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. 2000 Apr;105(8):1085-93.
doi: 10.1172/JCI8641.

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation

A Montero  1 , Y Okada, M Tomita, M Ito, H Tsurukami, T Nakamura, T Doetschman, J D Coffin, M M Hurley
Affiliations

Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation

A Montero et al. J Clin Invest. 2000 Apr.

Abstract

Basic fibroblast growth factor (FGF-2), an important modulator of cartilage and bone growth and differentiation, is expressed and regulated in osteoblastic cells. To investigate the role of FGF-2 in bone, we examined mice with a disruption of the Fgf2 gene. Measurement of trabecular bone architecture of the femoral metaphysis of Fgf2(+/+) and Fgf2(-/-) adult mice by micro-CT revealed that the platelike trabecular structures were markedly reduced and many of the connecting rods of trabecular bone were lost in the Fgf2(-/-) mice. Dynamic histomorphometry confirmed a significant decrease in trabecular bone volume, mineral apposition, and bone formation rates. In addition, there was a profound decreased mineralization of bone marrow stromal cultures from Fgf2(-/-) mice. This study provides strong evidence that FGF-2 helps determine bone mass as well as bone formation.

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Figures

Figure 1
Figure 1
Northern analysis for Fgf2 mRNA expression in primary calvarial osteoblasts prepared from 7-day-old Fgf2+/+ and Fgf2–/– mice. Total RNA was extracted from cells after 7, 9, 14, and 21 days of culture. A 6-kb Fgf2 mRNA transcript was expressed in calvarial osteoblast cultures from Fgf2+/+ mice (lanes 1, 3, 5, and 7). No Fgf2 mRNA transcript was detected in osteoblasts from Fgf2–/– mice (lanes 2, 4, 6, and 8).
Figure 2
Figure 2
Comparison of thymidine incorporation into DNA in calvarial osteoblasts from 8-week-old Fgf2+/+ and Fgf2–/– mice. Osteoblastic cells were prepared from calvariae of 8-week-old Fgf2+/+ and Fgf2–/– mice and plated in 100-mm dishes in DMEM with 10% heat inactivated FCS for 12 days. Cells were harvested and replated at a density of 5,000 cells/cm2 for 7 days. Media were changed every 3 days. Cultures were treated with fresh media in the absence or presence of FGF-2 (10 nM) for the last 24 hours of the culture period. For labeling studies, [3H]thymidine (10 μCi/well) was added for the last 4 hours of the culture to measure thymidine incorporation into DNA. Values are the mean ± SEM for 6 determinations per group. ASignificantly different from control cultures; P < 0.05. BSignificantly different from Fgf2+/+; P < 0.05.
Figure 3
Figure 3
(a) Effect of FGF-2 on colony area in mouse bone marrow cultures from Fgf2+/+ and Fgf2–/– mice. Cells were plated at a density of 1 million cells per well in αMEM containing penicillin/streptomycin and 10% heat inactivated FCS in the absence or presence of FGF-2 (10 nM). On day 3, media were changed and cells were cultured in differentiation media (αMEM, 10 nM dexamethasone, 10% FCS, 8 mM β-glycerophosphate, 50 μg/mL ascorbic acid) for an additional 11 days. ASignificantly different from control cultures; P < 0.05. BSignificantly different from Fgf2+/+; P < 0.05. (b) Comparison of the ability to form ALP colonies and mineralized nodules as determined by von Kossa staining in mouse bone marrow cultures from Fgf2+/+ and Fgf2–/– mice. Cells were plated at a density of 20 million cells per well in αMEM containing penicillin/streptomycin and 10% heat inactivated FCS. On day 3, media were changed and cells were cultured in differentiation media (αMEM, 10 nM dexamethasone, 10% FCS, 8 mM β-glycerophosphate, 50 μg/mL ascorbic acid) for the indicated times.
Figure 4
Figure 4
Morphological study by micro-CT scanning of trabecular bone of the femurs of 4.5-month-old Fgf2+/+ and Fgf2–/– mice. Three-dimensional trabecular bone architecture of distal femoral metaphysis of 4.5-month-old male Fgf2+/+ and Fgf2–/– mice were analyzed by micro-CT. Note that the platelike architecture of the trabecular bone is markedly reduced and the connecting rods of trabeculae are disrupted in Fgf2–/– mice compared with Fgf2+/+.
Figure 5
Figure 5
Three-dimensional microstructural parameters calculated using 2-dimensional data obtained from micro-CT of femoral bones from 4.5-month-old Fgf2+/+ and Fgf2–/– mice. Calculated morphometric indices included bone volume density (bone volume [BV]/trabecular volume [TV]), trabecular number [Tb.N = (BV/TV)/Tb.Th], and trabecular separation [Tb.Sp = (1/Tb.N)–Tb.Th]. ASignificantly different from Fgf2+/+ group; P < 0.05, Tukey-Kramer multiple comparison test (ANOVA).
Figure 6
Figure 6
Undecalcified sections of the distal femur of (a) an 8-month-old Fgf2+/+ mouse and (b) a Fgf–/– mouse (dark-field illumination). Trabecular number is decreased in the secondary spongiosa of the Fgf2–/– mouse.
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