[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.

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.

New sites of cellular vitronectin receptor immunoreactivity detected with osteoclast-reacting monoclonal antibodies 13C2 and 23C6.

The immunohistochemical profile of osteoclast-reacting monoclonal antibodies, 13C2 and 23C6, known to detect the alpha-chain of the vitronectin receptor, is described. Both antibodies reacted with several cell types apart from osteoclasts including megakaryocytes, smooth muscle cells, endothelial cells, thyroid follicular epithelium, renal glomeruli and tubular epithelium, myoepithelial and epithelial cells in the breast and prostate, and both cytotrophoblast and syncytiotrophoblast. In addition, macrophage polykaryons, synovial lining cells, a small number of mononuclear cells in buffy coats, and a few macrophage-like cells in the stroma of various tissues were also stained. The epitopes recognized by these antibodies are thus not osteoclast-specific and are present on other cells of the mononuclear phagocyte system. The implications of these results for osteoclast ontogeny, the nature of the antigens described and the question of osteoclast-specific antibodies are discussed.

Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin.

We investigated the effects of calcitonin (CT) and parathyroid hormone (PTH) on the distribution of actin, tubulin, vimentin, and on cell size in cultured chick osteoclasts. In addition, we studied the effects of colchicine on intracellular acidity. Osteoclasts were isolated from the endosteum of 2-3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with CT for 30 min or PTH for 60 min and were observed after immunocytochemical staining of cytoskeletal proteins. In untreated cells, actin was found in both a filamentous and a punctate staining pattern, with indented or invaginated regions free of punctate spots. The tubulin distribution in untreated cells was characterized by a pattern of microtubules radiating from the cell center and running parallel to the cell edge. Vimentin staining was usually localized to the perinuclear area. There were no changes in cytoskeletal element distribution or morphology attributable to PTH treatment. Osteoclasts treated with CT were more irregularly shaped, contained more retraction fibers, and were more rounded, with a denser array of cytoskeletal elements in the cell center. In addition, the mean area of the CT-treated cells was significantly less than that of the untreated cells. The actin distribution after CT treatment was still characterized by both a filamentous and a punctate pattern. After CT treatment, vimentin staining appeared more centrally localized than in untreated cells and tubulin staining revealed microtubules which now extended to the retracted cell margin. These results indicate that isolated osteoclasts respond to CT by significant morphological changes which are reflected in the distribution of the major cytoskeletal elements. Disruption of the microtubular system by colchicine treatment also resulted in an initial increase in intracellular acidity, suggesting the involvement of microtubules in the movement of acid-laden vesicles to the exterior.

Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.

Cells showing osteoclastic characteristics have not been identified outside bone. Because osteoclasts originate from an extraosseous source, this suggests that identifiable osteoclastic features do not develop until the precursors enter bone, where the local microenvironment may signal osteoclastic differentiation or maturation. We assessed the influence of bone matrix on osteoclastic differentiation by incubating bone marrow cells, after removal of pre-existing osteoclasts, on plastic coverslips or slices of devitalized cortical bone. We found that there was a threefold increase in the number of osteoclast-specific MAb-positive cells on the bone matrix compared with plastic coverslips. The number of MAb-positive cells correlated with the extent of excavation of the surface of the bone slices. Multinuclearity correlated with MAb-positive cell density, and for any given density the proportion of MAb-positive cells that were multinucleate was similar on plastic and bone. We conclude that, in the presence of 1,25-(OH)2 vitamin D3, bone matrix stimulates the generation of osteoclasts but has no demonstrable influence on the fusion of mononuclear osteoclastic precursors.

In vivo calvarial bone cell responses to dietary perturbations and the implications for mineral homeostasis.

Calvariae from small animals have been an important source for in vitro studies of bone. However, few in vivo studies have been undertaken on quantitative cell changes in calvariae. In the present study of mineral perturbations, rats were first deprived of calcium. After 18 days endosteal osteoclasts and nuclei/osteoclast in the parietal bone had increased 120% (P less than 0.001) and 26% (P less than 0.001), respectively, the marrow space had increased 141% (P less than 0.001), and the bone area experienced a 49% decrease (P less than 0.001). This thinning and weakening of the calvaria was accompanied by a compensatory increase in the number of endosteal osteoblasts (297%, P less than 0.001). These rats were then replenished with calcium, and after 14 days the number of endosteal osteoclasts had decreased to 86% (P less than 0.001) below the control and the endosteal surface was almost completely covered by osteoblasts (866% above the control, P less than 0.001). Bone area was increased by 51% (P less than 0.01). Similarly, in calcium-deficient rats in the tibial diaphysis at the fibular junction, the number of endosteal osteoclasts and the medullary space increased 1606% (P less than 0.001) and 63% (P less than 0.001), respectively, which were accompanied by a 32% decrease (P less than 0.001) in cortical bone area. After calcium replenishment, most endosteal osteoclasts in the tibial diaphysis disappeared from the endosteal surface and were replaced by osteoblasts (increased 487%, P less than 0.001). These results indicate that changes in bone cell activity in response to calcium deficiency are similar in calvariae and long bones, and that mobilization of calcium from the calvaria during calcium deficiency occurs at the expense of the protective action of the calvaria. Therefore, long bones as well as membranous bones are apparently important for the maintenance of mineral homeostasis.

Isolation and characterization of skeletal acid ATPase--a new osteoclast marker?

A tartrate-resistant, iron-activated and vanadate-sensitive nucleotide tri- and diphosphatase has been purified from rat bone. The purified enzyme (1,400-fold, 45% yield) has an Mr on SDS-PAGE of 30,000 Da. Hydrodynamic properties include a Stokes radius of 24A, a sedimentation coefficient of 3.2 S and a partial specific volume of 0.748 ml/g. The calculated Mr from hydrodynamic data is 32,000 and the enzyme binds 4 mol Triton X-100/mol enzyme. Substrate specificity studies demonstrate that the enzyme is active against nucleotide tri- and diphosphates and phosphotyrosine, but not against phosphoserine or phosphothreonine. Based on the purification profile and enzyme histochemistry, showing labelling of fewer mononuclear cells using ATP compared to conventional acid phosphatase substrates, it is suggested that the acid ATPase constitutes a unique form in the family of tartrate-resistant acid phosphatases and may thus have the potential as a marker for osteoclast ontogeny and function.

Beta 3 subunit of vitronectin receptor is present in osteoclast adhesion structures and not in other monocyte-macrophage derived cells.

The distribution of extracellular matrix receptors in human osteoclasts has been studied; a beta 3 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells and not in macrophages from the same source.

Dentin resorption mediated by odontoclasts in physiological root resorption of human deciduous teeth.

Extracted human deciduous teeth undergoing physiological root resorption were fixed with a mixture of formaldehyde and glutaraldehyde and processed for scanning (SEM) and analytical transmission electron microscopy, as well as acid trimetaphosphatase cytochemistry. The granulated tissues, which are rich in multinucleated odontoclasts and capillary vessels, formed various resorption lacunae on the resorbing dentin surfaces. SEM observations of dentin surfaces treated with sodium hypochlorite revealed two types of resorption lacunae: deep, round lacunae in which the peritubular matrix of dentinal tubules was strongly dissolved; and shallow, irregular lacunae with intact peritubular matrix. In trypsin-treated materials, the resorption surfaces were characterized by the presence of numerous collagen fibers in both the peritubular and intertubular matrices, suggesting demineralization of the surface dentin. Odontoclasts were characterized by the presence of abundant mitochondria, perinuclear stacks of Golgi membranes, various lysosomes, numerous endocytotic vacuoles, and a well-developed ruffled border against the resorption lacunae. Most endocytotic vacuoles were distributed in the cytoplasm between the ruffled border and the nuclei. In undemineralized ultrathin sections, the surface dentin of resorption lacunae consisted of collagen fibers and apatite crystals and had a lower packing density than those in unresorbed, deeper dentin. Many apatite crystals were demonstrated to be present in the extracellular channels of the ruffled border and in adjacent endocytotic vacuoles derived from it. Lysosomes located in the perinuclear cytoplasm of odontoclasts contained amorphous dense material and/or a small amount of crystals. An energy-dispersive x-ray microanalysis of apatite crystals in undemineralized sections indicated that the energy spectrum peaks of Ca and P detected from crystals in resorbing dentin were much lower than those in unresorbed dentin. Similarly, lower spectrum peaks of Ca and P were obtained from crystals found in the ruffled border and endocytotic vacuoles of odontoclasts. A slight trace Ca peak also was detected in the amorphous dense material in lysosomes of odontoclasts. The enzyme cytochemistry of lysosomal acid trimetaphosphatase indicated that odontoclasts had intense enzymatic activity in the Golgi membranes, endoplasmic reticulum cisternae, lysosomes, and endocytotic vacuoles. Dense reaction precipitates of enzymatic activity also were found along the dentin surfaces of resorption lacunae occupied by odontoclast ruffled borders.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of platelet antigens and fibrinogen on osteoclasts.

The antigenic phenotype of the human osteoclast, which is known to be derived from a circulating mononuclear precursor cell of haemopoietic origin, is controversial. Recent studies have shown that macrophage as well as megakaryocyte/platelet antigens are expressed by osteoclasts. In this study, we have sought to define, by immunohistochemistry, the nature and possible function of platelet antigens expressed by human osteoclasts in foetal and adult bone specimens. Monoclonal antibodies to platelet glycoprotein IIIa (gpIIIa) and CD9 antibodies stained osteoclasts in all bone specimens examined. Fibrinogen was also localized to the osteoclast membrane in foetal bone imprints. In addition, we found that CD9 and gpIIIa antibodies reacted weakly with monocytes in buffy coat smears. Antibodies to factor 8 and glycoproteins Ib and IIb/IIIa did not react with osteoclasts. These results show that osteoclasts, monocytes, macrophages, megakaryocytes and platelets possess common antigens and that fibrinogen is present on the surface of osteoclasts. By analogy with platelets, CD9 and gpIIIa may play a role in fibrinogen binding by osteoclasts. Possible mechanisms by which platelet antigens and fibrinogen binding could affect osteoclast function are proposed.

Aluminium overload of parathyroid glands in haemodialysed patients with hyperparathyroidism: effect on bone remodelling.

In the course of chronic renal failure, aluminium may deposit and accumulate in different tissues. The aluminium content of parathyroid glands was measured in 31 haemodialysis patients at the time of a parathyroidectomy. The values were compared with those obtained from ten control patients with primary hyperparathyroidism without renal failure, and were related to bone remodelling. Of the 31 patients, 27 had a bone biopsy after double tetracycline labelling, at the time of parathyroidectomy. Twenty-one patients had severe hyperparathyroidism, three patients had hyperparathyroidism associated with osteomalacia, three patients had mild hyperparathyroidism with reduced bone formation. Seven patients had bone aluminium deposits, associated with osteomalacia in one case. The parathyroid aluminium was 62 +/- 35.7 (mumol/g glandular dry weight) in haemodialysis patients and 14.3 +/- 6.3 in control patients (P less than 0.001). A significant positive correlation existed between parathyroid aluminium and serum aluminium (P less than 0.01). The parathyroid aluminium was not different in the patients with and without bone aluminium deposits. A weak correlation was found between parathyroid aluminium and plasma parathyroid hormone. A significant negative correlation existed between parathyroid aluminium and osteoblastic surfaces (P less than 0.05), but no correlation was found with bone formation rate at tissue and bone multicellular units levels. We conclude that aluminium accumulates in parathyroid glands of dialysed patients. Severe hyperparathyroidism may coexist with aluminium overload of parathyroid glands. A marked aluminium overload, however, may cut short the course of hyperparathyroidism and may decrease parathyroid function and cellular activity in bone.

Immunocytochemical localization of gamma-carboxyglutamic acid-containing proteins (osteocalcin) in rat bone and dentin.

Gla-protein or osteocalcin is one of the most abundant noncollagenous matrix proteins found in bone and dentin. The present study describes, with high resolution, the intracellular and extracellular distribution of Gla-protein in alveolar bone and incisor dentin. Sections of tissues embedded in Lowicryl K4M were incubated with rabbit antibodies to rat dentin Gla-protein. The site of the specific antigen-antibody reaction was revealed by the protein A-gold complex. Labeling was detected over bone and dentin while fewer gold particles were present over prebone and predentin. Gold particles were also seen over the protein synthetic organelles (rough endoplasmic reticulum, Golgi apparatus) of osteoblasts and odontoblasts. These findings confirm, with improved resolution, previous light immunohistochemical studies, and offer the possibility to examine the secretory pathway of the protein.

Chicken osteoclasts do not possess calcitonin receptors.

Osteoclasts freshly isolated from embryonic chicks have been examined for calcitonin receptors using radio-iodine-labeled salmon calcitonin. Calcitonin binding to chick osteoclasts could not be shown by either autoradiography or biochemical binding studies. Furthermore, calcitonin did not stimulate cyclic AMP production. By contrast, rat osteoclasts have abundant calcitonin receptors, and a sensitive cyclic AMP response to calcitonin has been shown previously. It is concluded that chick osteoclasts do not possess calcitonin receptors, a finding which could explain the lack of calcitonin responsiveness observed in other avian osteoclast culture systems.

Osteoclasts contain macrophage and megakaryocyte antigens.

The origin and mechanism of formation of the osteoclast remains controversial. Although it is known to be derived from a circulating mononuclear percursor, the identity of this cell is unknown. Using a panel of monoclonal antibodies raised against macrophage and other marrow-derived cells, we determined the immunocytochemical staining of human osteoclasts in both fetal bone metaphyseal imprints and frozen sections. Osteoclasts and marrow mononuclear cells were stained by three broad spectrum antimacrophage antibodies, EBM-11, Y182a and BM2. T310, an antibody which stains macrophages and T helper cells, and C17, an antimegakaryocyte antibody, also stained osteoclasts. EBM-11, Y182a and BM2 also stained megakaryocytes in bone imprints as well as normal bone marrow smears. The presence of macrophage-associated antigens in osteoclasts, megakaryocytes and bone marrow mononuclear cells indicates that they are phenotypically similar to macrophages.

Postparathyroidectomy hypocalcemia as an accurate indicator of preparathyroidectomy bone histology in the uremic patient.

19 chronic renal failure patients underwent iliac crest bone biopsy prior to total parathyroidectomy with autotransplantation. The preoperative serum calcium concentration did not correlate with the number of osteoclasts/mm2 present on the preparathyroidectomy iliac biopsy. However, the postparathyroidectomy decrement in serum calcium (mg/dl and percent change) and the osteoclasts/mm2 were strongly correlated (p less than 0.001). In addition, the postoperative fall in serum calcium also correlated with the postoperative change in serum alkaline phosphatase (p less than 0.001). The nadir in postparathyroidectomy serum calcium was attained in a mean of 4.4 +/- 2.7 days. Our results indicate that the preoperative serum calcium concentration does not necessarily reflect active bone resorption, but the postoperative decrement in serum calcium provides an accurate index of preoperative histologic activity. The available data do not provide information with respect to the mechanism of postparathyroidectomy hypocalcemia since either the cessation of bone resorption, continued bone deposition, or a combination of both may be operative.

Osteoclasts and monocytes have similar cytoskeletal structures and adhesion property in vitro.

The distribution of some cytoskeletal structures (microtubules, microfilaments, intermediate filaments) has been studied by indirect immunofluorescence microscopy and affinity purified antibodies in osteoclasts isolated from medullary bone of laying hens and in hen blood monocytes cultured in vitro. Both cell types show similar patterns of distribution of cytoskeletal structures and this further supports the concept that these cells are closely related. Osteoclasts and monocytes are also similar in their adhesion patterns, because they adhere to fibronectin-free areas and show closely comparable cell-to-substrate interactions when observed with interference reflection microscopy.

Bone morphometry in alcoholics.

The osteoclast activity (number of osteoclasts per surface bone section) and the osteoid abundance (percentage of trabecular bone surface covered with osteoid) were measured in iliac crest biopsies from 38 men with alcoholism. These patients had histories of decades of over-consumption of alcohol and repeated injuries, as well as medical complications. In older individuals, the bone mineral content of the forearms decreased so that the reduction in relation to age was comparable to that of women rather than men. Osteoid seams increased in thickness only in alcoholics who had previously undergone gastric resection. Otherwise, there were no differences in osteoid seams in alcoholics and control nonalcoholism patients. Osteoclasts, however, were more abundant in the iliac crest biopsies from alcoholics than from the nonalcoholic group. Osteoclasts were also more numerous in alcoholics who had undergone gastric resection although, in the other cases, there were also significant increases above normal. In any case, the nutritional effects of alcoholism on the skeleton were relatively slight inasmuch as there was no case of advanced osteomalacia. Nevertheless, alcoholism causes bone changes, both systemic (possibly hormonal) and local in nature, and is characterized by bone resorption.

Histomorphometric analysis of primary teeth during the process of resorption and shedding.

The state of pulp tissue and periodontal root surface of 90 non-carious human primary teeth during the process of resorption and shedding was assessed by histomorphometric means. The teeth were classified into preshedding, shedding and delayed-shedding groups according to age of the child at the time of extraction. The root surface length measured from the cementoenamel junction was related to stages of shedding. Inflammatory cells in the pulp tissue were observed in all three groups (p less than 0.005). Odontoclasts in the pulp tissue could be demonstrated in the shedding and delayed-shedding stages (p less than 0.05). Polymorphonuclear leukocytes and odontoclasts were not observed before resorption had occurred approximately 1 mm subjacent to the cementoenamel junction. Active resorption of the periodontal root surface was observed in all stages. Deposition of cementum-like tissue in resorption lacunae on the root surface was most pronounced in the delayed-shedding stage showing repair in 30% of resorbing root surface length. Findings indicate that the process of resorption occurs mainly during the shedding stage and repair tends to accelerate in the delayed-shedding stage. The predentin appears to have more power of resistance than any other part of the tooth and there is no reason to believe that the pulp participates in the process of resorption of human primary teeth under physiologic conditions.

[Separation of osteoclasts from bird medullary bone by sedimentation at unit gravity]. / Isolamento di osteoclasti di osso follicolinico di uccelli mediante sedimentazione a gravita' unitaria.

A procedure for the isolation of osteoclasts from the heterogeneus population of medullary bone and marrow cells is presented. Cell suspensions were prepared from medullary bone of laying hens by mechanical dispersion. Following an osmotic shock and two 90 min unit gravity sedimentations, fractions highly enriched in osteoclasts were collected and subsequently cultivated in MEM with FCS. Ara C was added to the cultures for 48h to remove all the cells entering the mitotic cycle. The advantages and the possible applications of the method are discussed.