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Endocrinology Vol. 143, No. 7 2515-2526
Copyright © 2002 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Sequential Treatment with Basic Fibroblast Growth Factor and PTH Is More Efficacious than Treatment with PTH Alone for Increasing Vertebral Bone Mass and Strength in Osteopenic Ovariectomized Rats

U. T. Iwaniec, Li. Mosekilde, N. G. Mitova-Caneva, J. S. Thomsen and T. J. Wronski

Department of Physiological Sciences, University of Florida (U.T.I., N.G.M.-C., T.J.W.), Gainesville, Florida 32610; and Department of Cell Biology, Institute of Anatomy, University of Aarhus (Li.M., J.S.T.), Aarhus DK-8000, Denmark

Address all correspondence and requests for reprints to: Urszula Iwaniec, Ph.D., Department of Physiological Sciences, P.O. Box 100144, JHMHC, University of Florida, Gainesville, Florida 32610-0144. E-mail: . iwaniecu{at}mail.vetmed.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The study was designed 1) to determine whether treatment with basic fibroblast growth factor (bFGF) and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic ovariectomized rats, and 2) to assess whether prior and concurrent administration of the antiresorptive agents estrogen and risedronate suppresses the bone anabolic response to treatment with bFGF alone and sequential treatment with bFGF and PTH. Three-month-old female Sprague Dawley rats were ovariectomized (OVX) or sham-operated (sham) and maintained untreated for 1 yr. Baseline sham and OVX rats were killed at this time (15 months of age). Groups of rats were injected sc with estrogen (10 µg/kg, 4 d/wk), risedronate (5 µg/kg, 2 d/wk), or vehicle. At the end of the second week of antiresorptive treatment, catheters were inserted into the jugular veins of all rats, and vehicle or bFGF at a dose of 250 µg/kg was injected daily for 14 d. Three groups of rats were killed at the end of bFGF treatment. The remaining rats were continued on their respective antiresorptive therapy and injected sc with vehicle or synthetic human PTH-(1–34) at a dose of 80 µg/kg, 5 d/wk, for 8 wk. Lumbar vertebrae were processed for cancellous bone histomorphometry and biomechanical testing.

Ovariectomy resulted in a decrease in vertebral bone mass and strength. Treatment of OVX rats for 14 d with bFGF markedly increased osteoblast surface, osteoid surface, and osteoid volume compared with vehicle treatment of sham and OVX rats. Furthermore, osteoid bridges were observed extending between preexisting trabeculae in bFGF-treated OVX rats. Prior and concurrent administration of estrogen and risedronate did not suppress these bone anabolic effects of bFGF. Treatment of OVX rats with PTH alone increased vertebral cancellous bone mass and strength to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH further augmented vertebral bone mass and strength to a level above that observed in OVX rats treated with PTH alone. The improvements in bone mass and strength were associated with an increase in trabecular thickness in OVX rats treated with PTH alone and with an increase in trabecular thickness and node to terminus ratio, an index of trabecular connectivity, in OVX rats treated sequentially with bFGF and PTH. Cotreatment with estrogen and risedronate did not suppress the anabolic response of bone to bFGF and PTH. In fact, a trend for an even greater increase in cancellous bone mass and node to terminus ratio was observed in OVX rats treated with risedronate, bFGF, and PTH. These findings indicate that sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength and improving trabecular microarchitecture in osteopenic OVX rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POSTMENOPAUSAL osteoporosis is a heterogeneous disease of skeletal fragility characterized by low bone mass and bone microarchitectural deterioration that leads to a reduction in bone strength and increased fracture risk (1). It results from an increase in the activation frequency of bone remodeling units and an imbalance between osteoclastic bone resorption and osteoblastic bone formation leading to cancellous bone loss (2). In addition, excessive osteoclastic activity during the early postmenopausal period results in perforation of trabeculae and loss of trabecular connectivity (3, 4, 5). Currently available therapies for the treatment of osteoporosis include antiresorptive agents such as estrogen, selective ER modulators, calcitonin, and the bisphosphonates (6). These maintain existing bone mass by decreasing rates of bone turnover and loss. Potential therapies that increase bone mass by stimulating bone formation include PTH and sodium fluoride (7, 8). Recent clinical trials show that intermittent administration of PTH increases bone mineral density and decreases fracture risk in women with postmenopausal osteoporosis (9). However, because the anabolic action of agents such as PTH and sodium fluoride appears dependent on the presence of bone templates, they may not be as effective for treating osteoporotic patients in whom the number of trabecular templates is low, and connectivity between trabeculae has been lost (7, 10, 11). A more ideal anabolic agent for the treatment of cancellous osteopenia would not only augment cancellous bone mass by increasing the thickness of existing trabeculae, but also reestablish trabecular connectivity disrupted during the development of osteoporosis.

Using the ovariectomized (OVX) rat as a model, we have previously shown that basic fibroblast growth factor (bFGF) induces formation of abundant osteoid within the marrow cavity and has the potential to improve trabecular connectivity (12). Because bFGF is a general mitogen with adverse side-effects, long-term administration is not practical (13). Consequently, one strategy for treating cancellous osteopenia may be to reconnect trabeculae by short-term treatment with bFGF and subsequently treat with an agent such as PTH to further increase the mass and strength of the newly formed bony connections. We have previously shown that sequential administration of bFGF and PTH increases bone mass and strength at a severely osteopenic site in aged OVX rats (14), although not significantly more than treatment with PTH alone. The purpose of the current investigation was to assess the effect of sequential treatment with bFGF and PTH vs. treatment with PTH alone on bone mass and strength at a moderately osteopenic site (lumbar vertebral body) in aged OVX rats. This skeletal site, which is more comparable to adult human bone in terms of remodeling activity than is the proximal tibial metaphysis (15), was chosen to better evaluate the effects of treatment on cancellous bone microarchitecture including indexes of spatial connectivity not evaluated in our previous studies. Another purpose of this study was to determine whether prior and concurrent administration of the antiresorptive agents estrogen and the bisphosphonate risedronate suppresses the bone anabolic response to treatment with bFGF alone as well as to sequential treatment with bFGF and PTH. This area of investigation is important, as osteopenic patients are likely to have had a course of antiresorptive therapy before or during bone anabolic therapy. Furthermore, concurrent treatment with antiresorptive agents may be necessary to preserve the trabecular connections induced initially by bFGF treatment during subsequent PTH treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Three-month-old, virgin, female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA), initially weighing 265 ± 12 g, were used in the experiment. The rats were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the institutional animal care and use committee at University of Florida (Gainesville, FL). The rats were housed individually in plastic shoebox cages at 21 C with a 12-h light, 12-h dark cycle. Food (22/5 rodent diet, Teklad, Madison, WI) and water were provided ad libitum to the sham animals. The food consumption of the OVX rats was restricted to that of the sham control rats (16).

Experimental protocol
The experimental time course is outlined in Table 1Go. Rats were ovariectomized (OVX) from a dorsal approach under ketamine (50 mg/kg) and xylazine (10 mg/kg) anesthesia administered im. Control rats were subjected to sham surgeries during which the ovaries were exteriorized, but replaced intact. All animals were maintained for 1 yr after surgery to allow for aging, the predominance of bone remodeling in the lumbar vertebra (15), and the development of cancellous osteopenia in OVX rats. Untreated baseline sham and OVX rats (groups 1 and 2) were killed at this time (15 months of age). The remaining rats were anesthetized as described above, and polyurethane catheters (Braintree Scientific, Inc., Braintree, MA) were inserted in their right jugular veins. Three groups of OVX rats (groups 3–5) were treated iv with bFGF (Chiron Corp., Emeryville, CA) dissolved in PBS at a daily dose of 250 µg/kg for 14 d. During this time, each catheter was flushed twice daily with 0.2 ml of a 2% heparin saline solution. In addition, OVX rats in groups 3–5 were treated sc with vehicle, estrogen, or the bisphosphonate risedronate, respectively, for 2 wk before bFGF treatment and during the 2 wk of treatment with the growth factor. Estrogen (17ß-E2, Sigma, St. Louis, MO) was dissolved in a vehicle of 95% corn oil and 5% benzyl alcohol and injected sc 4 d/wk at a dose of 10 µg/kg. Risedronate (Procter \|[amp ]\| Gamble, Cincinnati, OH) was dissolved in saline vehicle and injected 2 d/wk at a dose of 5 µg/kg. All solutions for iv and sc treatments were prepared to deliver the desired dose in a volume of 1 ml/kg body weight. OVX rats in groups 3–5 were killed at the end of the 2 wk of treatment with bFGF. This part of the study was designed to confirm the stimulatory effects of bFGF on bone formation and to determine whether prior and concurrent treatment with the antiresorptive agents estrogen and risedronate affects the bone anabolic response to the growth factor.


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  		Table 1. Experimental protocol

 
Polyurethane catheters were also implanted in the right jugular veins of rats comprising groups 6–11. Sham control rats in group 6 and OVX rats in groups 7 and 8 were injected iv with PBS daily for 14 d. OVX rats in group 8 were also injected sc with synthetic human (h) PTH-(1–34) 5 d/wk at a dose of 80 µg/kg during this time (wk 0–2) and for the following 8 wk. hPTH was obtained from Bachem (Torrance, CA) and dissolved in a vehicle of acid saline and 2% heat-inactivated rat serum. OVX rats from groups 9–11 were treated iv with bFGF for 14 d as described above. These animals were then withdrawn from bFGF treatment and injected sc with PTH as described above for 8 wk. During the 10 wk of treatment with bFGF and PTH and for 2 wk preceding these treatments, OVX rats in groups 10 and 11 were also treated with estrogen and risedronate, respectively, as described above. The main goal of this part of the study was to determine whether sequential treatment with bFGF and PTH (group 9) is more efficacious than treatment with PTH alone (group 8) for restoration of lost cancellous bone in the estrogen-deplete skeleton and to assess whether prior and concurrent treatment with antiresorptive agents (groups 10 and 11) affects the bone anabolic response to bFGF and PTH.

A double fluorochrome labeling technique was used to determine active mineralization sites and rates of bone formation. Each rat was injected with demeclocycline (15 mg/kg; Sigma) on the 17th (sc) and 16th (ip) d and with calcein (15 mg/kg; Sigma) on the 7th (sc) and 6th (ip) d before necropsy. For sample collection, all rats were anesthetized with ketamine/xylazine as described above, and death was induced by exsanguination from the abdominal aorta, followed by cervical dislocation. Successful ovariectomy was confirmed by observation of lack of ovarian tissue and atrophied uterine horns. Estrogen treatment was observed to reverse the atrophic effects of estrogen depletion on the uterine horns.

Hematocrit was measured at the time of necropsy with a microhematocrit reader (Clay Adams, Division of Dickinson and Co., Parsippany, NJ). Serum samples were collected and stored at -80 C until assay. Lumbar vertebrae 1–4 were excised and cleaned of soft tissue. Bodies of lumbar vertebrae 1 and 2 were scraped to expose bone marrow, placed in 10% phosphate-buffered formalin for 24 h, and subsequently transferred to 70% ethanol for histological processing. Lumbar vertebrae 3 and 4 were wrapped in saline-soaked gauze and stored frozen in vials (-20 C) for biomechanical assessment.

Serum analysis
Serum calcium and phosphorus levels were analyzed by the o-cresolphthalein compliance method and the ammonium molybdate method, respectively, using a Hitachi 911 Chemistry Analyzer (Roche, Indianapolis, IN).

Bone histomorphometry
The lumbar vertebrae were dehydrated in graded ethanols and xylene, and embedded undecalcified in modified methyl methacrylate (17). Frontal sections (4 and 8 µm thick) were cut with vertical bed microtomes (Leica Corp./Jung 2065 and 2165, Rockleigh, NJ) and affixed to slides precoated with a 1% gelatin solution. Two nonconsecutive, 4-µm-thick sections per animal were stained according to the Von Kossa method with a tetrachrome counterstain (Polysciences, Warrington, PA) and used for determining bone microarchitectural and cellular end points. Two nonconsecutive, 8-µm-thick sections were left unstained and used for assessing fluorochrome labeling and dynamic measurements of bone formation. Histomorphometric data were collected with the Bioquant Bone Morphometry System (R&M Biometrics Corp., Nashville, TN) and the Trabecular Analysis System (TAS; OsteoMetrics, Inc., Atlanta, GA), which measures microarchitectural end points (18). The data are reported in accordance with standard bone nomenclature (19).

For data collection, the measurement area consisted of secondary spongiosa at distances greater than 0.5 mm from the cranial and caudal growth plates. An average of 6.7 mm2 of cancellous bone tissue (including marrow) and 37 mm of cancellous bone perimeter were measured in each rat. Cancellous bone volume was measured at a magnification of x20 in the 4-µm stained sections and expressed as a percentage of bone tissue area. Microarchitectural variables in these sections, including trabecular thickness (microns), number (per millimeter), and separation (microns), were calculated with TAS software from measures of bone surface and area (4, 18). In addition, indexes of trabecular connectivity, including number of trabecular nodes (N.Nd/mm2), number of node to node connections (Nd.Nd/mm2), number of trabecular termini (N.Tm/mm2), number of node to terminus connections (Nd.Tm/mm2), and node to terminus ratio (N.Nd/N.Tm), were measured with the TAS software using an algorithm that reduces (thins) trabecular bars to strings of single pixels and allows for determination of connections between trabeculae (4, 18). A trabecular node was defined as a point where three trabecular struts intersect, and a terminus was defined as a free end of a trabecular strut. Osteoid volume was measured at x200 in the 4-µm stained sections and expressed as a percentage of bone tissue area. Osteoclast, osteoblast, and osteoid surfaces were also measured at x200 in the 4-µm stained sections and expressed as percentages of total cancellous bone surface. Fluorochrome-based indexes of bone formation, including mineralizing surface (percentage of cancellous bone surface with double label) and mineral apposition rate, were measured at x200 in the 8-µm unstained sections. Bone formation rate (total surface referent) was calculated by multiplying mineralizing surface by mineral apposition rate.

Bone biomechanics
The lumbar vertebrae were thawed, mounted with superglue on wooden blocks, and fixed in the holder of a diamond precision-parallel saw (Exakt, Apparatebau, Otto Herrmann, Norderstedt, Germany). The cranial and caudal surfaces were sawed off, removing the cartilaginous growth plates and primary spongiosa and leaving a central specimen with plano-parallel ends. Vertebral processes were removed with a fine electric saw [Minimot 40(E), Proxxon, Niersbach/Eifel, Germany]. The length of the vertebral cylinder was measured with a micrometer, and volume was estimated by weighing the bone specimen before and during immersion in water with an electronic balance (Mettler AG245, Mettler-Toledo, Nanikon-Greifensee, Switzerland) equipped to measure volumes. The average cross-sectional area of each sample was calculated by dividing the bone volume by the specimen length. The bone specimens were kept in Ringer’s solution until testing. The vertebral body cylinders were tested in compression along the cranio-caudal axis in a materials testing machine (Alwetron TCT5, Lorentzen and Wettre, Stockholm, Sweden) at a constant deformation rate of 2 mm/min. During compression, load-deformation curves were recorded and stored on a PC (ProLinea 4/33, Compaq, Houston, TX). The load-deformation curves were analyzed with an in-house-developed computer program specially designed for the analysis of biomechanical data. After biomechanical testing, the bone samples were ashed (105 C for 2 h and 580 C for 24 h), and ash weight was determined with the electronic balance. The data are defined as follows: 1) ash density (milligrams per cubic millimeter) is the ash weight divided by the total tissue volume (bone + marrow); 2) maximum load, Fmax (N), is the maximum load applied at fracture of the specimen and was determined directly from the stress-strain curves; 3) maximum stress, {sigma}max (MPa), is the maximum compressive load (Fmax) per unit cross-sectional area; 4) Young’s modulus, E (MPa), is the maximum slope of the load-deformation curve.

Statistical analysis
Data for groups 1–5 and 6–11 were analyzed separately with the Kruskal-Wallis test, followed by a nonparametric post hoc test (t statistic adjusted for the number of groups and comparisons) (20). The level of statistical significance was set at 0.05. The data are expressed as the mean ± SD. All statistical analyses were performed using Crunch software (Crunch Software Corp., Oakland, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight did not differ between sham (454 ± 76 g) and OVX (434 ± 30 g) rats at the initiation of treatments (wk -2). Although all rats lost weight after catheter surgery, the percentage of weight lost differed (P < 0.02) among the treatment groups. Whereas body weight decreased by 3% in the vehicle-treated OVX rats, it decreased by 6–7% in OVX rats treated with bFGF, either singly or in combination with antiresorptive agents. The difference among the three bFGF-treated groups was not significant, and all differed from OVX rats treated with vehicle. Body weight decreased by 4% in sham rats treated with vehicle during the same time period. This decrease in weight was not significantly different from any of the other groups. After termination of bFGF treatment (wk 2–10), body weight increased in all but the estrogen plus bFGF plus PTH group.

Treatment of OVX rats with bFGF for 2 wk resulted in reduced blood hematocrit levels compared with those after treatment of sham and OVX rats with vehicle (P < 0.0001). Hematocrit measured 21.6 ± 2.9%, 24.3 ± 2.7%, and 22.8 ± 2.5% in OVX rats treated with bFGF alone, estrogen plus bFGF, and risedronate plus bFGF, respectively, compared with a mean of 38.1 ± 2.7% in sham rats treated with vehicle and a mean of 42.6 ± 1.1% in OVX rats treated with vehicle.

Serum samples from baseline OVX rats were lost due to technical problems. The mean values for serum calcium and phosphorus were 10.1 ± 0.7 and 5.1 ± 1.4 mg/dl, respectively, in baseline sham rats. Similar mean values were obtained for OVX rats treated with bFGF alone and estrogen plus bFGF. The mean value for serum calcium (8.8 ± 0.4 mg/dl) was significantly decreased in OVX rats treated with risedronate plus bFGF. Although serum phosphorus (4.3 ± 0.7 mg/dl) in OVX rats treated with risedronate plus bFGF tended to be lower than that in baseline sham rats, statistical significance was not achieved.

Serum calcium was lower in vehicle-treated OVX rats than in vehicle-treated sham rats (9.5 ± 0.3 vs. 10.4 ± 0.7 mg/dl; P < 0.05). Treatment of OVX rats with PTH alone or sequentially with bFGF plus PTH had no effect on serum calcium (9.1 ± 0.6 and 9.1 ± 0.6 mg/dl, respectively) compared with vehicle treatment of OVX rats. In contrast, OVX rats treated with either estrogen or risedronate in conjunction with bFGF plus PTH had mean serum calcium values near the level of vehicle-treated sham rats. Mean values for serum phosphorus for the vehicle-treated sham and OVX groups and the various treatment groups ranged from 5.3 ± 0.8 to 5.7 ± 0.8 mg/dl with no significant differences among them.

Effects of antiresorptive agents on the skeletal response to bFGF
Effects of OVX and treatment with bFGF on cancellous bone volume, microarchitecture, and turnover in the lumbar vertebra are presented in Figs. 1Go and 2Go and Table 2Go. Ovariectomy had a negative effect on cancellous bone volume (BV/TV; Fig. 1AGo and Fig. 3Go, A and B). At baseline or 1 yr post-OVX, cancellous BV/TV was approximately 40% lower in the OVX rats than in the sham rats. The change in cancellous BV/TV was accompanied by changes in trabecular microarchitecture. Although trabecular thickness was not affected by ovariectomy (Fig. 1BGo), trabecular number (Fig. 1CGo), and various indexes of connectivity, including number of trabecular nodes, number of node to node junctions, number of trabecular termini, number of terminus to node junctions (Table 2Go), and node to terminus ratio (Fig. 1EGo), were lower, and trabecular separation (Fig. 1DGo) was greater in the OVX than in the sham rats at baseline. Treatment of OVX rats with bFGF for 2 wk had no effect on cancellous BV/TV (Fig. 1AGo) and most indexes of microarchitecture and connectivity regardless of cotreatment with the antiresorptive agents estrogen or risedronate.



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  		Figure 1. Mean values (±SD) for cancellous bone volume (A), trabecular thickness (B), trabecular number (C), trabecular separation (D), and node to terminus ratio (E) in lumbar vertebrae of 11 groups of rats. Baseline sham (BSL SHAM) and baseline OVX (BSL OVX) rats were killed at 15 months of age (1 yr post-OVX). The remaining groups were treated with bFGF or its vehicle for 2 wk. For 2 wk before bFGF treatment and during the 2-wk treatment with bFGF, the rats were also treated with vehicle (VEH), estrogen (EST), or risedronate (RIS). At the end of bFGF treatment, three groups were killed (OVX+VEH+FGF, OVX+EST+FGF, and OVX+RIS+FGF) to determine whether prior and concurrent administration of antiresorptive agents affects the bone anabolic response to bFGF. The remaining six groups were treated with PTH or its vehicle in addition to continued treatment with estrogen, risedronate, or their vehicles. This part of the study was designed to determine whether sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for restoring lost bone and whether continued treatment with antiresorptive agents affects the bone anabolic response to treatment with bFGF and PTH. a, Different from BSL SHAM rats, P < 0.05; b, different from BSL OVX rats, P < 0.05; c, different from OVX+VEH+FGF rats, P < 0.05; d, different from SHAM+VEH+VEH+VEH rats, P < 0.05; e, different from OVX+VEH+VEH+VEH rats, P < 0.05; f, different from OVX+VEH+VEH+PTH rats, P < 0.05; g, different from OVX+VEH+FGF+PTH rats, P < 0.05.

 


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  		Figure 2. Mean values (±SD) for osteoclast surface (A), osteoblast surface (B), osteoid surface (C), and bone formation rate (D) in lumbar vertebrae of 11 groups of rats. See Fig. 1Go for details. a, Different from BSL SHAM rats, P < 0.05; b, different from BSL OVX rats, P < 0.05; c, different from OVX+VEH+FGF rats, P < 0.05; d, different from SHAM+VEH+VEH+VEH rats, P < 0.05; e, different from OVX+VEH+VEH+VEH rats, P < 0.05; f, different from OVX+VEH+VEH+PTH rats, P < 0.05; g, different from OVX+VEH+FGF+PTH rats, P < 0.05.

 

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  		Table 2. Indexes of trabecular michroarchitecture in OVX rats treated with bFGF, estrogen plus bFGF, and risedronate plus bFGF

 


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  		Figure 3. Cancellous bone tissue from the lumbar vertebra of a baseline sham rat (A); a baseline OVX rat (B); an OVX rat treated with PTH alone (C); an OVX rat treated with bFGF and PTH (D); an OVX rat treated with estrogen, bFGF, and PTH (E); and an OVX rat treated with risedronate, bFGF, and PTH (F). Note the reduction in black-stained cancellous bone mass indicative of osteopenia in the baseline OVX rat in comparison to the baseline sham rat. Treatment of OVX rats with PTH alone restored cancellous bone mass by thickening existing trabeculae. Sequential treatment of OVX rats with bFGF and PTH further augmented cancellous bone mass by thickening existing trabeculae and increasing connectivity between trabeculae. Similar bone structural changes were observed in OVX rats cotreated with estrogen or risedronate. In fact, a trend for an even greater increase in cancellous bone mass was observed in OVX rats treated with risedronate, bFGF, and PTH. Von Kossa/tetrachrome stain; magnification, x40.

 
Osteoid volume (OV/TV) was negligible (<0.1%) in both sham and OVX rats at baseline (data not shown). Treatment of OVX rats with bFGF for 2 wk increased osteoid volume regardless of cotreatment with estrogen or risedronate. OV/TV was 3.5 ± 2.2% in OVX rats treated with bFGF alone, 5.6 ± 3.7% in OVX rats treated with estrogen plus bFGF, and 3.9 ± 3.8% in OVX rats treated with risedronate plus bFGF. The difference in OV/TV between rats treated with bFGF alone and rats treated with bFGF and the antiresorptive agents was not significant. Although the majority of osteoid was deposited along mineralized bone surfaces, osteoid bridges that appear to be extensions of osteoid connecting mineralized trabeculae, were also evident. The abundance of osteoid, including examples of osteoid bridges, in response to treatment with bFGF is clearly seen in Fig. 4Go.



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  		Figure 4. Cancellous bone tissue in the lumbar vertebra from an aged OVX rat treated with estrogen and bFGF. A, Note the abundant osteoid (o) deposited on the black-stained cancellous bone surfaces in response to treatment with bFGF and the osteoid bridge extending between two preexisting trabeculae. B, At higher magnification, numerous osteoblasts (arrows) can be seen lining the surfaces of an osteoid bridge (o) extending between trabeculae. This observation suggests that bFGF improves trabecular connectivity in the osteopenic skeleton. Von Kossa/tetrachrome stain; magnification: A, x100; B, x200.

 
The effects of OVX and treatment on osteoclast surface, osteoblast surface, osteoid surface, and bone formation rate are presented in Fig. 2Go. Osteoclast surface (Oc.S/BS), an index of bone resorption, did not differ between sham and OVX rats at baseline (Fig. 2AGo). Treatment of OVX rats with bFGF for 2 wk had no effect on Oc.S/BS. However, cotreatment of bFGF-treated rats with estrogen decreased Oc.S/BS, whereas cotreatment with risedronate increased Oc.S/BS compared with baseline sham, baseline OVX, and OVX rats treated with bFGF alone.

Osteoblast surface (Ob.S/BS) was higher in OVX than in sham rats at baseline (Fig. 2BGo). Treatment of OVX rats with bFGF for 2 wk increased Ob.S/BS relative to that in baseline sham and OVX rats. Cotreatment of bFGF-treated rats with estrogen or risedronate did not suppress the effects of bFGF on Ob.S/BS. In fact, the administration of estrogen to bFGF-treated OVX rats resulted in a significant increase in Ob.S/BS in these rats compared with OVX rats treated with bFGF alone.

Osteoid surface (OS/BS) was also higher in OVX rats compared with sham rats at baseline (Fig. 2CGo). The 2-wk treatment of OVX rats with bFGF increased OS/BS relative to that in baseline sham and OVX rats. Administration of estrogen or risedronate to bFGF-treated rats did not affect the observed increase in OS/BS.

The bone formation rate (BFR/BS) did not differ between sham and OVX rats at baseline (Fig. 2DGo). OVX rats treated with bFGF had markedly decreased values for BFR/BS relative to baseline sham and OVX rats regardless of cotreatment with estrogen or risedronate. Fluorochrome labels were almost totally lacking in cancellous bone of all bFGF-treated OVX rats.

OVX and treatment effects on vertebral ash density and determinants of vertebral strength are presented in Fig. 5Go. Ash density was approximately 30% lower in the OVX than in the sham rats at baseline (Fig. 5AGo). Treatment of OVX rats for 2 wk with bFGF, estrogen plus bFGF, or risedronate plus bFGF had no effect on ash density at the termination of treatment.



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  		Figure 5. Mean values (±SD) for ash density (A), maximum load (B), maximum stress (C), and Young’s modulus of elasticity (D) in lumbar vertebrae of 11 groups of rats. See Fig. 1Go for details. a, Different from BSL SHAM rats, P < 0.05; b, different from BSL OVX rats, P < 0.05; c, different from OVX+VEH+FGF rats, P < 0.05; d, different from SHAM+VEH+VEH+VEH rats, P < 0.05; e, different from OVX+VEH+VEH+VEH rats, P < 0.05; f, different from OVX+VEH+VEH+PTH rats, P < 0.05; g, different from OVX+VEH+FGF+PTH rats, P < 0.05.

 
The effects of OVX and treatment with bFGF and antiresorptive agents on vertebral maximum load (Figs. 5BGo), maximum stress (Fig. 5CGo), and Young’s modulus of elasticity (Fig. 5DGo) were very similar to those described above for ash density, although some trends did not achieve statistical significance.

Comparison of the skeletal response to treatment with PTH alone and sequential treatment with bFGF and PTH
Cancellous BV/TV was lower in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 1AGo). Treatment of OVX rats with PTH alone increased cancellous BV/TV to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH also increased BV/TV to at least the level of vehicle-treated sham rats. In addition, BV/TV in rats treated with bFGF and PTH was higher than that in rats treated with PTH alone. Cotreatment with estrogen or risedronate in rats treated sequentially with bFGF and PTH did not suppress the augmentation of bone mass by bFGF and PTH. In fact, BV/TV in OVX rats cotreated with risedronate was higher than that in both vehicle-treated sham rats and OVX rats treated with PTH alone. Differences in cancellous bone mass among the groups can be seen in Fig. 3Go.

Trabecular thickness did not differ between vehicle-treated OVX and sham rats (Fig. 1BGo). Treatment of OVX rats with PTH alone; bFGF and PTH; estrogen, bFGF, and PTH; or risedronate, bFGF, and PTH increased trabecular thickness relative to that in vehicle-treated OVX rats as well as vehicle-treated sham rats.

Trabecular number (Tb.N) was lower in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 1CGo). Treatment of OVX rats with either PTH alone or bFGF and PTH had no effect on Tb.N. However, treatment of OVX rats with either estrogen, bFGF, and PTH or with risedronate, bFGF, and PTH increased Tb.N relative to that in OVX rats treated with vehicle and OVX rats treated with PTH alone.

Trabecular separation (Tb.Sp) was greater in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 1DGo). Treatment of OVX rats with PTH alone tended to decrease Tb.Sp, but statistical significance was not achieved. However, treatment of OVX rats with bFGF and PTH; estrogen, bFGF, and PTH; or risedronate, bFGF, and PTH resulted in lower Tb.Sp than in OVX rats treated with either vehicle or PTH alone.

The N.Nd and Nd.Nd were lower in vehicle-treated OVX rats than in vehicle-treated sham rats (Table 3Go). Treatment of OVX rats with PTH alone had no effect on N.Nd or Nd.Nd, whereas treatment of OVX rats with bFGF plus PTH increased both end points above those observed in OVX rats treated with vehicle. Treatment of OVX rats with either estrogen, bFGF, and PTH or risedronate, bFGF, and PTH increased N.Nd and Nd.Nd relative to those in OVX rats treated with vehicle as well as OVX rats treated with PTH alone. Although N.Tm and Nd.Tm tended to be lower in all OVX rats compared with those in vehicle-treated sham rats regardless of treatment, the overall differences did not achieve statistical significance (P < 0.1).


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  		Table 3. Indexes of trabecular microarchitecture in OVX rats treated with PTH, bFGF plus PTH; estrogen, bFGF, plus PTH; and risedronate, bFGF, plus PTH

 
The N.Nd/N.Tm was lower in vehicle-treated OVX rats than in vehicle-treated sham rats, but the difference did not reach statistical significance (Fig. 1EGo). Treatment of OVX rats with PTH alone had no significant effect on the N.Nd/N.Tm, whereas treatment of OVX rats with bFGF and PTH as well as estrogen, bFGF, and PTH increased this measure of trabecular connectivity above that observed in vehicle-treated OVX rats. Treatment of OVX rats with risedronate, bFGF, and PTH increased the N.Nd/N.Tm to a level above that observed in sham rats treated with vehicle, OVX rats treated with vehicle, and OVX rats treated with PTH alone.

Oc.S/BS was higher in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 2AGo). Oc.S/BS in all other treatment groups did not differ from that of vehicle-treated OVX rats.

Ob.S/BS was greater in OVX rats treated with vehicle compared with that in sham rats treated with vehicle (Fig. 2BGo). Treatment of OVX rats with PTH alone or with bFGF and PTH increased Ob.S/BS to a level above that in vehicle-treated OVX rats. Ob.S/BS in OVX rats treated with estrogen, bFGF, and PTH was nearly identical to that in OVX rats treated with bFGF and PTH. Ob.S/BS in OVX rats treated with risedronate, bFGF, and PTH was greater than that in vehicle-treated sham rats but lower than that in OVX rats treated with PTH alone or with bFGF and PTH.

OS/BS was also greater in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 2CGo). OS/BS in OVX rats treated with PTH alone, bFGF and PTH, or estrogen, bFGF, and PTH tended to be greater than that in vehicle-treated OVX rats, but statistical significance was not achieved. OS/BS in rats treated with risedronate, bFGF, and PTH did not differ from that in vehicle-treated sham or OVX rats, but was lower than that in OVX rats treated with PTH alone or with bFGF and PTH.

The BFR/BS did not differ between vehicle-treated sham and OVX rats (Fig. 2DGo). Treatment of OVX rats with PTH alone, bFGF and PTH, or estrogen, bFGF, and PTH increased BFR/BS relative to that in vehicle-treated sham and OVX rats. Although BFR/BS in OVX rats treated with risedronate, bFGF, and PTH was also increased relative to that in vehicle-treated sham rats, it was lower than that in OVX rats treated sequentially with bFGF and PTH.

Vertebral ash density was lower in vehicle-treated OVX rats than in vehicle-treated sham rats (Fig. 5AGo). Treatment of OVX rats with PTH alone resulted in a mean value for ash density that was between those observed in vehicle-treated sham and OVX rats, but not different from that in either group. Sequential treatment of OVX rats with bFGF and PTH increased ash density to the level of vehicle-treated sham rats. Cotreatment with estrogen or risedronate did not suppress the effect of treatment with bFGF and PTH on ash density.

The Fmax was nearly 50% lower in vehicle-treated OVX rats than in vehicle-treated sham rats, but this difference was not statistically significant (Fig. 5BGo). Administration of PTH alone to OVX rats increased Fmax to the level of vehicle-treated sham rats. Sequential treatment of OVX rats with bFGF and PTH increased Fmax above the level observed in vehicle-treated sham rats and in OVX rats treated with PTH alone. Treatment of OVX rats with estrogen, bFGF, and PTH increased Fmax above the level of vehicle-treated sham and OVX rats, whereas treatment of OVX rats with risedronate, bFGF, and PTH increased Fmax above the level of vehicle-treated sham rats as well as above the level of OVX rats treated with PTH alone.

Maximum stress was approximately 40% lower in vehicle-treated OVX rats compared with that in vehicle-treated sham rats, but the difference was not significant (Fig. 5CGo). Treatment of OVX rats with PTH alone; estrogen, bFGF, and PTH; or risedronate, bFGF, and PTH increased maximum stress to at least the level of vehicle-treated sham rats, whereas treatment of OVX rats with bFGF and PTH increased maximum stress to a level above that in vehicle-treated sham rats. Significant differences due to OVX or the various treatments were not detected (P < 0.2) for Young’s modulus of elasticity (Fig. 5DGo), although trends were similar to those described above for maximum stress.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that sequential treatment with bFGF and PTH is more efficacious than treatment with PTH alone for increasing bone mass and strength in osteopenic OVX rats. Whereas treatment of OVX rats with PTH alone increased vertebral cancellous bone mass and strength to the level of sham rats, sequential treatment of OVX rats with bFGF and PTH further augmented vertebral cancellous bone mass and strength above that observed in PTH-treated OVX rats. The greater efficacy of the former treatment appears to be associated with bFGF-induced formation of osteoid bridges between disconnected trabeculae (Fig. 4Go), which results in improved trabecular connectivity and greater bone strength. This beneficial effect appears to have been enhanced by cotreatment with the bisphosphonate risedronate, which apparently preserved the bFGF-induced bone connections during subsequent PTH treatment. In any case, prior and concurrent administration of the antiresorptive agents estrogen and risedronate certainly did not inhibit the bone anabolic response to treatment with bFGF and to sequential treatment with bFGF and PTH.

The mechanical competence of cancellous bone is determined not only by its mass, but also by its microarchitecture, including connectivity of the trabecular network (21). Loss of trabecular connectivity due to trabecular perforation and complete loss of trabeculae is a well established characteristic of cancellous osteoporosis (4, 5). Measures of trabecular microarchitecture evaluated in the current study included trabecular thickness, trabecular number, trabecular spacing, and node to terminus ratio, the latter an index of trabecular connectivity. The intermittent administration of PTH alone improved trabecular microarchitecture somewhat in OVX rats, as indicated by increased trabecular thickness. However, there were no significant changes in trabecular number and node to terminus ratio in OVX rats treated with PTH alone compared with those in OVX rats treated with vehicle. These results are consistent with the majority of studies in rats which show that the main process by which PTH restores cancellous bone mass is by thickening existing trabeculae without significantly increasing trabecular connectivity (10). The latter is especially true if PTH treatment is started after half of the original trabecular connections have been lost (22). In the current study trabecular number and node to terminus ratio tended to be increased in OVX rats treated sequentially with bFGF and PTH compared with PTH alone. These trends became statistically significant when the former animals were cotreated with risedronate. This finding suggests that antiresorptive agents preserve the trabecular connections formed initially in response to bFGF treatment during subsequent PTH treatment.

In our previous study (14) sequential treatment with bFGF and PTH was not found to have a greater bone restorative effect than treatment with PTH alone in the proximal tibia of aged, OVX rats. This finding is not consistent with our current results from the lumbar vertebral body. The age of the animals and the duration of treatment were the same in the two studies. However, the dose of bFGF differed in these studies (200 vs. 250 µg/kg in the previous and current studies, respectively). Despite the use of the higher dose of bFGF, we confirmed the results of the previous study in the proximal tibia (data not shown). The most likely explanation for the divergent results in the proximal tibia and lumbar vertebra is that the former skeletal site is more severely osteopenic than the latter skeletal site. For example, tibial cancellous bone volume in baseline OVX rats was less than 5% compared with a vertebral cancellous bone volume of 20–25%. Therefore, the few bone spicules in the proximal tibia were too widely separated to be connected by the osteoid formed in response to bFGF treatment. In contrast, vertebral trabecular separation was much less, which allowed bFGF treatment to form osteoid bridges between preexisting bone spicules. This phenomenon resulted in greater bone mass and strength in the lumbar vertebrae of aged OVX rats treated with bFGF and PTH compared with treatment with PTH alone.

The bone formed as a result of treatment with bFGF is woven bone (12, 23) and thus biomechanically inferior to lamellar bone. However, in the current study vertebral strength in OVX rats treated sequentially with bFGF and PTH was not only increased relative to that in OVX rats treated with vehicle, it was also increased relative to that in vehicle-treated sham rats as well as in OVX rats treated with PTH alone. Consequently, the inferior biomechanical quality of the woven bone deposited during bFGF administration is not associated with decreased bone strength when treatment with the growth factor is followed by treatment with PTH. Deposition of lamellar bone induced by PTH increases trabecular thickness, thereby strengthening the intertrabecular connections initially formed during bFGF treatment. Nevertheless, formation of woven bone may be a necessary prerequisite to reestablish trabecular connectivity in the osteopenic skeleton by bridging the marrow space between disconnected trabeculae. Deposition of lamellar bone on preexisting bone surfaces is probably inadequate for this purpose.

As demonstrated in previous studies (12, 14), treatment of OVX rats with bFGF for 14 d markedly increased cancellous bone formation, as indicated by substantial increases in osteoblast surface, osteoid surface, and osteoid volume. These indexes of bone formation were not suppressed by prior and concurrent administration of either estrogen or risedronate. Osteoclast surface, an index of bone resorption, was not affected by treatment with bFGF in the current study. In previous studies indexes of bone resorption, including eroded surface and osteoclast surface, have been shown to be either unaffected (24) or decreased (12, 14, 25) by short-term administration of bFGF. As osteoclasts are rarely found adjacent to unmineralized bone surfaces, the reported decreases in osteoclast surface may be secondary to the extensive osteoid surface in animals treated with bFGF (12, 14). In the current study cotreatment of OVX rats with estrogen and bFGF resulted in suppressed osteoclast surface, whereas cotreatment with risedronate and bFGF was associated with an increase in osteoclast surface, suggesting that bone resorption was decreased and increased, respectively, during the specified treatments. However, because bisphosphonates inhibit bone resorption primarily by decreasing the function of osteoclasts (26, 27), the increase in osteoclast surface in OVX rats cotreated with risedronate and bFGF should not be interpreted as evidence for increased bone resorption (28).

As in previous studies (12, 14), treatment with bFGF impaired cancellous bone mineralization, as indicated by the almost complete lack of fluorochrome labeling, despite markedly increased osteoblast surface. However, the mineralization defect is temporary, and bone mineralization resumes after withdrawal of bFGF treatment (12). This sequence of events results in augmentation of cancellous bone mass not at the end of bFGF treatment, but only during the withdrawal period when the abundant osteoid calcifies (12). In agreement with this contention, osteoid volume, but not cancellous bone volume, was increased at the end of 14 d of treatment with bFGF in the current study.

Despite its potential as an osteoporosis treatment, systemic administration of bFGF is associated with various adverse side-effects. bFGF is a broad spectrum mitogen that affects the proliferation and differentiation of numerous cell types and tissues (29). Reported adverse side-effects of treatment with bFGF include kidney and lung hypertrophy (30, 31), increased or decreased longitudinal bone growth, depending on dose, in growing animals (32), retarded weight gain (32), and anemia (30, 31). In the current study OVX rats treated with bFGF for 14 d lost some (<10%) weight compared with OVX rats treated with vehicle. Weight loss in association with bFGF treatment was not observed in our previous studies (12, 14) of OVX rats treated with a lower (200 µg/kg) dose of bFGF than that used in the current study (250 µg/kg). Anemia resulting from bFGF administration is another major concern. In the current study hematocrit levels in bFGF-treated OVX rats were approximately 50% lower than those in vehicle-treated OVX rats. However, as with the other adverse side-effects, termination of bFGF treatment results in a normalization of hematocrit levels (30). Although these adverse side-effects make systemic use of bFGF in humans problematic, the current study provides proof of concept that a bone anabolic therapy with the potential to induce formation of osteoid bridges within bone marrow may be useful for restoring trabecular connectivity and bone strength in the osteopenic skeleton.

In summary, the results of this study indicate that sequential administration of bFGF and PTH to aged, osteopenic, OVX rats increases vertebral cancellous bone mass and strength and improves trabecular microarchitecture to a greater extent than administration of PTH alone. Furthermore, the anabolic response of bone to bFGF and sequential treatment with bFGF and PTH is not suppressed by cotreatment with either estrogen or risedronate. In fact, these antiresorptive agents appear to enhance the skeletal effects of treatment with bFGF and PTH by preserving bone formed in response to the initial treatment with bFGF. These findings in an animal model for postmenopausal bone loss provide support for a novel treatment strategy for osteoporotic patients who are unresponsive to conventional treatments.


   Acknowledgments
 
The authors gratefully acknowledge Anna Ratkus, Christina Florio, Carolyn Thomson, Dr. Qiming Vulcan, and Dr. Rachel Power from the University of Florida and Birthe Gylling-Jorgensen from the University of Aarhus for their technical assistance. We thank Dr. Judith Abraham of Chiron Corp. (Emeryville, CA) for supplying the bFGF used in this experiment. Risedronate was obtained through the generosity of Dr. Roger Phipps of Procter \|[amp ]\| Gamble Pharmaceuticals (Cincinnati, OH).


   Footnotes
 
This work was supported by NIH Grant R37-09241 from the NIA.

Abbreviations: bFGF, Basic fibroblast growth factor; BFR/BS, bone formation rate/bone surface; BV/TV, cancellous bone volume; Fmax, maximum load; hPTH, human PTH; Nd.Nd, number of node to node connections; Nd.Tm, number of node to terminus connections; N.Nd, number of trabecular nodes; N.Nd/N.Tm, node to terminus ratio; N.Tm, number of trabecular termini; Ob.S./BS, osteoblast surface/bone surface; Oc.S/BS, osteoclast surface/bone surface; OS/BS, osteoid surface/bone surface; OV/TV, osteoid volume; OVX, ovariectomized, ovariectomy; TAS, Trabecular Analysis System; Tb.N, trabecular number; Tb.Sp, trabecular separation.

Received October 30, 2001.

Accepted for publication March 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kanis JA, Melton III LJ, Christiansen C, Johnston CC, Khaltaev N 1994 The diagnosis of osteoporosis. J Bone Miner Res 9:1137–1141[Medline]
  2. Parfitt AM 1996 Skeletal heterogeneity and the purposes of bone remodeling. In: Marcus R, Feldman D, Kelsey J, eds. Osteoporosis. San Diego: Academic Press; 315–329
  3. Aaron JE, Shore PA, Shore RC, Beneton M, Kanis JA 2000 Trabecular architecture in women and men of similar bone mass with and without vertebral fracture. II. Three-dimensional histology. Bone 27:277–282[Medline]
  4. Parfitt AM, Mathews CHE, Villanueva AR, Kleerekoper M, Frame B, Rao DS 1983 Relationship between surface, volume, and thickness of iliac trabecular bone in aging and osteoporosis. J Clin Invest 72:1396–1409
  5. Recker RR 1993 Architecture and vertebral fracture. Calcif Tissue Int 53:S139–S142
  6. Rodan GA, Martin TJ 2000 Therapeutic approaches to bone diseases. Science 289:1508–1514[Abstract/Free Full Text]
  7. Hodsman AB, Fraher LJ, Watson PH 1998 Parathyroid hormone: the clinical experience and prospects. In: Whitfield JF, Morley P, eds. Anabolic treatments for osteoporosis. Boca Raton, FL: CRC Press; 83–108
  8. Lau K-HW, Akesson K, Libanati CR, Baylink DJ 1998 Osteogenic actions of fluoride: its therapeutic use for established osteoporosis. In: Whitfield JF, Morley P, eds. Anabolic treatments for osteoporosis. Boca Raton, FL: CRC Press; 207–254
  9. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster J, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH 2001 Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 344:1434–1441[Abstract/Free Full Text]
  10. Wronski TJ, Li M 1998 PTH: Skeletal effects in the ovariectomized rat model for postmenopausal bone loss. In: Whitfield JF, Morley P, eds. Anabolic treatments for osteoporosis. Boca Raton, FL: CRC Press; 59–81
  11. Zerwekh JE, Hagler HK, Sakhaee K, Gottschalk F, Peterson RD, Pak CY 1994 Effect of slow release sodium fluoride on cancellous bone histology and connectivity in osteoporosis. Bone 15:691–699[Medline]
  12. Liang H, Pun S, Wronski TJ 1999 Bone anabolic effects of basic fibroblast growth factor in ovariectomized rats. Endocrinology 140:5780–5788[Abstract/Free Full Text]
  13. Wronski TJ 2001 Skeletal effects of systemic treatment with basic fibroblast growth factor. J Musculoskel Neuron Interact 2:9–14
  14. Wronski TJ, Ratkus AM, Thomsen JS, Vulcan Q, Mosekilde L 2001 Sequential treatment with basic fibroblast growth factor and parathyroid hormone restores cancellous bone mass and strength in the proximal tibia of aged ovariectomized rats. J Bone Miner Res 16:1399–1407[Medline]
  15. Erben RG 1996 Trabecular and endocortical bone surfaces in the rat: modeling or remodeling? Anat Rec 246:39–46[CrossRef][Medline]
  16. Wronski TJ, Schenck PA, Cintrón M, Walsh CC 1987 Effect of body weight on osteopenia in ovariectomized rats. Calcif Tissue Int 40:155–159[Medline]
  17. Baron R, Vignery A, Neff L, Silverglate A, Santa Maria A 1983 Processing of undecalcified bone specimens for bone histomorphometry. In: Recker RR, ed. Bone histomorphometry: techniques and interpretation. Boca Raton, FL: CRC Press; 13–35
  18. Aaron JE, Johnson DR, Kanis JA, Oakley BA, O’Higgins P, Paxton SK 1992 An automated method for the analysis of trabecular bone structure. Comput Biomed Res 25:1–16[CrossRef][Medline]
  19. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: standardization of nomenclature, symbols, and units. J Bone Miner Res 2:595–610[Medline]
  20. Conover WJ 1980 Practical nonparametric statistics. 2nd ed. New York: Wiley & Sons
  21. Mosekilde L 1998 Osteoporosis: mechanisms and models. In: Whitfield JF, Morley P, eds. Anabolic treatments for osteoporosis. Boca Raton, FL: CRC Press; 31–58
  22. Lane NE, Thompson JM, Strewler GJ, Kinney JH 1995 Intermittent treatment with human parathyroid hormone (hPTH[1–34]) increased trabecular bone volume but not connectivity in osteopenic rats. J Bone Miner Res 10:1470–1477[Medline]
  23. Amizuka N, Yamada M, Watanabe J, Hoshi K, Fukushi M, Oda K, Ikehara Y, Ozawa H 1998 Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow. Microsc Res Tech 41:313–322[Medline]
  24. Nakamura T, Hanada K, Tamura M, Shibanushi T, Nigi H, Tagawa M, Fukumoto S, Matsumoto T 1995 Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats. Endocrinology 136:1276–1284[Abstract]
  25. Nakamura K, Kurokawa T, Aoyama I, Hanada K, Tamura M, Kawaguchi H 1998 Stimulation of bone formation by intraosseous injection of basic fibroblast growth factor in ovariectomized rats. Int Orthop 22:49–54[CrossRef][Medline]
  26. Miller SC, Jee WSS 1979 The effect of dichloromethylene diphosphonate, a pyrophosphate analog, on bone and bone cell structure in the growing rat. Anat Rec 193:439–445[CrossRef][Medline]
  27. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA 1991 Bisphosphonate action: alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88:2095–2105
  28. Wronski TJ, Dann LM, Qi H, Yen C-F 1993 Skeletal effects of withdrawal of estrogen and diphosphonate treatment in ovariectomized rats. Calcif Tissue Int 53:210–216[CrossRef][Medline]
  29. Gospodarowicz D 1991 Biological activities of fibroblast growth factors. In: Baird A, Klagsbrun M, eds. The fibroblast growth factor family. New York: New York Academy of Sciences; vol 638:1–8
  30. Mazue G, Bertolero F, Garofano L, Brughera M, Carminati P 1992 Experience with the preclinical assessment of basic fibroblast growth factor (bFGF). Toxicol Lett 64–65:329–338
  31. Mayahara H, Ito T, Nagai H, Miyajima H, Tsukuda R, Taketomi S, Mizoguchi J, Kato K 1993 In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats. Growth Factors 9:73–80[Medline]
  32. Nagai H, Tsukuda R, Mayahara H 1995 Effects of basic fibroblast growth factor (bFGF) on bone formation in growing rats. Bone 16:367–373[Medline]



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