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.     

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.     

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.     

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.     

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.     

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.     

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.     

Observations on the glycosaminoglycans of aging bronchial cartilage studied with Alcian Blue

Synopsis The changes in the distribution of acidic glycosaminoglycans in the extracellular matrix of aging human bronchial cartilage have been studied with Alcian Blue using the critical electrolyte technique described by Scott & Dorling (1965). Keratan sulphate was detected in the interterritorial matrix early in the first decade. The factors initiating the synthesis of keratan sulphate by chondrocytes are discussed and a hypothesis is proposed to explain the subsequent localization of this glycosaminoglycan in the extracellular matrix.     

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.