DIFFERENTIAL-EFFECTS OF IGF-1 AND TGF-BETA-2 ON THE ASSEMBLY OF PROTEOGLYCANS IN PERICELLULAR AND TERRITORIAL MATRIX BY CULTURED BOVINE ARTICULAR CHONDROCYTES

Objectives: Knowledge of matrix assembly is necessary to understand th e pathogenesis of disease processes and to find solutions for repair o f articular cartilage lesions. The influence of growth factors on matr ix assembly is largely unknown. We investigated whether, and to what d egree, insulin-like growth factor (IGF-1) and transforming growth fact or beta-2 (TGF beta-2) influence proteoglycan synthesis and accumulati on in the cell-associated matrix compartment (consisting of pericellul ar and territorial matrix) compared to the further-removed matrix comp artment (consisting of the interterritorial matrix). Design: Bovine ar ticular chondrocytes were cultured in alginate beads for day 13. The e ffect of addition of 25 ng/ml IGF-1 or 26 ng/ml TGF beta-2 during the last 7 days in culture was determined. Cell-associated and further-rem oved matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment. The amount of DNA, the total amount of proteo glycans and the amount of newly synthesized proteoglycans were analyze d biochemically. Morphometric analysis on electron micrographs was use d 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 m atrix compartment compared with 75 +/- 8% in the further-removed matri x compartment. The cell-associated matrix compartment in intact beads could be recognized in electron microscopy by a delineation of dense a morph material. Morphometric evaluation showed a relative volume of th e 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 morphome tric results showed that the concentration of proteoglycans in the cel l-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 a nd newly synthesized [S-35]proteoglycans at day 13 in culture increase d. In addition to an overall rise in proteoglycan content, IGF-1 signi ficantly 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-8 significantly decreased (24%) the relative amount of proteoglycans in the cell-associated matrix compartment which was paralleled by a reduc tion of the relative volume from 5.2 +/- 0.6 to 3.6 +/- 1.4%. This lea ds to a significant increase of 87% of the proteoglycan concentration in the cell-associated matrix compartment. Conclusions: This study dem onstrates that bath IGF-1 and TGF beta-8 significantly but differently influence proteoglycan synthesis and accumulation in the cell-associa ted matrix compartment of cultured bovine chondrocytes in alginate. Bo th 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 peric ellular matrix compartment compared to controls, indicating difference s in assembly of the matrix.