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.     

Demonstration of bone resorbing cells in elasmobranchs: Comparison with osteoclasts

We have previously shown that Elasmobranchs-characterized by a partially calcified cartilaginous endoskeleton-presented a bony vertebral arch containing osteoblasts, osteocytes and resorbing cells. The aim of this study is to test the ability of Elasmobranchs to resorb bone tissue. The subcutaneous implantation in dogfish (Scyliorhinus canicula) of devitalized mineral-containing bone particles, obtained from a bony fish (the eel, Anguilla anguilla) resulted, after 21 2 months, in the formation of mononucleated as well as multinucleated cells around and between the bone fragments. By light microscopy, the multinucleated giant cells presented the general aspect of osteoclastic cells whereas, by transmission electron microscopy they never showed ruffled borders which are considered as the typical features of osteoclasts. Except for this character, the mononucleated and multinucleated cells exhibited the typical ultrastructural aspects leading us to say that these cells are involved in the resorption of the bone fragments. This study shows that Elasmobranchs are able to resorb implanted bone.     

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.     

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.     

Osteoclasts in teleost fish: Light-and electron-microscopical observations

Summary This paper reports the common occurrence of osteoclasts during normal and experimental bone resorption in a number of teleost fishes. Light-microscopical observations on osteoclasts are presented in resorption areas on perichondral bone (mandibula and pharyngeal jaws of cichlids and vertebrae of gymnotids), on dermal bone (mandibula of salmonids and characoids and frontal bone of cichlids), on chondroid bone (pharyngeal jaws of cichlids), and on elasmoid body scales (eichlids and gymnotids). Osteoclasts acting along the bone surface usually lie in a Howship's lacuna whereas others are wrapped around bone extremities. Electronmicroscopical observations reveal that teleost osteoclasts show features similar to those of higher vertebrate osteoclasts, c.g., the presence of a ruffled border and the occurrence of numerous vacuoles, lysosomes and mitochondria. The multinucleated aspect that characterizes osteoclasts in other vertebrate groups is not a distinct feature of teleost osteoclasts since some are possibly mononucleated. Teleost osteoclasts are also able to resorb uncalcified tissues adjoining bone resorption areas, either as a primary process directed toward the tissue (basal plate of elasmoid scale) or as a secondary phenomenon (cartilage).     

Specific immunocytochemical localization of cathepsin E at the ruffled border membrane of active osteoclasts

The immunocytochemical localization of cathepsin E, a non-lysosomal aspartic proteinase, was investigated in rat osteoclasts using the monospecific antibody to this protein. At the light-microscopic level, the preferential immunoreactivity for cathepsin E was found at high levels in active osteoclasts in the physiological bone modeling process. Neighboring osteoblastic cells were devoid of its immunoreactivity. At the electron-microscopic level, cathepsin E was exclusively confined to the apical plasma membrane at the ruffled border of active osteoclasts and the eroded bone surface. Cathepsin E was also concentrated in some endocytotic vacuoles of various sizes in the vicinity of the ruffled border membrane, some of which appeared to be secondary lysosomes containing the phagocytosed materials. These results strongly suggest that this enzyme is involved both in the extracellular degradation of the bone organic matrix and in the intracellular breakdown of the ingested substances in osteoclasts.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

RANKL induces formation of avian osteoclasts from macrophages but not from macrophage polykaryons

We have shown that chick macrophages express RANK at their surface and human RANKL (hRANKL) triggers the formation of osteoclasts able to degrade dentine. As described for mammalian osteoclasts, hRANKL also stimulates the resorbing activity of chick bone-derived osteoclasts. In other hands, in culture, chick macrophages spontaneously form polykaryons sharing most of the osteoclast markers but unable to resorb bone. Since both bone-resorbing osteoclasts and macrophage polykaryons found in inflammatory tissues are multinucleated cells deriving from the fusion of macrophages, we examined whether macrophage polykaryons could be induced toward bone-resorbing osteoclasts. Long-term exposure of macrophage polykaryons to hRANKL failed to activate any resorbing activity, indicating that although deriving from the same precursors macrophage polykaryons and osteoclasts are independent cell types and polykaryons are not immature osteoclasts.     

THE EFFECTS OF PARATHYROID HORMONE, COLCHICINE, AND CALCITONIN ON THE ULTRASTRUCTURE AND THE ACTIVITY OF OSTEOCLASTS IN ORGAN CULTURE

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium (⁴⁵Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased ⁴⁵Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%). Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in ⁴⁵Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state. Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 Å filaments, suggesting that synthesis of microtubular subunits had increased.     

Bone resorption and bone remodelling in juvenile carp, Cyprinus carpio L.

The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte‐containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone‐resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.     

The unobtrusive majority: mononucleated bone resorbing cells in teleost fish and mammals

The canonical view of a mammalian (usually shown as human) bone resorbing cell is that of a giant macrophage‐like cell (osteoclast) that dissolves bone minerals and digests bone matrix proteins. The cells’ presence and activity is easily recognised based on three distinct morphological features: (i) multinuclearity, (ii) a multiply folded apical cell membrane (ruffled border), and (iii) deep lacunae (Howship’s lacunae) that the cells eroded into the bone surface. Mononucleated osteoclasts without these features are considered to be inactive precursors. We challenge the view that bone resorbing cells must be multinucleated giant cells, based on our comparative studies on the teleost skeleton, on what is currently known – but often disregarded – about mononucleated mammalian osteoclasts, and on what is know about osteocytic osteolysis.     

Osteoclast cell-surface changes during the egg-laying cycle in Japanese quail

The medullary bone serves as a source of labile calcium mobilized during calcification of the egg shell in birds. Quantitative histological methods demonstrate that the numbers of medullary bone osteoclasts and nuclei per osteoclast remain unchanged during the egg cycle in the Japanese quail (Coturnix). Therefore, cyclic changes in bone resorption cannot be explained by modulations of osteoclasts from and into other bone cells, a mechanism previously suggested for certain species of birds. Rather, dramatic changes in osteoclast cell-surface features occur during the egg cycle, which might account for cyclic variations in resorptive activity. During egg shell calcification, osteoclasts with ruffled borders are closely apposed to bone surfaces; the cytoplasm is rich in vacuoles that contain mineral crystals and seem to derive from the ruffled border. At the completion of egg shell calcification, the ruffled borders and vacuoles move away from the bone surface, although the osteoclast remains attached to the bone along the filamentous or "clear" zone. Associated with the disappearance of the ruffled borders is the appearance of extensive interdigitated cell processes along the peripheral surface of the osteoclast away from the bone. These unusual structures, which may serve as a reservoir of membrane, largely disappear when ruffled borders and associated structures reappear. Therefore, in these hens, the osteoclasts modulate their cell surface rather than their population during the egg cycle.     

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Cellular response to ectopically implanted silk sutures and osteopetrotic bone

Summary Faulty osteoclasts, characteristic of the incisors-absent (ia) rat mutation of osteopetrosis, cause a resorptive defect which results in the persistence of immature, highly mineralized bone matrix. We implanted osteopetrotic bone subcutaneously into normal andia rats to determine ifia bone could induce functionally active and morphologically identifiable osteoclasts at the implant surface. Assays of⁴⁵Ca released from the preparations showed that normal andia recipients were capable of equivalent cell-mediated release of Ca over a 2-week implant period, indicating that theia resorptive defect was not reproduced at the subcutaneous site. Freeze-thawed osteopetrotic bone released twice as much⁴⁵Ca as normal bone. This difference was eliminated by collagenase treatment. Cellular profiles were similar in both normal andia animals regardless of the implant preparation. At 3 days after implantation, both bone and suture were surrounded by mononuclear cells. By 14 days, multinucleated cells appeared at the implant surfaces. Morphological comparison of implant-induced multinucleated cells and tibial osteoclasts indicated that bone-elicited multinucleated cells lacked the ruffled borders characteristic of normal osteoclasts or the extensive clear zones typical ofia osteoclasts, but more closely resembled suture-induced macrophage-polykaryons. We conclude that ectopically implantedia bone as compared to normal bone elicits a different functional response from structurally similar cell populations. Bone-elicited multinucleated cells could not be classified as active osteoclasts despite evidence of release of⁴⁵Ca. Release of labeled Ca was probably due to the action of mononuclear phagocytes and macrophage-polykaryons rather than to osteoclastic resorption.     

A novel monoclonal antibody recognizing a unique antigen of rat osteoclasts induced by the calcified matrices

The morphology of osteoclasts, primary cells that resorb bone, is well documented; however, the precise details of their terminal differentiation remains obscure. To date, the only morphological criterion for identifying activated functional osteoclasts has been the presence of ruffled borders. We have developed a rat bone marrow culture system in which osteoclast-like cells formed. These cells fulfilled most of the criteria of osteoclasts, and when they were reseeded on calcified tissue, formed numerous resorption lacunae in vitro. To find an immunological marker for functional osteoclasts, we have used these cells in a functional state as antigens for the preparation of monoclonal antibodies (mAb) that reacted with rat osteoclasts; we obtained mAb Ch1 and Ch2. Interestingly, these mAbs reacted with the marginal portion of authentic osteoclasts, where they attached to the bone surface on frozen sections. The reactivity of Ch1 to rat osteoclasts was more restricted than that of Ch2: Ch1 reacted with few tartrate-resistant acid phosphatase (TRAP)-positive cells on a culture plate. These TRAP-positive cells (including mono- and multinucleated cells) were, however, converted to Ch1-positive cells when they were reseeded on calcified tissues. These findings suggested that the antigen recognized by the Ch1 antibody was induced by some factors of matrix proteins released from calcified tissues.     

Autophagy proteins regulate the secretory component of osteoclastic bone resorption

Osteoclasts resorb bone via the ruffled border, whose complex folds are generated by secretory lysosome fusion with bone-apposed plasma membrane. Lysosomal fusion with the plasmalemma results in acidification of the resorptive microenvironment and release of CatK to digest the organic matrix of bone. The means by which secretory lysosomes are directed to fuse with the ruffled border are enigmatic. We show that proteins essential for autophagy, including Atg5, Atg7, Atg4B, and LC3, are important for generating the osteoclast ruffled border, the secretory function of osteoclasts, and bone resorption in?vitro and in?vivo. Further, Rab7, which is required for osteoclast function, localizes to the ruffled border in an Atg5-dependent manner. Thus, autophagy proteins participate in polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the plasma membrane. These findings are in keeping with a putative link between autophagy genes and human skeletal homeostasis.     

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.     

v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation

Matrix-producing osteoblasts and bone-resorbing osteoclasts maintain bone homeostasis. Osteoclasts are multinucleated, giant cells of hematopoietic origin formed by the fusion of mononuclear pre-osteoclasts derived from myeloid cells. Fusion-mediated giant cell formation is critical for osteoclast maturation; without it, bone resorption is inefficient. To understand how osteoclasts differ from other myeloid lineage cells, we previously compared global mRNA expression patterns in these cells and identified genes of unknown function predominantly expressed in osteoclasts, one of which is the d2 isoform of vacuolar (H(+)) ATPase (v-ATPase) V(0) domain (Atp6v0d2). Here we show that inactivation of Atp6v0d2 in mice results in markedly increased bone mass due to defective osteoclasts and enhanced bone formation. Atp6v0d2 deficiency did not affect differentiation or the v-ATPase activity of osteoclasts. Rather, Atp6v0d2 was required for efficient pre-osteoclast fusion. Increased bone formation was probably due to osteoblast-extrinsic factors, as Atp6v02 was not expressed in osteoblasts and their differentiation ex vivo was not altered in the absence of Atp6v02. Our results identify Atp6v0d2 as a regulator of osteoclast fusion and bone formation, and provide genetic data showing that it is possible to simultaneously inhibit osteoclast maturation and stimulate bone formation by therapeutically targeting the function of a single gene.     

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.