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Endocrinology Vol. 139, No. 6 2900-2904
Copyright © 1998 by The Endocrine Society

ARTICLES

Effects of Fibroblast Growth Factor-2 on Longitudinal Bone Growth1

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

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: Jeffrey_Baron{at}nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LONGITUDINAL 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, 3, 4, 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, 8, 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 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 µg/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 µCi/ml, and the rudiments were incubated for an additional 3 h. The metatarsals were then washed three times for 10 min each time and 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-µm 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 µCi/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%.

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-µm sections were stained with toluidine blue. Hypertrophic cells were defined by a height along the longitudinal axis greater than 9 µm. 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 ± SEM. Statistical significance was determined by ANOVA and post-hoc Fisher’s protected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Longitudinal growth
Fetal rat metatarsal bones were cultured for 4 days in serum-free medium containing 0–1000 ng/ml FGF-2 (n = 15–19/group). In the absence of FGF-2, fetal metatarsals grew an average of 102 ± 8 µm/day (mean ± SEM; Fig. 1Go). Bone rudiments cultured with 10 and 100 ng/ml FGF-2 showed growth curves indistinguishable from the control curve (Fig. 1Go). Bones treated with 1000 ng/ml FGF-2 showed a slower growth rate for the first 2 days of culture (mean ± SEM, 30 ± 15 µm/day; P < 0.001; Fig. 1Go). 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.



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  		Figure 1. Longitudinal bone growth (mean ± SEM). Fetal rat metatarsals [embryonic day 20 (e20); n = 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.

 
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 = 31–32 per group; P < 0.0001; Fig. 2Go). To determine which cell types were responsible for this increased incorporation, we performed autoradiography after labeling with [3H]thymidine (n = 19–24/group; Fig 3Go). 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. 4Go). FGF-2 also caused a significant increase in the perichondrial thickness (mean ± SEM, 84 ± 8 vs. 80 ± 7 vs. 98 ± 9 vs. 116 ± 11 µm, at 0 vs. 10 vs. 100 vs. 1000 ng/ml FGF-2, respectively; P < 0.03, by ANOVA).



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  		Figure 2. Total [3H]thymidine incorporation (mean ± SEM). Fetal rat metatarsals (n = 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 µCi/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).

 


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  		Figure 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 µCi/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-µm 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.

 


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  		Figure 4. [3H]Thymidine labeling indexes (mean ± SEM). Fetal rat metatarsals (n = 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. 3Go. 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).

 
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 = 6–8/group; P < 0.001; Fig. 5Go). No hypertrophic chondrocytes were observed in bone rudiments treated with 1000 ng/ml FGF-2 (Fig. 6Go).



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  		Figure 5. Quantitation of hypertrophic chondrocytes (mean ± SEM). Fetal rat metatarsals (n = 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-µm longitudinal sections were obtained. Hypertrophic cells were operationally defined by a height along the longitudinal axis greater than 9 µm. 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)].

 


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  		Figure 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.

 
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 = 15–16/group; Fig 7Go). At a low concentration of FGF-2 (10 ng/ml), glycosaminoglycan synthesis was significantly increased (P < 0.001), whereas at a high concentration (1000 ng/ml), synthesis was significantly decreased (P < 0.001).



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  		Figure 7. Sulfate incorporation into glycosaminoglycans (mean ± SEM). Fetal rat metatarsals (n = 15–16/group) were cultured for 2 days in serum-free medium containing 0–1000 ng/ml FGF-2. Bones were labeled with 5 µCi/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)].

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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, 8, 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, 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.

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, 20, 21, 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.


   Footnotes
 
1 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. Back

Received November 3, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Iannotti JP 1990 Growth plate physiology and pathology. Orthop Clin North Am 21:1–17
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  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[Abstract]
  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[CrossRef][Medline]
  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[Abstract]
  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[CrossRef][Medline]
  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[Medline]
  9. Webster MK, D’Avis PY, Robertson SC, Donogue DJ 1996 Profound ligand-independent 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[Abstract]
  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[CrossRef][Medline]
  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[CrossRef][Medline]
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  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[Medline]
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