The effects of trypsin treatment on proteoglycan biosynthesis by bovine articular cartilage.
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ABSTRACT:
SUBMITTER: Bartholomew JS
PROVIDER: S-EPMC1144862 | biostudies-other | 1985 Apr
REPOSITORIES: biostudies-other
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Project description:Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.
| S-EPMC1147052 | biostudies-other
Project description:The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.
| S-EPMC1147434 | biostudies-other
Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.
| S-EPMC4778950 | biostudies-literature
Project description:The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (?(PG)), and the collagen network (CN) provides the restraining forces to counterbalance ?(PG). Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-?(PG) relationship; 2), compute ?(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on ?(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and ?(PG) increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and ?(PG) decreased. The ?(PG)-values were close to equilibrium stress (?(EQ)) in all bovine and young human cartilage, but were only approximately half of ?(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), ?(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-?(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage.
| S-EPMC3175069 | biostudies-literature
Project description:Articular cartilage is the soft tissue that covers contacting surfaces of bones in synovial joints. Cartilage is composed of chondrocytes and an extracellular matrix containing numerous biopolymers, cations and water. Healthy cartilage functions biomechanically to provide smooth and stable joint movement. Degenerative joint diseases such as osteoarthritis involve cartilage deterioration, resulting in painful and cumbersome joint motion. Temperature is a fundamental quantity in mechanics, yet the effects of temperature on cartilage mechanical behavior are unknown. This study addressed the questions of whether cartilage stiffness and stress relaxation change with temperature. Samples of middle-zone bovine calf patellofemoral cartilage were tested in unconfined compression first at 24°C and then again after heating to 60°C. The data reveal that when temperature increases: (1) both peak and equilibrium stiffness increase by 150 and 8%, respectively, and (2) stress relaxation is faster at higher temperature, as shown by a 60% decrease in the time constant. The increases in temperature-dependent stiffness are consistent with polymeric mechanisms of matrix viscoelasticity but not with interstitial fluid flow. The changes in the time constant are consistent with a combination of both fluid flow and matrix viscoelasticity. Furthermore, we discovered a novel phenomenon: at stress-relaxation equilibrium, compressive stress increased with temperature. These data demonstrate a rich area of cartilage mechanics that has previously been unexplored and emphasize the role of polymer dynamics in cartilage viscoelasticity. Further studies of cartilage polymer dynamics may yield additional insight into mechanisms of cartilage material behavior that could improve treatments for cartilage degeneration.
| S-EPMC2966351 | biostudies-literature
Project description:1. Gel electrophoresis of proteoglycans extracted with use of 4 M-guanidinium chloride from baboon (Papio papio) articular cartilage and purified on DEAE-cellulose in 8 M-urea yielded three bands on electrophoresis in polyacrylamide/agarose gels: two wide bands close together (I and II) and a third, thinner and more rapidly moving band (III). 2. Gel electrophoresis of fractions from direct 'dissociative' gradients showed that these bands were partially separated (buoyant density of I greater than II greater than III). 3. Reduction and alkylation of proteoglycans did not alter either the gel-electrophoretic pattern or the distribution of the bands in the fractions of the gradient. 4. Band III was found in the upper third of 'associative' gradients but not in the bottom fraction, which yielded after dissociation only bands I and II. 5. The third band was completely extracted for 24h with an iso-osmotic solution, but was contaminated with bands I and II. The second extraction step with 4M-guanidinium chloride yielded only bands I and II. 6. The data strongly suggest the presence in the articular cartilage of several populations of dissociated proteoglycans differing in gel-electrophoretic migration, buoyant density and aggregation capacity.
| S-EPMC1164664 | biostudies-other
Project description:Proteoglycans were prepared from human femoral-head articular cartilage by using either guanidinium hydrochloride or MgCl2 as extractant, followed by density-gradient centrifugation. The proteoglycan subunit had a particle weight of 2.6 x 10(6), with a radius of gyration, RG, of 68.5 nm in 150 mM-NaCl/20 mM-sodium phosphate buffer, pH 7.0. The proteoglycan aggregate had a particle weight of 3.7 x 10(6) (RG 84 nm) for guanidinium hydrochloride extracts and 8.7 x 10(6) (RG 118 nm) for MgCl2 extracts in the same buffer. The addition of excess of high-molecular-weight hyaluronate did not significantly alter the particle size of the aggregate. The small increase in size probably reflects a rapid equilibrium between hyaluronate and proteoglycan monomers, and is not due to proteolytic cleavage producing non-aggregating units. Experiments that support the rapid-interaction hypothesis include analytical ultracentrifugation and column chromatography. This interaction does not appear to be pressure-sensitive at 20 degrees C, but is sensitive to temperature variation near the physiological range.
| S-EPMC1163387 | biostudies-other
Project description:Water, collagen, and proteoglycans determine articular cartilage functionality. If altered, susceptibility to premature degeneration is increased. This study investigated the effects of enzymatic proteoglycan depletion on cartilage functionality as assessed by advanced Magnetic Resonance Imaging (MRI) techniques under standardized loading. Lateral femoral condylar cartilage-bone samples from patients undergoing knee replacement (n?=?29) were serially imaged by Proton Density-weighted and T1, T1?, T2, and T2* mapping sequences on a clinical 3.0 T MRI scanner (Achieva, Philips). Using pressure-controlled indentation loading, samples were imaged unloaded and quasi-statically loaded to 15.1 N and 28.6 N, and both before and after exposure to low-concentrated (LT, 0.1 mg/mL, n?=?10) or high-concentrated trypsin (HT, 1.0 mg/mL, n?=?10). Controls were not treated (n?=?9). Responses to loading were assessed for the entire sample and regionally, i.e. sub- and peri-pistonally, and zonally, i.e. upper and lower sample halves. Trypsin effects were quantified as relative changes (?), analysed using appropriate statistical tests, and referenced histologically. Histological proteoglycan depletion was reflected by significant sub-pistonal decreases in T1 (p?=?0.003) and T2 (p?=?0.008) after HT exposure. Loading-induced changes in T1? and T2* were not related. In conclusion, proteoglycan depletion alters cartilage functionality and may be assessed using serial T1 and T2 mapping under loading.
| S-EPMC7492285 | biostudies-literature
Project description:INTRODUCTION: The zone of calcified cartilage (ZCC) anchors articular cartilage (AC) to subchondral bone through a layer of intermediate stiffness. The regulation and functional consequences of cartilage calcification may vary with depth from the articular surface. The hypothesis of this study was that the in vitro calcification of immature AC occurs selectively in the deep region and is associated with a local increase in stiffness. METHODS: AC and growth plate cartilage (GPC) from calves were incubated in DMEM, 1% FBS, 100?g/mL ascorbate, and ±10mM ß-glycerophosphate (ßGP) for up to 3 weeks. To assess the time course and effects of cell viability and ßGP, full-depth strips of AC and GPC were analyzed by histology, indentation, and (45)Ca(++) uptake. To assess the effect of tissue zone, disks harvested from surface and deep zone AC and from reserve and hypertrophic zone of GPC were incubated independently and analyzed by compression and for (45)Ca(++) uptake and biochemical components. RESULTS: The deep ~20% of immature AC calcified within 3 weeks, with calcification dependent on cell viability and ßGP. Mineral was deposited continuously around cells in AC but only between cell columns in GPC. The deep zone of AC exhibited a compressive modulus of 0.53 MPa after ßGP-induced calcification, ~4-fold stiffer than AC incubated without ßGP. CONCLUSIONS: Cartilage explants exhibit inherent zone-specific calcification processes, resulting in an increase in stiffness associated with cartilage calcification. Such properties may be useful for engineering a biomimetic ZCC tissue to integrate cartilaginous tissue to bone, thereby forming a mechanically functional osteochondral unit.
| S-EPMC3382981 | biostudies-literature
Project description:Osteoarthritis (OA), the commonest form of arthritis and a major cause of morbidity, is characterized by progressive degeneration of the articular cartilage. Along with increased production and activation of degradative enzymes, altered synthesis of cartilage matrix molecules and growth factors by resident chondrocytes is believed to play a central role in this pathological process. We used an ovine meniscectomy model of OA to evaluate changes in chondrocyte expression of types I, II and III collagen; aggrecan; the small leucine-rich proteoglycans (SLRPs) biglycan, decorin, lumican and fibromodulin; transforming growth factor-beta; and connective tissue growth factor. Changes were evaluated separately in the medial and lateral tibial plateaux, and were confirmed for selected molecules using immunohistochemistry and Western blotting. Significant changes in mRNA levels were confined to the lateral compartment, where active cartilage degeneration was observed. In this region there was significant upregulation in expession of types I, II and III collagen, aggrecan, biglycan and lumican, concomitant with downregulation of decorin and connective tissue growth factor. The increases in type I and III collagen mRNA were accompanied by increased immunostaining for these proteins in cartilage. The upregulated lumican expression in degenerative cartilage was associated with increased lumican core protein deficient in keratan sulphate side-chains. Furthermore, there was evidence of significant fragmentation of SLRPs in both normal and arthritic tissue, with specific catabolites of biglycan and fibromodulin identified only in the cartilage from meniscectomized joints. This study highlights the focal nature of the degenerative changes that occur in OA cartilage and suggests that altered synthesis and proteolysis of SLRPs may play an important role in cartilage destruction in arthritis.
| S-EPMC1175037 | biostudies-literature