Lacuno-canalicular Network Research Articles

OR29-04 TGFβ Regulates Bone Perilacunar/Canalicular Remodeling in a Sexually Dimorphic Manner

Sexual dimorphism in the material properties of bone extracellular matrix (ECM) and geometry, which are parameters of bone quality have been recognized (Alswat, 2017; Nieves et al., 2005). However, the molecular mechanisms responsible for dimorphic bone quality remain unclear, limiting our ability to therapeutically target fragility, which is determined by bone quality. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFβ signaling (TβRIIocy-/-), suppresses perilacunar/canalicular remodeling (PLR) and compromises bone quality (Dole et al., 2017). Since we previously demonstrated that female bone mass is more sensitive to TGFβ (Mohammad et al., 2009), we hypothesized that loss of osteocytic TGFβ in females will lead to more severe deterioration of PLR and bone quality.Analysis of female bones, however, surprisingly revealed sexual dimorphism in the TGFβ regulation of osteocyte function. While the male TβRIIocy-/- mice demonstrated reduced trabecular bone mass and repression in expression of requisite PLR genes, including matrix metalloproteinases (Mmp2, Mmp13, and Mmp14), cathepsin K (Ctsk), and tartrate-resistant acid phosphatase (Acp5/TRAP) (Qing et al., 2012), the female TβRIIocy-/- bones were intact. With absence of osteocytic TGFβ signaling, the canaliculi of male TβRIIocy-/- osteocytes were truncated and a severe deterioration of lacuno-canalicular network (LCN) was observed. Unlike the male TβRIIocy-/- mice that exhibited poor bone quality, female TβRIIocy-/- bones were completely protected from bone quality defects and macromechanical bone fragility. Thus, TGFβ-dependent regulation PLR and bone quality is sexually dimorphic.Since, lactation is a potent PLR agonist, we also evaluated the lactation-induced PLR response of TβRIIocy-/- female mice. Compared to WT, lactation-induced bone loss and upregulation of PLR genes was significantly attenuated in the TβRIIocy-/- female mice. Lactation-mediated increases in osteocyte lacunar volume were suppressed in TβRIIocy-/- bones as well. This mitigation in PLR of lactating TβRIIocy-/- mice was analogous to that observed in mice with osteocyte-specific ablation of parathyroid hormone type I receptor (PTHR1) (Qing et al., 2012). Remarkably, lactation-inducible PTHR1 expression was also diminished in TβRIIocy-/- osteocytes. Therefore, osteocyte-intrinsic TGFβ signaling is critical for the maximal induction of PLR during lactation, and TGFβ is an upstream regulator of PTHR1-dependent activation of osteocytic PLR. Indeed, we also found sexual dimorphism in the regulation of osteocytic PTHR1 expression by TGFβ, such that male, but not female, TβRIIocy-/- mice had reduced PTHR1 compared to WT.In summary, we identify a novel PTH-dependent mechanism that protects female bone against the severe bone fragility that results from suppressed TGFβ signaling in osteocytes.

TGFβ Regulation of Perilacunar/Canalicular Remodeling Is Sexually Dimorphic.

Bone fragility is the product of defects in bone mass and bone quality, both of which show sex-specific differences. Despite this, the cellular and molecular mechanisms underpinning the sexually dimorphic control of bone quality remain unclear, limiting our ability to effectively prevent fractures, especially in postmenopausal osteoporosis. Recently, using male mice, we found that systemic or osteocyte-intrinsic inhibition of TGFβ signaling, achieved using the 9.6-kb DMP1 promoter-driven Cre recombinase (TβRIIocy-/- mice), suppresses osteocyte perilacunar/canalicular remodeling (PLR) and compromises bone quality. Because systemic TGFβ inhibition more robustly increases bone mass in female than male mice, we postulated that sex-specific differences in bone quality could likewise result, in part, from dimorphic regulation of PLR by TGFβ. Moreover, because lactation induces PLR, we examined the effect of TGFβ inhibition on the female skeleton during lactation. In contrast to males, female mice that possess an osteocyte-intrinsic defect in TGFβ signaling were protected from TGFβ-dependent defects in PLR and bone quality. The expression of requisite PLR enzymes, the lacunocanalicular network (LCN), and the flexural strength of female TβRIIocy-/- bone was intact. With lactation, however, bone loss and induction in PLR and osteocytic parathyroid hormone type I receptor (PTHR1) expression, were suppressed in TβRIIocy-/- bone, relative to the control littermates. Indeed, differential control of PTHR1 expression, by TGFβ and other factors, may contribute to dimorphism in PLR regulation in male and female TβRIIocy-/- mice. These findings provide key insights into the sex-based differences in osteocyte PLR that underlie bone quality and highlight TGFβ signaling as a crucial regulator of lactation-induced PLR. © 2020 American Society for Bone and Mineral Research.

Hox genes maintain critical roles in the adult skeleton

Hox genes are indispensable for the proper patterning of the skeletal morphology of the axial and appendicular skeleton during embryonic development. Recently, it has been demonstrated that Hox expression continues from embryonic stages through postnatal and adult stages exclusively in a skeletal stem cell population. However, whether Hox genes continue to function after development has not been rigorously investigated. We generated a Hoxd11 conditional allele and induced genetic deletion at adult stages to show that Hox11 genes play critical roles in skeletal homeostasis of the forelimb zeugopod (radius and ulna). Conditional loss of Hox11 function at adult stages leads to replacement of normal lamellar bone with an abnormal woven bone-like matrix of highly disorganized collagen fibers. Examining the lineage from the Hox-expressing mutant cells demonstrates no loss of stem cell population. Differentiation in the osteoblast lineage initiates with Runx2 expression, which is observed similarly in mutants and controls. With loss of Hox11 function, however, osteoblasts fail to mature, with no progression to osteopontin or osteocalcin expression. Osteocyte-like cells become embedded within the abnormal bony matrix, but they completely lack dendrites, as well as the characteristic lacuno-canalicular network, and do not express SOST. Together, our studies show that Hox11 genes continuously function in the adult skeleton in a region-specific manner by regulating differentiation of Hox-expressing skeletal stem cells into the osteolineage.

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.

Jawbone remodeling: a conceptual study based on Synchrotron High-resolution Tomography

One of the most important aspects of bone remodeling is the constant turnover mainly driven by the mechanical loading stimulus. The remodeling process produces changes not only in the bone microarchitecture but also in the density distribution of the mineralized matrix - i.e. in calcium concentrations- and in the osteocyte lacunar network. Synchrotron radiation-based X-ray microtomography (microCT) has proven to be an efficient technique, capable to achieve the analysis of 3D bone architecture and of local mineralization at different hierarchical length scales, including the imaging of the lacuno-canalicular network. In the present study, we used microCT within a conceptual study of jawbone remodeling, demonstratively focusing the investigation in two critical contexts, namely in the peri-dental and the peri-implant tissues. The microCT analysis showed that a relevant inhomogeneity was clearly present in both peri-dental and peri-implant biopsies, not only in terms of microarchitecture and mineralization degree, but also considering the lacunar network, i.e. size and numerical density of the osteocyte lacunae. The correlated histological results obtained on the same samples confirmed these observations, also adding information related to non-mineralized tissues. Despite its demonstrative nature, it was concluded that the proposed method was powerful in studying jawbone remodeling because it revealed a direct correlation of its rate with the lacunar density, as achieved by the analysis of the osteocyte lacunar network, and an inverse correlation with the local bone mineral density, as revealed with the Roschger approach.

Interaction of ultrasound waves with bone remodelling: a multiscale computational study.

Ultrasound stimulation is thought to influence bone remodelling process. But recently, the efficiency of ultrasound therapy for bone healing has been questioned. Despite an extensive literature describing the positive effect of ultrasound on bone regeneration-cell cultures, animal models, clinical studies-there are more and more reviews denouncing the inefficiency of clinical devices based on low-intensity pulsed ultrasound stimulation (LIPUS) of the bone healing. One of the reasons to cause controversy comes from the persistent misunderstanding of the underlying physical and biological mechanisms of ultrasound stimulation of bone repair. As ultrasonic waves are mechanical waves, the process to be studied is the one of the mechanotransduction. Previous studies on the bone mechanotransduction have demonstrated the key role of the osteocytes in bone mechano-sensing. Osteocytes are bone cells ubiquitous inside the bone matrix; they are immersed in the interstitial fluid (IF) inside the lacuno-canalicular network (LCN). They are considered as particularly sensitive to a particular type of mechanical stress: wall shear stress on osteocytes due to the IF flow in the LCN. Inspired from these findings and observations, the present work investigates the effect of LIPUS on the cortical bone from the tissue to the osteocytes, considering that the impact of the ultrasound stimulation applied at the tissue scale is related to the mechanical stress experimented by the bone cells. To do that simulations based on the finite element method are carried out in the commercial software Comsol Multiphysics to assess the wall shear stress levels induced by the LIPUS on the osteocytes. Two formulations of the wall shear stress were investigated based on two IF flow models inside the LCN and associated with two different values of the LCN permeability. The wall shear stress estimate is very different depending on the assumption considered. One of these two models provides wall shear stress values in accordance with previous works published on bone mechanotransduction. This study presents the preliminary results of a computational model that could provide keys to understanding the underlying mechanisms of the LIPUS.

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.

Network architecture strongly influences the fluid flow pattern through the lacunocanalicular network in human osteons

A popular hypothesis explains the mechanosensitivity of bone due to osteocytes sensing the load-induced flow of interstitial fluid squeezed through the lacunocanalicular network (LCN). However, the way in which the intricate structure of the LCN influences fluid flow through the network is largely unexplored. We therefore aimed to quantify fluid flow through real LCNs from human osteons using a combination of experimental and computational techniques. Bone samples were stained with rhodamine to image the LCN with 3D confocal microscopy. Image analysis was then performed to convert image stacks into mathematical network structures, in order to estimate the intrinsic permeability of the osteons as well as the load-induced fluid flow using hydraulic circuit theory. Fluid flow was studied in both ordinary osteons with a rather homogeneous LCN as well as a frequent subtype of osteons—so-called osteon-in-osteons—which are characterized by a ring-like zone of low network connectivity between the inner and the outer parts of these osteons. We analyzed 8 ordinary osteons and 9 osteon-in-osteons from the femur midshaft of a 57-year-old woman without any known disease. While the intrinsic permeability was 2.7 times smaller in osteon-in-osteons compared to ordinary osteons, the load-induced fluid velocity was 2.3 times higher. This increased fluid velocity in osteon-in-osteons can be explained by the longer path length, needed to cross the osteon from the cement line to the Haversian canal, including more fluid-filled lacunae and canaliculi. This explanation was corroborated by the observation that a purely structural parameter—the mean path length to the Haversian canal—is an excellent predictor for the average fluid flow velocity. We conclude that osteon-in-osteons may be particularly significant contributors to the mechanosensitivity of cortical bone, due to the higher fluid flow in this type of osteons.

Abnormal lacuno-canalicular network and negative correlation between serum osteocalcin and Cobb angle indicate abnormal osteocyte function in adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a prevalent spinal deformity occurring during peripubertal growth period that affects 1–4% of adolescents globally without clear etiopathogenetic mechanism. Low bone mineral density is an independent and significant prognostic factor for curve progression. Currently, the cause underlying low bone mass in AIS remains elusive. Osteocytes play an important role in bone metabolism and mineral homeostasis, but its role in AIS has not been studied. In the present study, iliac bone tissues were harvested from 21 patients with AIS (mean age of 14.3 ± 2.20 yr old) with a mean Cobb angle of 55.6 ± 10.61° and 13 non-AIS controls (mean age of 16.5 ± 4.79 yr old) intraoperatively. Acid-etched scanning electron microscopy (SEM) images of AIS demonstrated abnormal osteocytes that were more rounded and cobblestone-like in shape and were aligned in irregular clusters with shorter and disorganized canaliculi. Further quantitative analysis with FITC-Imaris technique showed a significant reduction in the canalicular number and length as well as an increase in lacunar volume and area in AIS. SEM with energy-dispersive X-ray spectroscopy analysis demonstrated a lower calcium-to-phosphorus ratio at the perilacunar/canalicular region. Moreover, microindentaion results revealed lower values of Vickers hardness and elastic modulus in AIS when compared with controls. In addition, in the parallel study of 99 AIS (27 with severe Cobb angle of 65.8 ± 14.1° and 72 with mild Cobb angle of 26.6 ± 9.1°) with different curve severity, the serum osteocalcin level was found to be significantly and negatively associated with the Cobb angle. In summary, the findings in this series of studies demonstrated the potential link of abnormal osteocyte lacuno-canalicular network structure and function to the observed abnormal bone mineralization in AIS, which may shed light on etiopathogenesis of AIS.—Chen, H., Zhang, J., Wang, Y., Cheuk, K.-Y., Hung, A. L. H., Lam, T.-P., Qiu, Y., Feng, J. Q., Lee, W. Y. W., Cheng, J. C. Y. Abnormal lacuno-canalicular network and negative correlation between serum osteocalcin and Cobb angle indicate abnormal osteocyte function in adolescent idiopathic scoliosis.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 μm non-motile cocci, S. aureus deforms smaller than 0.5 μm in the sub-micron channels of the OLCN. Here we present the μSiM-CA (Microfluidic – Silicon Membrane – Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The μSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the μSiM-CA. However the μSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

Canalicular Junctions in the Osteocyte Lacuno-Canalicular Network of Cortical Bone.

The osteocyte lacuno-canalicular network (LCN) is essential for bone remodeling because osteocytes regulate cell recruitment. This has been proposed to occur through liquid-flow-induced shear forces in the canaliculi. Models of the LCN have thus far assumed that it contains canaliculi connecting the osteocyte lacunae. However, here, we reveal that enlarged spaces occur at places where several canaliculi cross; we name these spaces canalicular junctions. We characterize them in detail within mice cortical bone using synchrotron nanotomography at two length scales, with 50 and 130 nm voxel size, and show that canalicular junctions occur at a density similar to that of osteocyte lacunae and that canalicular junctions tend to cluster. Through confocal laser scanning microscopy, we show that canalicular junctions are widespread as we have observed them in cortical bone from several species, even though the number density of the canalicular junctions was not universal. Fluid flow simulations of a simple model system with and without a canalicular junction clearly show that liquid mass transport and flow velocities are altered by the presence of canalicular junctions. We suggest that these canalicular junctions may play an important role in osteocyte communication and possibly also in canalicular fluid flow. Therefore, we believe that they constitute an important component in the bone osteocyte network.

Changes in the osteocyte lacunocanalicular network with aging

Osteoporosis is an aging-related disease of reduced bone mass that is particularly prevalent in post-menopausal women, but also affects the aged male population and is associated with increased fracture risk. Osteoporosis is the result of an imbalance whereby bone formation by osteoblasts no longer keeps pace with resorption of bone by osteoclasts. Osteocytes are the most abundant cells in bone and, although previously thought to be quiescent, they are now known to be active, multifunctional cells that play a key role in the maintenance of bone mass by regulating both osteoblast and osteoclast activity. They are also thought to regulate bone mass through their role as mechanoresponsive cells in bone that coordinate adaptive responses to mechanical loading. Osteocytes form an extensive interconnected network throughout the mineralized bone matrix and receive their nutrients as well as hormones and signaling factors through the lacunocanalicular system. Several studies have shown that the extent and connectivity of the lacunocanalicular system and osteocyte networks degenerates in aged humans as well as in animal models of aging. It is also known that the bone anabolic response to loading is decreased with aging. This review summarizes recent research on the degenerative changes that occur in osteocytes and their lacunocanalicular system as a result of aging and discusses the implications for skeletal health and homeostasis as well as potential mechanisms that may underlie these degenerative changes. Since osteocytes are such key regulators of skeletal homeostasis, maintaining the health of the osteocyte network would seem critical for maintenance of bone health. Therefore, a more complete understanding of the structure and function of the osteocyte network, its lacunocanalicular system, and the degenerative changes that occur with aging should lead to advances in our understanding of age related bone loss and potentially lead to improved therapies.

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone.

The lacuno-canalicular network (LCN) hosting the osteocytes in bone tissue represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. Using third-harmonic generation (THG) microscopy with sub-micrometer resolution, we investigate the impact of microgravity on the 3D LCN structure in mice following space flight. A specific analytical procedure to extract the LCN characteristics from THG images is described for ex vivo studies of bone sections. The analysis conducted in different anatomical quadrants of femoral cortical bone didn’t reveal any statistical differences between the control, habitat control and flight groups, suggesting that the LCN connectivity is not affected by one month space flight. However, significant variations are systematically observed within each sample. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone

Third harmonic generation imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone

Ptychographic X-ray CT characterization of the osteocyte lacuno-canalicular network in a male rat's glucocorticoid induced osteoporosis model

Ptychographic X-ray computed tomography (PXCT) is a quantitative imaging modality that non-destructively maps the 3D electron density inside an object with tens of nanometers spatial resolution. This method provides unique access to the morphology and structure of the osteocyte lacuno-canalicular network (LCN) and nanoscale density of the tissue in the vicinity of an osteocyte lacuna. Herein, we applied PXCT to characterize the lacunae and LCN in a male Wistar rat model of glucocorticoid-induced osteoporosis (GIO). The ptychographic images revealed significant (p < 0.05) differences in the number of canaliculi originating from the lacuna per ellipsoidal surface unit, Ca.Nb (p = 0.0106), and the 3D morphology of the lacuna (p = 0.0064), between GIO and SHAM groups. Moreover, the mean canalicular diameter, Ca.Dm, was slightly statistically un-significantly smaller in GIO (152 ± 6.5) nm than in SHAM group (165 ± 8) nm (p = 0.053). Our findings indicate that PXCT can non-destructively provide detailed, nanoscale information on the 3D organization of the LCN in correlative studies of pathologies, such as osteoporosis, leading to improved diagnosis and therapy.

A Review of the Impact of Implant Biomaterials on Osteocytes

In lamellar bone, a network of highly oriented interconnected osteocytes is organized in concentric layers. Through their cellular processes contained within canaliculi, osteocytes are highly mechanosensitive and locally modulate bone remodeling. We review the recent developments demonstrating the significance of the osteocyte lacuno-canalicular network in bone maintenance around implant biomaterials. Drilling during implant site preparation triggers osteocyte apoptosis, the magnitude of which correlates with drilling speed and heat generation, resulting in extensive remodeling and delayed healing. In peri-implant bone, osteocytes physically communicate with implant surfaces via canaliculi and are responsive to mechanical loading, leading to changes in osteocyte numbers and morphology. Certain implant design features allow peri-implant osteocytes to retain a less aged phenotype, despite highly advanced extracellular matrix maturation. Physicochemical properties of anodically oxidized surfaces stimulate bone formation and remodeling by regulating the expression of RANKL (receptor activator of nuclear factor–κB ligand), RANK, and OPG (osteoprotegerin) from implant-adherent cells. Modulation of certain osteocyte-related molecular signaling mechanisms (e.g., sclerostin blockade) may enhance the biomechanical anchorage of implants. Evaluation of the peri-implant osteocyte lacuno-canalicular network should therefore be a necessary component in future investigations of osseointegration to more completely characterize the biological response to materials for load-bearing applications in dentistry and orthopedics.

Acid-etching technique of non-decalcified bone samples for visualizing osteocyte-lacuno-canalicular network using scanning electron microscope

ABSTRACTThe aim of this study was to define the acid-etching technique for bone samples embedded in polymethyl metacrylate (PMMA) in order to visualize the osteocyte lacuno-canalicular network (LCN) for scanning electron microscopy (SEM). Human jaw bone tissue samples (N = 18) were collected from the study population consisting of patients having received dental implant surgery. After collection, the bone samples were fixed in 70% ethanol and non-decalcified samples embedded routinely into polymethyl metacrylate (PMMA). The PMMA embedded specimens were acid-etched in either 9 or 37% phosphoric acid (PA) and prepared for SEM for further analysis. PMMA embedded bone specimens acid-etched by 9% PA concentration accomplishes the most informative and favorable visualization of the LCN to be observed by SEM. Etching of PMMA embedded specimens is recommendable to start with 30 s or 40 s etching duration in order to find the proper etching duration for the samples examined. Visualizing osteocytes and LCN provides a tool to study bone structure that reflects changes in bone metabolism and diseases related to bone tissue. By proper etching protocol of non-decalcified and using scanning electron microscope it is possible to visualize the morphology of osteocytes and the network supporting vitality of bone tissue.

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.

The small world of osteocytes: connectomics of the lacuno-canalicular network in bone

Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization.