Osteoclasts and their relationship to bone as studied by electron microscopy

Summary Osteoclasts in metaphyses from young rats were systematically sectioned at different levels. Two types of osteoclasts were recognized. One type had no ruffled border while the other, and predominant type contained a ruffled border in a part of its length; some of the latter contained two ruffled borders. The closest contact between osteoclast and bone occurred at the level of the ruffled border and this bone under the border showed characteristic changes indicative of resorption. In some osteoclasts the ruffled border consisted of numerous slender cytoplasmic projections separated by very narrow spaces or channels while in other osteoclasts it was more open. The ruffled border was commonly surrounded by a transitional zone containing numerous thin filaments. The osteoclast usually had its greatest dimension at the level of the ruffled border and the cytoplasm here contained many bodies and vacuoles but a sparse endoplasmic reticulum. Away from the level of the ruffled border the cytoplasmic vacuoles and bodies were fewer while the endoplasmic reticulum was often more pronounced. Parts of the osteoclasts were usually situated close to a vessel. It is suggested that there is a correlation between the development of the ruffled border and the degree of bone resorption and that osteoclasts without a ruffled border are, at least temporarily, inactive with respect to bone resorption. The numerous cytoplasmic bodies, interpreted as lysosomes, are presumed to be important in the resorption process. The closely adjacent positioning of osteoclasts and vessels may facilitate the transport of resorption products to the blood.This research was supported by the Danish Research Council. Grant no. 512–727, 512–819 and 512–1545.I wish to thank Professor Arvid B. Maunsbach for valuable discussions.     

Some morphological observations on osteoclasts

Summary Osteoclasts of the peripheral portions of the endocranial aspect of young rat parietal and frontal bones were studied by scanning electron microscopy of glutaraldehyde fixed, critical point dried specimens. These studies show Osteoclasts to have a much more complicated form than has previously been realised. Extensively branching, elongated, smooth-surfaced cells, which are for the most part elevated above the level of the surrounding bone matrix surface and sometimes above portions of osteoblasts or other osteoclasts, were identified as motile non-resorbing cells. Portions of the former and other entire cells may be embowered in Howship's lacunae, have microvilli on their dorsal surface, and are surrounded by a serrated border of microprojections which have an apparently firm attachment to the matrix surface. Osteoclasts in short term culture show additional free surface ruffles which are not encountered in specimens taken fresh from the animal. No evidence of recruitment of osteoblasts or osteocytes into osteoclasts was found. Disinterred osteocytes retained an ability to migrate from their lacunae on to surrounding bone matrix surface.We would like to thank Elaine Maconnachie for expert technical assistance and Dr. Martin J. Evans for the use of his tissue culture laboratory. These studies have been supported in part by a grant from the Medical Research Council     

Ultrastructure of the giant cell infiltrate of subcutaneously implanted bone particles in rats and mice

The giant cells of soft tissues and those of mineralized tissues (osteoclasts) have distinctly different cell surface receptors and ultrastructural characteristics. Recently, the removal of dead bone particles in a subcutaneous environment has been described as a prototype of bone resorption, and a major issue is whether the giant cells that surround these ectopic bone implants and the processes involved in the disruption of bone surfaces are the same as those in the skeleton. We have compared the cytology and ultrastructure of giant cells recruited to subcutaneously implanted isogeneic bone particles with similar features of osteoclasts in metaphyseal bone of young normal rats and mice. Giant cells on surfaces of bone particles 2, 3, and 4 weeks after implantation were multinucleated, had a homogeneous, nonvacuolated cytoplasm, and had a bone surface interface unremarkable by light microscopy. In a few cells randomly distributed, small cytoplasmic vacuoles were present and large vacuoles were noted next to the bone surface at high magnification. By transmission electron microscopy, folded membrane configurations forming extensive interdigitations with adjacent cells were prominent features on most surfaces of giant cells. In instances where these interdigitations abutted bone surfaces, configuration resembling a ruffled border were noted, but these regions were always part of two different cells when examined at lower magnification or in serial sections. Breakdown of bone particles appeared to be by phagocytosis of small pieces and subsequent intracellular digestion in electron-dense cytoplasmic vacuoles. Osteoclasts from these same young animals were smaller with fewer nuclei, had cytoplasmic vacuoles concentrated next to bone surfaces, and had characteristic ruffled borders and clear zones. These results confirm those of others that native osteoclasts and multinucleated giant cells on dead bone particles are distinctly different with respect to both ultrastructure and mechanism of disruption of bone surfaces.     

Osteoclasts secrete the chemotactic cytokine mim-1

Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.     

Myeloblastic cell line expresses osteoclastic properties following coculture with marrow stromal adipocytes

Osteoclasts are derived from hemopoietic precursors in the marrow. Their differentiation pathway is still underfined, but an important role was obserned for the marrow micrienvironment in the regulation of osteoclasto genesis. various marrow stromal cell subtypes were used to study their possible role in the formation of osteoclasts from myeloblast (M1) cells. Interactions between M1 cell and the 14F1.1 endothelial-adipocyte stromal cell line were demonstrated in a coculture model. M1 cells attached to the adherent layer of 14F1.1 cells and formed distinct focireminiscente of “cobblestone areas.” Follwing these inteactions, M1 Cells developed specific enzymatic activites and became multinucleated. Both monouclear M1 cells became positive to tartrate-resistant acid phosphatase (TRaP) and ATPase, a feature charactreistic of osteoclasts, and were also responsive to calcitonin. Furthermore, they attached to mineralized bone particles and their membrane changed into a ruffled border at the zone of interaction with the bone matrix. We thus demonstrated that marrow endothelial-adipocytes may play a role in regulating the differentiation of myeloblast into osteoclasts.     

Uptake of peroxidase by calcitonin inhibited osteoclasts

Summary The present electron microscopic histochemical study demonstrates that osteoclasts from calcitonin treated bone are able to take up organic macromolecules even though the ruffled border has disappeared. The absorption occurs around the entire periphery of the osteoclast, but the amount of absorbed peroxidase seems to be reduced in comparison with that of untreated cells. It is concluded that the effect of calcitonin on osteoclasts is primarily a cessation of the exocytosis and the concomitant disappearance of the ruffled border.This research was supported by the Danish Medical Research Council, grant No. 512-7184     

Ultrastructural evidence in vitro of osteoclast-induced degradation of calcium phosphate ceramic by simultaneous resorption and phagocytosis mechanisms

Osteoclasts are physiological polykaryons specialized in the resorption of calcified tissue. In the context of the clinical use of calcium-phosphate (CaP) ceramics as bone substitutes, this study used transmission electron microscopy to investigate the in vitro mechanisms of CaP ceramic degradation by osteoclastic cell types. Osteoclasts cultured on CaP ceramic developed typical ultrastructural features of bone osteoclasts, such as a polarized dome shape, a clear zone and a ruffled border. Modification of the shape and density of CaP crystals under the ruffled border indicated an acidic microenvironment. Moreover, osteoclasts were able to degrade ceramic by simultaneous resorption and phagocytosis mechanisms. Phagocytosis did not alter the ability of osteoclasts to resorb CaP ceramic. The phagocytosis mechanism consisted of three steps: crystal phagocytosis, disappearance of the endophagosome envelope membrane and fragmentation of phagocytosed crystals within the cytoplasm. The common mechanism of phagocytosis described here is similar to that observed with the monocyte/macrophage lineage, confirming that osteoclasts are part of the mononuclear phagocyte system. Osteoclasts are thus clearly involved in CaP degradation by means of resorption and phagocytosis.     

CELLULAR BIOLOGY OF BONE RESORPTION

Past knowledge and the recent developments on the formation, activation and mode of action of osteoclasts, with particular reference to the regulation of each individual step, have been reviewed. The following conclusions of consensus have emerged.
1. The resorption of bone is the result of successive steps that can be regulated individually.
2. Osteoclast progenitors are formed in bone marrow. This is followed by their vascular dissemination and the generation of resting preosteoclasts and osteoclasts in bone.
3. The exact pathways of differentiation of the osteoclast progenators to mature osteoclasts are debatable, but there is clear evidence that stromal cells support osteoclast generation.
4. Osteoclasts are activated following contact with mineralized bone. This appears to be controlled by osteoblasts that expose mineral to osteoclasts and/or release a factor that activates these cells.
5. Activated osteoclasts dissolve the bone mineral and digest the organic matter of bone by the action of agents secreted in the segregated microcompartments underlying their ruffled borders. The mineral is solubilized by protons generated from CO, by carbonic anhydrase and secreted by an ATP-driven vacuolar H⁺-K⁺-ATPase located at the ruffled border. The organic matrix of the bone is removed by acid proteinases, particularly cysteine-proteinases that are secreted together with other lysosomal enzymes in the acid environment of the resorption zone.
6. Osteoclastic bone resorption is directly regulated by a polypeptide hormone, calcitonin (CT), and locally, by ionized calcium (Ca²⁺) generated as a result of osteoclastic bone resorption.
7. There is new evidence that osteoclast activity may also be influenced by the endothelial cells via generation of products including PG, NO and endothelin.     

Localization of CD44, the hyaluronate receptor; on the plasma membrane of osteocytes and osteoclasts in rat tibiae

CD44 is a multifunctional adhesion molecule that binds to hyaluronic acid, type I collagen, and fibronectin. We have studied the immunohistochemical localization of CD44 in bone cells by confocal laser scanning microscopy and transmission electron microscopy in order to clarify its role in the cell-cell and/or cell-matrix interaction of bone cells. In round osteoblasts attached to bone surfaces, immunoreactivity is restricted to their cytoplasmic processes. On the other hand, osteocytes in bone matrices show intense immunoreactivity on their plasma membrane. Intense immunoreactivity for CD44 can be detected on the basolateral plasma membranes of osteoclasts. There is considerably less reactivity observed in the area of the plasma membrane that is in direct contact with bone. The pre-embedding electron-microscopical method has revealed that CD44 is mainly localized on the basolateral plasma membrane of osteoclasts. However, the ruffled border and clear zone show little immunoreactivity. A CD44-positive reaction can be detected on both plasma membranes in the contact region between osteoclasts and osteocytes. These findings suggest that: 1) cells of the osteoblast lineage express CD44 in accordance with their morphological changes from osteoblasts into osteocytes; 2) osteoclasts express CD44 on their basolateral plasma membrane; 3) CD44 in osteoclasts and osteocytes may play an important role in cell-cell and/or cell-matrix attachment via extracellular matrices.     

Immunocytochemical localization of cathepsin D in the rat osteoclast.

We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 micron) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsin-negative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Osteogenic periosteum esterase activity: a comparative morphological and cytochemical study of bone cells in situ on rat proximal tibiae and in smears

Mononucleate phagocytes are presently thought to participate, along with osteoclasts, in bone resorption. Similar cells may also be precursors to osteoclasts. Esterase is a hydrolytic enzyme commonly found in most phagocytes and cells such as these have been shown to degrade bone matrix in vitro. Therefore, periosteal cells of remodeling proximal tibiae in rats were examined for esterase activity. Stain reactions were compared both in situ and in smears. Percentages of different cell types were obtained by counting cells in Wright-Giemsa-stained smears. Osteoclasts and mononucleate phagocyte-like cells made up approximately 20% of the isolated cells. Cells resembling preosteoblasts, mononucleate phagocytes, and osteoblasts stained for esterase activity with light to moderate intensity. Osteoclasts and mononucleate osteoclast-like cells were strongly esterase positive. The esterase was characterized as being type B by using nonorganophosphorus inhibitors of the various esterase types. The addition of esterase to the list of enzyme activities of cells in close proximity to osteoclasts lends further support to the hypothesis that mononucleate cells also participate in bone resorption.     

Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteoclasts formed from both types of precursors. In contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. The substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption.     

Evidence that failure of osteoid bone matrix resorption is caused by perturbation of osteoclast polarization

Osteoclasts resorb bone by a complex dynamic process that initially involves attachment, polarization and enzyme secretion, followed by their detachment and migration to new sites. In this study, we postulated that mineralized and osteoid bone matrix signal osteoclasts differently, resulting in the resorption of mineralized bone matrix only. We, therefore, compared the cytoplasmic distribution of cytoskeletal proteins F-actin and vinculin using confocal laser-scanning microscopy in osteoclasts cultured on mineralized and demineralized bone slices and correlated the observations with their functional activity. Our results have demonstrated significant differences in F-actin and vinculin staining patterns between osteoclasts cultured on mineralized bone matrix and those on demineralized bone matrix. In addition, the structural variations were accompanied by significant differences in bone resorbing activity between osteoclasts grown on mineralized bone matrix and those on demineralized bone matrix after 24 h of culture -- resorption only occurring in mineralized bone but not in demineralized bone. These results indicated that failure of osteoid bone resorption is caused by perturbation of osteoclast polarization. © 1998 Chapman & Hall     

A simplified method for the generation of human osteoclasts in vitro

Osteoclasts are large, multinucleated cells responsible for the resorption of mineralized bone matrix. These cells are critical players in the bone turnover involved in bone homeostasis. Osteoclast activity is connected to the establishment and expansion of skeletal metastases from a number of primary neoplasms. Thus, the formation and activation of osteoclasts is an area of research with many potential avenues for clinical translation. Past studies of osteoclast biology have utilized primary murine cells cultured in vitro. Recently, techniques have been described that involve the generation of osteoclasts from human precursor cells. However, these protocols are often time-consuming and insufficient for generating large numbers of osteoclasts. We therefore developed a simplified protocol by which human osteoclasts may be easily and reliably generated in large numbers in vitro. In this study, osteoclasts were differentiated from bone marrow cells that had been aliquotted and frozen. Cells were generated by culture with recombinant macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). Both human and murine RANKL were shown to efficiently generate osteoclasts, although higher concentrations of murine RANKL were required. Formation of osteoclasts was demonstrated qualitatively by tartrate-resistant acid phosphatase (TRAP) staining. These cells were fully functional, as confirmed by their ability to form resorption pits on cortical bone slices. Functional human osteoclasts can be difficult to generate in vitro by current protocols. We have demonstrated a simplified system for the generation of human osteoclasts in vitro that allows for large numbers of osteoclasts to be obtained from a single donor.     

Immunocytochemical localization of cathepsin D in the rat osteoclast

Summary We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 m) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections.At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface.These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.     

Treatment with tacrolimus enhances alveolar bone formation and decreases osteoclast number in the maxillae: a histomorphometric and ultrastructural study in rats

Recent studies have suggested that tacrolimus monotherapy is a beneficial therapeutic alternative for the normalization of cyclosporin-induced bone loss in animal models and humans. The mechanism accounting for this action is unclear at present. In the present study, we attempted to determine the effect of tacrolimus monotherapy on alveolar bone using histological, histomorphometric and transmission electron microscopy (TEM). Groups of rats (n=10 each) were treated with either tacrolimus (1mg/kg/day, s.c.) or drug vehicle for 60 days. Fragments containing maxillary molars were processed for light microscopy to investigate the alveolar bone volume, trabecular separation, number of osteoclasts and osteoblasts, and transmission electron microscopy to investigate their ultrastructural basic phenotype. Treatment with tacrolimus monotherapy during 60 days may induce increases in alveolar bone volume (BV/TV,%; P<0.05) and a non-significant decrease in trabecular separation (Tb.Sp,mm; P>0.05), represented by a decrease in osteoclast number (N.Oc/BS; P<0.05) and maintenance of osteoblast number (N.Ob/BS; P>0.05). Osteoblasts were often observed as a continuous layer of active cells on the bone surface. Osteoclasts appeared to be detached from the resorbed bone surface, which was often filled by active osteoblasts and collagen-rich matrix. Moreover, osteoclasts in the treated group were frequently observed as inactive cells (without ruffled border, clear zone and detached from the bone surface). Within the limits of the present study, we conclude that tacrolimus leads to an increase in alveolar bone formation, which probably exerts action on osteoclasts. Tacrolimus could, therefore, play a crucial role in the control of both early osteoclast differentiations from precursors, as well as in functional activation.     

Electron microscopic and histochemical studies of the mononuclear osteoclast of the mouse.

Osteoclasts collected from the long bones of mice were cultured on dentin slices. To identify osteoclasts, the tartrate-resistant acid phosphatase (TRACPase) activity of cultured cells was histochemically examined by the azo dye method. The TRACPase-positive cells could be distinguished from other cells by light microscopy. The cells were sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional structure. TRACPase activity was detected both in multi-nucleated osteoclasts and in mononuclear cells. Most of the mononuclear TRACPase-positive cells had features similar to preosteoclasts. A mononuclear TRACPase-positive cell was a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell possessed a large clear zone and small ruffled border. Under the ruffled border, no lacuna was apparent; but there was disruption of the dentin surface. The results suggest that this cell was a mononuclear osteoclast and that it might have been in the process of making a new lacuna.     

Cloning and function of rabbit peroxisome proliferator-activated receptor delta/beta in mature osteoclasts

Osteoclasts modulate bone resorption under physiological and pathological conditions. Previously, we showed that both estrogens and retinoids regulated osteoclastic bone resorption and postulated that such regulation was directly mediated through their cognate receptors expressed in mature osteoclasts. In this study, we searched for expression of other members of the nuclear hormone receptor superfamily in osteoclasts. Using the low stringency homologous hybridization method, we isolated the peroxisome proliferator-activated receptor delta/beta (PPARdelta/beta) cDNA from mature rabbit osteoclasts. Northern blot analysis showed that PPARdelta/beta mRNA was highly expressed in highly enriched rabbit osteoclasts. Carbaprostacyclin, a prostacyclin analogue known to be a ligand for PPARdelta/beta, significantly induced both bone-resorbing activities of isolated mature rabbit osteoclasts and mRNA expression of the cathepsin K, carbonic anhydrase type II, and tartrate-resistant acid phosphatase genes in these cells. Moreover, the carbaprostacyclin-induced bone resorption was completely blocked by an antisense phosphothiorate oligodeoxynucleotide of PPARdelta/beta but not by the sense phosphothiorate oligodeoxynucleotide of the same DNA sequence. Our results suggest that PPARdelta/beta may be involved in direct modulation of osteoclastic bone resorption.     

THE FINE STRUCTURE OF BONE CELLS

An electron microscopic study of Araldite-embedded, undecalcified human woven and chick lamellar bone is presented. The fine structure of the cells of bone in their normal milieu is described. Active osteoblasts possess abundant granular endoplasmic reticulum, numerous small vesicles, and a few secretion droplets. Their long cytoplasmic processes penetrate the osteoid. The transition of osteoblasts into osteoid osteocytes and then into osteocytes is traced and found to involve a progressive reduction of cytoplasmic organelles. Adjoining the osteocytes and their processes is a layer of amorphous material which is interposed between the cell surfaces and the bone walls of their respective cavities. Osteoclasts contain numerous non-membrane-associated ribosomes, abundant mitochondria, and little granular endoplasmic reticulum, thus differing markedly from other bone cells. The brush border is a complex of cytoplasmic processes adjacent to a resorption zone in bone. No unmineralized collagen is seen at resorption sites and it appears that collagen is removed before or at the time of mineral solution. All bone surfaces are covered by cells, some of which lack distinctive qualities and are designated endosteal lining cells. The structure of osteoid, bone, and early mineralization sites is illustrated and discussed.