Electron microscopic observations on the fate of hypertrophic chondrocytes in condylar cartilage of rat mandible

The present study focused on the hypertrophic cell zone and the adjacent region of primary spongiosa in the mandibular condylar cartilage in growing rats (3 to 7 weeks old). In this cartilage, chondrocytes were not arranged in columns, and there was no clear distinction between longitudinal and transverse septum. The hypertrophic chondrocytes were not surrounded entirely by calcified matrix, and capillaries were in close contact with cartilage cells. The staining intensity of the pericellular matrix decreased in the lower hypertrophic cell zone in comparison with that in the upper part of the hypertrophic cell zone. Electron microscopic examinations indicated that the lowest hypertrophic cells contained lysosomes and pinocytotic vesicles. Some hypertrophic chondrocytes appeared to have been released from their lacunae and were observed in the region of the primary spongiosa. Hence it is suggested that the lowest hypertrophic chondrocytes in the rat mandibular condyle do not die but are released from their lacunae into the bone marrow. Further study is needed to determine whether or not these cells do indeed become osteoblasts and/or chondroclasts.     

Electron microscopy of cell surfaces of Paramecium caudatum stained with ruthenium red

The cell surface of Paramecium caudatum, syngen 3, was examined by electron microscopy using ruthenium red (RR) staining. The RR-positive surface coat is of uniform thickness and is found on the entire surface of paramecia, including the gullet area. The surface coat is also observed on the membranes in the tightly united region of conjugating cells. Measurements of the thickness of the surface coat in six stocks of P. caudatum demonstrate a significant difference between complementary mating types: cells of mating type VI have a thicker layer than those of mating type V. No detectable differences in morphology of the surface coat are observed between mating reactive and unreactive cells. Observation in detached cilia indicated that changes in the morphology of the surface coat provoked by the detachment procedure have no effect on mating reactivity. RR stainable substances are detected on both sides of ciliary membranes.     

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.     

Electron microscopic study of isolated proteoglycans]

Chemically isolated separate preparations of the non-aggregating protein-chondroitin-keratin sulphate (PCKS) fraction from the hyaline cartilage and hyaluronic acid (HUA) of the vitreous body and of the umbilicus were investigated by electron microscopy. PCKS and HUA in films without cytochrome c were present in the form of granules and differed by structural organization. The proteoglycans of the cytochrome c films were seen as finely-filamentous-cellular network, and were distinctly differentiated by their macromolecular organization. A mixture of both proteoglycans formed complexes as a result of a noncovalent interaction. Uranyl acetate ensured a good contrasting of proteoglycans, especially of PCKS, without cytochrome c.     

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.     

Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.     

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods

Two histochemical methods for visualization of zinc in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata. In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining. Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type. However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.     

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods

Summary Two histochemical methods for visualization of zine in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata.In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining.Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type, However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.     

Replica immunoelectron microscopic study of the upper surface layer in rat mandibular condylar cartilage by a quick-freezing method

A quick-freezing and deep-etching method in combination with replica immunoelectron microscopy was applied for examining localization of hyaluronic acid and fibronectin on the upper surface layer of rat mandibular condylar cartilage. Rat temporomandibular joints were dissected with articular disks in order to leave the articular cartilage surface intact. The disks were slightly cut with razor blades for exposing the condylar articular cartilage surface. They were quickly frozen with the isopentane-propane cryogen (–193°C) and prepared for freeze-fracturing and deep-etching replica membranes. They were additionally treated with 5% SDS and 0.5% collagenase to keep some antigens attached on the replica membranes. After such a treatment, a routine immunogold method was applied for clarifying the localization of hyaluronic acid and fibronectin in the upper surface layer. Small immunogold particles for hyaluronic acid were mainly localized around upper filamentous networks covered with amorphous materials, but large immunogold ones for fibronectin were localized on deep thicker fibrils. We have revealed the native architecture of the upper surface layer of mandibular condylar cartilage on the replica membranes and also three-dimensional localization of hyaluronic acid and fibronectin by the immunogold method.     

Electron microscopic studies of negatively stained liposomes and membrane intrinsic polypeptide

High resolution electron microscopy has been applied to the study of the permeability properties of liposomes reconstituted with lens fibre protein. The experiments showed that liposomes formed in the presence of main intrinsic polypeptide (MIP) of bovine lens fibres appeared to become modified by changes in shape; also the bilayers seen in the electron micrographs of negatively stained specimens showed evidence of some structural changes or granularity. The morphological appearance of liposomes formed in the presence of MIP were interpreted as being consistent with the results of experiments performed by other methods.     

Immunohistochemical study of the upper surface layer in rat mandibular condylar cartilage

Both hyaluronic acid and fibronectin localizations were examined in the upper surface layer of rat mandibular condylar cartilages by immunohistochemical techniques. Their delicate structure was successfully preserved by preparation procedures of joint condyles with disks. Paraformaldehyde-fixed cartilaginous tissues were cut in a cryostat, and cryosections were analyzed using streptavidin-peroxidase and indirect immunofluorescence methods. Another immunogold method with conventional preparation procedures and a quick-freezing method was performed for their ultrastructural analyses. Both hyaluronic acid-binding protein and anti-fibronectin antibody were used to localize hyaluronic acid and fibronectin in the mandibular condylar cartilage, respectively. Some cryosections were pre-treated with hyaluronidase and chondroitinase before such labeling. The upper surface layer was composed of double laminar structures. One bordered with the cartilage matriceal surface, which was positive for fibronectin. The hyaluronic acid was localized over the fibronectin layer. Therefore, the hyaluronic acid in vivo was bound with fibronectin in the cartilaginous matrix, performing lubrication for the mandibular joint movement.     

Atlas of rat fetal skeleton double stained for bone and cartilage

BACKGROUND: The double staining of fetal skeleton for bone and cartilage is a very useful method to evidence skeletal abnormalities in laboratory animals. However, this method has been rarely used in routine developmental toxicity tests. One reason could be the difficulty of comparing the single skeletal pieces and of having reference points. In this paper the fetal rat skeleton double stained with Alizarin red S and Alcyan Blue is described in detail to produce an atlas for developmental toxicity laboratories.     

Ultrastructural study of upper surface layer in rat mandibular condylar cartilage by quick-freezing method

The purpose of the present study is to clarify native ultrastructures of upper surface layers of the rat mandibular condylar cartilage in vivo by a quick-freezing method. The mandibular cartilaginous tissues were removed with their articular discs attached without opening the lower joint cavity. The specimens were processed for light microscopy, transmission or scanning electron microscopy. Deep-etching replica membranes were also prepared after the routine quick-freezing method. The upper surface layer was well preserved by the quick-freezing method. The cartilaginous tissues, which were fixed without opening their articular discs, appeared to keep better morphology than those after opening them. The upper surface layer was thicker than the corresponding layer as reported before. It consisted of atypical extracellular matrices with lots of apparently amorphous components, which were distributed over typical collagen fibrils, by conventional electron microscopy. As revealed with the replica membranes, it also consisted of variously sized filaments and tiny granular components localized on the typical collagen fibrils. A pair of stereo-replica electron micrographs three-dimensionally showed compact filaments within the upper surface layer. The quick-freezing method was useful for keeping native ultrastructures of the fragile upper surface layer in the mandibular condylar cartilage, which may be functionally important to facilitate smooth movement of the temporomandibular joint.     

Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.     

Electron microscopic studies on the uptake of colloidal thorium dioxide particles by isolated fetal guinea-pig chondrocytes and the distribution of labeled lysosomes in cartilage formed by transplanted chondrocytes

Summary Chondrocytes isolated from fetal, guinea-pig epiphyses were grown in monolayer culture, exposed to thorium dioxide particles, and studied ultrastructurally after varying intervals. The exogenous marker was ingested by endocytosis and subsequently accumulated in lysosomes. After intramuscular injection into young guinea pigs, the thorium dioxidelabeled chondrocytes formed a typical hyaline cartilage. This consisted mainly of rounded or polygonal cells with large, eccentrically located nuclei. The cytoplasm showed an extensive granular endoplasmic reticulum and a well-developed Golgi complex, suggesting active synthesis and secretion of matrix components. Among the other cytoplasmic organelles, lysosomes containing variable amounts of marker particles were observed. After 2–3 weeks the transplants showed signs of cellular degeneration and disintegration. During these processes, lysosomes remained structurally intact and, furthermore, retained the incorporated marker. Thus, thorium dioxide-labeled bodies were found in former chondrocyte lacunae and in the intercellular substance proper. In the latter location labeled bodies could be observed in close proximity to early mineral deposits. These results are discussed with special reference to the cellular origin and lysosomal nature of matrix vesicles in calcifying cartilages.The skilled technical assistance of Mrs. Eva Lundberg and Miss Karin Askfors and the secretarial assistance of Mrs. Ingrid Wäälma are gratefully acknowledged.Financial support was obtained from the Swedish Medical Research Council (proj. no. 12X-3355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundations, the C. B. Nathhorst Foundation, the A. O. Swärd Foundation, and from the Funds of Karolinska Institutet.