The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

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.     

Eccentric localization of osteocytes expressing enzymatic activities, protein, and mRNA signals for type 5 tartrate-resistant acid phosphatase (TRAP).

Enzymatic activity of type 5 tartrate-resistant acid phosphatase (TRAP) has been regarded as one of the reliable markers for osteoclasts and their precursors. The presence of TRAP activity in osteocytes near the bone resorbing surface has also been pointed out in some reports. However, the significance of TRAP reactions in osteocytes remains controversial and, in fact, there is no agreement as to whether the histochemical enzyme reactions in osteocytes represent the TRAP enzyme generated by the respective osteocytes or is a mere diffusion artifact of the reaction products derived from the nearby osteoclasts. Current histochemical, immunohistochemical, and in situ hybridization studies of rat and canine bones confirmed TRAP enzyme activity, TRAP immunoreactivity, and the expression of Trap mRNA signals in osteocytes located close to the bone-resorbing surface. TRAP/Trap- positive osteocytes thus identified were confined to the areas no further than 200 microm from the bone-resorbing surface and showed apparent upregulation of TRAP/Trap expression toward the active osteoclasts. Spatial and temporal patterns of TRAP/Trap expression in the osteocytes should serve as a valuable parameter for further analyses of biological interactions between the osteocytes and the osteoclasts associated with bone remodeling.     

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.     

Osteocyte messages from a bony tomb

Until recently, osteocytes have escaped the limelight, embedded within their cave-like lacunae in the mineralized matrix of bone. In this issue of Cell Metabolism, Tatsumi et al. (2007) present evidence that this "third bone cell" can send either inhibitory signals to osteoclasts to maintain bone mass with normal loading or stimulatory signals to osteoclasts to initiate bone loss upon immobilization, thereby playing an indispensable role in skeletal homeostasis.     

Cell-specific paracrine actions of IL-6 family cytokines from bone,marrow and muscle that control bone formation and resorption

Bone renews itself and changes shape throughout life to account for the changing needs of the body; this requires co-ordinated activities of bone resorbing cells (osteoclasts), bone forming cells (osteoblasts) and bone’s internal cellular network (osteocytes). This review focuses on paracrine signaling by the IL-6 family of cytokines between bone cells, bone marrow, and skeletal muscle in normal physiology and in pathological states where their levels may be locally or systemically elevated. These functions include the support of osteoclast formation by osteoblast lineage cells in response to interleukin 6 (IL-6), interleukin 11 (IL-11), oncostatin M (OSM) and cardiotrophin 1 (CT-1). In addition it will discuss how bone-resorbing osteoclasts promote osteoblast activity by secreting CT-1, which acts as a “coupling factor” on osteocytes, osteoblasts, and their precursors to promote bone formation. OSM, produced by osteoblast lineage cells and macrophages, stimulates bone formation via osteocytes. IL-6 family cytokines also mediate actions of other bone formation stimuli like parathyroid hormone (PTH) and mechanical loading. CT-1, OSM and LIF suppress marrow adipogenesis by shifting commitment of pluripotent precursors towards osteoblast differentiation. Ciliary neurotrophic factor (CNTF) is released as a myokine from skeletal muscle and suppresses osteoblast differentiation and bone formation on the periosteum (outer bone surface in apposition to muscle). Finally, IL-6 acts directly on marrow-derived osteoclasts to stimulate release of “osteotransmitters” that act through the cortical osteocyte network to stimulate bone formation on the periosteum. Each will be discussed as illustrations of how the extended family of IL-6 cytokines acts within the skeleton in physiology and may be altered in pathological conditions or by targeted therapies.     

Evidence for osteocyte regulation of bone homeostasis through RANKL expression

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.     

Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon--a proposal

The concept of bone remodelling by basic multicellular units is well established, but how the resorbing osteoclasts find their way through the pre-existing bone matrix remains unexplained. The alignment of secondary osteons along the dominant loading direction suggests that remodelling is guided by mechanical strain. This means that adaptation (Wolff's Law) takes place throughout life at each remodelling cycle. We propose that alignment during remodelling occurs as a result of different canalicular flow patterns around cutting cone and reversal zone during loading. Low canalicular flow around the tip of the cutting cone is proposed to reduce NO production by local osteocytes thereby causing their apoptosis. In turn, osteocyte apoptosis could be the mechanism that attracts osteoclasts, leading to further excavation of bone in the direction of loading. At the transition between cutting cone and reversal zone, however, enhanced canalicular flow will stimulate osteocytes to increase NO production, which induces osteoclast retraction and detachment from the bone surface. Together, this leads to a treadmill of attaching and detaching osteoclasts in the tip and the periphery of the cutting cone, respectively, and the digging of a tunnel in the direction of loading.     

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.     

Transient versus sustained phosphorylation and nuclear accumulation of ERKs underlie anti-versus pro-apoptotic effects of estrogens

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.     

The osteopetrotic rabbit: skeletal cytology and ultrastructure

The lethal, autosomal recessive osteopetrotic mutation in the rabbit, osteosclerosis (os/os), has recently been made available for experimental investigation. We have examined the cytology and ultrastructure of skeletal cells in mutants and report abnormalities in osteoblasts, osteocytes, and osteoclasts. Mutant osteoclasts lack a well-defined ruffled border and show few morphological signs of bone resorption. Osteoblasts in mutants form bone in neonatal life but show signs of degeneration by 2 weeks after birth. Mutant osteoblasts and osteocytes contain large, electron-dense cytoplasmic inclusions. External surfaces of mutant long bones show no evidence of bone resorption by scanning electron microscopy, and fibrosis of intertrabecular spaces is a prominent feature in mutants. These data, considered with recent evidence that the functions of osteoblasts and osteoclasts are interrelated, suggest that reduced bone resorption, a characteristic feature of osteopetrosis, may be related to osteoblast incompetence in this mutation.     

Immunolocalization of the prostaglandin E2 receptor subtypes in human bone tissue: differences in foetal, adult normal, osteoporotic and pagetic bone

PGE(2) is an important mediator of bone metabolism, but the precise localization of its receptors in human bone remains unknown. The present study used specific antibodies against EP(1), EP(2), EP(3) and EP(4) receptors for immunolocalization in normal, osteoporotic and pagetic human adult bone and in human foetal bone. No labelling was obtained for the EP(1) and EP(2) receptors. The EP(3) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes, but only in osteoclasts and some osteoblasts from adult bone. The EP(4) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes and in adult osteoclasts and osteoblasts, but not in adult osteocytes. Our results show differences in PGE(2) receptor expression in foetal and adult human bone but no difference in adult normal compared to pathologic bone. Finally, these results show that the distribution of EP receptors in human osteoblasts in bone corresponds in part to what we recently described in human osteoblasts in culture.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism

Vertebrate bone is composed of three main cell types: osteoblasts, osteoclasts and osteocytes, the latter being by far the most numerous. Osteocytes are thought to play a fundamental role in bone physiology and homeostasis, however they are entirely absent in most extant species of teleosts, a group that comprises the vast majority of bony ‘fishes’, and approximately half of vertebrates. Understanding how this acellular (anosteocytic) bone appeared and was maintained in such an important vertebrate group has important implications for our understanding of the function and evolution of osteocytes. Nevertheless, although it is clear that cellular bone is ancestral for teleosts, it has not been clear in which specific subgroup the osteocytes were lost. This review aims to clarify the phylogenetic distribution of cellular and acellular bone in teleosts, to identify its precise origin, reversals to cellularity, and their implications. We surveyed the bone type for more than 600 fossil and extant ray‐finned fish species and optimised the results on recent large‐scale molecular phylogenetic trees, estimating ancestral states. We find that acellular bone is a probable synapomorphy of Euteleostei, a group uniting approximately two‐thirds of teleost species. We also confirm homoplasy in these traits: acellular bone occurs in some non‐euteleosts (although rarely), and cellular bone was reacquired several times independently within euteleosts, in salmons and relatives, tunas and the opah (Lampris sp.). The occurrence of peculiar ecological (e.g. anadromous migration) and physiological (e.g. red‐muscle endothermy) strategies in these lineages might explain the reacquisition of osteocytes. Our review supports that the main contribution of osteocytes in teleost bone is to mineral homeostasis (via osteocytic osteolysis) and not to strain detection or bone remodelling, helping to clarify their role in bone physiology.     

Role of the heat shock protein family in bone metabolism

Heat shock proteins (HSPs) are a family of proteins produced by cells in response to exposure to stressful conditions. In addition to their role as chaperones, they also play an important role in the cardiovascular, immune, and other systems. Normal bone tissue is maintained by bone metabolism, particularly by the balance between osteoblasts and osteoclasts, which are physiologically regulated by multiple hormones and cytokines. In recent years, studies have reported the vital role of HSPs in bone metabolism. However, the conclusions remain largely controversial, and the exact mechanisms are still unclear, so a review and analyses of previous studies are of importance. This article reviews the current understanding of the roles and effects of HSPs on bone cells (osteoblasts, osteoclasts, and osteocytes), in relation to bone metabolism.     

Probing the tissue to subcellular level structure underlying bone's molecular sieving function

Transport of fluorescent probes between 300 and 2,000,000 Da was studied in mechanically loaded and unloaded ulnae of skeletally mature rats to characterize the permeability of the pericellular space of the lacunocanalicular system (LCS), and the microporosity of the bony matrix. The mineral matrix porosity allowed for penetration of the 300 Da probe but impeded transport of larger probes. The pericellular space of the LCS was permeable up to 10 kDa; above 10 kDa, diffusion was ineffective for transport through the pericellular space. Convective transport via load-induced fluid flow increased penetration of all probes up to 70 kDa. Above this threshold, probes were excluded from bone, both with and without loading. This exploratory study suggests that bone acts as a molecular sieve and that mechanical loading modulates transport of solutes through the pericellular space that links osteocytes deep within the tissue to the blood supply and to osteoblasts and osteoclasts on bone forming and resorbing surfaces. This provides support for the postulate of transport modulated bone remodeling in which osteocytes are influenced by and modulate the local permeability of their surroundings as a means for survival (Knothe Tate et al. 1998, [28]) and has profound implications for osteocyte viability and intercellular communication in bone.     

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.     

Ultrastructural and lanthanum tracer examination of rapidly resorbing rat alveolar bone suggests that osteoclasts internalize dying bone cells

Glutaraldehyde-formaldehyde fixed undecalcified alveolar bone from 7-day-old rats was prepared for light and electron microscopy. Colloidal lanthanum was used as an ultrastructural tracer, and both random and semi-serial sections were examined. Lanthanum penetrated the infoldings of the ruffled border and some nearby vacuoles and vesicles. The majority of vacuoles and vesicles were lanthanum-free. Some osteoclast profiles contained a large vacuole with a cell enclosed in its interior. The enclosed cell exhibited an irregular nucleus containing condensed peripheral chromatin, intact cytoplasmic organelles, conspicuous rough endoplasmic reticulum and large blebs on the cell surface. These features are characteristic of osteoblasts or bone-lining cells or immature osteocytes which may be undergoing apoptosis or necrosis. The observation of remnants of cellular structures within internalized osteoclast vacuoles, together with the above results, suggests that osteoclasts engulf and probably degrade dying osteoblasts/bone-lining cells or immature osteocytes. Received: 29 April 1997 / Accepted: 20 February 1998     

Pulsed electromagnetic fields regulate osteocyte apoptosis,RANKL/OPG expression,and its control of osteoclastogenesis depending on the presence of primary cilia

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).