Explant Cultures Of Articular Cartilage Research Articles

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-Length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA).

Probing the adsorption and lubrication behavior of lubricin, also known as proteoglycan 4 (PRG4), is important for understanding the ultralow friction of cartilage lubrication. Most previous research has focused on native lubricin either purified from synovial fluid or articular cartilage explant culture media. In this work, the adsorption behavior and lubrication mechanism of full-length recombinant human PRG4 (rhPRG4) on mica as well as the effect of adding hyaluronic acid (HA, a polysaccharide) were systematically investigated using a surface forces apparatus (SFA) technique. A low friction coefficient (μ ∼ 0.04) was measured when multilayer rhPRG4 (∼31 nm) was confined in between mica surfaces, even when the load increased to ∼1.2 MPa. Intriguingly, a previously unreported ultralow friction coefficient (μ < 0.005) was observed at a low sliding velocity ( v = 0.14 μm/s) with the applied load P reaching ∼3.6 MPa when a diluted rhPRG4 solution (∼90 μg/mL) was used. The distinct friction behavior is likely due to the smooth and more close-packed lubricin coating, as made evident by the atomic force microscope imaging. Adding HA onto multilayer rhPRG4-coated mica increased the friction coefficient μ to ∼0.1; however, the load bearing property increased, indicating potential synergistic effect between rhPRG4 and HA, which was further demonstrated by the weak adhesion observed when separating rhPRG4-coated mica and HA-coated aminopropyltriethoxysilane-mica (APTES-mica). Alternatively, adding premixed rhPRG4-HA on mica had a friction coefficient (μ ∼ 0.1) close to that of injecting concentrated rhPRG4 (∼450 μg/mL) with lower load sustainability. Our results provide fundamental insights into the adsorption and lubrication behavior of lubricin and its interaction with HA, with useful implications for the underlying mechanism of ultralow friction provided by synovial fluid.

Comparison of the Effects of Interleukin-1 on Equine Articular Cartilage Explants and Cocultures of Osteochondral and Synovial Explants.

Osteoarthritis (OA) is a ubiquitous disease affecting many horses. The disease causes chronic pain and decreased performance for patients and great cost to owners for diagnosis and treatment. The most common treatments include systemic non-steroidal anti-inflammatory drugs and intra-articular injection of corticosteroids. There is excellent support for the palliative pain relief these treatments provide; however, they do not arrest progression and may in some instances hasten advancement of disease. Orthobiologic treatments have been investigated as potential OA treatments that may not only ameliorate pain but also prevent or reverse pathologic articular tissue changes. Clinical protocols for intra-articular use of such treatments have not been optimized; the high cost of in vivo research and concerns over humane use of research animals may be preventing discovery. The objective of this study was to evaluate a novel in vitro articular coculture system for future use in OA treatment research. Concentrations and fold increases in various markers of inflammation (prostaglandin E2 and tumor necrosis factor-alpha), degradative enzyme activity [matrix metalloproteinase-13 (MMP-13)], cartilage and bone metabolism (bone alkaline phosphatase and dimethyl-methylene blue), and cell death (lactate dehydrogenase) were compared between IL-1-stimulated equine articular cartilage explant cultures and cocultures comprised of osteochondral and synovial explants (OCS). Results suggested that there are differences in responses of culture systems to inflammatory stimulation. In particular, the IL-1-induced fold changes in MMP-13 concentration were significantly different between OCS and cartilage explant culture systems after 96 h. These differences may be relevant to responses of joints to inflammation in vivo and could be important to the biological relevance of in vitro research findings.

Modulation of Superficial Zone Protein/Lubricin/PRG4 by Kartogenin and Transforming Growth Factor-β1 in Surface Zone Chondrocytes in Bovine Articular Cartilage.

Superficial zone protein (SZP)/lubricin/PRG4 functions as a boundary lubricant in articular cartilage to decrease friction and wear. As articular cartilage lubrication is critical for normal joint function, the accumulation of SZP at the surface of cartilage is important for joint homeostasis. Recently, a heterocyclic compound called kartogenin (KGN) was found to induce chondrogenic differentiation and enhance mRNA expression of lubricin. The objective of this study was to determine whether KGN can stimulate synthesis of SZP in superficial zone, articular chondrocytes. We investigated the effects of KGN and transforming growth factor-β1 (TGF-β1) on articular cartilage and synovium of the bovine knee joint by evaluating SZP secretion by enzyme-linked immunosorbent assay analysis. Monolayer, micromass, and explant cultures of articular cartilage, and monolayer culture of synoviocytes, were treated with KGN. SZP accumulation in the medium was evaluated and mRNA expression was measured through quantitative polymerase chain reaction. TGF-β1 stimulated SZP secretion by superficial zone chondrocytes in monolayer, explant, and micromass cultures as expected. In addition, SZP secretion was inhibited by IL-1β in explant cultures, and enhanced by TGF-β1 in synoviocyte monolayer cultures. Although KGN elicited a 1.2-fold increase in SZP mRNA expression in combination with TGF-β1, KGN neither stimulated any significant increases in SZP synthesis nor prevented catabolic decreases in SZP production from IL-1β. These data suggest that the chondrogenic effects of KGN depend on cellular phenotype and differentiation status, as KGN did not alter SZP synthesis in differentiated, superficial zone articular chondrocytes.

Andrographolide Exerts Chondroprotective Activity in Equine Cartilage Explant and Suppresses Interleukin-1 β -Induced MMP-2 Expression in Equine Chondrocyte Culture.

Cartilage erosion in degenerative joint diseases leads to lameness in affected horses. It has been reported that andrographolide from Andrographis paniculata inhibited cartilage matrix-degrading enzymes. This study aimed to explore whether this compound protects equine cartilage degradation in the explant culture model and to determine its effect on matrix metalloproteinase-2 (MMP-2) expression, a matrix-degrading enzyme, in equine chondrocyte culture. Equine articular cartilage explant culture was induced by 25 ng/mL interleukin-1β, a key inducer of cartilage degeneration, in cultures with or without andrographolide ranging from 10 to 50 μM. After 3–21 days, they were analyzed for the markers of cartilage degradation. It was found that interleukin-1β increased the release of sulfated glycosaminoglycans and hyaluronan from the explants into the culture media consistently with the decrease in uronic acid and collagen content in the cartilage explants. These catabolic effects were inhibited when cotreated with interleukin-1β and andrographolide. In primary equine chondrocytes, andrographolide suppressed interleukin-1β-induced MMP-2 mRNA expression and MMP-2 activity in the culture medium. These results confirmed the in vitro potent chondroprotective activities of this compound which were performed in cartilage explants and on a cellular level. These may indicate the application of andrographolide for therapeutic use in equine degenerative joint diseases.

Deiodinase 2 upregulation demonstrated in osteoarthritis patients cartilage causes cartilage destruction in tissue-specific transgenic rats

Chondrocyte hypertrophy followed by cartilage destruction is a crucial step for osteoarthritis (OA) development, however, the underlying mechanism remains largely unknown. The objectives of this study are to identify the gene that may cause cartilage hypertrophy and to elucidate its role on OA pathogenesis. Gene expression profiles of cartilages from OA patients and normal subjects were examined by microarray analysis. Expression of deiodinases, enzymes for regulation of triiodothyronine (T3) biosynthesis, in human and rat articular cartilage (AC) were examined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Rat ACs and chondrocytes were treated with T3 to investigate its role on chondrocyte hypertrophy and inflammatory reaction. Cartilage-specific Type II deiodinase (DIO2) transgenic rats were generated using bacterial artificial chromosome harboring the entire rat Col2a1 and human DIO2 gene. An experimental OA model was created in the animal to examine the role of DIO2 on cartilage degeneration. DIO2 is highly expressed in OA patient AC compared to normal control. In rat AC, DIO2 is specifically expressed among deiodinases and dominantly expressed the same as in brown adipose tissue. T3 induces hypertrophic markers in articular chondrocyte and cartilage explant culture, and enhances the effect of IL-1α on induction of cartilage degrading enzymes. Importantly, cartilage-specific DIO2 transgenic rats are more susceptible to knee joint destabilization and develop severe AC destruction. Our findings demonstrate that upregulated expression of DIO2 in OA patient cartilage might be responsible for OA pathogenesis by enhancing the chondrocyte hypertrophy and inflammatory response.

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures.

Development of an in vitro model of feline cartilage degradation.

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1alpha, or IL-1beta. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1beta and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1beta and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1beta dose-response studies showed that an IL-1beta dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1beta did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine

Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Global analysis of nuclear receptor expression and dysregulation in human osteoarthritic articular cartilage: Reduced LXR signaling contributes to catabolic metabolism typical of osteoarthritis

Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRalpha and LXRbeta, and their heterodimeric partners retinoid X receptor (RXR)alpha and RXRbeta were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

Review of In Vitro Models and Development and Initial Validation of a Novel Co-Culture Model for the Study of Osteoarthritis

Objective: Provide an overview of models used for the study of osteoarthritis (OA) and describe a novel in vitro model of OA using synovial and cartilage explants from dogs which we compared to spontaneously occurring OA in dogs. Design: Controlled laboratory study. Methods: Articular cartilage and synovial explants were created from canine cadaveric tissues (n=38 dogs) or tissues excised during surgery on clinical canine patients (n=17 hips). Matched cartilage and synovial explant co-cultures were compared to articular cartilage explant culture. Four groups were created based on presence or absence of synovium and presence or absence of IL-1β: Cartilage Culture Control (n=48), Cartilage Culture OA (n=48), Co-Culture Control (n=76), Co-Culture OA (n=76). Explants and media were evaluated on days 1, 3, 6, 12, and/or 20 of culture. For comparison to spontaneously occurring OA, articular cartilage and synovial tissues from dogs that had no evidence of OA, Clinical Control, and dogs that had OA, Clinical OA, were evaluated. Outcome measures included molecular, biochemical, and histologic assessments. Results: Gene expression levels in articular tissues correlated well between Co-Culture and Clinical Control and OA groups. GAG and collagen in normal and OA cartilage also corresponded well between Co-Culture and Clinical groups. Subjective histologic assessment revealed consistent similarities in cell and matrix characteristics between these groups. Changes in the media of the Co-Culture OA group were consistent with what has been reported for changes in synovial fluid of OA in dogs and humans. Conclusions: This co-culture model allows for assessment of cartilage and synovium, and can be used to provide a relatively comprehensive picture of spontaneously occurring OA in dogs. This model may have advantages over other models of OA in that the influences of both synovium and articular cartilage can be investigated at many levels and initial validation of the model using tissues from spontaneously occurring OA in dogs as the gold standard has been performed.

Chondroprotective Potential of Fruit Extracts of Phyllanthus emblica in Osteoarthritis

There is a need for effective nutraceuticals for osteoarthritis care. The fruit of Phyllanthus emblica is used as a powerful rejuvenator in Ayurvedic medicine. This study measured the chondroprotective potential of P. emblica (‘Amalaki’) fruits in vitro. We used aqueous extracts of unprocessed P. emblica fruit powder (powder A), and the powder obtained after hot water extraction and drying of powder A (powder B). Chondroprotection was measured in three different assay systems. First, we tested the effects of both fruit powders on the activities of the enzymes hyaluronidase and collagenase type 2. Second, an in vitro model of cartilage degradation was set-up with explant cultures of articular knee cartilage from osteoarthritis patients. Cartilage damage was assayed by measuring glycosaminoglycan release from explants treated with/without P. emblica fruit powders. Aqueous extracts of both fruit powders significantly inhibited the activities of hyaluronidase and collagenase type 2 in vitro. Third, in the explant model of cartilage matrix damage, extracts of glucosamine sulphate and powder B (0.05 mg/ml) exhibited statistically significant, long-term chondroprotective activity in cartilage explants from 50% of the patients tested. This result is important since glucosamine sulphate is the leading nutraceutical for osteoarthritis. Powder A induced a statistically significant, short-term chondroprotective activity in cartilage explants from all of the patients tested. This is the first study to identify and quantitate new chondroprotective activities of P. emblica fruits. These data provide pilot pre-clinical evidence for the use of P. emblica fruits as a chondroprotective agent in osteoarthritis therapy.

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195–218), but not synthetic peptide CB12-IV (residues 231–254), potently and consistently induced in explant cultures at 10 μM and 25 μM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.

Kinetics of collagen crosslinking in adult bovine articular cartilage

Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies. Explant cultures of adult bovine articular cartilage were radiolabeled with [14C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, beta-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking. Tissue was hydrolyzed and analyzed for [14C]metabolites in the forms of lysine, hydroxylysine, dehydrodihydroxylysinonorleucine (DeltaDHLNL), and hydroxylysyl pyridinoline (HP). Explant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, DeltaDHLNL and HP. During chase, [14C]hydroxylysine maintained steady-state levels, [14C]DHLNL rose to a plateau, and [14C]HP increased gradually. Addition of BAPN inhibited formation of [14C]DHLNL. Analysis of raw data and that normalized to [14C]hydroxylysine gave characteristic time constants for formation of DeltaDHLNL and HP crosslinks of 1-2 and 7-30 days, respectively. The distribution of [14C]lysine metabolites in collagen crosslinks was described by peak values in [14C]DHLNL/[14C]hydroxylysine of 0.047-0.064 and in [14C]HP/[14C]hydroxylysine of 0.03. Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.

Lectin‐like oxidized low‐density lipoprotein receptor 1 mediates matrix metalloproteinase 3 synthesis enhanced by oxidized low‐density lipoprotein in rheumatoid arthritis cartilage

To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture. Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay. The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels. Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH2-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1 µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10 µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10 µM SB203580 strongly inhibited the collagen cleavage compared with 1 µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH(2)-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1 microM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10 microM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10 microM SB203580 strongly inhibited the collagen cleavage compared with 1 microM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.

ADAMTS‐4 and ADAMTS‐5 sequestration and activity in chondrocyte‐agarose cultures

Introduction A primary event in the destruction of cartilage in arthritic diseases is the loss of aggrecan from the extracellular matrix of articular cartilage. During aggrecan breakdown, cleavage sites are utilized, which reside within the IGD of the aggrecan core protein. The Asn341–Phe342 bond is cleaved by members of the MMP family, whereas the second of the two cleavage sites, the Glu373–Ala374 bond, is cleaved by the aggrecanases which are all members of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Both ADAMTS‐4 and ‐5 have been shown to readily cleave aggrecan at this so‐called aggrecanase site (Tortorella et al. 1999; Abbaszade et al. 1999; Sandy et al. 2000). An in vitro model of cartilage degradation has also shown that these enzymes are responsible for the loss of aggrecan from explant cultures of articular cartilage stimulated with IL‐1‐a or TNF‐a (Tortorella et al. 2001). Both ADAMTS‐4 and ‐5 are thought to be synthesized as inactive zymogens that are activated via removal of their propeptide domain by the Golgi enzyme furin. The secreted active ADAMTS‐4 and ‐5 have predicted molecular weights of 67.9 and 73.6 kDa, respectively. The objective of this study was to monitor ADAMTS‐4 and ‐5 secretion and sequestration in the extracellular matrix of chondrocyte‐agarose cultures.Methods Porcine articular chondrocytes were isolated and embedded in agarose (Hughes et al. 1997) before preculture in DMEM + 50 µg/ml gent. with 10% FBS and 25 µg/ml Phos.C for 21 days. The plates were washed, then cultured in serum‐free DMEM with or without IL‐1‐α for 96 h. GAG release to the medium was measured using the DMMB assay. Media samples were analysed by Western blotting for aggrecan metabolites using mAb BC‐3 to recognize the aggrecanase‐generated neoepitope ARGSV. The presence of ADAMTS‐4 in the media was analysed using the mAb anti‐TS‐4N, which recognizes the metalloproteinase domain, and commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐4. The presence of ADAMTS‐5 was detected using commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐5. Agarose plugs were extracted in detergent buffer and analysed by Western blotting for ADAMTS‐4 and ‐5 using the same monoclonal and polyclonal antibodies.Results In control cultures, only 20–30% of the total GAG was released into the medium after 96 h of culture. In contrast, 80–90% of the total GAG was released in cultures exposed to IL‐1. Western blot analysis showed aggrecanase‐generated aggrecan metabolites in the IL‐1‐treated cultures but none in the control cultures. Sequestered forms of both ADAMTS‐4 and ‐5 are present in the matrix prior to treatment in serum‐free conditions, and following treatment with or without IL‐1 for 96 h, there are no differences in the high molecular weight isoforms of the enzymes sequestered in the matrix. Western blots of partially purified media samples showed no differences in the zinc chelator‐bound isoforms of either ADAMTS‐4 or ‐5 between control and IL‐1‐treated cultures. However, the predominant heparin sepharose‐bound isoforms of ADAMTS‐4 and ‐5 co‐migrate at approximately 37 kDa. Each of the heparin‐bound 37‐kDa isoforms of ADAMTS‐4 and ‐5 are detected in increased amounts in IL‐1α‐treated cultures compared to controls.Discussion The increased amounts of the 37‐kDa isoforms of both ADAMTS‐4 and ‐5 in the IL‐1‐treated cultures suggest a role for these smaller isoforms in the increased aggrecanase activity seen in the IL‐1‐treated cultures compared to controls. This study has identified multiple isoforms of putative aggrecanase activity that could be responsible for increased aggrecan catabolism that leads to cartilage degradation in arthritis.

Effects of long-term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage

Objective: To investigate the effect of long-term exposure to glucosamine or mannosamine on the catabolism of aggrecan by explant cultures of bovine articular cartilage maintained in the presence of retinoic acid. Design: The kinetics of loss of 35S-labeled and total aggrecan from explant cultures of bovine articular cartilage maintained in the presence of 1 μM retinoic acid and exposed to varying concentrations of glucosamine or mannosamine was investigated over a 9-day culture period. In other experiments, the reversibility of the inhibition of aggrecan catabolism by glucosamine or mannosamine was investigated in cultures exposed to these amino sugars for the first 5 days of a 15-day culture period. The metabolism of chondrocytes exposed to these amino sugars was evaluated by measurement of lactate production or 3H-serine and 35S-sulfate incorporation into protein and glycosaminoglycans, respectively. The direct effect of these amino sugars on soluble aggrecanase activity was determined from immunoblots of aggrecan digests. Results: Glucosamine at 5 mM concentration and mannosamine at 2 mM concentration inhibited degradation of radiolabeled and chemical levels of aggrecan. At concentrations of up to 10 mM amino sugars, the metabolism of chondrocytes was not impaired, as determined by lactate production, protein synthesis and the incorporation of 35S-sulfate into proteoglycans. These amino sugars did not inhibit soluble aggrecanase activity. The exposure of articular cartilage explants to 5 mM glucosamine or mannosamine for 5 days in culture in the presence or absence of retinoic acid did not provide long-term suppression of stimulated aggrecan loss. Conclusions: This study indicates that continuous presence of amino sugars is required to protect cartilage from stimulated loss of aggrecan.

Recombinant adeno-associated virus vectors efficiently and persistently transduce chondrocytes in normal and osteoarthritic human articular cartilage.

Successful gene transfer into articular cartilage is a prerequisite for gene therapy of articular joint disorders. In the present study we tested the hypothesis that recombinant adeno-associated virus (rAAV) vectors are capable of effecting gene transfer in isolated articular chondrocytes in vitro, articular cartilage tissue in vitro, and sites of articular damage in vivo. Using an rAAV vector carrying the Escherichia coli beta-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate-early promoter/enhancer (rAAV-lacZ), transduction efficiency exceeded 70% for isolated normal human adult articular chondrocytes, and osteoarthritic human articular chondrocytes. These were comparable to the transduction efficiency obtained with neonatal bovine articular chondrocytes. Transduction of explant cultures of articular cartilage resulted in reporter gene expression within the tissue of all three cartilage types to a depth exceeding 450 microm, which remained present until 150 days. When rAAV-lacZ vectors were applied to femoral chondral defects and osteochondral defects in vivo in a rat knee model, reporter gene expression was achieved for at least 10 days after transduction. These data suggest that AAV-based vectors can efficiently transduce and stably express foreign genes in articular chondrocytes, including chondrocytes of normal and osteoarthritic human articular cartilage. The data further suggest that the same rAAV vectors are capable of transducing chondrocytes in situ within their native matrix to a depth sufficient to be of potential clinical significance. Finally, the data demonstrate that these rAAV vectors are capable of effectively delivering recombinant genes to chondral and osteochondral defects in vivo.