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Rapid determination of cancellous bone mineral loss in ovariectomized rats by a subtraction technique.

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THE ANATOMICAL RECORD 230:169-174 (1991)
Rapid Determination of Cancellous Bone Mineral
Loss in Ovariectomized Rats by a
Subtraction Technique
Center for Hard Tissue Research, Department of Medicine, Creighton University, Omaha,
Nebraska (E.E., D.B.K.); Department of Physiological Sciences, College of Veterinary
Medicine, University of Florida, Gainesville, Florida 32091 (T.J.W.)
We present a rapid technique for determining cancellous bone
mineral changes in small experimental animals. We used the distal centimeter of
the right femur from ovariectomized (OX) (N = 30) and shamovariectomized
(ShOX) (N = 28) rats, aged 90 days a t surgery and killed a t times from 125-540
days postsurgery. We used dual photon absorptiometry to scan the segment three
times: intact, after parasagittal splitting, and after removing all cancellous bone.
We equated the difference between the second and third scans to cancellous bone
mineral content (Cn.BMC). To validate this, we compared i t with histomorphometrically determined bone volume (BVITV) of the proximal tibia1 metaphysis of
the same rat. Parasagittally splitting the segment removed no detectable mineral.
OX rats had 40% less Cn.BMC than ShOX rats. However, OX rats had 80% lower
BVITV than ShOX rats. The subtraction technique not only makes a rapid, reasonable assessment of cancellous bone loss in OX rats but permits a smaller sample
size than histomorphometry. The histomorphometric technique finds a greater
difference between OX and ShOX rats because it examines a region where cancellous bone loss is more marked than does the scanning technique. The current
technique measures bone of not only the central secondary spongiosa but also the
juxtacortical region and the primary spongiosa, where OX-related differences are
less prominent. The principles of this subtraction technique proved workable.
However, for the future, we recommend a two-scan technique using a dual energy
X-ray scanner. It is likely to take only 20-30 min per specimen to assess cancellous
bone mineral.
A frequent clinical symptom of postmenopausal osteoporosis is vertebral crush fractures, often associated
with low cancellous bone strength (Cummings et al.,
1985). Part (Weaver and Chalmers, 1966; Mosekilde
and Mosekilde, 1988) but not all (Kleerekoper et al.,
1985) of cancellous bone’s strength is due to its mineral. Studying cortical and cancellous bone mass separately in vivo in humans is possible by several quantitative computed tomography (CT) techniques (Jones et
al., 1987; Cann, 1988; Liliequist et al., 1979; Ruegsegger et al., 1981). Cody and Flynn (1989) even subdivide
spinal cancellous bone measured in vivo into regions
containing 1.6 cm’. Separating cancellous and cortical
bone is necessary because their rates of loss may be
different (Meier et al., 1984; Riggs et al., 1986).
Investigators using experimental animals to test
regimens to increase cancellous bone mass could benefit from a quick, accurate technique. The above-described CT techniques are often unavailable or lacking
sufficient resolution for measuring small animal bones.
However, investigators using small animals have the
advantage of harvesting and dissecting apart bones for
analysis. They measure bone quantity by weighing and
ashing (Yamazaki and Yamaguchi, 1989; Nottestad et
al., 1987; Gong et al., 19641, neutron activation (Parks
e t al., 1986), photon absorptiometry (Kiebzak et al.,
1988; Kimmel and Wronski, 1990), or quantitative histology (Kimmel and Jee, 1983). Physically separating
and quantitating cancellous and cortical bone remains
troublesome, but necessary (Nottestad et al., 1987;
Gunness-Hey and Hock, 19841, if the analysis is to approach the level of in vivo human applications (Jones et
al., 1987; Cann, 1988; Liliequist e t al., 1979; Ruegsegger et al., 1981). The purpose of this work is to report
and validate a quick, reliable technique for measuring
cancellous bone mass in small experimental animals.
Experimental Design
We obtained 58 female Sprague-Dawley rats 85 days
of age weighing roughly 210 g (Charles River Co.,
Cambridge, MA). They were caged individually and fed
ad libitum in a room with a 12 h r on112 h r off light
cycle. We randomized them by weight into experimental and control groups. At age 100 days, the rats were
Received September 14, 1990; accepted November 5, 1990.
Scanning and Analysis Procedure
ovariectomized (OX) (N = 30) or sham-ovariectomized
(ShOX) (N = 28) under ketaset/xylazine anesthesia by
a dorsal approach as described by Waynforth (1980).
We sacrificed them a t 125,180,270,360, and 540 days
postovariectomy. Bone loss in the proximal tibia1 metaphysis after estrogen depletion in rats reaches a steady
state by 100 days (Wronski e t al., 1989). At autopsy, we
removed the right tibias and femurs. We placed the
femurs immediately into 70% EtOH. We placed the tibias in formalin for 48 h r and then moved them into 70%
EtOH. We blind coded all specimens before any manipulation.
We used a Norland 2600 Dichromatic Bone Densitometer (Norland Corp., Ft Atkinson, WI; Software
Version 2.23W), previously validated a s accurate for
small animal bones (Kimmel and Wronski, 1990). We
placed the bone specimens in a n 18.5 cm2 weighing
dish on a 2.5-cm-thick piece of Plexiglass to provide the
appropriate soft tissue equivalent. We used a scan
width of 1.9 cm and a line and point “resolution” of 0.4
mm a t 1 m d s e c scan speed. Each scan lasted 10-12
min, including set-up. We analyzed the raw data averaging over 13 “points.”
Strategy for Measuring Cancellous Bone
Quantity (Cn.BMC)
Cancellous Bone Quantity Measured by
Histomorphometry (BV/TV)
We used a subtraction technique to find cancellous
bone quantity. First, we removed the distal 1cm of the
femur, a region containing both cancellous and cortical
bone and measured its mineral content (BMC). Next,
we split it parasagitally and remeasured its BMC. Finally, we removed the cancellous bone tissue from both
halves and remeasured BMC of the remaining piece.
We determined mineral lost to opening the bone as the
difference between BMCs of the first and second scans.
We determined cancellous mineral content (Cn.BMC)
as the difference between BMCs of the second and third
scans. To validate Cn.BMC as a bone mass index, we
compared Cn.BMC of the distal femur to BV/TV of the
proximal tibia in each animal, as reported by Wronski
et al. (1989).
We dehydrated the proximal 1 cm of the tibia in
graded alcohols and embedded i t in modified methyl
methacrylate (Wronski et al., 1989). We cut 4 pm frontal sections with a Reichert-Jung 1150 microtome,
from a region midway through the bone in a n anteroposterior direction. We stained them with the Goldner
(1938) method. Finally, we measured cancellous bone
volume (BV/TV) in a region located 1mm distal to the
growth cartilage-metaphyseal junction, which measured 2 mm transversely by 3 mm proximodistally
(Wronski et al., 1989).
Specific Steps for Mineral Determination
1. First, we cut the femur transversely 1cm from its
distal end with a Hyperflex diamond disk (Brassler
USA, 940-220, Savannah, GA) in a straight dental
handpiece. The diamond disk was 22 mm in diameter
and 0.15 mm thick. The time taken to slice each bone
was about 1 min.
2. We scanned the piece by dual photon absorptiometry (DPA) (see Scanning and Analysis Procedure)
(scan 1).
3. Next, we cut the 1 cm piece parasagitally into two
halves with the diamond disk. We cleaned the halves of
bone“crumbs” in a n ultrasonicator for 30 sec and reapposed the pieces in their anatomically correct relationship. The time taken to slice and reappose each bone
was about 1 min.
4. We scanned the reapposed pieces (scan 2).
5. Next, we removed the cancellous bone from each
half with a slowly rotating (100-200RPM) round burr
(Brassler USA, No. 4, 014, Savannah, GA) and a discoid-cleoid dental carver (G.C. International, 3-COOO5,
Scottsdale, AZ). We worked under a 2 x magnifier, removing all cancellous bone distal to the growth cartilage-metaphysealjunction but leaving the cartilage intact. We left a smooth endocortical surface. We again
cleaned both halves in the ultrasonicator and reapposed the pieces as in step 3 (scan 3). The time taken to
remove the cancellous bone and clean the pieces was
5-10 min. We reapposed the two halves and scanned
them a third time. Much of this step was done during
other scans.
6. Finally, to find Cn.BMC, we subtracted BMC of
scan 3 from BMC of scan 2. We assumed t h a t bone
marrow contained negligible mineral.
Statistical Methods
We applied a paired t test when comparing scans 1
and 2 or scans 2 and 3 of the same femoral specimen.
We applied a t test of means when comparing ShOX
and OX rats (Sokal and Rohlf, 1969). To find the minimum detectable change in Cn.BMC and BV/TV, we
applied this formula from Snedecor and Cochran
where N is the sample size, (Z, - ZP)’ is a multiplier
reflecting the probabilities of Type I and Type I1 errors
(a = .05 and @ = 0.2), with a value of 7.9, aD2 is the
stochastically determined variance of the differences,
and s is the absolute minimum detectable difference.
All values are expressed as mean SD.
We estimate the time for this three-scan analysis at
40-50 min per bone.
Mineral Lost to Parasagittal Cut
(Difference of Scans 1 and 2)
For OX rats, BMC changed from 115.5 -t 9.6 mg to
113.8 t 9.4 mg between the first two scans (Fig. 1).For
ShOX rats, BMC changed from 130.7 -t 16.1 mg to
129.5 2 17.7 mg between the first two scans (Fig. 1).
These changes within the OX and ShOX groups of rats
were not significant.
Cn.BMC (BMC of Cancellous Bone)
(Difference of scans 2 and 3)
In OX rats, BMC decreased from 113.8 -t 9.4 mg in
the second scan to 99.7 2 11.2 mg in the third scan
(Fig. 1).In ShOX rats, BMC decreased from 129.5 2
17.7 mg in the second scan to 106.2 -t 16.2 mg in the
Distal Femoral
Cancellous Bone Endpoints
(Mg) (*SEMI
ap 2 5
-5” 15
1 1 3
Fig. 1. Distal femoral bone mineral content (BMC) in all scans.
There is no difference between the first two scans of the distal 1 cm of
the femur in either group. However, BMC was significantly less in
scan 3 than in scan 2 in both OX and ShOX rats (bothP < ,001).
the difference of scans 1 and 2 represents the mineral lost during a
parasagittal slice of the bone with a thin diamond disc, we conclude
that scan 1 can be omitted. OX, ovariectomized rats; ShOX, shamovariectomized rats; e, P < .001.
third scan (Fig. 1). These changes within the OX and
ShOX groups of rats were significant (P < .001).
Comparison of Cancellous Bone Quantity in ShOX
and OX Rats
By subtraction technique
The difference in BMC between the second and third
scans is distal femoral cancellous bone mineral
(Cn.BMC). Cn.BMC of OX rats was 14.1 2 6.1 mg, 39%
less than the ShOX rats’ 23.2 2 5.8 mg ( P < .001)
(Fig. 2).
By histomorphometry
Cancellous bone volume (BVITV) of the proximal
tibia in OX rats was 4.8% 2 4.2%, 80% less ( P < .001)
than the ShOX rats’ 24.2% 2 10.3% (Fig. 2).
The difference between Cn.BMC (mg) and BV/TV
(%) in the ShOX rats was -1.0 2 9.4, whereas the
difference between Cn.BMC and BV/TV in the OX rats
was greater at 9.5 2 6.2 ( P < .001). Cn.BMC detected
a significantly smaller difference in bone mass than did
By percent of distal femoral mineral in cancellous bone
In OX rats, 12.5% 2 5.6% of the mineral in the distal
centimeter of the femur was in cancellous bone. In
ShOX rats, 18.0% -t 4.1% of that mineral was in cancellous bone. The difference between OX and ShOX
rats was 31% (P < .001).
Minimum Detectable Difference
To find reliably a 20% change in Cn.BMC of intact
rats, that is, from 23.2 to 27.8 mg, the minimum group
size would be 12. On the other hand, to find a 20%
change in BVITV of intact rats, from 24.2% to 29.0%,
Fig. 2. Cancellous bone mass endpoints in distal femur of ovariectomized and sham-ovariectomized rats. Cn.BMC is the difference of
scans 2 and 3 for the OX and ShOX groups of rats. There is less
cancellous bone in OX rats than ShOX rats whether measured by
Cn.BMC or BVITV. However, the histomorphometric measurement
shows a much greater difference than the subtraction technique. It
probably measures a n area of the metaphysis where cancellous bone
loss is most marked. Cn.BMC, cancellous bone mineral content of
distal femur by subtraction technique; BV/TV, bone volume of proximal tibia1 metaphysis by histomorphometry; ShOX, sham-ovariectomized rats; OX, ovariectomized rats; e, P < .001.
the minimum group size would be 36. We have made
additional calculations in Table 1.
We present a quick, accurate technique for measuring cancellous bone mineral in bones of small experimental animals. We used i t to find differences in cancellous bone mass between groups of sham-operated
and ovariectomized rats and validated it by comparing
its results to histomorphometric studies of the proximal tibia of the same animal.
There was no significant difference between scans 1
and 2 for either group. The only difference between the
specimen in scans 1and 2 was that a parasagittal slice
was made through its central part. We conclude that
making this slice with a thin diamond disc removes a n
insignificant amount of mineral. It is suitable to begin
this subtraction technique without a scan of the intact
This technique is faster than histomorphometry. Under the best of circumstances, quantitative histology,
with its embedding and sectioning, would take 4-6
weeks for a 40 r a t experiment. With this technique, the
time to find Cn.BMC is less than 1 h r per specimen.
Even allowing time for errors, a 40 rat study can be
completed within 1week. One might envision quickly
screening for cancellous BMC changes with this technique, saving the more comprehensive histomorphometric studies of cell endpoints for experiments in
which a certain difference exists.
TABLE 1. Sample size to find changes in cancellous bone quantity by subtraction
and histomorphometric techniques'
Sample size to find change of
'Abbreviations: Cn.BMC, cancellous bone mineral content of distal femur; BV/TV, bone volume of proximal tibial metaphysis; ShOX, sham-ovariectomized rats; OX, ovariectomized rats.
It seems straightforward. All techniques for determining cancellous bone mineral in small animals must
rely on anatomic dissection and separation of cancellous and cortical bone (Nottestad et al., 1987; Gong e t
al., 1964; Parks et al., 1986; Gunness-Hey and Hock,
1984). However, most require saving the marrow and
small pieces of removed cancellous bone so that they
can be evaluated. Saving those small pieces can be difficult, particularly when few exist. The nondestructive
nature of absorptiometry makes possible this subtraction technique, which does not require saving the small
The mineral assessment technology seems widely
available. Past studies of mineral content in cortical
and cancellous bone have relied on ashing or neutron
activation for mineral analysis. Ashing would not work
for the subtraction technique, and neutron activation is
available only at several locations worldwide. Photon
absorptiometry is now widely available and well validated for small animal bones (Kiebzak et al., 1988;
Kimmel and Wronski, 1990). Dual energy X-ray absorptiometry, with its fourfold reduction in scan time
and improved precision over DPA, seems likely to be
even better (Stein et al., 1988).
Less difference between cancellous bone of OX
and ShOX rats
We detected a 39% difference in Cn.BMC of OX and
ShOX rats but a n 80% difference in BVITV of OX and
ShOX rats. The change found with Cn.BMC was significantly less than that found with BVITV (P< .001).
The histomorphometric change in cancellous bone
mass of the proximal tibia for OX rats was greater than
that seen with our technique. Four possible reasons for
these discrepancies exist.
1. The technique of removing cancellous bone probably removed some cortical bone at the endocortical
surface. We used 2 x magnification, which we believe
is too low to ensure that the burr does not occasionally
gouge the endocortical surface. In the future, we recommend doing all work with the round burr and discoid-cleoid, under a dissecting microscope at 5-10 x .
2. The technique of removing cancellous bone removed cancellous bone from the primary spongiosa, but
the histomorphometric method did not study the primary spongiosa. If the difference of primary spongiosa
mineral content between OX and ShOX rats is less
than in the secondary spongiosa, the subtraction technique will reveal a smaller difference.
3. Other work suggests that cancellous bone loss in
estrogen deplete rats is most marked in the area of the
secondary spongiosa most distant from the endocortical
surface (Okumura et al., 1987). BVITV samples only
the central section; Cn.BMC measures the whole metaphysis (and probably part of the inner one-third of the
cortex). Since the difference of juxtacortical cancellous
mineral content between OX and ShOX rats is less
than in the central secondary spongiosa, the subtraction technique would be expected to reveal a smaller
4. We measured Cn.BMC of the distal femur, but
BVITV of the proximal tibia. Cancellous bone behavior
in the distal femur and proximal tibia may differ. If
proximal tibial cancellous bone is more sensitive to estrogen loss than that of the distal femur, then our results are as expected.
The subtraction technique seems more sensitive
than histomorphometry. It can detect a 30% change in
Cn.BMC of OX rats with a group size of 16. It can even
detect a 20% change in Cn.BMC of ShOX rats with a
group size of 12. Using a histomorphometric technique
in the same animals would require group sizes of 67
and 36, respectively, to find the same relative changes
(Table 1).The reason for the lower sample size is the
decrease in variance with Cn.BMC compared with BVI
No assessment of cell function
This quick technique, which does not permit studies
of cell function, is no replacement for histomorphometry and biochemistry. However, since for now bone
mass improvement is the critical endpoint in animal
studies evaluating osteoporosis interventions, i t may
enable a rapid decision about the need for studies of
bone cell activity.
Poor site specificity
The subtraction technique is not adequate to separate adjacent macroanatomic sites within bone. The
histomorphometric examination was easily limited to
the central secondary spongiosa, a n area interesting
for its bone behavior similar to the adult human skeleton (Wronski et al., 1989). In contrast, the subtraction
technique measured mineral in both the primary and
secondary spongiosa. We believe it also lacked the ability to delineate the endocortical surface properly. Furthermore, the subtraction technique would probably be
imprecise for assaying a bone formation response at the
Ability to detect poorly mineralized bone
With the subtraction technique, detecting woven
bone tissue is likely to be problematic because its mineral content is low. This could be critical because several interesting agents, including prostaglandin E,
(Jee et al., 1985), fluoride (Eriksen et al., 1985), aluminum (Quarles et al., 19891, and newer bisphosphonates (Fujimoto et al., 19901, and interventions (Rubin
and Lanyon, 1985; Burr et al., 1989) have their earliest
effects by causing accumulation of poorly mineralized
lamellar or frankly woven bone.
Physiologic alterations of endocortical surface possibly
affecting identification of cancellous bone
Duncan et al. (1973) showed that corticosteroids
cause endosteal tunneling into the inner one-third of
the cortex. This could create a weak bone layer at the
endocortical surface that might be penetrated easily
with the burr. This would lead to spurious identification of cancellous bone. Some studies suggest that the
remodeling activity and loss of bone a t the endocortical
surface is the most critical site affected by estrogen
depletion (Parfitt, 1989).
We present a rapid technique that detects differences
in cancellous bone mineral in a relevant small animal
model of egtrogen depletion bone loss. Because negligible mineral is lost during opening the bone, we believe
that one scan can be eliminated. We now recommend
using dual energy X-ray scanning (Stein e t al., 1988)
instead of DPA. The following is a n idealized two-scan
technique for finding Cn.BMC.
Step 1: With a band saw and premeasured 1cm scale,
cut off the distal 1 cm of the femur. There is little to
choose between the current technique and a similar
one t h a t removes the epiphysis a t this point (GunnessHey and Hock, 1984). However, removing the epiphysis
could prove more difficult and less reproducible in older
rats, which more resemble the aged human skeleton,
than in the younger rats usually used with that method
(Gunness-Hey and Hock, 1984). With a thin diamond
disc, split the specimen parasagitally into medial and
lateral halves. Lightly ultrasonicate both halves, then
reappose them in their anatomically correct relationship. Scan the reapposed “distal femur” (scan A).
Step 2: Working with a dissecting microscope at 510 x , use a slowly rotating round burr and discoidcleoid carver to remove all cancellous bone from each
half. The higher magnification will help ensure accurate removal of cancellous bone a t the endocortical surface. It is the operator’s choice whether to remove the
growth cartilage (Gunness-Hey and Hock, 1984).
Step 3: Lightly ultrasonicate both halves, then reappose them in their anatomically correct relationship.
Scan the reapposed “distal femur (scan B).”
Step 4: Subtract BMC of scan B from that in scan A
to find Cn.BMC.
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