scholarly journals Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification.

1983 ◽  
Vol 31 (9) ◽  
pp. 1089-1100 ◽  
Author(s):  
M Takagi ◽  
R T Parmley ◽  
F R Denys
Keyword(s):  
Chondroitin Sulfate ◽  
Tannic Acid ◽  
Keratan Sulfate ◽  
Matrix Vesicles ◽  
Enzyme Digestion ◽  
Epiphyseal Cartilage ◽  
Fe Method ◽  
Cartilage Calcification ◽  
Testicular Hyaluronidase ◽  
Chondroitin Sulfate A

Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.

Effects of EDTA, Hyaluronidase and Collagenase on Epiphyseal Cartilage Matrix

1968 ◽  
Vol 26 ◽  
pp. 56-57
Author(s):  
H. Clarke Anderson ◽  
Priscilla R. Coulter
Keyword(s):  
Light And Electron Microscopy ◽  
Matrix Vesicles ◽  
Cartilage Matrix ◽  
Collagen Fibrils ◽  
Epiphyseal Cartilage ◽  
Dense Matrix ◽  
Type Iv ◽  
Bovine Testicular Hyaluronidase ◽  
The Matrix ◽  
Testicular Hyaluronidase

Epiphyseal cartilage matrix contains fibrils and particles of at least 5 different types: 1. Banded collagen fibrils, present throughout the matrix, but not seen in the lacunae. 2. Non-periodic fine fibrils <100Å in diameter (Fig. 1), which are most notable in the lacunae, and may represent immature collagen. 3. Electron dense matrix granules (Fig. 1) which are often attached to fine fibrils and collagen fibrils, and probably contain protein-polysaccharide although the possibility of a mineral content has not been excluded. 4. Matrix vesicles (Fig. 2) which show a selective distribution throughout the epiphysis, and may play a role in calcification. 5. Needle-like apatite crystals (Fig. 2).Blocks of formalin-fixed epiphysis from weanling mice were digested with the following agents in 0.1M phosphate buffer: a) 5% ethylenediaminetetraacetate (EDTA) at pH 8.3, b) 0.015% bovine testicular hyaluronidase (Sigma, type IV, 750 units/mg) at pH 5.5, and c) 0.1% collagenase (Worthington, chromatograhically pure, 200 units/mg) at pH 7.4. All digestions were carried out at 37°C overnight. Following digestion tissues were examined by light and electron microscopy to determine changes in the various fibrils and particles of the matrix.

scholarly journals Keratan Sulfate Epitopes Exhibit a Conserved Distribution During Joint Development That Remains Undisclosed on the Basis of Glycosaminoglycan Charge Density

2002 ◽  
Vol 50 (8) ◽  
pp. 1039-1047 ◽  
Author(s):  
Emma Kavanagh ◽  
Anne C. Osborne ◽  
Doreen E. Ashhurst ◽  
Andrew A. Pitsillides
Keyword(s):  
Chondroitin Sulfate ◽  
Alcian Blue ◽  
Functional Role ◽  
Keratan Sulfate ◽  
Epiphyseal Cartilage ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Joint Development ◽  
Joint Cavity ◽  
And Cartilage

Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development. However, their precise character has not been established. We have used immunohistochemistry (IHC) and “critical electrolyte” Alcian blue staining to assess such changes in developing chick and rabbit joints. IHC showed chondroitin sulfate labeling in chick epiphyseal cartilage but not in interzones. In contrast, prominent labeling for keratan sulfate (KS) was restricted to chick cartilage–interzone interfaces. In rabbit knees, KS labeling was also prominent at presumptive cavity borders, but weak in interzone and cartilage. Selective pre-digestion produced appropriate loss of label and undersulfated KS was undetectable. Quantification of Alcian blue staining by scanning and integrating microdensitometry showed prominent hyaluronan-like (HA-like) interzone staining, with chondroitin sulfate and weaker KS staining restricted to epiphyseal cartilage. Hyaluronidase decreased HA-like staining in the interzone. Surprisingly, keratanases also reduced HA-like but not sulfated GAG (sGAG-like) staining in the interzone. Chondroitinase ABC had little effect on HA-like staining but decreased sGAG staining in all regions. Rabbit joints also showed HA-like but not KS staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage. Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints. Alcian blue staining does not detect this moiety. Therefore, it appears that although histochemistry allows relatively insensitive quantitative assessment of GAGs, IHC increases these detection limits. This is particularly evident for KS, which exhibits immunolabeling patterns in joints from different species that is consistent with a conserved functional role in chondrogenesis.

Immunocytochemical Study of Proteoglycans in Vocal Folds

1996 ◽  
Vol 105 (1) ◽  
pp. 6-11 ◽  
Author(s):  
Agnieszka S. Pawlak ◽  
Elizabeth Hammond ◽  
Thomas Hammond ◽  
Steven D. Gray
Keyword(s):  
Chondroitin Sulfate ◽  
Lamina Propria ◽  
Vocal Fold ◽  
Keratan Sulfate ◽  
Vocal Folds ◽  
Basement Membrane Zone ◽  
Immunocytochemical Study ◽  
Frozen Sections ◽  
Intense Staining ◽  
Immunocytochemical Techniques

We evaluated the proteoglycan composition of normal vocal folds using immunocytochemical techniques. Frozen sections of 14 normal cadaveric vocal folds were obtained within 12 hours of death and sectioned immediately. Vocal fold sections were stained with antibodies against keratan sulfate, chondroitin sulfate, heparan sulfate proteoglycan (HSPG), decorin, and hyaluronate receptor. We found that the lamina propria has diffuse staining of fibrillar components with keratan sulfate and decorin. Intense staining was observed in the vocal ligament area with keratan sulfate. The HSPG was localized to the basement membrane zone. Chondroitin sulfate, HSPG, and hyaluronate receptor were detected in the cytoplasm of interstitial cells with immunocytochemical characteristics of macrophages. The keratan sulfate distribution suggests that fibromodulin may be significant in normal vocal folds. Production of HSPG and probably versican occurs in macrophages and fibroblasts in the lamina propria.

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