The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.

Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.

There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

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.

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.

Deep infection is a devastating complication of total joint arthroplasty. In a significant proportion of cases it remains a diagnostic challenge. Haematological tests are not specific, particularly in chronic cases, and radiological investigations such as bone scan and radiographs are of only limited value. The most common infective organisms are staphylococcus and some streptococcus species. Acidity is a well established occurrence in infective processes and is caused by the direct production of acid by the organism or by enzymatic degradation of tissues ^1,2. In wound infections, peritonitis and some other conditions pH is used as an indicator of infection in clinical practice³. The aim was to assess whether fluid biochemistry (pH, pCO2, pO2, Lactate and Glucose) is altered in infected total knee replacements and whether it could be used as a diagnostic test.

Nineteen consecutive patients undergoing either revision total knee replacement (TKR) or arthroscopic synovial biopsy were included in the study. All had had their primary joint replacement within the previous 3 years. All had a painful total knee replacement and some had evidence of loosening of the prosthesis on radiological investigations. The following investigations were performed on each patient, White cell count (WCC), Erythrocyte Sedimentation Rate (ESR), C-Reactive Protein (CRP), interface synovial biopsy for histology and microbiology and a synovial fluid aspiration from the affected joint prior to application of a tourniquet. A blood gas analyser was used to measure pH, pCO2, pO2, Lactate and Glucose in all synovial fluid specimens.

Seven patients were diagnosed as having an infected TKR on clinical and laboratory investigations. The mean synovial fluid biochemistry results were pH = 7.09, pO2= 5.08kPa, pCO2=10.40kPa, Lactate = 5.33 mmol/l, Glucose = 2.30 mmol/l. In the non-infected group the results were pH = 7.23, pO2 = 7.72kPa, pCO2 = 8.41kPa, Lactate = 4.03 mmol/l, Glucose = 3.42 mmol/l. The differences in pH, pCO2/pO2 ratio, and glucose levels were statistically significant (t-Test p < 0.05) between the two groups. Lactate levels were not significantly different. There was no correlation between high WCC’s and synovial fluid biochemistry or laboratory results for infected cases. Using laboratory results as a gold standard and a synovial fluid pH of less than 7.20, the sensitivity was 85% and specificity 77% for diagnosing an infection. Using a combination of the synovial fluid biochemistry results these values were greater.

Synovial fluid biochemistry is significantly altered in infected total knee replacements. pH levels below 7.2, pCO2/pO2 ratio above 2.5 and Glucose levels below 2.5 mmol/l are strong indicators of an infected TKR. Synovial pH assessment may prove to be a quick, cheap and effective method of diagnosing an infected TKR and may also apply to other joints. Further studies using non-problematic TKR’s as controls are required.