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Effects of daily administration of prostaglandin E2 and its withdrawal on the lumbar vertebral bodies in male rats.

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THE ANATOMICAL RECORD 234:172-182 (1992)
Effects of Daily Administration of Prostaglandin E, and Its
Withdrawal on the Lumbar Vertebral Bodies in Male Rats
HUA ZHU KE AND WEBSTER S.S. JEE
Division of Radiobiology, University of Utah School of Medicine, Salt Lake City, Utah
(H.Z.K., W.S.S.J.) and Department of Anatomy, Zhanjiang Medical College, Zhanjiang,
Guangdong, People$ Republic of China (H.Z.K.)
ABSTRACT
The effects of daily prostaglandin E, (PGE,) treatment (on)
and PGE, treatment followed by withdrawal (on-off)on cancellous bone in
lumbar vertebral bodies were studied in 7-month-oldmale Sprague-Dawley
rats. The first groups of rats were given daily subcutaneous injections of 0,
1,3, and 6 mg PGE,/kg/d for 60,120, and 180 days, and the second group of
rats were given PGE, for 60 days followed by withdrawal for 60 and 120
days. Histomorphometric analyses were performed on double-fluorescent
labeled undecalcified sections of fourth lumbar vertebral bodies. Systemic
PGE, treatment elevated cancellous bone mass of lumbar vertebral bodies
2640% above control levels within 60 days and continued treatment maintained it for another 120 days, but the excess bone was lost after the treatment was withdrawn. PGE, treatment for 60 days increased trabecular
bone area, trabecular width, and bone formation parameters, and shortened remodeling periods in a dose-response manner. These changes were
sustained at the levels achieved by 60-day treatment in the rats treated for
120 and 180 days. The eroded perimeter increased at day 60 and further at
day 120 and then plateaued. In the on-off treated rats, the cancellous bone
area, bone formation, and resorption parameters returned to near agerelated controls by 60 days after withdrawal and were maintained there
after 120 days of withdrawal. Therefore we conclude that the continuous
treatment is needed in order to maintain the PGE,-induced bone gain.
When these findings were compared to those previously reported for the
proximal tibial metaphyses, we found that the proximal tibial spongiosa
was much more responsive to PGE, treatment than the fourth lumbar vertebral body. o 1992 Wiley-Liss, Inc.
Key words: Prostaglandin E,, Long-term treatment, Withdrawal, Cancellous bone
Daily administration of prostaglandin E, (PGE,) can
exert a n anabolic effect on bone by increasing bone
turnover and bone mass in dogs (Shih and Norrdin,
1987a,b; High, 1988; Norrdin and Shih, 1988; Li et al.,
1990b), rats (Ueno et al., 1985; Jee et al., 1985, 1987,
1990; Furuta and Jee, 1986; Mori et al., 1990, 1992),
and humans (Sone et al., 1980; Ueda et al., 1980; Lund
et al., 1982; Ringel et al., 1982). When 7-month-old
male rats were given daily subcutaneous injection for
60, 120, and 180 days, PGE, significantly increased
proximal tibial metaphyseal cancellous and tibial shaft
cortical bone mass (Jee et al., 1991, Ke et al., 1992).
However, after stopping the PGE, treatment, there
was a loss of cancellous and cortical bone to near control levels (Ke et al., 1991; Jee et al., 1992).The present
report was designed to examine two issues. First, does
the pattern of the maintenance of increased cancellous
bone mass induced by continuous PGE, treatment in
long bone metaphyses, and the subsequent loss of the
extra bone mass after withdrawal hold true in the can0
1992 WILEY-LISS, INC.
cellous bone of the axial skeleton (e.g., lumbar vertebral body)? Second, how do the responses to PGE, and
its withdrawal compare between two cancellous bone
sites, the proximal tibial metaphysis that is intermittently loaded and the fourth lumbar vertebral body
that is continuously loaded?
MATERIALS AND METHODS
A total of 110 male Sprague-Dawley rats (7 months
old), weighing 622 g (Charles River Laboratory, Inc.,
Portage, MI), were acclimated to local vivarium conditions for 16 days. The rats were allowed free access to
water and pelleted commercial natural product diet
(Rodent Laboratory Chow 5001, Ralston-Purina Co.,
-
Received December 11, 1991; accepted February 6, 1992.
Address reprint requests to Webster S.S. Jee, Building 586, Division of Radiobiology, University of Utah, Salt Lake City, UT 84112.
EFFECTS OF ON AND ON-OFF PGE, ON RAT CANCELLOUS BONE
173
Fig. 1. Microradiograph of typical fourth lumbar vertebral body (LVB)with outline marking areas of
histomorphometry analyses. The area of each zone (the cranial, caudal, and center zones) is 1.08mmz (0.6
x 1.8 mm2),total for all three zones was 3.24 mm'.
St. Louis, MO) containing 1.46%of calcium, 0.99%of
phosphorus, and 4.96 IU of vitamin D,/gram. They
were divided into six groups of five to seven rats as
follows:
I. Controls: Vehicle injections for 0,60, 120, or 180
days.
11. 60 days on: 1, 3, or 6 mg/kg/d of PGE, injections
for 60 days.
111. 120 days on: 1,3, or 6 mg/kg/d of PGE, injections
for 120 days.
IV. 180 days on: 1,3, or 6 mglkg/d of PGE, injections
for 180 days.
V. 60 days on plus 60 days off: 1 , 3 , or 6 mglkgid of
PGE, iniections for first 60 days, and then vehicle iije&ons for another 60 diys.
VI. 60 days on plus 120 days off 1,3, or 6 mglkgld of
PGE, injections for first 60 days, and then vehicle injections for another 120 days.
~~
tration was 20%)for 0 , 1, 3, or 6 mglkg/d groups. The
injections were given subcutaneously on the back. The
rats were weighed weekly and the volume of the injection solution was adjusted accordingly. All rats received a subcutaneous injection of 25 mglkg of Achromycin-tetracycline hydrochloride (Lederle Laboratory,
Pearl River, NY) 12 days before sacrifice, and 10 mg/kg
of calcein (Sigma Chemical Co., St. Louis, MO) 2 days
before sacrifice.
All animals were sacrificed by cardiac puncture under ketamine anesthesia. Blood samples were collected
and serum osteocalcin levels were determined (Patterson-Allen et al., 1982). The lungs, liver, adrenal
glands, thymus, spleen, and kidneys were removed and
weighed.
During autopsy, the fourth lumbar vetebral body
was removed, dissected, and cut parasagitally to expose
the bone marrow using a low speed metallurgical saw.
They were fixed in 10% phosphate-buffered formalin
(pH 7.2) for 24 hours and then dehydrated in graded
ethanol, defatted in acetone, and embedded in methyl
methacrylate (Eastman Organic Chemicals, Rochester,
NY). Sagittal sections were cut at 220 p m thickness
using a low speed saw and then ground to 100 pm using
a precision lapping machine (Maruto, Co., Tokyo,
Japan). Microradiographs were prepared on spectroscopic plates (Ultra Fla lA, Imtec, Salt Lake City, UT)
at 12 Kv, 25 mA for 7 min. Thereafter, sections were
mounted on plastic slides and further ground to a
thickness of 20 pm and stained with 0.1% toluidine
blue 0 (Fisher Scientific, Co., Fair Lawn, NJ), then
coverslipped (Jee et al., 1983) for histomorphometric
analyses.
This study is one part of our larger study on the
skeletal responses to continuous and on/off PGE, treatment in the rats. The effects of continuous daily administration on proximal tibial metaphyseal cancellous
and tibial diaphyseal cortical bone involving Groups
I-IV and the effects of onloff PGE, treatment on these
bones involving Groups I, 11, V, and VI have been previously reported (Ke et al., 1991, 1992; Jee et al., 1991,
1992). The current study involved all groups (1-VI), to
detect the effects of continual daily (on) and oxdoff
PGE, treatment on lumbar vertebral body cancellous
bone.
All rats, except those killed at day 0, received injections of 1 mllkg PGE, solution. Powdered PGEz (UpHistomorphometricAnalyses
john Co., Kalamazoo, MI) was first dissolved in 100%
Undecalcified sections (20 pm thick) were viewed
ethanol and further diluted with deionized water into
desired PGE, concentrations (the final ethanol concen- qualitatively and quantitatively. The static and kinetic
H.Z. KE AND W.S.S. JEE
174
TABLE 1. Histomorphometreic measurements and calculations
Parameters
I. Static measurements and calculations
Total tissue area
Trabecular bone area
Trabecular bone perimeter
Eroded perimeter
Osteoid perimeter
Wall width
Trabecular width
Trabecular number
Trabecular separation
Percent trabecular bone area
Percent eroded perimeter
Percent osteoid perimeter
Percent remodeling perimeter
11. Dynamic measurements and calculations
Single labeled perimeter
Double labeled perimeter
Interlabel width
Mineral apposition rate
Labeled perimeter
Percent labeled perimeter
Bone formation rate (bone area referent)
Bone formation rate (tissue area referent)
Formation period
Resorption period
Remodeling period
Bone balance
Formed bone area
Resorbed bone area
Abbreviations
T.Ar
Tb.Ar
Tb.Pm.
E.Pm
O.Pm
W.Wi
Tb.Wi
Tb.N.
Tbsp
%Tb.Ar
%E.Pm
%O.Pm
%Rm.Pm
sL.Pm
dL.Pm
IrL.Wi
MAR
L.Pm
%L.Pm
BFR/BV
BFRJTV
F.P
R.P
Rm.P
B.B
F.B.Ar
R.B.Ar
Formulae
1
1
1
1
1
(2000/1.199) x Tb.Ar/Tb.Pm
1.199/2 x Tb.Pm/T.Ar
(2000/1.199) x (T.Ar-Tb.Ar)/Tb.Pm
Tb.Ar/T.Ar x 100
Er.Pm/Tb.Pm x 100
O.Pm/Tb.Pm x 100
(E.Pm + O.Pm)/Tb.Pm x 100
IrL. Wi/interva12
s L . P d 2 + dL.Pm
L.Pm/Tb.Pm x 100
L.PmxMAR/Tb.Ar x 365 x 100
L.PmxMAR/T.Ar x 365 X 100
W.Wi/MAR x L.Pm/O.Pm
F.P x E.Pm/O.Pm
F.P + R.P
Tb.Ar, - Tb.Ar,
(L.Pm, x MAR, + L.Pm, x MAR,)/2 x period
F.B.Ar - B.B
Units
mm2
mm2
mm
mm
mm
)Lm
I*m
/mm
)Lm
%
%
70
%
mm
mm
I*m
Pdd
mm
%
%ly
%/y
days
days
days
mm2
mm2
mm2
~~~
'Measured parameter.
'Int: Labeling interval defined as the interval between first and second labeling date.
measurements were performed on the 20-pm-thick undecalcified sections using a digitizing image analyzing
system, which consists of a light or epifluorescent microscope, a n Apple Macintosh Computer with a digitizing pad, and a morphometry program named "Stereology" (KSS Computer Engineers, Magna, UT). We
selected three zones for the histomorphometric measurements of the lumbar vertebral body: the cranial,
caudal, and center zones. The area of each zone is 1.08
mm2 (0.6 x 1.8 mm2),total for all three zones was 3.24
mm2 (Fig. 1).The 0.6 mm metaphyseal region adjacent
to the growth cartilage metaphyseal junction was
avoided in order to exclude the primary spongiosa from
the measurements. All measurements were performed
a t 156 x or 312 x magnification. Static and dynamic
histomorphometric measurements and calculations are
summarized in Table 1 according to Jee e t al. (1983)
and Parfitt et al. (1987).
dependent response, 60, 120, and 180 days) by linear
regression (Sokal and Rohlf, 1980).
RESULTS
Previous Findings
Changes in body weights, serum osteocalcin level,
and soft tissue weights have been reported previously
(Ke et al., 1991, 1992). Briefly, these animals lost
about 20% of their body weight after 60 days of PGE,
and their weight remained depressed at that level
thereafter. They regained some of the weight after
PGE, was withdrawn. The adrenals, kidneys, liver,
and lungs were heavier in animals given PGE,. However, these same organs weighed within the normal
range after the withdrawal of PGE,. Serum osteocalcin
levels were elevated in PGE, animals but fell to control
level after withdrawal.
Statistical Analyses
Statistical differences between age-matched control
and treatment groups were evaluated for each time
period using Analysis of Variance with Dunnett's t-test
(Neter et al., 1982). Statistical differences between 60
day on and odoff treated groups were evaluated using
two-tail Student's t-test. The data were further analyzed a s a function of dose (dose-dependent response, 0,
1, 3, and 6 mg/kg/d) and as a function of time (time-
Aging Bone Changes
No age-related differences were found between 7 and
13months of age in percentage of trabecular bone area,
trabecular bone structure (width, number and separation), and bone formation and resorption parameters of
the fourth lumbar vertebral bodies (Tables 2,3). Linear
regression analyses further confirmed the above observations (see Table 5).
E F F E C T S OF ON AND ON-OFF PGE, ON RAT CANCELLOUS BONE
175
TABLE 2. Static histomorphometricparameters of 4th lumbar vertebral body cancellous bone'
Trabecular
bone area
Trabecular
number
(#lmm)
Trabecular
separation
( pm)
Osteoid
perimeter
60.7
6.6
3.89
0.41
286
35
5.12
1.47
4.14
0.63
9.3
1.6
14.1
1.1
0.0011
21.5
4.6
5 26.4
4.6
6 33.5#
3.8
7 37.0#
8.5
0.0002
57.8
6.0
74.7#
2.4
89.5#
7.8
87.4#
17.0
0.1720
3.70
0.52
3.55
0.68
3.75
0.26
4.23
0.55
0.0415
312
60
311
86
257
29
219#
48
0.3241
7.41
1.29
9.42
3.66
10.63
1.71
9.91
3.66
0.1182
4.35
1.37
4.20
1.46
5.36
0.71
5.87
1.51
0.1552
11.8
1.8
13.6
4.8
16.0
2.2
15.8
3.5
0.0003
16.9
2.6
20.4
0.8
21.8#
2.9
28.1#
5.5
0.0159
20.9
3.2
5 27.9#
4.4
6 30.7#
6.7
7 30.7#
4.2
0.0062
58.1
5.1
71.3
7.4
75.1#,@
11.3
83.3#
12.9
0.3374
3.59
0.42
3.90
0.24
4.09
0.68
3.70
0.38
0.1113
322
50
267
30
251#
59
272@
34
0.0015
5.68
0.90
8.96
1.29
11.1#
2.33
13.4#
4.39
0.0309
4.39
1.19
6.70#,@
0.93
7.10#
2.28
6.90#
0.76
0.0001
10.1
1.7
15.7#
1.4
18.1#
2.6
20.3#
4.1
0.0001
13.6
0.7
15.5@
1.5
24.5#
1.2
22.9#
3.5
0.0001
23.0
2.5
5 28.0#
1.9
4 31.1#
3.5
3 37.3#
1.9
0.0323
65.0
10.0
77.7
11.2
89.4#
9.3
82.8#
14.6
0.0559
3.58
0.47
3.64
0.41
3.48
0.13
4.63#
1.09
0.0090
314
45
288
34
285
20
202#
48
0.0011
7.73
2.09
12.8#
3.25
18.6#,@
3.94
18.1#
4.91
0.1114
4.75
0.80
6.50
2.21
7.24@
1.70
8.10#
2.48
0.0035
12.5
2.8
19.3#
5.0
25.9#,@~
5.6
26.1#
6.7
0.0135
17.5
1.4
19.1
2.6
23.4#
3.4
22.5#
2.8
SD
0.4663
20.9
3.2
7 23.4
2.5
7 23.7@
3.4
5 24.1@
5.0
0.2420
58.1
5.1
64.6
10.8
65.4@
5.2
68.6@
9.7
0.8991
3.59
0.42
3.67
0.42
3.62
0.36
3.5@
0.29
0.8754
322
50
303
32
306@
41
314@
41
0.0020
5.68@
0.90
7.30#
1.01
7.01@
1.03
9.20#
1.84
0.1293
4.39
1.19
4.97
1.08
5.92
2.33
6.70#
1.20
0.0022
10.1
1.7
12.3
1.6
12.9@
2.0
16.0#
2.8
0.4191
13.6
0.7
15.1@
1.1
15.2@
2.8
15.06~
0.7
Mean
6
0.7440
23.0
2.5
6 24.3
3.0
8 24.7@
2.2
5 24.2@
0.0136
65.0
10.0
68.8
5.5
80.3#,@
6.9
71.2@
2.1
0.1340
3.58
0.47
3.52
0.17
3.09#,@
0.32
4.01@
0.40
0.3382
314
45
310
26
354
50
325@
48
0.2560
7.73
2.09
9.40
1.96
10.39
1.96
8.79
2.57
0.7878
4.75
0.80
4.22
0.98
4.42
1.02
4.81
1.33
0.5460
12.5
2.8
13.6
2.0
14.8
2.2
13.6
3.5
0.2190
17.5
1.4
16.5
0.9
18.9
2.8
18.0Cu~
0.9
(%I
n
day 0
0 mglkgld
Remodeling
perimeter
Trabecular
width
(pm)
Mean
SD
5
23.5
2.6
(%I
Eroded
perimeter
(%I
(%)
Wall
width
(pm)
day 60
ANOVA
0 mglkgld
Mean
SD
1 mglkgid
3 mglkgld
Mean
SD
Mean
6 mglkgld
Mean
SD
SD
5
day 120
ANOVA
0 mgikgld
Mean
SD
Mean
1 mglkgld
SD
3 mglkgld
Mean
SD
Mean
6 mglkgld
SD
5
day 180
ANOVA
0 mglkgld
Mean
SD
Mean
1 mglkgld
SD
3 mglkgld
Mean
SD
Mean
6 mglkgld
SD
60 on-60
off
ANOVA
0 mglkgld
Mean
SD
Mean
SD
Mean
SD
Mean
1 mglkgld
3 mglkgld
6 mglkgld
60 on-120
5
off
ANOVA
0 mglkgld
SD
1 mglkgld
Mean
SD
3 mglkgld
Mean
SD
6 mglkgid
~~
6
Mean
Sn
_~
2.6
~-
~
'Key: N = Number of animals; #: P < 0.05 vs. age-related controls; @: P < 0.05 vs. the value of 60 days PGE2 treatment at the same dose levels;
on: PGE2 treated period, off: vehicle injection period.
Consequence of Continuous Daily PGE, Administration
Figure 2 shows the responses to daily subcutaneous
administration of graded doses of PGE, for 60,120, and
180 days. PGE, treatment increased trabecular bone
mass in a dose-dependent manner by the first 60 days
(Fig. 2D, G, J), and the increased bone mass was maintained by continuous treatment a t days 120 and 180
(pictures not shown).
H.Z. KE AND W.S.S. JEE
176
TABLE 3. Dynamic histomorphometric parameters of 4th lumbar vertebral body cancellous bone'
n
day 0
0 mglkgld
Mean
day 60
ANOVA
0 mglkgld
Mean
1mglkgld
Mean
5
SD
5
SD
5
SD
3 mglkgld
Mean
6
SD
6 mglkgld
Mean
7
SD
day 120
ANOVA
0 mglkgld
Mean
5
SD
1 mglkgld
3 mglkgld
6 mglkgld
Mean
SD
Mean
SD
Mean
5
6
7
SD
day 180
ANOVA
0 mglkgld
Mean
6
SD
1 mglkgld
3 mglkgld
6 mglkgld
Mean
SD
Mean
SD
Mean
5
4
3
SD
60 on-60 off
ANOVA
0 mglkgld
1 mglkgld
3 mglkgld
Mean
SD
Mean
SD
Mean
5
7
7
SD
6 mglkgld
60 on-120 off
ANOVA
0 mglkgld
1 mglkgld
Mean
SD
5
Mean
SD
Mean
6
6
SD
3 mglkgld
Mean
8
SD
6 mglkgld
Mean
5
Mineral
apposition
rate
(@d)
Labeling
perimeter
Bone
formation
rate1BV
(%ly)
Bone
formation
rate1TV
(%ly)
Formation
period
(days)
Resorption
period
(days)
Remodel'ng
period
(days)
0.85
0.10
6.2
1.3
52
11
12
3
16.2
12.1
12.2
5.6
28.4
17.3
0.0004
0.99
0.23
1.15
0.11
1.53#
0.29
1.58#
0.38
0.0002
7.7
2.0
16.0#
3.4
18.9#
4.2
21.3#
5.5
0.0041
82
30
151
42
198#
61
242#
98
0.0001
18
10
39
9
65#
14
86#
31
0.0408
19.3
11.6
10.8#
3.8
8.7#
3.6
8.9#
3.9
0.0021
10.2
3.6
4.7#
1.2
4.5#
1.9
5.3#
2.2
0.0013
29.6
14.4
15.5#
4.7
13.2#
5.4
14.2#
5.0
0.0001
0.80
0.10
1.19#
0.08
1.42#
0.15
1.43#
0.20
0.0001
7.4
1.3
14.0#
2.8
23.4#
2.8
21.6#
3.6
0.0001
64
20
150#
45
274#
63
231#
65
0.0001
13
4
40#
9
82#
16
70#
17
0.0231
13.5
3.4
8.5#
1.0
8.4#
2.3
10.5#
3.3
0.0026
10.2
2.5
6.6#
2.1
5.4#
2.0
5.2#
0.8
0.0013
23.7
4.7
15.1#
3.0
13.7#
3.2
15.3#
3.2
0.0003
0.87
0.13
1.26#
0.21
1.55#
0.22
1.49#
0.16
0.0002
7.8
2.0
12.9#
1.8
21.8#
2.9
21.6#
8.3
0.0002
63
13
129
31
236#
65
239#
88
0.0001
15
4
36
10
73#
19
90#
36
0.2064
20.2
3.3
15.7
6.5
13.1
3.2
13.9
6.3
0.0067
12.8
3.3
8.1#
3.5
5.1#
1.6
5.9#
2.0
0.0476
33.0
6.0
23.8
9.6
18.2#
4.8
19.8#
8.1
0.0041
0.80
0.10
0.4@
0.17
1.05#,@
0.18
1.20#,@
0.14
0.2340
7.4
1.3
10.1
4.9
9.4@'
2.9
11.8@
2.8
0.0809
64
20
91(d
43
940
38
131#,@
46
0.0184
13
4
21@)
11
21@
7
30#,@
17
0.4101
13.5
3.4
13.9
5.9
11.7
4.1
10.0
1.6
0.5230
10.2
2.5
9.2@
3.1
10.1(@!
4.7
7.4
1.7
0.4367
23.7
4.7
23.1
8.5
21.865
7.7
17.3
3.1
0.3580
0.87
0.13
0.86@
0.08
0.90@
0.08
0.9863
0.0614
7.8
2.0
8.5@
1.4
11.4#,@
3.1
10.463
1.4
0.1552
63
13
64@
10
7963
25
87@
15
0.2087
15
4
l6@
4
19@
6
21@
3
0.4907
20.2
3.3
22.4@
8.0
20.3@
7.4
15.8@
4.3
0.1210
12.8
3.3
9.8@
2.9
8.4#,@
2.6
8.8@
3.1
0.4341
33.0
6.0
32.2@
9.4
28.76
9.5
24.6@
6.5
(%I
'Key: n = number of animals; #: P < 0.05 vs. age-related controls; (u : P < 0.05 vs. the value of 60 days PGE2 treatment a t the same dose levels;
on: PGE2 treated period, off: vehicle injection period.
When compared to age-related controls, PGE, treatment for 60 days significantly increased percent trabecular bone area ( + 56% and + 72% at 3 and 6 mg/
kgld dose levels) and trabecular width ( + 55% and
+44% at 3 and 6 mg/kgld dose levels) (Table 2, Fig. 3
A, B), and significantly decreased trabecular bone separation (Table 2). There was no significant difference
in trabecular number (Table 2; Fig. 3C). The bone mass
gain was due to bone formation exceeding bone resorption: a significant increase in percent osteoid perime-
E F F E C T S OF ON AND ON-OFF PGE, ON RAT CANCELLOUS BONE
177
Fig. 2. Microradiographs showing cancellous bone changes in fourth
lumbar vertebral bodies from age-matched controls at day 60 (A), 120
(B) and 180 (C);and from rats treated daily with PGE, at 1mg/kg/d
dose level for 60 (D), 60 od60 off (E) and 60 od120 off (F) days; at 3
mgkg/d dose level for 60 (G),60 od60 off (H) and 60 o d l 2 0 off (I)
days; and a t 6 mg/kg/d dose level for 60 (J), 60 od60 off (K)and 60
o d l 2 0 off (L) days; 100 pm undecalcified sections. x 36.
ter, percent labeling perimeter, mineral apposition
rate, and bone formation rates (BFWBV and BFR/TV)
along with a nonsignificant increase in percentage of
eroded perimeter (Table 2; Fig. 3 D, E). Furthermore,
there was a significant shortening of the bone remodeling period (a decrease in formation, resorption, and
remodeling periods, Table 3; Fig. 3F). Except for continual increases in percent osteoid perimeter and
eroded perimeter, all other parameters were sustained
at the levels achieved by 60 days of treatment with
continuous daily administration (Tables 2, 3; Figs. 2,
3). During the first 60 days of PGE, treatment, the
formed bone area (representing bone formation) increased significantly (Fig. 4A) and the resorbed bone
area (representing bone resorption) was unchanged
(Fig. 4B) in the PGEz treated rats compared to agerelated controls. The net result was a significant increase in positive bone balance (Fig. 4C). Thereafter,
the formed bone area remained elevated (Fig. 4A), but
the resorbed bone area (bone resorption) increased significantly in the PGEz treated rats over age-related
controls during the 120 and 180 days of treatment. This
resulted in bone formation equaling bone resorption,
and a nonsignificant difference in bone balance (Fig.
H.Z. KE AND W.S.S. JEE
178
B. Trabecular width (bm)
A. Percent trabecular bone area (%)
40 -
35
J
-
30 -
/
I
:
20
0
Controls
1
1
I
60
120
180
50
0
60
120
180
D. Bone formation ratelBV (%Is)
C. Trabecular number (#him)
.
6 rng on
u
4.5
1
0
I
120
180
F. Remodeling period (days)
E. Percent eroded perimeter (%)
'1
60
35
1
.3 mg on
I m g on
Controls
7 rng off
-1 rng off
0
60
120
180
n
u
h0
120
10
0
180 Days
Fig. 3. Time course of (A) percent trabecular area, (B) trabecular
width, ( C ) trabecular number, (D) bone formation rate/BV, (E)percent eroded perimeter, and (F) remodeling period changes in controls,
continuous and o d o f fPGE, treated fourth lumbar vertebral body cancellous bone. #P < 0.05 compared with age-related controls; @P <
0.05 compared with day 60 at the same dose levels.
4C). Thus a new steady state was established with
more bone and higher bone turnover rates after 60
days or more of treatment (Tables 2, 3; Figs. 2-4).
The linear regression analyses of dose-dependent responses ( 0 , 1 , 3 , and 6 mglkgld dose levels) showed that
all parameters listed in Tables 2 and 3 were significant
at days 60, 120, and 180 (data not shown). The linear
regression analyses of time-dependent responses between 60, 120, and 180 days of PGE, administration
indicated that static and dynamic histomorphometry
were unchanged between these time periods. This further reinforces the finding that the cancellous bone of
EFFECTS OF ON AND ON-OFF PGE, ON RAT CANCELLOUS BONE
U>
A. Formed bone a r u (rnrn"2)
08
U. R c w r b c d bone LreJ frnm"2)
C. Ilone I u l n n c e
06
n,
04
9:
02
00
00
00
02
02
02
60 on
06
04
02
04
04
06
06
120 on
60 nn
120 on
180 on
60 on
E. Resorbed bone a r e a (mrn"2)
D. Formed bone area (mm"2)
0.8
0.8
1) I,
0.6
0.6
0.;
0.4
0.4
01
02
0.2
00
0.0
-0 2
-0 2
-0 2
-0:;
-0.4
.I),*
60 off
120 oif
so
0"
0.0
-0.6
60 on
120 o~
F. Bone balance ( m m " ? )
05
-0.6
mmA?
08
iib
11 6
179
-0.6
60 on
(A "If
120off
60on
69 0:T
,
t L-< l Z > . .
Fig. 4. Changes in formed bone area, resorbed bone area and bone balance, between 0 to 60,60 to 120,
and 120 to 180 days for continuous and odoff PGE, treatment. #P < 0.05 compared with age-related
controls; @P < 0.05 compared with day 60 at the same dose levels.
the lumbar vertebral bodies had more or less settled
into a new steady state after 60 days of daily PGE,
administration (data not shown).
Consequence of PGE, Withdrawal
PGE, treatment increased trabecular bone mass in a
dose-dependent manner by the first 60 days (Fig. 2D,
G, J).But the increased bone mass was nearly lost to
age-related control levels (Fig. 2B) in the first 60 days
of PGE, withdrawal (Figs. 2 E, H, K, 3A) and the same
levels of bone mass were maintained for another 60
days after withdrawal (120 days of withdrawal) (Figs. 2
F, I, L, 3A).
After 60 days of withdrawal, trabecular bone area
and trabecular number decreased significantly, and
separation increased significantly compared to 60-day
treated levels, and all of these parameters returned to
age-related controls (Table 2; Fig. 3A, B). Some histomorphometric parameters were still significantly
above the age-related control levels. These include osteoid and eroded perimeters (Fig. 3E), mineral appositional and bone formation rates (Fig. 3F), especially at
the 6 mg PGE,/kg/d on/off dose levels (Tables 2, 3).
However, after 120 days of withdrawal, these parameters had fallen, the remodeling period had risen to agerelated controls (Tables 2, 3; Figs. 4B,C, D).
After the first 60 days of withdrawal of PGE, treatment, the formed bone area remained at the same elevated level seen for the 60 day PGE, treated group (up
to a factor of 3, Fig. 4D). However, the area of bone
resorbed was 2 to 6 times higher than controls (Fig.
4E), creating a negative bone balance (Fig. 4F). Subsequent withdrawal for 120 days caused formed and resorbed bone area to fall to control (typical steady state)
levels. Thus the withdrawal of PGE, for 60 days rids
the lumbar vertebral body of cancellous bone and this
reestablishes a new steady state in which the bone
mass and turnover rates nearly match normal age-related control levels (Tables 2, 3).
Linear regression analyses of the time course responses for odoff PGEz treatment further supported
the reduction in static and dynamic histomorphometry
reported above. It was clearest in animals treated on/
off with 6 mg PGE,/kg/d for 60 days than for those a t
lower doses. Trabecular width and osteoid, eroded and
remodeling perimeters were the only parameters not
exhibiting significant depression (Table 4).
DISCUSSION
The current study further confirmed that PGEz is a
powerful anabolic agent. It stimulated lamellar bone
formation, increased bone turnover, and shortened the
bone remodeling cycle to create a positive net bone balance (Jee et al., 1985, 1987, 1990; Ueno et al., 1985;
Furuta and Jee, 1986; Mori et al., 1990,1992; Ke et al.,
1992). After daily administration of PGE, for 60 days,
a new steady state was achieved in which there were
increases in cancellous bone mass and bone turnover
with a shortened bone remodeling cycle. After the
withdrawal of PGE, administration, the new bone
H.Z. KE AND W.S.S. JEE
180
TABLE 4. Linear regression anal ses of 4th lumbar vertebral trabecular bone histomorphometry (on-off
geatment. time deDendent resDonses)'
Feature
1 rnglkgld'
Trabecular bone area
Trabecular width
Trabecular number
Trabecular separation
Osteoid perimeter
Eroded perimeter
Remodeling perimeter
Wall width
Mineral apposition rate
Labeling perimeter
Bone formation rate/BV
Bone formation rate/TV
Formation period
Resorption period
Remodeling period
~~
Units
Intercept
Slope
SE
R
P
26.931
75.579
3.604
309.923
8.823
4.414
13.237
21.455
1.276
19.102
190.102
48.700
4.179
2.712
6.891
-0.019
-0.053
-0.0002
-0.016
-0.001
0.001
-0.001
-0.350
-0.002
-0.063
-0.736
-0.196
0.095
0.044
0.139
0.018
0.040
0.002
0.277
0.013
0.006
0.016
0.010
0.001
0.019
0.183
0.045
0.030
0.013
0.037
0.266
0.324
0.019
0.015
0.025
0.022
0.013
0.678
0.707
0.661
0.720
0.745
0.636
0.648
0.692
NS
NS
NS
NS
NS
NS
NS
c0.01
c0.01
c0.01
c0.01
<0.001
c0.01
<0.01
<0.01
35.970
86.892
4.149
207.882
9.461
6.211
15.672
21.395
1.780
20.488
240.486
80.000
1.966
3.870
5.836
-0.074
-0.077
-0.005
0.816
-0.002
-0.008
-0.010
-0.024
-0.005
-0.062
-0.987
-0.379
0.096
0.033
0.129
0.020
0.057
0.002
0.191
0.011
0.008
0.011
0.018
0.001
0.021
0.231
0.068
0.025
0.018
0.036
0.665
0.310
0.647
0.720
0.043
0.237
0.205
0.308
0.778
0.577
0.720
0.810
0.677
0.398
0.655
c0.01
42.034
93.297
4.577
174.686
10.451
6.695
17.145
31.544
1.857
25.988
313.107
113.700
4.895
3.666
8.561
-0.114
-0.147
-0.007
-9.310
-0.010
-0.007
-0.017
-0.094
-0.005
-0.096
-1.335
-0.570
0.055
0.029
0.084
0.037
0.070
0.002
0.262
0.015
0.008
0.017
0.028
0.001
0.023
0.365
0.122
0.190
0.012
0.026
0.650
0.505
0.639
0.701
0.176
0.245
0.263
0.675
0.710
0.758
0.712
0.790
0.633
0.561
0.671
~
3 mg/kg/d2
Trabecular bone area
Trabecular width
Trabecular number
Trabecular separation
Osteoid perimeter
Eroded perimeter
Remodeling perimeter
Wall width
Mineral apposition rate
Labeling perimeter
Bone formation rate/BV
Bone formation r a t e l m
Formation period
Resorption period
Remodeling period
6 mg/kgld2
Trabecular bone area
Trabecular width
Trabecular number
Trabecular separation
Osteoid perimeter
Eroded perimeter
Remodeling perimeter
Wall width
Mineral apposition rate
Labeling perimeter
Bone formation rate/BV
Bone formation rate/TV
Formation period
Resorption period
Remideling period
%
Pm
Imm
Pm
%
%
%
Pm
Pdd
%
%/y
%ly
days
days
days
%
Pm
/mm
Pm
%
%
%
Pm
d d
%
%/y
%/y
days
days
days
%
Pm
/mm
Pm
%
%
%
Pm
Pdd
%
%/y
%/y
days
days
days
NS
c0.01
<0.001
NS
NS
NS
NS
<0.001
<0.01
co.001
<0.001
c0.01
NS
c0.01
c0.01
NS
c0.05
c0.01
NS
NS
NS
c0.01
<0.01
<0.01
c0.01
co.001
<0.05
<0.05
c0.01
'Untreated lumbar vertebral body (0 mg/kg/d) showed no significant response with time.
'60 on, 60 on - 60 off, 60 on - 120 off.
NS = No significant difference (P > 0.05).
mass induced by 60 days of PGE, treatment was lost.
Percentage of trabecular bone area returned to near
age-related control levels after 60 and 120 days of withdrawal. The loss of new bone mass after withdrawal
was due to the cessation of PGE,-stimulated bone formation and increased bone resorption above the level
established by PGE, treatment.
The detailed mechanism of the in vivo effects of exogenous PGE, on bone metabolism is unknown. Frost's
mechanostat indicated that the threshold (set point) of
bone modeling and remodeling controls bone mass
(Skeletal adaptation t o mechanical usage; Frost, 1983,
1986a,b, 1987, 1988a,b, 1990a,b).The PGE, treatment
may decrease the set point of bone modeling and remodeling, which induces an increase in bone modeling
and remodeling in formation mode (switching on a positive bone balance mechanism). Theoretically, as long
as treatment continues, there should be a positive bone
balance and bone should be added indefinitely. However, under continued treatment, there was no indefinite continued addition of bone, because the addition of
bone eventually made the mechanostat sense that
E F F E C T S OF ON AND ON-OFF PGE, ON RAT CANCELLOUS BONE
181
TABLE 5. Comparison between 4th lumbar vertebral body and proximal tibial metaphyseal cancellous bone
between 7 and 13 months of age
Trabecular
Age
bone area
(months)
(%)
Lumbar vertebral body
7
23.48
9
21.54
11
20.85
13
23.01
Intercept
22.531
Slope
-0.003
SE
0.001
R
0.072
P
NS
Proximal tibial metaphyses
7
13.21
9
11.60
11
10.62
13
8.94
Intercept
13.07
Slope
-0.02
SE
0.01
R
0.46
P
<0.05
NS
=
Trabecular
width
(#/mm)
Trabecular
number
(pm)
60.69
57.84
58.07
65.05
58.413
0.022
0.024
0.212
NS
3.89
3.70
3.59
3.58
3.843
-0.002
0.001
0.27
NS
57.24
51.46
69.16
59.69
55.75
0.04
0.04
0.25
2.30
2.20
1.61
1.50
2.35
-0.01
0.00
0.54
<0.05
NS
Bone
Bone
formation
rate/BV
formation
rate/TV
(%)
(%/Y)
(%/Y)
4.14
4.35
4.39
4.75
4.128
0.003
0.003
0.217
52
82
64
63
63.008
0.025
0.072
0.08
Eroded
perimeter
Remodeling period
(days)
Perimeter
12
18
13
15
14.4
0.003
0.02
0.039
28
30
24
33
28
0
0.013
0.079
area
(ratio)
NS
NS
NS
NS
27.77
29.13
28.91
26.12
28.90
-0.01
0.01
0.19
NS
6.26
7.09
9.51
10.05
6.165
0.023
0.004
0.790
<0.001
330
404
385
308
371
-0.174
0.003
0.090
NS
42
41
41
25
45
-0.090
0.040
0.420
NS
42
42
43
54
39.9
0.063
0.038
0.360
NS
29.15
33.16
24.89
28.30
30.71
-0.02
0.01
0.26
NS
no significant difference (P > 0.05).
there is more bone than needed and it proceeded to
remove the bone as fast as it was added. As a consequence, as long as the PGE2 treatment continued, the
bone mass should plateau at a higher level and the
local turnover rate would be elevated. The relative increase in bone mass a t the new steady state should
provide a clue as to the relative decrease in the setpoint
of the threshold that controls switching on the positive
bone balance responses. Apparently, the mechanostat
somehow can dominate drug action at the cell level in
order to prevent bone mass and architecture from departing too far from the optimum relative to typical
mechanical usage.
Once the PGE, treatment was withdrawn, the set
point returned to the higher control level. Trabecular
bone was underloaded (more bone than needed). The
above change shuts off the positive bone balance responses and activates the mechanostat to rid itself of
the unneeded bone to adapt its typical mechanical usage (Frost, 1987, 1988a,b, 1989a,b; Li et al., 1990a; Ke
et al., 1991; Jee et al., 1992).
We have studied two different cancellous bone sites
of the skeleton's response to PGE, treatment in the
same animals. One site came from the appendicular
skeleton, the proximal tibial metaphysis and the other
from the axial skeleton, the lumbar vertebral body.
The long-term PGEz and odoff PGE, responses in the
proximal tibial metaphyses have been reported previously (Ke et al., 1991, 1992). In general, the two cancellous bone sites exhibit similar effects to continuous
(on) and odoff PGE, administration. The cancellous
bone areas in both bone sites were significantly increased by the first 60 days of PGE, treatment. Continuous treatment was able to maintain the increased
bone mass but was unable to increase bone mass further (Ke et al., 1992).The increased bone mass was lost
after 60 days withdrawal of PGE, (Ke et al., 1991).
However, there were some differences in the behavior
of cancellous bone in two bone sites during the first 60
days of PGE, treatment (Table 6). First, the proximal
tibial metaphysis added nearly three times ( + 248%)as
much bone with 6 mg PGE,/kg/d than did the lumbar
vertebral body ( + 72%).This could be partly attributed
to the woven bone formation seen in the proximal tibia
(Ke et al., in press) but not seen in the lumbar vertebral body. Second, both trabecular width and number
were significantly increased in the proximal tibial
metaphyses, but only trabecular width was significantly increased in the lumbar vertebral body (Table
6). The above differences indicate that the proximal
tibial spongiosa is much more responsive to PGE,
treatment than the lumbar vertebral body.
We can only speculate on why the proximal tibial
metaphysis is more responsive than the lumbar vertebral body to PGE, treatment. One explanation is found
in the properties of the mechanostat. In terms of mean
tissue age, the vertebral spongiosa is older than the
tissue underneath the growth plate of the tibial metaphysis so the vertebral spongiosa has had longer to
adapt to its typical mechanical usage. The tibial spongiosa must be considered to be still in the process of
adapting. Given this difference, lowering the threshold
that switches lamellar bone drifts to woven bone then
should do several things. In the vertebra where the
bones are reasonably well adapted so that only little or
no modeling in the formation mode would be going on,
lowering the threshold would start lamellar bone
drifts, which would thicken the existing trabeculae but
not necessarily add woven bone. In the tibial metaphysis, however, where the spongiosa has had less time to
adapt to typical mechanical usage, some activity in the
modeling mode should already be going on so that low-
H.Z. KE AND W.S.S. J E E
182
TABLE 6. Two cancellous bone sites response to 6 mg
PGE2kgld treated for 60 days’
Trabecular bone area (%)
Trabecular width (pm)
Trabecular number (#/mm)
Trabecular separation (pm)
LV4B
FTM
t ( + 72%)
t ( + 44%)
t ( + 248%)
0
J. (-30%)
t ( + 53%)
‘Key: LV4B: Fourth lumbar vertebral body; PTM: Proximal tibial
metaphyses; t : Significantly increased from age-related controls; 1:
Significantly decreased from age-related controls; 0: no significant
difference from age-related controls; 0:Percent change from age-related controls.
ering the threshold would more readily initiate woven
bone formation (a higher level of formation drift than
lamellar bone formation) as well as lamellar bone formation on the existing bone surface. There are numerous other factors that must be taken into consideration
when evaluating the ability of an anabolic agent to
increase bone mass a t these two sites. These include the
availability of osteoblastic precursor cells, the role of
intermittent loading of the extremities versus the continuous loading of the axial skeleton, the differences in
mechanical loading, and other unknown factors.
ACKNOWLEDGMENTS
This study was supported by research grants from
the National Institutes of Health (AR-38346),the Department of Energy (DE-FG02-89ER60764 AOOl), and
the National Aeronautics and Space Administration
(NAG-2-4351,as well as by a contract from the Department of Energy (DE-AC02-76EV 00119). The authors
thank Dr. Charles Hall and Ronald Lane of Upjohn Co.
for the generous supply of prostaglandin E,.
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