P. Mocetti

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.

A Histomorphometric, Structural, and Immunocytochemical Study of the Effects of Diet-induced Hypocalcemia on Bone in Growing Rats

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal–metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell–matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone–calcified cartilage interface in some cases were thickened but generally did not appear affected at bone–bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption. (J Histochem Cytochem 48:1059–1077, 2000)

Morphological and Immunocytochemical Analyses on the Effects of Diet-induced Hypocalcemia on Enamel Maturation in the Rat Incisor

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the “hardest” tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid–Schiff- and periodic acid–silver nitrate–methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the amelo-blast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal “ghosts.” The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation. (J Histochem Cytochem 48:1043–1057, 2000)

Combined use of malachite green fixation and PLA2-gold complex technique to localize phospholipids in areas of early calcification of rat epiphyseal cartilage and bone

Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.

Localization of the glucocorticoid receptor mRNA in cartilage and bone cells of the rat. An in situ hybridization study

The in vivo localization of glucocorticoid receptor (GR) mRNA expression was studied in the cartilage and bone cells of the femur of young adult rats to compare its distribution with that of the GR protein, which had previously been shown histochemically in the same areas. To achieve this, we used a synthetic oligodeoxynucleotide as a probe, in line with the published human GR (hGR) cDNA sequence. The probe was coupled to fluorescein (FL), applying a rapid Fast-Tag TM FL nucleic acid labeling method. Negative controls were achieved by using sense sequences of the hGR oligoprobe, similarly coupled by using the Fast-Tag TM FL labeling kit. Dewaxed sections were treated for in situ hybridization (ISH) histochemistry with the antisense and sense oligoprobes. The ISH reaction product was more intense in the cytoplasm of proliferative and maturative chondrocytes of the growth plate cartilage than in that shown in the hypertrophic ones. In the metaphyseal secondary ossification zone, osteoblasts (OBs) and osteocytes (OCs) were variably labeled, whereas osteoclasts (OCLs) were always intensely stained. The labeling was also visible in some bone marrow cells, in articular chondrocytes, in the cells of tendon-bone junctions, and in the perichondrium and periosteal cells. Our results confirm a cellular co-location of GR protein and mRNA. In agreement with GR immunolocalization, the variability of labeling appeared to be related to the cell cycle, the stage of differentiation and cell-type differences.