Osteocyte Processes Research Articles

Differential distribution of microtubules in immature osteocytes in vivo

ObjectivesThe transition from osteoblasts to osteocytes is associated with dramatic changes in the cytoskeleton. We previously showed that the formation of osteoblast cell processes in 3D culture is microtubule dependent. However, the distribution of microtubules during the transition from osteoblasts to osteocytes in vivo is unknown. In this study, we investigated the distribution of microtubules in osteocytes in vivo. MethodsWe observed the microtubules in osteocytes in chick embryonic calvaria via fluorescence staining of microtubules and confocal laser scanning microscopy. ResultsMicrotubules were observed throughout the cytoplasm in all examined osteoblasts. In immature osteocytes, several cell processes contained microtubules, whereas in mature osteocytes, microtubules were localized only in the cell body. ConclusionThese results suggest that the early arrangement of microtubules may be correlated with the initial development of osteocyte processes.

Effects of pericellular microenvironment on the deformation of osteocyte processes induced by interstitial fluid flow

Effects of pericellular microenvironment on the deformation of osteocyte processes induced by interstitial fluid flow

Simulation of flow-induced deformation of osteocyte processes in a poroelastic trabecula

Simulation of flow-induced deformation of osteocyte processes in a poroelastic trabecula

Potential role for a specialized β3 integrin-based structure on osteocyte processes in bone mechanosensation.

Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αV β3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αV β3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to β3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2 × 7R and the low voltage transiently opened T-type calcium channel CaV3.2-1 all reside in close proximity to β3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:642-652, 2018.

Bone repair and ultrasound stimulation: An insight into the interaction of LIPUS with the bone callus through a multiscale computational study

In the 1950s, the effect of ultrasound stimulation on bone healing has been discovered. Nowadays, Low Intensity Pulsed Ultrasound Stimulation (LIPUS) is admitted to influence the mechanotransduction of bone. Nevertheless, despite a growing literature—cell cultures, animal models, and clinical studies—the underlying physical and biological mechanisms of LIPUS on bone healing are still misunderstood. Inspired from previous studies on the mechanotransduction induced by physiological loading, this work focuses on the effect of LIPUS on the osteocytes. These bone cells are thought to be the principal mechanosensors of bone. They are ubiquitous inside the bone matrix, immersed in the lacuno-canalicular network (LCN) filled with interstitial fluid (IF). The goal is to relate the ultrasound stimulation applied at the tissue scale, to the biological response at the cell scale. To tackle this question, two finite element models were implemented in the commercial software Comsol Multiphysics. The tissue-scale model considers an anisotropic poroelastic matrix to evaluate the IF pressure gradient induced by LIPUS into the LCN. Then, in the cell-scale model, the IF shear stress magnitude and the induced drag forces applied on osteocyte process are calculated and compared with levels of cell activation recorded in literature.

Coalignment of osteocyte canaliculi and collagen fibers in human osteonal bone.

During bone formation osteocytes get connected with each other via a dense network of canaliculi within the mineralized bone matrix. Important functions attributed to the osteocyte network include the control of bone remodeling and a contribution to mineral homeostasis. To detect structural clues of the formation and functionality of the network, this study analyzes the structure and orientation of the osteocyte lacuno-canalicular network (OLCN), specifically in relation to the concentric bone lamellae within human osteons. The network structure within 49 osteons from four samples of cortical bone from the femoral midshaft of middle-aged healthy women was determined by a combination of rhodamine staining and confocal laser scanning microscopy followed by computational image analysis. A quantitative evaluation showed that 64±1% of the canalicular length has an angle smaller than 30° to the direction towards the osteon center, while the lateral network - defined by an orientation angle larger than 60° - comprises 16±1%. With the same spatial periodicity as the bone lamellae, both radial and lateral network show variations in the network density and order. However, only the preferred orientation of the lateral network twists when crossing a lamella. This twist agrees with the preferred orientation of the fibrous collagen matrix. The chirality of the twist was found to be individual-specific. The coalignment between network and matrix extends to the orientation of the elongated osteocyte lacunae. The intimate link between OLCN and collagen matrix implies an interplay between osteocyte processes and the arrangement of the surrounding collagen fibers during osteoid formation.

Effect of pericellular matrix on the flow-induced deformation of osteocyte process

Effect of pericellular matrix on the flow-induced deformation of osteocyte process

Direct communication between osteocytes and acid-etched titanium implants with a sub-micron topography.

The osteocyte network, through the numerous dendritic processes of osteocytes, is responsible for sensing mechanical loading and orchestrates adaptive bone remodelling by communicating with both the osteoclasts and the osteoblasts. The osteocyte network in the vicinity of implant surfaces provides insight into the bone healing process around metallic implants. Here, we investigate whether osteocytes are able to make an intimate contact with topologically modified, but micrometre smooth (S a < 0.5 µm) implant surfaces, and if sub-micron topography alters the composition of the interfacial tissue. Screw shaped, commercially pure (cp-Ti) titanium implants with (i) machined (S a = ~0.2 µm), and (ii) two-step acid-etched (HF/HNO3 and H2SO4/HCl; S a = ~0.5 µm) surfaces were inserted in Sprague Dawley rat tibia and followed for 28 days. Both surfaces showed similar bone area, while the bone-implant contact was 73 % higher for the acid-etched surface. By resin cast etching, osteocytes were observed to maintain a direct intimate contact with the acid-etched surface. Although well mineralised, the interfacial tissue showed lower Ca/P and apatite-to-collagen ratios at the acid-etched surface, while mineral crystallinity and the carbonate-to-phosphate ratios were comparable for both implant surfaces. The interfacial tissue composition may therefore vary with changes in implant surface topography, independently of the amount of bone formed. Implant surfaces that influence bone to have higher amounts of organic matrix without affecting the crystallinity or the carbonate content of the mineral phase presumably result in a more resilient interfacial tissue, better able to resist crack development during functional loading than densely mineralised bone.

A Single Chance to Contact Multiple Targets: Distinct Osteocyte Morphotypes Shed Light on the Cellular Mechanism Ensuring the Robust Formation of Osteocytic Networks.

The formation of the complex osteocytic network relies on the emission of long cellular processes involved in communication, mechanical strain sensing, and bone turnover control. Newly deposited osteocytic processes rapidly become trapped within the calcifying matrix, and, therefore, they must adopt their definitive conformation and contact their targets in a single morphogenetic event. However, the cellular mechanisms ensuring the robustness of this unique mode of morphogenesis remain unknown. To address this issue, we examined the developing calvaria of the amphibian Xenopus tropicalis by confocal, two-photon, and super-resolution imaging, and described flattened osteocytes lying within a woven bone structured in lamellae of randomly oriented collagen fibers. While most cells emit peripheral and perpendicular processes, we report two osteocytes morphotypes, located at different depth within the bone matrix and exhibiting distinct number and orientation of perpendicular cell processes. We show that this pattern is conserved with the chick Gallus gallus and suggest that the cellular microenvironment, and more particularly cell-cell contact, plays a fundamental role in the induction and stabilization of osteocytic processes. We propose that this intrinsic property might have been evolutionarily selected for its ability to robustly generate self-organizing osteocytic networks harbored by the wide variety of bone shapes and architectures found in extant and extinct vertebrates.

Alternation in the gap-junctional intercellular communication capacity during the maturation of osteocytes in the embryonic chick calvaria

IntroductionThe intercellular network of cell-cell communication among osteocytes is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in the integration and synchronization of bone remodeling. To further understand the mechanism of bone development it is important to quantify the difference in the GJIC capacity of young and developmentally mature osteocytes. Materials and methodsWe first established an embryonic chick calvaria growth model to show the growth of the calvaria in embryos at 13 to 21days of age. We then applied a fluorescence recovery after photobleaching (FRAP) technique to compare the difference in the GJIC capacity of young osteocytes with that of developmentally mature osteocytes. Finally, we quantified the dye (Calcein) diffusion from the FRAP data using a mathematic model of simple diffusion which was also used to identify simple diffusion GJIC pattern cells (fitted model) and accelerated diffusion GJIC pattern cells (non-fitted model). ResultsThe relationship between the longest medial-lateral length of the calvaria (frontal bone) and the embryonic age fit a logarithmic growth model: length=5.144×ln(day)−11.340. The morphometric data during osteocyte differentiation showed that the cellular body becomes more spindle-shaped and that the cell body volume decreased by approximately 22% with an increase in the length of the processes between the cells. However, there were no significant differences in the cellular body surface area or in the distance between the mass centres of the cells. The dye-displacement rate in young osteocytes was significantly higher than that in developmentally mature osteocytes: dye displacement only occurred in 26.88% of the developmentally mature osteocytes, while it occurred in 64.38% of the young osteocytes. Additionally, in all recovered osteocytes, 36% of the developmentally mature osteocytes comprised non-fitted model cells while 53.19% of the young osteocytes were the non-fitted model, which indicates the active transduction of dye molecules. However, there were no statistically significant differences between the young and developmentally mature osteocytes with regard to the diffusion coefficient, permeability coefficient, or permeance of the osteocyte processes, which were 3.93±3.77 (×10−8cm2/s), 5.12±4.56 (×10−5cm2/s) and 2.99±2.47 (×10−13cm2/s) (mean±SD), respectively. ConclusionsThese experiments comprehensively quantified the GJIC capacity in the embryonic chick calvaria and indicated that the cell-cell communication capacity of the osteocytes in the embryonic chick calvaria was related to their development.

Overexpression of BCLXL in Osteoblasts Inhibits Osteoblast Apoptosis and Increases Bone Volume and Strength.

The Bcl2 family proteins, Bcl2 and BclXL, suppress apoptosis by preventing the release of caspase activators from mitochondria through the inhibition of Bax subfamily proteins. We reported that BCL2 overexpression in osteoblasts using the 2.3 kb Col1a1 promoter increased osteoblast proliferation, failed to reduce osteoblast apoptosis, inhibited osteoblast maturation, and reduced the number of osteocyte processes, leading to massive osteocyte death. We generated BCLXL (BCL2L1) transgenic mice using the same promoter to investigate BCLXL functions in bone development and maintenance. Bone mineral density in the trabecular bone of femurs was increased, whereas that in the cortical bone was similar to that in wild-type mice. Osteocyte process formation was unaffected and bone structures were similar to those in wild-type mice. A micro-CT analysis showed that trabecular bone volume in femurs and vertebrae and the cortical thickness of femurs were increased. A dynamic bone histomorphometric analysis revealed that the mineralizing surface was larger in trabecular bone, and the bone-formation rate was increased in cortical bone. Serum osteocalcin but not TRAP5b was increased, BrdU-positive osteoblastic cell numbers were increased, TUNEL-positive osteoblastic cell numbers were reduced, and osteoblast marker gene expression was enhanced in BCLXL transgenic mice. The three-point bending test indicated that femurs were stronger in BCLXL transgenic mice than in wild-type mice. The frequency of TUNEL-positive primary osteoblasts was lower in BCLXL transgenic mice than in wild-type mice during cultivation, and osteoblast differentiation was enhanced but depended on cell density, indicating that enhanced differentiation was mainly owing to reduced apoptosis. Increased trabecular and cortical bone volumes were maintained during aging in male and female mice. These results indicate that BCLXL overexpression in osteoblasts increased the trabecular and cortical bone volumes with normal structures and maintained them majorly by preventing osteoblast apoptosis, implicating BCLXL as a therapeutic target of osteoporosis. © 2016 American Society for Bone and Mineral Research.

Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20nm and a diameter of 2–4nm. Force pulling revealed a strong protein core that can withstand over 100pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890pN and a corresponding Young's modulus of 71MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.

High-Resolution Visualization of the Osteocyte Lacuno-Canalicular Network Juxtaposed to the Surface of Nanotextured Titanium Implants in Human.

Osseointegration is controlled by a number of cellular mechanisms. Although factors governing bone formation are well-understood, the maintenance of bone at the bone-implant interface is less clear. Of some interest is the role of osteocytes, which via mechanotransduction are believed to be responsible for mechanical loading-based remodelling events in bone. Using a resin cast etching technique, we investigated the osteocyte lacuno-canalicular network adjacent to nanostructured titanium human dental implants after four years in clinical function. Correlative electron microscopy showed nanoscale osteocyte processes extending directly onto the implant surface. Calcium signal mapping via electron energy loss spectroscopy (EELS) showed apatite ingrowth into the nanotextured surface, while the apatite platelet c-axis was oriented approximately parallel to the collagen fibril direction. Furthermore, Z-contrast electron tomography demonstrated that natural bone-osteocyte and engineered bone-implant interfaces are similar in ultrastructural morphology. The present ultrastructural observation of multiple connections between osteocyte canaliculi and the nanotextured surface oxide suggests that osteocytes contribute toward the maintenance of osseointegration in long-term clinical function.

Quantifying the osteocyte network in the human skeleton

Osteocytes form an extensive cellular network throughout the hard tissue matrix of the skeleton, which is known to regulate skeletal structure. However due to limitations in imaging techniques, the magnitude and complexity of this network remain undefined.We have used data from recent papers obtained by new imaging techniques, in order to estimate absolute and relative quantities of the human osteocyte network and form a more complete understanding of the extent and nature of this network.We estimate that the total number of osteocytes within the average adult human skeleton is ~42 billion and that the total number of osteocyte dendritic projections from these cells is ~3.7 trillion. Based on prior measurements of canalicular density and a mathematical model of osteocyte dendritic process branching, we calculate that these cells form a total of 23 trillion connections with each other and with bone surface cells. We estimate the total length of all osteocytic processes connected end-to-end to be 175,000km. Furthermore, we calculate that the total surface area of the lacuno-canalicular system is 215m2. However, the residing osteocytes leave only enough space for 24mL of extracellular fluid. Calculations based on measurements in lactation-induced murine osteocytic osteolysis indicate a potential total loss of ~16,000mm3 (16mL) of bone by this process in the human skeleton. Finally, based on the average speed of remodelling in the adult, we calculate that 9.1 million osteocytes are replenished throughout the skeleton on a daily basis, indicating the dynamic nature of the osteocyte network.We conclude that the osteocyte network is a highly complex communication network, and is much more vast than commonly appreciated. It is at the same order of magnitude as current estimates of the size of the neural network in the brain, even though the formation of the branched network differs between neurons and osteocytes. Furthermore, continual replenishment of large numbers of osteocytes in the process of remodelling allows therapeutic changes to the continually renewed osteoblast population to be rapidly incorporated into the skeleton.

Optical clearing in dense connective tissues to visualize cellular connectivity in situ.

Visualizing the three-dimensional morphology and spatial patterning of cells embedded deep within dense connective tissues of the musculoskeletal system has been possible only by utilizing destructive techniques. Here we utilize fructose-based clearing solutions to image cell connectivity and deep tissue-scale patterning in situ by standard confocal microscopy. Optical clearing takes advantage of refractive index matching of tissue and the embedding medium to visualize light transmission through a broad range of bovine and whole mount murine tissues, including cartilage, bone, and ligament, of the head and hindlimb. Using non-destructive methods, we show for the first time intercellular chondrocyte connections throughout the bulk of cartilage, and we reveal in situ patterns of osteocyte processes and the lacunar-canalicular system deep within mineralized cortical bone. Optical clearing of connective tissues is expected to find broad application for the study of cell responses in normal physiology and disease pathology.

Dark horse in osteocyte biology: Glycocalyx around the dendrites is critical for osteocyte mechanosensing.

Osteocytes are considered as the major mechanosensory cells of the bone tissue that control the bone remodeling process. Since osteocytes are buried inside mineralized matrix, they maintain a strong communication network with other cells. Long dendritic processes of the osteocytes act as communication cables, conveying mechanical signals to the neighboring osteocytes and the cells on the bone surface; like osteoblasts and osteoclasts. Gap junctions and hemichannels formed by Connexin (Cx) 43 are observed to be involved in responding to the mechanical stimulus and in communicating the mechano-responsive biochemical signals. The contrast in the arrangement of the osteocyte cell body and the dendrites raises an important question of how these parts of the osteocyte respond to mechanical stimulation. We addressed this issue in our recent report through the stimulation of either osteocyte cell body or dendrites and our findings suggest that the osteocyte dendritic processes are sensitive to mechanical stimulation in comparison to the cell body. Most importantly, we observed that the dendritic processes are capable of conveying the mechanical signals to the cell body. Our findings also suggested that the glycocalyx surrounding the dendrites is required for sensing and conveying the mechanical signals. Degradation of the glycocalyx also leads to poor integrin attachment, thereby, affecting dendritic stiffness. These results suggest that the osteocyte dendritic processes are highly responsive towards mechanical stimulation and the glycocalyx surrounding the dendrites is critical in transducing these mechanical signals.

Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions.

The integrity of the interface between the osteocyte (Ot) process and the canalicular wall was investigated in terms of change in the lateral dimensions of the Ot process in relation to the canalicular width, i.e., widening of the pericellular space. This has been interpreted as shrinkage of the Ot process relative to the canalicular wall during sample preparation stages of fixation, dehydration, and resin embedding. Sprague-Dawley rat tibial cross-sections were prepared for transmission electron microscopy (TEM). Four different fixative preparations: paraformaldehyde (PF), modified Karnovsky's (MK), glutaraldehyde (GRR) with ruthenium red (GRR), and zinc formalin (ZF); and two different embedding resins: LR Gold (LRG) and Epon812 (Epon) were evaluated. It was found that for LRG embedding, formalin-only fixatives (PF and ZF) induced lower shrinkage than GRR-containing fixatives (MK and GRR). In contrast, for Epon embedding, MK showed the highest shrinkage, while no differences were found between the remaining fixatives (PF, ZF, and GRR). All formalin-containing fixatives (MK, PF, and ZF) induced similar shrinkage in both embedding media. The most dramatic difference was for GRR fixation, which in combination with LRG embedding showed ∼ 62% more shrinkage than with Epon embedding, suggesting that the combination of GRR fixation and LRG embedding synergistically amplifies Ot shrinkage. These differences likely suggest a role of the resin in secondarily influencing the tissue structure following fixation. Further, the work confirms LRG as a poor embedding medium for bone specimens, as it causes large variations in shrinkage depending on fixation.

Fracture and fatigue in osteocytes

Fatigue is a common mode of mechanical failure which occurs when a material is subjected to repeated cycles at a strain level less than that needed for monotonic fracture. Fatigue has been observed and measured in many different materials but, until recently, not in cells. We devised a novel experiment which allowed us to create both monotonic failure and fatigue in the cellular processes of osteocytes within samples of bone (Dooley et al., European Cells and Materials 2014). In the present paper, we describe the results of further experiments and a computer simulation, which has allowed us to estimate the strain history of each sample tested and thus present, for the first time, strain/life data for cells. Failure occurred during the first cycle at strains of 0.1–0.2; at lower strains failure occurred after a number of cycles which depended inversely on the applied strain range. Scatter in the strain/life data was reduced when we allowed for the effects of mean stress using the Smith–Watson–Topper parameter. We confirmed that aspects of our experimental method (the types of microcrack used and the testing of fresh versus frozen samples) did not affect the results. Such information is useful because many cell types, including the cellular processes of osteocytes, experience cyclic strain in vivo.

Polarized mechanotransduction in osteocytes: role of αVβ3 integrin on the cell processes (1180.9)

Osteocyte processes attach to the bony canalicular wall at discrete integrin sites and loading‐induced fluid flow in the lacunar‐canalicular system results in focally high membrane strains around these attachments, but not at the cell body. While the presence of integrin in the attachment sites to the canalicular wall suggests its participation in osteocyte mechano‐transduction, such a role still remains to be demonstrated. The goal of this study was to address these points by using the novel Stokesian fluid stimulus probe (SFSP) to achieve the spatial resolution required to stimulate MLO‐Y4 osteocyte processes and cell body with physiologically relevant hydrodynamic forces at pN levels. Ca2+ imaging studies demonstrated that osteocyte processes are uniquely mechanosensitive. 1‐5 pN level forces applied to the cell processes triggered Ca2+ signaling while comparable stimulation of cell bodies did not. Inhibition of αVβ3 integrin attachment sites with IntegriSense probe blocked the Ca2+ response to SFSP stimulation. Furthermore, we found that ATP is a major autocrine/paracrine signaling messenger that amplifies spread of Ca2+ signals from the mechanically stimulated process to cell body and from one osteocyte to another in the network. We thus validated the essential role of αVβ3 integrin on osteocyte processes in focal mechanosensing and transduction.Grant Funding Source: Supported by AR057139, DK091466

Fatigue failure of osteocyte cellular processes: implications for the repair of bone

The physical effects of fatigue failure caused by cyclic strain are important and for most materials well understood. However, nothing is known about this mode of failure in living cells. We developed a novel method that allowed us to apply controlled levels of cyclic displacement to networks of osteocytes in bone. We showed that under cyclic loading, fatigue failure takes place in the dendritic processes of osteocytes at cyclic strain levels as low as one tenth of the strain needed for instantaneous rupture. The number of cycles to failure was inversely correlated with the strain level. Further experiments demonstrated that these failures were not artefacts of our methods of sample preparation and testing, and that fatigue failure of cell processes also occurs in vivo. This work is significant as it is the first time it has been possible to conduct fatigue testing on cellular material of any kind. Many types of cells experience repetitive loading which may cause failure or damage requiring repair. It is clinically important to determine how cyclic strain affects cells and how they respond in order to gain a deeper understanding of the physiological processes stimulated in this manner. The more we understand about the natural repair process in bone the more targeted the intervention methods may become if disruption of the repair process occurred. Our results will help to understand how the osteocyte cell network is disrupted in the vicinity of matrix damage, a crucial step in bone remodelling.