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 STUDIES ON ULTRASTRUCTURAL IDENTIFICATION AND DISTRIBUTION OF PROTEIN-POLYSACCHARIDE IN CARTILAGE MATRIX

1967 ◽  
Vol 32 (2) ◽  
pp. 365-377 ◽  
Author(s):  
Victor J. Matukas ◽  
Bernard J. Panner ◽  
J. Lowell Orbison
Keyword(s):  
Articular Cartilage ◽  
Cartilage Matrix ◽  
Epiphyseal Cartilage ◽  
Ground Substance ◽  
Dense Matrix ◽  
Chick Embryos ◽  
Chemical Analyses ◽  
Electron Microscopic ◽  
Colloidal Iron ◽  
The Matrix

Previous reports on the ultrastructure of cartilage matrix have described fibers, amorphous ground substance and, in some instances, dense matrix granules. The fibers are presumably collagen, but the nature of the granules is unknown. The primary purpose of this study has been to investigate the ultrastructure of cartilage matrix ih chick embryos with particular emphasis on the distribution and composition of these granules. In matrix of the zone of articular cartilage, mature collagen fibers can be seen but granules are not present. In matrix of all other zones of cartilage, fibers are smaller and granules are present. When the matrix of epiphyseal cartilage is compared to that of the zone of hypertrophic cells, fibers are similar but the granules in the latter zone are larger and more numerous. The granules in both zones were digested by hyaluronidase and positive to colloidal iron staining. Chemical analyses of cartilage from these zones indicate the hexosamine and radiosulfate content of the zone of hypertrophic cells to be higher than that of the zone of epiphyseal cartilage. The increased hexosamine was shown by column chromatography to be principally sulfated mucopolysaccharide, thereby indicating a direct correlation between size and number of granules and sulfated mucopolysaccharide content in the two zones. These data and the results of the electron microscopic histochemical studies are consistent with the concept that the granules in cartilage matrix contain acidic mucopolysaccharide.

RADIOAUTOGRAPHIC STUDY OF S35O4 UPTAKE BY EPIPHYSEAL CARTILAGE IN EXPERIMENTAL LATHYRISM

1962 ◽  
Vol 40 (1) ◽  
pp. 565-570 ◽  
Author(s):  
Yurika K. Shintani ◽  
H. E. Taylor
Keyword(s):  
Normal Diet ◽  
Cartilage Matrix ◽  
Epiphyseal Cartilage ◽  
Acid Mucopolysaccharides ◽  
The Matrix ◽  
Endochondral Growth ◽  
Experimental Lathyrism

Disturbances in the uptake of radiosulphate were observed in radioautographs of the epiphysis of rats made lathyritic by giving either beta-aminopropionitrile or semicarbazide. There was a decreased uptake of sulphate which became more marked as the lesions advanced and an increased uptake was noted as the lesions regressed when the rats were returned to a normal diet. In normal rats, the radio-sulphate shifted to the zone of calcifying cartilage by the 4th day postinjection and, by the 7th day, it was concentrated over the ossifying trabeculae. In lathyrism this shift was delayed and the radiosulphate image was still concentrated in the cartilage matrix at days 4 and 7 postinjection. It is believed these findings reflect a disturbance in the matrix acid mucopolysaccharides and an interference with endochondral growth in lathyrism.

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.

RADIOAUTOGRAPHIC STUDY OF S35O4 UPTAKE BY EPIPHYSEAL CARTILAGE IN EXPERIMENTAL LATHYRISM

1962 ◽  
Vol 40 (5) ◽  
pp. 565-570 ◽  
Author(s):  
Yurika K. Shintani ◽  
H. E. Taylor
Keyword(s):  
Normal Diet ◽  
Cartilage Matrix ◽  
Epiphyseal Cartilage ◽  
Acid Mucopolysaccharides ◽  
The Matrix ◽  
Endochondral Growth ◽  
Experimental Lathyrism

Disturbances in the uptake of radiosulphate were observed in radioautographs of the epiphysis of rats made lathyritic by giving either beta-aminopropionitrile or semicarbazide. There was a decreased uptake of sulphate which became more marked as the lesions advanced and an increased uptake was noted as the lesions regressed when the rats were returned to a normal diet. In normal rats, the radio-sulphate shifted to the zone of calcifying cartilage by the 4th day postinjection and, by the 7th day, it was concentrated over the ossifying trabeculae. In lathyrism this shift was delayed and the radiosulphate image was still concentrated in the cartilage matrix at days 4 and 7 postinjection. It is believed these findings reflect a disturbance in the matrix acid mucopolysaccharides and an interference with endochondral growth in lathyrism.

scholarly journals COMPARISON OF THE EFFECTS OF PAPAIN AND VITAMIN A ON CARTILAGE

1960 ◽  
Vol 111 (5) ◽  
pp. 705-718 ◽  
Author(s):  
Lewis Thomas ◽  
Robert T. McCluskey ◽  
Jacobus L. Potter ◽  
Gerald Weissmann
Keyword(s):  
Vitamin A ◽  
Chondroitin Sulfate ◽  
Cartilage Matrix ◽  
Epiphyseal Cartilage ◽  
Blue Staining ◽  
Daily Injection ◽  
Alcian Blue Staining ◽  
The Matrix ◽  
Cartilage Cells ◽  
Ear Cartilage

The administration of large amounts of vitamin A to rabbits has been shown to result in depletion of cartilage matrix. The normal basophilic, metachromatic, and Alcian blue staining properties of the matrix are lost, especially in articular and epiphyseal cartilage. The cartilage cells remain intact, but are reduced in size. These changes sometimes appeared as early as 48 hours after the initiation of daily injection of 1 million units of vitamin A, and were usually well established by 5 days. Some rabbits failed to show changes in cartilage, even after 5 daily injections. Increased amounts of material presumed to be chondroitin sulfate were present in the sera of vitamin A-treated rabbits, usually by 72 hours after the first injection. This was demonstrated by a turbidimetric procedure using hexamminecobaltic chloride. In rabbits given sulfur-35 (Na2S35O4) 5 days before the initiation of vitamin A treatment, it was shown that sulfur-35 was lost from articular and epiphyseal cartilage. This was associated with an increase in the non-dialyzable sulfur-35 in both serum and in the cobalt-precipitable material. These rabbits also excreted more sulfur-35 than rabbits not given vitamin A. There was a reduction in sulfur-35 activity in chondromucoprotein extracted from the ear cartilage of vitamin A-treated rabbits. The changes are interpreted as indicating that the administration of large amounts of vitamin A to rabbits results in removal of chondroitin sulfate from cartilage matrix. The administration of small amounts of crude papain causes histologic changes in cartilage that are remarkably similar to those seen in vitamin A-treated rabbits. The possibility is suggested that the changes in cartilage produced by administration of vitamin A to rabbits may be the result of activation of a proteolytic enzyme or enzymes, with properties similar to those of papain.

scholarly journals Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

1976 ◽  
Vol 24 (5) ◽  
pp. 621-629 ◽  
Author(s):  
N Shepard ◽  
N Mitchell
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Toluidine Blue ◽  
Light And Electron Microscopy ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Epiphyseal Cartilage ◽  
Fixation Method ◽  
Fixed Tissue

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.

Freeze-etch studies of epiphyseal growth plate cartilage reveal matrix vesicles associated with calcification

1978 ◽  
Vol 36 (2) ◽  
pp. 670-671
Author(s):  
Russell N. A. Cecil ◽  
H. Clarke Anderson
Keyword(s):  
Chromic Acid ◽  
Light And Electron Microscopy ◽  
Matrix Vesicles ◽  
Matrix Vesicle ◽  
Glycerol Solution ◽  
Sprague Dawley ◽  
Epiphyseal Growth Plate ◽  
Growth Plates ◽  
Sprague Dawley Rats ◽  
Tibial Epiphyseal

Unfixed proximal tibial epiphyseal growth plates were studied by freeze-etch to confirm the presence of extracellular calcifying matrix vesicles and to determine the substructure of matrix vesicle membranes as compared to plasma and other membranes of intact chondrocytes. Growth plates from 6-10 week old Sprague-Dawley rats were cut into 1x3 mm blocks whose long dimension was oriented either perpendicular or parallel to the long axis of the tibia. Some blocks were fixed at pH 7. 0 in 0. 2M cacodylate - buffered 2. 5% glutaraldehyde for 1 hour at 4ÅC. The blocks were immersed in 30% glycerol solution at 4ÅC for 1 hour, frozen in liquid nitrogen, and then fractured, etched for 2 minutes, and coated with platinum, carbon and 0. 2% Formvar solution. The replicas were cleaned with chromic acid, floated onto Formvar coated grids, and examined with a Phillips EM 300 electron microscope.Fixed and unfixed specimens appeared similar in ultrastructure. Chondrocytes, matrix, and matrix vesicles were identified. In specimens fractured parallel to the long axis of the tibia, the reserve, proliferative, hypertrophic, and calcifying zones could be discerned as described by light and electron microscopy.

Biodegradable polyester neuroprostheses promote the reconnection of separated peripheral nerve stubs

1992 ◽  
Vol 50 (2) ◽  
pp. 1094-1095
Author(s):  
Beverly L. Giammara ◽  
Jennifer S. Stevenson ◽  
Peggy E. Yates ◽  
Robert H. Gunderson ◽  
Jacob S. Hanker
Keyword(s):  
Image Analysis ◽  
Basement Membrane ◽  
Periodic Acid ◽  
Light And Electron Microscopy ◽  
Computer Assisted ◽  
Type Iii Collagen ◽  
Image Analysis System ◽  
Biodegradable Polyester ◽  
Adult Rats ◽  
Type Iv

An 11mm length of sciatic nerve was removed from 10 anesthetized adult rats and replaced by a biodegradable polyester Vicryl™ mesh sleeve which was then injected with the basement membrane gel, Matrigel™. It was noted that leg sensation and movement were much improved after 30 to 45 days and upon sacrifice nerve reconnection was noted in all animals. Epoxy sections of the repaired nerves were compared with those of the excised segments by the use of a variation of the PAS reaction, the PATS reaction, developed in our laboratories for light and electron microscopy. This microwave-accelerated technique employs periodic acid, thiocarbohydrazide and silver methenamine. It stains basement membrane or Type IV collagen brown and type III collagen (reticulin), axons, Schwann cells, endoneurium and perineurium black. Epoxy sections of repaired and excised nerves were also compared by toluidine blue (tb) staining. Comparison of the sections of control and repaired nerves was done by computer-assisted microscopic image analysis using an Olympus CUE-2 Image Analysis System.

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