Objectives: Knowledge of matrix assembly is necessary to understand the pathogenesis of disease processes and to find solutions for repair of articular cartilage lesions. The influence of growth factors on matrix assembly is largely unknown. We investigated whether, and to what degree, insulin-like growth factor (IGF-1) and transforming growth factor beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment (consisting of pericellular and territorial matrix) compared to the further-removed matrix compartment (consisting of the interterritorial matrix).
Design: Bovine articular chondrocytes were cultured in alginate beads for day 13. The effect of addition of 25 ng/ml IGF-1 or 25 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteoglycans and the amount of newly synthesized proteoglycans were analyzed biochemically. Morphometric analysis on electron micrographs was used to calculate the volumes of the cell-associated and further-removed matrix components.
Results: It was demonstrated in control beads that 25 +/- 8% of the proteoglycans were laid down in the cell-associated matrix compartment compared with 75 +/- 8% in the further-removed matrix compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense amorph material. Morphometric evaluation showed a relative volume of the cell-associated matrix compartment of 5.2 +/- 0.6% compared with 91.3 +/- 0.8% of the further-removed matrix compartment and 3.5 +/- 0.3% of the area occupied by cells. Combination of biochemical and morphometric results showed that the concentration of proteoglycans in the cell-associated matrix compartment was 3.63 +/- 0.32 mg/ml. By adding IGF-1 or TGF beta-2, the amount of both total accumulated proteoglycans and newly synthesized [35S]proteoglycans at day 13 in culture increased. In addition to an overall rise in proteoglycan content, IGF-1 significantly increased (24%) the percentage of proteoglycans laid down in the cell-associated matrix compartment while not changing the relative volume of this compartment (5.2 +/- 0.8%). This leads to a 82% (P < 0.05) increase in the proteoglycan concentration in the cell-associated matrix compartment compared to control beads. In contrast, TGF beta-2 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduction of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This leads to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment.
Conclusions: This study demonstrates that both IGF-1 and TGF beta-2 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associated matrix compartment of cultured bovine chondrocytes in alginate. Both growth factors increase the concentration of proteoglycans in the cell-associated matrix compartment. However, addition of TGF beta-2 to bovine articular chondrocytes cultured in alginate beads for 13 days results in a significant reduction of the relative volume of the pericellular matrix compartment compared to controls, indicating differences in assembly of the matrix.