Objective: To evaluate the effects of high molecular weight hyaluronan (HA) on the distribution and movement of proteoglycan (PG) formed around rabbit chondrocytes cultured in alginate beads. Design: Rooster comb-derived HA (MW 8x105 Da) was co-polymerized in alginate gel to study the direct effects of extrinsic HA on chondrocytes. PG metabolism of rabbit chondrocytes cultured in alginate beads was examined by measuring the incorporation of [35S]sulfate into glycosaminoglycan in two distinct regions, the cells with their cell-associated matrix (CM) and the further-removed matrix (FRM). Immunohistochemical analysis was performed using monoclonal antibodies against chondroitin sulfate and keratan sulfate. Autoradiography using degenerated cartilage tissue from the rabbit osteoarthritis (OA) model was performed to discover the effect of HA on the distribution of newly-synthesized PG in the cartilage tissue. Results: The incorporation of [35S]sulfate into newly-synthesized PG in the cells with CM decreased with the addition of 0.125-1.0 mg/ml HA, while the incorporation in the FRM increased. These effects of HA on the distribution of newly-synthesized PG were the same either in chondrocytes with CM or chondrocytes without CM. Immunohistochemical analysis showed that staining of PG in the CM was decreased and staining in the FRM was increased in the HA treated group compared to the control group. Autoradiography using degenerated cartilage tissue from the rabbit OA model indicated that [35S]-labeled macromolecules showed a more diffuse distribution in the HA treated group compared with the control group. Conclusion: These results indicate that extrinsic HA could affect the movement of newly-synthesized PG from the CM to the FRM in both alginate beads and cartilage tissue.
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