Ultrastructure of hypertrophic cartilage: histochemical procedures compared with high pressure freezing and freeze substitution.

The effect of cationic dyes on the ultrastructure of hypertrophic cartilage was compared with results obtained with modern cryotechniques in studies on rat epiphyseal growth plate. Addition of alcian blue, acridine orange, cupromeronic blue, ruthenium hexamine trichloride, ruthenium red, or safranin O to conventional glutaraldehyde/osmium tetroxide fixatives to a large extent resulted in prevention of chondrocyte shrinkage except for alcian blue which showed poor tissue penetration. The fine structure of the matrix in pericellular and territorial compartments appeared very coarse with areas of high contrast in tissue exposed to fixatives containing cationic dyes. This indicates structural collapse and precipitation of electron-dense material, a pattern clearly differing from that observed in specimens prepared by the cryotechniques. The dyes giving a pattern most similar to that seen after high pressure freezing, freeze substitution, and low temperature embedding were acridine orange and safranin O. It is concluded that studies of matrix ultrastructure down to the molecular level necessitate the application of cryotechniques.

Parathyroid cell number and size in hypocalcemic young rats.

Weanling rats were fed diets with normal (1%) or low (0.08% or 0.02%, respectively) Ca content for 28 days prior to sacrifice. The total volume of the parathyroids was estimated from serial sections. Volume density of secretory cells was calculated according to conventional stereological techniques, whereas cell number and cell size were estimated by the dissector method. Compared with controls the animals of the experimental groups developed moderate and severe hypocalcemia and their parathyroids were enlarged with a proportional growth of parenchyma and interstitium. Related to the body weight, secretory cell volume was highest in animals with severe hypocalcemia. In the enlarged glands the size of parathyroid secretory cells was increased by 30-40%, whereas total cell number was unaltered. Thus, the increased parathyroid size was due to cell hypertrophy rather than hyperplasia.

Ultrastructural localization of a tartrate-resistant acid ATPase in bone.

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.