The osteocyte lineage

The osteocyte resides in the lacuna/canalicular system in bone and has been hypothesized to orchestrate local bone remodeling. Certainly the identification of the osteocyte as the source of Sclerostin, a molecule that regulates osteoblast function, has supported this possibility. As our understanding of this cell increases it has become clear that it has more far reaching influence than simply local bone turnover activity. The osteocyte is also the source of DMP-1 and FGF-23, the later being a hormone that regulates kidney function in terms of phosphate uptake. We now see the osteocyte as having important roles both locally in the skeleton and also in other distant tissues. The study of osteocyte biology has reached a particularly exiting level of maturity and illustrates the value of this cell type as a drug discovery target.     

Shear stress inhibits while disuse promotes osteocyte apoptosis

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.     

Three‐dimensional characterization of osteocyte volumes at multiple scales,and its relationship with bone biology and genome evolution in ray‐finned fishes

Osteocytes, cells embedded within the bone mineral matrix, inform on key aspects of vertebrate biology. In particular, a relationship between volumes of the osteocytes and bone growth and/or genome size has been proposed for several tetrapod lineages. However, the variation in osteocyte volume across different scales is poorly characterized and mostly relies on incomplete, two‐dimensional information. In this study, we characterize the variation of osteocyte volumes in ray‐finned fishes (Actinopterygii), a clade including more than half of modern vertebrate species in which osteocyte biology is poorly known. We use X‐ray synchrotron micro‐computed tomography (SRµCT) to achieve a three‐dimensional visualization of osteocyte lacunae and direct measurement of their size (volumes). Our specimen sample is designed to characterize variation in osteocyte lacuna morphology at three scales: within a bone, among the bones of one individual and among species. At the intra‐bone scale, we find that osteocyte lacunae vary noticeably in size between zones of organized and woven bone (being up to six times larger in woven bone), and across cyclical bone deposition. This is probably explained by differences in bone deposition rate, with larger osteocyte lacunae contained in bone that deposits faster. Osteocyte lacuna volumes vary 3.5‐fold among the bones of an individual, and this cannot readily be explained by variation in bone growth rate or other currently observable factors. Finally, we find that genome size provides the best explanation of variation in osteocyte lacuna volume among species: actinopterygian taxa with larger genomes (polyploid taxa in particular) have larger osteocyte lacunae (with a ninefold variation in median osteocyte volume being measured). Our findings corroborate previous two‐dimensional studies in tetrapods that also observed similar patterns of intra‐individual variation and found a correlation with genome size. This opens new perspectives for further studies on bone evolution, physiology and palaeogenomics in actinopterygians, and vertebrates as a whole.     

Extracellular NO signalling from a mechanically stimulated osteocyte

Bone remodelling is a dynamic process that requires the coordinated interaction of osteocytes, osteoblasts, and osteoclasts, collaborating in basic multicellular units (BMUs). Communication between these cells can be by extracellular soluble molecules as well as directly propagating intercellular signalling molecules. Key to the understanding of bone remodelling is osteocyte mechanosensing and chemical signalling to the surrounding cells, since osteocytes are believed to be the mechanosensors of bone, responding to mechanical stresses. Nitric oxide (NO) is an important parameter to study osteocyte activation following mechanical loading. It is a small short-lived molecule, which makes its real-time, quantitative monitoring difficult. However, recently we demonstrated that DAR-4M AM chromophore can be used for real-time quantitative monitoring of intracellular NO production in individual cells following mechanical loading. Here we studied if a single mechanically stimulated osteocyte communicates with, and thus activates its surrounding cells via extracellular soluble factors. We monitored quantitatively intracellular NO production in the stimulated osteocyte and in its surrounding osteocytes, which were not interconnected. Mechanical stimulation by microneedle of a single-MLO-Y4 osteocyte-like cell upregulated the average intracellular NO production by 94% in the stimulated cell, and by 31-150% in the surrounding osteocytes. In conclusion, a single osteocyte can disseminate a mechanical stimulus to its surrounding osteocytes via extracellular soluble signalling factors. This reinforces the putative mechanosensory role of osteocytes, and demonstrates a possible mechanism by which a single mechanically stimulated osteocyte can communicate with other cells in a BMU, which might help to better understand the intricacies of intercellular interactions in BMUs and thus bone remodelling.     

Adaptive filtering for enhancement of the osteocyte cell network in 3D microtomography images

The osteocyte cell network in bone tissue is thought to orchestrate tissue adaptation and remodeling, thus holding responsibility for tissue quality. Previously, this structure has been studied mainly in 2D and its architecture and functions are not fully elucidated. The assessment of the osteocyte system is prerequisite for deeper understanding of bone remodeling and for advances in management of bone diseases. Our goal is to enable 3D isotropic imaging of bone at cellular level and to develop algorithms for quantitative image analysis of the cell network. We recently demonstrated accurate 3D imaging of this cell structure with synchrotron radiation tomography at submicrometric scale. Due to the limited spatial resolution of the imaging system and the constraints in terms of radiation dose, the images suffer from low signal to noise ratio and the detection of the cell dendrites is challenging. Here we detail a method for enhancement of the osteocyte network in human bone from 3D microtomography images. The approach combines Hessian-based 3D line enhancement and bilateral filtering. Our method enables extraction of the interconnected cells from noisy images, preserving the integrity of the cells and of their slender dendrites. Qualitative and quantitative results are presented.     

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.     

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes: role of integrinβ1 and focal adhesion kinase signaling cues

Introduction

Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function.

Methods

De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies.

Results

The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell’s attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression.

Conclusion

This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.     

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

     

Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes

The morphological changes undergone by the osteoblast at the ultrastructural level, during its differentiation into osteocyte, were studied in the primary parallel-fibred bone of the newborn rabbit by means of incomplete three-dimensional reconstruction from partially serial-sectioned preosteocytes. The findings obtained suggest that the formation of osteocyte cytoplasmic processes is an asynchronous and asymmetrical phenomenon that seems to precede the mineralization of the organic matrix and to give rise to an asymmetrical mature osteocyte. The functions of cytoplasmic processes as regards bone formation, cell nutrition and osteoblast modulation are discussed. The mechanism by which the osteoblast 'enters' the bone matrix is hypothesized.     

Fluid flow in the osteocyte mechanical environment: a fluid–structure interaction approach

Osteocytes are believed to be the primary sensor of mechanical stimuli in bone, which orchestrate osteoblasts and osteoclasts to adapt bone structure and composition to meet physiological loading demands. Experimental studies to quantify the mechanical environment surrounding bone cells are challenging, and as such, computational and theoretical approaches have modelled either the solid or fluid environment of osteocytes to predict how these cells are stimulated in vivo. Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. This represents a most challenging multi-physics problem in which fluid and solid domains interact, and as such, no previous study has accounted for this complex behaviour. The objective of this study is to employ fluid–structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. Fluorescent staining of osteocytes was performed in order to visualise their native environment and develop geometrically accurate models of the osteocyte in vivo. By simulating loading levels representative of vigorous physiological activity ( $3,000\,\upmu \upvarepsilon $ compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities $(\sim 60.5\,\upmu \text{ m/s })$ and average maximum shear stresses $(\sim 11\, \text{ Pa })$ surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. Significantly our results suggest that the greatest mechanical stimulation of the osteocyte occurs in the cell processes, which, cell culture studies have indicated, is the most mechanosensitive area of the cell. These are the first computational FSI models to simulate the complex multi-physics mechanical environment of osteocyte in vivo and provide a deeper understanding of bone mechanobiology.     

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.     

Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation

Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.     

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.     

The influence of mechanical stimulation on osteocyte apoptosis and bone viability in human trabecular bone

It has been shown previously using in vivo and ex vivo animal models, that cyclical mechanical stimulation is capable of maintaining osteocyte viability through the control of apoptotic cell death. Here we have studied the effect of mechanical stimulation on osteocyte viability in human trabecular bone maintained in a 3-D bioreactor system. Bone samples, maintained in the bioreactor system for periods of 3, 7 and 27 days, were subjected to either cyclical mechanical stimulation which engendered a maximum of 3,000 microstrain in a waveform corresponding to physiological jumping exercise for 5 minutes daily or control unloading. Unloading resulted in a decrease in osteocyte viability within 3 days that was accompanied by increased levels of cellular apoptosis. Mechanical stimulation significantly reduced apoptosis (p< or =0.032) and improved the maintenance of osteocyte viability in bone from all patient samples. The percentage Alkaline Phosphatase (ALP) labelled bone surface was significantly increased (p< or =0.05) in response to mechanical stimulation in all samples as was the Bone Formation Rate (BFR/BS) (p=0.005) as determined by calcein label incorporation in the 27-day experiment. These data indicate that in this model system, mechanical stimulation is capable of maintaining osteocyte viability in human bone.     

Structural differences in the osteocyte network between the calvaria and long bone revealed by three-dimensional fluorescence morphometry, possibly reflecting distinct mechano-adaptations and sensitivities

The structural features of osteocytes and their cellular process network are thought to allow for mechanotransduction from the bone tissue to these cells. This study applied three-dimensional fluorescence microscopy to fixed and decalcified bone specimens to quantitatively compare the osteocytes and their networks between mouse parietal bone and tibia that are physiologically enforced by distinct mechanical loads. The subsequent morphometric analysis by the surface rendering of osteocyte cell bodies revealed the tibia to have relatively enriched cytoplasm in the osteocyte cell body in comparison to the parietal bone. Furthermore, quantitative tracing of the cellular processes in silico demonstrated that the numbers of the cellular processes and their bifurcation points per osteocyte in the tibia were significantly higher than those in the parietal bone. Though the total length of the processes per osteocyte in the tibia was two times longer, its total surface area and total volume were smaller than those in the parietal bone, due to its thinner diameter. These architectural differences in the osteocytes and their networks are thus implicated in the adaptation to physiologically different loading, and may also induce distinct mechanosensitivities.     

Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro

Age-related skeletal changes is closely associated with imbalanced bone remodeling characterized by elevated osteocyte apoptosis and osteoclast activation. Since osteocytes are the commander of bone remodeling, attenuating increased osteocyte apoptosis may improve age-related bone loss. Exosomes, derived from mesenchymal stem cells, hold promising potential for cell-free therapy due to multiple abilities, such as promoting proliferation and suppressing apoptosis. We aimed to explore the effect of exosomes derived from adipose mesenchymal stem cell (ADSCs-exo) on osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro. The osteocyte-like cell line MLO-Y4 was used as a model, and apoptosis was induced by hypoxia and serum deprivation (H/SD). Our results showed that ADSCs-exo noticeably reduced H/SD-induced apoptosis in MLO-Y4 cells via upregulating the radio of Bcl-2/Bax, diminishing the production of reactive oxygen species and cytochrome c, and subsequent activation of caspase-9 and caspase-3. Additionally, ADSCs-exo lowered the expression of RANKL both at the mRNA and protein levels, as well as the ratio of RANKL/OPG at the gene level. As determined by tartrate-resistant acid phosphatase staining, reduced osteoclastogenesis was further validated in bone marrow monocytes cultured under conditioned medium from exosome-treated MLO-Y4. Together, ADSCs-exo could antagonize H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis, indicating the therapeutic potential of ADSCs-exo in age-related bone disease.     

Dual recombinases-based genetic lineage tracing for stem cell research with enhanced precision

Science China Life Sciences - Stem cell research has become a hot topic in biology, as the understanding of stem cell biology can provide new insights for both regenerative medicine and clinical...     

Functions of the osteocyte network in the regulation of bone mass

Osteocytes establish an extensive intracellular and extracellular communication system via gap-junction-coupled cell processes and canaliculi throughout bone and the communication system is extended to osteoblasts on the bone surface. The osteocyte network is an ideal mechanosensory system and suitable for mechanotransduction. However, the overall function of the osteocyte network remains to be clarified, since bone resorption is enhanced by osteocyte apoptosis, which is followed by a process of secondary necrosis attributable to the lack of scavengers. The enhanced bone resorption is caused by the release of intracellular content, including immunostimulatory molecules that activate osteoclastogenesis through the canaliculi. Therefore, a mouse model is required in which the osteocyte network is disrupted but in which no bone resorption is induced, in order to evaluate the overall functions of the osteocyte network. One such model is the BCL2 transgenic mouse, in which the osteocyte network, including both intracellular and extracellular networks, is disrupted. Another model is the osteocyte-specific Gja1 knockout mouse, in which intercellular communication through gap junctions is impaired but the canalicular system is intact. Combining the findings from these mouse models with previous histological observations showing the inverse linkage between osteocyte density and bone formation, we conclude that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions. Further, studies with BCL2 transgenic mice show that these osteocyte functions are augmented in the unloaded condition. In this condition, Rankl upregulation in osteoblasts and Sost upregulation in osteocytes are, at least in part, responsible for enhanced bone resorption and suppressed bone formation, respectively.