Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)     

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)     

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit articular cartilage.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.     

Osteogenic protein 1 in synovial fluid from patients with rheumatoid arthritis or osteoarthritis: relationship with disease and levels of hyaluronan and antigenic keratan sulfate

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1 – mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.     

Regulation of osteogenic proteins by chondrocytes

The purpose of this review is to summarize the current scientific knowledge of bone morphogenetic proteins (BMPs) in adult articular cartilage. We specifically focus on adult cartilage, since one of the major potential applications of the members of the BMP family may be a repair of adult tissue after trauma and/or disease. After reviewing cartilage physiology and BMPs, we analyze the data on the role of recombinant BMPs as anabolic agents in tissue formation and restoration in different in vitro and in vivo models following with the endogenous expression of BMPs and factors that regulate their expression. We also discuss recent transgenic modifications of BMP genes and subsequent effect on cartilage matrix synthesis. We found that the most studied BMPs in adult articular cartilage are BMP-7 and BMP-2 as well as transforming growth factor-beta (TGF-beta). There are a number of contradicting reports for some of these growth factors, since different models, animals, doses, time points, culture conditions and devices were used. However, regardless of the experimental conditions, only BMP-7 or osteogenic protein-1 (OP-1) exhibits the most convincing effects. It is the only BMP studied thus far in adult cartilage that demonstrates strong anabolic activity in vitro and in vivo with and without serum. OP-1 stimulates the synthesis of the majority of cartilage extracellular matrix proteins in adult articular chondrocytes derived from different species and of different age. OP-1 counteracts the degenerative effect of numerous catabolic mediators; it is also expressed in adult human, bovine, rabbit and goat articular cartilage. This review reveals the importance of the exploration of the BMPs in the cartilage field and highlights their significance for clinical applications in the treatment of cartilage-related diseases.     

IL-1beta induction of IL-6 and LIF in normal articular human chondrocytes involves the ERK, p38 and NFkappaB signaling pathways

Interleukin-1 (IL-1) is an important catabolic cytokine in rheumatoid and osteoarthritic joint disease. Besides inducing a catabolic response in articular chondrocytes it also strongly induces synergistic mediators such as leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). The molecular basis of this is so far hardly understood. The aim of our study was to evaluate in vitro and in vivo whether IL-6 and LIF are differentially expressed in normal human and osteoarthritic adult articular chondrocytes and to investigate the potential intracellular signaling pathways of IL-1 involved in these gene regulation events. IL-6 and LIF mRNA expressions were found only at low levels in normal adult articular cartilage. Neither IL-6 nor LIF was strongly over-expressed in osteoarthritic cartilage degeneration. Clearly, both IL-6 and LIF can be very efficiently induced by IL-1beta in articular chondrocytes in vitro. However, this induction was somewhat less in osteoarthritic cells, which were overall activated in terms of expression of both cytokines without stimulation. Experiments using pathway selective inhibitors showed that intracellular signaling of IL-1beta for IL-6 and LIF is mediated by a mixture of the IL-1 signaling cascades. However, the ERK-pathway appeared to be particularly important and might be, therefore, of particular potential if one intends to block induction of these molecules by IL-1 in arthritic joint disease.     

Del1: a new protein in the superficial layer of articular cartilage

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.     

Regulation of chondrocyte gene expression by osteogenic protein-1

Introduction  

The objective of this study was to investigate which genes are regulated by osteogenic protein-1 (OP-1) in human articular chondrocytes using Affimetrix gene array, in order to understand the role of OP-1 in cartilage homeostasis.     

Glenohumeral joint cartilage contact in the healthy adult during scapular plane elevation depression with external humeral rotation

The shoulder (glenohumeral) joint has the greatest range of motion of all human joints; as a result, it is particularly vulnerable to dislocation and injury. The ability to non-invasively quantify in-vivo articular cartilage contact patterns of joints has been and remains a difficult biomechanics problem. As a result, little is known about normal in-vivo glenohumeral joint contact patterns or the consequences that surgery has on altering them. In addition, the effect of quantifying glenohumeral joint contact patterns by means of proximity mapping, both with and without cartilage data, is unknown. Therefore, the objectives of this study are to (1) describe a technique for quantifying in-vivo glenohumeral joint contact patterns during dynamic shoulder motion, (2) quantify normal glenohumeral joint contact patterns in the young healthy adult during scapular plane elevation depression with external humeral rotation, and (3) compare glenohumeral joint contact patterns determined both with and without articular cartilage data. Our results show that the inclusion of articular cartilage data when quantifying in-vivo glenohumeral joint contact patterns has significant effects on the anterior–posterior contact centroid location, the superior–inferior contact centroid range of travel, and the total contact path length. As a result, our technique offers an advantage over glenohumeral joint contact pattern measurement techniques that neglect articular cartilage data. Likewise, this technique may be more sensitive than traditional 6-Degree-of-Freedom (6-DOF) joint kinematics for the assessment of overall glenohumeral joint health. Lastly, for the shoulder motion tested, we found that glenohumeral joint contact was located on the anterior–inferior glenoid surface.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Topographic localization of a 116,000-dalton protein in cartilage

A disulfide-bonded greater than 400,000-dalton (greater than 400-kD) protein with 116-kD subunits in hyaline cartilage from several species has recently been described. It constitutes 2-4% of the total noncollagenous protein in 4 M guanidinium chloride extracts of normal articular cartilage and accounts for most of the total noncollagen, nonproteoglycan protein synthesized in short-term organ cultures of canine articular cartilage. In the present study, immunofluorescence techniques were used to examine the topographic distribution of the 116-kD subunit protein in normal cartilage. In specimens of normal adult articular cartilage from several species, the protein was located throughout the matrix. More intense staining was observed at the articular surface than in the remainder of the uncalcified cartilage. In contrast, in fetal cartilage, the protein was uniformly distributed throughout the matrix without a marked increase in surface staining. Normal canine menisci and annulus fibrosus also demonstrated moderate fluorescence after incubation with the antiserum to the 116-kD subunit protein. Normal canine nucleus pulposus, synovium, aorta, and monolayer cultures of canine synovial cells exhibited only weak immunofluorescence after incubation with the antiserum. Therefore, the 116-kD subunit protein appears to be a ubiquitous matrix protein in cartilage.     

Lectin-binding in normal and osteoarthrotic articular cartilage from STR/1N-mouse knee joints

Fluorescein-isothiocyanate (FITC) labeled lectins were used to study the distribution pattern of specific binding-sites in histological sections of normal and osteoarthrotic articular cartilage from the mouse knee joint. Male inbred mice of the STR/1N-strain develop spontaneous arthrotic articular cartilage lesions on the medial condyle of tibia and femur. The varus-deformity of the knee joint leads to a recurrent medial patellar luxation with osteoarthrotic defects on the medial part of the facies patellaris femoris. It was demonstrated that the lectin staining pattern of osteoarthrotic articular cartilage, especially on the facies patellaris femoris, was different from that of normal articular cartilage. The differences in lectin staining corresponded to those observed between normal and fibrillated articular cartilage from human patellae. The normal articular cartilage of the mouse knee joint possessed lectin binding-sites for Concanavalin A (ConA) and wheat germ agglutinin (WGA), but not for Ulex europaeus agglutinin (UEA), soy bean agglutinin (SBA) and peanut agglutinin (PNA). In addition to the completely changed distribution pattern of ConA and WGA in osteoarthrotic cartilage, SBA, PNA and UEA developed distinct staining patterns particular to the fibrillated areas of arthrotic cartilage. The increased lectin-binding to arthrotic articular cartilage may be due to unmasking of sugars in the course of bondage breakdown in fibrillated cartilage or the production of pathological glycoproteins. It is evident that lectins can demonstrate minute differences between normal and arthrotic cartilage and it is concluded, therefore, that lectins are sensitive and specific tools for the study of degenerative joint diseases.     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

Identification of a high-molecular-weight (> 400 000) protein in hyaline cartilage

A high-molecular-weight (> 400 000) non-collagenous protein has been identified in normal articular cartilage from several mammalian species and in bovine tracheal cartilage. This protein is reduced by 2-mercaptoethanol to subunits with a molecular weight of 116 000, which appear to constitute approx. 2–4% of the total protein detectable by the Lowry assay in 4 M guanidinium chloride extracts of normal bovine and canine articular cartilage. Antiserum to the 116 kDa subunit protein from bovine articular cartilage cross-reacts with the intact and subunit proteins from bovine trachea and from normal canine, porcine and human articular cartilage. This protein is not found in non-cartilagenous tissues, suggesting that it is a cartilage-specific protein. We conclude that the > 400 kDa protein and its subunit are ubiquitous and quantitatively significant proteins in hyaline cartilage.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.