Background

Proteoglycans (PGs) have long been known to be important to the functioning of the intervertebral disc. The most common PG is aggrecan, but there are also small leucine-rich proteoglycans (SLRPs) which constitute only a small percentage of the total PGs. However, they have many important functions, including organising the collagen, protecting it from degradation and attracting growth factors to the disc. We have examined how the core proteins of these molecules vary in intervertebral discs from patients with different pathologies.

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.

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.

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.

Introduction: The search continues for ideal markers and methods of monitoring cartilage degeneration. Various cartilage components, whole or fragmented, have been measured in synovial fluids. A common problem in quantitating these markers is often the unknown dilution of synovial fluid which can occur in obtaining the samples. In this study we have used urea (ratio in synovial fluid:serum) as a method to correct for the dilution of synovial fluid, and hence to quantify enzyme levels in patients with a spectrum of cartilage degradation, in addition to identifying aggrecan degradation products, many of them for the first time in such samples.

Methods: Forty synovial fluid samples were obtained from 4 groups of individuals (10 in each):

normal,

Levels of matrix metalloproteinases (MMPs) 2 and 3 and the inhibitor, TIMP 1, were measured in the fluids via ELISA assays. Urea levels were measured in blood and synovial fluids and enzymes and their inhibitors were normalized according to the ratio of serum:SF urea, to account for the dilution factor of the SF (Kraus et al 2001). Western blotting was used to identify the presence of aggrecan components (chondroitin-4-sulphate: 2B6 antibody; C-6-S: 3B3 and C-0-S: 1B5; keratan sulphate: BKS-1; the G1 domain: 7D1; interglobular domain: 6B4) and also enzyme degradation products of MMPs (BC14) and aggrecanases (BC3; BC-13).

Results: MMPs 2 and 3 and TIMP 1 were all significantly increased in the synovial fluids from OA patients compared to normals (P< 0.01, 0.001 and 0.01 respectively) and MMP3 was greater in the grade IV chondral and osteochondral defect groups than the normals (P< 0.01). Western blotting demonstrated fragmented aggrecan components with a range of molecular weights. Aggrecanase activity was seen in the OA and grade IV chondral damage groups but not in the osteochondral or normal groups, whereas MMP activity was seen in all 3 groups showing cartilage damage but not in the normals.

Conclusion: Dilution of the synovial fluid, either due to inflammation or joint lavage, is often a problem in quantitating metabolites and markers in joint cavities. This pilot study of a limited number of samples from well characterized patient groups indicates that using urea concentrations in synovial fluid relative to serum provides a mechanism to overcome this. It confirms elevated enzyme activity, both aggrecanase and MMPs, in the joints of patients with degenerate cartilage, compared to normals.

Introduction: Autologous chondrocyte implantation is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogenous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.

Methods: Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into poly vinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM+10% FBS within 15ml conical tubes rotated at 37°C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H& E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X, and to the glycosaminoglycans, chondroitin-4- and -6-sulphate and keratan sulphate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein, and small leucine-rich PGs.

Results: H& E staining of 10μm-thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining revealed the presence of GAGs, increasing with time. Ki-67 revealed approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X and the glycosaminoglycans, chondroitin-4-, -6 and keratan sulphate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.

Conclusions: Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.

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.

Although the function of proteoglycans within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies. In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon, indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side-effects which involve tendonitis and “musculoskeletal syndrome”. Such side effects may relate to non-specific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate turnover of tendon and cartilage proteoglycans derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 M) or actinonin (0–200 M). Proteoglycan and lactate quantification, Western blot analysis of degradation products and RT-PCR analyses were performed on these samples. In a separate experiment for measurement of proteoglycan turnover, explants were set up as described above then pulse chase labelled with [35S] sulphate. The rate of turnover of 35S-labelled proteoglycans from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of 35S-labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. Proteoglycan turnover (presumably predominantly aggrecan) was markedly higher in tendon versus cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase-mediated proteoglycan catabolism in both tendon and cartilage explants. As expected mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by addition of these inhibitors to the culture medium. Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this proteoglycan in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half-life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase-mediated proteoglycan catabolism in tendon cultures. This suggests that the occurrence of “musculoskeletal syndrome” in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function.

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

Introduction: Proteoglycans are found both in the annulus fibrosus and nucleus pulposus of the intervertebral disc and contribute to the hydration of the tissue (aggrecan) and the regulation of matrix assembly (small proteoglycans) [1]. Whilst loss of proteoglycan is the main chemical change in disc degeneration seen in back pain patients, little is known of the events leading to and controlling this loss. In this study the metabolism of the most common proteoglycan, aggrecan, and others including decorin, biglycan, lumican, fibromodulin and versican, together with collagen types I and II were studied in diseased and normal discs.

Methods: Ten discs from patients aged 11–57 years (mean:39±15) with scoliosis (n=1), spondylolisthesis (n=1) and low back pain (n=8), were graded for macroscopic degeneration (Grades 1–4). Three ‘normal’ cadaveric discs from 3 individuals aged 25–27 years (mean 26±1) were also investigated. Disc was either snap-frozen (for RNA isolation) or the proteoglycans extracted with 4M GuHCl. Total RNA was isolated and RT-PCR performed using various oligonucleotide primers. GuHCl-extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and small leucine-rich proteoglycans.

Results: Intervertebral discs contain a very heterogenous population of proteoglycans demonstrating extensive enzymic degradation, particularly with increasing age and macroscopic degeneration such as is seen in back pain patients. Younger, less degenerate discs contained more biglycan than the older, more degenerate discs. However, the mRNA gene expression analyses demonstrated little cellular activity and potential synthetic response, there was very little expression of particularly in comparison to osteoarthritic cartilage cells which show considerable synthetic capability for all the major matrix components.

Discussion: Our analyses indicate that several biochemical, catabolic and biosynthetic changes occur in disc matrix molecules which are likely to contribute to loss of disc function with ageing and degeneration. The loss of biosynthetic capability of cells is very important in considering the potential of newer therapeutic modalities such as cellular repair and genetic engineering for the treatment of degenerative disc disease.

Introduction: Previous studies have demonstrated that exposure of normal bovine and human osteoarthritic cartilage to n-3 polyunsaturated fatty acids (PUFAs) such as those present in fish oils can modulate the expression and activity of the degradative and inflammatory factors that are responsible for cartilage destruction [1,2]. In these studies, supplementation of cartilage explant cultures with n-3 PUFAs resulted in an abrogation of aggrecanase activity as well as mRNA expression of mediators of inflammation. To date, few studies have examined the effect of PUFAs on the metabolism of other tissues within the musculoskeletal system, therefore the present work examines the effect of n-3 PUFA supplementation on tendon metabolism.

Methods: Bovine deep digital flexor tendon explants were obtained from the compressed region of young metacarpophalangeal joints (2-week-old) and supplemented with eicosapentaenoic acid (EPA), as previously described [2]. Release of proteoglycan metabolites was analysed using Western blotting whilst RT-PCR analysis was used to examine the mRNA expression patterns of matrix proteases and inflammatory agents.

Results: Exposure to the n-3 fatty acid, EPA, markedly changed the overall lipid composition profile of the tendon with major changes occurring in the supplemented fatty acid (i.e., EPA), with a concomitant percentage reduction in other polyunsaturated fatty acids. Aggrecanase activity was present in the media from control cultures, as expected [3]. However, supplementation with EPA had no effect on this activity, in contrast to articular cartilage where aggrecanase catabolites were absent from the conditioned media following treatment with n-3 PUFAs [1,2]. mRNA expression for the inflammatory mediators (COX-2, IL-1β, TNF), ADAMTS-5, MMPs and TIMPs was also unchanged following supplementation with EPA, again contrasting with articular cartilage where mRNA expression was abolished.

Discussion: This study demonstrates that exposure of bovine tendon explant cultures to an n-3 PUFA, EPA, had no effect on the mRNA expression or activity of aggrecanases; similarly, expression of the inflammatory mediators was also unaffected. Importantly, within this musculoskeletal tissue, aggrecanases are constitutively active and appear to be involved in normal, everyday turnover of aggrecan, in contrast to non-pathological articular cartilage where aggrecanase-generated metabolites are only detected following treatment with catabolic agents. Similarly, COX-2 mRNA expression is present constitutively within tendons whereas in cartilage it is absent under basal (unstimulated) conditions. These data demonstrate that the incorporation of n-3 PUFAs have a differential effect on the regulatory mechanisms which control gene expression within articular cartilage versus tendon.

Aim: To evaluate the functional outcome of patients following intra-osseous suturing for repair of distal biceps tendon ruptures, using the Mayo scoring system. Subsequent analysis of mRNA expression; in the ruptured biceps tendons was performed.

Methods: We operated on 8 patients who had ruptured their biceps tendon. The average ages of the patients were 36 (Range 22–50). The technique involved using intrasosseous suturing via a single anterior skin crease incision. The functional outcome of these patients was scored by using the Mayo elbow performance score. The average follow-up was 7 months. (Range 5–8 months). The tendons were processed for RNA isolation and reverse -transcription – polymerase chain reaction (RT-PCR).

Results: The average subjective assessment (pain and function) of these patients was 63/70 (Range 57–68). The average objective assessment (motion and stability) was 24/30 (Range 22–27). The overall average was 87/100. None of the patients had any complications postoperatively. Our results showed that in the samples of ruptured biceps tendon there was mRNA expression of ECM structural components, especially aggrecan and the small proteoglycans biglycan and decorin. Interestingly, these samples also showed a high expression for the enzymes commonly involved in articular cartilage degradation and turnover, the aggrecanases (ADAMTS-4 and ADAMTS-5) and the matrix metalloproteinases (MMP-3 and MMP-13).

Conclusion: We demonstrated that intrasosseous suturing via a single anterior incision, in-patients with ruptured biceps tendons could provide a good functional outcome. This technique should therefore be considered as one of the surgical options in the management of this condition. We know clinically that patients can rupture their biceps tendon either due to trauma if not due to degenerative conditions. In our study we wanted to know if the subset of patients how ruptured their tendons traumatically had any pre-existing degenerative conditions leading on to the rupture compared to the normal subjects. Interestingly our study has shown that there is mRNA expression of degradative enzymes (aggrecanases and MMPs) in the samples of ruptured biceps tendon. Furthermore, our samples also showed mRNA expression for factors involved in the inflammatory response. In conclusion, mRNA expression of the factors involved in degradation and inflammation may suggest a phenotype that predisposes the biceps tendon to rupture, although further studies are required in order to investigate this.

Aim: To study mRNA expression in ruptured biceps tendon.

Methods: Our study was carried out in the University College of Medicine. We took the biceps tendon of 5 patients who had traumatic ruptures. The age of the patients ranged from 35–53. The tendons were processed for RNA isolation and reverse-transcription-polymerase chain reaction (RT-PCR) carried out in order to investigate the mRNA gene expression in ruptured biceps tendon of extra cellular matrix (ECM) components (e.g. proteoglycans and collagens); ECM degradative components (e.g. aggrecanases and MMPs); inflammatory components (e.g. cytokines and cyclooxygenases); and factors involved in the apoptotic response.

Results: Our results showed that in the samples of ruptured biceps tendon there was a good mRNA expression of ECM structural components, especially aggrecan and the small proteoglycans biglycan and decorin. Interestingly, these samples also showed a high expression for the enzymes commonly involved in articular cartilage degradation and turnover, the aggrecanases (ADAMTS-4 and –5) and the matrix metalloproteinases (MMP-3 and –13). As has been recently reported for Achilles tendon rupture (Cetti et al, 2003), an inflammatory reaction was also observed in these ruptured bicep tendons with expression of the inflammatory cytokines IL-1α and TNFα and the enzyme cyclooxygenase-2.

Conclusion: We know clinically that patients can rupture their biceps tendon either due to trauma if not due to degenerative conditions. In our study we wanted to know if the subset of patients who ruptured their tendons traumatically had any pre-existing degenerative conditions leading on to the rupture compared to the normal subjects. Interestingly our study has shown that there is mRNA expression of degradative enzymes (aggrecanases and MMPs) in the samples of ruptured biceps tendon. Whether these mRNA levels equate to increased enzyme activity of these molecules warrants further investigation. Furthermore, our samples also showed mRNA expression for factors involved in the inflammatory response. In conclusion, mRNA expression of the factors involved in degradation and inflammation may suggest a phenotype that predisposes the bicep tendon to rupture, although further studies are required in order to investigate this further.