Lacuno-canalicular Network Research Articles

Bone canalicular network segmentation in 3D nano-CT images through geodesic voting and image tessellation

Recent studies emphasized the role of the bone lacuno-canalicular network (LCN) in the understanding of bone diseases such as osteoporosis. However, suitable methods to investigate this structure are lacking. The aim of this paper is to introduce a methodology to segment the LCN from three-dimensional (3D) synchrotron radiation nano-CT images. Segmentation of such structures is challenging due to several factors such as limited contrast and signal-to-noise ratio, partial volume effects and huge number of data that needs to be processed, which restrains user interaction. We use an approach based on minimum-cost paths and geodesic voting, for which we propose a fully automatic initialization scheme based on a tessellation of the image domain. The centroids of pre-segmented lacunæ are used as Voronoi-tessellation seeds and as start-points of a fast-marching front propagation, whereas the end-points are distributed in the vicinity of each Voronoi-region boundary. This initialization scheme was devised to cope with complex biological structures involving cells interconnected by multiple thread-like, branching processes, while the seminal geodesic-voting method only copes with tree-like structures. Our method has been assessed quantitatively on phantom data and qualitatively on real datasets, demonstrating its feasibility. To the best of our knowledge, presented 3D renderings of lacunæ interconnected by their canaliculi were achieved for the first time.

Mechanism of bone mass regulation by mechanical stress

Osteocytes establish an extensive intercellular and extracellular communication system via gap junction-coupled cell processes and canaliculi, through which cell processes pass throughout bone, and the communication system is extended to osteoblasts on the bone surface. The lacunocanalicular network formed by osteocytes is thought to be an ideal mechanosensory system and suitable for mechanotransduction, by which mechanical energy is converted into electrical and/or biochemical signals. The function of osteocytes cannot be estimated based on the events caused by osteocyte death, because apoptotic osteocytes are not phagocytosed, undergo secondary necrosis, and trigger a process of repair to replace the damaged bone. The analysis of the mice, in which both intercellular and extracellular communication systems are disturbed, shows that the osteocyte network mildly inhibits bone formation and mildly stimulates bone resorption in physiological condition. In unloaded condition, the functions of the osteocyte network are augmented, and it strongly inhibits bone formation and strongly stimulates bone resorption, at least in part, through the induction of Sost in osteocytes and Rankl in osteoblasts.

Preliminary analysis of osteocyte lacunar density in long bones of tetrapods: all measures are bigger in sauropod dinosaurs.

Osteocytes harbour much potential for paleobiological studies. Synchrotron radiation and spectroscopic analyses are providing fascinating data on osteocyte density, size and orientation in fossil taxa. However, such studies may be costly and time consuming. Here we describe an uncomplicated and inexpensive method to measure osteocyte lacunar densities in bone thin sections. We report on cell lacunar densities in the long bones of various extant and extinct tetrapods, with a focus on sauropodomorph dinosaurs, and how lacunar densities can help us understand bone formation rates in the iconic sauropod dinosaurs. Ordinary least square and phylogenetic generalized least square regressions suggest that sauropodomorphs have lacunar densities higher than scaled up or comparably sized mammals. We also found normal mammalian-like osteocyte densities for the extinct bovid Myotragus, questioning its crocodilian-like physiology. When accounting for body mass effects and phylogeny, growth rates are a main factor determining the density of the lacunocanalicular network. However, functional aspects most likely play an important role as well. Observed differences in cell strategies between mammals and dinosaurs likely illustrate the convergent nature of fast growing bone tissues in these groups.

Talbot-defocus multiscan tomography using the synchrotron X-ray microscope to study the lacuno-canalicular network in mouse bone

The three-dimensional network of lacunae and canaliculi that regulates metabolism in bone contains osteocytes and their dendritic processes. We constructed a synchrotron radiation X-ray microscope for sequential tomography of mouse tibia first by using a Talbot interferometer to detect the degree of bone mineralization and then by using absorption contrast under a slightly defocused setting to enhance outline contrast thereby visualizing structures of the osteocyte lacuno-canalicular network. The resultant pair of tomograms was precisely aligned with each other, allowing evaluation of mineral density in the vicinity of each osteocyte lacuna and canaliculus over the entire thickness of the cortical bone. Thus, multiscan microscopic X-ray tomography is a powerful tool for analyzing bone mineralization in relation to the lacuno-canalicular network at the submicron resolution level.

Histomorphometric Study and Three-Dimensional Reconstruction of the Osteocyte Lacuno-Canalicular Network One Hour after Applying Tensile and Compressive Forces

The occurrence of very early morphological changes in the osteocyte lacuno-canalicular network following application of tensile and/or compressive forces remains unknown to date. Thus, the aim of this study was to perform a morphological and morphometric evaluation of the changes in the three-dimensional structure of the lacuno-canalicular network and the osteocyte network of alveolar bone that take place very early after applying tensile and compressive forces in vivo, conducting static histomorphometry on bright-field microscopy and confocal laser scanning microscopy images. Our results showed that both the tensile and compressive forces induced early changes in osteocytes and their lacunae, which manifested as an increase in lacunar volume and changes in lacunar shape and orientation. An increase in canalicular width and a decrease in the width and an increase in the length of cytoplasmic processes were also observed. The morphological changes in the lacuno-canalicular and osteocyte networks that occur in vivo very early after application of tensile and compressive forces would be an indication of an increase in permeability within the system. Thus, both compressive and tensile forces would cause fluid displacement very soon after being applied; the latter would in turn rapidly activate alveolar bone osteocytes, enhancing transmission of the signals to the entire osteocyte network and the effector cells located at the bone surface.

The effect of osteocyte apoptosis on signalling in the osteocyte and bone lining cell network: a computer simulation

Osteocytes play a critical role in the regulation of bone remodelling by translating strain due to mechanical loading into biochemical signals transmitted through the interconnecting lacuno-canalicular network to bone lining cells (BLCs) on the bone surface. This work aims to examine the effects of disruption of that intercellular communication by simulation of osteocyte apoptosis in the bone matrix. A model of a uniformly distributed osteocyte network has been developed that simulates the signalling through the network to the BLCs based on strain level. Bi-directional and asymmetric communication between neighbouring osteocytes and BLCs is included. The effect of osteocyte apoptosis is examined by preventing signalling at and through the affected cells. The simulation shows that apoptosis of only 3% of the osteocyte cells leads to a significant reduction in the peak signal at the BLCs. Furthermore, experiments with the model confirm how important the location and density of the apoptotic osteocytes are to the signalling received at the bone surface. With 5% and 9% osteocyte apoptosis, the mean peak BLC levels were reduced by 25% and 37% respectively. Such a significant reduction in the signal at the BLCs may explain a possible mechanism that leads to the increased remodelling and eventual bone loss observed with osteoporosis. More generally, it provides a unique framework for a broader exploration of the role of osteocyte and bi-directional and asymmetric cell–cell communication in mechanotransduction, and the effects of disruption to that communication.

X-Ray Phase Nanotomography Resolves the 3D Human Bone Ultrastructure

Bone strength and failure are increasingly thought to be due to ultrastructural properties, such as the morphology of the lacuno-canalicular network, the collagen fiber orientation and the mineralization on the nanoscale. However, these properties have not been studied in 3D so far. Here we report the investigation of the human bone ultrastructure with X-ray phase nanotomography, which now provides the required sensitivity, spatial resolution and field of view. The 3D organization of the lacuno-canalicular network is studied in detail over several cells in osteonal and interstitial tissue. Nanoscale density variations are revealed and show that the cement line separating these tissues is hypermineralized. Finally, we show that the collagen fibers are organized as a twisted plywood structure in 3D.

Nanoscale imaging of the bone cell network with synchrotron X‐ray tomography: optimization of acquisition setup

The fundamental role of the osteocyte cell network in regulating the bone remodeling has become evident in the last years. This has raised the necessity to explore this complex three-dimensional interconnected structure, but the existing investigation methods cannot provide an adequate assessment. The authors propose to use parallel beam synchrotron radiation computed tomography at the nanoscale to image in three dimensions the osteocyte lacunocanalicular network. To this aim, the authors study the feasibility of this technique and present an optimized imaging protocol suited for the bone cell network. Moreover, they demonstrate the multifaceted information provided by this method. The high brilliance of synchrotron radiation combined with state of art detectors permits reaching nanoscale spatial resolution. With a nominal pixel size of 280 nm, the parallel beam computed tomography setup at the ID19 experimental station of the ESRF is capable of imaging the bone lacunocanalicular network, considering that the reported diameter of canaliculi is in the range 300-600 nm. However, the actual resolution is limited by the detector and by the radiation dose causing sample damage during the scan. The authors sought to overcome these limitations by optimizing the imaging setup and the acquisition parameters in order to minimize the necessary radiation dose to create the images and to improve the spatial resolution of the detector. The authors achieved imaging of the osteocyte cell network in human bone. Due to the optimization of the imaging setup and acquisition parameters, they obtained simultaneously a radiation dose reduction and an increase of the signal to noise ratio in the images. This permitted the authors to generate the first three-dimensional images of the lacunocanalicular network in an area covering several osteons, the fundamental functional units in the bone cortex. The method enables assessment of both architectural parameters of the microporosity and of mineralization degree in the bone matrix. The authors found that the cell network is dense and connected inside osteonal tissue. Conversely, the cell lacunae are sparse, unorganized, and disconnected in interstitial tissue. The authors show that synchrotron radiation computed tomography is a feasible technique to assess the lacunocanalicular network in three dimensions. This is possible due to an optimal imaging setup in which the detector plays an important role. The authors could establish two valid setups, based on two different insertion devices. These results give access to new information on the bone cell network architecture, covering a number of cells two orders of magnitude greater than existing techniques. This enables biomedical studies on series of samples, paving the way to better understanding of bone fragility and to new treatments for bone diseases.

Bone researches using synchrotron radiation

Bone researches with synchrotron radiation have now expanded from a simple mineralization measurement to more complicated studies such as strain mapping and tissue engineering. The most common techniques are high resolution X-ray tomography and X-ray diffraction, but the spectroscopic techniques such as fluorescence analysis, infrared FTIR and XAFS are also available. High resolution X-ray tomography has been improved in the time and spatial resolution. In vitro tomographic experiments have become common and been applied to 3D imaging of a live mouse. As a new imaging method, coherent diffraction imaging has been applied to visualize the lacunocanalicular network. As each of these techniques provides unique information and the sample condition depends much on the technique, the choice of the technique is important to achieve a scientific goal.

Fluid flow induced calcium response in osteoblasts: Mathematical modeling

Fluid flow in the bone lacuno–canalicular network can induce dynamic fluctuation of intracellular calcium concentration ([Ca2+]i) in osteoblasts, which plays an important role in bone remodeling. There has been limited progress in the mathematical modeling of this process probably due to its complexity, which is controlled by various factors such as Ca2+ channels and extracellular messengers. In this study we developed a mathematical model to describe [Ca2+]i response induced by fluid shear stress (SS) by integrating the major factors involved and analyzed the effects of different experimental setups (e.g. [Ca2+]i baseline, pretreatment with ATP). In this model we considered the ATP release process and the activities of multiple ion channels and purinergic receptors. The model was further verified quantitatively by comparing the simulation results with experimental data reported in literature. The results showed that: (i) extracellular ATP concentration has more significant effect on [Ca2+]i baseline (73% increase in [Ca2+]i with extracellular ATP concentration varying between 0 and 10μM), as compared to that induced by SS (25% variation in [Ca2+]i with SS varying from 0 to 3.5Pa); (ii) Pretreatment with ATP-medium results in different [Ca2+]i response as compared to the control group (ATP-free medium) under SS; (iii) Relative [Ca2+]i fluctuation over baseline is more reliable to show the [Ca2+]i response process than the absolute [Ca2+]i response peak. The developed model may improve the experimental design and facilitate our understanding of the mechanotransduction process in osteoblasts.

Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuno-canalicular network

Up to now, a quantitative three-dimensional (3D) assessment of the lacuno-canalicular network (LCN) within bone has not been achieved in a comprehensive way and the LCN has mostly been investigated using two-dimensional imaging methods only. First attempts for the 3D assessment of the osteocytes and their cell processes have been reported using different imaging techniques. Nevertheless, various experimental limitations allowed for assessment of isolated or incompletely interconnected osteocytes only. On the other hand, serial focused ion beam/scanning electron microscopy (FIB/SEM) currently seems to be a promising imaging method for quantitative 3D assessment of the LCN. However, combined 3D visualization and quantification of the LCN using serial FIB/SEM imaging has not been reported so far. The aim of this study was to provide a proof of concept that serial FIB/SEM meets all requirements for quantitative 3D imaging of the LCN. To this end, we developed a new bone sample preparation protocol for serial FIB/SEM imaging providing a resolution on the order of 30nm. This technique was successfully applied to the mid-diaphysis of a mouse femur. Moreover, we devised and applied novel measures for subsequent quantitative 3D morphometry of the LCN. Briefly, serial FIB/SEM was shown to be an appropriate technique to quantify the morphology of the LCN truly in 3D. This will allow investigating bone matrix changes on an ultrastructural level, which result from aging, disease, and treatment.

Imaging Resin-Cast Osteocyte Lacuno-Canalicular System at Bone-Bioactive Glass Interface by Scanning Electron Microscopy

The morphology of the osteocyte lacuno-canalicular system at the bone-biomaterial implant-interface has not been fully investigated. In this study, the resin-cast scanning electron microscopy technique was used, for the first time, to image the lacuno-canalicular network within neoformed bone around bioactive glass (BG) particles implanted in rat tibia bone marrow. The most salient finding was that the osteocyte canaliculi pass through the calcium-phosphorus layer formed at the bone-BG interface and reach the silica-rich layer of the reacted BG.

Asymmetric intercellular communication between bone cells: Propagation of the calcium signaling

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.

Role of osteocytes in the adaptation of bone to mechanical loading

The mechanical performance of our skeleton is secured by the constant adaptation of bone to its mechanical loading environment. Mechanical adaptation of bone is brought about by the coordinated actions of osteoclasts and osteoblasts, which are orchestrated by the most mechano-sensitive cells in bone, the osteocytes. Loading on bone generates a flow of interstitial fluid through the lacuno-canalicular network in which the osteocytes are positioned. This flow is sensed by the osteocytes, which respond by the release of signaling factors such as nitric oxide, prostaglandins and Wnts, which alter the recruitment and activity of osteoblasts and osteoclasts, thereby affecting bone mass. Thus, any factor that alters the response of osteocytes to mechanical loading potentially affects bone mass. This paradigm could have implications for the field of rheumatology, where proinflammatory cytokines might affect bone mass by altering the response of osteocytes to mechanical loading.

Intravital Microlesions in the Human Otic Capsule

Purpose of the Study: To verify microcracks in human temporal bones by a method which distinguishes intravital lesions from artifacts, to revisit previous information derived from decalcified materials on their morphology and classification and to discuss the possible clinical significance. Procedures: Histological analysis of 210 undecalcified adult human temporal bones bulk-stained by basic fuchsin in ethanol prior to embedding in methyl methacrylate and processing by the cutting and grinding method. Results: Intravital crack lesions appeared in all specimens and could be distinguished from artifacts regardless of crack morphology and size. Particularly, large microfissures were found around the labyrinthine windows, while smaller microfissures originating at the perilymphatic space were found throughout perilabyrinthine bone. Small noncommunicating fatigue microdamage was identified for the first time. All lesions were reorganized according to morphology, topography and possible clinical significance. Conclusion: Microfissures accumulate in the adult otic capsule as expected when bone remodeling is low. Any capsular microlesion may introduce a possible barrier in the recently discovered antiresorptive signaling pathway from inner ear structures via the lacunocanalicular osteocytic network to perilabyrinthine bone. For this reason intravital microfissures may interfere with in vivo inhibition of temporal bone remodeling and consequently offer another pathogenetic factor in otosclerosis.

Morphologic changes associated with functional adaptation of the navicular bone of horses

Failure of functional adaptation to protect the skeleton from damage is common and is often associated with targeted remodeling of bone microdamage. Horses provide a suitable model for studying loading-related skeletal disease because horses are physically active, their exercise is usually regulated, and adaptive failure of various skeletal sites is common. We performed a histologic study of the navicular bone of three groups of horses: (1) young racing Thoroughbreds (n = 10); (2) young unshod ponies (n = 10); and (3) older horses with navicular syndrome (n = 6). Navicular syndrome is a painful condition that is a common cause of lameness and is associated with extensive remodeling of the navicular bone; a sesamoid bone located within the hoof which articulates with the second and third phalanges dorsally. The following variables were quantified: volumetric bone mineral density; cortical thickness (Ct.Th); bone volume fraction, microcrack surface density; density of osteocytes and empty lacunae; and resorption space density. Birefringence of bone collagen was also determined using circularly polarized light microscopy and disruption of the lacunocanalicular network was examined using confocal microscopy. Remodeling of the navicular bone resulted in formation of transverse secondary osteons orientated in a lateral to medial direction; bone collagen was similarly orientated. In horses with navicular syndrome, remodeling often led to the formation of intracortical cysts and development of multiple tidemarks at the articular surface. These changes were associated with high microcrack surface density, low bone volume fraction, low density of osteocytes, and poor osteocyte connectivity. Empty lacunae were increased in Thoroughbreds. Resorption space density was not increased in horses with navicular syndrome. Taken together, these data suggest that the navicular bone may experience habitual bending across the sagittal plane. Consequences of cumulative cyclic loading in horses with navicular syndrome include arthritic degeneration of adjacent joints and adaptive failure of the navicular bone, with accumulation of microdamage and associated low bone mass, poor osteocyte connectivity, and low osteocyte density, but not formation of greater numbers of resorption spaces.

Development of Pore Pressure Measurement System in Lacunocanalicular Network of Trabeculae Using MEMS Based Micro-Pressure Transducer

Experimental measurement of pore pressure generation in lacunocanalicular network of trabeculae is never measured, although the characteristics could be important for bone remodeling. In this study, the pore pressure generation in micro-trabecular specimens within the elastic range was measured in vitro using a specially designed micro-experimental setup and a MEMS based micro-pressure transducer. Then, a quasi-static loading (9㎛/min) was applied up to the strain of 0.4 % with measuring pore pressure generations in the undrained and drained conditions. 49.2 ± 4.45 KPa of pore pressure generation at the 0.4% strain was found in the undrained condition. In contrast, no pore pressure generation was measured in the drained condition. The result could let us know the amount of a possible maximum pore pressure generation in lacunocanalicular network of trabeculae within the elastic range.

Knee-Loading Modality Drives Molecular Transport in Mouse Femur

Mechanical loading is well known to stimulate bone remodeling. Load-driven interstitial fluid flow and molecular transport have been postulated to play a role in the enhancement of bone formation. In order to evaluate load-driven molecular transport in a lacunocanalicular network, we conducted fluorescence recovery after photobleaching (FRAP) experiments using lacunae stained with uranine (376 Da). Loads were applied to a mouse femur ex vivo with a novel knee-loading modality, where the distal epiphysis was loaded with a sinusoidal force at 2 Hz. The lacunae in the diaphysis located 25% (approximately 4 mm) proximal to the loading site were photobleached and sequentially imaged, and a time constant for fluorescence recovery was determined both with and without knee loading. The time constant was estimated as the period to recover 63% of fluorescent intensity using a best-fit exponential curve. The results reveal that the applied loads shortened the time constant from 33 +/- 9 s with non-loading control to 25 +/- 11 s with knee loading (p = 0.0014). The strain in the measurement site was <100 microstain along the femoral midshaft, which was an order of magnitude smaller than the minimum effective strain threshold for bone remodeling. Taken together, the current study supports the notion that molecular transport in cortical bone is enhanced by the loads applied to the epiphysis without inducing significant in situ strain in the diaphysis.

Release of nitric oxide, but not prostaglandin E2, by bone cells depends on fluid flow frequency

Loading frequency is an important parameter for the stimulation of bone formation in vivo. It is still unclear how the information of external loading characteristics is conveyed to osteoblasts and osteoclasts. Osteocytes are thought to detect mechanical loads by sensing fluid flow through the lacuno-canalicular network within bone and to translate this information into chemical signals. The signaling molecules nitric oxide (NO) and prostaglandin E2 (PGE2) are known to play important roles in the adaptive response of bone to mechanical loads. We have investigated the effects of fluid flow frequency on the production of PGE2 and NO in bone cells in vitro. Pulsatile fluid flow with different frequencies stimulated the release of NO by MC3T3-E1 osteoblasts in a dose-dependent manner. In contrast, PGE2 production was enhanced consistently by all fluid flow regimes, independent of flow frequency. This implies that the NO response may play a role in mediating the differential effects of the various loading patterns on bone.

Mechanobiology of bone tissue

In order to obtain bones that combine a proper resistance against mechanical failure with a minimum use of material, bone mass and its architecture are continuously being adapted to the prevailing mechanical loads. It is currently believed that mechanical adaptation is governed by the osteocytes, which respond to a loading-induced flow of interstitial fluid through the lacuno-canalicular network by producing signaling molecules. An optimal bone architecture and density may thus not only be determined by the intensity and spatial distribution of mechanical stimuli, but also by the mechanoresponsiveness of osteocytes. Bone cells are highly responsive to mechanical stimuli, but the critical components in the load profile are still unclear. Whether different components such as fluid shear, tension or compression may affect cells differently is also not known. Although both tissue strain and fluid shear stress cause cell deformation, these stimuli might excite different signaling pathways related to bone growth and remodeling. In order to define new approaches for bone tissue engineering in which bioartificial organs capable of functional load bearing are created, it is important to use cells responding to the local forces within the tissue, whereby biophysical stimuli need to be optimized to ensure rapid tissue regeneration and strong tissue repair.