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Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes - PubMed

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Basic fibroblast growth factor inhibits the anabolic activity of insulin-like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

Richard F Loeser et al. Arthritis Rheum. 2005 Dec.

Abstract

Objective: To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1).

Methods: Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum-free medium with or without IGF-1 (100 ng/ml), OP-1 (100 ng/ml), or bFGF (0-100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants.

Results: Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF-1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF-1 and OP-1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF-1 or OP-1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF-1 and OP-1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures.

Conclusion: The anabolic activity of IGF-1 and OP-1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.

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Figures

Figure 1
Figure 1

Chondrocyte cell number during culture in the presence of basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1), and osteogenic protein 1 (OP-1). Human articular chondrocytes were cultured in alginate in serum-free medium with mini-ITS+ (insulin–transferrin–selenium) (control) or the control medium plus bFGF (0–100 ng/ml), IGF-1 (100 ng/ml), and/or OP-1 (100 ng/ml). Cell numbers at the end of 21 days of culture were measured in triplicate samples using a DNA assay and are expressed as a percentage of the control cultures (mean and SEM). Results are for cultures with bFGF alone (A), bFGF plus IGF-1 (B), bFGF plus OP-1 (C), and bFGF plus IGF-1 and OP-1 (“combo”) (D). Numbers in parentheses are the number of donors used for each culture condition.* = P < 0.05 versus control; ** = P < 0.001 versus control; † = P = 0.02 versus 1 ng/ml bFGF plus IGF-1; †† = P = 0.007 versus 1 ng/ml bFGF plus IGF-1 and OP-1.

Figure 2
Figure 2

Chondrocyte appearance and pericellular matrix production after alginate culture in the presence of bFGF, IGF-1, and OP-1. Chondrocytes were cultured for 21 days in alginate beads in serum-free mini-ITS+ medium (control) or the control medium supplemented with 50 ng/ml bFGF, 100 ng/ml each of IGF-1 and OP-1, or the combination of all 3. At the end of the culture period, the beads were incubated with Calcein AM and ethidium bromide homodimer, as described in Materials and Methods, to assess survival. The beads were dissolved in sodium citrate, and a sample of the cells from each condition was observed using a fluorescence microscope (top). Dissolved alginate beads from separate cultures were pelleted by centrifugation, resuspended in Dulbecco’s modified Eagle’s medium, and placed in the bottom of microwell plates, followed by the addition of fixed erythrocytes, as described in Materials and Methods. A representative sample was photographed using an inverted phase-contrast microscope (bottom). The cell-associated (pericellular) matrix can be seen excluding the erythrocytes from the chondrocyte plasma membrane in the cells treated with IGF-1 plus OP-1. The clusters of cells treated with bFGF revealed by Calcein AM staining were partially broken up by the processing for the particle exclusion assay. See Figure 1 for definitions. (Original magnification × 400.)

Figure 3
Figure 3

Chondrocyte proteoglycan production after culture in the presence of bFGF, IGF-1, and OP-1. Human articular chondrocytes were cultured in alginate in serum-free medium with mini-ITS+ (control) or the control medium plus bFGF (0–100 ng/ml), IGF-1 (100 ng/ml), and/or OP-1 (100 ng/ml). The amount of proteoglycan in the alginate beads (cell-associated and further-removed matrix) was measured by the dimethylmethylene blue (DMMB) assay and normalized to cell numbers using DNA measurements (DMMB/DNA). Samples were measured in triplicate and are expressed as a percentage of the day 21 control cultures (mean and SEM). Numbers in parentheses are the number of donors used for each culture condition. Results are for cultures with A, bFGF alone (* = P < 0.05 versus control); B, bFGF plus IGF-1 (* = P = 0.02 versus control; † = P = 0.04 versus IGF-1; †† = P = 0.002 versus IGF-1); C, bFGF plus OP-1 (* = P < 0.008 versus control; † = P = 0.02 versus OP-1; †† = P = 0.001 versus OP-1); and D, bFGF plus IGF-1 and OP-1 (“combo”) (* = P < 0.05 versus control; † = P < 0.05 versus combo; †† = P < 0.001 versus combo). E, Results of the same experiments shown in D, but for the total DMMB in the beads, without DNA correction (** = P < 0.0001 versus control; † = P < 0.001 versus combo). See Figure 1 for other definitions.

Figure 4
Figure 4

Chondrocyte proteoglycan production when human des(–3) IGF-1 was substituted for IGF-1 in bFGF- and OP-1–treated cultures. Experiments were performed as described in Figure 3, except that equal amounts of human des(–3) IGF-1 were compared with IGF-1. Samples from 1 donor were measured (in triplicate) by the dimethylmethylene blue (DMMB) assay and normalized to cell numbers using DNA measurements (DMMB/DNA) (mean and SEM). * = P < 0.0001 versus day 21 control; † = P < 0.0001 for comparison of each growth factor with and without bFGF. See Figure 1 for other definitions.

Figure 5
Figure 5

Chondrocyte proteoglycan synthesis after culture in the presence of bFGF, IGF-1, and OP-1. Human articular chondrocytes were cultured in alginate for 21 days or as cartilage explants for 10–21 days in serum-free medium with mini-ITS+ (control) or the control medium plus bFGF (50 ng/ml), IGF-1 (100 ng/ml), and/or OP-1 (100 ng/ml). Proteoglycan synthesis was measured during the last 4 hours of culture using sulfate incorporation and was normalized to cell numbers by DNA assay (for alginate cultures) or by wet weight (for explants). Results are expressed as a percentage of control for triplicate samples (mean and SEM); n = 2 donors for alginate cultures and 3 donors for explants. A, Alginate cultures (* = P < 0.05 versus control; † = P = 0.03 versus IGF-1 + OP-1). B, Cartilage explants (* = P < 0.05 versus control; ** = P < 0.001 versus control; † = P < 0.0001 versus each growth factor without bFGF). See Figure 1 for definitions.

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