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Effects of prostaglandin E2 and risedronate administration on cancellous bone in older female rats
Bone, Vol . 15, No . 5, pp. 489-496, 1994 Copyright C 1994 Elsevier Science Ltd Printed in the USA . All rights reserved 8756-328294 $6 .00 + . 00
Pergamon 8756-3282(94)EO010-V
Effects of Prostaglandin E 2 and Risedronate Administration on Cancellous Bone in Older Female Rats t,2 1,5 B . Y . LIN, W . S . S . JEE,' Y . F . MA,' H . Z . KE" 3 D . B . KIMMEL, 4 and X . J . LI Division of Radiobiology, University of Utah School of Medicine, Salt Lake City, UT, USA Bone Biology Laboratory, Guangdong Medical College, Zhanjiang City, Guangdong, P .R . China ' Department of Metabolic Diseases, Pfizer Central Research, Groton, CT, USA 4 Center for Hard Tissue Research, Department of Medicine, Creighton University, College of Medicine, Omaha, NE, USA ` Procter & Gamble Pharmaceuticals, Inc ., Miami Valley Laboratories, Ross, OH, USA 2
Address for correspondence and reprints : Webster S . S . lee, PhD, Radiobiology Division, Building 586, University of Utah, Salt Lake City, UT 84112, USA .
Abstract
PGE, alone in old bone without endochondral ossification . However, the tissue mechanisms by which PGE, alone and PGE,, + Ris treatments accumulated bone differed in that the latter allowed the same bone mass to accumulate with lower levels of cell recruitment and activity .
The effects of Prostaglandin E, (PGE,,) and Risedronate (Ris) both separately and in combination (PGE, + Ris) were studied on the intact aged female rat skeleton to determine whether the combination of PGE, with an antiresorptive agent is more effective anabolically than PGE, alone . Ninemonth-old Sprague-Dawley rats were injected subcutaneously either with vehicle, 6 mg PGE,/kg per day, 1 or 5 µg Ris/kg twice a week, or 6 mg PGE,Ikg per day plus 1 or 5 µg Ris/kg twice a week (PGE, + I Ris or PGE, + 5 Big) for 60 days . After the treatment, we determined the longitudinal bone growth rate, the qualitative appearance of the primary spongiosa (PS), and the static and dynamic bone histomorphometry of the secondary spongiosa (SS) of the proximal tibial metaphysis (PTM) by examining undecalcifled longitudinal sections after double-fluorescent labeling . The relative effects of these treatments on longitudinal bone growth were ranked as follows : PGE, + 5 Ris > PGE, + 1 Ris = basal > PGE, > 1 p.g Ris = 5 pig Ris = aging . The density of the PS was ranked as follows: PGE, + 5 Ris > PGE, + 1 Ris = PGE, = 5 µg Ris = 1 µg Ris > basal = aging . The increase in density of the PS was the result of stimulated longitudinal growth and the action of bisphosphonate . Bone mass in the SS was ranked as follows : PGE, + S Ris = PGE, + 1 Ris = PGE, > 5 µg Ris = I µg Ris = aging = basal . However, PGE, alone and its cotreatment with Ris accumulated bone by different tissue mechanisms . PGE, alone created new bone by increasing activation frequency 8 .3-fold and the formation to resorption ratio 1 .3-fold from the controls . The combination of PGE, and Ris depressed activation frequency (-54% to -74%n), and bone formation rate (tissue-based -31%, and bone-based -42%) and eroded surface (-79% to -81%), so as to increase the formation to resorption ratio (three- to four-fold) over PGE, alone . The increased ratio was due primarily to a greater decrease in eroded perimeter than in labeled perimeter . The major finding of this study is that the combination of PGE, and a bisphosphonate (Ris) is more anabolic than PGE, or Ris alone when endochondral ossification is active, but PGE, + Ris is no more anabolic than
Key Words : Prostaglandin E zRisedronate-CotreatmentCancellous bone-Bone formation-Bone resorption .
Introduction It has been postulated that a concurrent treatment with an inhibitor of bone resorption and a stimulator of bone formation would be more beneficial for increasing bone mass than the effects of either agent alone . Yet previous findings in support of this hypothesis conflict (Lauritzen et al . 1993 ; Wronski et al . 1993) . Several studies have suggested that concurrent treatments of osteoporotic patients with estrogen and sodium fluoride induce a greater increase in bone density than treatment with either agent alone (Alois et al . 1982 ; Lueg 1988) . However, Wronski et al . (1993) reported that concurrent treatment of parathyroid hormone (PTH) with estrogen or Risedronate (Ris), a bisphosphonate, did not offer significant advantages over treatment with PTH alone in a long-term estrogen-deplete rat study . Furthermore, Lauritzen ct al . (1993) showed that cotreatment of prostaglandin E, (PGE,) with Alendronate, a bisphosphonate, for 25 days was no more effective than PGE, alone in restoring bone to estrogen-depleted, osteopenic rat skeletons . These studies were limited to rats aged 3 months or younger ; studies with minimal bone growth in older rats are needed . Thus, we designed the current study to determine whether an antiresorptive agent (Ris) or in combination with an anabolic agent and an antiresorptive agent (PGE, and Ris) would be more effective in adding new cancellous bone mass than an anabolic agent (PGE,) treated alone in 9-month-old female rats . We qualitatively reported on the effects of primary and secondary spongiosa and limited our static and dynamic histomorphometry measurements to canceltons bone in the secondary spongiosa of the proximal tibial metaphysis (PTM) . 489
490
B . Y . Lin et al .. Effects of PGE, and Ris treatment on rat skeleton
Materials and Methods
disodiumi, a highly potent, nitrogen-containing bisphosphonate (Procter and Gamble Pharmaceuticals Inc ., Cincinnati, OH) were prepared as previously reported (Ke et al . 1992; Tang et al . 1992) . We used a 20% ethanol as PGE 2 vehicle and a saline as Ris vehicle . The aging group received subcutaneously 20% ethanol daily and saline twice a week, The rats were weighed weekly and the volume of the injection solution was adjusted accordingly throughout the experiment . All rats were sacrificed by cardiac puncture under ketamine hydrochloride (50 mglkg) and xylazine (10 mg/kg) anesthesia . The right tibiae were removed and dissected free of soft tissue . The proximal one third of tibiae were stained with Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded undecalcified in methyl methacrylate . The bone was sectioned frontally using a low-speed metallurgical saw to 230-µm sections, then ground to 100-µm sections for microradiographs and microstructure analysis, and then further processed to 20 µm sections for static and dynamic histomorphometry (Jee et al . 1983, 1993 ; Tang et al . 1992; Fig . 1) . In the 9-month-old rat, we determined the effects of our various treatments on the amount of the primary spongiosa and performed static and dynamic histomorphometric analyses of the secondary spongiosa . Previously we reported the longitudinal hone growth rates in 9- and 12-month-old virgin female rats as 3 .74 and 1 .74 gm/d, respectively (Li et al . 1991) . Therefore, these rats formed a new primary spongiosa of less than 500 µm in length in 60 days, well within the I mm length designated for the primary spongiosa in a growing rat (Kimmel & Jee 1980) . Thus, when performing our analysis in the secondary spongiosa, a region of the metaphysis 1-4 mm from the growth platemetaphyseal junction (GPMJ), we analyzed established spongiosa that received treatment throughout the entire experiment .
Animal care and study protocol
Fifty-two 9-month-old Sprague-Dawley virgin female rats, weighing 283 ± 25 g (Sasco Laboratories, Inc ., Omaha, NE) . were acclimated to local vivarium conditions for 2 weeks . The rats were housed in pairs in plastic cages (21 x 32 X 20 cm) at 22°C with a 12 h/12 h light-dark cycle . A diet containing 1 .46% calcium, 0-99% phosphorous, and 4.96 IU/g of Vitamin D3 (Rodent Laboratory Chow 5001, Ralston Purina Co ., St . Louis, MO) and water were given to the animals ad libitum . I'hey were divided into seven groups with 6-8 rats in each as follows : 1 . Basal controls (Basal, 6 rats) : no treatment and killed at day I . IL Age-related controls (Aging, 6 rats ; : PGE2 and Ris vehicles subcutaneous (SC) injections for 60 days . III . Six milligrams PGE, (PGE,, 8 rats) : 6 mg PGE,/kg per day SC injections for 60 days . IV . One microgram Ris (I Ris, 8 rats) : I pg Ris/kg twicea-week SC injections for 60 days . V . Five micrograms Ris (5 Ris, 8 rats) : 5 µg Ris/kg twice a week SC injections for 60 days . VI . PGE2 + I µg Ris (PGE, + I Ris, 8 rats) : 6 mg PGE,/ kg per day plus I jig Ris/kg twice-a-week SC injections for 60 days . VII . PGEz + 5 Vg Ris (PGEz + 5 Ris, 8 rats) : 6 mg PGE 2/ kg per day plus 5 µg Ris/kg twice-a-week SC injections for 60 days . Group I animals were killed on the first day of the study as basal controls, while others were injected subcutaneously on the back either with vehicle, PGE2 . Ris or PGE, + Ris . All rats received subcutaneous injections of 25 mg/kg of tetracycline (achromycin-tetracycline hydrochloride ; Lederle Laboratory, Pearl River, NY) at 14 and 13 days, and 10 mg/kg of calcein (Sigma Chemical Co ., St- Louis, MO) at 4 and 3 days before killing . Both PGE 2 (Upjohn Co ., Kalamazoo, MI) and Ris (NE58095,2-(3-pyridinyl)-2-hydroxyethylidene-1,1-bisphosphonate
Histomorphometrv measurements and calculations
A digitizing image analysis system (DIAS) was used for the static and dynamic histomorphometric measurements of the secondary spongiosa . The area of the PTM measured was as de-
Table 1 . Comparison of treatments with PGE, alone and concurrent with Ris
POE,
Versus aging controls I Ris 5 Ris P- I Ris
P+ 5 Ris
Versus PGE, P+ I Ris P+ 5 Ris
Versus P + I Ris P+ 5 Ris
Primary spongiosa
Trabecular bone area
T
(60)
T
(43)
T (64)
1
(87)
t (129)
1
(48)
T
T (44)
T (25)
Secondary spongiosa
Trabecular bone area Trabecular width Trabecular number Node/free end Labeled perimeter Osteoid perimeter Eroded perimeter Labeled/eroded Mineral apposition rate Bone formation rate/BV Bone formation rate/TV Activation frequency Formation period Resorption period Remodeling period Longitudinal growth rate T ,
(46) 1 (24) 1
I
T (24) 1 1
1 T 1 1 1
(187) (645) (130) (59) (272) (436) (843)
j (-87) j (-48) 1 (52)
(190) T (210) ( 75) 1 (1085) 1 (58) T (285) T (459) 1 (335) .( (-93) j (-54) 1 (95)
1 1 1
1 T T 1
T
(68) (26) (34) (145) (116) (142) (-78) (831) (28) (115) (272) (141)
1 ( -90) j (-38) 1 (120)
I : Significant increase or decrease ; -: nonsignificant change ; P : PGE,; Ris : Risedronate .
-
b (-25)
1 (--5s)
1 (-68)
b (-79)
1 (-81) 1 (305)
T
(416) --
1(-54)
1
(28)
1 (-25)
1 (-19) T (-42)
b (-is)
1 (-31)
1 (-33)
1 (-74)
1(-45)
T (44)
y (-44)
B . Y . Lin et al . : Effects of PGE2 and Ris treatment on rat skeleton scribed above . Static parameters included total tissue area, trabecular bone area, and perimeter . Dynamic parameters included eroded perimeter, osteoid perimeter, single- and double-labeled perimeters, interlabeled width (at the trabecular surface and metaphysis), and trabecular wall width . The trabecular wall width measurements were performed on all completed basicmulticellular remodeling units . The distance between the scalloped cement line to resting bone surface was measured . There were about 3-6 in each animal . The above parameters were used to calculate percent trabecular bone area, trabecular width, number and separation, and percent eroded perimeter, percent osteoid perimeter, percent labeled perimeter (double label + onehalf single label based), mineral apposition rate, bone formation rates at bone and tissue levels, the ratio of labeled to eroded perimeters, formation period, resorption period, activation frequency, and longitudinal growth rate . The definitions of the measured parameters and the formulae of the calculated parameters have been published previously (Frost 1983 ; Jee et al . 1983 ; Parfitt et al . 1987 ; Thorngren & Hansson 1973) . Using microradiograph slides of the proximal tibia, and a video image analysis system (VIAS), the bone mass of the primary spongiosa (a region of the metaphysis 1 mm from the GPMJ) and the microstructural composition of the cancellous bone in the secondary spongiosa from the PTM was determined . The microstructural parameters measured were the number of trabecular nodes, node
49 1 to free end, free end to free end, and cortical to free end, but only calculated for the ratio of node number to free end (Compston et al . 1987, 1989 ; Garrahan et al, 1986) . Statistical analyses One-way analysis of variance (ANOVA) with Dunnett's 1-test was used for multiple comparisons to compare between the means of different treatment groups (Neter et al . 1982) . Twoway analysis of variance (ANOVA) was used to examine if the changes in the cotreatments were interacted by PGE 2 or Ris (Snedecor & Cochran 1980) . Probabilities (p) < 0 .05 were considered significant . Results are presented as means ± standard deviation (SD) .
Results Body weight No significant changes in body age-related controls and treated periment (data not shown) . The reported by others (Mori et al .
weight were observed groups throughout the results are consistent 1992 ; Wronski et al .
among all entire exwith those 1991) .
Fig. 1 . Microradiographs of proximal tibiae from the age-related controls at day 0 (a) and day 60 (b); and from the rats treated either with I .tg Rivkg twice weekly (c), 5 µg Ris/kg twice weekly (d), 6 mg PGE 2/kg per day (e), 6 mg PGE2/kg per day + 1 ag Ris/kg twice weekly (f), and 6 mg PGE2/kg per day + 5 .pg Ris/kg twice weekly (g) for 60 days . The primary spongiosa thickens more, in a dose-response manner, in the rats treated with Ris alone than in the basal and aging controls (c, d vs . a, b) and more in those treated with PGE 2 plus 1 or 5 tag Ris than in those treated with PGE 2 alone (f, g vs . e) . There was no significant difference in the bone mass of the secondary spongiosa between that treated with Ris alone and that of the control rats (c, d vs . a, b) or between those treated with ?GE, plus 1 or 5 pg Ris and PGE2 alone (f, g vs . c) (x7) .
49 2
B . Y Lin et al . : Effects of PGE 2 and Ris treatment on rat skeleton
Trabecular bone area (%) (Primary spongiosa) 100 -
ab ct
< 0 .05) (Table 1, Fig . 4F) . Combining PGE2 with I or 5 ag Ris further stimulated longitudinal bone growth rates by 28% and 44% above that of the group treated with PGE, alone (Interaction p - 0 .0452 and 0 .0034, respectively) .
goEffects on primary spongiosa: Bone mass findings 60-
010
All the treatments resulted in increased density of the primary spongiosa compared with that of the age-related controls : PGE 2 + 5 Ris (129%), PGE, + 1 Ris (87%), PGE z (60%), 5 .pg Ris (64%), and I µg Ris (43%) . One PGE2 + 5 Ris was significantly greater than those in all other treatment groups (Table I, Figs . 1 and 2) .
op
3
40 20 0
Effects on secondary spongiosa : Static and dynamic histomorphomerry
a N
C
m
00 d
U a
-
+ 0 .
Fig . 2 . Trabecular bone area changes of primary spongiosa (PS) in the PTM of the controls and treated rats after 60 days of the treatment. Y error bar represents standard deviation, a : p < 0 .01, compared with those of basal controls ; # : p < 0 .05 ; h: p < 0 .01, compared with those of age-related controls: c : p <= 0 .01, compared with those of PGE z treated alone ; t : p < 0 .01, compared with those of P + I Ris group . All treatment groups exhibited more PS than those in the basal and agerelated controls, and only PGEz + 5 Ris contained more PS than those in all other groups .
Effects of aging . There was no significant difference in all bone histomorphometric parameters of the secondary spongiosa between 9 to I I months of age (Figs . 3, 4, and 5) . These findings are similar to a previous study by Li et al . (1991) on aging female rats .
Effects, on longitudinal bone growth
Effects of PGE2 . PGE2 treatment added 46% more new trabecular bone mass than that in the age-related controls, There were increases in bone mass and trabecular width accompanied by increased bone turnover (a 8 .3-fold increase in activation frequency) with bone formation exceeding bone resorption (a 1 .3-fold increase in the ratio of labeled to eroded perimeters) and
Longitudinal growth rate in controls was diminished by 45% between 9 and 11 months . One or 5 µg Rislkg twice a week had no effect on bone elongation ; however, the PGE, treatment alone stimulated bone elongation to 52% over age-related controls (p
Effects of Ris . Doses of I or 5 µg Ris/kg twice a week for 60 days produced no significant changes in bone histomorphometry from that of the age-related controls (Figs . 3-5, Table I) .
B . Trabecular bone width (pm) 88 . x
A . Trabecular bone area (%)
1I
50-
66 , 40
C. Trabecular bone number (#/mm
D . Ratio of node to free end b
7-
14 1
2-
0806 0,40,20
a
d
w ,~
rc m" T
rc
„
`s'
n
n
Fig. 3 . Trabecular bone area, width, number, and microstructure changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y error bar represents standard deviation . * : p < 0 .05 and a : p < 0 .01, compared with those of basal controls . # : p < 0 .05 and b : p < 0 .01, compared with those of age-related controls_ No significant difference in above indices was found between the PGE z alone and concurrent groups .
B . Y . Lin et al . : Effects of PGE2 and Ris treatment on rat skeleton
493
B . Osteoid perimeter (%) 35
a b a b c
25 20
b 15
la 5 0C
D
D. Ratio or labeled to eroded perimeters ma a 50b b c 40
c
3020-
m n
ido
Fig. 4 . The percent labeled perimeter, osteoid perimeter, eroded perimeter, ratio of labeled-to-eroded perimeters, mineral apposition rate, and longitudinal growth rate changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y e„o, bar represents standard deviation . * : p < 0 .05 and a: p < 0 .01, compared with those of basal controls . # : p < 0 .05 and b: p < 0 .01, compared with those of age-related controls . @ : p < 0 .05 and c: p < 0 .01, compared with those of PGE 2 treated alone . t : p < 0 .05 and d : p < 0 .01, compared with those of PGE2 + I Ris group . shortened bone resorption (- 87%) and remodeling (- 48%) periods than those in the age-related controls (Figs . 3-5, Table I) . Effects of the combined PGE z and I pig Ris (PGEz + I Ris) treatment . The group treated with PGE 2 + 1 Ris did not differ in bone mass, architecture (trabecular bone area, width, number, and node to free end ratio), and remodeling period from the group treated with PGE2 alone . However, there were other differences . The osteoid (-58%) and eroded (-79%) perimeters and activation frequency (-54%) decreased while the labeled to eroded ratio (416%) increased (Interaction p = 0 .0007-0 .0001) (Figs . 3-5 ; Table 1) . Effects of the combined PGE2 and 5 pig Ris (PGE2 + Ris) treatment . The PGE2 + 5 Ris group also did not differ significantly in bone mass, architecture, and remodeling period from the PGE2 alone group . However, the following dynamic histomorphometric differences were found : depressed labeled perimeter (-25%), osteoid perimeter (-68%), eroded perimeter (-81%), mineral apposition rate (-19%), tissue- and bonelevel-based bone formation rates (-31% and -42%, respec-
tively), activation frequency (-74%), and elevated labeled to eroded ratio (305%) (interaction p = 0 .0471-0 .0001) . Nevertheless, the PGE2 + 5 Ris treatment lowered bone formation parameters (labeled perimeter - 25%), mineral apposition rate (- 18%), tissue- and bone-level formation rates (- 33% and -44%, respectively), and activation frequency (-45%) further than that observed in the PGE2 + 1 Ris treatment group (Figs . 3-5, Table I) .
Discussion The current study confirms the earlier reports showing that the bisphosphonate treatment increased primary spongiosa (Miller & lee 1975 ; Schenk et al . 1973 ; Wronski et al . 1989, 1991) and that PGE2 stimulated longitudinal bone growth (Mori et al . 1992) and new bone formation (fee et al . 1985 ; Ke et al . 1992; Mori et al, 1992 ; Tang et al . 1992) . New findings of this study include : I) the histomorphometric effects of PGE 2 , Ris, and the combination of PGE2 and Ris treatments in the PTM of 9-month-old virgin female rats ; 2) 1 or 5 pig Ris/kg twice a week for 60 days
494
B . Y . Lin et al . : Effects of PGE, and Ris treatment on rat skeleton A. Bone formation rate/BV (%Iy)
B . Bone formation rate/PV (%/y)
450-
M_
c
C . Formation period (day) 40 -
D . Resorption period (day) w
11011
E. Remodeling period (day)
-
km
tion frequency (eycle/y)
70 60' 50
Fig . 5 . The bone formation rates at bone and tissue levels, formation period, resorption period, remodeling period, and activation frequency changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y error bar represents standard deviation . * : p < 0 .05 and a : p < 0 .01, compared with those of basal controls: # : p < 0 .05 and b: p < 0.01 . compared with those of age-related controls : r : p < 0 .05 and c : p < 0 .01, compared with those of PGE, treated alone- t : p < 0 .05 and d : p < 0 .01, compared with those of PGE, + I Ris group . did not significantly alter bone formation and resorption rates in the secondary spongiosa from controls ; 3) the combined PGE, plus Ris treatment stimulated longitudinal bone growth more than PGE, alone while Ris alone did nothing ; 4) the amount of primary spongiosa formed was driven by stimulated longitudinal bone growth and the concentration of bisphosphonatc ; 5) the combination of PGE, and Ris treatment formed the same amount of secondary spongiosa mass as PGE, alone in intact female rats ; and 6) the histomorphometry profile of the PGE,, plus Ris treated secondary spongiosa differed by showing lower formation rates from that treated with PGE, alone . We were surprised that the bisphosphonate treatment did not depress bone resorption and formation or increase bone mass in the secondary spongiosa (SS) of the proximal finial metaphysis (PTM) of our 9-I t-month-old rats treated with I or 5 pg Ris/kg twice a week . Nevertheless, Wronski et al . (1989) reported a depression in bone resorption and formation and an increase in bone mass in the SS of the PTM in 3-month-old SpragueDawley female rats treated with 5 p .g Ris/kg per day for 35 and 75 days . Although they administered the same dose per day, their protocol differs from the present study in that the Ris was given according to a 1-week-on, 3-week-off regime . Also, the
age of animals in the two studies differ . The Wronski study dealt with 3-month-old while the present employed 9-month-old rats . The difference in age would greatly influence the turnover and responsiveness of the SS of the PCM . It has been reported the tissue level bone formation rate for this site is 266 *_ 99%/year (SD) in 3-month-old (Li et al . 1990) and 24 .4 ± 2 .6%/year in our study, a factor of greater than ten times . Therefore, the lack of responsiveness to Ris in the present study may be caused by the less responsive SS of the PTM of the older animals and the difference in treating 5 .pg Ris/kg per day for 5 consecutive days instead of administering it every other day . It was surprising to find the combined PGE, plus Ris treatment stimulated longitudinal bone growth more than PGE, alone while Ris alone did nothing to this parameter (Table I) . Possibly, the inhibition of cattilagenous matrix mineralization allowed PGE, to fully express its stimulatory effects on the growth cartilage . Whether an anabolic agent like parathyroid hormone given with a bisphosphonate will produce the same result should be investigated . We found Ris and PGE, alone and PGE, plus Ris increased primary spongiosa bone mass and the stimulatory effect of the latter to be additive (Table 1, Figs . I and 2) . Because we did not
B . Y . Lin et al . : Effects of PGEZ and Ris treatment on rat skeleton
perform a dynamic histomorphometry analysis of the primary spongiosa, we can only speculate on how the various treatments
Ris
induced bone gain:
alone may depress bone resorption (re-
sorption drift and remodeling dependent bone loss) greater than bone formation to increase mass ; PGE Z alone would stimulate
49 5
research grants from the Department of Energy (DE-AC02-76EVOOI19) . We thank Dr. Charles Hall and Mr . Ronald E. Lane of the Upjohn Company for the generous supply of PGE Z , and Dr . Barbara Miller of Norwich Eaton Pharmaceuticals, Inc ., for the generous supply of Ris (NE-58095) . We also thank R . B . Setterberg and Drs . Liya Tang and Qing Oiling Zeng for their expert assistance and advice .
bone formation more than resorption and stimulate longitudinal growth to increase mass ; and the combination of PGE Z and Ris would depress bone resorption (resorption drift and remodeling dependent bone loss) and stimulate bone formation (formation drift) and longitudinal growth to add more bone than either agents alone . Treating 9-month-old female rats for 60 days with 6 mg PGE2/kg per day increased the secondary spongiosa (trabecular bone mass) by controls (Table
46% when compared with that in the age-related 1) . In general, these findings are in agreement
with those reported for male and female younger rats (Jee et al . 1985 ; Ke et al . 1992) . However, the above result was much less than the addition of 248% more trabecular bone gained with the same dose and duration of PGE 2 given to 7-month-old male rats (Ke et al . 1992) . Although there was a larger response to PGEZ in the males than in the females, the final percent trabecular bone area of the secondary spongiosa for both sexes plateaued at about 40% . This suggests that there is a limit to the amount of bone that can be produced by a given dose level of PGEZ , even though the male
PTM
initially contains 13% trabecular bone in 7-month-old
rats, compared with the 28% in 9-month-old female rats (males from 13-40% and females from 28-40%) . Although our animals were older female rats, the tissue and cell level mechanisms determined by histomorphometry by which PGE Z added more bone to the secondary spongiosa was similar to those reported in younger female and older male rats (ice et al . 1985 ; Ke et al . 1992) . Increased cancellous bone mass and trabecular width were caused by increasing bone turnover (i .e ., activation frequency), bone formation, and resorption with the former greater than the latter (i .e ., the ratio of labeled to eroded perimeters increased) and shortening of resorption and remodeling periods .
6 mg PGEZ/kg per day alone and its or 5 . pg Ris/kg twice a week for 60 days
The treatment with combination with
1
resulted in the same quantity and architecture of secondary spongiosa . However, their histomorphometry profile differed from that
of
PGE2 alone . The PGEZ
+ I
Ris treatment accumulated bone while decreasing activation frequency (-54%) and resorption (-79%) which dramatically elevated the formation-toresorption ratio (416%) from PGE Z alone (Table I) . In other words, there were few total active bone surfaces, but many more forming than resorbing ones . On the other hand, the POE, + 5 Ris histomorphometric profile showed the same decrease in activation frequency (-45% of PGEZ
+ 1
Ris) ; but, in addition, it also showed
+ I Ris and PGEZ These responses suggest less bone
depressed bone formation (- 33%) of PGEZ treated alone groups (Table
1) .
should have accumulated . Therefore, one can only speculate that the 5-µg
Ris
dose quickly depressed bone turnover by depressing
resorption more than formation, which resulted in an earlier occurrence of a more favorable labeled-to-eroded perimeter ratio that accumulated bone mass . The observed increase in this ratio was due primarily to a greater decrease in eroded perimeter than in labeled perimeter . Only a short-term study, like killing animals at
2
weeks, could tell if indeed this actually occurs .
References Aloia, I . F . ; Zanzi, .I ; Vaswani, A . : Ellis, K . ; Cuba, S . H . Combination therapy for osteoporosis with estrogen, fluoride, and calcium . J . Am . Geriat Soc . 30 :13-17; 1982 . Compston, J . E. ; Mellish, R . W . E . ; Garrahan, N. 1 . Age-related changes in iliac crest rrabecular microanatomic bone structure in man . Bone 8:289-292 ; 1987 . Compaton, J . E . ; Mellish, R . W . E . : Croucher, P . ; Newcombe, R . ; Garrahan, N . J . Structural mechanisms of trabecularbone loss in man . Bane Min . 6:339350 ; 1989 . Frost, H . M . Bone histomorphometry : Analysis of trabecular bone dynamics . Recker, R . R ., ed . Bone histomorphometry: Techniques and interpretation . Boca Raton, FL: CRC Press, 1983; 109, 131 . Garrahan, N . 1 . ; Mellish, R. W . E . ; Compston, J . E . A new method for the twodimensional analysis of bone structure in human iliac crest biopsies . J . Microsc .143 :341-349,1986 . Jee, W . S . S. ; Inane, 1 . ; Joe, K . W . ; Haba, T . Histomorphometric assay of the growing long bone . Takahashi, H ., ed. Handbook of bane morphology . Niigata City, Japan: Nishimura Co., Ltd . ; 1993 ; 101, 122 lee, W . S . S . ; Ueno, K . ; Deng, Y . P . ; Woodbury, D. M . The effects of prostaglandin E a in rapidly growing rats : Increased metaphyseal hard tissue and cortico-endosteal bone formation . Calcif. Tissue Ins . 37 :148-157 ; 1985 . Joe, W . S . S . ; Tang, L. Y . ; Ke, H . Z. ; Setterberg, R . B . ; Kimmel, D . 8 . Mainmining restored bone with bisphosphonate in the ovariectomized rat skeleton: Dynamic histomorphometry, of changes in bone mass . Bone 14:493-498; 1993 . Ke, H . 7, . ; Jee, W . S S . ; Mori, S . ; LL X. J . ; Kimmel, D . B . Effect of tong term daily administration of prostaglandin-E, on maintaining elevated proximal tibia] memphyseal cancellous bone mass in male rats. Calcif. Tissue Jar . 50 :245252 ; 1992 . Kimmel, D . B . ; Jee, W . S . S . A quantitative histologic analysis of the growing long bone metaphysis . Calcif. Tissue Int . 32:113-122 ; 1980 . Li, X . 1 . ; Jee, W. S . S . ; Ke, H . Z . ; Mod, S. ; Akamine, T. Age-related changes of cancellous and conical bone histomorphometry in female Sprague-Dawtey rats . Celts Mater . Sappl . 1 :25-35 ; 1991 . Lueg, M . C . Postmenopausal osteoporosis : Treatment with low-dose sodium Fluoride and estrogen . So. Med . J . 81 :597-600 ; 1988 . Laudtzen, D . B . ; Bale. ., R . ; Shea, M . : Seeder, 1 . G. ; Markatos, A . ; Le, H . M . ; Toolan, B . C . ; Myers, E R . ; Rodan, G . A . ; Hayes W. C . Effects of combined prostaglandin and alendronate treatment on the histomorphornetry and biomechanical properties of bone in ovariectondzed rats . J . Bone Min . Res . 8 :871-880 ;1993 . Miller, S . C . ; lee, W. S . S . Ethane-]-hydroxy-l,l-diphospho nate (EHDP) effects on growth and modeling of them tibia . Calcif. Tissue Res . 18 :215-231 ; 1975 . Mori, S . ; Jee, W . S . S . ; Li, X . J . Production of new trabecular bone in osteopenic ovariectomized rats by prostaglandin E z. Calcif . Tissue In : . 50:80-87 ; 1992 . Neter, I . ; Wasserman, W . ; Whitmore, G . A. Applied statistics, Boston : Allyn and Bacon ; 1982 ; 544, 572 . Norrdin, R . W . ; Jee, W . S . S . : High, W . B . The role of prostaglandin in bone in vivo . Prostaglandins, Leukotrienes and Essential Acids 41 :139-149 ; 1990. Partin, A . M . ; Drezner, M . K . ; Gloricux, F . H . ; Kanis, J . A. ; Malluche, H. ; Meunier, P . 1 . ; On, S . M . ; Recker, R. R . Bone histomorphometry : Standardization of nomenclature, symbols, and units . J . Bone Min . Res. 2 :595410 ; 1987 . Schenk, R . ; Men, W . A . ; Milhlbauer, R . ; Russell, R . G . G . ; Fleisch, H . Effect of ethane-l-hydroxy-1,1-diphosphonete (EHDP) and dichloromethylene diphosphonate (Cl2MDP) on the calcification and resorption of cartilage and bone in the tibia) epiphysis and metaphysis of rats . Calcif. Tissue Res . 11 :196-214 ; 1973 . Sued= .,, G . W . ; Cochran, W. G . Statistical methods. (7th ed .) Ames IA: Iowa State University Press; 1980 .
Acknowledgments : This work was supported mainly by a grant from the National Aeronautics and Space Administration (NAG-2-435) and National Institutes of Health (AR-38346) . It was also partially supported by
Tang, L . Y . ; Jee, W . S. S . ; Kr, H. Z. ; Kimmel, D . B . Restoring and maintaining bone in osteopenic female mt skeleton: 1 . Changes in bone mass and structure . J . Bone Min . Res . 7 :1093-1104 ; 1992 . Thomgren, K . G . ; Hansson, L . 1 . Cell kinetics and morphology of the growth plate
496 in the normal and hypophysectomized mt . Cold). Tissue Res. 13 :113-129 ; 1973 . Wronski, T . J. ; Dunn, L . M. ; Scott, K . S . : Crooke, L . P . Endocrine and pharmacological suppressors of bone turnover protect against osteopenia in ovaries mmized rats . Endocrinology 125(2) :810 .816; 1989. Wronski, T . J . ; Yen, C .-F . ; Scott, K . S . Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats . J . Bone Min . Res. 6 :387-394 ; 1991 .
B . Y . Lin et al . : Effects of PGE 2 and Ris treatment on rut skeleton Wronski, T . L ; Yen, C .-F . ; Qi, H. ; Dann, L . M . Parathyroid hormone is more effective than estrogen or bisphosphonates for restoration of lost bone mass in ovariectomized rats . Endocrinology 132(2) :823-831 ; 1993 .
Due, Received: September 28, 1993 Dare Revised: January 5, 1994 Date Accepted: January 27, 1994
Pergamon 8756-3282(94)EO010-V
Effects of Prostaglandin E 2 and Risedronate Administration on Cancellous Bone in Older Female Rats t,2 1,5 B . Y . LIN, W . S . S . JEE,' Y . F . MA,' H . Z . KE" 3 D . B . KIMMEL, 4 and X . J . LI Division of Radiobiology, University of Utah School of Medicine, Salt Lake City, UT, USA Bone Biology Laboratory, Guangdong Medical College, Zhanjiang City, Guangdong, P .R . China ' Department of Metabolic Diseases, Pfizer Central Research, Groton, CT, USA 4 Center for Hard Tissue Research, Department of Medicine, Creighton University, College of Medicine, Omaha, NE, USA ` Procter & Gamble Pharmaceuticals, Inc ., Miami Valley Laboratories, Ross, OH, USA 2
Address for correspondence and reprints : Webster S . S . lee, PhD, Radiobiology Division, Building 586, University of Utah, Salt Lake City, UT 84112, USA .
Abstract
PGE, alone in old bone without endochondral ossification . However, the tissue mechanisms by which PGE, alone and PGE,, + Ris treatments accumulated bone differed in that the latter allowed the same bone mass to accumulate with lower levels of cell recruitment and activity .
The effects of Prostaglandin E, (PGE,,) and Risedronate (Ris) both separately and in combination (PGE, + Ris) were studied on the intact aged female rat skeleton to determine whether the combination of PGE, with an antiresorptive agent is more effective anabolically than PGE, alone . Ninemonth-old Sprague-Dawley rats were injected subcutaneously either with vehicle, 6 mg PGE,/kg per day, 1 or 5 µg Ris/kg twice a week, or 6 mg PGE,Ikg per day plus 1 or 5 µg Ris/kg twice a week (PGE, + I Ris or PGE, + 5 Big) for 60 days . After the treatment, we determined the longitudinal bone growth rate, the qualitative appearance of the primary spongiosa (PS), and the static and dynamic bone histomorphometry of the secondary spongiosa (SS) of the proximal tibial metaphysis (PTM) by examining undecalcifled longitudinal sections after double-fluorescent labeling . The relative effects of these treatments on longitudinal bone growth were ranked as follows : PGE, + 5 Ris > PGE, + 1 Ris = basal > PGE, > 1 p.g Ris = 5 pig Ris = aging . The density of the PS was ranked as follows: PGE, + 5 Ris > PGE, + 1 Ris = PGE, = 5 µg Ris = 1 µg Ris > basal = aging . The increase in density of the PS was the result of stimulated longitudinal growth and the action of bisphosphonate . Bone mass in the SS was ranked as follows : PGE, + S Ris = PGE, + 1 Ris = PGE, > 5 µg Ris = I µg Ris = aging = basal . However, PGE, alone and its cotreatment with Ris accumulated bone by different tissue mechanisms . PGE, alone created new bone by increasing activation frequency 8 .3-fold and the formation to resorption ratio 1 .3-fold from the controls . The combination of PGE, and Ris depressed activation frequency (-54% to -74%n), and bone formation rate (tissue-based -31%, and bone-based -42%) and eroded surface (-79% to -81%), so as to increase the formation to resorption ratio (three- to four-fold) over PGE, alone . The increased ratio was due primarily to a greater decrease in eroded perimeter than in labeled perimeter . The major finding of this study is that the combination of PGE, and a bisphosphonate (Ris) is more anabolic than PGE, or Ris alone when endochondral ossification is active, but PGE, + Ris is no more anabolic than
Key Words : Prostaglandin E zRisedronate-CotreatmentCancellous bone-Bone formation-Bone resorption .
Introduction It has been postulated that a concurrent treatment with an inhibitor of bone resorption and a stimulator of bone formation would be more beneficial for increasing bone mass than the effects of either agent alone . Yet previous findings in support of this hypothesis conflict (Lauritzen et al . 1993 ; Wronski et al . 1993) . Several studies have suggested that concurrent treatments of osteoporotic patients with estrogen and sodium fluoride induce a greater increase in bone density than treatment with either agent alone (Alois et al . 1982 ; Lueg 1988) . However, Wronski et al . (1993) reported that concurrent treatment of parathyroid hormone (PTH) with estrogen or Risedronate (Ris), a bisphosphonate, did not offer significant advantages over treatment with PTH alone in a long-term estrogen-deplete rat study . Furthermore, Lauritzen ct al . (1993) showed that cotreatment of prostaglandin E, (PGE,) with Alendronate, a bisphosphonate, for 25 days was no more effective than PGE, alone in restoring bone to estrogen-depleted, osteopenic rat skeletons . These studies were limited to rats aged 3 months or younger ; studies with minimal bone growth in older rats are needed . Thus, we designed the current study to determine whether an antiresorptive agent (Ris) or in combination with an anabolic agent and an antiresorptive agent (PGE, and Ris) would be more effective in adding new cancellous bone mass than an anabolic agent (PGE,) treated alone in 9-month-old female rats . We qualitatively reported on the effects of primary and secondary spongiosa and limited our static and dynamic histomorphometry measurements to canceltons bone in the secondary spongiosa of the proximal tibial metaphysis (PTM) . 489
490
B . Y . Lin et al .. Effects of PGE, and Ris treatment on rat skeleton
Materials and Methods
disodiumi, a highly potent, nitrogen-containing bisphosphonate (Procter and Gamble Pharmaceuticals Inc ., Cincinnati, OH) were prepared as previously reported (Ke et al . 1992; Tang et al . 1992) . We used a 20% ethanol as PGE 2 vehicle and a saline as Ris vehicle . The aging group received subcutaneously 20% ethanol daily and saline twice a week, The rats were weighed weekly and the volume of the injection solution was adjusted accordingly throughout the experiment . All rats were sacrificed by cardiac puncture under ketamine hydrochloride (50 mglkg) and xylazine (10 mg/kg) anesthesia . The right tibiae were removed and dissected free of soft tissue . The proximal one third of tibiae were stained with Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded undecalcified in methyl methacrylate . The bone was sectioned frontally using a low-speed metallurgical saw to 230-µm sections, then ground to 100-µm sections for microradiographs and microstructure analysis, and then further processed to 20 µm sections for static and dynamic histomorphometry (Jee et al . 1983, 1993 ; Tang et al . 1992; Fig . 1) . In the 9-month-old rat, we determined the effects of our various treatments on the amount of the primary spongiosa and performed static and dynamic histomorphometric analyses of the secondary spongiosa . Previously we reported the longitudinal hone growth rates in 9- and 12-month-old virgin female rats as 3 .74 and 1 .74 gm/d, respectively (Li et al . 1991) . Therefore, these rats formed a new primary spongiosa of less than 500 µm in length in 60 days, well within the I mm length designated for the primary spongiosa in a growing rat (Kimmel & Jee 1980) . Thus, when performing our analysis in the secondary spongiosa, a region of the metaphysis 1-4 mm from the growth platemetaphyseal junction (GPMJ), we analyzed established spongiosa that received treatment throughout the entire experiment .
Animal care and study protocol
Fifty-two 9-month-old Sprague-Dawley virgin female rats, weighing 283 ± 25 g (Sasco Laboratories, Inc ., Omaha, NE) . were acclimated to local vivarium conditions for 2 weeks . The rats were housed in pairs in plastic cages (21 x 32 X 20 cm) at 22°C with a 12 h/12 h light-dark cycle . A diet containing 1 .46% calcium, 0-99% phosphorous, and 4.96 IU/g of Vitamin D3 (Rodent Laboratory Chow 5001, Ralston Purina Co ., St . Louis, MO) and water were given to the animals ad libitum . I'hey were divided into seven groups with 6-8 rats in each as follows : 1 . Basal controls (Basal, 6 rats) : no treatment and killed at day I . IL Age-related controls (Aging, 6 rats ; : PGE2 and Ris vehicles subcutaneous (SC) injections for 60 days . III . Six milligrams PGE, (PGE,, 8 rats) : 6 mg PGE,/kg per day SC injections for 60 days . IV . One microgram Ris (I Ris, 8 rats) : I pg Ris/kg twicea-week SC injections for 60 days . V . Five micrograms Ris (5 Ris, 8 rats) : 5 µg Ris/kg twice a week SC injections for 60 days . VI . PGE2 + I µg Ris (PGE, + I Ris, 8 rats) : 6 mg PGE,/ kg per day plus I jig Ris/kg twice-a-week SC injections for 60 days . VII . PGEz + 5 Vg Ris (PGEz + 5 Ris, 8 rats) : 6 mg PGE 2/ kg per day plus 5 µg Ris/kg twice-a-week SC injections for 60 days . Group I animals were killed on the first day of the study as basal controls, while others were injected subcutaneously on the back either with vehicle, PGE2 . Ris or PGE, + Ris . All rats received subcutaneous injections of 25 mg/kg of tetracycline (achromycin-tetracycline hydrochloride ; Lederle Laboratory, Pearl River, NY) at 14 and 13 days, and 10 mg/kg of calcein (Sigma Chemical Co ., St- Louis, MO) at 4 and 3 days before killing . Both PGE 2 (Upjohn Co ., Kalamazoo, MI) and Ris (NE58095,2-(3-pyridinyl)-2-hydroxyethylidene-1,1-bisphosphonate
Histomorphometrv measurements and calculations
A digitizing image analysis system (DIAS) was used for the static and dynamic histomorphometric measurements of the secondary spongiosa . The area of the PTM measured was as de-
Table 1 . Comparison of treatments with PGE, alone and concurrent with Ris
POE,
Versus aging controls I Ris 5 Ris P- I Ris
P+ 5 Ris
Versus PGE, P+ I Ris P+ 5 Ris
Versus P + I Ris P+ 5 Ris
Primary spongiosa
Trabecular bone area
T
(60)
T
(43)
T (64)
1
(87)
t (129)
1
(48)
T
T (44)
T (25)
Secondary spongiosa
Trabecular bone area Trabecular width Trabecular number Node/free end Labeled perimeter Osteoid perimeter Eroded perimeter Labeled/eroded Mineral apposition rate Bone formation rate/BV Bone formation rate/TV Activation frequency Formation period Resorption period Remodeling period Longitudinal growth rate T ,
(46) 1 (24) 1
I
T (24) 1 1
1 T 1 1 1
(187) (645) (130) (59) (272) (436) (843)
j (-87) j (-48) 1 (52)
(190) T (210) ( 75) 1 (1085) 1 (58) T (285) T (459) 1 (335) .( (-93) j (-54) 1 (95)
1 1 1
1 T T 1
T
(68) (26) (34) (145) (116) (142) (-78) (831) (28) (115) (272) (141)
1 ( -90) j (-38) 1 (120)
I : Significant increase or decrease ; -: nonsignificant change ; P : PGE,; Ris : Risedronate .
-
b (-25)
1 (--5s)
1 (-68)
b (-79)
1 (-81) 1 (305)
T
(416) --
1(-54)
1
(28)
1 (-25)
1 (-19) T (-42)
b (-is)
1 (-31)
1 (-33)
1 (-74)
1(-45)
T (44)
y (-44)
B . Y . Lin et al . : Effects of PGE2 and Ris treatment on rat skeleton scribed above . Static parameters included total tissue area, trabecular bone area, and perimeter . Dynamic parameters included eroded perimeter, osteoid perimeter, single- and double-labeled perimeters, interlabeled width (at the trabecular surface and metaphysis), and trabecular wall width . The trabecular wall width measurements were performed on all completed basicmulticellular remodeling units . The distance between the scalloped cement line to resting bone surface was measured . There were about 3-6 in each animal . The above parameters were used to calculate percent trabecular bone area, trabecular width, number and separation, and percent eroded perimeter, percent osteoid perimeter, percent labeled perimeter (double label + onehalf single label based), mineral apposition rate, bone formation rates at bone and tissue levels, the ratio of labeled to eroded perimeters, formation period, resorption period, activation frequency, and longitudinal growth rate . The definitions of the measured parameters and the formulae of the calculated parameters have been published previously (Frost 1983 ; Jee et al . 1983 ; Parfitt et al . 1987 ; Thorngren & Hansson 1973) . Using microradiograph slides of the proximal tibia, and a video image analysis system (VIAS), the bone mass of the primary spongiosa (a region of the metaphysis 1 mm from the GPMJ) and the microstructural composition of the cancellous bone in the secondary spongiosa from the PTM was determined . The microstructural parameters measured were the number of trabecular nodes, node
49 1 to free end, free end to free end, and cortical to free end, but only calculated for the ratio of node number to free end (Compston et al . 1987, 1989 ; Garrahan et al, 1986) . Statistical analyses One-way analysis of variance (ANOVA) with Dunnett's 1-test was used for multiple comparisons to compare between the means of different treatment groups (Neter et al . 1982) . Twoway analysis of variance (ANOVA) was used to examine if the changes in the cotreatments were interacted by PGE 2 or Ris (Snedecor & Cochran 1980) . Probabilities (p) < 0 .05 were considered significant . Results are presented as means ± standard deviation (SD) .
Results Body weight No significant changes in body age-related controls and treated periment (data not shown) . The reported by others (Mori et al .
weight were observed groups throughout the results are consistent 1992 ; Wronski et al .
among all entire exwith those 1991) .
Fig. 1 . Microradiographs of proximal tibiae from the age-related controls at day 0 (a) and day 60 (b); and from the rats treated either with I .tg Rivkg twice weekly (c), 5 µg Ris/kg twice weekly (d), 6 mg PGE 2/kg per day (e), 6 mg PGE2/kg per day + 1 ag Ris/kg twice weekly (f), and 6 mg PGE2/kg per day + 5 .pg Ris/kg twice weekly (g) for 60 days . The primary spongiosa thickens more, in a dose-response manner, in the rats treated with Ris alone than in the basal and aging controls (c, d vs . a, b) and more in those treated with PGE 2 plus 1 or 5 tag Ris than in those treated with PGE 2 alone (f, g vs . e) . There was no significant difference in the bone mass of the secondary spongiosa between that treated with Ris alone and that of the control rats (c, d vs . a, b) or between those treated with ?GE, plus 1 or 5 pg Ris and PGE2 alone (f, g vs . c) (x7) .
49 2
B . Y Lin et al . : Effects of PGE 2 and Ris treatment on rat skeleton
Trabecular bone area (%) (Primary spongiosa) 100 -
ab ct
< 0 .05) (Table 1, Fig . 4F) . Combining PGE2 with I or 5 ag Ris further stimulated longitudinal bone growth rates by 28% and 44% above that of the group treated with PGE, alone (Interaction p - 0 .0452 and 0 .0034, respectively) .
goEffects on primary spongiosa: Bone mass findings 60-
010
All the treatments resulted in increased density of the primary spongiosa compared with that of the age-related controls : PGE 2 + 5 Ris (129%), PGE, + 1 Ris (87%), PGE z (60%), 5 .pg Ris (64%), and I µg Ris (43%) . One PGE2 + 5 Ris was significantly greater than those in all other treatment groups (Table I, Figs . 1 and 2) .
op
3
40 20 0
Effects on secondary spongiosa : Static and dynamic histomorphomerry
a N
C
m
00 d
U a
-
+ 0 .
Fig . 2 . Trabecular bone area changes of primary spongiosa (PS) in the PTM of the controls and treated rats after 60 days of the treatment. Y error bar represents standard deviation, a : p < 0 .01, compared with those of basal controls ; # : p < 0 .05 ; h: p < 0 .01, compared with those of age-related controls: c : p <= 0 .01, compared with those of PGE z treated alone ; t : p < 0 .01, compared with those of P + I Ris group . All treatment groups exhibited more PS than those in the basal and agerelated controls, and only PGEz + 5 Ris contained more PS than those in all other groups .
Effects of aging . There was no significant difference in all bone histomorphometric parameters of the secondary spongiosa between 9 to I I months of age (Figs . 3, 4, and 5) . These findings are similar to a previous study by Li et al . (1991) on aging female rats .
Effects, on longitudinal bone growth
Effects of PGE2 . PGE2 treatment added 46% more new trabecular bone mass than that in the age-related controls, There were increases in bone mass and trabecular width accompanied by increased bone turnover (a 8 .3-fold increase in activation frequency) with bone formation exceeding bone resorption (a 1 .3-fold increase in the ratio of labeled to eroded perimeters) and
Longitudinal growth rate in controls was diminished by 45% between 9 and 11 months . One or 5 µg Rislkg twice a week had no effect on bone elongation ; however, the PGE, treatment alone stimulated bone elongation to 52% over age-related controls (p
Effects of Ris . Doses of I or 5 µg Ris/kg twice a week for 60 days produced no significant changes in bone histomorphometry from that of the age-related controls (Figs . 3-5, Table I) .
B . Trabecular bone width (pm) 88 . x
A . Trabecular bone area (%)
1I
50-
66 , 40
C. Trabecular bone number (#/mm
D . Ratio of node to free end b
7-
14 1
2-
0806 0,40,20
a
d
w ,~
rc m" T
rc
„
`s'
n
n
Fig. 3 . Trabecular bone area, width, number, and microstructure changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y error bar represents standard deviation . * : p < 0 .05 and a : p < 0 .01, compared with those of basal controls . # : p < 0 .05 and b : p < 0 .01, compared with those of age-related controls_ No significant difference in above indices was found between the PGE z alone and concurrent groups .
B . Y . Lin et al . : Effects of PGE2 and Ris treatment on rat skeleton
493
B . Osteoid perimeter (%) 35
a b a b c
25 20
b 15
la 5 0C
D
D. Ratio or labeled to eroded perimeters ma a 50b b c 40
c
3020-
m n
ido
Fig. 4 . The percent labeled perimeter, osteoid perimeter, eroded perimeter, ratio of labeled-to-eroded perimeters, mineral apposition rate, and longitudinal growth rate changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y e„o, bar represents standard deviation . * : p < 0 .05 and a: p < 0 .01, compared with those of basal controls . # : p < 0 .05 and b: p < 0 .01, compared with those of age-related controls . @ : p < 0 .05 and c: p < 0 .01, compared with those of PGE 2 treated alone . t : p < 0 .05 and d : p < 0 .01, compared with those of PGE2 + I Ris group . shortened bone resorption (- 87%) and remodeling (- 48%) periods than those in the age-related controls (Figs . 3-5, Table I) . Effects of the combined PGE z and I pig Ris (PGEz + I Ris) treatment . The group treated with PGE 2 + 1 Ris did not differ in bone mass, architecture (trabecular bone area, width, number, and node to free end ratio), and remodeling period from the group treated with PGE2 alone . However, there were other differences . The osteoid (-58%) and eroded (-79%) perimeters and activation frequency (-54%) decreased while the labeled to eroded ratio (416%) increased (Interaction p = 0 .0007-0 .0001) (Figs . 3-5 ; Table 1) . Effects of the combined PGE2 and 5 pig Ris (PGE2 + Ris) treatment . The PGE2 + 5 Ris group also did not differ significantly in bone mass, architecture, and remodeling period from the PGE2 alone group . However, the following dynamic histomorphometric differences were found : depressed labeled perimeter (-25%), osteoid perimeter (-68%), eroded perimeter (-81%), mineral apposition rate (-19%), tissue- and bonelevel-based bone formation rates (-31% and -42%, respec-
tively), activation frequency (-74%), and elevated labeled to eroded ratio (305%) (interaction p = 0 .0471-0 .0001) . Nevertheless, the PGE2 + 5 Ris treatment lowered bone formation parameters (labeled perimeter - 25%), mineral apposition rate (- 18%), tissue- and bone-level formation rates (- 33% and -44%, respectively), and activation frequency (-45%) further than that observed in the PGE2 + 1 Ris treatment group (Figs . 3-5, Table I) .
Discussion The current study confirms the earlier reports showing that the bisphosphonate treatment increased primary spongiosa (Miller & lee 1975 ; Schenk et al . 1973 ; Wronski et al . 1989, 1991) and that PGE2 stimulated longitudinal bone growth (Mori et al . 1992) and new bone formation (fee et al . 1985 ; Ke et al . 1992; Mori et al, 1992 ; Tang et al . 1992) . New findings of this study include : I) the histomorphometric effects of PGE 2 , Ris, and the combination of PGE2 and Ris treatments in the PTM of 9-month-old virgin female rats ; 2) 1 or 5 pig Ris/kg twice a week for 60 days
494
B . Y . Lin et al . : Effects of PGE, and Ris treatment on rat skeleton A. Bone formation rate/BV (%Iy)
B . Bone formation rate/PV (%/y)
450-
M_
c
C . Formation period (day) 40 -
D . Resorption period (day) w
11011
E. Remodeling period (day)
-
km
tion frequency (eycle/y)
70 60' 50
Fig . 5 . The bone formation rates at bone and tissue levels, formation period, resorption period, remodeling period, and activation frequency changes of secondary spongiosa in the PTM of the controls and treated rats after 60 days of the treatment . Y error bar represents standard deviation . * : p < 0 .05 and a : p < 0 .01, compared with those of basal controls: # : p < 0 .05 and b: p < 0.01 . compared with those of age-related controls : r : p < 0 .05 and c : p < 0 .01, compared with those of PGE, treated alone- t : p < 0 .05 and d : p < 0 .01, compared with those of PGE, + I Ris group . did not significantly alter bone formation and resorption rates in the secondary spongiosa from controls ; 3) the combined PGE, plus Ris treatment stimulated longitudinal bone growth more than PGE, alone while Ris alone did nothing ; 4) the amount of primary spongiosa formed was driven by stimulated longitudinal bone growth and the concentration of bisphosphonatc ; 5) the combination of PGE, and Ris treatment formed the same amount of secondary spongiosa mass as PGE, alone in intact female rats ; and 6) the histomorphometry profile of the PGE,, plus Ris treated secondary spongiosa differed by showing lower formation rates from that treated with PGE, alone . We were surprised that the bisphosphonate treatment did not depress bone resorption and formation or increase bone mass in the secondary spongiosa (SS) of the proximal finial metaphysis (PTM) of our 9-I t-month-old rats treated with I or 5 pg Ris/kg twice a week . Nevertheless, Wronski et al . (1989) reported a depression in bone resorption and formation and an increase in bone mass in the SS of the PTM in 3-month-old SpragueDawley female rats treated with 5 p .g Ris/kg per day for 35 and 75 days . Although they administered the same dose per day, their protocol differs from the present study in that the Ris was given according to a 1-week-on, 3-week-off regime . Also, the
age of animals in the two studies differ . The Wronski study dealt with 3-month-old while the present employed 9-month-old rats . The difference in age would greatly influence the turnover and responsiveness of the SS of the PCM . It has been reported the tissue level bone formation rate for this site is 266 *_ 99%/year (SD) in 3-month-old (Li et al . 1990) and 24 .4 ± 2 .6%/year in our study, a factor of greater than ten times . Therefore, the lack of responsiveness to Ris in the present study may be caused by the less responsive SS of the PTM of the older animals and the difference in treating 5 .pg Ris/kg per day for 5 consecutive days instead of administering it every other day . It was surprising to find the combined PGE, plus Ris treatment stimulated longitudinal bone growth more than PGE, alone while Ris alone did nothing to this parameter (Table I) . Possibly, the inhibition of cattilagenous matrix mineralization allowed PGE, to fully express its stimulatory effects on the growth cartilage . Whether an anabolic agent like parathyroid hormone given with a bisphosphonate will produce the same result should be investigated . We found Ris and PGE, alone and PGE, plus Ris increased primary spongiosa bone mass and the stimulatory effect of the latter to be additive (Table 1, Figs . I and 2) . Because we did not
B . Y . Lin et al . : Effects of PGEZ and Ris treatment on rat skeleton
perform a dynamic histomorphometry analysis of the primary spongiosa, we can only speculate on how the various treatments
Ris
induced bone gain:
alone may depress bone resorption (re-
sorption drift and remodeling dependent bone loss) greater than bone formation to increase mass ; PGE Z alone would stimulate
49 5
research grants from the Department of Energy (DE-AC02-76EVOOI19) . We thank Dr. Charles Hall and Mr . Ronald E. Lane of the Upjohn Company for the generous supply of PGE Z , and Dr . Barbara Miller of Norwich Eaton Pharmaceuticals, Inc ., for the generous supply of Ris (NE-58095) . We also thank R . B . Setterberg and Drs . Liya Tang and Qing Oiling Zeng for their expert assistance and advice .
bone formation more than resorption and stimulate longitudinal growth to increase mass ; and the combination of PGE Z and Ris would depress bone resorption (resorption drift and remodeling dependent bone loss) and stimulate bone formation (formation drift) and longitudinal growth to add more bone than either agents alone . Treating 9-month-old female rats for 60 days with 6 mg PGE2/kg per day increased the secondary spongiosa (trabecular bone mass) by controls (Table
46% when compared with that in the age-related 1) . In general, these findings are in agreement
with those reported for male and female younger rats (Jee et al . 1985 ; Ke et al . 1992) . However, the above result was much less than the addition of 248% more trabecular bone gained with the same dose and duration of PGE 2 given to 7-month-old male rats (Ke et al . 1992) . Although there was a larger response to PGEZ in the males than in the females, the final percent trabecular bone area of the secondary spongiosa for both sexes plateaued at about 40% . This suggests that there is a limit to the amount of bone that can be produced by a given dose level of PGEZ , even though the male
PTM
initially contains 13% trabecular bone in 7-month-old
rats, compared with the 28% in 9-month-old female rats (males from 13-40% and females from 28-40%) . Although our animals were older female rats, the tissue and cell level mechanisms determined by histomorphometry by which PGE Z added more bone to the secondary spongiosa was similar to those reported in younger female and older male rats (ice et al . 1985 ; Ke et al . 1992) . Increased cancellous bone mass and trabecular width were caused by increasing bone turnover (i .e ., activation frequency), bone formation, and resorption with the former greater than the latter (i .e ., the ratio of labeled to eroded perimeters increased) and shortening of resorption and remodeling periods .
6 mg PGEZ/kg per day alone and its or 5 . pg Ris/kg twice a week for 60 days
The treatment with combination with
1
resulted in the same quantity and architecture of secondary spongiosa . However, their histomorphometry profile differed from that
of
PGE2 alone . The PGEZ
+ I
Ris treatment accumulated bone while decreasing activation frequency (-54%) and resorption (-79%) which dramatically elevated the formation-toresorption ratio (416%) from PGE Z alone (Table I) . In other words, there were few total active bone surfaces, but many more forming than resorbing ones . On the other hand, the POE, + 5 Ris histomorphometric profile showed the same decrease in activation frequency (-45% of PGEZ
+ 1
Ris) ; but, in addition, it also showed
+ I Ris and PGEZ These responses suggest less bone
depressed bone formation (- 33%) of PGEZ treated alone groups (Table
1) .
should have accumulated . Therefore, one can only speculate that the 5-µg
Ris
dose quickly depressed bone turnover by depressing
resorption more than formation, which resulted in an earlier occurrence of a more favorable labeled-to-eroded perimeter ratio that accumulated bone mass . The observed increase in this ratio was due primarily to a greater decrease in eroded perimeter than in labeled perimeter . Only a short-term study, like killing animals at
2
weeks, could tell if indeed this actually occurs .
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Acknowledgments : This work was supported mainly by a grant from the National Aeronautics and Space Administration (NAG-2-435) and National Institutes of Health (AR-38346) . It was also partially supported by
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Due, Received: September 28, 1993 Dare Revised: January 5, 1994 Date Accepted: January 27, 1994