Aims

Adductor canal block (ACB) has emerged as an alternative to femoral nerve block (FNB) for analgesia after total knee arthroplasty (TKA). The optimal duration of maintenance of the ACB is still questionable. The purpose of this study was to compare the analgesic benefits and physiotherapy (PT) outcomes of single-shot ACB to two different regimens of infusion of the continuous ACB, 24-hour and 48-hour infusion.

This study aimed to verify the accuracy of the DIERS Formetric Scan when assessing vertebral rotation of the apical vertebrae in Adolescent Idiopathic Scoliosis (A.I.S) patients, to determine whether the DIERS Formetric Scans can be used instead of or alongside radiographs when assessing A.I.S patients.

Both the radiographs and the DIERS Formetric Scans of 60 Preoperative A.I.S patients. All patients included in our study had predominant thoracic curves using the Lenke classification method, Cobb angle range 33° – 85°. Each radiograph was categorised into groups according to the severity of Nash-Moe rotation score of the apical vertebrae. Three groups were formed Nash-Moe +1 (20 patients), Nash-Moe +2 (27 patients), Nash-Moe +3 (13 patients). Each result was then compared to the maximal rotation analysed by the DIERS Formetric Scan, which took place on the same day as the radiographs. The results were then assessed using a Pearson Correlation Coefficient and a One-Way ANOVA with Post-Hoc Tukey HSD Analysis.

The Nash-Moe +1 Group scored a mean maximal rotation of 14.65° ±6.56 (11.82 – 17.48) (95% Confidence Interval), Nash-Moe +2 mean maximal rotation was 19.6° ±7.1 (16.92 – 22.28) and Nash-Moe +3 scored 21.53° ±8.9 (16.99 – 26.37). The Pearson Correlation Coefficient of this assessment was +0.342 (p value 0.07) demonstrating a weak positive correlation. The One-Way ANOVA analysis with Post-Hoc Tukey HSD analysis. The results of this analysis was an F value score of +4.115 (p Value 0.021) for the overall One-Way ANOVA test. The Post-Hoc Tukey HSD tests demonstrate that there is a statistical difference between Group 1 and Group 3 (p value 0.030) but there is no statistical difference between Group 1 and Group 2 (p value 0.068) as well as no statistical difference between Group 2 and Group 3 (p value 0.716).

DIERS Formetric Scan assessment of vertebral rotation shows a positive correlation with the Nash-Moe method. This allows us to rely on the Formetric scans and thus a possible reduction in radiographs when assessing A.I.S, this reduces the exposure to ionising radiation in A.I.S patients.

Introduction

Novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) have been shown to be putative biomarkers of progenitor/stem cell sub-populations (Hayes et al., 2007; Dowthwaite et al., 2005). Also, recent studies show that unique CS sulphation motifs are localized in putative stem/progenitor cell niches at sites of incipient articular cartilage & other musculoskeletal tissues (Hayes et al., 2011), which indicates their potential importance in cell differentiation during development. In this study, we investigated the importance of CS in the differentiation of bone marrow stem cells to the chondrogenic phenotype in vitro using p-nitrophenyl xyloside (PNPX) as a competitive inhibitor of CS substitution on matrix PGs.

Introduction

Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism.

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development.

Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA.

Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA.

Introduction: In a previous study (Hayes et al., 2007)we reported that novel chondroitin sulphate (CS) sulphation motifs on cell-associated proteoglycans (PGs) may be putative biomarkers of progenitor/stem cell sub-populations resident within the superficial zone of articular cartilage (Dowthwaite et al., 2005). In this study, using the same panel of antibodies, we examine the distribution of novel CS sulphation epitopes in a more clinically relevant model – the developing human knee joint.

Methods: Twelve-14 week human foetal knee joint rudiments were processed into paraffin wax then de-waxed and immunoperoxidase-stained with mAbs 3B3(−), 7D4 and 4C3 using the Vector Universal Elite kit with Nova Red, Mayers Haematoxylin, mounted under coverslips and then photographed.

Results: All three CS sulphation motif epitopes localised prominently at sites of incipient articular cartilage formation at a stage before there was any histological evidence of secondary ossification at the epiphysis. Interestingly, these CS epitopes were also detectable in very defined regions within the perichondrium; growth plate; the fibrocartilage of both meniscus and enthesis; vasculature; and at sites of capillary invasion, with subtle differences in their distribution; for example, 3B3(−) identified the cellular lining of cartilage canals within the epiphyses, whereas 7D4 labelled more their cellular contents.

Discussion: The results of this study show that novel CS sulphation motifs on cell and matrix PGs play important and diverse roles in the development of a wide range of musculoskeletal connective tissues, including articular cartilage. We hypothesize that the unique sulphation sequences on CS-containing PGs are involved in regulating cell proliferation and differentiation events, through interaction with soluble signalling molecules (e.g. growth factors) in the extracellular milieu. These antibodies show considerable promise for uses in tissue engineering applications for identifying and sorting stem/progenitor cells for regeneration of musculoskeletal tissues.

Background: Surgery in adolescent idiopathic scoliosis is done mainly for cosmesis and outcomes are reported in terms of radiological measurements (Cobb angle), outcome questionnaires (SRS-22) and back surface measurements (Scoliometer & Quantec). Previous studies have shown correlations between SRS-22 and objective radiological and back surface measures at a point in time (Asher et al 2003 & 2004).

Aim: of the study was to evaluate the association between subjective and objective outcomes in posterior instrumented scoliosis correction.

Patients and Methods: 43 patients with late-onset thoracic idiopathic scoliosis were included in the study with 39 girls and 4 boys with a mean age 13.2 years. Mean pre-operative Cobb angle was 71o. The objective assessment of back surface was done using a scoliometer and the POTSI & Suzuki Hump Sum scores. The subjective assessment was done using the Scoliosis Research Society (SRS)-22 score. The assessments were done pre-operatively and then post-operatively at 8-weeks and one year.

Results: The average percentage improvement in various outcomes after surgery was as follows: Cobb angle (71%), Maximum Angle of Trunk Inclination (Max. ATI) (52 % at 8 weeks and 39 % at 1 year), POTSI (57%), Hump Sum (24%), SRS-Total (14%), SRS-self image (14%). Pre-operatively, there were good inter-correlations (r= 0.4–0.7) between the objective measures (Max. ATI, POTSI and Hump Sum). Significant correlation was found between SRS-22 total versus Cobb angle (p-0.001, r=0.41). No significant correlation was found between the SRS-22 (total & domains) versus the Max. ATI, POTSI or the Hump Sum scores. Post-operatively, good correlation (r=0.6) was again found between the objective measures (Max. ATI, POTSI and Hump Sum) of back surface measurements (absolute and percentage). No significant correlation was found between SRS-22 (domains & total) versus Cobb angle correction, Max. ATI, POTSI, or Hump Sum.

Conclusion: In this study SRS-22 was found to be responsive to change with surgery, especially the self-image/appearance domain. However the SRS-22 score after surgery and the change in this score did not correlate with the change in objective measures of back surface deformity after surgery.

In the mid-1980s we produced and characterised several monoclonal antibodies ‘mAbs 3-B-3(−); 4-C-3, 6-C-3 & 7-D-4) that recognised unique native sulphation motifs in chondroitin sulphate (CS) glycosaminoglycan (GAG) chains on connective tissue proteoglycans (PGs).

These antibodies were shown to specifically locate CS-PGs in the pericellular regions surrounding putative sites where haemopoietic stem cells were undergoing lymphopoiesis in the Bursa of Fabricius of embryonic chicks. In later studies, we also observed immunostaining for some of these mAbs ‘3-B-3(-) & 7-D-4’ in chondrocyte clusters present in tissue sections from late-stage osteoarthritic cartilage from canine and human patients. In a recent study ‘Hayes et al (2008), J. Histochem Cytochem. 56: 125–128’ we have used these anti-CS sulphation motif mAbs to specifically identify stem/chondroprogenitor cells in the surface/superficial zone of hyaline articular cartilage. Furthermore, we used these mAbs in FACS analyses to sort and isolate chondroprogenitor cells for potential pluripotent cell enrichment in tissue engineering/tissue regeneration technologies. We have also used several of these mAbs to identify stem/progenitor cells in different anatomical and functional regions of the tendon; i.e. where the tendon wraps around bone in compressed regions where the cells exhibit a more chondrogenic phenotype and also in the outer zones of the tendon surrounding pericytes where vascularisation occurs. In the developing intervertebral some of these mAbs specifically recognise stem/progenitor cells at the interzone between the outer and inner anulus an also the boundary of the nucleus with the inner annulus, these results indicating their use for stem/progenitor cell identification and isolation in other musculoskeletal tissues. Interestingly, these mAbs also immunostained the pericellular environment (stem cell niche) in the crypts of the gut and the limbus of the eye where stem cells reside. Collectively, this data strongly suggests that these mAbs recognising CS sulphation motifs can be used as biomarkers to identify stem cell niches in numerous tissues of the body and that they can be used for stem/progenitor cell isolation for use in tissue engineering/regeneration procedures.

This work was supported by BBSRC and ARC funding.

We identified eight patients of 2900 with a primary malignant bone tumour who had coexisting neurofibromatosis type 1. This was a much higher incidence than would be expected by chance. The patients had a mean age of 22.4 years (9 to 54): five were male. Two patients subsequently developed a second bone sarcoma, one of which was radiation induced. Four of the primary tumours were osteosarcomas, four were spindle-cell sarcomas and one a Ewing’s sarcoma. All the patients were treated with chemotherapy and surgery: six of the eight appear to be cured.

This study suggests a possible relationship between neurofibromatosis type 1 and the development of a bone sarcoma, the increased risk being estimated at eight times that of the normal population. We recommend that further research into this possible link should be considered.

Introduction: Monoclonal antibodies (mAbs) recognizing linear sulphation motifs in keratan sulphate (KS) were first developed in the early 1980’s. Over the years, ELISAs using 5-D-4 or other related anti-KS mAbs have been used in many studies monitoring increased cartilage aggrecan degradation with the onset of degenerative joint diseases. However, whilst these studies have in general been useful for monitoring some aspects of disease progression (usually in parallel with other biomarker assays), many longitudinal studies have shown efficacy in only the transient (early, mid or late) stages of the degenerative joint disease process. During the onset of degenerative joint disease, the pathological tissue attempts to repair/regenerate the cartilage, the chondrocytes thus synthesizing cartilage aggrecan with KS substitution [and chondroitin sulphate (CS) isomer composition] that is more like that found in developing or immature cartilage. This immature cartilage aggrecan contains much less KS substitution with shorter chain size and less linear sulphation motifs. Thus, during the different stages of degenerative joint disease progression one would expect to find variable changes in different linear sulphation epitopes present in the serum or synovial fluids. The aim of this study was to investigate the use of several monoclonal antibodies that recognise different sulphation epitopes [high sulphation (5-D-4), low sulphation (1-B-4) and KS-stubs (BKS-1)] to see if patterns of their expression could be used to distinguish different stages of degenerative joint disease. We have also developed ELISAs using mAbs recognising the KS-proteoglycans, keratocan (Ker 1) and lumican (Lum 1) for their quantification as potential biomarkers of osteoarthritis.

Methods: Competitive ELISAs were developed using monoclonal antibodies (mAbs) 5-D-4, 1B4, BKS-1, Ker-1 and Lum-1. Bovine corneal KS-proteoglycans pre-treated with keratanase were used as both the coating antigen and “standard” antigen on the same ELISA plate. Blood, synovial fluid and cartilage samples (surgical waste) obtained from patients undergoing arthroplasty with different Kellgren & Lawrence grades were analysed.

Results and Discussion: 5-D-4 and BKS-1 showed similar inhibition curves and relative 50% inhibition points. However, the curve obtained with 1B4 indicated lower relative expression of 1B4 epitope. Analysis of serum and synovial fluid sample with 5-D-4 mAb showed the presence of the epitope in both samples, but there was significantly less KS in serum than in the synovial fluid. Our results show that competitive ELISA for quantification of several different KS sulphation or “stub” epitopes and two KS-proteoglycans can all be quantified and compared using the same experimental conditions. These studies are ongoing as part of an Arthritis Research Campaign (UK) funded study. In addition the data indicates that keratocan and lumican are also increased in their expression with the progression of disease. Future studies will be performed in an attempt to quantify increased keratocan and lumican expression as potential biomarkers of degenerative joint disease.

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.

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: 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