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.

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study.

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.

Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats.

The expression of Bcl-2 and Bax has been evaluated by immunohistochemistry in normal rats, and in rats after treatment with high-dose corticosterone (CORT). Proliferative (PC) and maturative/hypertrophic (MaHC) chondrocytes of the growth plate have been examined, as well as osteoblasts (Obs), osteocytes (Ots) and osteoclasts (Ocs) of the metaphyseal secondary spongiosa. For each cell type, the Bcl-2 and Bax immunopositive cells were expressed as a percentage of the total number of cells. Bcl-2 and Bax expression was considered to be enhanced when the percentage of positive cells rose. Bcl-2 and Bax were expressed in all cell types, and two main kinds of labeling distribution, both suggestive of association with intracellular organelles, were observed in the cytoplasm: scarce and spotty labeling (type 1) or abundant, granular and diffuse labeling (type 2). In some cases, nuclear membranes could also be seen to be positive. Positive PCs and Obs generally showed a labeling of type 1, MaHCs and Ocs of type 2, while Ots varied with labeling of type 1 or type 2. CORT administration induced a fall in the percentage of Bcl-2 immunopositive cells, and a rise in that of Bax immunopositive cells, in PCs and Ots. The same trend was observed in MaHCs, although the Bcl-2 decrease was not significant. The percentage of Bcl-2 and Bax immunopositive Obs rose, and their labeling distribution shifted from type 1- to type 2-labeled cells. Ocs showed the highest immunopositivity for both Bcl-2 and Bax, which did not change after CORT administration. These data suggest that CORT treatment, by lowering Bcl-2, and raising Bax expression, may promote the apoptotic process in PCs, MaHCs and Ots. Obs, however, do not undergo the same variations. This finding, together with the results of a previous study showing that CORT administration raises the frequency of apoptotic Obs, does not support a direct relationship between apoptosis and Bax overexpression, at least in Obs. The CORT effect might be related to cell types and their state of differentiation, so that Bcl-2 and Bax might regulate not only the machinery of cell death, but also cell proliferation and differentiation.

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 ameloblast 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.

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.

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.

Cytochemical demonstration of the glucocorticoid receptor in skeletal cells of the rat.

The in vivo localization of the glucocorticoid receptor (GR) was studied in cartilage and bone cells of femurs of young adult rats. Deparaffinized sections were treated with a polyclonal antibody raised against the amino-terminus of human GR; the immunoreaction was detected with the streptavidin-biotin amplification method. Histomorphometric, computer-assisted analysis of GR-positive cells was performed by counting the percentage of GR-immunostained cells in the proliferative and maturative/hypertrophic zone of the epiphyseal growth plate cartilage, and of the percentage of positive osteoblasts (OBs), osteoclasts (OCLS) and osteocytes (OCs) in the metaphyseal secondary ossification zone. Numbers of OBs and OCLs per mm of metaphyseal endosteal perimeter, and numbers of OCs per mm2 of trabecular area were also counted. Immunopositive cells were found both in cartilage and bone, with variable degree of nuclear and/or cytoplasmic immunostaining; immunonegative cells were present among the positive ones. Histomorphometry showed that about 54% of chondrocytes in the proliferative zone, and 55% of chondrocytes in the maturative/hypertrophic zone of the growth plate were labeled; in metaphyseal bone, 68% of OBs, 65% of OCs, and 98% of OCLs were GR-positive. The density of positive cells was 12.06 OBs/mm, 3.32 OCLs/mm, and 520.40 OCs/mm2. These results, for the first time obtained in vivo, show that GR is present in cartilage and bone cells, and that the degree of GR-immunostaining is variable in the same type of cell. This may be dependent on the cell cycle and stage of differentiation, and may reflect a variable cellular sensitivity to the stimulation of the glucocorticoid hormone.

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia.

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.

MC22-33F monoclonal antibody shows unmasked polar head groups of choline-containing phospholipids in cartilage and bone.

The importance of the role lipids may have in biological calcification, and the paucity and poor specificity of the methods available for studying their ultrastructural localization, call for further investigations involving new techniques. The monoclonal antibody MC22-33F displays a specific reaction with phosphatidylcholine, sphingomyelin and dimethylphosphatidylethanolamine, as confirmed by its reaction with synthetic lyposomes of pure phosphatidylcholine. For this reason, it was used to demonstrate the presence and ultrastructural localization of choline-containing phospholipids in calcifying cartilage and bone. The MC22-33F MAb immunoreaction was found in the cytoplasm of maturing and hypertrophic and, to a lesser extent, proliferating and degenerating chondrocytes; it was strongly positive in matrix vesicles, at the periphery of calcification nodules, and at the periphery of calcified matrix. In bone, the immunoreaction was especially strong along the peripheral membrane of the osteoblasts, in the interfibrillary spaces of the osteoid border, in matrix vesicles and in the peripheral zone of the calcification nodules. The central zone of the nodules, the fully calcified matrix and most of the intracellular membranes were negative in both cartilage and bone. These results confirm the presence of choline-containing phospholipids in early areas of calcification, including matrix vesicles. They also show that the intracellular membrane phospholipids are not accessible to the antibody, possibly because they are masked by other substances. These are not proteoglycans, because their enzymatic digestion does not increase or modify the reactivity of MC22-33F MAb. In spite of this limitation, MC22-33F MAb offers considerable advantages in the study of the calcification process, because the positive immunoreaction of the calcifying bone matrix, and the negativity of calcified matrix, confirm without doubt that phospholipids have an active role in biological calcification.

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.

Morphological studies of hypomineralized enamel of rat pups on calcium-deficient diet, and of its changes after return to normal diet.

BACKGROUND: Micro-hardness investigations have shown that rat pups nursed by mothers on a low calcium diet and weaned with the maternal calcium-deficient diet develop hypomineralized enamel. The inorganic and organic components of this enamel, their relationships, and their changes after return to normal diet have been studied by light and electron microscopy. METHODS: The maturation zone of incisor enamel has been studied in: (1) rats nursed for 20 days by mothers on a low calcium diet and weaned for 30 days with the same diet (E1 enamel); (2) rats that after the calcium-deficient diet were fed normal diet for 10 days (E2 enamel); and (3) rats nursed for 20 days by mothers on a normal diet and weaned for 30 days with a normal diet (controls). RESULTS: The results showed that E1 enamel was hypomineralized, as noted by its Azure II-Methylene blue stainability in undecalcified sections, its light staining with the von Kossa method, and its ultrastructure. E1 crystallites, although present throughout the whole enamel, were thinner than those of E2 enamel, which were similar to those of controls. E1 interrod crystallites were thicker in the intermediate than in the dentinal zone and were thicker than rod crystallites. Organic matrix was present throughout the whole E1 enamel. Its organic components (crystal ghosts) had the same shape, arrangement, and organization as those of inorganic crystallites. Crystal ghosts were greatly reduced in E2 enamel and in controls. CONCLUSIONS: The results lead to the conclusions that: (1) E1 enamel is hypomineralized, and its degree of calcification is restored by return to a normal calcium diet; (2) intra- and interprismatic calcification occurs in a different way; (3) crystallite thickness is initially greater in dentinal than in the superficial zone and is reversed as crystallite growth is completed; and (4) loss of enamel proteins is necessary for completion of crystallite growth and not for crystallite formation.

Neoplastic infiltration of laryngeal cartilages: histocytochemical study.

The relationship between cartilage and invading neoplastic cells was studied in 32 cases of laryngeal cancer by histological and cytochemical methods. Cartilage invasion was present in 12 cases, 10 of which were in proximity or in contact with areas of calcification and ossification. It was significantly correlated only to tobacco consumption (P less than .05) and, in regard to glottic tumors, to tumor diameter greater than 3 cm (P less than .01). Histologically, neoplastic invasion in cartilage was massive in 2 cases, occurred in areas of ossification in 4, between cartilage and bone in 4, and in epiglottic cartilage in 2. In 3 of the cases with bone invasion, there was also new bone formation. Hyaline cartilage and bone resorption was due to tartrate-resistant acid phosphatase (TRAP)-positive giant cells; in epiglottic cartilage only mononuclear cells were present, some of which were TRAP-positive. These results show that neoplastic cells can promote not only resorption and formation of bone, but also resorption of cartilage, which is considered resistant to neoplastic invasion. The different types of resorbing cells in contact with hyaline cartilage and bone in laryngeal cancer, and elastic cartilage in epiglottic cancer, suggest that the structure of the tissue being resorbed can influence the type of resorbing cells.

Experimental aluminum intoxication and parathormone: effects on the mineralization process.

In order to study the effect of aluminum intoxication on bone and bone cells in normal animals and its relationship with hyperparathyroidism, and so to obtain further data on a pathogenetic role of this condition in inducing osteomalacia in uremic patients, 31 rats divided in four groups were injected intraperitoneally for 11 weeks with: Al (75.6 mg); Al-PTH (Al = 75.6 mg + PTH = 200 USP during the last week); C (saline solution), and C-PTH (saline solution + PTH = 200 USP during the last week). Al injection induced a consistent increase in the element in serum and tibia. PTH administration further enhanced Al content in tibia. The trabecular bone surfaces of Al-administered rats were stained by aluminon; the endosteal borders of their compact bone were always negative. Rib histomorphometry showed absence of osteomalacia in Al group and increase in osteoid in Al-PTH group, with development of mild osteomalacia. In these groups osteoclasts were less numerous than in controls. Dynamic bone parameters showed no separation of double tetracycline labels in trabecular bone of both Al-administered groups. Cortical bone was only slightly affected by treatment. All these data indicate that Al alone, in the quantity administered, does not induce osteomalacia in normal rats and that PTH, although given for a few days, enhances Al content in bone and induces osteoid increment. The reduction of tetracycline labels in all Al-treated animals is due to reduction of calcification and formation rate, which might be an index of osteoblast inhibition. The decrease in the number of osteoclasts suggests that Al might inhibit their formation.

Distribution of aluminium following intraperitoneal injection of aluminium lactate in the rat.

An animal experiment was performed to evaluate the absorption and distribution of aluminium in serum and tissues of normal rats. The animals were intraperitoneally injected with an aluminium lactate solution at a pH adjusted to 7.0. Before starting, a short preliminary study was carried out in order to verify the validity of the treatment with aluminium lactate instead of aluminium chloride at endogenous pH 3.4. Thirty-one rats were used in the main experiment, divided in four groups. In treated animals, the total Al-administered dose was 75.6 mg during 78 days of treatment. Furthermore, to evaluate the influence of the parathyroid hormone on Al absorption and/or distribution, 200 USP/rat of parathyroid hormone extract (PTH) were also administered during the last 5 days of the experiment. Aluminium content in serum, tibia, rib, brain, liver, muscle, kidney and spleen was determined. Calcium analysis in serum and bone was also performed. The highest concentrations of aluminium were found in liver and spleen, whereas the lowest level was found in the brain. The PTH effect on Al absorption was evident in brain and bone.

Aluminum bone content in dialysis osteodystrophy with secondary hyperparathyroidism.

To assess whether parathyroid hormone (PTH) has any effect on bone aluminium (A1) accumulation, we evaluated the relationship between bone quantitative histomorphometry and bone A1 content in 25 dialysis patients with clinical features of secondary hyperparathyroidism. By means of bone biopsy, patients were classified as showing predominant hyperparathyroidism in 17 cases and mixed lesions in 8. The A1 burden was low, judging from the dialysate A1 and the oral A1 assumption. Bone A1 content and serum A1 levels were higher than normal in both groups, particularly so in the patients with predominant hyperparathyroidism. Significant positive correlations were found between bone indexes of hyperparathyroidism and bone A1 content. Moreover, bone and serum A1 concentrations were directly related. Aluminium staining appeared faintly positive only in a minority of bone samples, and was mainly localized at the neutral bone surfaces. We conclude that in dialysis osteodystrophies with features of secondary hyperparathyroidism and in a clinical setting characterized by low-grade A1 burden, PTH may favour A1 accumulation into the bone.