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Objectives: To determine (1) if interleukin-1 alpha (IL-1alpha), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-beta1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion., Methods: Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10ng/ml IL-1alpha, 300ng/ml IGF-I, or 10ng/ml TGF-beta1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot., Results: PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1alpha, stimulated by TGF-beta1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion., Conclusions: Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair.

The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules, found both in synovial fluid (SF) and bound to the articular cartilage surface. Currently the mechanism by which PRG4 binds to the articular surface is not well understood. The objectives of this study were to determine (1) the effect of bathing fluid contents on PRG4 concentration at the articular surface ([PRG4](cart)), and (2) whether native PRG4 can be removed from the surface and subsequently repleted with PRG4 from synovial fluid. In one experiment, cylindrical cartilage disks were stored in solutions of various PRG4 concentrations, either in phosphate-buffered saline (PBS) or SF as the carrier fluid. In a separate experiment, cartilage disks were stored in solutions expected to remove native PRG4 from the articular surface and allow subsequent repletion with PRG4 from SF. [PRG4](cart) was independent of PRG4 concentration of the bathing fluid, and was similar for both carrier fluids. PRG4 was removed from cartilage by treatment with hyaluronidase, reduction/alkylation, and sodium dodecyl sulphate, and was repleted fully by subsequent bathing in SF. These results suggest that the articular surface is normally saturated with tightly bound PRG4, but this PRG4 can exchange with the PRG4 in SF under certain conditions. This finding suggests that all tissues surrounding the joint cavity that secrete PRG4 into the SF may help to maintain lubrication function at the articular surface.

Articular cartilage is a heterogeneous tissue with superficial (S), middle (M), and deep (D) zones. Chondrocytes in the S zone secrete the lubricating PRG4 protein, while chondrocytes from the M and D zones are more specialized in producing large amounts of the glycosaminoglycan (GAG) component of the extracellular matrix. Soluble and insoluble chemicals and mechanical stimuli regulate cartilage development, growth, and homeostasis; however, the mechanisms of regulation responsible for the distinct PRG4-positive and negative phenotypes of chondrocytes are unknown. The objective of this study was to determine if interaction between S and M chondrocytes regulates chondrocyte phenotype, as determined by coculture in monolayer at different ratios of S:M (100:0, 75:25, 50:50, 25:75, 0:100) and at different densities (240,000, 120,000, 60,000, and 30,000 cells/cm(2)), and by measurement of PRG4 secretion and expression, and GAG accumulation. Coculture of S and M cells resulted in significant up-regulation in PRG4 secretion and the percentage of cells expressing PRG4, with simultaneous down-regulation of GAG accumulation. Tracking M cells with PKH67 dye in coculture revealed that they maintained a PRG4-negative phenotype, and proliferated less than S cells. Taken together, these results indicate that the up-regulated PRG4 expression in coculture is a result of preferential proliferation of PRG4-expressing S cells. This finding may have practical implications for generating a large number of phenotypically normal S cells, which can be limited in source, for tissue engineering applications., ((c) 2007 Orthopaedic Research Society.)

Distinguishing between implanted and host-derived cells, as well as between distinct cell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. The fluorescent tracker dye, PKH26, was previously applied to several cell types to assess proliferation in vitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 for tracking chondrocytes from superficial and middle zones and their proliferation, and determine the effects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan 4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated in either low- or high-density monolayer culture and analyzed for retention of PKH26 by flow cytometry and fluorescence microscopy at days 0 and 7. Cell suspensions and conditioned media were analyzed for DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so that the number of cells in each doubling generation contributing to the final cell population could be estimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles of division. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could be distinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cell fluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis of PRG4 suggest that PKH26 can be useful in determining the fate and function of implanted chondrocytes in vivo, as well as monitoring proliferation in vitro., (Copyright (c) 2006 Orthopaedic Research Society.)

Objective: The boundary lubrication function of articular cartilage is mediated in part by proteoglycan 4 (PRG4) molecules at the articular surface and in synovial fluid. The objective of this study was to determine the effects of dynamic shear stimulation on PRG4 biosynthesis by bovine cartilage explants., Methods: Cartilage disks with intact articular surfaces were harvested from immature bovines. Some disks were subjected to 24 hours (day 1) of loading, consisting of a step load of 20% static compression either alone or with superimposed dynamic shear (3% amplitude and 0.1 Hz), while other disks were cultured free-swelling as controls. After the 24-hour loading period, disks were terminated or were further incubated for up to 72 hours (days 2-4) in free-swelling culture to assess chondrocyte responses to, and following, unloading. PRG4 products secreted into culture medium were quantified by enzyme-linked immunosorbent assay and characterized by Western blotting. Chondrocytes expressing PRG4 were localized by immunohistochemistry, and depth-associated variations in chondrocyte PRG4 expression were quantified by image analysis., Results: Dynamic shear stimulation increased PRG4 secretion to 3-4 times that of unloaded controls and statically compressed samples. Sheared cartilage secreted more PRG4 of 345 kd relative to smaller molecular weight species, as compared with unloaded controls. Immunohistochemistry revealed that shear stimulation also increased the total number of cells expressing PRG4 by inducing expression by cells at a depth of 200-400 microm., Conclusion: The paradigm that certain mechanical stimuli up-regulate biosynthesis in cartilage appears operative not only for load-bearing matrix constituents, but also for PRG4 molecules that mediate lubrication.

Articular cartilage provides a low-friction surface for joint articulation, with boundary lubrication facilitated by proteoglycan 4 (PRG4), which is secreted by chondrocytes of the superficial zone. Chondrocytes from different zones are phenotypically distinct, and their phenotypes in vitro are influenced by the system in which they are cultured. We hypothesized that culturing cells from the superficial (S) zone in two-dimensional monolayer or three-dimensional alginate would affect their synthesis of PRG4, and that subsequently seeding them atop alginate-recovered cells from the middle/ deep (M) zone in various proportions would result in tissue-engineered constructs with varying levels of PRG4 secretion and matrix accumulation. During monolayer culture, S cells retained their PRG4-secreting phenotype, whereas in alginate culture the percentage of cells secreting PRG4 decreased with time. Constructs formed with increasing percentages of S cells decreased in thickness and matrix accumulation, depending on both the culture conditions before construct formation and the S-cell density. PRG4-secreting cells were localized to the S-cell seeded construct surface, with secretion rates of 0.1-4 pg/cell/day or 0.1-1 pg/cell/day for constructs formed with monolayer-recovered or alginate-recovered S cells, respectively. Tailoring secretion of PRG4 in cartilage constructs may be useful for enhancing low-friction properties at the articular surface, while maintaining other surfaces free of PRG4 for enhancing integration with surrounding tissues.

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.

Proteoglycan 4 (PRG4) is synthesized and secreted into the synovial fluid by articular chondrocytes and synovial cells, lining the cavity of joints. A thin layer of PRG4 is also present at the articular surface, where it appears to be involved in boundary lubrication. This study investigated if PRG4 is also synthesized and secreted by the cells within meniscus, and if PRG4 is also present in, and at the surface of, meniscus. PRG4 was visualized in sections of bovine calf menisci by immunohistochemistry. PRG4 was detected in two regions: (1) at the femoral and tibial surfaces of the meniscus, and within cells below these surfaces; and (2) within and near cells along the radial tie fibers and circumferential fibers. From meniscus tissue harvested from these surfaces, PRG4 was extracted with 4M GuHCl and quantified by ELISA. There was 0.20 +/- 0.01 and 0.25 +/- 0.04 microg PRG4/cm(2) area of lateral and medial meniscus surface, respectively. ELISA analysis of spent medium from other samples of meniscus surface tissue incubated in medium supplemented with serum and ascorbate showed that 8.1 +/- 1.1 microg PRG4/cm(2) area of meniscus surface was secreted over six days. These results demonstrate that PRG4 is synthesized and secreted by certain cell populations in the meniscus, and that PRG4 is present in the meniscus at surfaces and also internal fibers where it may contribute to boundary lubrication.

Objective: To determine the effects of the articular cartilage surface, as well as synovial fluid (SF) and its components, specifically proteoglycan 4 (PRG4) and hyaluronic acid (HA), on integrative cartilage repair in vitro., Methods: Blocks of calf articular cartilage were harvested, some with the articular surface intact and others without. Some of the latter types of blocks were pretreated with trypsin, and then with bovine serum albumin, SF, PRG4, or HA. Immunolocalization of PRG4 on cartilage surfaces was performed after treatment. Pairs of similarly treated cartilage blocks were incubated in partial apposition for 2 weeks in medium supplemented with serum and (3)H-proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength, and protein biosynthesis and deposition were determined by incorporated (3)H-proline., Results: Samples with articular surfaces in apposition exhibited little integrative repair compared with samples with cut surfaces in apposition. PRG4 was immunolocalized at the articular cartilage surface, but not in deeper, cut surfaces (without treatment). Cartilage samples treated with trypsin and then with SF or PRG4 exhibited an inhibition of integrative repair and positive immunostaining for PRG4 at treated surfaces compared with normal cut cartilage samples, while samples treated with HA exhibited neither inhibited integrative repair nor PRG4 at the tissue surfaces. Deposition of newly synthesized protein was relatively similar under conditions in which integration differed significantly., Conclusion: These results support the concept that PRG4 in SF, which normally contributes to cartilage lubrication, can inhibit integrative cartilage repair. This has the desirable effect of preventing fusion of apposing surfaces of articulating cartilage, but has the undesirable effect of inhibiting integrative repair.

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems., Methods: Bovine calf articular cartilage discs or isolated cells were used in 1 of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells., Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from approximately 25 microg/cm(2)/day on days 0-1 to approximately 3 microg/cm(2)/day on days 5-9), while increasing in monolayer culture (from approximately 1 pg/cell/day on days 0-1 to approximately 7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching approximately 2 microg/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells., Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

We have performed cDNA sequencing and homology analyses to elucidate the complete amino acid composition for a superficial zone protein (SZP) from human and bovine cartilage which has previously been shown to be a proteoglycan specifically synthesized by chondrocytes located at the surface of bovine articular cartilage and also some synovial lining cells. The results of this study indicate that cartilage SZP is homologous with a glycoprotein first described as the precursor protein of a megakaryocyte stimulating factor (MSF). Sequence comparisons and analyses indicate that (i) the amino acid composition of SZP is highly conserved between bovine and human species, (ii) SZP contains structural motifs at the N- and C-termini which are similar to those found in vitronectin and which may impart cell-proliferative and matrix-binding properties to the molecule, and (iii) SZP contains large and small mucin-like repeat domains composed of the sequences KEPAPTTT/P (76-78 repeats) and XXTTTX (6-8 repeats), respectively, which occur within a large central region of approximately 940 amino acids. The mucin-like domains are likely to be substituted with O-linked oligosaccharides which would impart lubricating properties to SZP which in part accumulates at the articular cartilage-synovial fluid interface. Additionally, we have shown that interleukin-1 inhibits the biosynthesis of chondrocyte SZP, while TGF-beta and IGF-1 increase its biosynthesis, and that in pathological (osteoarthritic) human articular cartilage SZP mRNA can be expressed as an alternatively spliced variant lacking exons 4 and 5 which encode a potential heparin binding domain. The occurrence of different SZP alternative splice variants and the differential expression of SZP in the presence of cytokines and growth factors suggest that SZP may play an important cytoprotective role by preventing cellular adhesion to the articular cartilage surface in normal cartilage metabolism. Modifications to the structure of SZP, coupled with inhibition of SZP synthesis during inflammation, may account for the attachment and invasion of pannus observed in inflammatory joint diseases., (Copyright 1999 Academic Press.)

We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.

A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4 degrees C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.

Human and bovine adult articular chondrocytes cultured in alginate beads or agarose gel retain their spherical shape and typical chondrocytic appearance for at least 5 weeks. Aggrecan is always the major population of proteoglycans (PGs) synthesized; its size varies depending upon the age of the cartilage from which the cells are derived but it is not influenced by the culture system used. Studies of human chondrocytes cultured in alginate showed that the majority of the newly-synthesized aggrecan molecules are rapidly incorporated into aggregates which can be extracted from the gel in their native form. About one-third of the aggregates formed during a 4-hour period of incubation in the presence of [35S]-sulfate are packed at high concentration in the cell-associated matrix; the remainder is present in areas of the gel further removed from the cells. Aggrecan appears to be turned over more slowly by human than by bovine cells in both culture systems; in both species, the rate of turnover is slower in alginate than in agarose. Decorin is also synthesized in small amounts but it is rapidly lost from the agarose or alginate gel.