from the
furnace to the mass spectrometer was not blocked. An empty
crucible was heated under the same conditions as the
samples and the resulting thermogram subtracted from those
obtained from the samples to enable buoyancy effects to be
Results from the three groups were compared using analysis
of variance or its non-parametric equivalent, Kruskal-Wallis
analysis of variance on ranks, where data were not normally
distributed. Normality of the distributions was assessed using
the Kolmogorov-Smirnov test with p=0.05. Mean values
and their associated standard deviations are shown for data
that are normally distributed, otherwise values are medians.
Ages are shown as median and maximum and minimum.
Pairwise comparisons of groups following analysis of
variance were performed using Tukey’s test. All tests were
performed using SigmaStat software (version 2.01; SPSS Inc,
Chicago, IL, USA).
x ray diffraction
The mean values of the unit cell dimensions and the
crystallite size are shown in table 2. No significant difference
was found in the unit cell dimensions between either the OA
or the OP patient groups and the control group (analysis of
variance p=0.12, for both a and c). Similarly, no significant
differences were found between median values of crystallite
size (p=0.36, analysis of variance on ranks). Two of the OP
samples had large crystals, (crystallite sizes of 37.9 nm and
39.9 nm) that were outliers to the normal distribution, but
no reason for these unusual values was evident.
A typical trace of mass as a function of time, from TGA in air,
and the corresponding MS recordings of water and carbon
dioxide are shown in fig 1. The traces from all three patient
groups were similar. The first mass loss, at about 100˚C, was
due to adsorbed water and was not considered further. The
baseline dry mass was recorded after this loss had occurred
for determining fractional compositions. The decomposition
of the organic component occurred between 300 and 500˚C,
and gave rise to one water peak and two, often imperfectly
resolved, CO2peaks in the MS trace. The change in slope of
the mass trace also indicated that mass was lost in two
stages. There was a significant difference (p,0.001, two way
Details of groups from which bone was
Measurement of crystallite size
73 (90, 50)*
86 (92, 66)*
77 (93, 64)
Unit cell dimensions
73.5 (81, 60)
73.5 (87, 66)
71.5 (93, 56)
73 (90, 50)*
85 (92, 66)*?
69 (93, 37)?
Analysis of sex matching was performed using a x2test. Age distributions
were analysed using one way analysis of variance followed by Tukey’s
test for pairwise comparisons, when normally distributed, or Kruskal-
Wallis one way analysis of variance on ranks if data failed the
Kolmogorov-Smirnov test for normality with p,0.05 (p.0.05 indicates
no significant differences between groups). *?Pairs of variables that are
significantly different (p,0.05).
samples from the various patient groups
Unit cell dimensions and crystallite size for bone
OA Control OP
a dimension (nm) 0.9415
(21.6 to 24.2)
c dimension (nm)
Crystallite size (nm)
Values shown are mean (SD) except where data were not normally
distributed, in which case the median (25 to 75%) values are shown.
Material properties of OA and OP bone mineral 223
analysis of variance) between the group means for the water
peak and the first CO2 peak (339 (5)˚C v 349 (9)˚C),
indicating that these are not being driven off simultaneously.
No significant difference was found between patient groups.
The position of the second CO2peak was found to be slightly
higher in the OA group than the control group (436 (11)˚C v
428 (7)˚C) (p=0.04), indicating that there may be a small
increase in the temperature at which this mass is lost in the
OA group. The masses of the organic components lost during
this process from each patient group are shown in table 3,
with OA bone containing a greater organic fraction than
normal or OP.
There was a statistically significant difference between the
groups for the mineral and organic components (p=0.038)
but not for the carbonate content of the mineral (p=0.16)
(one way analysis of variance).
The mineral content was derived from the mass remaining
at about 600˚C after all the organic material had been
removed. This was lowest in the OA and greatest in the OP
bone, with the control samples in between (table 3). At
temperatures greater than 600˚C, the mineral starts to
decompose. The peaks in the CO2trace, which occurred at
about 700–800˚C, arise from carbonate being driven out of
the crystal structure.28 29The two peaks occurred at about
700˚C and 760˚C in all three groups. Corresponding to one of
these there was absorption of water, shown by the dip in the
water trace at about 700˚C (fig 1b, inset). There was then an
extended loss of CO2up to a temperature of about 1100˚C, as
can be seen from the inset MS trace in fig 1b, and a loss of
water at about 1300˚C. Assuming the CO2 arises from
breakdown of carbonate in the mineral, the mass of
carbonate can be calculated from the change in mass
between 600˚C and 1200˚C multiplied by 1.36 (the relative
molecular masses of CO3 and CO2) and expressed as a
percentage of the mineral mass. The resulting carbonate
contents are shown in table 3, but any differences were not
This study confirms previous results that reported a reduction
in the mineral content of cancellous bone from the hip of
patients with osteoarthritis.2 4 14Studies of bone from the iliac
crest have reported higher mineral contents by density
fractionation of cortical bone30and back scattered electron
microscopy.31These data indicate that site and bone type may
be important factors governing the changes caused by OA.
The slightly higher temperature required to obtain carbon
dioxide from the OA bone, shown by the second low
temperature peak in the MS trace, suggests a greater stability
of the organic matrix, but the origins of this are unknown.
The results also show that ashing at 600˚C, as performed
previously,2is a reliable way of determining mineral content,
as no carbonate is lost from the mineral until higher
temperatures are reached. There are few previous studies of
human bone using thermogravimetry, and the stages of
material loss and the carbonate content found here are
similar to those reported elsewhere.32 33The smaller mineral
content of OA bone accords with the lower hardness and, by
implication, stiffness, found by indentation testing,34but
otherwise the lack of differences provides no evidence for
disease related processes in the mineral phase.
These data are from the bulk material and provide a basis
for further studies in which heterogeneities on a microscopic
scale may be explored in more detail. Studies using Fourier
transform infrared microscopy on human OP bone35and
ovariectomised cynomolgus macaques,36an animal model of
the disease, have indicated that OP bone mineral has an
increased crystallinity and a higher ratio of carbonate to
phosphate. These studies used thin sections of bone and
recorded spectra from highly localised regions and it may be
that in our study, where the bone was homogenised, these
subtle changes are masked.
Carbonate starts to be lost from the mineral at around
650˚C as shown by the double peak plus an absorption of
water in fig 1. The origin of the double peak is unclear, but
the strong water absorption would support that at least one
of these peaks corresponds to loss of carbonate on a hydroxyl
site (A site carbonate), picking up water to maintain the
0 1400 12001000 800600400 200
Ionisation current (pA)
0 14001200 1000800 600400200
Ionisation current (pA)
function of temperature, measured using TGA, and (b) the
corresponding traces of water and carbon dioxide recorded on the mass
spectrometer (the inset is an enlarged version of the traces above
600˚C). All samples were air dried, so no account was taken of the first
mass lost, which can be explained by loss of residual water. Organic
components are lost between 200–550˚C. Carbonate was evolved in
two stages: a doublet peak between 700–800˚C then an extended loss
up to 1100˚C. Final decomposition of the mineral occurs at about
A typical trace, from a control sample, of (a) the mass as a
components as a percentage of the dry mass, and
carbonate as a fraction of the mineral, for each patient
Masses attributed to organic and mineral
224 Mkukuma, Imrie, Skakle, et al
mineral (to the non-hydroxyapatite phases) is heralded by Download full-text
the water loss at ,1300˚C (fig 1). Carbonate can substitute
for either hydroxyl (A site) or phosphate (B site) groups in
carbonate hydroxyapatite (CHA) but is known to affect the
unit cell dimensions: in B site CHA, a decreases and c
increases, whereas in A site CHA, the opposite is true. The
unit cell dimensions measured, however, are the same as
those reported for pure hydroxyapatite.38This lack of change
in dimensions may indicate that there is a balance between A
and B site substitution. This view is supported by the TGA-
MS results in which the double peak is followed by a small,
but steady, loss of carbonate from 800˚C up to 1200˚C.29
In conclusion, these results confirm the hypomineralisa-
tion of cancellous bone from the femoral head in OA. They
also show that the mineral in cancellous bone is the same in
both OA and OP and in individuals without either of these
disorders. Furthermore, the mineral comprises a carbonate
substituted hydroxyapatite in which the carbonate appears to
be distributed over both the possible sites of substitution.
We thank the Engineering and Physical Sciences Research Council
for financial support of this project (GR/L67066) and the Medical
Research Council for a Senior Fellowship for R M Aspden. We are
grateful to Dr I R Gibson for allowing us to benefit from his
considerable knowledge of biological apatites, the Orthopaedic
Surgeons of Grampian University Hospitals Trust for kindly donating
tissues from their patients, and L A Bestwick and B Paterson for
expert technical assistance.
L D Mkukuma, R M Aspden, Department of Orthopaedic Surgery,
University of Aberdeen, Aberdeen AB25 2ZD, UK
C T Imrie, J M S Skakle, Department of Chemistry, University of
Aberdeen, Aberdeen AB25 2ZD, UK
D W L Hukins, Department of Bio-Medical Physics and Bio-Engineering,
University of Aberdeen, Aberdeen AB25 2ZD, UK
D W L Hukins, Current address: School of Engineering, Mechanical
Engineering, University of Birmingham, Edgbaston, Birmingham B15
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Material properties of OA and OP bone mineral225