Aims

During total knee replacement (TKR), surgeons can choose whether or not to resurface the patella, with advantages and disadvantages of each approach. Recently, the National Institute for Health and Care Excellence (NICE) recommended always resurfacing the patella, rather than never doing so. NICE found insufficient evidence on selective resurfacing (surgeon’s decision based on intraoperative findings and symptoms) to make recommendations. If effective, selective resurfacing could result in optimal individualized patient care. This protocol describes a randomized controlled trial to evaluate the clinical and cost-effectiveness of primary TKR with always patellar resurfacing compared to selective patellar resurfacing.

Background

Specifically designed control interventions can account for expectation effects in clinical trials. For the interpretation of efficacy trials of physical, psychological, and self-management interventions for people living with pain, the design, conduct, and reporting of control interventions is crucial.

Introduction:

Historically the incidence of Achilles re-ruptures has been described as around 5% after surgical repair and up to 21% after conservative management. In 2008 we commenced a dedicated Achilles tendon rupture clinic for both conservative and surgically managed patients using new standardised operating procedures (SOP). We have evaluated the impact of this new service, particularly with regard to re-rupture rate.

Osteotomy through the bare area of olecranon minimises the damage to articular cartilage in the trans-olecranon approach to the distal humerus. In this study we have identified a reliable and easily reproducible anatomical land mark to make sure that the osteotomy passes through the bare area.

Two methods were used to determine the line for the osteotomy, in the first a line from the lateral epicondyle perpendicular to the olecranon and in the second an intra-articular marker was used to determine the osteotomy. In 5 cadavers the osteotomy with lateral epicondylar line as a marker went 2 mm proximal to the bare area. Of the 5 cadavers dissected with a marker passed to the angle of olecranon the osteotomy went through the bare area of olecranon in three specimens and just proximal in the other two.

In conclusion a cheveron osteotomy with the base of the chevron on the lateral epicondylar line will be the ideal site to make sure that the osteotomy passes through the bare area of the olecranon.

Introduction

The Bristol Knee Group has prospectively followed the results of over 500 isolated patellofemoral arthroplasties. Initial experience with the Lubinus prosthesis was disappointing. The main causes of failure were mal-tracking and instability leading to excessive polythene button wear and disease progression in the tibio femoral joint.

This experience resulted in the design of a new prosthesis to correct the tracking problems and improve the wear. We have now performed over 425 Avon arthroplasties with a maximum follow-up of 9 years.

Purpose

The importance of interpreting clinical trial results in terms of the benefits a treatment may offer to individuals with chronic pain is becoming more widely recognized. The clinical meaningfulness of group differences can better be described by looking at the percentages of responders in each treatment group, rather than between group mean differences. We have reassessed the outcomes of a clinical trial for chronic low back pain (LBP) from this new perspective.

Current orthopedic practice requires consideration of apparently contradictory recommendations regarding VTE prevention among THR/TKR patients. American College of Chest Physicians (ACCP) 8th Clinical Practice Guidelines for the Prevention of Venous Thromboembolism recommend against aspirin for VTE prophylaxis in any patient1. The American Academy of Orthopedic Surgeons (AAOS) Guideline recommends pulmonary embolism risk stratification, then implementation of one of many possible courses including the use of aspirin2.

We conducted a prospective observational study among consecutive patients presenting for total hip or knee arthroplasty. Pre-operative PE risk stratification was performed at the discretion of the surgeon. Patients identified as usual risk for PE received aspirin. Patients considered being at elevated risk for PE received warfarin. This observational study protocol called for one year of data collection. At approximately 8 months 656 patients were enrolled, and the surgeon principally implementing PE risk stratification and administration of aspirin chose to stop enrolling patients due to a high incidence of pulmonary emboli. One hundred fifty five patients received thromboprophylaxis with aspirin 600 mg PR in the PACU, then 325 mg BID for one month (reduced to 81 mg daily if GI symptoms were present). The remaining 501 patients received an ACCP-based warfarin protocol managed by a pharmacist anticoagulation management service.

Our hypothesis is the null hypothesis; that an AAOS-based approach to hromboembolism prevention is not inferior to an ACCP-based approach. The a priori primary endpoints of the AVP Study are clinically overt VTE, DVT, PE, major bleeding, and death. All patients will receive a 90 day follow-up questionnaire in person or by telephone. Additionally, the electronic medical record of Intermountain Healthcare will be interrogated for ICD-9 codes germane to the outcomes of interest.

Ninety day follow-up has been completed for approximately 140 patients. The dataset will be locked upon completion of the 90 day follow-up among those patients who last received PE risk stratification and aspirin therapy (data lock early June, 2009). We anticipate preliminary data available for report by July, 2009.

Classification of osteoarthritis of the hip is fraught with difficulty Although different patterns of disease are recognised, there is no accepted classification or grading system. We aim to develop a classification system that reflects both the radiological changes, and the local disease process within the joint.

After ethical permission and consent tissue was taken from 20 patients undergoing primary hip replacement surgery. Intra-operative tissue samples were taken from each patient and the steady state gene expression of several cytokines (TNF-α, IL1-β, IFN-γ, IL-6, RANKL and OPG) measured quantitatively using Taqman RT-PCR. Relative expression was calculated for each sample using standard curves and normalised to 18S expression. The technique was consistent with high correlations for repeated measures from the same tissue type (κ=0.99) and from different tissue types in the same joint (κ=0.92). Intra-observer (κ=0.93) and inter-observer (κ=0.89) reliability for the technique were also found to be high.

Preoperative radiographs were scored by two independent observers and joint space narrowing, cysts, osteophytes and sclerosis noted in each of the DeLee-Charnley zones on the femoral and acetabular side. Based on these scores patients were then classified to either lytic or sclerotic type and subclassified into either hypertrophic or atrophic.

Subgroup analysis of cytokine expression by radiographic type was performed. There were statically significant differences in expression of macrophage stimulating cytokines (IL-1γ and OPG) in the lytic group as compared to the sclerotic group (p< 0.05). Conversely, the sclerotic group expressed significantly higher levels of IL-6. Individuals with atrophic subtype demonstrated significantly higher levels of IL-1β and IL-6, but lower levels of IFN-γ

Our results demonstrate greatly differing patterns of disease within osteoarthritic hip joints. These changes are reflected in radiographic appearances of osteoarthritis. Our proposed classification system can be used grade and classify osteoarthritis in a manner that reflects the disease process.

We undertook a prospective randomised trial to determine the outcome of locked intramedullary fixation vs. plating of displaced shortened mid-shaft clavicle fractures. The primary outcome measure was the Constant shoulder score, while secondary outcome measures included the Oxford shoulder score, union rate, and complication rates.

Thirty-two patients were recruited to the trial; 17 randomised to locked intramedullary fixation and 15 randomised to plating. Mean age was 29.3years (13 to 53 years). Mean follow-up was 12.4 months (5 to 28 months). There was no significant difference in Constant scores (p = 0.365) and no significant difference in Oxford scores (p = 0.686). There was 100% union in both groups. In the intramedullary group, there was one case of soft tissue irritation that settled after the pin was removed, one pin backed out and had to be revised with another pin. There were three superficial wound infections resulting in plate removal and 8 plates (53%) were removed.

Locked intramedullary fixation and plating are equally effective in the management of shortened displaced mid-shaft clavicle fractures.

Introduction- Proximal humeral fractures remain a challenging problem. Most authors agree that anatomical reduction and stable fixation are essential to allow early range of motion. A variety of techniques have been described such as threaded pins, tension band wiring, screws, nails, plates and primary prosthesis. Locking plates score over other implants by the virtue of providing greater angular stability and better biomechanical properties. The Aim of the Study is to evaluate the functional outcome of PHILOS plate Osteosynthesis of displaced proximal humeral fractures.

Materials and Methods- A retrospective study of 50 patients treated with PHILOS plating for the 2 part, 3part and 4 part proximal humeral fractures with a minimum follow up of 1 year. All the patients were assessed in clinic by Constant Murley and ASES scoring systems. X-ray evaluation was done for fracture healing, AVN, mal-union, non-union, collapse of head, screw penetration and impingement of plate.

Results- Total of 50 acute displaced fractures of proximal humerus treated with PHILOS plating between 2003–2005 were assessed. Mean age was 64 years (15–86) Male to female ratio was 12:38, dominant to non-dominant ratio was 32:18. According to Neer’s classification 16 fractures were 2 part, 24 fractures were 3 part and 10fractures were 4 part. The overall mean Constant score was 73.4(range20–100) and ASES score was 71.7(range 25–98). Under 60 years of age the mean Constant and ASES scores were 83.5 and 83, over 60 years of age scores were 63.1 and 60.4 respectively. The complications include two deep infections which needed excision arthroplasty, one malunion, one subacromial impingement which needed plate removal after fracture healing. No mechanical failure, no non-union, no ANV was noted.

Conclusions- PHILOS plate Osteosynthesis is a reliable method of treating complex proximal humeral fractures. It provides good mechanical stability and allows rapid mobilization with out compromising fracture healing.

Since 1996 over 400 isolated patellofemoral replacements using the Avon prosthesis have been undertaken in Bristol. As a result of the usually gratifying outcomes seen in patients over 55 years old, the indications for this surgery were soon extended to include the many younger patients who have severely disabling patellofemoral disease. We present results from a prospective cohort study of patients under 55 years of age at the time of surgery.

110 knees in 86 patients (median age 47 years, range 24–54) have been treated with Avon patellofemoral replacement (88 in females and 22 in males). Diagnoses included lateral facet OA (59 knees), patella dislocation (25 knees), trochlear dysplasia (41 knees) and post patellectomy instability (7 knees). 79 knees had undergone previous surgery. 17 knees required additional intra-operative procedures including 11 lateral releases and 2 patella realignments. All patients were assessed pre-operatively and at regular intervals using the Oxford, Bristol and WOMAC scores.

All knees were scored preoperative and only one knee has been lost to follow-up due to the patient’s death, which was unrelated to surgery. Post-operative Oxford knee scores have been obtained for 106 knees with follow-up between 8 months and 8 years (mean follow-up 24 months). The mean Oxford score improved from 18 preoperatively to 31 at latest review. Bristol and WOMAC scores showed similar improvements. 16 knees required post-operative additional procedures including 6 lateral releases, 3 patella realignments and 11 revisions. Of the revisions 5 were for progression of tibiofemoral OA but none of these were knees with trochlear dysplasia. Equally good mean scores were seen when comparing patients with the 3 main underlying pathologies (trochlear dysplasia, patellar dislocation and lateral facet OA). At least 37% of the patients studied had pre-existing trochlear dysplasia and the majority of these patients report onset of symptoms, often patellar dislocation, in the first three decades of life. More than 90% of patients were overweight or obese according to their BMI at the time of surgery.

Many of this type of patient, with disabling symptoms, wish to “live now”. The short-term improvements are frequently dramatic. As yet there is no suggestion of prosthetic failure. Revision presents little difficulty since minimal bone is resected in the primary proceedure. Radiological deterioration of the tibio femoral joint is seen in some cases of primary OA but not with trochlear dysplasia.

Objective: To review the results of the Avon patellofemoral arthroplasty at 5 to 8 years.

Methods: The Avon patellofemoral arthroplasty was introduced in clinical practice in September 1996. We present a prospective cohort review of all patients treated in the first three years. Patients were evaluated using the Bristol knee score (BSK), the Melbourne patella score (MPS) and the Oxford knee score (OKS).

Results: 109 patellofemoral arthroplasties were performed in 92 patients between September 1996 and November 1999. The median age was 68 years (range 46 to 86 years). Nine patients (12 knees) died and two patients (two knees) were unfit for follow-up. Ten knees in 9 patients were lost to follow-up giving a follow-up rate of 89%. The mean period of follow-up was 5.6 years.

The median pain score rose from 15/40 pre-operatively to 40 points at eight years. The median (MPS) rose from 10/30 points pre-operatively to 25 points at eight years. The median (OKS) rose from 18/48 pre-operatively to 38 at eight years. 87% of knees had mild or no pain at eight years. There were no cases of failure of the prosthesis itself. All 15 revisions resulted from progression of arthrititis in the tibio-femoral joint. The five-year survival rate for all causes with 86 cases at risk was 96%.

Conclusions: The results show that this type of arthroplasty can give predictable pain relief and excellent functional improvement in patients with isolated patello-femoral arthritis. Disease progression is the main reason for revision to total knee replacement and great care is required in assessing the indications for this procedure.

Purpose of Study: Fixed bearing unicompartmental knee replacement (UKR) has become popular since several series have shown good 10 year survivorship and excellent function. However little is known about survival during the second decade.

Method: From the Bristol database of over 4000 knee replacements 203 St. George Sled UKR’s which had already survived 10 years were identified. The mean age at surgery was 67 years (48–85), with 64% being female. This cohort has been further reviewed at an average of 14.8 years (10–30) from surgery to determine survivor-ship and function.

Results: Survivorship during the second decade was 87.5%. 58 patients (69 knees) had deceased with implant in situ, only 2 after revision. A further 15 UKR’s have been revised at an average of 13 years post op; 7 for progression of disease, 4 for tibial loosening, 3 for polyethylene wear, 2 for femoral component fracture and 2 for infection.

99 knees were followed for 15 years and 21 knees for 20 years. The average Bristol knee score of the surviving knees fell from 86 to 79 during the second decade.

A previous study showed an 89% 10 year survivor-ship and this is now extended to 82% at 15 years and 76.5% at 20 years.

Conclusion: Satisfactory survival of fixed bearing UKR can be achieved in the second decade suggesting that the indications for mobile bearings require careful definition since there is a higher incidence of complications in many people’s hands.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different soft and hard musculosk-eletal tissues. In the intervertebral disc (IvD) the major SLRPs involved in regulation of types I & II collagen fibril size are believed to be decorin, fibromodulin and lumican. Research into IvD degeneration and backpain is hampered by a lack of specific biomarkers to detect and monitor the disease process. We have discovered that two keratan sulphate (KS) substituted members of the SLRP family, Keratocan and Lumican (that are major KS-pro-teoglycans found in cornea) were unusually expressed in extracts from degenerative disc tissues.

Methods: Non-degenerate disc tissue (n=10) was obtained from 2 scoliosis patients and degenerate disc tissue from 11 patients undergoing surgery. The degenerate discs were graded using criteria described by Pfir-rman et al (Spine26: 1873; 2001). Tissue samples were extracted with 4M guanidine HCl and after dialysis subjected to SDS-PAGE and Western blot analyses using monoclonal antibodies that recognise epitopes on kera-tocan and lumican.

Results & Discussion: Keratocan was not found in the non-degenerate disc tissue but was present in all degenerate IvD tissues tested. Lumican showed and increased expression in extracts of degenative IvD tissues. Our working hypothesis is that the increased expression of these two SLRPs in degenerative disc tissue results from a reparative depostion of a type I collagen fibrillar ‘scar’. This unusual expression suggests their potential as biomarkers for detecting the onset of degenrative disc disease.

We report the mid-term results of a new patellofemoral arthroplasty for established isolated patellofemoral arthritis. We have reviewed the experience of 109 consecutive patellofemoral resurfacing arthroplasties in 85 patients who were followed up for at least five years.

The five-year survival rate, with revision as the endpoint, was 95.8% (95% confidence interval 91.8% to 99.8%). There were no cases of loosening of the prosthesis. At five years the median Bristol pain score improved from 15 of 40 points (interquartile range 5 to 20) pre-operatively, to 35 (interquartile range 20 to 40), the median Melbourne score from 10 of 30 points (interquartile range 6 to 15) to 25 (interquartile range 20 to 29), and the median Oxford score from 18 of 48 points (interquartile range 13 to 24) to 39 (interquartile range 24 to 45). Successful results, judged on a Bristol pain score of at least 20 at five years, occurred in 80% (66) of knees. The main complication was radiological progression of arthritis, which occurred in 25 patients (28%) and emphasises the importance of the careful selection of patients. These results give increased confidence in the use of patellofemoral arthroplasty.

Introduction: Kashin-Beck disease (KBD) is a special endemic osteoarthropathy whose main pathologic changes occur in growth plate cartilage and articular cartilage of human limbs and joints where it is manifested as cartilage degeneration and necrosis. Past and current research suggests that KBD, and its endemic geographic distribution in China, is due to the combined presence of fungal mycotoxins (on stored food ingested by affected populations) and a regional selenium deficiency in the environment providing local food sources. Thus, we hypothesise that the presence of fungal mycotoxins and the absence of selenium in the diet specifically affects chondrocyte metabolism in the growth plate during limb and joint development and in articular cartilage of adults, which leads to localised tissue necrosis, and the onset of degenerative joint disease. The aim of this study was to examine the effects of mycotoxins; e.g. Nivalenol (NIV), selenium and NIV in the presence of selen! ium in in vitro chondrocyte culture systems to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD.

Methods: Chondrocyte tissue cultures were established using cartilage explant cultures either in the presence or absence of selenium (0.5–1.5 microg/ml) and the mycotoxin nivalenol (0.5–1.5 microg/ml) and culture for 1 to 4 days. Medium was harvested daily at day 1 through 4 and analysed for glycosaminoglycan (GAG) release and the presence of aggrecanase or MMP activity using RT-PCR for gene expression and monoclonal antibodies that detect their respective enzyme-generated neo-epitopes on cartilage aggrecan metabolites.

Results: Our studies to date have shown that NIV exposure induces catabolic changes in chondrocyte metabolism with an increased expression of aggrecanase activity. Addition of selenium did not affect mRNA expression of the aggrecanases ADAMTS-4 & 5. Parallel studies involving immunohistochemical analyses of articular cartilage from KBD showed an increase in aggrecanase activity.

Conclusions: These studies demonstrate that induction of aggrecanase activity as one of the molecular mechanisms involved is the pathogenesis of KBD. However, the addition of selenium does not alter aggrecanase gene expression indicating that its beneficial effects are occurring in other areas of cartilage metabolism.

Degenerative joint disease (DJD) involves the proteolysis of many extracellular matrix molecules (ECM) present in articular cartilage and other joint tissues such as tendon, meniscus and ligaments. Recent research has identified key enzymes involved in the catabolism of ECM. Two classes of enzyme the Matrix Metalloproteinases (MMP’s) MMP-2, MMP-3, MMP-13 and the ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) of proteinases most notably, ADAMTS-1, -4 and −5, have been shown to be involved in the catabolism of ECM (such as type II collagen and cartilage aggrecan). The presence of several MMPs in the synovial fluid has been reported; however, little data has yet been gathered on the presence of ADAMTS-1, -4 or −5 (the aggrecanases) in synovial fluids. In this study we have used a recombinant artificial substrate and specific neoepitope antibodies that recognise either MMP- generated or aggrecanase -generated degradation products to measure the relative activity of these two enzyme families in the synovial fluid from human patients.

Methods: A recombinant substrate containing the interglobular domain of cartilage aggrecan , flanked by a complement regulator and the Fc region of IgG has been stably transfected into CHO cells. The recombinant protein has been purified from the medium using a Protein A column followed by gel chromatography using a Superose 12 column. Synovial fluid samples were depleted of serum immunoglobulin by pre-absorption with ProSepA. The recombinant substrate was then added to synovial fluid samples and incubated overnight as 37?C. The recombinant substrate was recovered from samples using ProsepA and then separated by SDS-PAGE (10% gels). Gels were transferred to nitrocellulose membranes and immunoblotted with antibodies recognising the undigested substrate and using neoeptiope antibodies specifically recognising MMP or aggrecanase –generated catabolites.

Results: Preliminary analysis by Western blot using the anti IGD neoepitopes BC-14 (detecting cleavage at the major MMP site) and BC-3 (detecting cleavage at the aggrecanase site) demonstrated that enzymes in human synovial fluid collected from patients diagnosed with rheumatoid arthritis cleaved the pro-drug at the MMP site with little or no evidence of aggrecanase catabolism. In contrast, synovial fluid collected from patients diagnosed with osteoarthritis indicted that there was cleavage at the aggrecanase site. In these preliminary studies we have also examined the enzyme activity in a set of clinical samples collected from patients that have undergone knee replacement surgery having been given either n-3 fatty acids or a placebo 10 weeks prior to surgery. Results indicate that aggrecanase generated fragments were found in synovial fluid from placebo patients, and reduced levels of enzyme activity were apparent in fluids tested from patients that had received n-3 fatty acids prior to surgery.

Discussion: This data suggests that the recombinant substrate will aid in the detection of MMP or aggrecanase activities in synovial fluid samples. The ratio of MMP to aggrecanase activity has potential as a biomarker for the severity of cartilage degeneration in degenerative joint diseases.

Introduction: Several small leucine-rich proteoglycans (SLRPs) are involved in the regulation of collagen fibril size(s) in a variety of different musculoskeletal tissues. In hyaline articular cartilage the major SLRPs involved in regulation of type II collagen fibrils are believed to be decorin and fibromodulin. These two SLRPs along with another family member, lumican, have also been identified in intervertebral disc tissues. In recent studies, we serendipitously discovered that, keratocan and lumican [two keratan sulphate (KS) substituted members of the SLRP family] were unusually expressed in extracts from degenerative joint and degenerative disc tissues. The object of this study has been to further investigate this finding with a view to examining the increased expression of keratocan and lumican using qualitative Western blot analysis and quantitative ELISA methods. Our working hypothesis is that the increased expression of these two SLRPs in degenerative joint and disc tissue results from a reparative deposition of a type I collagen fibrillar ¡®scar¡^-.

Methods: Monoclonal antibodies were produced to core protein epitopes in lumican and keratocan. Degenerate cartilage was obtained from patients undergoing routine joint replacement for either hip or knee joints, whilst normal articular cartilage tissue was obtained from surgical knee procedures. In addition, disc samples were obtained from patients undergoing a variety of spinal procedures and were Graded I-IV using a modified Thompson score. The tissue was diced and extracted in a 4M guanidine HCl buffer, pH6.8 containing an inhibitor cocktail for 48 h at 4¢ªC. Samples were then dialysed exhaustively against Milli Q water and assayed for glycosaminoglycan (GAG) content using the DMMB assay. Cartilage extracts containing equal amounts of GAG were then separated by SDS-PAGE and transferred to nitrocellulose for Western blotting using mMAbs to either keratocan or lumican. In addition, a competitive ELISA has been developed for quantifying keratocan and lumican.

Results: Western blot analysis of normal and degenerative articular cartilage revealed the presence of both keratocan and lumican. However, the presence of these SLRPs was substantially increased in the degenerate articular cartilge extracts. In addition, these proteins were also present in extracts of intervertabral disc with an increase being apparent in those disc samples with increased pathology. Preliminary data for the development of a quantitative ELISA for these two SLRPs shows promise.

Discussion: The unexpected increase in the detection of keratocan and lumican in degenerative articular cartilage and disc suggests their potential as biomarkers for the onset of degenerative joint and disc disease. However, this will involve the development of a quantitative assay and the investigation of the presence of these molecules in synovial fluid and serum.

Introduction: Kashin-Beck disease (KBD) is an endemic osteoarthropathy with pathological changes occurring in growth plate and articular cartilage in humans. It manifests as cartilage degeneration and necrosis. It is postulated that KBD is due to fungal mycotoxins infiltrating the diet and a regional selenium deficiency in the environment providing food sources in a broad belt across China. Previous work has established an in vitro system in which chondrocytes are cultured and an ex vivo cartilage graft is produced. Subjecting these chondrocytes to either selenium (SEL), Nivalenol (NIV) or in combination during the growth of the graft was found to alter the morphology of the cartilage graft. In addition, the quantity of the large aggregating proteoglycan, was significantly reduced in a dose dependent manner in the presence of Nivalenol. This study aimed to examine the composition of aggrecan from grafts grown in the presence of NIV or SEL alone, or in combination to better understand cellular and molecular mechanisms underlying the pathogenesis of KBD.

Methods: Chondrocytes (from 7 day old bovine cartilage) were seeded at high density in MilliCell filter inserts (12mm diameter; Millipore, MA). Cultures were maintained for 4 weeks in DMEM supplemented with 20% heat–inactivated FBS, ascorbate (100μg/ml) and TGFß2 (5ng/ml) or additionally supplemented with either SEL , NIV or both at concentrations of 0.01, 0.05 and 0.1μg/ml. Media was refreshed thrice weekly and later analysed. At 4 weeks the cartilage grafts were harvested, weighed and extracted in 4M guanidium chloride (with an inhibitor cocktail) for biochemical analysis of matrix molecules. Residues were papain digested. Glycosaminoglycan concentration was determined using the DMMB assay in all media samples, guanidine extracts and papain digests. Aggrecan and GAG composition was determined using Western blotting with a panel of antibodies recognising chondroitin sulphate (CS), keratan sulphate (KS) and protein core epitopes present in aggrecan.

Results: The total GAG synthesised in a 4week period was substantially reduced in chondrocytes cultured in the presence of NIV at 0.05 and 0.1μg/ml and to a lesser extent in those cultures exposed to the highest dose of SEL. However, the amount of GAG released into the media remained fairly constant within the treatment groups, but a marked reduction was apparent in the guanidine extracts of the cartilage grafts. Western blot analysis with a series of antibodies on guanidine extracted aggrecan showed no substantial changes in the core protein molecular weights however analysis demonstrated that KS was reduced in NIV treated cultures. Results also indicated that NIV treated cultures appeared to contain less CS substitutions on the aggrecan core protein.

Discussion: The GAG concentration data indicates that there is an inability of the GAG to remain within the cartilage grafts extracellular matrix. when treated with NIV. Western blot analysis indicates minor changes in the composition of the aggrecan in relation to protein core length and CS/KS side chain substitutions or length. Further work will investigate the proportion of aggrecan able to form high molecular weight aggregates, the metabolism of link protein and hyaluronan.

Introduction: Osteoarhthritis is a degenerative disease affecting a large proportion of the population. Recently, there has been renewed interest in the use of neutraceuticals (such as glucosamine) for the treatment of symptomatic pain and pathology in arthritic joints. However, little research has been carried out to assess the biochemical mechanisms by which glucosamine imparts its effects on the disease process. Biochemically, an early change in the cartilage metabolism is a loss of the large aggregating proteoglycan, aggrecan. Functionally, this loss results in a decreased capacity for the tissue to sustain mechanical loading that leads to cartilage destruction and a painful joint. The enzymes responsible for the loss of aggrecan from the tissue are commonly referred to as the aggrecanases and are members of the ADAMTS family of enzymes. Degradation of aggrecan by the aggrecanases can be detected using a specific neoepitope monoclonal antibody BC-3 (1). Model systems using cartilage explant cultures that mimic the degradative processes seen in osteoarthritis have been developed in which cytokine such as IL-1 are used to initiate the catabolic processes leading to cartilage degradation.

Methods: Cartilage explant cultures (bovine) were established using published methodologies (1). Explants were then incubated in either DMEM, DMEM supplemented with a chemically modified glucosamine (0.5–15mM) or DMEM supplemented with glucosamine hydrochloride (0.5–15mM) for 1 hour. IL-1 (10ng/ml) was then added to half of the explant cultures in each experimental group. Cultures were maintained for 4 days in the experimental media after which media and explants were harvested for analysis. Glycosaminoglycan (GAG) concentrations of media samples and cartilage extracts were determined using the DMMB assay. RNA was extracted from cartilage explants and RT-PCR was performed using primers to cartilage matrix molecules, ADAMTS and MMPs. Western blot analysis was performed on the experimental media using MAb BC-3 to determine the presence of aggrecanase-generated aggrecan catabolites.

Results: Experiments show that glucosamine hydrochloride (0.5–15mM) was unable to inhibit the release of GAG from explant cultures induced by treatment with IL-1. However, explant cultures preincubated with 10–15mM chemically-modified glucosamine were able to inhibit the release of GAG induced by IL-1 to that of control culture levels. The decreased release of GAG corresponded to a decrease in the detection of aggrecanase-generated aggrecan catabolites as assessed by Western blotting with MAb BC-3.

Discussion: This data questions the effectiveness of glucosamine hydrochloride in the inhibition of biochemical mechanisms involved in the IL-1 induced degradation of aggrecan in articular cartilage. However, the data suggests a role for a chemically modified glucosamine in the IL-1 induced degradative pathways involved in the loss of aggrecan from cartilage. The use of glucosamine in the treatment of arthritic diseases is controversial, however, the modified form of glucosamine used in this study helps to support the potential use of the dietary ingestion of glucosamine and its beneficial effects in arthritis patients. 1. Hughes, C.E., et al. (1995). Biochem. Journal. 305, 799–80