Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT).

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT)

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Morphological and histochemical study of supragingival human calculus and dental plaque using ruthenium hexammine trichloride and acridine orange.

Ruthenium hexammine trichloride (RHT) and acridine orange were used to preserve and visualize anionic groups in human plaque and dental calculus. RHT-reacting material was present on the membrane of micro-organisms and in intermicrobial spaces of the calcifying areas, and seems to correspond to, and derive from, acidic glyco- and phospholipids of the plasma membrane of the micro-organisms. However, the presence of acidic salivary peptidoglycans cannot be ruled out. Two types of calcification were found: extramicrobial and intramicrobial. The former consisted of calcified deposits irregularly scattered in the intermicrobial matrix. They were in close relationship with RHT-reacting material, or were placed inside vesicular structures delimited by a membrane. Intramicrobial calcification consisted of small aggregates of needle-shaped crystals and/or of granular deposits; in both cases, they either masked the whole cytoplasm of the micro-organisms, or were located only over the plasma membrane. These results suggest that mineral deposition occurs in connection with acidic components of intermicrobial matrix, microbial plasma membranes, and cytoplasms. The addition of RHT and acridine orange to fixing and decalcifying solutions yields satisfactory preservation of dental calculus and plaque, and apparently reduces loss of their anionic organic components and increases their electron density. However, these substances are not sufficient to preserve all ultrastructural details in decalcified areas, probably because the inorganic substance prevents reaction of acridine orange and RHT with the organic components of the calcified matrix.     

Structure of the pericellular matrix: Association of heparan and chondroitin sulfates with fibronectin-procollagen fibers

Immunofluorescent staining of a pericellular matrix produced by cultured human embryonic skin fibroblasts showed a codistribution among fibronectin, heparan sulfate proteoglycans and part of the chondroitin sulfate in a fibrillar network. Isolated matrix in an “intact” form could be scraped off the dish after detergent solubilization of the cells. On centrifugation In cesium chloride density gradients, most sulfated glycosaminoglycans and matrix proteins remained associated and were recovered at a density of 1.34 g/cm³ (≥2 M CsCI). However, when 4 M guanidine hydrochloride was included in the gradient medium, the components dissociated, suggesting that the sulfated glycosaminoglycans are bound to matrix proteins by strong noncovalent linkages. Interactions between sulfated glycosaminoglycans produced by the fibroblasts and fibronectin could also be demonstrated by affinity chromatography on immobilized plasma fibronectin and by immunoprecipitation of fibronectin in conditioned culture medium, which resulted in a coprecipitation of the sulfated glycosaminoglycans. In these two systems, the fibronectin glycosaminoglycan bonds were broken at 0.2 M salt and were apparently weaker than the bonds responsible for the structural integrity of the matrix. These findings Implicate heparan and chondroitin sulfate proteoglycans as Integral components of the pericellular matrix fibers and suggest that the association of the proteoglycans with the fibronectin-procollagen matrix is stabilized by multiple molecular Interactions.     

The dynamic structure of the pericellular matrix on living cells

Although up to several microns thick, the pericellular matrix is an elusive structure due to its invisibility with phase contrast or DIC microscopy. This matrix, which is readily visualized by the exclusion of large particles such as fixed red blood cells is important in embryonic development and in maintenance of cartilage. While it is known that the pericellular matrix which surrounds chondrocytes and a variety of other cells consists primarily of proteoglycans and hyaluronan with the latter binding to cell surface receptors, the macromolecular organization is still speculative. The macromolecular organization previously could not be determined because of the collapse of the cell coat with conventional fixation and dehydration techniques. Until now, there has been no way to study the dynamic arrangement of hyaluronan with its aggregated proteoglycans on living cells. In this study, the arrangement and mobility of hyaluronan-aggrecan complexes were directly observed in the pericellular matrix of living cells isolated from bovine articular cartilage. The complexes were labeled with 30- to 40-nm colloidal gold conjugated to 5-D-4, an antibody to keratan sulfate, and visualized with video-enhanced light microscopy. From our observations of the motion of pericellular matrix macromolecules, we report that the chondrocyte pericellular matrix is a dynamic structure consisting of individual tethered molecular complexes which project outward from the cell surface. These complexes undergo restricted rotation or wobbling. When the cells were cultured with ascorbic acid, which promotes production of matrix components, the size of the cell coat and the position of the gold probes relative to the plasma membrane were not changed. However, the rapidity and extent of the tethered motion were reduced. Treatment with Streptomyces hyaluronidase removed the molecules that displayed the tethered motion. Addition of hyaluronan and aggrecan to hyaluronidase-treated cells yielded the same labeling pattern and tethered motion observed with native cell coats. To determine if aggrecan was responsible for the extended configuration of the complexes, only hyaluronan was added to the hyaluronidase-treated cells. The position and mobility of the hyaluronan was detected using biotinylated hyaluronan binding region (b- HABR) and gold streptavidin. The gold-labeled b-HABR was found only near the cell surface. Based on these observations, the hyaluronan- aggrecan complexes composing the cell coat are proposed to be extended in a brush-like configuration in an analogous manner to that previously described for high density, grafted polymers in good solvents.     

Electron microscopic study of condylar cartilage of rat mandible stained with ruthenium red: proteoglycans and hypertrophic chondrocytes

Ultrastructure of hypertrophic chondrocytes and extracellular matrix in condylar cartilage of rat mandible was studied in conjunction with ruthenium red staining. Special care was given to the preservation of proteoglycans in the extracellular matrix. Ruthenium red-positive granules were observed in the pericellular matrix of condylar chondrocytes, and their size and number increased around the hypertrophic cells. However, these granules disappeared in the lowest hypertrophic zone, in which uncalcified cartilage matrix was also disintegrated prior to initiation of ossification. Moreover, hypertrophic chondrocytes observed at the lowest zone appeared intact in their ultrastructural features, i.e., containing numbers of lysosomes and coated vesicles in the cytoplasm facing the blood capillaries. The results strongly suggest that the lowest hypertrophic chondrocytes in rat condylar cartilage may have an active role in the degradation and resorption of the pericellular matrix, especially proteoglycans, and uncalcified matrix, which changes seem an essential step for the initiation of endochondral ossification.     

Morphologic complexity of the pericellular matrix in the annulus of the human intervertebral disc

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.     

Morphologic complexity of the pericellular matrix in the annulus of the human intervertebral disc

The pericellular region of the extracellular matrix (ECM) contains collagens, proteoglycans and other noncollagenous matrix proteins. Although such specialized pericellular ECM has been well studied in articular cartilage, little is known about the pericellular matrix in the disc. In the study reported here, pericellular matrix was studied in annulus tissue from 52 subjects ranging in age from 17-74 years. In aging/degenerating intervertebral discs, cells were identified that formed a distinctive cocoon of encircling pericellular ECM. Immunohistochemical studies identified types I, II, III and VI collagen in these pericellular sites with diverse morphological features. Similar types of changes in the pericellular matrix were observed in both surgical specimens and control donor discs. Results indicate the need for future studies to address why such specialized matrix regions form around certain disc cells and to determine the consequences of these unusual matrix regions on annular lamellar organization and function.     

Proteoglycan production by immortalized human chondrocyte cell lines cultured under conditions that promote expression of the differentiated phenotype

Large and small proteoglycans are essential components of articular cartilage. How to induce chondrocytes to repair damaged cartilage with normal ratios of matrix components after their loss due to degenerative joint disease has been a major research focus. We have developed immortalized human chondrocyte cell lines for examining the regulation of cartilage-specific matrix gene expression. However, the decreased synthesis and deposition of cartilage matrix associated with a rapid rate of proliferation has presented difficulties for further examination at the protein level. In these studies, proteoglycan synthesis was characterized in two chondrocyte cell lines, T/C-28a2 and tsT/AC62, derived, respectively, from juvenile costal and adult articular cartilage, under culture conditions that either promoted or decreased cell proliferation. Analysis of proteo[36S]glycans by Sepharose CL-4B chromatography and SDS-PAGE showed that the large proteoglycan aggrecan and the small, leucine-rich proteoglycans, decorin and biglycan, were produced under every culture condition studied. In monolayer cultures, a high initial cell density and conditions that promoted proliferation (presence of serum for T/C-28a2 cells or permissive temperature for the temperature-sensitive tsT/AC62 cells) favored cell survival and ratios of proteoglycans expected for differentiated chondrocytes. However, the tsT/AC62 cells produced more proteoglycans at the nonpermissive temperature. Culture of cells suspended in alginate resulted in a significant decrease in proteoglycan production in all culture conditions. While the tsT/AC62 cells continued to produce a larger amount of aggrecan than small proteoglycans, the T/C-28a2 cells lost the ability to produce significant amounts of aggrecan in alginate culture. In addition, our data indicate that immortalized chondrocytes may alter their ability to retain pericellular matrix under changing culture conditions, although the production of the individual matrix components does not change. These findings provide critical information that will assist in the development of a reproducible chondrocyte culture model for the study of regulation of proteoglycan biosynthesis in cartilage.     

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.     

Ultrastructural localization of heparan sulfate and chondroitin sulfates associated with granulopoiesis in embryonic chick bone marrow

Sulfated glycoconjugates were ultrastructurally localized within embryonic chick marrow by using the high iron diamine-silver proteinate stain. Stain was concentrated in the extravascular, granulopoietic compartment, indicating that granulopoiesis, but not erythropoiesis, proceeded in a highly sulfated environment. It was likely that most of the stainable material represented sulfated proteoglycans since staining was abrogated by predigesting tissue with enzymes and other treatments known to degrade specific glycosaminoglycan chains. Chondroitinase/hyaluronidase digestion resulted in the removal of most of the stainable material associated with the extracellular matrix and a portion of the stainable material associated with fibroblastic cell surfaces. Unaffected material lay in close proximity to fibroblastic cell membranes. Heparitinase/heparinase digestion had essentially the opposite effect. Sulfated material associated with matrix components was largely unaffected, but the fibroblastic plasmalemmal material was now absent. These results suggest that there are at least two categories of sulfated proteoglycans in the granulopoietic compartment, each differentially distributed. The plasmalemmal material likely represented heparan sulfate which in this tissue appeared to be associated in a uniform layer with fibroblastic stromal cell membranes and not with blood or endothelial cell membranes. Material identified as chondroitin sulfates was found within patches of amorphous matrix that was located on fibroblastic stromal cell surfaces and that was interspersed with fibrils in the extracellular matrix. Chondroitin sulfates were sparsely distributed on granulocytic cell surfaces.     

Differential extraction of proteoglycans from cartilage tissue matrix compartments in isotonic buffer salt solutions and commercial tissue-culture media

Small tissue blocks of native rat growth plate cartilage were incubated for short periods in one of several generally used isotonic buffer salt solutions or commercial tissue-culture media. The total percentage (approximately 12) of [35S]-labeled proteoglycans (PG) extracted from cartilage matrix under these conditions was not significantly influenced by either the chemical composition of the medium or the presence of a protease inhibitor. Morphological examination of incubated tissue after fixation in the presence of ruthenium hexamine trichloride (RHT) (included to preserve PG in situ) revealed, however, that the PG staining profiles across cartilage matrix varied with the composition of the incubation medium used. The various susceptibilities exhibited by PG within the different matrix compartments to selective extraction was estimated semi-quantitatively. The observed effects may prove useful in extracting these molecules differentially from cartilage matrix compartments.     

Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.     

The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining

The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method. © 1998 Chapman & Hall     

In vitro biosynthesis by articular chondrocytes of a specific low molecular size proteoglycan pool

Proteoglycans synthesized by articular and epiphyseal chondrocytes in culture were compared. Proteoglycans extruded by the two types of cells into the culture medium are of identical Mr. On the other hand, the proteoglycans of cells or pericellular matrix synthesized by the articular chondrocytes are characterized by an heterogeneous fraction of low-Mr which is not present in the material derived from epiphyseal chondrocytes. There are at least two components in this fraction: the first seems to be a precursor of aggregated proteoglycans, the other may represent a component of cell coat. Stimulation of the cell cultures with vitamin D metabolites and somatomedin enhances proteoglycan biosynthesis but no modification is observed in the proteoglycan Mr.     

Light and electron microscopical immunohistochemical localization of the small proteoglycan core proteins decorin and biglycan in human knee joint cartilage

Summary The distribution of decorin and biglycan was investigated at the light and electron microscopical level in adult human articular cartilage. In general, the amount of decorin and biglycan was found to decrease with the depth of the layer of the cartilage. Decorin was found in the interterritorial matrix where most of the collagen is located. This fits in well with the assumption that decorin may modulate collagen metabolism. Biglycan was found next to the chondrocytes in the pericellular matrix and is assumed to be responsible for cellular activities. At the ultrastructural level, decorin was localized in the interterritorial matrix and in vesicles in chondrocytes. Biglycan was found, usually though not exclusively in the pericellular matrix. Both small proteoglycans were detected close to and on the collagen fibres and also associated with the more globular structures of the matrix between the fibrils. A double-staining approach revealed that the two molecules could be located along the same collagen fibril. However, staining for biglycan and decorin was not observed simultaneously within the same region of the fibre.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

Heparan sulfate proteoglycans: coordinators of multiple signaling pathways during chondrogenesis

Heparan sulfate proteoglycans are abundantly expressed in the pericellular matrix of both developing and mature cartilage. Increasing evidence indicates that the action of numerous chondroregulatory molecules depends on these proteoglycans. This review summarizes the current understanding of the interactions of heparan sulfate chains of cartilage proteoglycans with both soluble and nonsoluble ligands during the process of chondrogenesis. In addition, the consequences of mutating genes encoding heparan sulfate biosynthetic enzymes or heparan sulfate proteoglycan core proteins on cartilage development are discussed.     

CD44-Anchored Hyaluronan-Rich Pericellular Matrices: An Ultrastructural and Biochemical Analysis

The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.     

The Nucleus of the Intervertebral Disc from Development to Degeneration

The nucleus of the intervertebral disc in humans shows the mostdramatic changes with age of any cartilaginous tissue. It originatesfrom the notochord. In the foetus and infant, the nucleus containsactively dividing and biosynthetically active notochordal cells.The proteoglycans and other matrix components produced havea high osmotic pressure, imbibe water and maintain a hydratedstructure which, though it has little mechanical strength, hasa high swelling pressure which maintains disc turgor. In somespecies, the notochordal cells and the mucoid nucleus pulposuspersist throughout adult life. However by about 4 yr of agein humans, the notochordal cells have disappeared to be replacedby those of chondrocytic appearance but of unknown origin. Thesecells continue to produce proteoglycans but also synthesizesignificant amounts of collagen. The nucleus becomes firmerand less hydrated and loses its transparent appearance. Thecell density of the adult nucleus is very low with cells occupyingless than 0.5% of tissue volume; each cell thus has to turnover and maintain a large domain of extracellular matrix. Thedensity of living cells decreases with age, possibly becauseof problems with nutrient supply to this large avascular tissue.Proteoglycan concentration also falls, and nucleus hydrationdecreases markedly, the disc discolours and in many cases cleftsand fissures form. In most adults, the disc nucleus degenerateseventually to a stage where it can no longer fulfil its mechanicalrole.