Lacuno-canalicular Porosity Research Articles

Interstitial fluid transport in cortical bone porosities: Effects of blood pressure and mass exchange using porous media theory

This research focuses on the importance of understanding bone adaptation for gaining insights into diseases such as osteoporosis. The study investigates the role of fluid flow in cortical bone adaptation, with a specific focus on the network of osteocyte canaliculi and the chemical reactions involved in bone resorption. Prior studies have implemented methods such as poroelasticity theory and dual porosity, but these models did not account for mass exchange between the solid and fluid phases within the bone or the pressure exerted by arteries. The movement of interstitial fluid between the vascular porosity and lacuno-canalicular porosity in cortical bone, as well as the velocity of the fluid, are affected by both the pressure exerted by arteries and the exchange of mass between the solid and fluid components of the bone. To incorporate these effects in the model, the authors propose a dual porosity model based on the theory of porous media. The model considers the transfer of mass between vascular and lacunar-canalicular porosities, as well as between solid and fluid phases, while also accounting for blood pressure variations. To verify the proposed model, a numerical example is solved and compared with the results of previous studies. The model enables the measurement and analysis of the interstitial fluid velocity and the pressure in the respective porosities. The results reveal that accounting for blood pressure pulses and mass exchange between solid and fluid phases can alter the velocity and pressure fields of interstitial fluid within cortical bone. In addition, this study investigates transient examples and various scenarios of the bone remodeling. Overall, this research provides a more accurate understanding of the bone remodeling and the effects of blood pressure variation and mass exchange between solid and fluid phases of cortical bone.

Computational framework for analyzing flow-induced strain on osteocyte as modulated by microenvironment

Osteocytes buried in bone matrix are major mechanosensory cells that regulate bone remodeling in response to interstitial fluid flow in a lacuno-canalicular porosity. To gain an understanding of the mechanism of osteocyte mechanosensing, it is important to be able to evaluate the local strain on the osteocyte process membrane induced by the interstitial fluid flow. The microenvironment of the osteocytes, including the pericellular matrix (PCM) and canalicular ultrastructure, is a key modulator of the flow-induced strain on the osteocyte process membrane because it produces heterogeneous flow patterns in the pericellular space. To investigate the effect of changes in the microenvironment of osteocytes on the flow-induced strain, we developed a novel computational framework for analyzing the fluid–structure interaction. Computer simulations based on the proposed framework enabled evaluation of the spatial distribution of flow-induced strain on the osteocyte process membrane according to changes in the PCM density and canalicular curvature. The simulation results reveal that a decrease in PCM density and an increase in canalicular curvature, each of which is associated with aging and bone disease, have the notable effect of enhancing local flow-induced strain on the osteocyte process membrane. We believe that the proposed computational framework is a promising framework for investigating cell-specific mechanical stimuli and that it has the potential to accelerate the mechanobiological study of osteocytes by providing a deeper understanding of their mechanical environment in living bone tissue.

Quantification of the bone lacunocanalicular network from 3D X‐ray phase nanotomography images

There is a growing interest in developing 3D microscopy for the exploration of thick biological tissues. Recently, 3D X-ray nanocomputerised tomography has proven to be a suitable technique for imaging the bone lacunocanalicular network. This interconnected structure is hosting the osteocytes which play a major role in maintaining bone quality through remodelling processes. 3D images have the potential to reveal the architecture of cellular networks, but their quantitative analysis remains a challenge due to the density and complexity of nanometre sized structures and the need to handle and process large datasets, for example, 20483 voxels corresponding to 32 GB per individual image in our case. In this work, we propose an efficient image processing approach for the segmentation of the network and the extraction of characteristic parameters describing the 3D structure. These parameters include the density of lacunae, the porosity of lacunae and canaliculi, and morphological features of lacunae (volume, surface area, lengths, anisotropy etc.). We also introduce additional parameters describing the local environment of each lacuna and its canaliculi. The method is applied to analyse eight human femoral cortical bone samples imaged by magnified X-ray phase nanotomography with a voxel size of 120 nm, which was found to be a good compromise to resolve canaliculi while keeping a sufficiently large field of view of 246 μm in 3D. The analysis was performed on a total of 2077 lacunae showing an average length, width and depth of 17.1 μm × 9.2 μm × 4.4 μm, with an average number of 58.2 canaliculi per lacuna and a total lacuno-canalicular porosity of 1.12%. The reported descriptive parameters provide information on the 3D organisation of the lacuno-canalicular network in human bones.

Assessment of the human bone lacuno-canalicular network at the nanoscale and impact of spatial resolution

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of β-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released β-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

Capturing microscopic features of bone remodeling into a macroscopic model based on biological rationales of bone adaptation.

To understand Wolff's law, bone adaptation by remodeling at the cellular and tissue levels has been discussed extensively through experimental and simulation studies. For the clinical application of a bone remodeling simulation, it is significant to establish a macroscopic model that incorporates clarified microscopic mechanisms. In this study, we proposed novel macroscopic models based on the microscopic mechanism of osteocytic mechanosensing, in which the flow of fluid in the lacuno-canalicular porosity generated by fluid pressure gradients plays an important role, and theoretically evaluated the proposed models, taking biological rationales of bone adaptation into account. The proposed models were categorized into two groups according to whether the remodeling equilibrium state was defined globally or locally, i.e., the global or local uniformity models. Each remodeling stimulus in the proposed models was quantitatively evaluated through image-based finite element analyses of a swine cancellous bone, according to two introduced criteria associated with the trabecular volume and orientation at remodeling equilibrium based on biological rationales. The evaluation suggested that nonuniformity of the mean stress gradient in the local uniformity model, one of the proposed stimuli, has high validity. Furthermore, the adaptive potential of each stimulus was discussed based on spatial distribution of a remodeling stimulus on the trabecular surface. The theoretical consideration of a remodeling stimulus based on biological rationales of bone adaptation would contribute to the establishment of a clinically applicable and reliable simulation model of bone remodeling.

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation.

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions' orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young's modulus [Formula: see text]GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young's modulus in the preferential direction of 9-16GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.

骨細管内の間質液流れによるシグナル分子移流拡散シミュレーション

Interstitial fluid flow in the lacuno-canalicular porosity is closely associated with osteocyte mechanosensing followed by bone remodeling via the interstitial fluid flow. The lacuno-canalicular structure is one of important factors that influences intercellular signal transport. Structure of the lacuno-canalicular porosity is known to be different among types of bone tissue. In this study, we investigated the influence of the structural difference of lacuno-canalicular porosity on advection-diffusion of signal molecules enhanced by the interstitial fluid flow through computer simulation based on a poroelastic theory. The results showed that the diffusion of signal molecules in a single trabecula was promoted by the increase in the bone porosity. This suggests that bone remodeling induced by the transport of signal molecules also depends on the porosity of bone tissue.

Poroelastic analysis of interstitial fluid flow in a single lamellar trabecula subjected to cyclic loading.

Trabecula, an anatomical unit of the cancellous bone, is a porous material that consists of a lamellar bone matrix and interstitial fluid in a lacuno-canalicular porosity. The flow of interstitial fluid caused by deformation of the bone matrix is believed to initiate a mechanical response in osteocytes for bone remodeling. In order to clarify the effect of the lamellar structure of the bone matrix--i.e., variations in material properties--on the fluid flow stimuli to osteocytes embedded in trabeculae, we investigated the mechanical behavior of an individual trabecula subjected to cyclic loading based on poroelasticity. We focused on variations in the trabecular permeability and developed an analytical solution containing both transient and steady-state responses for interstitial fluid pressure in a single trabecular model represented by a multilayered two-dimensional poroelastic slab. Based on the obtained solution, we calculated the pressure and seepage velocity of the interstitial fluid in lacuno-canalicular porosity, within the single trabecula, under various permeability distributions. Poroelastic analysis showed that a heterogeneous distribution of permeability produces remarkable variations in the fluid pressure and seepage velocity in the cross section of the individual trabecula, and suggests that fluid flow stimuli to osteocytes are mostly governed by the value of permeability in the neighborhood of the trabecular surfaces if there is no difference in the average permeability in a single trabecula.

ELASTIC PROPERTIES OF WOVEN BONE: EFFECT OF MINERAL CONTENT AND COLLAGEN FIBRILS ORIENTATION

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus \(E = 1.90\) GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.

The Viability of Perilabyrinthine Osteocytes: A Quantitative Study Using Bulk‐Stained Undecalcified Human Temporal Bones

Bone remodeling is highly inhibited around the inner ear space, most likely by the anti-resorptive action of the inner ear cytokine osteoprotegerin (OPG) entering perilabyrinthine bone through the lacuno-canalicular porosity (LCP). This extracellular signaling pathway depends on the viability of individual osteocytes. The objective of this study was to evaluate the patency of the LCP at different ages. Sixty-five bulk-stained undecalcified human temporal bones and 19 ribs were selected to span the ages from the 30th gestational week to 95 years. Osteocytes from inside a 2-mm wide perilabyrinthine zone of bone were identified by 3D vector calculations and the numerical densities estimated with an optical dissector and compared to age-matched ribs. From a high fetal count of 90,000 cells/mm(3), the density of viable capsular osteocytes declined rapidly to 73,000 cells/mm(3) at three years of age, and non-viable osteocytes increased inversely. After 3 years, this decline/increase continued at a much slower rate. The densities of viable as well as non-viable osteocytes and the rates of change were much higher in perilabyrinthine bone compared to ribs. Only after the age of 80 years had the density of viable capsular osteocytes declined to the level of ribs. The bi-phasic osteocyte kinetics reflects different development stages. The high initial density of viable osteocytes may secure a life-long anatomical route for inner-ear OPG despite the unique accumulation of non-viable osteocytes. Clustering of non-viable osteocytes may cause local aberrations in the signaling system by closure of the LCP.

Otosclerosis: A perilabyrinthine threshold phenomenon

This paper is a review of our most recent findings concerning the osteo-dynamics of the bony otic capsule and pathogenesis of otosclerosis. By exploring the spatial relationship between normal perilabyrinthine bone remodeling, the viability and spatial distribution of labyrinthine osteocytes, and the location of otosclerosis, a unique spatial pattern emerged. Bone remodeling is highly inhibited around the inner ear space. Most likely, inner ear anti-resorptive signals enter the bony otic capsule through the lacuno-canalicular porosity. The patency of this signaling pathway depends on the viability of individual osteocytes. In the young otic capsule the density of viable osteocytes is high and centripetally distributed. This arrangement may sustain a life-long osseus pathway for anti-resorptive signals even within a bone where a considerable loss of viable osteocytes must be expected, as demonstrated by a centripetal accumulation of dead osteocytes with age. The spatial distribution of dead osteocytes follows the same general pattern as otosclerosis. We suggest that clustering of dead osteocytes may impede the transmission of anti-resorptive signals locally, leaving such ghost regions susceptible to focal bone remodeling as in human otosclerosis. The preserved network of viable osteocytes around the depleted ghost regions may contain the process and distort the structure of bone remodeling into an abnormal otosclerotic pattern.

Possible role of calcium permselectivity in bone adaptation

According to the core activity of calcium in the bone cellular expression, a new hypothesis linking calcium transport with the mechanical loading is proposed to explain the mechano-adaptation of bone tissue. Due to the piezoelectric coupling, the tensile and compressive areas of bone produce different electrical environments for the osteocytic cells that are embedded in the lacuno-canalicular porosity. This electrical asymmetry engenders a calcium enrichment–exclusion effect that strongly changes the calcium concentration in the lacuno-canalicular fluid and thus modifies the remodelling process. A bibliographic body of evidence supporting this idea is given and its experimental validation is suggested.

BONE ADAPTATION AND REGENERATION – NEW DEVELOPMENTS

Bone is a dynamic tissue that is constantly renewed and adapts to its local loading environment. Mechanical loading results in adaptive changes in bone size and shape that strengthen bone structure. The mechanisms for adaptation involve a multistep process called mechanotransduction, which is the ability of resident bone cells to perceive and translate mechanical energy into a cascade of structural and biochemical changes within the cells. The transduction of a mechanical signal to a biochemical response involves pathways within the cell membrane and cytoskeleton of the osteocytes, the professional mechansensor cells of bone. During the last decade the role of mechanosensitive osteocytes in bone metabolism and turnover, and the lacuno-canalicular porosity as the structure that mediates mechanosensing, is likely to reveal a new paradigm for understanding the bone formation response to mechanical loading, and the bone resorption response to disuse. Strain-derived fluid flow of interstitial fluid through the lacuno-canalicular porosity seems to mechanically activate the osteocytes, as well as ensures transport of cell signaling molecules, nutrients and waste products. Cell-cell signaling from the osteocyte sensor cells to the effector cells (osteoblasts or osteoclasts), and the effector cell response – either bone formation or resorption, allow an explanation of local bone gain and loss as well as remodeling in response to fatigue damage as processes supervised by mechanosensitive osteocytes. The osteogenic activity of cultured bone cells has been quantitatively correlated with varying stress stimulations highlighting the importance of the rate of loading. Theoretically a possible mechanism for the stress response by osteocytes is due to strain amplification at the pericellular matrix. Single cell studies on molecular responses of osteocytes provide insight on local architectural alignment in bone during remodeling. Alignment seems to occur as a result of the osteocytes sensing different canalicular flow patterns around cutting cone and reversal zone during loading, thus determining the bone's structure. Disturbances in architecture and permeability of the 3D porous network will affect transduction of mechanical loads to the mechanosensors. Uncovering the cellular and mechanical basis of the osteocyte's response to loading represents a significant challenge to our understanding of cellular mechanotransduction and bone remodeling. In view of the importance of mechanical stress for maintaining bone strength, mechanical stimuli have great potential for providing a therapeutic approach for bone (re)generation.

Effects of loading frequency on the functional adaptation of trabeculae predicted by bone remodeling simulation

The process of bone remodeling is regulated by metabolic activities of many bone cells. While osteoclasts and osteoblasts are responsible for bone resorption and formation, respectively, activities of these cells are believed to be controlled by a mechanosensory system of osteocytes embedded in the extracellular bone matrix. Several experimental and theoretical studies have suggested that the strain-derived interstitial fluid flow in lacuno-canalicular porosity serves as the prime mover for bone remodeling. Previously, we constructed a mathematical model for trabecular bone remodeling that interconnects the microscopic cellular activities with the macroscopic morphological changes in trabeculae through the mechanical hierarchy. This model assumes that fluid-induced shear stress acting on osteocyte processes is a driving force for bone remodeling. The validity of this model has been demonstrated with a remodeling simulation using a two-dimensional trabecular model. In this study, to investigate the effects of loading frequency, which is thought to be a significant mechanical factor in bone remodeling, we simulated morphological changes of a three-dimensional single trabecula under cyclic uniaxial loading with various frequencies. The results of the simulation show the trabecula reoriented to the loading direction with the progress of bone remodeling. Furthermore, as the imposed loading frequency increased, the diameter of the trabecula in the equilibrium state was enlarged by remodeling. These results indicate that our simulation model can successfully evaluate the relationship between loading frequency and trabecular bone remodeling.

The viability and spatial distribution of osteocytes in the human labyrinthine capsule: A quantitative study using vector-based stereology

Background Bone remodeling is highly inhibited around the labyrinthine space, most likely by the action of the anti-resorptive cytokine osteoprotegerin (OPG). Inner ear OPG may enter the bony otic capsule through the lacuno-canalicular porosity (LCP). The aim of the study was to investigate the patency of this extracellular signaling pathway by exploring the viability, age dependency and spatial distribution of capsular osteocytes. Methods Sixty-five bulk-stained undecalcified human temporal bones were selected to span the ages from 30th gestational week to 95 years. Osteocytes within 1000 μm wide iso-concentric perilabyrinthine zones of bone were identified by 3-D vector calculations and the number of cells per unit of bone volume estimated within each zone by optical dissectors. Results From a high initial numerical density and a centripetal distribution of viable osteocytes, the density declined over time. This effect was higher towards the inner ear space and shifted viable osteocytes into to a centrifugal distribution with age. Contrary to this, non-viable osteocytes accumulated centripetally around the inner ear space and accounted for 50% of all lacunae at 80 years of age. Non-viable osteocytes were heterogeneously distributed forming islets of varying size surrounded by the intact and viable parts of the LCP. Conclusion The simultaneous presence of high numbers of non-viable osteocytes within a dense network of viable osteocytes is unique for the bony otic capsule. Viable osteocytes may sustain a life-long anti-resorptive signaling pathway for inner ear OPG. Clustering of non-viable osteocytes may locally impede the effect of OPG leaving the ghost regions unprotected against focal bone remodeling, as in human otosclerosis.

Estimation of bone permeability considering the morphology of lacuno-canalicular porosity

Load-induced interstitial fluid flow in lacuno-canalicular porosity is believed to play an important role in cellular activities regulating adaptive bone remodeling. To investigate interstitial fluid behavior based on poroelasticity, it is important to determine the anisotropic permeability tensor reflecting the morphological features of the lacuno-canalicular porosity as fluid channels. In this study, we presented an estimation method of trabecular permeability by describing the analytical relationship between the volume orientation (VO) fabric tensor, which represents the canalicular orientation, and the permeability tensor. The relationship showed that the trabecular permeability tensor is proportional to the product of the volume fraction of the interstitial fluid and the VO fabric tensor of the canaliculi. We applied the proposed method to a two-dimensional fluorescent image of a trabecular cross section to quantify the canalicular anisotropy and the trabecular permeability tensor. The results indicated that the canaliculi are predominantly oriented in the radial direction of the trabecula, and the permeability depends strongly on the canalicular morphology.

Influence of vascular porosity on fluid flow and nutrient transport in loaded cortical bone

Load-induced fluid flow is a key factor in triggering bone modeling and remodeling processes that maintain bone mass and architecture. To provide an enhanced understanding of fluid flow in bone, unique computational models of a tibial section were developed. The purpose of the study was to examine the effects of incorporating vascular porosity on pore fluid pressure and resulting lacunocanalicular flow and to determine the role of load-induced fluid flow in tracer transport. Simulations revealed large local pressure gradients surrounding the vascular canals that were dependent on the magnitude and state (i.e., compressive or tensile) of the stress. Fluid velocity magnitudes were increased by over an order of magnitude in the dual-porosity model, relative to the single-porosity model. Fluid flow had a marked effect on tracer perfusion within the cortex. After 10 loading cycles, a 9-fold increase in tracer concentration, relative to diffusion alone, was observed in the compressive region where fluid exchange was greatest between the lacunocanalicular porosity and the vascular canals. Agreement was achieved between computational results and experimental investigations of electrokinetic phenomenon, tracer transport, cellular stimulation, and functional adaptation. The models produced substantial improvements in bone fluid flow simulation and underscored the significance of incorporating vascular porosity in models designed to quantify fluid pressure and flow characteristics within mechanically loaded cortical bone.

Osteocytes: Mechanosensors of Bone and Orchestrators of Mechanical Adaptation

Significant progress has been made in the field of mechanotransduction in bone cells. The knowledge about the role of osteocytes as the professional mechanosensor cells of bone as well as the lacuno-canalicular porosity as the structure that mediates mechanosensing is increasing. New insights might result in a paradigm for understanding the bone formation response to mechanical loading, and the bone resorption response to disuse. Under physiological loading conditions the strain-derived flow of interstitial fluid through the lacuno-canalicular porosity seems to mechanically activate the osteocytes, which subsequently alter the bone remodeling activity of osteoblasts and/or osteoclasts. Fatigue loading results in local microdamage, disruption of normal flow patterns, and osteocyte apoptosis. Apoptotic osteocytes likely attract osteoclasts to resorb the damaged bone. This concept allows explanation of local bone gain and loss, as well as remodeling in response to fatigue damage, as processes supervised by mechanosensitive osteocytes. Uncovering the cellular and mechanical basis of the osteocyte’s response to loading would greatly contribute to our understanding of the cellular basis for bone remodeling, and could contribute to the discovery of new treatment modalities for bone mass disorders, such as osteoporosis.

Osteocytes subjected to fluid flow inhibit osteoclast formation and bone resorption

Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 ± 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-κB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N G -nitro- l-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.