THE EFFECTS OF HYALURONIC ACID ON ARTICULAR CHONDROCYTES

Abstract

The use of intra-articular hyaluronic acid injections for the treatment of early osteoarthritis is in widespread clinical use. Hyaluronate (HA) is a major component of connective tissue¹ and is available commercially for the intra-articular injective treatment of osteoarthritis of the knee and periarthritis of the shoulder. Although it is known to improve intra-articular lubrication it is also thought to promote articular cartilage structure and prevent catabolism of matrix proteoglycans in osteoarthritis. Clinical studies have shown beneficial effects lasting for many months after cessation of therapy unlike anti-inflammatory drugs that have relatively short term relieving effects^2,3 . Documentation of the true chondroprotective effects of hyaluronic acid (HA) at the cellular level is lacking and therefore this study aimed to identify the effects of HA on chondrocytes cultured in vitro.

Bovine articular chondrocytes were isolated by sequential digestion with pronase and collagenase and seeded in 2% alginate at 1x10⁷ cells/ml. The constructs were cultured for up to 14 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of HA (Sigma), including doses equivalent to those found in vivo. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycans and Collagen. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

Chondrocytes cultured in the HA system proliferated (increase in DNA) at a faster rate than the controls. There was a 2.2 fold increase in cell concentration at 14 days compared to a 1.2 fold increase in the controls. Total GAG levels at each time point were significantly greater for cells cultured in HA than in controls. Histologically, constructs were characterised by extensive cell cluster formation and intense Safranin-O staining. The newly synthesised matrix also stained positive for type II collagen. By contrast, control constructs exhibited minimal cluster formation, Safranin-O and type II collagen staining.

Cells maintained with HA exhibited a significantly greater rate of proliferation and matrix production. The presence of matrix rich in type II collagen indicates maintenance of chondrocytic phenotype. By contrast, cells cultured without HA did not show these features. These results support the use of intra-articular injections for the treatment of osteoarthritis. The benefits of HA injections may be due to cellular mechanisms as well as mechanical.