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.

The use of intra-articular corticosteroid injections for their anti-inflammatory effects is widespread amongst clinicians. Despite their use in both rheumatoid arthritis and osteoarthritis, the effect of these agents on articular chondrocytes is not fully established. Previous reports suggest a detrimental effect on cartilage explants resulting from inhibition of matrix synthesis¹. However it has also been suggested that the beneficial effects in vivo may be due to prevention of inflamed synovium causing cartilage degradation². Our aim was to assess the effect of a commercially available preparation of methylprednisolone (MP), at clinical doses, on articular 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 15 days in standard culture medium (DMEM + 20% Fetal calf serum) containing varying concentrations of MP, 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 and glycosaminoglycans. Further constructs were fixed in 4% paraformaldehyde for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

Chondrocytes cultured in MP containing medium showed a significant abnormality in cell morphology compared to controls at the day 15 time point. Histologically there was evidence of cell necrosis, reduced amounts of extracellular matrix and loss of collagen type II staining. The effects were dose dependant, with significant damage occurring even at clinical doses. Biochemical analysis revealed a reduction in DNA content and an inhibition of glycosaminoglycan and collagen type II synthesis. In contrast, in the controls, there was cell proliferation with a cell doubling time of 14 days, collagen type II containing extracellular matrix synthesis occurred and the chondrocytes maintained their phenotype throughout the culture period.

Methylprednisolone has a significant detrimental effect on cultured articular chondrocytes in vitro. There was significant cell necrosis associated with inhibition of extracellular matrix synthesis. Based on these results, intra-articular corticosteroid injections should be used with extreme caution.

Nicotine is a constituent of tobacco smoke and is present in the body fluids of smokers^1,2. Numerous studies have confirmed that smoking is a strong risk factor for back pain³. The most widely accepted explanation for the association is that smoking leads to malnutrition of spinal discs due to carboxyhaemoglobin formation. However, other constituents of smoke, such as nicotine, may also be responsible for intervertebral disc (IVD) degeneration by leading to cell necrosis in both the nucleus pulposus and annulus fibrosis. Despite evidence suggesting the detrimental effect on a variety of tissues, the effect of nicotine on IVD cells has not previously been investigated. This study investigated the influence of nicotine on the metabolism and viability of IVD cells cultured in vitro.

Bovine nucleus pulposus (NP) intervertebral disc cells were isolated by sequential digestion of caudal spinal disc nuclei with pronase and collagenase and seeded in 2% alginate at 5x10⁶ cells/ml. The constructs were cultured for 21 days in standard culture medium (DMEM + 20% Fetal calf serum) containing free base nicotine (Sigma) at concentrations ranging from 25nM and 300nM, which reflected the normal physiological concentrations found in the serum of smokers. The medium was replaced every 3 days and representative constructs were removed from culture, digested and assayed for DNA, glycosaminoglycan (GAG) and hydroxyproline content at time points 3, 7, 14 and 21 days. Further constructs were processed for standard histology and immunolocalisation of collagen types I, II and chondroitin-6-sulphate.

The results were analysed statistically using an ANOVA test followed by a non-parametric Dunnit’s test. NP cells demonstrated a dose dependent response. At 25nM dose of nicotine there was a significant increase (p< 0.05) in DNA content, GAG and collagen synthesis in the constructs. At 100nM, 200nM and 300nM doses, there was a significant dose dependent decrease (p< 0.05) in all of these parameters compared to controls cultured under nicotine free conditions. In addition, adverse morphological changes were observed on histology, which included reduced cell proliferation, disrupted cell architecture, disintegration of cells and extracellular matrix. Immunohistochemistry showed the production of type I collagen rather than type II collagen as in the controls.

Nicotine has an overall detrimental effect on cultured nucleus pulposus disc cells in vitro. There was significant inhibition of cell proliferation and extracellular matrix synthesis. Nicotine in tobacco smoke may therefore play a role in the aetiology of disc degeneration that leads to back pain in smokers.