Figure 6

Fibroblast growth factor 2 antagonizes BMP7-mediated stimulation of proteoglycan accumulation in the cell-associated matrix. (a) Nucleus pulposus cells isolated from bovine intervertebral disc were cultured for 21 days in 1.2% alginate beads in serum-free medium with mini-insulin–transferrin–selenium (control) or the control medium plus 10 ng/ml fibroblast growth factor 2 (FGF2), 100 ng/ml BMP7, or 10 ng/ml FGF2 combined with 100 ng/ml BMP7. At the end of the culture period the beads were dissolved in sodium citrate, and cell pellets containing the cells and their cell-associated matrix (CM) were separated by centrifugation. The amount of proteoglycan in the CM was measured by dimethylethylene blue assay and normalized to cell numbers using DNA measurement (DMMB/DNA). Samples were measured in triplicate and expressed as a percentage of the day 21 control cultures. Error bars represent three different donors in three separate experiments (Fig 6A). (b) Nucleus pulposus cell pericellular matrix production after alginate culture for 21 days in the presence or absence of FGF2, BMP7 or the combination of both factors was measured in an exclusion assay as described in Materials and methods. A representative sample was photographed using an inverted phase-contrast microscope. The CM can be seen excluding the erythrocytes from the cell plasma membrane (original magnification × 400).