0013-7227/98/$03.00/0
Endocrinology
Copyright © 1998 by The Endocrine Society
Vol. 139, No. 6
Printed in U.S.A.
Effects of Fibroblast Growth Factor-2 on Longitudinal
Bone Growth*
EDNA E. MANCILLA, FRANCESCO DE LUCA, JENNIFER A. UYEDA,
FRANK S. CZERWIEC, AND JEFFREY BARON
Developmental Endocrinology Branch, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
In vivo, fibroblast growth factor-2 (FGF-2) inhibits longitudinal
bone growth. Similarly, activating FGF receptor 3 mutations impair
growth in achondroplasia and thanatophoric dysplasia. To investigate the underlying mechanisms, we chose a fetal rat metatarsal
organ culture system that would maintain growth plate histological
architecture. Addition of FGF-2 to the serum-free medium inhibited
longitudinal growth. We next assessed each major component of longitudinal growth: proliferation, cellular hypertrophy, and cartilage
matrix synthesis. Surprisingly, FGF-2 stimulated proliferation, as
assessed by [3H]thymidine incorporation. However, autoradiographic
studies demonstrated that this increased proliferation occurred only
in the perichondrium, whereas decreased labeling was seen in the
L
ONGITUDINAL bone growth occurs at the growth plate
by a process termed endochondral ossification (1).
Chondrogenesis results from growth plate chondrocyte proliferation, hypertrophy, and extracellular matrix secretion.
Simultaneously, the metaphyseal border of the growth plate
is invaded by blood vessels and bone cell precursors that
remodel the growing cartilage into bone.
Fibroblast growth factor-1 (FGF-1) and FGF-2 and FGF
receptors 1, 2, and 3 are expressed by growth plate chondrocytes (2–5). Overexpression of FGF-2 in mice slows longitudinal growth (6). Similarly, in humans, activating mutations in FGF receptor 3 inhibit bone growth in
achondroplasia and thanatophoric dysplasia (7–9). Conversely, inactivating knock-out mutations in FGF receptor 3
increase longitudinal bone growth in mice (10, 11). Thus, in
vivo, the FGF system appears to inhibit longitudinal bone
growth. However, in vitro, FGFs stimulate growth; addition
of FGF-2 to growth plate chondrocyte culture increases proliferation (12, 13). This discrepancy may reflect the loss of
tissue architecture and intercellular interactions that occur
when chondrocytes are removed from the growth plate and
placed in cell culture.
Therefore, to study the role of FGFs in the growth plate,
we chose an organ culture system that maintains cellular
Received November 3, 1997.
Address all correspondence and requests for reprints to: Jeffrey
Baron, Developmental Endocrinology Branch, National Institute of
Child Health and Human Development, National Institutes of Health,
Building 10, Room 10N262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862. E-mail: [email protected].
* Presented in part at the 5th Joint Meeting of the European Society
for Pediatric Endocrinology and the Lawson Wilkins Pediatric Endocrine Society and at the 79th Annual Meeting of The Endocrine Society.
proliferative and epiphyseal chondrocytes. FGF-2 also caused a
marked decrease in the number of hypertrophic chondrocytes. To
assess cartilage matrix synthesis, we measured 35SO4 incorporation
into newly synthesized glycosaminoglycans. Low concentrations (10
ng/ml) of FGF-2 stimulated cartilage matrix production, but high
concentrations (1000 ng/ml) inhibited matrix production. We conclude
that FGF-2 inhibits longitudinal bone growth by three mechanisms:
decreased growth plate chondrocyte proliferation, decreased cellular
hypertrophy, and, at high concentrations, decreased cartilage matrix
production. These effects may explain the impaired growth seen in
patients with achondroplasia and related skeletal dysplasias. (Endocrinology 139: 2900 –2904, 1998)
relationships. Fetal rat metatarsals on embryonic day 20 were
placed in serum-free medium with varying concentrations of
FGF-2. We assessed the effects on the rate of longitudinal
growth. To elucidate the mechanism of action, we studied the
effects on chondrocyte proliferation, hypertrophy, and cartilage matrix formation in this organ culture system.
Materials and Methods
Organ culture
The second, third, and fourth metatarsal bone rudiments were dissected from Sprague-Dawley rat fetuses at 20 days postconception and
cultured individually in 24-well plates. Each well contained 0.5 ml MEM
(Life Technologies, Gaithersburg, MD) supplemented with 0.05 mg/ml
ascorbic acid (Life Technologies), 0.3 mg/ml l-glutamine (Life Technologies), 1 mm sodium glycerophosphate (Sigma Chemical Co., St.
Louis, MO), 0.2% BSA (Sigma), 100 U/ml penicillin and 100 mg/ml
streptomycin (Life Technologies), and FGF-2 (Life Technologies) at concentrations of 0 –1000 ng/ml. Plates were incubated in humidified air
containing 5% CO2 at 37 C. Medium was changed daily. Animal procedures were approved by the NICHHD animal care and use committee.
Animal care was in accordance with the Guide for the Care and Use of
Laboratory Animals [DHEW Publication (NIH) 85-23, revised 1988].
Measurement of longitudinal growth
The length of each bone rudiment was measured daily using an
eyepiece micrometer in a dissecting microscope. Culture medium was
briefly removed immediately before each measurement.
Assessment of cell proliferation
Cell proliferation was assessed by measuring [3H]thymidine incorporation into newly synthesized DNA as previously described (14).
After 2 days of culture, [3H]thymidine (Amersham, Arlington Heights,
IL; SA, 25 Ci/mmol) was added to the culture medium at a concentration
of 5 mCi/ml, and the rudiments were incubated for an additional 3 h.
The metatarsals were then washed three times for 10 min each time and
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FGF-2 AND LONGITUDINAL BONE GROWTH
2901
solubilized using NCS-II Tissue Solubilizer (0.5 n solution; Amersham)
overnight. Total [3H]thymidine incorporation was then measured by
liquid scintillation counting. Each metatarsal bone was treated as an
individual sample and assayed once. The intraassay coefficient of variation was 2.5%.
[3H]Thymidine incorporation was localized to specific populations of
chondrocytes by autoradiography. After 2 days of culture, bone rudiments were labeled with [3H]thymidine as described above and fixed in
10% phosphate-buffered formalin. After embedding in paraffin, 5-mm
longitudinal sections were prepared. Autoradiography was performed
by dipping the slides in Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY), exposing for 1 week, and developing with Kodak D-19
developer (15). Sections were counterstained with hematoxylin and
eosin. The labeling index was determined by a single observer blinded
to the treatment category.
Assessment of glycosaminoglycan synthesis
Glycosaminoglycan synthesis was assessed by measuring 35SO4 incorporation as previously described (16). After 2 days of culture, bones
were labeled with 5 mCi/ml Na235SO4 (Amersham; SA, up to 100 mCi/
mmol) for 3 h. The bone rudiments were rinsed three times for 10 min
each time in Puck’s saline solution and digested in 1.5 ml fresh medium
with 0.3% papain at 60 C for 16 h. Then, 0.5 ml 10% cetyl pyridinium
chloride (Sigma) in 0.2 m NaCl was added to precipitate glycosaminoglycans, and the samples were incubated at room temperature for 18 h.
The precipitate was collected by vacuum filtration through filter paper
(Whatman, Clifton, NJ; catalogue no. 1001090), washed three times with
a solution of 0.1% cetyl pyridinium chloride in 0.2 m NaCl, and dissolved
in 0.5 ml 23 n formic acid. The samples were counted by liquid scintillation. Each metatarsal bone was treated as an individual sample and
assayed once. The intraassay coefficient of variation was 7.5%.
FIG. 1. Longitudinal bone growth (mean 6 SEM). Fetal rat metatarsals [embryonic day 20 (e20); n 5 15–19/group] were cultured for 4
days in serum-free medium containing 0 –1000 ng/ml FGF-2. The
lengths of the bone rudiments were measured daily using an eyepiece
micrometer in a dissecting microscope. By the third day of culture,
metatarsals incubated with 1000 ng/ml FGF-2 had become curved,
preventing accurate measurement of linear growth.
Assessment of cellular hypertrophy and perichondrial
thickness
At the end of the second day of culture, metatarsals were fixed in 10%
buffered formalin for 24 h. After routine processing, the metatarsals
were embedded in plastic, and longitudinal 5-mm sections were stained
with toluidine blue. Hypertrophic cells were defined by a height along
the longitudinal axis greater than 9 mm. The same histological sections
were used to evaluate perichondrial thickness. This thickness was measured midway between the center of ossification and the end of the
metatarsal rudiment. All quantitative histology was performed by a
single observer blinded to the treatment category.
Statistics
All data were expressed as the mean 6 sem. Statistical significance
was determined by ANOVA and post-hoc Fisher’s protected least significant difference test.
Results
Longitudinal growth
Fetal rat metatarsal bones were cultured for 4 days in
serum-free medium containing 0 –1000 ng/ml FGF-2 (n 5
15–19/group). In the absence of FGF-2, fetal metatarsals
grew an average of 102 6 8 mm/day (mean 6 sem; Fig. 1).
Bone rudiments cultured with 10 and 100 ng/ml FGF-2
showed growth curves indistinguishable from the control
curve (Fig. 1). Bones treated with 1000 ng/ml FGF-2 showed
a slower growth rate for the first 2 days of culture (mean 6
sem, 30 6 15 mm/day; P , 0.001; Fig. 1). By the third day of
culture, these bones had become curved, preventing accurate
measurement of linear growth. At this stage of development,
longitudinal bone growth has three principal components:
cell proliferation, hypertrophy, and cartilage matrix synthesis. We therefore assessed each of these components.
FIG. 2. Total [3H]thymidine incorporation (mean 6 SEM). Fetal rat
metatarsals (n 5 31–32/group) were cultured for 2 days in serum-free
medium containing 0 –1000 ng/ml FGF-2. [3H]Thymidine, at a concentration of 5 mCi/ml, was added to the culture medium, and the
rudiments were incubated for an additional 3 h. The metatarsals were
washed and solubilized using NCS-II Tissue Solubilizer (0.5 N solution; Amersham) overnight. Total [3H]thymidine incorporation was
measured by liquid scintillation counting. *, P , 0.001 vs. control (0
ng/ml FGF-2).
Cell proliferation
To assess chondrocyte proliferation, we measured incorporation of [3H]thymidine in metatarsal rudiments after 2
days of culture. FGF-2 caused a concentration-dependent
increase in [3H]thymidine incorporation (n 5 31–32 per
group; P , 0.0001; Fig. 2). To determine which cell types were
responsible for this increased incorporation, we performed
autoradiography after labeling with [3H]thymidine (n 5 19 –
24/group; Fig 3). FGF-2 caused a significant decrease in the
labeling index in the epiphyseal (P , 0.005) and proliferative
zones (P , 0.001), and a significant increase in the perichondrium (P , 0.001; Fig. 4). FGF-2 also caused a significant
increase in the perichondrial thickness (mean 6 sem, 84 6 8
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FGF-2 AND LONGITUDINAL BONE GROWTH
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Vol 139 • No 6
FIG. 3. Autoradiography of [3H]thymidine incorporation. Fetal rat metatarsals were cultured for 2 days in serum-free medium without FGF-2
(A and B) or with 1000 ng/ml FGF-2 (C and D). [3H]Thymidine, at a concentration of 5 mCi/ml, was added to the culture medium, and the
rudiments were incubated for an additional 3 h and fixed in 10% phosphate-buffered formalin. After routine processing, samples were embedded
in paraffin, and 5-mm longitudinal sections were prepared. Autoradiography was performed by standard techniques using a 1-week exposure
time. Sections were counterstained with hematoxylin and eosin. Representative sections are shown in brightfield (A and C) and darkfield (B
and D) views. pe, Perichondrium; e, epiphyseal region; pr, proliferative zone; h, hypertrophic zone; o, primary center of ossification. The primary
center of ossification could be distinguished from the hypertrophic zone by the presence of cells incorporating [3H]thymidine, multiple cells per
lacuna, differential staining with Masson-Trichrome stain and toluidine blue stain (data not shown), and avid 45Ca uptake (data not shown).
The apparent width of the bone rudiments in a particular section depends on the precise plane of that section and does not necessarily reflect
the full width of the bone rudiment.
vs. 80 6 7 vs. 98 6 9 vs. 116 6 11 mm, at 0 vs. 10 vs. 100 vs.
1000 ng/ml FGF-2, respectively; P , 0.03, by ANOVA).
Cellular hypertrophy
After 2 days of culture in the presence or absence of FGF-2,
metatarsal rudiments were examined histologically. The
number of hypertrophic chondrocytes per slide was quantitated. FGF-2 caused a concentration-dependent decrease in
the number of hypertrophic cells per section (n 5 6 – 8/
group; P , 0.001; Fig. 5). No hypertrophic chondrocytes were
observed in bone rudiments treated with 1000 ng/ml FGF-2
(Fig. 6).
FIG. 4. [3H]Thymidine labeling indexes (mean 6 SEM). Fetal rat
metatarsals (n 5 19 –24/group) were cultured for 2 days in 0 –1000
ng/ml FGF-2, labeled with [3H]thymidine, and prepared for autoradiography as described in Fig. 3. Labeling index (number of labeled
cells per total cells) was determined by a single observer blinded to the
treatment category. *, P , 0.05; **, P , 0.001 vs. control (0 ng/ml
FGF-2).
Glycosaminoglycan synthesis
To assess cartilage matrix formation, we measured 35SO4
incorporation into newly synthesized glycosaminoglycans.
We observed a biphasic effect of FGF-2 on glycosaminoglycan synthesis (n 5 15–16/group; Fig 7). At a low concentration of FGF-2 (10 ng/ml), glycosaminoglycan synthesis was
FGF-2 AND LONGITUDINAL BONE GROWTH
significantly increased (P , 0.001), whereas at a high concentration (1000 ng/ml), synthesis was significantly decreased (P , 0.001).
Discussion
In fetal rat metatarsal organ culture, FGF-2 inhibited longitudinal bone growth. This effect is consistent with the
inhibition of longitudinal growth seen in vivo. Overexpression of FGF-2 in mice results in decreased growth of long
bones (6). Similarly, activating mutations in FGF receptor 3
inhibit longitudinal bone growth in achondroplasia and
thanatophoric dysplasia (7–9). Conversely, inactivating mutations in FGF receptor 3 in mice cause increased longitudinal
bone growth (10, 11). Thus, the fetal rat metatarsal organ
culture system appears to provide a good model to study the
role of FGF-2 in longitudinal bone growth.
During fetal development, longitudinal bone growth has
three principal components: cell proliferation, hypertrophy,
FIG. 5. Quantitation of hypertrophic chondrocytes (mean 6 SEM).
Fetal rat metatarsals (n 5 6 – 8/group) were cultured for 2 days in
serum-free medium with the indicated concentrations of FGF-2. After
routine histological processing, bones were embedded in plastic, and
5-mm longitudinal sections were obtained. Hypertrophic cells were
operationally defined by a height along the longitudinal axis greater
than 9 mm. Quantitation was performed by a single observer blinded
to the treatment category. *, P , 0.005; **, P , 0.001 [vs. control (0
ng/ml FGF-2)].
2903
and cartilage matrix synthesis. We therefore assessed each of
these major components. FGF-2 caused a concentration-dependent increase in total proliferation, as assessed by
[3H]thymidine incorporation. This observation is consistent
with findings in isolated growth plate chondrocyte culture.
In the current study, the highest concentration of FGF-2
caused the greatest increase in proliferation, yet decreased
longitudinal bone growth. This apparent discrepancy was
resolved by the autoradiographic findings. The increased
proliferation occurred exclusively in the perichondrium,
which does not contribute significantly to longitudinal bone
growth. In the mature growth plate, longitudinal growth
depends on replication of the proliferative chondrocytes.
However, at this earlier stage in development, replication of
both the proliferative zone and the epiphyseal chondrocytes
contributes to growth. FGF-2 inhibited proliferation of chondrocytes in the epiphyseal and proliferative zones of the bone
rudiments. The decreased proliferation in these zones provides one explanation for the decreased overall growth.
FIG. 7. Sulfate incorporation into glycosaminoglycans (mean 6 SEM).
Fetal rat metatarsals (n 5 15–16/group) were cultured for 2 days in
serum-free medium containing 0 –1000 ng/ml FGF-2. Bones were
labeled with 5 mCi/ml Na235SO4 for the last 3 h of culture. The bone
rudiments were then rinsed and digested with papain. Glycosaminoglycans were precipitated with 10% cetyl pyridinium chloride, and
the precipitate was counted by liquid scintillation. *, P , 0.05; **, P ,
0.001 [vs. control (0 ng/ml FGF-2)].
FIG. 6. Micrographs of fetal rat metatarsals. Bone rudiments were cultured for 2 days in serum-free medium without FGF-2 (A) or with 1000
ng/ml FGF-2 (B). A representative hypertrophic chondrocyte is labeled (h). No hypertrophic chondrocytes are present in micrograph B.
2904
FGF-2 AND LONGITUDINAL BONE GROWTH
FGF-2 decreased the number of hypertrophic chondrocytes present in the growth plate. Similar inhibition of hypertrophy has been observed in mice overexpressing FGF-2
and in cultured growth plate chondrocytes treated with
FGF-2 (6, 13, 17). A similar decrease in the hypertrophic zone
has also been observed in growth plates of patients with
achondroplasia (18).
Addition of FGF-2 had a biphasic effect on glycosaminoglycan synthesis. A low dose (10 ng/ml) of FGF-2 significantly increased matrix synthesis, whereas a high dose (1000
ng/ml) of FGF-2 decreased matrix synthesis. In different
published studies, FGF-2 has been reported to cause either
increased or decreased glycosaminoglycan synthesis in primary cultures of growth plate chondrocytes (19 –22). The
effects in isolated cell culture may depend on the confluence
of cells, the presence of other growth factors, the concentration of FGF-2, or other conditions in vitro.
Thus, at higher concentration of FGF-2, the decreased longitudinal bone growth could be explained by three mechanisms: decreased replication of proliferative and epiphyseal
chondrocytes, decreased cellular hypertrophy, and decreased matrix production. At lower concentrations, the divergent effects on proliferation, hypertrophy, and matrix
production produced little net effect on the overall rate of
longitudinal growth. Similarly, in mice receiving iv FGF-2, a
high dose decreased the rate of bone growth (23). A low
concentration actually increased the growth rate, suggesting
that the net effect of low FGF-2 concentrations can actually
be positive under some circumstances.
We conclude that FGF-2 inhibits longitudinal bone growth
by three mechanisms: decreased growth plate chondrocyte
proliferation, decreased hypertrophy, and, at high concentrations, decreased cartilage matrix production. These effects
may also explain the impaired growth seen in patients with
hypochondroplasia, achondroplasia, and thanatophoric
dysplasia.
References
1. Iannotti JP 1990 Growth plate physiology and pathology. Orthop Clin North
Am 21:1–17
2. Leach RM, Sokol C, McMurtry JP 1997 Immunolocalization of basic fibroblast
growth factor in porcine epiphyseal growth plate. Dom Anim Endocrinol
14:129 –132
3. Jingushi S, Scully SP, Joyce ME, Sugioka Y, Bolander ME 1995 Transforming
growth factor b and fibroblast growth factors in rat growth plate. J Orthop Res
13:761–768
Endo • 1998
Vol 139 • No 6
4. Peters KG, Werner S, Chen G, Wiliams LT 1992 Two FGF receptor genes are
differentially expressed in epithelial and mesenchymal tissues during limb
formation and organogenesis in the mouse. Development 114:233–243
5. Peters K, Ornitz D, Werner S, Williams L 1993 Unique expression pattern of
the FGF receptor 3 gene during mouse organogesis. Dev Biol 155:423– 430
6. Coffin JD, Florkiewicz RZ, Neumann J, Mort-Hopkins T, DornII GW, Lightfoot P German R, Howles PN, Kier A, O’Toole BA, Sasse J, Gonzalez AM,
Baird A, Doetschman T 1995 Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol
Biol Cell 6:1861–1873
7. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M,
Winokur ST, Wasmuth JJ 1994 Mutations in the transmembrane domain of
FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell
78:335–342
8. Webster MK, Donogue DJ 1996 Constitutive activation of fibroblast growth
factor receptor 3 by the transmembrane domain point mutation found in
achondroplasia. EMBO J 15:520 –527
9. Webster MK, D’Avis PY, Robertson SC, Donogue DJ 1996 Profound ligandindependent kinase activation of fibroblast receptor 3 by the activation loop
mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia
type II. Mol Cell Biol 16:4081– 4087
10. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P 1996 Fibroblast growth
factor receptor 3 is a negative regulator of bone growth. Cell 84:911–921
11. Colvin JS, Bohne BA, Harding GW, McEwen DG, Ornitz DM 1996 Skeletal
overgrowth and deafness in mice lacking fibroblast growth factor receptor 3.
Nat Genet 12:390 –397
12. Hill DJ, Logan A, De Sousa D 1991 Stimulation of DNA and protein synthesis
in epiphyseal growth plate chondrocytes by fibroblast growth factors. Interactions with other growth factors. Ann NY Acad Sci 638:449 – 452
13. Wroblewski J, Edwall-Arvidsson C 1995 Inhibitory effects of basic fibroblast
growth factor on chondrocyte differentiation. J Bone Miner Res 10:735–742
14. Bagi CM, Miller SC 1992 Dose-related effects of 1,25-dihydroxyvitamin D3 on
growth, modeling, and morphology of fetal rat metatarsals cultured in serumfree medium. J Bone Miner Res 7:29 – 40
15. Walker, KVR, Kember NF 1972 Cell kinetics of growth cartilage in the rat tibia.
I. Measurement in young male rats. Cell Tissue Kinet 5:401– 408
16. Bagi C, Burger EH 1989 Mechanical stimulation by intermittent compression
stimulates sulfate incorporation and matrix mineralization in fetal mouse
long-bone rudiments under serum-free conditions. Calcif Tissue Int 45:342–347
17. Kato Y, Iwamoto M 1990 Fibroblast growth factor is an inhibitor of terminal
differentiation. J Biol Chem 265:5903–5909
18. Ponseti IV 1970 Skeletal growth in achondroplasia. J Bone Joint Surg
52:701–716
19. Trippel SB, Wroblewsky J, Makower AM, Whelan MC, Schoenfield D,
Doctrow SR 1993 Regulation of growth plate chondrocytes by insulin-like
growth factor I and basic fibroblast growth factor. J Bone Joint Surg
75A:177–189
20. Kato Y, Hiraki Y, Inoue H, Kinoshita M, Yutani Y, Suzuki F 1983 Differential
and synergistic actions of somatomedin-like growth factors, fibroblast growth
factor and epidermal growth factor in rabbit costal chondrocytes. Eur J Biochem 129:685– 690
21. Kato Y, Gospodarowicz D 1985 Sulfated proteoglycan synthesis by confluent
cultures of rabbit costal chondrocytes grown in the presence of fibroblast
growth factor. J Cell Biol 100:477– 485
22. Inoue H, Kato Y, Iwamoto M, Hiraki Y, Sakuda M, Suzuki F 1989 Stimulation
of cartilage-matrix proteoglycan synthesis by morphologically transformed
chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-b. J Cell Physiol 138:329 –337
23. Nagai H, Tsukuda R, Mayahara H 1995 Effects of basic fibroblast growth factor
(bFGF) on bone formation in growing rats. Bone 16:367–373