Deficiency of hyaluronan synthase 1 (Has1) results in chronic joint inflammation and widespread intra-articular fibrosis in a murine model of knee joint cartilage damage.

OBJECTIVE: Articular cartilage defects commonly result from traumatic injury and predispose to degenerative joint diseases. To test the hypothesis that aberrant healing responses and chronic inflammation lead to osteoarthritis (OA), we examined spatiotemporal changes in joint tissues after cartilage injury in murine knees. Since intra-articular injection of hyaluronan (HA) can attenuate injury-induced osteoarthritis in wild-type (WT) mice, we investigated a role for HA in the response to cartilage injury in mice lacking HA synthase 1 (Has1(-/-)). DESIGN: Femoral groove cartilage of WT and Has1(-/-) mice was debrided to generate a non-bleeding wound. Macroscopic imaging, histology, and gene expression were used to evaluate naïve, sham-operated, and injured joints. RESULTS: Acute responses (1-2 weeks) in injured joints from WT mice included synovial hyperplasia with HA deposition and joint-wide increases in expression of genes associated with inflammation, fibrosis, and extracellular matrix (ECM) production. By 4 weeks, some resurfacing of damaged cartilage occurred, and early cell responses were normalized. Cartilage damage in Has1(-/-) mice also induced early responses; however, at 4 weeks, inflammation and fibrosis genes remained elevated with widespread cartilage degeneration and fibrotic scarring in the synovium and joint capsule. CONCLUSIONS: We conclude that the ineffective repair of injured cartilage in Has1(-/-) joints can be at least partly explained by the markedly enhanced expression of particular genes in pathways linked to ECM turnover, IL-17/IL-6 cytokine signaling, and apoptosis. Notably, Has1 ablation does not alter gross HA content in the ECM, suggesting that HAS1 has a unique function in the metabolism of inflammatory HA matrices.

Human genome-wide expression analysis reorients the study of inflammatory mediators and biomechanics in osteoarthritis.

A major objective of this article is to examine the research implications of recently available genome-wide expression profiles of cartilage from human osteoarthritis (OA) joints. We propose that, when viewed in the light of extensive earlier work, this novel data provides a unique opportunity to reorient the design of experimental systems toward clinical relevance. Specifically, in the area of cartilage explant biology, this will require a fresh evaluation of existing paradigms, so as to optimize the choices of tissue source, cytokine/growth factor/nutrient addition, and biomechanical environment for discovery. Within this context, we firstly discuss the literature on the nature and role of potential catabolic mediators in OA pathology, including data from human OA cartilage, animal models of OA, and ex vivo studies. Secondly, due to the number and breadth of studies on IL-1ß in this area, a major focus of the article is a critical analysis of the design and interpretation of cartilage studies where IL-1ß has been used as a model cytokine. Thirdly, the article provides a data-driven perspective (including genome-wide analysis of clinical samples, studies on mutant mice, and clinical trials), which concludes that IL-1ß should be replaced by soluble mediators such as IL-17 or TGF-ß1, which are much more likely to mimic the disease in OA model systems. We also discuss the evidence that changes in early OA can be attributed to the activity of such soluble mediators, whereas late-stage disease results more from a chronic biomechanical effect on the matrix and cells of the remaining cartilage and on other local mediator-secreting cells. Lastly, an updated protocol for in vitro studies with cartilage explants and chondrocytes (including the use of specific gene expression arrays) is provided to motivate more disease-relevant studies on the interplay of cytokines, growth factors, and biomechanics on cellular behavior.

The relationship between fibrogenic TGFß1 signaling in the joint and cartilage degradation in post-injury osteoarthritis.

OBJECTIVE: To review the literature on modulation of chondrocyte activities in the osteoarthritic joint, and to discuss these changes in relation to established hard and soft tissue repair paradigms, with an emphasis on transforming growth factor beta (TGFß1)-mediated signaling which can promote either a chondrogenic or fibrogenic phenotype. METHODS: Papers addressing the close relationship between repair in general, and the specific post-injury response of joint tissues are summarized. Different interpretations of the role of TGFß1 in the emergence of an "osteoarthritic" chondrocyte are compared and the phenotypic plasticity of "reparative" progenitor cells is examined. Lastly, emerging data on a central role for A-Disintegrin-And-Metalloproteinase-with-Thrombospondin-like-Sequences-5 (ADAMTS5) activity in modulating TGFß1 signaling through activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5) pathways is discussed. RESULTS: The review illustrates how a transition from ALK5-mediated fibrogenic signaling to ALK1-mediated chondrogenic signaling in joint cells represents the critical transition from a non-reparative to a reparative cell phenotype. Data from cell and in vivo studies illustrates the mechanism by which ablation of ADAMTS5 activity allows the transition to reparative chondrogenesis. Multiple large gene expression studies of normal and osteoarthritis (OA) human cartilages (CAs) also support an important role for TGFß1-mediated pro-fibrogenic activities during disease progression. CONCLUSIONS: We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFß1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo, but also improve the properties of tissue-engineered CA for implantation.

The effects of oral glucosamine on joint health: is a change in research approach needed?

OBJECTIVE: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions. METHODS: An editorial "narrative" review of peer-reviewed publications is organized into four topics (1) Chemistry and pharmacokinetics of GlcN salts (2) Biological effects of GlcN salts in vitro (3) Therapeutic effects of GlcN salts in animal models of OA and (4) GlcN salts in the treatment of clinical OA. RESULTS: Data reporting potent pleiotropic activities of GlcN in in vitro cell and explant cultures are discussed in the context of the established pharmacokinetic data in humans and animals. The available clinical trial data are discussed to place the patient in the context of controlled research on disease management. CONCLUSIONS: Future research to determine therapeutic mechanisms of GlcN salt preparations will require use of standardized and clinically relevant in vitro assay systems and in vivo animal models for testing, as well as development of new outcome measures for inflammation and pain pathways in human OA.

The human pharmacokinetics of oral ingestion of glucosamine and chondroitin sulfate taken separately or in combination.

OBJECTIVE: As part of the National Institutes of Health (NIH)-sponsored Glucosamine/Chondroitin sulfate Arthritis Intervention Trial (GAIT) our objective here was to examine (1) the pharmacokinetics (PK) of glucosamine (GlcN) and chondroitin sulfate (CS) when taken separately or in combination as a single dose in normal individuals (n=29) and (2) the PK of GlcN and CS when taken as a single dose after 3 months daily dosing with GlcN, CS or GlcN+CS, in patients with symptomatic knee pain (n=28). METHODS: The concentration of GlcN in the circulation was determined by established fluorophore-assisted carbohydrate electrophoresis (FACE) methods. The hydrodynamic size and disaccharide composition of CS chains in the circulation and dosage samples was determined by Superose 6 chromatography and FACE. RESULTS: We show that circulating levels of CS in human plasma are about 20 microg/ml. Most significantly, the endogenous concentration and CS disaccharide composition were not detectably altered by ingestion of CS, when the CS was taken alone or in combination with GlcN. On the other hand, the Cmax (single-dose study) and AUC values (multiple-dose study) for ingested GlcN were significantly reduced by combination dosing with CS, relative to GlcN dosing alone. CONCLUSIONS: We conclude that pain relief perceived following ingestion of CS probably does not depend on simultaneous or prior intake of GlcN. Further, such effects on joint pain, if present, probably do not result from ingested CS reaching the joint space but may result from changes in cellular activities in the gut lining or in the liver, where concentrations of ingested CS, or its breakdown products, could be substantially elevated following oral ingestion. Moreover, since combined dosing of GlcN with CS was found to reduce the plasma levels seen with GlcN dosing alone, any improved pain relief by combination dosing cannot be explained by higher circulating concentrations of GlcN.

Removal of O-linked and N-linked oligosaccharides is required for optimum detection of NITEGE neoepitope on ADAMTS4-digested fetal aggrecans: implications for specific N-linked glycan-dependent aggrecanolysis at Glu373-Ala374.

OBJECTIVES: We have observed that Western blot analysis with an anti-G1 antibody detects G1-NITEGE product in a disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4)-digested fetal and mature human and bovine aggrecan, but the neoepitope-specific anti-NITEGE antibody only detects this product in digests of mature aggrecan. Our objective was to determine whether enzymatic removal of O- and/or N-linked oligosaccharides from the fetal products would enable detection of the NITEGE neoepitope with anti-NITEGE antibody. METHODS: Aggrecan was purified from fetal and mature human and bovine cartilage and digested with: (1) ADAMTS4, (2) ADAMTS4, sialidase II, and N-glycanase, (3) ADAMTS4, sialidase II, and O-glycanase, or (4) ADAMTS4, sialidase II, and both N- and O-glycanases. Western blot analysis was performed using anti-G1 and anti-NITEGE antibodies. RESULTS: When fetal G1-NITEGE products were treated with a combination of ADAMTS4, sialidase II, O-glycanase and N-glycanase, the resultant products migrated faster on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the NITEGE neoepitope was rendered detectable. CONCLUSIONS: It appears that the NITEGE neoepitope is blocked on Western blots by oligosaccharide structures present on Asn368 and Thr370 of fetal human and bovine aggrecans. Such masking structures do not appear to be present on mature aggrecans from these species. We suggest that when anti-NITEGE antibody is used in Western analysis, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC), removal of oligosaccharides with appropriate glycosidases may unmask reactivity that would otherwise go undetected. The implications of these findings for the much-studied effect of Asn368-linked keratan sulfate (KS)-based structures on ADAMTS4 and ADAMTS5 activity are discussed.

Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury.

Superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter-alpha-trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin.

OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.

The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Synovial fluid was collected from patients with recent knee injury and from patients with early or late stage osteoarthritis. Chondroitin sulfate-substituted aggrecan fragments present in these fluids, and in normal bovine synovial fluid, were purified by cesium chloride gradient centrifugation, enzymically deglycosylated and fractionated by gel filtration on Superose-12. Each sample contained two major aggrecan core protein populations with apparent molecular masses of approximately 90 kD and 150 kD. For all samples, NH2-terminal analysis of both populations gave a single major sequence beginning ARGSV. This NH2 terminus results from cleavage of the human aggrecan core protein at the Glu 373-Ala 374 bond within the interglobular domain between the G1 and G2 domains. Cleavage at this site also occurs during control and interleukin-1 stimulated aggrecan catabolism in bovine cartilage explant cultures (Sandy, J., P. Neame, R. Boynton, and C. Flannery. 1991. J. Biol. Chem. 266:8683-8685). These results indicate that the major aggrecan fragments present in both osteoarthritic human synovial fluid and in normal bovine synovial fluid are large, being composed of a short NH2-terminal stretch of the interglobular domain, the G2 domain, the keratan sulfate domain, and variable lengths of the chondroitin sulfate domain(s). We conclude that the release of aggrecan fragments from articular cartilage into the synovial fluid seen at all stages of human osteoarthritis (Lohmander, L. S. 1991. Acta Orthop. Scand. 62:623-632) is promoted by the action of a normal cartilage proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain.

Intact aggrecan and fragments generated by both aggrecanse and metalloproteinase-like activities are present in the developing and adult rat spinal cord and their relative abundance is altered by injury.

Aggrecan is a large proteoglycan (PG) that has been grouped with different PG families on the basis of its physical characteristics. These families include the chondroitin sulfate PGs, which appear to inhibit the migration of cells and axons during development. Although aggrecan has been studied primarily in cartilage, in the present study, tissue samples from developing, mature, and injured-adult rat spinal cords were used to determine whether aggrecan is present in the mammalian spinal cord. By the use of Western blot analysis, tissues were probed with aggrecan-specific antibodies (ATEGQV, TYKHRL, and LEC-7) and aggrecan-specific neoepitope antibodies (NITEGE, FVDIPEN, and TFKEEE) to identify full-length aggrecan and several fragments. Unlike many other aggrecan gene family members, aggrecan species were similar in embryonic day 14, postnatal day 1, and adult spinal cords. Spinal cord injury caused significant decreases in aggrecan. Partial recovery in some aggrecan species was evident by 2 weeks after injury. The presence of specific aggrecan neoepitopes suggested that aggrecan is cleaved in the spinal cord by both a disintegrin and metalloproteinase thrombospondin (also known as aggrecanase) and metalloproteinase-like activities. Many aggrecan species found in the spinal cord were similar to species in cartilage. Additional antibodies were used to identify two other aggrecan gene family members, neurocan and brevican, in the adult spinal cord. These studies present novel information on the aggrecan core protein species and enzymes involved in aggrecan cleavage in vivo in the rat spinal cord throughout development and after injury. They also provide the basis for investigating the function of aggrecan in the spinal cord.

Degradation of proteoglycan in articular cartilage.

Adult rabbit articular cartilage was labelled in vivo over 48 h with [35S]sulphate and was then incubated in organ culture at pH 7.2. Approx. 65% of the tissue content of [35S]proteoglycan was released into the culture medium during the first 48 h of incubation. The average molecular size of the released proteoglycans, as assessed by fractionation on Sepharose 2B/CL and 4B/Cl, was only slightly smaller than that of the proteoglycans extracted from non-cultured cartilage with 4 M guanidine HCl. The percentage of released proteoglycans and extracted proteoglycans which formed aggregates with hyaluronic acid was approx. 25% and 75%, respectively. The results indicate that proteoglycan degradation in adult articular cartilage is initiated by a limited proteolysis of subunit core protein, with the production of non-aggregating species which diffuse readily from the tissue.

Structural studies on proteoglycan catabolism in rabbit articular cartilage explant cultures.

Mature rabbit articular cartilage cultures have been used to study the catabolism of aggregating proteoglycan monomers in normal cartilage. During the first 4 days of culture, about 40% of monomers are degraded and lose the ability to bind to hyaluronate. The non-aggregating products (NAgg-PG) have been isolated and compared structurally and immunologically to aggregating monomers (Agg-PG) purified from fresh tissue. The results show that: (1) NAgg-PG are smaller, more heterogeneous in size and have a lower protein/glycosaminoglycan ratio than Agg-PG. (2) NAgg-PG and Agg-PG have a very similar chondroitin sulfate/keratan sulfate ratio. (3) NAgg-PG have 25-50% lower disulfide content than Agg-PG. (4) NAgg-PG have only about 20% of the reactivity of Agg-PG towards a monoclonal antibody (12-20/1-C-6) specific for the hyaluronate binding region of the core protein. These results provide further evidence that proteoglycan catabolism in cartilage explants involves proteolysis of core protein resulting in separation of the hyaluronate binding region from the glycosaminoglycan-rich regions.

Aggrecan in bovine tendon.

Large proteoglycans were purified by ion-exchange chromatography, gel filtration and CsCl gradient centrifugation from the compressed and tensional regions of adult bovine deep flexor tendon. Tryptic peptide maps of proteoglycan from the compressed region were very similar to maps of aggrecan from bovine articular cartilage, with evidence for the presence of all fifteen previously identified markers from the G1, G2 and G3 domains. The presence of aggrecan in these samples was confirmed by sequencing the G1 peptide YPIHTPR. The equivalent maps for large proteoglycan from tensional tendon were also consistent with the presence of aggrecan, and this was confirmed by sequencing three marker peptides from each of the G2 and G3 domains. However, G1 marker peptides were conspicuously absent from tensional samples. Northern blots for aggrecan mRNA showed high levels in cells from compressed tendon and articular cartilage. Extended exposure revealed a lower level of hybridization to RNA from tensional tendon as well. The results confirm that aggrecan, which is similar in core protein structure to articular cartilage aggrecan, is the predominant chondroitin sulfate-bearing large proteoglycan of compressed tendon. The results also indicate that aggrecan fragments lacking the G1 domain can account for the small amounts of chondroitin sulfate-bearing large proteoglycan in tensional regions of adult tendon.

Association between protease-specific proteolytic cleavage of brevican and synaptic loss in the dentate gyrus of kainate-treated rats.

Proteolytic fragments generated by ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identified, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEAVESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS-cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate-putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate.Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by specific matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS-induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region.

Physical and biological regulation of proteoglycan turnover around chondrocytes in cartilage explants. Implications for tissue degradation and repair.

The development of clinical strategies for cartilage repair and inhibition of matrix degradation may be facilitated by a better understanding of (1) the chondrocyte phenotype in the context of a damaged extracellular matrix, and (2) the roles of biochemical and biomechanical pathways by which matrix metabolism is mediated. Using methods of quantitative autoradiography, we examined the cell-length scale patterns of proteoglycan deposition and turnover in the cell-associated matrices of chondrocytes in adult bovine and calf cartilage explants. Results highlight a rapid turnover in the pericellular matrix, which may indicate spatial organization of PG metabolic pools, and specific biomechanical roles for different matrix regions. Subsequent to injurious compression of calf explants, which resulted in grossly visible tissue cracks and caused a decrease in the number of viable chondrocytes within explants, cell-mediated matrix catabolic processes appeared to increase, resulting in apparently increased rates of proteoglycan turnover around active cells. Furthermore, the influences of cell-stimulatory factors such as IL-1 beta appeared to be delayed in their effects subsequent to injurious compression, suggesting interactions between biomechanical and biochemical pathways of PG degradation. These results may provide a useful reference point in the development of in vitro models for cartilage injury and disease, and hint at possible new approaches in the development of cartilage repair strategies.

Age-related changes in the kinetics of release of proteoglycans from normal rabbit cartilage explants.

The release of proteoglycans from explant cultures of articular cartilage from immature and mature rabbits has been studied with the following results. At both ages the tissue proteoglycan was released in two phases: an initial extensive release (day 0 to 3) and a period of slow release (day 4 to 15). The percentage released in the initial phase was, however, significantly greater for mature (55%) than immature (38%) explants. At both ages the newly synthesized proteoglycans (in vivo labeled) were also released in two kinetic pools. Thus, graphical analysis of release data readily resolved the disappearance curves into two linear components with in vitro half-lives of 1 day and 22 days. Again, the percentage in the short half-life pool was much greater for mature (70%) than immature (40%) explants. At both ages the initial release was largely chondrocyte-mediated since freeze-thawing the tissue before culture markedly reduced proteoglycan release. At both ages the released proteoglycans were smaller than equivalent preparations of extracted proteoglycans and they were much less capable of forming aggregates with hyaluronate. The results show that there are age-dependent changes in rabbit articular cartilage that increase the proportion of proteoglycans, both total and newly synthesized, that are susceptible to rapid chondrocyte-mediated catabolism in explant cultures.

Structure of newly synthesised (35S)-proteoglycans and (35S)-proteoglycan turnover products of cartilage explant cultures from dogs with experimental osteoarthritis.

The structure of newly synthesised proteoglycans from explant cultures of cartilage from joints subjected to transection of the anterior cruciate ligament (osteoarthritic) and from normal (non- or sham-operated) joints was examined. The structure of the products of proteoglycan turnover was also examined using explants of normal and osteoarthritic cartilage maintained in culture for a 48 h chase period. The findings were as follows: Newly synthesised (35S)-proteoglycans extracted from cartilage explants from osteoarthritic joints whether examined 3 weeks, 3 months, or 6 months after surgery were larger than those from corresponding normal cartilage. This can be explained by the synthesis in osteoarthritic cartilage of abnormally long chondroitin sulphate chains on newly synthesised proteoglycans. The extracts also contained a newly formed small proteoglycan species that was unable to interact with hyaluronic acid. The proportion of this species was higher in osteoarthritic cartilage compared with normal, examined 3 weeks after surgery, but was generally absent from cartilage obtained 3 and 6 months after surgery. Compared with controls, a smaller proportion of the (35S)-proteoglycans released into the maintenance medium of explant cultures of osteoarthritic cartilage during a 48 h chase period was able to interact with hyaluronic acid. However, although furnished with longer (35S)-glycosaminoglycan chains, these proteoglycans were smaller than those from control explants.

The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage.

We have examined extracts of articular cartilage from rabbits aged 3-100 weeks for evidence of age-related changes in the structure and content of link protein (LP) in this tissue, with the following findings: (a) Two major molecular weight forms of LP were seen on SDS-PAGE (41 and 48 kDa) and the proportion of these changed markedly with age. The 48 kDa species was predominant in young animals (representing about 78% of the total LP at 5 weeks) whereas the 41 kDa species increased in amount with age (representing 35% of the total LP at 100 weeks). A minor form of about 43 kDa, representing less than 20% of the total, was present only during the growth phase. A small amount of fragmented link protein (less than 5% of the total) of about 25-30 kDa was present in samples from mature and aged rabbits only. (b) The quantitation of LP in guanidinium: HCl extracts of cartilage, by radioimmunoassay with monoclonal antibody 8-A-4, was markedly influenced by the conditions of preparation and pretreatment of samples. Assays of dialyzed guanidine extracts following treatment at 80 degrees C for 15 min in 0.025% (w/v) SDS indicated that immature and mature cartilage contains about 50 and 180 micrograms of LP/g of tissue, respectively. On the other hand, assays following treatment at 100 degrees C for 20 min in 0.1% (w/v) SDS suggested that rabbit cartilage contains about 300 micrograms of LP/g of tissue at all ages; finally, assay of CsCl purified proteoglycan samples under these conditions indicated a content of about 500 micrograms of LP/g at all ages. (c) Calculations based on the analysis of proteoglycan preparations for aggregating monomer and link protein suggest that a LP:aggregating monomer molar ratio of about 0.9 is maintained in the articular cartilage throughout maturation and aging in the rabbit.

Demonstration of increased proteoglycan turnover in cartilage explants from dogs with experimental osteoarthritis.

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants.

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.