Objective. To investigate the effects of hyaluronic acid (HA) on the release of proteoglycan by cultured rabbit chondrocytes.Methods. Articular cartilage chondrocytes were isolated from the knee joints of New Zealand white rabbits. Proteoglycan synthesis after incubation with HA was determined by measuring "S-sulfate incorporation. Cells incubated with HA were labeled with 3H-glucosamine and applied to a Sepharose CL-2B column. After incubation of confluent cells with "Ssulfate and then with HA in various concentrations in the presence or absence of cytokines, proteoglycan release from the cell matrix layer was measured.Results. HA ( M , 3 x lo5 to 19 x lo5), at 10 pg/ml ~ From the Department of Biochemistry, School of Dentistry, Hiroshima University, Hiroshima-city. the Departments of Biochemistry and Radiology, Faculty of Dentistry, Osaka University. Osaka, the Department of Orthopaedic Surgery, National Defense Medical College, Tokorozawa, and the Department of Pathology, Medical School, K a n a i w a University. Kanazawa, Japan.Atsushi Address reprint requests to Yukio Kato, PhD, DDS, Department of Biochemistry, School of Dentistry, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima-city. 734, Japan.Submitted for publication June 9, 1992; accepted in revised form October I , 1992.to 1 mg/ml, had little effect on the incorporation of 35S-sulfate or 3H-glucosamine into cartilage matrix proteoglycans, or on the hydrodynamic size of proteoglycan monomers, in rabbit chondrocyte cultures. However, at 1&1,000 pg/ml, HA suppressed the release of "Sproteoglycans from the cell matrix layer into the medium in the presence and absence of interleukin-1, tumor necrosis factor a, or basic fibroblast growth factor.Conclusion. These results suggest that HA is a potent inhibitor of the displacement of matrix proteoglycan into culture medium.The synthesis of proteoglycans by chondrocytes and the incorporation of these newly synthesized proteoglycans into the matrix are regulated by macromolecules in the extracellular matrix. Hyaluronic acid (HA) has been shown to cause a >50% reduction in the incorporation of "S-sulfate into proteoglycans synthesized by chick embryo chondrocytes ( l-3), adult pig chondrocytes (4), and Swarm rat chondrosarcoma cells (3, although the various studies have shown differences in the effective dose ( I ng/ml to I mg/ml). HA has also been found to inhibit chondrogenesis in vitro (6). Furthermore, HA or HA oligomers have been shown to stimulate the displacement of newly synthesized proteoglycans into the medium in chondrocyte cultures (3).These studies emphasized the importance of the inhibitory effect of HA on the accumulation of a proteoglycan matrix by developing chondrocytes. N o stimulatory effects of H A on chondrocytes or cartilage matrix have been demonstrated in chondrocyte cultures, even though cartilage contains 0.5-2.5 mg of HA