Age-related changes in bone in the dog: fluid spaces and their potassium content.

We measured erythrocyte, plasma, osteocyte, extravascular extracellular, and total water fluid spaces and calculated their K content in cortical bone of dogs of various age. Total and exchangeable K were measured by atomic absorption spectroscopy and by volume of distribution techniques, respectively. All fluid spaces in bone decreased with increasing age of the dogs. The total and exchangeable K contents of cortical bone and the K content calculated from the fluid spaces were within the same range in each age group. These values all fell with increasing age.

Steroid-induced accumulation of lipid in the osteocytes of the rabbit femoral head. A histochemical and electron microscopic study.

A high dose of methylprednisolone was administered intramuscularly to rabbits, and we studied the accumulation of lipid in the osteocytes of the femoral head by histochemical methods and electron microscopy. Advanced hyperlipidemia and a fatty liver were observed in four weeks. Electron microscopy was used to define the ultrastructural changes in osteocytes, which showed small lipid droplets that gradually enlarged, finally resulting in the formation of vacuolated vesicles. An increase in the size of the lipid droplets caused them to compress the nucleus to one side of the lacuna, resulting in discontinuities of the cell membrane followed by cell disintegration. These observations demonstrate the effect on osteocytes of a disturbance in lipid metabolism caused by the administration of large doses of steroids.

Histochemical reactions for mucopolysaccharides in the dinosaur bone. Studies on Epon- and methacrylate-embedded semithin sections as well as on isolated osteocytes and ground sections of bone.

The dinosaur bone was examined with the aim to detect in it the presence of mucopolysaccharides. The methacrylate- and Epon-embedded semithin sections of the studied bone were found to contain substances giving positive reaction on application of histochemical procedures used for identification of mucopolysaccharides (mucosubstances). These substances were also shown to be localized in the perivascular space, within the vascular canal, and around the osteocytes. The distribution of mucopolysaccharides in the dinosaur bone turned out to be comparable with their distribution in the bones of contemporary animals.

Submicroscopic structure of glycosaminoglycans in osteocyte capsule of human embryonic bone as revealed by polarization microscopy.

Using qualitative and quantitative polarization microscopical techniques, an axiparallel orientation of chondroitin sulfate molecules and collagen fibrils could be detected in the osteocyte capsule of human embryonic and fetal bones. It is suggested that this spatially oriented microstructure plays a role in ana- and catabolic transport processes of the bone tissue.

Demonstration of cyclic AMP in bone cells by immuno-histochemical methods.

By utilizing immunohistochemical procedures it was possible to locate bone cells that contained adenosine 3'5'-monophosphate (cyclic AMP) in frozen, undecalcified cat jaw sections. An immunoglobulin-enzyme bridge method was found to be superior to other techniques, including immunofluorescence. Differences in staining pattern were found between bone cells of kittens and young adult cats, indicating different metabolic levels related to cellular maturity, and different intracellular compartmentalization of cyclic AMP in the two age groups.

Positivity to glial fibrillary acidic protein in bone, cartilage, and chordoma.

Twenty vertebral bones, 11 costal, 11 epiglottic, six tracheal, and five bronchial cartilages and seven chordomas were evaluated by the application of peroxidase-antiperoxidase (PAP) indirect immunohistochemical method for localization of glial fibrillary acidic protein (GFAP). Positive immunostaining for GFAP was observed in osteocytes of normal bone (13/20), chondrocytes of normal epiglottis (5/11), costal cartilage (3/11), trachea (2/6), and bronchus (4/5). Four of seven chordomas had neoplastic cells that exhibited cytoplasmic positivity to GFAP. These findings suggested that osteocytes, chondrocytes, and chordoma cells have cytoskeletal intermediate filaments that are antigenically identical to or similar to or associated with GFAP.

Ultrastructural immunocytochemical localization of endogenous 1,25-dihydroxyvitamin D3 and its receptors in osteoblasts and osteocytes from neonatal mouse and rat calvaria.

Immunoreactivity to 1,25-dihydroxyvitamin D3 receptors and endogenous 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) were studied in osteoblasts and osteocytes from calvaria of neonatal mice and rats by immunocytochemistry with the use of ultrathin sections obtained by cryo-ultramicrotomy. Tissue samples were fixed in glutaraldehyde, postfixed in osmium tetroxide and frozen under liquid nitrogen. 1,25(OH)2D3 and 1,25(OH)2D3 receptor-like immunoreactivities were observed in osteoblasts and osteocytes. In both types of cell, 1,25(OH)2D3 and its receptors were similarly located in the cytoplasmic matrix but not in organelles, and mainly in the nucleus (primarily in the chromatin and sometimes near the nuclear membrane or in the nucleolus). Reaction products, however, were never seen at the plasma membrane level. These results provide immunocytological evidence for the presence of 1,25(OH)2D3 and its receptors in osteoblasts and osteocytes. The similar localization of the hormone and its receptors in osteoblasts and osteocytes supports the hypothesis of a direct action of 1,25(OH)2D3 in these bone cells. The fact that the main localization of 1,25(OH)2D3 receptors was nuclear, implies, as postulated for other steroid receptors, that 1,25(OH)2D3 receptors occur primarily in the nucleus.

[Lectin histochemistry of bone cells and bone tumors]. / Csontsejtek és csonttumorok lektin-hisztokémiája.

The use of lectins in histochemical research substantially contributed to the knowledge on carbohydrates. This paper is a preliminary report of findings with lectin histochemistry on normal and pathological bone tissue.

Guanosine 3',5'-monophosphate in bone: microscopic visualization by an immuno-histochemical technique.

Guanosine 3',5'-monophosphate (cyclic GMP, cGMP) was localized in bone cells by the use of an immunoglobulin-enzyme bridge method. We observed that in cat alveolar bone most osteoblasts did not stain for cGMP, while adjacent periodontal cells displayed cytoplasmic as well as nuclear staining. Numerous osteocytes contained diffuse reaction products over most or all of the cellular area. The method used in this study may be helpful in identifying specific hard tissue cell types whose function(s) involve cGMP.

Cellular localization of cyclic AMP in periodontal tissues during experimental tooth movement in cats.

Using an immune-histochemical method, cyclic AMP was localized in cells of periodontal tissues in orthodontically-treated cats. Sixteen cats were treated for periods ranging from 1 hour to 4 weeks. Fresh, frozen, undecalcified 6 mu sections of the tissues were incubated with rabbit anti-cyclic AMP antibodies, followed by sequential incubations with sheep anti-rabbit IgG, rabbit anti-peroxidase IgG and horseradish peroxidase. In the final step, the peroxidase was demonstrated by the diaminobenzidine (DAB) method. It was found that the number of intensely stained cells increased within a short time in areas in which bone resorption or apposition occurred later. However, differences in the pattern of cellular activation were found to exist between areas of compression and tension. The alveolar osteocytes appeared to be affected to only a slight degree by the applications of mechanical forces. These results indicate: (a) that our immune-histochemical method was useful in following the cellular distribution of cyclic AMP during bone remodeling; and (b) that mechanical forces may affect only a small part of the bone cell population and therefore cannot be regarded as an efficient means to bring about extensive bone remodeling.

Observations on pigment granules in the bones of silky fowls.

The distribution of melanin pigment-containing cells in the bones of both young and adult silky fowls was observed. Melanin pigment was detected not only in melanocytes which were mainly distributed in the periosteum, but also in all the other types of cells in the periosteum and bone. The continuity of the number of pigment granules in melanocytes and that in the other pigment-containing cells could not be recognized because the granules in the latter cells were much fewer than those in the former. In young fowls, the pigment-containing cells were distributed in all layers of the periosteum and bone, but their number was low. On the other hand, in aged fowls, most of the cells in the periosteum had pigment granules. In the bone, however, pigment granules were observed only in osteocyte situated near the surface. These findings suggest that the pigment granules which are observed in osteocytes have been transferred from melanocytes to osteogenic cells or osteoblasts before they differentiate to osteocytes, where they are presumed to be digested.

A comparative ultrahistochemical study of glycosaminoglycans with cuprolinic blue in bone formed in vivo and in vitro.

Histochemical and morphological studies have shown that proteoglygans (PG) are involved in mineralization process in vivo but such studies have not yet been conducted in vitro. A comparative histochemical study in electronic microscopy of the localization, organization, and morphology of the PG was performed with bones of calvaria rat formed in vivo and bone nodules formed in vitro from osteoblastic cells in culture. For this investigation, we used a cationic phthalocyanin dye, cuprolinic blue, in a critical electrolyte concentration which simultaneously stained the glycosaminoglycans and demineralized the bone. This histochemical technique demonstrated (1) osteoblast cells in vitro synthesized PG which were included in the matrix formed. (2) These PG were found in the calcified and uncalcified matrix both in vivo and in vitro. In the uncalcified matrix, PG were either free with a granular or rodlike structure or tightly connected to the periphery of the collagen fiber. Contrarily, in the calcified matrix, PG formed dense filamentous reticular patches between the collagen fibers. (3) Similarities in localization, organization, and morphology were noted in PG of bone formed de novo in vitro and in vivo with the exception of the mineralization front, where the staining in vivo compared with in vitro was faint or absent.