Histochemistry of glycosaminoglycans in cartilage ground substance. Alcian-blue staining and lectin-binding affinities in semithin Epon sections

The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex europaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding sites (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive for keratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.     

Ultrastructural demonstration of cartilage acid glycosaminoglycans

Synopsis A method for the demonstration of cartilage acid glycosaminoglycans by light and electron microscopy is described. Rabbit ear cartilage was fixed in cacodylate buffered 2.5% methanol-free formaldehyde with 0.001 M Ruthenium Red andp-chloromercuribenzoate (PCMB). Dehydration was carried out in ethylene glycol followed by embedding in the water-soluble glycol methacrylate (GMA). In some experiments unfixed cartilage was rapidly dehydrated. Sections, 1 thick, and ultrathin sections from the same blocks were stained with 0.001 M Ruthenium Red. Semi-thin sections from cartilage fixed without heavy metal additives were, in addition, stained with the acidophilic fluorochrome Berberine sulphate. It was found that Ruthenium Red intensely stained the same pericellular zone that stained metachromatically with Toluidine Blue or fluoresced after staining with Berberine sulphate. Prior treatment with 0.05% cetylpyridinium chloride entirely blocked the three reactions. Previous digestion with 0.2 mg hyaluronidase/ml for 30 min at 37°C led to the abolition of the fluorescence reaction with Berberine sulphate. It is concluded that Ruthenium Red selectively stains cartilage acid glycosaminoglycans. With the electron microscope the pericellular zones were found to be built up of a three-dimensional branched meshwork of fibrils covered with a mantle of electron-dense material, presumably acid glycosaminoglycans bound to Ruthenium Red.     

Structure of the interstitial space and ground substance of the human articular cartilage

By means of transmissive and scanning electron microscopy (investigation of ultra-replicas) three-dimensional organization of the interstitial (interfibrillar) space of the articular cartilage has been demonstrated; it repeats, to some extent, construction of the fibrous base. By means of mercury porometry quantitative characteristics of various parameters of the interfibrillar space are obtained. Their specific volume is 0.96 cm3/g of the dehydrated cartilage, space with equivalent diameters from 300 up to 5 nm makes 94%. By means of the gas adsorption method it has been stated that the specific internal surface is 23.8 m2 per 1 g of the dehydrated articular cartilage. Transmissive and scanning electron histochemistry has revealed several various forms of structured proteoglycans, demonstrated their spatial organization and interconnection with collagenous fibrils. The methodical complex applied can be used for investigating the connective tissue interstitial spaces in other parts of the human locomotor apparatus.     

Structuro-functional organization of the fibrillar component and ground substance of human rib cartilage

A complex morphological investigation (histology, histochemistry, scanning and transmissive electron microscopy, electron histochemistry) has been performed to study the intercellular substance of the costal hyalinous cartilage. It has been demonstrated that the fibrillar framework of the costal cartilage consists of branching collagenous fibrillae, chaotically scattering. The fibrillae are surrounded with the ground substance; one of its components is the reticular ruthenium-positive structure.     

The glycosaminoglycans of human tracheobronchial cartilage

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5-1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.     

Histochemistry of the extracellular matrix of aging hyaline cartilage

Cartilage proteoglycans (PGs) exhibit marked structural changes with increasing age. There is an increase in small PGs rich in KS as compared to larger PGs rich in chondroitin sulfate (CS) with increasing age. In the present study investigations have been performed to obtain more detailed information about the distribution of different glycosaminoglycans (GAGs). Changes were observed in the interterritorial matrix by means of ultrastructural visualization of PGs with acridin orange. The changes in the ultrastructural organization of the interterritorial matrix of costal cartilage are followed by significant changes in its mechanical properties.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.     

The structure and physicochemical properties of human hyaline cartilage in the disorganization of the carbohydrate-protein complexes of the ground substance]

In the experiments in vitro we studied the influence of the process of disorganization of the carbohydrate-protein complexes of the ground substance on the structure, water content and biomechanical properties of the human hyaline cartilage. It was shown that the disorganization process of the cartilage ground substance and the subsequent removal of the formed products resulted in the increasing porosity of the cartilaginous tissue. This is expressed in the exposure of the fibrillar frame of the cartilage and formation of cavities of various volumes between its elements. The mentioned changes of the cartilage structure are followed by the reduction of the amount of monomolecular-bound water and simultaneous increase in swelling in water and water vapor sorption at maximal relative humidity. The removal of about 25% of glycosaminoglycanes of the ground substance resulted in the reduction of the rigidity of the cartilagenous tissue, and the increase in the residual deformation. The examination of the hyaline cartilage did not reveal any interrelation between the contents of proteoglycanes and the water content of the cartilagenous tissue.     

Age changes in the localization and distribution of glycosaminoglycans in human hyaline cartilage

Summary The localization and distribution of acid glycosaminoglycans, glycoproteins and basic proteins have been studied in human costal cartilage, trachea and primary bronchi from the fetal period through old age. Alcian blue and PAS were used for the staining of acid glycosaminoglycans and glycoprotein and/or sugar-containing compounds respectively. Bromphenol blue and/or bromsulfalein were utilized for the detection of basic proteins. In a further attempt to histochemically identify the various polyanions staining was carried out with AB containing different concentrations of electrolytes. Mild acid hydrolysis, hyaluronidase and proteolytic digestions were also employed. It appeared that the distribution, localization and concentration of the different macromolecules vary according to age, to the type of hyaline cartilage and to the different areas of the same segment. The results also suggested that in developing tissues the areas of appositional growth synthesize mainly chondroitinsulphate while in the territory and interterritory this acidic polyanion is found with keratansulphate. With advancing age the latter slowly disappears from the interterritorial matrix and is nearly exclusively found in the matrix adjacent to the chondrocytes.The susceptibility of these tissues to enzyme digestion also varied according to age and to the type of hyaline cartilage. Proteolytic and hyaluronidase treatment, which were extremely effective in the prenatal period and in young subjects, had practically no effect on mature and old ribs. In the bronchi, instead, the substrates were easily extracted even at an old age. The problems related to the different chemical state of the various macromolecules, their localization and distribution and their possible effect on senescence of hyaline cartilage are discussed.Supported by Research Grant DE-01952(03) of the National Institutes of Public Health, Bethesda, Md.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.