Osteocyte Processes Research Articles

Cross-scale mechanobiological regulation of cylindrical compressible liquid inclusion via coating

The double-bag theory in modern anatomy suggests that structures with coatings are commonly found in human body at various length scales, such as osteocyte processes covered by pericellular matrix and bones covered by muscle tissue. To understand the mechanical behaviors and physiological responses of such biological structures, we develop an analytical model to quantify surface effects on the deformation of a coated cylindrical compressible liquid inclusion in an elastic matrix subjected to remote loading. Our analytical solution reveals that coating can either amplify or attenuate the volumetric strain of the inclusion, depending on the relative elastic moduli of inclusion, coating, and matrix. For illustration, we utilize this solution to explore amplification/attenuation of volumetric strain in musculoskeletal systems, nerve cells, and vascular tissues. We demonstrate that coating often plays a crucial role in mechanical regulation of the development and repair of human tissues and cells. Our model provides qualitative analysis of cross-scale mechanical response of coated liquid inclusions, helpful for constructing mechanical microenvironment of cells.

Modeling and simulation of osteocyte process–fluid interaction in a canaliculus

An osteocyte is a bone cell situated inside a hard bone matrix in an interstice (lacuna). It has many dendritic structures called cellular processes that radiate outward from the cell through the bone matrix via cylindrical openings (canaliculi). Osteocytes can sense stress and strain applied by the interstitial fluid flow and respond by releasing biochemical signals that regulate bone remodeling. In vitro experiments have suggested that the stress and strain typically experienced at the macroscale tissue level have to be amplified 10× in order for osteocytes to have a significant response in vivo. This stress and strain amplification mechanism is not yet well understood. Previous studies suggest that the processes are the primary sites for mechanosensation thanks to the tethering elements that attach the process membrane to the canalicular wall. However, there are other potential factors which may also contribute to stress and strain amplification, such as canalicular wall geometry and osteocyte-associated proteins in the interstitial space called pericellular matrix. In this work, we perform computational studies to study how canalicular wall roughness affects stress and strain amplification. Our major finding is that the wall roughness induces significantly greater wall shear stress (WSS) on the process when the wall roughness increases flow resistance; and the roughness has relatively smaller influence on the WSS when the resistance remains the same.

Bundling of collagen fibrils influences osteocyte network formation during bone modeling

Osteocytes form a cellular network by gap junctions between their cell processes. This network is important since intercellular communication via the network is essential for bone metabolism. However, the factors that influence the formation of this osteocyte network remain unknown. As the early stage of osteocyte network formation occurs on the bone surface, we observed a newly formed trabecular bone surface by orthogonal focused ion beam-scanning electron microscopy. The embedding late osteoblast processes tended to avoid bundled collagen fibrils and elongate into sparse collagen fibrils. Then, we examined whether the inhibition of bundling of collagen fibrils using a potent lysyl oxidase inhibitor, β-aminopropionitrile (BAPN) changed the cellular network of the chick calvaria. The osteocyte shape of the control group was spindle-shape, while that of the BAPN group was sphere-shaped. In addition, the osteocyte processes of the control group were elongated vertically to the long axis of the cell body, whereas the osteocyte processes of the BAPN group were elongated radially. Therefore, it was suggested that the bundling of collagen fibrils influences normal osteocyte network formation during bone modeling.

Multiscale simulation of the effect of low-intensity pulsed ultrasound on the mechanical properties distribution of osteocytes

Low-intensity pulsed ultrasound (LIPUS) is a potential effective means for the prevention and treatment of disuse osteoporosis. In this paper, the effect of LIPUS exposure on the mechanical properties distribution of the osteocyte system (osteocyte body contains nucleus, osteocyte process, and primary cilia) is simulated. The results demonstrate that the mechanical micro-environment of the osteocyte is significantly improved by ultrasound exposure, and the mean von Mises stress of the osteocyte system increases linearly with the excitation sound pressure amplitude. The mechanical effect of LIPUS on osteocytes is enhanced by the stress amplification mechanism of the primary cilia and osteocyte processes.

Focused Low-Intensity Pulsed Ultrasound (FLIPUS) Mitigates Apoptosis of MLO-Y4 Osteocyte-like Cells

Long cytoplasmic processes of osteocytes orchestrate bone activity by integration of biochemical and mechanical signals and regulate load-induced bone adaptation. Low-Intensity Pulsed Ultrasound (LIPUS) is a clinically used technique for fracture healing that delivers mechanical impulses to the damaged bone tissue in a non-invasive and non-ionizing manner. The mechanism of action of LIPUS is still controversially discussed in the scientific community. In this study, the effect of focused LIPUS (FLIPUS) on the survival of starved MLO-Y4 osteocytes was investigated in vitro. Osteocytes stimulated for 10 min with FLIPUS exhibited extended dendrites, which formed frequent connections to neighboring cells and spanned longer distances. The sonicated cells displayed thick actin bundles and experienced increase in expression of connexin 43 (Cx43) proteins, especially on their dendrites, and E11 glycoprotein, which is responsible for the elongation of cellular cytoplasmic processes. After stimulation, expression of cell growth and survival genes as well as genes related to cell-cell communication was augmented. In addition, cell viability was improved after the sonication, and a decrease in ATP release in the medium was observed. In summary, FLIPUS mitigated apoptosis of starved osteocytes, which is likely related to the formation of the extensive dendritic network that ensured cell survival.

Multi-scale numerical simulation on mechano-transduction of osteocytes in different gravity fields

A three-dimensional model for osteocyte was established to research the mechanisms of mechano-transduction and amplification of primary cilium and osteocyte process in every gravity field. The results showed that significant stress concentration was observed in the area of physical connection between TES and the osteocyte process, where the fluid shear stress (FSS) was around two orders of magnitude higher than that in other areas. Due to the significant amplification effect of the TES structure on mechanical stimulation, making osteocyte process the “optimal mechanical receptor”. In microgravity, the mechanical signal conduction ability of the osteocyte decreased significantly.. Highlights At the micro-nano scale, a 3D model for single bone lacunae-osteocyte system is established. The stress amplification mechanism of the transverse element is verified. Compared with the primary cilium, osteocyte process is the ‘optimal mechanical receptor’. In microgravity, the mechanical signal conduction ability of osteocyte system decreased.

Application of machine learning for the semantic segmentation and morphological analysis of osteocyte processes and canaliculi

Application of machine learning for the semantic segmentation and morphological analysis of osteocyte processes and canaliculi

Osteocytes: Their Lacunocanalicular Structure and Mechanoresponses.

Osteocytes connect with neighboring osteocytes and osteoblasts through their processes and form an osteocyte network. Shear stress on osteocytes, which is induced by fluid flow in the lacunae and canaliculi, has been proposed as an important mechanism for mechanoresponses. The lacunocanalicular structure is differentially developed in the compression and tension sides of femoral cortical bone and the compression side is more organized and has denser and thinner canaliculi. Mice with an impaired lacunocanalicular structure may be useful for evaluation of the relationship between lacunocanalicular structure and mechanoresponses, although their bone component cells are not normal. We show three examples of mice with an impaired lacunocanalicular structure. Ablation of osteocytes by diphtheria toxin caused massive osteocyte apoptosis, necrosis or secondary necrosis that occurred after apoptosis. Osteoblast-specific Bcl2 transgenic mice were found to have a reduced number of osteocyte processes and canaliculi, which caused massive osteocyte apoptosis and a completely interrupted lacunocanalicular network. Osteoblast-specific Sp7 transgenic mice were also revealed to have a reduced number of osteocyte processes and canaliculi, as well as an impaired, but functionally connected, lacunocanalicular network. Here, we show the phenotypes of these mice in physiological and unloaded conditions and deduce the relationship between lacunocanalicular structure and mechanoresponses.

Differential Nanoscale Topography Dedicates Osteocyte-Manipulated Osteogenesis via Regulation of the TGF-β Signaling Pathway.

Osteocytes function as the master orchestrator of bone remodeling activity in the telophase of osseointegration. However, most contemporary studies focus on the manipulation of osteoblast and/or osteoclast functionality via implant surface engineering, which neglects the pivotal role of osteocytes in de novo bone formation. It is confirmative that osteocyte processes extend directly to the implant surface, but whether the surface physicochemical properties can affect the functionality of osteocytes and determine the fate of the osseointegration in the final remodeling stage remains to be determined. Titania nanotube arrays (NTAs) with distinct diameters were fabricated to investigate the relationship between the nanoscale topography and the functionality of osteocytes. In vitro results pinpointed that NTA with a diameter of 15 nm (NTA-15) significantly promote osteogenesis of osteocytes via the enhancement of spreading, proliferation, and mineralization. The osteocyte transcriptome of each group further revealed that the TGF-β signaling pathway plays a pivotal role in osteocyte-mediated osteogenesis. The in vivo study definitely mirrored the aforementioned results, that NTA-15 significantly promotes bone formation around the implant. Consequently, nanoscaled topography-induced osteocyte functionality is important in late osseointegration. This suggests that surface designs targeting osteocytes may, therefore, be a potential approach to solving the aseptic loosening of the implant, and thus strengthen osseointegration.

A morphometric analysis of the osteocyte canaliculus using applied automatic semantic segmentation by machine learning.

Osteocytes play a role as mechanosensory cells by sensing flow-induced mechanical stimuli applied on their cell processes. High-resolution imaging of osteocyte processes and the canalicular wall are necessary for the analysis of this mechanosensing mechanism. Focused ion beam-scanning electron microscopy (FIB-SEM) enabled the visualization of the structure at the nanometer scale with thousands of serial-section SEM images. We applied machine learning for the automatic semantic segmentation of osteocyte processes and canalicular wall and performed a morphometric analysis using three-dimensionally reconstructed images. Six-week-old-mice femur were used. Osteocyte processes and canaliculi were observed at a resolution of 2nm/voxel in a 4 × 4μm region with 2000 serial-section SEM images. Machine learning was used for automatic semantic segmentation of the osteocyte processes and canaliculi from serial-section SEM images. The results of semantic segmentation were evaluated using the dice similarity coefficient (DSC). The segmented data were reconstructed to create three-dimensional images and a morphological analysis was performed. The DSC was > 83%. Using the segmented data, a three-dimensional image of approximately 3.5μm in length was reconstructed. The morphometric analysis revealed that the median osteocyte process diameter was 73.8 ± 18.0nm, and the median pericellular fluid space around the osteocyte process was 40.0 ± 17.5nm. We used machine learning for the semantic segmentation of osteocyte processes and canalicular wall for the first time, and performed a morphological analysis using three-dimensionally reconstructed images.

Computational study of the mechanical influence of lacunae and perilacunar zones in cortical bone microcracking

The mechanical behavior of cortical bone is influenced by microstructural components such as osteons, Haversian canals, and osteocyte lacunae that arise from biological remodeling processes. This study takes a computational approach to investigate the role of the perilacunar zones formed by the local remodeling processes of lacunar-dwelling osteocytes by utilizing phase-field finite element models based on histological imaging of human bone. The models simulated the microdamage accumulation that occurs in cortical bone under transverse compression in bone without lacunae, with lacunae, and with a perilacunar zone surrounding lacunae in order to investigate the role of these features. The results of the simulations found that while lacunae create stress concentration which initiate further damage, perilacunar regions can delay or prevent the emergence and growth of microcracks.

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.

Estrogen depletion on In vivo osteocyte calcium signaling responses to mechanical loading.

Microstructural adaptation of bone in response to mechanical stimuli is diminished with estrogen deprivation. Here we tested in vivo whether ovariectomy (OVX) alters the acute response of osteocytes, the principal mechanosensory cells of bone, to mechanical loading in mice. We also used super resolution microscopy (Structured Illumination microscopy or SIM) in conjunction with immunohistochemistry to assess changes in the number and organization of "osteocyte mechanosomes" - complexes of Panx1 channels, P2X7 receptors and CaV3 voltage-gated Ca2+ channels clustered around αvβ3 integrin foci on osteocyte processes. Third metatarsals bones of mice expressing an osteocyte-targeted genetically encoded Ca2+ indicator (DMP1-GCaMP3) were cyclically loaded in vivo to strains from 250 to 3000 με and osteocyte intracellular Ca2+ signaling responses were assessed in mid-diaphyses using multiphoton microscopy. The number of Ca2+ signaling osteocytes in control mice increase monotonically with applied strain magnitude for the physiological range of strains. The relationship between the number of Ca2+ signaling osteocytes and loading was unchanged at 2days post-OVX. However, it was altered markedly at 28days post-OVX. At loads up to 1000 με, there was a dramatic reduction in number of responding (i.e. Ca2+ signaling) osteocytes; however, at higher strains the numbers of Ca2+ signaling osteocytes were similar to control mice. OVX significantly altered the abundance, make-up and organization of osteocyte mechanosome complexes on dendritic processes. Numbers of αvβ3 foci also staining with either Panx 1, P2X7R or CaV3 declined by nearly half after OVX, pointing to a loss of osteocyte mechanosomes on the dendritic processes with estrogen depletion. At the same time, the areas of the remaining foci that stained for αvβ3 and channel proteins increased significantly, a redistribution of mechanosome components suggesting a potential compensatory response. These results demonstrate that the deleterious effects of estrogen depletion on skeletal mechanical adaptation appear at the level of mechanosensation; osteocytes lose the ability to sense small (physiological) mechanical stimuli. This decline may result at least partly from changes in the structure and organization of osteocyte mechanosomes, which contribute to the distinctive sensitivity of osteocytes (particularly their dendritic processes) to mechanical stimulation.

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.

PDGF-AA promotes cell-to-cell communication in osteocytes through PI3K/Akt signaling pathway.

Osteocytes are the main sensitive cells in bone remodeling due to their potent functional cell processes from the mineralized bone matrix to the bone surface and the bone marrow. Neighboring osteocytes communicate with each other by these cell processes to achieve molecular exchange through gap junction channels. Platelet-derived growth factor-AA (PDGF-AA) has been reported to enhance bone tissue remodeling by promoting cell proliferation, migration, and autocrine secretion in osteoid cell linage. However, the effect of PDGF-AA on intercellular communication between osteocytes is still unclear. In the present study, we elucidated that PDGF-AA could enhance the formation of dendritic processes of osteocytes and the gap junctional intercellular communication by promoting the expression of connexin43 (Cx43). This modulation process was mainly dependent on the activation of phosphorylation of Akt protein by phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling. Inhibition of PI3K/Akt signaling decreased the Cx43 expression induced by PDGF-AA. These results establish a bridge between PDGF-AA and cell-cell communication in osteocytes, which could help us understand the molecular exchange between bone cells and fracture healing.

Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels

Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors.

Multi-scale mechanotransduction of the poroelastic signals from osteon to osteocyte in bone tissue

In order to quantify the poroelastic mechanical signals conduction and evaluate the biomechanical effectiveness of functional units (osteocyte processes, canaliculi and lacuna) in lacunar-canalicular system (LCS), a multiscale poroelastic finite element model was established by using the Comsol Multiphysics software. The poroelastic mechanical signals (pore pressure, fluid velocity, von-Mises stress, strain) were analyzed inside the osteon-osteocyte system. The effects of osteocyte (OCY)’s shape (ellipse and circle), long axis directions (horizontal and vertical) and mechanical properties (Elastic modulus and permeability) on its poroelastic responses were examined. It is found that the OCY processes is the best mechanosensor compared with the OCY body, lacunae and canaliculi. The mechanotransduction ability of the elliptic shaped OCY is stronger than that of circular shaped. The pore pressure and flow velocity around OCYs increase as the elastic modulus and permeability of OCY increase. The established model can be used for studying the mechanism of bone mechanotransduction at the multiscale level.

The Role of Connexin Channels in the Response of Mechanical Loading and Unloading of Bone.

The skeleton adapts to mechanical loading to promote bone formation and remodeling. While most bone cells are involved in mechanosensing, it is well accepted that osteocytes are the principal mechanosensory cells. The osteocyte cell body and processes are surrounded by a fluid-filled space, forming an extensive lacuno-canalicular network. The flow of interstitial fluid is a major stress-related factor that transmits mechanical stimulation to bone cells. The long dendritic processes of osteocytes form a gap junction channel network connecting not only neighboring osteocytes, but also cells on the bone surface, such as osteoblasts and osteoclasts. Mechanosensitive osteocytes also form hemichannels that mediate the communication between the cytoplasmic and extracellular microenvironment. This paper will discuss recent research progress regarding connexin (Cx)-forming gap junctions and hemichannels in osteocytes, osteoblasts, and other bone cells, including those richly expressing Cx43. We will then cover the recent progress regarding the regulation of these channels by mechanical loading and the role of integrins and signals in mediating Cx43 channels, and bone cell function and viability. Finally, we will summarize the recent studies regarding bone responses to mechanical unloading in Cx43 transgenic mouse models. The osteocyte has been perceived as the center of bone remodeling, and connexin channels enriched in osteocytes are a likely major player in meditating the function of bone. Based on numerous studies, connexin channels may present as a potential new therapeutic target in the treatment of bone loss and osteoporosis. This review will primarily focus on Cx43, with some discussion in other connexins expressed in bone cells.

TGF-\u03b21 facilitates cell\u2013cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions

Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.

Les fractures de fatigue du col fémoral et du bassin chez le sportif

Fatigue fractures are the result of repeated mechanical stresses above the resistance threshold on a healthy bone. Repeated mechanical stress may cause fatigue fracture in the dendritic processes of osteocytes leading to an activation of bone remodeling where osteoclastic destruction is greater than osteoblastic formation. Fatigue fractures generally occur in young, healthy, active subjects and represent the most frequent pathology of sport and occur mainly in the lower limbs. The activities most at risk are: marathon, cross-country running, basketball, gymnastics, and ballet dancing and among army recruits. Femoral neck and pelvis are not the most common sites of fatigue fractures. The risk factors are: abrupt increment in the intensity of training, female sex, low body mass index, estrogen deficiency, predisposing anatomical factors and footwear errors. The diagnosis is suspected on mechanical pain of increasing intensity at the time of loading in a subject at risk and requires confirmation by radiological investigations. While plain radiographs must be performed as a first-line procedure, MRI has now become the gold-standard second-line imaging investigation, even providing for professional athletes prognostic factors to predict a return to sports activity. Except for fractures of the femoral neck in areas of tension, which are at high risk of complications and require surgical management, treatment is mainly based on sports rest and the normalization, where appropriate, of vitamin, calcium and energy intake.