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Department of Physiological Sciences, University of Florida, Gainesville, Florida 32610
Address all correspondence and requests for reprints to: Thomas J. Wronski, Ph.D., Department of Physiological Sciences, Box 100144, JHMHC, University of Florida, Gainesville, Florida 32610. E-mail: wronskit{at}mail.vetmed.ufl.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bFGF is synthesized by osteoblasts and deposited in bone matrix (11, 12). This growth factor enhances the proliferation of osteoprogenitor and osteoblast-like cells (13, 14, 15, 16, 17) and induces formation of bone-like nodules in cultures of bone marrow cells (17, 18, 19, 20). Similar skeletal effects were observed in vivo in young and aged rats treated with bFGF. For example, systemic injections of the growth factor increased the osteoblast population and cancellous bone mass in intact rats (21, 22, 23). In addition, new bone spicules were observed in the marrow cavity of these animals.
The skeletal effects of bFGF in the OVX rat have not been studied extensively to date. Nakamura et al. (24) reported that a single intraosseous injection of bFGF in the ilium of OVX rats restored lost bone mass to normal levels. Dunstan et al. (25) recently described an increase in tibial osteoid and bone mass and the formation of new trabecular-like structures in OVX rats treated systemically with acidic FGF (aFGF). However, this study did not include an evaluation of the effects of aFGF on cellular and fluorochrome-based indices of bone formation and resorption in OVX rats. The purpose of the current study is to characterize more completely, by histomorphometric techniques, the bone anabolic response to bFGF in osteopenic OVX rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the day of surgery, all rats were anesthetized with an im injection of ketamine hydrochloride and xylazine at doses of 50 and 10 mg/kg BW, respectively. Fourteen rats were subjected to sham surgery, during which the ovaries were exteriorized but replaced intact. Bilateral ovariectomies were performed in the remaining 74 rats from a dorsal approach. Each rat was housed individually at 25 C with a 13-h light, 11-h dark cycle. Food (Teklad 22/5 Rodent Diet, Madison, WI), with Ca and PO4 contents of 0.95% and 0.67%, respectively, was available ad libitum to the sham-operated control rats. The food consumption of OVX rats was restricted to that of control rats (pair-feeding) to minimize the increase in body weight associated with ovariectomy (26). All rats were untreated for 2 months after surgery, to allow for the development of tibial cancellous osteopenia.
Baseline control (n = 6) and OVX groups (n = 6) were killed at the beginning of treatment, when all animals were approximately 5 months of age (2 months post surgery). The remaining rats were anesthetized, as described above, and implanted with polyurethane catheters (Braintree Scientific, Inc., Braintree, MA) in their right jugular veins. Six groups of OVX rats were treated iv with bFGF (Scios, Inc., Mountain View, CA) dissolved in PBS, at doses of 100 or 200 µg/kg BW, for 7 or 14 days. Another group of OVX rats and a group of sham-operated control rats were treated iv with vehicle alone for 14 days. Certain groups of bFGF-treated OVX rats were killed at 7 or 14 days after withdrawal of treatment. The various groups are as follows: 1) sham + vehicle (14 days, n = 8); 2) OVX + vehicle (14 days, n = 8); 3) OVX + bFGF (100 µg/kg·day for 7 days, n = 12); 4) OVX + bFGF (200 µg/kg·day for 7 days, n = 12); 5) OVX + bFGF (100 µg/kg·day for 14 days, n = 12); 6) OVX + bFGF (200 µg/kg·day for 14 days, n = 12); 7) OVX + bFGF (200 µg/kg·day for 14 days + 7 days off, n = 6); and 8) OVX + bFGF (200 µg/kg·day for 14 days + 14 days off, n = 6)
During the treatment period, the catheters were flushed twice daily with a 2% heparin saline solution. Each rat was injected sc with demeclocycline and calcein (Sigma, St. Louis, MO) at a dose of 15 mg/kg BW on the 10th and 3rd days before sacrifice, respectively, to label sites of bone formation.
All rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. At necropsy, failure to detect ovarian tissue and observation of marked atrophy of the uterine horns confirmed the success of ovariectomy. The right tibia from each animal was dissected free of muscle and cut in half, cross-sectionally, with a hand-held saw (Dremel Moto Tool, Racine, WI). The proximal tibiae were then placed in 10% phosphate-buffered formalin for 24 h for tissue fixation. Serum samples were stored at -80 C for future analyses.
Serum calcium and phosphorus
Serum samples were analyzed for their calcium and phosphorus
contents, by the arsenazo-3 dye and ammonium molybdate colorimetric
methods, respectively, with an Abbott Alcyon 300i Chemistry Analyzer
(Abbott Diagnostics, Abbott Park, IL).
Cancellous bone histomorphometry
The proximal tibiae were dehydrated in ethanol and embedded
undecalcified in methyl methacrylate (27). Longitudinal sections (4-
and 8-µm thick) were cut with AO Autocut/Jung 1150 or 2065
microtomes. The 4-µm-thick sections were stained according to the Von
Kossa method with a tetrachrome counterstain (Polysciences Inc.,
Warrington, PA). Some additional sections were stained with 1%
toluidine blue for observations under polarized light. Bone
measurements were performed in cancellous bone tissue of the proximal
tibial metaphysis, beginning at a distance of 1 mm from the growth
plate-metaphyseal junction to exclude the primary spongiosa. The sample
site also excluded cancellous bone tissue within 0.4 mm of the
endocortical surfaces. The proximal and distal regions of the secondary
spongiosa (1–2.5 mm and 2.5–4 mm from the growth plate, respectively)
were measured separately.
All measurements were performed with the Bioquant Bone Morphometry System (R&M Biometrics Corp., Nashville, TN), as previously described (28). Cancellous bone volume (as a percentage of bone tissue area) and osteoblast and osteoclast surfaces (as percentages of total cancellous perimeter) were measured in 4-µm thick, stained sections. In addition, osteoid volume (as a percentage of bone tissue area) and osteoid surface (as a percentage of total cancellous perimeter) were measured in the same manner.
Fluorochrome-based indices of bone formation were measured in unstained, 8-µm-thick sections of the proximal tibial metaphysis. The distance between the 2 fluorochrome labels distal to the growth plate was measured with the Bioquant system and divided by the time interval between their administration, to calculate the rate of longitudinal bone growth. The percentage of cancellous bone surface with a double fluorochrome label (mineralizing surface) and mineral apposition rate were also measured with the Bioquant system. In addition, bone formation rate (tissue level, total surface referent) was calculated by multiplying mineralizing surface by mineral apposition rate (29). Values for mineral apposition rate were not corrected for obliquity of the plane of section in cancellous bone (29).
Data are expressed as the mean ± SE for each group. Statistical differences among groups for body weight, serum calcium, and serum phosphorus were evaluated by one-way ANOVA, followed by the Fisher PLSD test for multiple comparisons (30). Statistical differences among groups for bone histomorphometric data were evaluated with the nonparametric Kruskal-Wallis test involving a multiple comparison procedure, which is valid for both normal and nonnormal data distributions (31). For both statistical analyses, P values less than 0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum calcium and phosphorus
Serum minerals were monitored for changes that may affect bone
mineralization. Mean serum calcium ranged from 9.5–11.4 mg/dl, with no
significant differences among groups. Mean serum phosphorus ranged from
5.8–9.1 mg/dl. OVX rats treated with the higher dose of bFGF for 7 or
14 days had the lowest mean values for serum phosphorus (5.9 ±
0.5 and 5.8 ± 0.3 mg/dl, respectively). These values were
significantly lower than those of vehicle-treated control and OVX rats
(8.0 ± 0.3 and 8.1 ± 0.4 mg/dl, respectively). Mean serum
phosphorus in OVX rats previously treated with bFGF returned to at
least the level of vehicle-treated control and OVX rats during the
withdrawal period (8.1 ± 0.2 mg/dl at 7 days and 9.1 ± 0.4
mg/dl at 14 days).
Cancellous bone histomorphometry
Bone histomorphometry was performed to determine the effects
of ovariectomy and bFGF treatment on cancellous bone mass and levels of
bone formation and resorption. Baseline and vehicle-treated OVX rats
exhibited significantly lower cancellous bone volumes, relative to
baseline and vehicle-treated control rats, in the proximal and distal
regions of the proximal tibia (Fig. 1
, A
and B). No significant change in cancellous bone volume was detected at
7 and 14 days of treatment with either dose of bFGF. However,
cancellous bone volume increased significantly above the level of
vehicle-treated OVX rats, after 7 days of withdrawal from bFGF
treatment, in both regions of the proximal tibia before declining
somewhat at 14 days of withdrawal. Some differences in cancellous bone
mass among the groups are seen in Fig. 2.
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, C and D) was negligible in baseline and
vehicle-treated control and OVX rats. In contrast, OVX rats treated
with both doses of bFGF exhibited significant increases in osteoid
volume in both regions of the proximal tibia. Treatment with the higher
dose of bFGF, for 14 days, induced a much greater increase in osteoid
volume than the lower dose of bFGF. This variable declined
substantially during the withdrawal period, coincident with the
observed increase in cancellous bone volume in bFGF-treated OVX rats at
this time.
The large increase in osteoid, in response to bFGF treatment, is seen
in
Figs. 2–5. Osteoid spicules were observed in bFGF-treated OVX rats,
extending into the marrow cavity from the endocortical surface of
cortical bone (Fig. 3). In addition, new
osteoid spicules formed in response to bFGF treatment near the middle
of the marrow cavity (Fig. 4). These new
spicules were most commonly observed in the distal regions of the
proximal tibial metaphysis and in the diaphysis. Observation of
histologic sections under polarized light revealed that the new osteoid
in bFGF-treated OVX rats was woven in nature (Fig. 5).
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, A and B), an
index of bone resorption, was significantly increased in baseline and
vehicle-treated OVX rats, relative to baseline and vehicle-treated
control rats, in both regions of the proximal tibia. Treatment of OVX
rats with both doses of bFGF markedly depressed osteoclast surface. The
greatest decrease in osteoclast surface occurred in OVX rats treated
with the higher dose of bFGF for 14 days. In the proximal region, after
withdrawal of bFGF treatment, osteoclast surface increased to the level
of vehicle-treated OVX rats at 14 days. However, in the distal region,
osteoclast surface increased somewhat, but remained lower than that of
vehicle-treated OVX rats, at 14 days after withdrawal.
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, C and D), an index of bone formation, was
significantly increased by ovariectomy in both regions of the proximal
tibia. Treatment with bFGF induced a dramatic increase in osteoblast
surface, which was maximally increased by approximately 17-fold in OVX
rats treated with the higher dose of bFGF, for 14 days, compared with
vehicle-treated OVX rats. Osteoblast surface then rapidly declined
toward the level of vehicle-treated OVX rats during the withdrawal
period.
Differences among groups in osteoid surface (Fig. 6, E and F) were
nearly the same as described above for osteoblast surface. OVX rats
treated for 14 days with the higher dose of bFGF had a remarkably high
mean value of at least 75% for osteoid surface in the proximal and
distal regions of the proximal tibia. Although osteoid surface
decreased substantially after withdrawal of bFGF treatment, it remained
above the level of vehicle-treated OVX rats at the end of the
study.
During the treatment period, mean values for the rate of longitudinal bone growth ranged from 10–15 µm/day, with no significant differences among groups. This variable declined to approximately 7.5 µm/day in bFGF-treated OVX rats during the withdrawal period.
Mean values for bone formation rate in the proximal region of the
proximal tibia are depicted in Fig. 7.
Baseline and vehicle-treated OVX rats had a significantly increased
bone formation rate, in comparison with baseline and vehicle-treated
control rats, which was associated primarily with a significant
increase in mineralizing surface (data not shown). Bone formation rate
was not significantly different in OVX rats treated with the lower dose
of bFGF, in comparison with vehicle-treated OVX rats. However, this
variable was markedly lower in OVX rats treated with the higher dose of
bFGF, because of an almost total lack of double fluorochrome labeling
in cancellous bone of these animals. Bone formation rate in
bFGF-treated OVX rats returned to at least the level of vehicle-treated
OVX rats at 7 and 14 days of withdrawal. Similar results were obtained
from the distal region of the proximal tibia (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the most part, the observed bone anabolic effects of bFGF in OVX rats are in agreement with the findings of previous in vitro studies (13, 14, 15, 16, 17, 18, 19, 20). For example, Hanada et al. (20) reported that bFGF markedly stimulated cell growth and induced osteoblastic differentiation, followed by calcium deposition and bone nodule formation, in cultures of rat bone marrow-derived mesenchymal stem cells. In the current in vivo study, marked increases in the osteoblast population and the formation of new osteoid spicules within bone marrow were induced by bFGF treatment, and the newly-formed osteoid became calcified by 7 days after withdrawal of bFGF treatment. Furthermore, our histomorphometric results in OVX rats are also consistent with previous in vivo studies in intact rats (21, 22, 23) that show osteoblast proliferation, osteoid formation, and increased cancellous bone mass in response to bFGF treatment. These marked increases in cancellous osteoid and bone mass, as well as formation of new trabecular-like structures, were also observed in OVX rats treated with aFGF (25). Therefore, both aFGF and bFGF seem to have similar anabolic effects in the skeleton of OVX rats.
Despite the above consistencies, some findings of the current study are in conflict with those of previous investigations. Hurley and co-workers (32) reported that bFGF stimulated osteoclast formation in murine bone marrow cultures. In contrast, osteoclast surface was clearly decreased in bFGF-treated OVX rats. However, it is important to note that this latter in vivo observation may not be a direct effect of bFGF but rather secondary to the extensive osteoid surface (>75%) in these animals. Osteoclasts are rarely found adjacent to such unmineralized surfaces.
Another point of contention seems to be the effect of bFGF on cartilage cell proliferation, which has been reported to be increased by the growth factor in vitro (33, 34, 35). Such a cellular effect would presumably result in an increased rate of longitudinal bone growth in vivo. This phenomenon has been observed by Nagai et al. (23) in young (8 weeks old) intact rats treated with bFGF for 7 days at a dose of 100 µg/kg·day. In the current study, the rate of longitudinal bone growth in OVX rats was not affected by treatment with the same dose of bFGF, but this finding may be attributable to use of older (5 months old), slowly growing rats. In any case, the bFGF-induced stimulation of longitudinal bone growth observed by Nagai et al. (23) seems to be transient, because this skeletal process returned to normal levels by 21 days of treatment with the growth factor.
The incidence of adverse side effects may limit systemic use of bFGF and other potential new therapies for human disorders. Prostaglandin E2 (PGE2) has a strong bone anabolic effect similar to that of bFGF, including de novo formation of new bone spicules within the bone marrow of at least some rats treated with the drug (36). Nevertheless, PGE2 has not received a great deal of clinical attention as an osteoporosis therapy, partly because of its gastrointestinal side effects, which may result in diarrhea and weight loss (37, 38). Systemic administration of platelet-derived growth factor increased bone mass in OVX rats, but it also induced extraskeletal fibrosis (39). This side effect is also a likely complication of bFGF treatment, in view of the strong mitogenic effects of the growth factor. However, bFGF-treated OVX rats of the current study were not examined closely for areas of extraskeletal fibrosis nor for potential tumor formation, although no gross abdominal tumors were observed during necropsy procedures. Young intact rats treated with a high dose of bFGF (300 µg/kg) exhibited undesirable side effects, such as a slower rate of weight gain and defective calcification at the growth plate-metaphyseal junction (23). Nonspecific mitogenic effects of this high dose of bFGF have also been observed in intact rats, including hypertrophy of the glomerular epithelium in the kidney and alveolar epithelium in the lung (21). In the current study, treatment of OVX rats with lower doses of bFGF (100 and 200 µg/kg) did not affect body weight, a general index of the health of an animal. However, OVX rats treated with the higher dose of bFGF did exhibit impaired bone mineralization, as indicated by an almost total lack of double fluorochrome labeling in cancellous bone, despite markedly increased osteoblast and osteoid surfaces. Nevertheless, bone mineralization quickly resumed during the withdrawal period in OVX rats previously treated with bFGF, as osteoid volume declined, cancellous bone volume increased, and double fluorochrome labeling returned to the level of vehicle-treated OVX rats. The mechanism for impaired bone mineralization in bFGF-treated OVX rats is unclear, but the observed hypophosphatemia in these animals may play a role. Hypophosphatemic humans and mice exhibit osteomalacia, despite normal or slightly decreased serum calcium levels (40). A similar serum mineral profile was detected in bFGF-treated OVX rats.
The peak cancellous bone volume in bFGF-treated OVX rats occurred at 7 days of withdrawal from the growth factor. This observation should not be considered a delayed anabolic effect of bFGF treatment but rather a consequence of conversion of osteoid to bone caused by a resumption of normal mineralization, as indicated by a precipitous drop in osteoid volume during the withdrawal period. Furthermore, the marked decline in osteoblast surface during the withdrawal period is not consistent with continued augmentation of bone mass by these bone-forming cells. By 14 days of withdrawal, however, OVX rats previously treated with bFGF exhibited a decline in their tibial cancellous bone volume associated with an increase in osteoclast surface. These findings indicate that the new bone formed in response to bFGF treatment would eventually be resorbed away after withdrawal of treatment, as has been observed in OVX rats after a single intraosseous injection of bFGF (24) and after withdrawal from other anabolic agents such as PGE2 and PTH (41, 42). In this situation, antiresorptive therapy with estrogen or bisphosphonates would be necessary during the withdrawal period to maintain the increase in cancellous bone mass induced by anabolic agents (41, 42).
Our histomorphometric analysis did not reveal any major differences in
the skeletal response to bFGF in the proximal and distal regions of the
proximal tibial metaphysis. Samuels et al. (43) also did not
detect obvious regional differences in the tibial anabolic response of
mice to a very high dose of estrogen, which induces de novo
medullary bone formation specifically in these rodents. However, the
endocortical response to bFGF treatment was observed to be more
pronounced in the more distal regions of the proximal tibial metaphysis
and the diaphysis, as indicated by numerous osteoid spicules extending
from this bone surface into the marrow cavity (Fig. 3). This
observation is not reflected in our histomorphometric data, because of
the sample site excluding areas of the proximal tibial metaphysis
adjacent to the endocortical surface. In agreement with previous
reports in intact rats (21, 22, 23), osteoid accumulation was not observed
along the periosteal surface of cortical bone in bFGF-treated OVX
rats.
In summary, the results of the current study indicate that bFGF not only stimulates bone formation on pre-existing bone surfaces but also induces de novo formation of bone spicules within the marrow cavity of OVX rats. These bone anabolic effects resulted in partial restoration of lost cancellous bone mass in osteopenic OVX rats after only 14 days of bFGF treatment. Our findings suggest that bFGF may be considered for development as a potential treatment for patients with severe osteopenia who are unresponsive to conventional osteoporosis therapies.
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Acknowledgments |
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Footnotes |
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Received May 12, 1999.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, OVX + vehicle; 
, OVX + bFGF (low dose); 
, OVX +
bFGF (high dose); a, significantly different from vehicle-treated
control group (P < 0.05); b, significantly
different from vehicle-treated OVX group (P <
0.05).
