what-when-how
In Depth Tutorials and Information
WT
OIM
HA
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2-Theta-Scale
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FIGURE 4.5
Wide-angle X-ray diffraction patterns of an oim
/
oim and WT femur at 3 months. The bottom line is a pattern of well crystalline
hydroxyapatite.
in overall shape to those in wild-type mice, but they
also appeared less well aligned than those in the wild
type. A similar study in the oim model as a function of
age also found smaller, less well-aligned crystals, and
confirmed changes by bone mineral density distribu-
tion (BMDD) measurements (below).
56
measured based on ash weight (the constant weight of
dry bone that remains after the bone is heated to 600°C).
Cassella, studying transgenic mice expressing a mutant
form of human type Iα1 collagen, found reduced ash
weight in the fractured bones compared to the normal
littermates' bones.
59
While calcium to phosphate (Ca
/
P)
ratios can be measured on ashed bone, hydroxypro-
line measurement requires dry tissue. Hydroxyproline,
essentially unique to collagen, is measured to assess col-
lagen content.
60
Such analyses were carried out for oim
/
oim and wild-type mice developed in two different back-
grounds
61
but the data only demonstrated that the Ca
/
P
ratio was consistently reduced in young oim
/
oim bones
and the hydroxyproline content was reduced in all oim
/
oim animals independent of age or background.
X-ray microanalysis (with calibrated standards)
also provides Ca
/
P ratios. In a transgenic mouse model
expressing a part of a human mutant Iα1 chain such anal-
yses showed a reduced Ca
/
P ratio.
44
Casella also used
X-ray microanalysis to determine the Ca
/
P ratio in type
I OI patient biopsies and reported Ca
/
P ratio values were
lower than normal bone.
62
Continuing this study with a
larger group of samples of different Sillence types of OI,
these investigators found the perinatal lethal form (type
II) had the lowest Ca
/
P ratio.
63
It must be noted that Ca
/
P
ratios increase with age, thus some of the variation in
those studies might be related to the age of the patient
from which the biopsy was obtained.
In agreement with the finding of reduced Ca
/
P in OI,
neutron activation analysis demonstrated the elemental
composition of oim
/
oim and wild-type mouse femurs
had significant differences in magnesium, fluoride and
sodium content compared to wild-type. The ratio of Ca
/
P
was also significantly reduced in the oim
/
oim mouse
femurs compared with wild-type femurs.
64
This study
QUANTITATIVE BACKSCATTERED ELECTRON
IMAGING
A related technique
57
that allows the measurement
of the distribution of atoms, in this case Ca, in thin tis-
sue sections is backscattered electron imaging. Here
the parameters quantified are BMDD across the speci-
men examined, and the width (heterogeneity) of this
distribution. Oim mice had increased BMDD relative to
age-matched controls
56
while studies of humans with
classical OI revealed significant increases in BMDD
for types I, III and IV OI.
57
Humans with type VII
OI and the mouse model lacking CrTAP (one of the
mutations contributing to type VII OI) showed a sig-
nificant increase in mean Ca concentration and most
frequent mineral concentration relative to wild-type lit-
termates and control children. The mineralization het-
erogeneity was reduced in CrTAP−
/
− mice but normal
in OI type VII patients.
58
Reduced heterogeneity has
been associated with increased micro-crack propagation
and increased fracture risk in patients with osteoporosis,
and this increased homogeneity may contribute to the
brittle phenotype in OI.
CHEMICAL ANALYSES
Historically, bone mineral composition was evalu-
ated chemically by analyzing the calcium, phosphate
and hydroxyproline (collagen) contents of whole pieces
or ground pieces of bone. Mineral content was initially
