Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures--also stained by hematoxylin--were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images--round/ ovoid bodies with dense crescent-like chromatin--on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis.     

Osteoclast-osteoblast communication

Cells in osteoclast and osteoblast lineages communicate with each other through cell-cell contact, diffusible paracrine factors and cell-bone matrix interaction. Osteoclast-osteoblast communication occurs in a basic multicellular unit (BMU) at the initiation, transition and termination phases of bone remodeling. At the initiation phase, hematopoietic precursors are recruited to the BMU. These precursors express cell surface receptors including c-Fms, RANK and costimulatory molecules, such as osteoclast-associated receptor (OSCAR), and differentiate into osteoclasts following cell-cell contact with osteoblasts, which express ligands. Subsequently, the transition from bone resorption to formation is mediated by osteoclast-derived ‘coupling factors’, which direct the differentiation and activation of osteoblasts in resorbed lacunae to refill it with new bone. Bidirectional signaling generated by interaction between ephrinB2 on osteoclasts and EphB4 on osteoblast precursors facilitates the transition. Such interaction is likely to occur between osteoclasts and lining cells in the bone remodeling compartment (BRC). At the termination phase, bone remodeling is completed by osteoblastic bone formation and mineralization of bone matrix. Here, we describe molecular communication between osteoclasts and osteoblasts at distinct phases of bone remodeling.     

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.     

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.     

Ossification of the antler in the Lapland reindeer (Rangifer tarandus tarandus)

A histological and histochemical study of biopsy specimens from the Lapland reindeer antler indicated that the intercellular matrix of the cartilage that forms the partitions of longitudinal channels becomes calcified at virtually the initial stage of formation. The lacunae at the peripheries of the cartilaginous partitions are invaded by osteoblasts in a process comparable to endochondral ossification. The very centres of the partitions evidently become directly converted into bone without the presence of e.g. osteoclasts. The osseous partitions are remodelled by osteoclasts. The antler increases in diameter by periosteal apposition.     

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.     

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.     

Regulation of osteoclast activity.

Osteoclasts are the primary cell type responsible for bone resorption. This paper reviews many of the known regulators of osteoclast activity, including hormones, cytokines, ions, and arachidonic acid metabolites. Most of the hormones and cytokines that inhibit osteoclast activity act directly on the osteoclasts. In contrast, most of the hormones and cytokines that stimulate osteoclast activity act indirectly through osteoblasts. Particularly interesting in this regard are agents that directly inhibit activity of highly purified osteoclasts yet stimulate activity of osteoclasts that are co-cultured with osteoblasts. Recent studies have demonstrated that the primary mechanism by which bone resorptive agents stimulate osteoclast activity indirectly is likely to be up-regulation of production of osteoclast differentiation factor/osteoprotegerin ligand (ODF/OPGL) by the osteoblasts. In addition to discussing regulators of osteoclast activity per se, this paper also reviews the role of osteoclast apoptosis to limit the extent of bone resorption.     

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.     

培养破骨细胞扫描电镜样本制备方法的探讨

目的:探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法:实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果:破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论:牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

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.     

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.     

培养破骨细胞扫描电镜样本制备方法的探讨

目的：探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法：实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果：破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论：牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

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.     

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.     

Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies

The properties of five monoclonal antibodies raised against isolated osteoclasts are described. Osteoclasts were isolated from medullary bone of egg-laying female quails. Mice were immunized with cell preparations consisting for about 10% of multinucleated osteoclasts. A large number of monoclonal antibodies against cell surface antigens were obtained, five of which were extensively characterized by their interactions with different tissues of the quail and their cross-reactivity with other species. Two monoclonals (OC 5.3 and OC 6.8), recognize surface antigens present on osteoclasts, monocytes, granulocytes and endothelial cells, but not on osteoblasts, osteocytes, fibroblasts, lymphocytes, erythrocytes and others. The three other monoclonal antibodies are specific for multinucleated osteoclasts in bone tissue but recognize some cell surface structures in other tissues. Antibody OC 6.9, which in bone tissue stains primarily the surface area of the osteoclast that is adjacent to the resorbing bone surface, also interacts with bile capillaries in the liver and with specific, but not yet identified parts of the nephron. The antibodies OC 6.1 and OC 6.3 interact with Kupffer cells in the liver and tissue macrophages of small intestine. In view of the possible fallacies inherent to the use of cell surface markers for the demonstration of cell relationship and origin, definite conclusions can not yet be made. The fact that the osteoclast, the Kupffer cell and the intestine macrophage are the only cells in bone, bone marrow, liver, kidney and intestine, that share the same surface antigen recognized by monoclonals OC 6.1 and OC 6.3, suggests, however, a common origin for osteoclasts and a number of well described tissue macrophages.     

Cell surface antigens on osteoclasts and related cells in the quail studied with monoclonal antibodies

Summary The properties of five monoclonal antibodies raised against isolated osteoclasts are described.Osteoclasts were isolated from medullary bone of egglaying female quails. Mice were immunized with cell preparations consisting for about 10% of multinucleated osteoclasts. A large number of monoclonal antibodies against cell surface antigens were obtained, five of which were extensively characterized by their interactions with different tissues of the quail and their cross-reactivity with other species. Two monoclonals (OC 5.3 and OC 6.8), recognize surface antigens present on osteoclasts, monocytes, granulocytes and endothelial cells, but not on osteoblasts, osteocytes, fibroblasts, lymphocytes, erythrocytes and others. The three other monoclonal antibodies are specific for multinucleated osteoclasts in bone tissue but recognize some cell surface structures in other tissues. Antibody OC 6.9, which in bone tissue stains primarily the surface area of the osteoclast that is adjacent to the resorbing bone surface, also interacts with bile capillaries in the liver and with specific, but not yet identified parts of the nephron. The antibodies OC 6.1 and OC 6.3 interact with Kupffer cells in the liver and tissue macrophages of small intestine. In view of the possible fallacies inherent to the use of cell surface markers for the demonstration of cell relationship and origin, definite conclusions can not yet be made. The fact that the osteoclast, the Kupffer cell and the intestine macrophage are the only cells in bone, bone marrow, liver, kidney and intestine, that share the same surface antigen recognized by monoclonals OC 6.1 and OC 6.3, suggests, however, a common origin for osteoclasts and a number of well described tissue macrophages.     

A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function

Osteoclasts, the multinucleated giant cells that resorb bone, develop from monocyte-macrophage lineage cells. Osteoblasts or bone marrow stromal cells have been suggested to be involved in osteoclastic bone resorption. The recent discovery of new members of the tumor necrosis factor (TNF) receptor-ligand family has elucidated the precise mechanism by which osteoblasts/stromal cells regulate osteoclast differentiation and function. Osteoblasts/stromal cells express a new member of the TNF-ligand family "osteoclast differentiation factor(ODF)/osteoprotegerin ligand (OPGL)/TNF-related activation-induced cytokine (TRANCE)/receptor activator of NF-kB ligand (RANKL)" as a membrane associated factor. Osteoclast precursors which possess RANK, a TNF receptor family member, recognize ODF/OPGL/TRANCE/RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage colony-stimulating factor. Mature osteoclasts also express RANK, and their bone-resorbingactivity is also induced by ODF/OPGL/TRANCE/RANKL which osteoblasts/stromal cells possess. Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF)/TNF receptor-like molecule 1 (TR1) is a soluble decoy receptor for ODF/OPGL/TRANCE/RANKL. Activation of NF-kB and c-Jun N-terminal kinase through the RANK-mediated signaling system appears to be involved in differentiation and activation of osteoclasts.     

Lysophosphatidic acid: A potential mediator of osteoblast–osteoclast signaling in bone

Osteoclasts (bone resorbing cells) and osteoblasts (bone forming cells) play essential roles in skeletal development, mineral homeostasis and bone remodeling. The actions of these two cell types are tightly coordinated, and imbalances in bone formation and resorption can result in disease states, such as osteoporosis. Lysophosphatidic acid (LPA) is a potent bioactive phospholipid that influences a number of cellular processes, including proliferation, survival and migration. LPA is also involved in wound healing and pathological conditions, such as tumor metastasis and autoimmune disorders. During trauma, activated platelets are likely a source of LPA in bone. Physiologically, osteoblasts themselves can also produce LPA, which in turn promotes osteogenesis. The capacity for local production of LPA, coupled with the proximity of osteoblasts and osteoclasts, leads to the intriguing possibility that LPA acts as a paracrine mediator of osteoblast–osteoclast signaling. Here we summarize emerging evidence that LPA enhances the differentiation of osteoclast precursors, and regulates the morphology, resorptive activity and survival of mature osteoclasts. These actions arise through stimulation of multiple LPA receptors and intracellular signaling pathways. Moreover, LPA is a potent mitogen implicated in promoting the metastasis of breast and ovarian tumors to bone. Thus, LPA released from osteoblasts is potentially an important autocrine and paracrine mediator — physiologically regulating skeletal development and remodeling, while contributing pathologically to metastatic bone disease. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.