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Bone measurement by enhanced contrast image analysis Ovariectomized and intact Macaca fascicularis as a model for human postmenopausal osteoporosis.

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Bone Measurement by Enhanced Contrast Image
Analysis: Ovariectomized and Intact Macaca fascicularis
as a Model for Human Postmenopausal Osteoporosis
Departments of Anthropology @,A.B.,D.S. W.,L C.M.),Biology (A.H. W ,
M.JJ.),and Comparadiue Medicine (D.S. U?,L C.M.), Wake Forest
Uniuersity, Winston-Salem, North Carolina 27109, Laboratory of TreeRing
Research, University of Arizona, Tucson,Arizona 85721 (Z? W.Z)
KEY WORDS Image analysis, Radiographic density,
Postmenopausal osteoporosis, Macaca fascicularis
ABSTRACT This paper presents a n image enhancement and analysis system (DARWIN) based on a n inexpensive microcomputer and applies the system
to two bone morphometry problems relevant to postmenopausal osteoporosis.
Using ovariectomized and intact female Macaca fascicularis as a model, we
examined the radiodensity of the sixth lumbar vertebra and the cross-section
area of the right femur. Significantly lower bone density was observed in the
vertebral segments of the ovariectomized animals. No significant differences
were observed in comparisons of the femoral cross sections. The reduction in
radiographic density of the ovariectomized animals' vertebrae is similar to
that observed in postmenopausal women, supporting the use of female cynomolgus macaques as models of bone loss in postmenopausal osteoporosis.
Osteoporosis in human beings is character- (Cameron and Sorenson, 1963; Gordan and
ized by the accelerated loss of bone, without Vaughan, 1976). Total mineral content in the
changes in chemical composition, ultimately axial skeleton without distinction between
often resulting in femoral neck, distal radius, cortical and trabecular bone can be meaand vertebral wedge fractures (Albanese, sured using dual photon absorptiometry and
1977). Osteoporosis is a serious health prob- neutron activation analysis (Cann et al.,
lem with considerable social and medical im- 1980). The absorptiometry methods inform
plications; the national cost associated with on the density of bone but provide no details
osteoporosis was estimated to be a t least 3.8 of the structure of the bone. Quantitative
billion dollars annually in the United States computed tomography can distinguish be(NIH,1984).Osteoporosis is most pronounced tween trabecular and cortical bone mineral
among post menopausal wamen and is asso- contents, which change at different rates in
ciated with the hormonal changes accompa- osteoporosis, and provides a better estimate
nying the cessation of ovarian function of the extent of vertebral osteoporosis (Cann
et al., 1980).
(Raisz, 1982).
To evaluate the histological effects of menEarly detection of the osteoporotic condition without painful intrusive methods is dif- opause in bone, intrusive bone biopsies must
ficult and fraught with error. Standard be performed. Standard morphometrics on
radiographs can be used to measure relative various bone types and bone microstructure
densities by radiogrammetry or micro- can be performed with manual or automatic
densitometry (Garn, 1970). However, bone digitizers and eyepiece micrometers (Teitelpositioning, tissue interference, film quality, baum et al., 1976; Dempster et al., 1983;
and film exposure present uncontrollable Smith and Jee, 1983). Microradiographic
variables in the radiographic methods of
analysis. Photon absorptiometry measuring
bone mineral content by absorption of radiaReceived December 16, 1983; revised October 26, 1984; action from the isotope 1-125 is a useful method cepted October 30,1984.
0 1985 ALAN R. LISS, INC.
Fig. 1. Digital AnaIyzer of Resolvable Whole-picturesby Image Numeration (DARWIN).
analysis can detect mineral content in individual bone microstructures (Stout and Simmons, 1979).
This paper will use a new microcomputerbased automated image analyzer, of local design, to examine the effects of ovariectomy
on radiographic bone density in a sample of
Macaca fmcicularis.
Thirty-eight adult female cynomolgus macaques (Macaca fmcicularis Medway) were
skeletonized by maceration. Seventeen of the
animals had been ovariectomized 22 months
prior to death. Housing
medical and diet
conditions were identical €or both the intact
and the ovariectomized animals. Mean body
weights of the two groups of animals (intact
and ovariectomized) were not significantly
different. Hormonal assays showed significantly lower serum estradiol and progesterone levels at necropsy in the ovariectomized
females (Weaver and Brodish, 1983).This paper will address one aspect of the project,
which was to assess the utility of Macaca
fmcicularis as a n animal model for human
postmenopausal osteoporosis by examining
relative bone density differences in the sixth
lumbar vertebrae and in bone surface area
differences in femoral cross sections.
To accomplish the assessment, the authors
used the Digital Analyzer of Resolvable
Whole-pictures by Image Numeration (DARWIN), a n image enhancement and analysis
system (see Fig. 1). The analog signal from a
Dage model VC-65-S vidicon camera is converted to a digital signal by a Colorado Video
model 270A digitizer which is in communication with a n Apple 11+ microcomputer
through a n interface board of local design
(Telewski et al., 1983). The digitizer has a
resolution of 512 lines horizontally by 480
lines vertically to produce 245,760 matrix
coordinates (pixels). This matrix can be accommodated by the microcomputer and the
camera, and has 256 gray-scale levels, a black
level adjustment, and amplitude adjustment.
The video image can be enhanced to optimize
contrast using the externalized video gain
and target and gamma functions of the camera. The DARWIN system can analyze microscopic and macroscopic objects, photographs, and negatives. After the external
controls are adjusted, the microcomputer is
calibrated to a standard length, and the scan
field “window” is preset. During the scan,
the digitized signal is read by the microcomputer according to the program in use. The
computer compares each pixel of interest to
the gray scale and saves its respective grayscale value. The duration of a scan depends
on the size of the scan window. The longest a
scan of a n entire screen would take is 17
To measure relative radiographic densities
of sixth lumbar vertebrae, the DENSITY
SCAN program was used. DENSITY SCAN
is a program of local design which allows
DARWIN to function as a scanning densitometer. Since gray-scale values were de-
sired, contrasts were not maximized; the
camera was adjusted to measure over the
entire range of gray-scale values.
DENSITY SCAN does not measure absolute density. The vertical scale of the density
profile (Fig. 2) is the percent gray scale or the
percent transmittance of light through the
measured object. In this study, a 100%F a y
scale value corresponded to complete opacity,
or highest bone density.
The sixth lumbar vertebra from each animal was bisected transversely, and the superior and inferior vertebral halves were
radiographed on a single large (35 x 28 cm)
radiographic plate at 45 kV, 0.01 mas, 1 m
focal distance. To reduce orientation error,
all vertebral halves were positioned with the
cut surface against the radiographic cassette.
The radiograph was placed on a light table
under the video camera. The scan window
was set to cover a 2-mm-widefield along the
midline of the vertebral image, from the dorsal to the ventral borders. A razor blade was
placed across the spinous process of the vertebra being measured to provide a black-level
standard (zero % on the gray scale).The video
amplitude was adjusted so the gray scale
value of the vertebral foramen equalled the
zero value of the razor blade. We averaged
three scans, for a total scan time for each
vertebra of 6 seconds. DENSITY SCAN then
displayed the density profile (Fig. 21, printed
the profile with numbered peaks, the peak
height of each numbered peak of interest, the
area under any designated segment of the
density profile, and stored the data.
Relative density differences between the
intact and ovariectomized groups of animals
were evaluated from the density profiles using various measures (Fig. 2). The dorsal and
ventral peak heights represent the regions of
greatest bone density through the vertebral
borders. The lowest trough height represents
the region of least bone density through the
vertebral body. These measures, standardized by dividing each by the thickness of the
cut vertebral body at its midpoint, were compared between the two groups.
Cross sections of midshaft right femora (0.5
mm thickness) were then scanned with DARWIN using the TOPOGRAPHER I1 program
to determine bone surface area. TOPOGRAPHER 11 is a program of local design which
recognizes and selects topographical features
of interest on the basis of preset gray-scale
limits. The image must be enhanced to provide sufficient contrast between the features
Fig. 2. Vertebral scan.
of interest and the background. The femoral
sections, mounted on glass slides, were placed
on the light table under the video camera.
The scan window was selected,the object and
background levels set, and the instrument
calibrated to a millimeter scale. After the
scan, which took approximately 7 seconds for
each cross section, a high-resolution reconstruction of the cross section was displayed
and compared to the actual image. Dirt or
other extraneous objects were eliminated before the scan data were stored. TOPOGRAPHER II displays, stores, and prints out the
area, perimeter, and gray scale value of each
topographical feature of interest, and the
percent of the total scan area occupied by the
objects of interest.
The DARWIN system has been tested for
precision in density and area measurements.
An EIA Logarithmic Reflectance Chart (Hale
Color Consultants), consisting of nine different gray-scale chips of known reflectance,
was scanned, producing a density trace with
“steps” corresponding to different chip reflectances. The average height of each density
trace step was compared to the % reflectance
of the corresponding chip to determine the
accuracy of the density measures. To test the
TABLE 1. Bensitometric measurements an sixth lumbar vertebral halves'
Superior half
Dorsal peak height
Lowesttrough height
Ventral peak height
Inferior half
Dorsal peak height
Lowest trough height
Ventral peak height
(n = 17)
(n = 21)
4.31 (0.53)
1.31 (0.23)
4.78 (0.37)
4.83 (0.33)
1.61 (0.18)
6.26 (0.52)
> .10
> .10
0.25 > P > ,010
4.72 (0.37)
1.82 (0.24)
6.33 (0.47)
6.15 (0.45)
2.42 (0.21)
7.95 (0.44)
,010 > P > .005
.05 > P > ,025
,010 > P
'Mean (S.E.).OVX, ovariectomized.
%tact density - OVX density
x 100.
intact density
TABLE 2. Femoral cross-section surface area'
Cross section (mm')
Body weight (kg)
Cross sectionhody weight
(n = 21)
(n = 17)
38.94 (1.10)
3.07 (0.11)
12.86 (0.37)
37.24 (1.0)
2.97 (0.11)
12.69 (0.37)
P > .10
P > .10
P > .10
'Mean (S.E.).
precision of the density measures, density
scans of seven radiographs of Plexiglas density wedge were performed. The mean step
height generated by each change in density
was correlated to the natural log of the actual height of the wedge step as measured
with dial calipers. To evaluate the precision
of the area measurements, a 100-mm2square
and a metal disk of known area (283.53 mm2)
were each scanned 20 times by the TOPOGRAPHER I1 program, and the results compared to the actual areas.
The DARWIN system of image analysis
provides a very high level of precision and
accuracy in density and area measurements.
After 20 scans, the average DARWIN area of
a 100-mm2square was found to be 99.91 mm2
& 0.11,with the average percent deviation
0.42 f 0.07. The average area of the metal
disk, after 20 trials, was 282.96 mm2 0.060
with an average percent deviation of 0.22 f
Using the EIA Logarithmic Reflectance
Chart, the correlation between the gray-scale
chip % reflectance and the natural log of the
mean step height of the chip on the density
trace was highly significant (r = .992, P <
.005). In the density precision analysis, the
correlation of the plexiglass wedge step
height to the density trace step height was
.9899 (P < .005).
The analysis of bone density of the sixth
lumbar vertebrae of the intact and ovariectomized animals showed differences in the
reIative amounts of bone present. The relative amount of bone present is represented
by the gray-scale density reading divided by
the animal's weight (kg). In all cases, the
ovariectomized female macaques had lower
mean densities for the three measurements
in both the superior and inferior vertebral
halves (Table 1). The superior half-ventral
peak heights in the ovariectomized animals
were significantly lower than those of the
intact group. All three inferior half measurements were significantly lower in the ovariectomized female macaques than in the
intact females. The ovariectomized macaques had lower mean femoral cross-section
area than the intact animals, but the difference was not statistically significant (Table 2).
The DARWIN system has proven useful in
evaluating the utility of Macaca fascicularis
as a potential animal model for human postmenopausal osteoporosis. The DARWIN system can produce highly accurate density and
area measurements. The high correlation between the density trace step height of the
density wedge on seven different radiographs
and the actual step heights shows that differences in radographic quality have little effect on density measures.
Although the between-groups difference of
the femoral cross-section surface area was
not statistically significant, the direction of
the difference, with the ovariedomized females having slightly less cortical bone, was
interesting, especially in light of the identical conditions under which the two groups
were housed. A longer postovariectomy experimentation duration in future studies
might result in greater differences in the
bone cross-section surface area.
The density data for the sixth lumbar vertebrae show a significantly lower amount of
bone in the ovariectomized animals. The percentage density difference between the two
groups ranges from 10.77% to 24.79% and is
consistent with the pattern of lumbar vertebral mineral loss in ovariectomized women
(Cann et al., 1980). The data on differential
bone density show less bone present in the
ventral region of the superior segment of the
vertebrae. This pattern of bone density would
encourage eventual wedge fractures, of the
kind occasionally seen in human postmenopausal osteoporotics (Raisz, 1982)
The use of a n image analyzer to examine
the degree of bone density difference in intact and ovariectomized female Macaca fas
cicularis indicates that the cynomolgus
macaque may prove a useful animal model
for postmenopausal osteoporosis. The cynomolgus macaque exhibits regular, monthly
ovarian cycles that are similar in length to
human females (Napier and Napier, 1967)
and shows depression of estrogen levels following ovariectomy (Weaver and Brodish,
19831, a s do human females. In this study,
the lumbar vertebrae of the ovariectomized
monkeys showed lower radiographic density
than those of the intact animals. The degree
of density difference agrees well with vertebral density measurements taken in studies
on ovariectomized human females. As in human females, substantial bone loss has occurred in the vertebral column of the
ovariectomized macaques with no appreciable effect in femoral midshaft cortical bone.
This research was supported by NIH grant
RROO919-10 and NHLBI grant HL 14164 to
the Department of Comparative Medicine
and by National Science Foundation grant
PCM-8003689 and National Aeronautics and
Space Administration grant NAGW-96 to
We are greatly indebted to Dr. R. Biro for
his help in performing the precision measurements on the DARWIN system.
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mode, intact, fascicularis, osteoporosi, contrast, postmenopausal, human, bones, measurements, image, enhance, analysis, macaca, ovariectomized
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