Extractable proteoglycan from human femoral-head articular cartilage.
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ABSTRACT:
SUBMITTER: Bullimore DW
PROVIDER: S-EPMC1163387 | biostudies-other | 1981 Nov
REPOSITORIES: biostudies-other
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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:Total hip arthroplasty is a common surgical technique, yet it has severe complications, such as loosening and repeated revision. Thus, hip-preserving surgical options should be considered first to treat cartilage defects in the femoral head, especially for younger patients. Current surgical options for chondral repair of the femoral head include microfracture, trapdoor procedure, transplantation of osteochondral allografts and autografts, and autologous chondrocyte implantation. Each of these techniques has unique advantages and limitations; however, none of them have been consented as the best practice for cartilage defects. In this review article, we also introduced a novel technique for repairing osteochondral defects of the femoral head using autologous costal cartilage grafts that may have good translational potential for cost-effective and safe applications. The translational potential of this article:This review updates current surgical options for reparing articular cartilage defects in the femoral head. We also introduce a novel technique for repairing osteochondral defects of the femoral head using autologous costal cartilage grafts.
| S-EPMC7152792 | biostudies-literature
Project description:Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a 'beaded' structure and a contiguous filamentous 'tail' at one end and others which appeared as round 'blobs', some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the 'blob-like' appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.
| S-EPMC1147373 | biostudies-other
Project description:ObjectiveIschemic osteonecrosis of the femoral head (ONFH) in piglets results in an ischemic injury to the immature articular cartilage. The molecular changes in the articular cartilage in response to ONFH have not been investigated using a transcriptomic approach. The purpose of this study was to perform a genome-wide transcriptomic analysis to identify genes that are upregulated in the immature articular cartilage following ONFH.MethodsONFH was induced in the right femoral head of 6-week old piglets. The unoperated femoral head was used as the normal control. At 24 hours (acute ischemic-hypoxic injury), 2 weeks (avascular necrosis in the femoral head) and 4 weeks (early repair) after surgery (n = 4 piglets/time point), RNA was isolated from the articular cartilage of the femoral head. A microarray analysis was performed using Affymetrix Porcine GeneChip Array. An enrichment analysis and functional clustering of the genes upregulated due to ONFH were performed using DAVID and STRING software, respectively. The increased expression of selected genes was confirmed by a real-time qRTPCR analysis.ResultsInduction of ONFH resulted in the upregulation of 383 genes at 24 hours, 122 genes at 2 weeks and 124 genes at 4 weeks compared to the normal controls. At 24 hours, the genes involved in oxidoreductive, cell-survival, and angiogenic responses were significantly enriched among the upregulated genes. These genes were involved in HIF-1, PI3K-Akt, and MAPK signaling pathways. At 2 weeks, secretory and signaling proteins involved in angiogenic and inflammatory responses, PI3K-Akt and matrix-remodeling pathways were significantly enriched. At 4 weeks, genes that represent inflammatory cytokines and chemokine signaling pathways were significantly enriched. Several index genes (genes that are upregulated at more than one time point following ONFH and are known to be important in various biological processes) including HIF-1A, VEGFA, IL-6, IL6R, IL-8, CCL2, FGF2, TGFB2, MMP1, MMP3, ITGA5, FN and Col6A1 were upregulated in the immature articular cartilage following ONFH. A qRTPCR analysis of selected genes confirmed the upregulated expression observed in the microarray analysis.ConclusionImmature articular cartilage responds to ONFH by the upregulation of genes involved in hypoxic stress response, angiogenesis, matrix remodeling and inflammation. This study provides novel insights into the multi-faceted role of immature articular cartilage, with inflammation as a key component, following ONFH in piglets.
| S-EPMC4821627 | 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:ObjectiveThe metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage.MethodFemoral head (hip) cartilage was isolated from 5- and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses.Results55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites.ConclusionThis study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.
| S-EPMC6702098 | biostudies-literature
Project description:Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.
| S-EPMC1134898 | biostudies-other
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:Tissue material properties are crucial to understanding their mechanical function, both in healthy and diseased states. However, in certain circumstances logistical limitations can prevent testing on fresh samples necessitating one or more freeze-thaw cycles. To date, the nature and extent to which the material properties of articular cartilage are altered by repetitive freezing have not been explored. Therefore, the aim of this study is to quantify how articular cartilage mechanical properties, measured by nanoindentation, are affected by multiple freeze-thaw cycles. Canine cartilage plugs (n = 11) from medial and lateral femoral condyles were submerged in phosphate buffered saline, stored at 3-5°C and tested using nanoindentation within 12h. Samples were then frozen at -20°C and later thawed at 3-5°C for 3h before material properties were re-tested and samples re-frozen under the same conditions. This process was repeated for all 11 samples over three freeze-thaw cycles. Overall mean and standard deviation of shear storage modulus decreased from 1.76 ± 0.78 to 1.21 ± 0.77MPa (p = 0.91), shear loss modulus from 0.42 ± 0.19 to 0.39 ± 0.17MPa (p=0.70) and elastic modulus from 5.13 ± 2.28 to 3.52 ± 2.24MPa (p = 0.20) between fresh and three freeze-thaw cycles respectively. The loss factor increased from 0.31 ± 0.38 to 0.71 ± 1.40 (p = 0.18) between fresh and three freeze-thaw cycles. Inter-sample variability spanned as much as 10.47MPa across freezing cycles and this high-level of biological variability across samples likely explains why overall mean "whole-joint" trends do not reach statistical significance across the storage conditions tested. As a result multiple freeze-thaw cycles cannot be explicitly or statistically linked to mechanical changes within the cartilage. However, the changes in material properties observed herein may be sufficient in magnitude to impact on a variety of clinical and scientific studies of cartilage, and should be considered when planning experimental protocols.
| S-EPMC5429396 | biostudies-literature
Project description:BACKGROUND:Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. OBJECTIVE:To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. STUDY DESIGN:Controlled laboratory study. METHODS:Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O-fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1? (IL-1?) levels in synovial fluid and serum. RESULTS:The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1? levels. CONCLUSION:All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. CLINICAL RELEVANCE:Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.
| S-EPMC5453820 | biostudies-literature