alcian-blue and Osteoarthritis

The temporomandibular joint (TMJ) is a exceptional joint involved in growth as well as mastication. In adult mice, it provides a model for age related natural osteoarthritis (OA). The insulin-like growth factor (IGF-) system plays was tested because it plays important roles in cartilage biology and OA pathogenesis. Decalcified and paraffin embedded TMJs of 48 NMRI mice sacrificed in groups of three male and females each at the ages of 3, 4, 5, 7, 9, 10, 12, and 18 months were prepared for histopathology and immunohistochemistry for IGF-I and -II, IGF1 receptor and IGF binding proteins -1, -2, -3, -4, -5, and -6. Histopathological signs of OA were obvious in the TMJ of all animals older than 5 months, but did not show a clear age-related staging. Immunoreactivity for all IGF components was found in unchanged anterior and posterior regions of the condyle and in regions of advanced OA lesions. Receptor immunostaining was obvious in all ages. Most IGFBPs showed immunostaining patterns similar to IGFs. While the anterior and posterior zones of the condylar cartilage appear to be specialized judging by structure and IGF immunostaining pattern, probably due to metabolic or biomechanical peculiarities, the central portion undergoes early degeneration. In advanced OA stages, the IGF system seems to be upregulated to induce repair processes. According to their mainly inhibiting functions, IGFBPs may suppress anabolic IGF activities.

Topics: Aging; Alcian Blue; Animals; Cartilage; Female; Immunohistochemistry; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Mice; Osteoarthritis; Receptor, IGF Type 1; Somatomedins; Temporomandibular Joint

The synthesis and contents of extracellular non-collagenous matrix macromolecules was studied in early and late human osteoarthritic (OA) cartilage obtained at surgery for sarcomas in the lower extremities (normal and early OA) or for total knee replacement (late stage OA). The early OA samples were those that had some fibrillation in the joint by visual examination. One group had fibrillation in the area sampled and the other group had no fibrillation. Cartilage was taken from the same topographical area on the medial femoral condyle in all the samples, labeled with [3H]leucine and [35S]sulfate for 4 h at 37 degrees C and extracted with 4 M guanidine-HCl. Analysis of the extracts showed that the total amount of proteoglycans relative to hydroxyproline content was higher in the early and late OA than in the normal cartilage. These proteoglycans showed a relatively lower [35S]sulfate incorporation into GAG chains and a higher [3H]leucine incorporation. The pattern of newly synthesized proteins was altered similarly in early and late OA. Notably, synthesis of cartilage oligomeric matrix protein (COMP), fibronectin, and cartilage intermediate layer protein (CILP) was increased, also reflected in their abundance as determined by enzyme-linked immunosorbent assay (ELISA). Collagen synthesis appeared significantly increased only in the late stage OA. The observed altered composition and pattern of biosynthesis indicate that the joint undergoes metabolic alterations early in the disease process, even before there is overt fibrillation of the tissue. The early OA samples studied appear to represent two distinct groups of early lesions in different stages of the process of cartilage deterioration as shown by their differences in relative rates of synthesis and abundance of proteins.

Topics: Aged; Alcian Blue; Body Weight; Cartilage; Cartilage, Articular; Chromatography; Chromatography, Ion Exchange; Collagen; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Female; Humans; Hydroxyproline; Leucine; Male; Middle Aged; Osteoarthritis; Procollagen; Proteoglycans; Temperature

To determine the concentrations of aggrecan fragments in synovial fluid from patients with knee joint injury, osteoarthritis (OA), or acute pyrophosphate arthritis (PPA; pseudogout), and to test their relative reactivity with the 846 epitope, a putative marker of cartilage aggrecan synthesis.. Samples of knee joint fluid from 385 patients and 9 healthy-knee volunteers were obtained in a cross-sectional study. Study groups were acute PPA/ pseudogout (n = 60), anterior cruciate ligament (ACL) rupture (n = 159), meniscus lesion (n = 129), and primary knee OA (n = 37). The 846 epitope on aggrecan was assayed by competitive solution-phase radioimmunoassay. Aggrecan fragments were assayed by enzyme-linked immunosorbent assay using a monoclonal antibody (1-F21). Cartilage oligomeric matrix protein (COMP), C-propeptide of type II collagen (CPII), bone sialoprotein, matrix metalloproteinases 1 and 3, and tissue inhibitor of metalloproteinases 1 were previously quantified by immunoassays.. Reactivity of the 846 epitope was increased in all study groups compared with the reference group, and was highest in patients with primary OA. The median levels (in microg fetal aggrecan equivalents/ml) of the epitope were 0.28 (range 0.24-0.47) in the reference group, 0.48 (range 0.26-1.32) in PPA/pseudogout, 0.61 (range 0.12-2.87) in ACL rupture, 0.53 (range 0.22-3.02) in meniscus lesion, and 0.68 (range 0.31-4.31) in primary OA. The 846 epitope reactivity per microg aggrecan fragments in the joint fluid was higher in late-stage OA than in early-stage OA. Epitope 846 reactivity correlated positively with several markers of matrix turnover, particularly with COMP (r(s) = 0.421) and CPII (r(s) = 0.307).. The observed differences in 846 epitope reactivity in synovial fluid, and its concentration in relation to aggrecan and other markers of matrix turnover, were consistent with marked ongoing changes in aggrecan turnover after joint injury and in the development of OA. OA is thus a disease characterized by dynamic changes in tissue macromolecule turnover, which is reflected by measurable changes in aggrecan epitopes in the synovial fluid.

Topics: Aggrecans; Alcian Blue; Autoantibodies; Biomarkers; Cartilage, Articular; Enzyme-Linked Immunosorbent Assay; Epitopes; Extracellular Matrix Proteins; Humans; Knee Injuries; Lectins, C-Type; Osteoarthritis; Proteoglycans; Regression Analysis; Synovial Fluid

Previous studies on osteoarthritic changes induced by intraarticular injections of estradiol benzoate (EB) suggest estrogen as a risk factor in the development of knee osteoarthritis (OA). The present study observed the anti-arthritic effects of tamoxifen (TMX). Oophorectomized rabbits were subjected to intraarticular injections of vehicle control, EB, TMX, or EB/TMX for 3 weeks. The cartilage changes were assessed by light and scanning electron microscopic examination, enzyme histochemical analysis, and the amount of alcian blue stain binding to glycosaminoglycans. EB injections resulted in cell necrosis, chondrocyte clonings, and pittings, whereas the vehicle control, TMX, and EB/TMX-injected groups showed no histologic abnormalities. Histochemical analysis showed that the numbers of lactate dehydrogenase (LDH)-reactive chondrocytes in the EB-injected group were significantly reduced when compared to other groups (p less than 0.001). The injections of EB/TMX significantly reduced the chondrocyte numbers in the lateral superficial layer (p less than 0.05), compared with the vehicle injection. TMX-injected group revealed slight although insignificant decreases in chondrocyte numbers. The amount of alcian blue stains, representing the relative amount of proteoglycans, significantly decreased only in the superficial layer of the EB- and EB/TMX-injected groups (p less than 0.05). TMX, when concurrently injected with EB, antagonized the chondrodestructive effects of estradiol at the early stage of knee OA in rabbits. The results suggest the potential therapeutic use of TMX at the early stage of OA.

Topics: Alcian Blue; Animals; Cartilage, Articular; Estradiol; Female; L-Lactate Dehydrogenase; Microscopy, Electron, Scanning; Osteoarthritis; Ovariectomy; Proteoglycans; Rabbits; Tamoxifen

We used Alcian Blue (AB) and dimethylmethylene blue (DMB) methods to measure glycosaminoglycan (GAG) excretion in the first morning urine specimens of patients with osteoarthritis (OA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA) in different stages of disease. By the AB method, urinary GAG excretion in patients with RA was not different from healthy control subjects. However, the DMB method showed significant differences (in milligrams of GAG per gram of creatinine) for OA (median 25.4, range 14.3-44.0, P less than 0.01, n = 23) and RA patients (median 33.0; range 10.0-147.6; P less than 0.001, n = 63) in comparison with unaffected individuals (median 20.2; range 8.9-41.4, n = 38). We noted a significant difference in urinary GAG excretion between RA and OA patients (P less than 0.01) and between RA and AS (P less than 0.01) patients. The DMB method was further investigated by clinical decision analysis. The DMB method is simple and rapid and may be useful in diagnosing RA by distinguishing between RA and OA or AS.

Topics: Adolescent; Adult; Aged; Alcian Blue; Arthritis, Rheumatoid; Glycosaminoglycans; Humans; Methylene Blue; Middle Aged; Osteoarthritis; Rheumatic Diseases; Spectrophotometry; Spondylitis, Ankylosing

An alcian blue precipitation method for quantifying the hyaluronic acid (HA) and sulphated glycosaminoglycan concentration (SGAG) in solutions containing both compounds was assessed. The assay was found to be rapid and reliable in solutions containing 0 to 200 mg of HA/dl and 50 to 1,000 micrograms of SGAG/dl, and was not affected by the presence of protein, hemoglobin, or methemoglobin in concentrations normally found in synovial fluid. The HA and SGAG concentrations in intercarpal synovial fluid from 13 clinically normal and 11 arthritic horses were evaluated. A relationship was not found between the concentration of HA and SGAG and any other synovial fluid variable. The SGAG concentration was found to be markedly high in several of the synovial fluid samples from arthritic horses, but did not correlate with the degree of articular cartilage erosion.

Topics: Alcian Blue; Animals; Glycosaminoglycans; Horse Diseases; Horses; Hyaluronic Acid; Osteoarthritis; Synovial Fluid

At least 80% of male STR/ORT mice naturally develop osteoarthritis that predominantly affects the medial tibial cartilage. Overt osteoarthritic changes, as judged by radiological and histological abnormalities, become apparent after 30 weeks of age. Consequently, mice less than 30 weeks of age were used to investigate early changes in the cartilage matrix related to the natural development of osteoarthritis, without the need for experimental intervention to induce this condition. Quantitative Alcian blue staining showed little change in the total amount of proteoglycans in mice of this age. Polarized light microscopy of the birefringence induced by such staining demonstrated a progressive decline in the orientation of the proteoglycans in the medial cartilage of these mice. This decline was not found in CBA mice, which only very rarely develop osteoarthritis of this joint. Such progressive disorganization of the proteoglycans would be likely to permit the increase free water-content characteristic of osteoarthritic cartilage.

Topics: Aging; Alcian Blue; Animals; Birefringence; Cartilage; Collagen; Male; Mice; Mice, Inbred Strains; Osteoarthritis; Proteoglycans; Rodent Diseases

Changes in the cellularity and in the nature of the matrix were studied in the cartilages of the tibial plateau in experimentally induced arthritis in the dog, 7 and 14 days after section of the anterior cruciate ligament. Samples from the different regions of these cartilages were chilled and sectioned in a cryostat, with a variable microtome chuck to allow precise orientation of the specimens. The samples were examined by normal light microscopy, by microscopic interferometry, and by quantitative polarized light microscopy. The orientation of the glycosaminoglycans was assessed by the new "induced birefringence" method. The results indicated that only the region of the medial tibial cartilage that was unprotected by the meniscus was affected, showing increased water content, loss of superficial cells, and a decrease in orientation of the glycosaminoglycans. Whereas the birefringence of the collagen was unaffected, the superficial area that lacked oriented glycosaminoglycans was markedly increased; this may be a useful indicator of early osteoarthritic changes.

Topics: Alcian Blue; Animals; Birefringence; Cartilage; Cartilage, Articular; Cell Count; Collagen; Dogs; Female; Glycosaminoglycans; Joints; Mathematics; Microscopy, Interference; Microscopy, Polarization; Microtomy; Osteoarthritis; Pyronine; Staining and Labeling; Tibia; Water

The formation of soluble complexes between alcian blue dye and sulfated glycosaminoglycans provides the basis for the quantitative spectrophotometric estimation of the total concentration of these polysaccharides. Samples containing microgram quantities of sulfated glycosaminoglycan are mixed with a stable dye solution prepared in 15% phosphoric/2% sulfuric acids and absorbance readings at 480 nm are compared to an appropriate standard curve. The method is rapid, convenient, and reproducible. Analyses are performed under conditions in which there is no interference from the non-sulfated glycosaminoglycan hyaluronic acid, or most other anionic macromolecules. In addition, estimations are not effected by small anions or individual monosaccharides. The method has been used for the determination of the purity of commercially available preparations of hyaluronic acid and for the estimation of the sulfated glycosaminoglycan content of various biological fluids including normal human urine and the synovial fluid of individuals with rheumatoid arthritis and osteoarthritis.

Topics: Alcian Blue; Arthritis, Rheumatoid; Chondroitin Sulfates; Glycosaminoglycans; Humans; Osteoarthritis; Spectrophotometry; Synovial Fluid

The distribution of sulphated glycosaminoglycans (GAG's) in the human temporomandibular joint disk and its relationship to sex, age and osteoarthrosis was studied by histochemical methods in autopsy material from 18 individuals. The disks were embedded in paraffin and frontal sections, 5-7 mu thick, were cut at different levels. Two staining methods were used, toluidine blue at pH 0.5 and alcian blue with different concentrations of MgCl2. The two methods gave comparable results. The sulphated GAG's as represented by metachromatic staining with toluidine blue at pH 0.5 and staining with alcian blue in concentrations of MgCl2 above 0.55-M were found in the central load-bearing part of the disks evenly distributed in the medio-lateral direction. The findings from the alcian blue staining method indicated the presence of sulphated GAG's with characteristics similar to chondroitin/dermatan sulphate and keratan sulphate. The latter finding was most frequent in a surface zone )10-100 mu) located mainly in the central part of the disks. In macroscopically thin areas of the disks judged as early osteoarthrosis a significant reduction in the staining of sulphated GAG's could be observed. No age or sex differences were found in the distribution of GAG's, either in normal or in osteoarthrotic disks.

Topics: Adult; Age Factors; Aged; Alcian Blue; Cartilage, Articular; Child; Female; Glycosaminoglycans; Humans; Infant, Newborn; Male; Middle Aged; Osteoarthritis; Sex Factors; Staining and Labeling; Temporomandibular Joint; Tolonium Chloride