A study of the viscoelastic effect in a bone remodeling model

The present paper addresses the following question can a simple regulatory bone remodeling model predict effects of viscosity on the trabecular morphology? For that, we propose an extension of a previous bone remodeling model by taking into account the viscosity properties of the tissue. Zener’s law is used to describe the mechanical behavior of the bone and a specific law of the apparent bone density rate is proposed. Based on stability analysis, numerical simulations are then performed to investigate the viscosity role on simulations of the bone remodeling process. We show that the viscous contribution affects the evolution of the apparent bone density, by slowing down the adaptation process, which seems to be confirmed by simulations with real data obtained from rat tibia.     

Death of osteocytes turns off the inhibition of osteoclasts and triggers local bone resorption

Osteocytes have been suggested to play a role in the regulation of bone resorption, although their effect on bone turnover has remained controversial. In order to study this open question, we developed an organ culture system based on isolated rat calvaria, where the osteocyte viability and its effect on osteoclastic bone resorption can be monitored. Our results suggest that osteocytes are constitutively negative regulators of osteoclastic activity. Osteoclasts, which were cultured on calvarial slices with living osteocytes inside, failed to form actin rings which are the hallmarks of resorbing cells. A similar inhibitory effect was also achieved by the conditioned medium obtained from calvarial organ culture, suggesting that living osteocytes produce yet unrecognized osteoclast inhibitors. On the contrary, when osteocyte apoptosis was induced, this inhibitory effect disappeared and strong osteoclastic bone resorption activity was observed. Thus, local apoptosis of osteocytes may play a major role in triggering local bone remodeling.     

Control of bone mass and remodeling by PTH receptor signaling in osteocytes

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.     

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.     

A sclerostin-based theory for strain-induced bone formation

Bone formation responds to mechanical loading, which is believed to be mediated by osteocytes. Previous theories assumed that loading stimulates osteocytes to secrete signals that stimulate bone formation. In computer simulations this 'stimulatory' theory successfully produced load-aligned trabecular structures. In recent years, however, it was discovered that osteocytes inhibit bone formation via the protein sclerostin. To reconcile this with strain-induced bone formation, one must assume that sclerostin secretion decreases with mechanical loading. This leads to a new 'inhibitory' theory in which loading inhibits osteocytes from inhibiting bone formation. Here we used computer simulations to show that a sclerostin-based model is able to produce a load-aligned trabecular architecture. An important difference appeared when we compared the response of the stimulatory and inhibitory models to loss of osteocytes, and found that the inhibitory pathway prevents the loss of trabeculae that is seen with the stimulatory model. Further, we demonstrated with combined stimulatory/inhibitory models that the two pathways can work side-by-side to achieve a load-adapted bone architecture.     

Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%–80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these “osteocyte-less” mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.     

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

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

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.     

Mechanical Loading Stimulates Expression of Connexin 43 in Alveolar Bone Cells in the Tooth Movement Model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.     

Mechanical loading stimulates expression of connexin 43 in alveolar bone cells in the tooth movement model

Bone osteoblasts and osteocytes express large amounts of connexin (Cx) 43, the component of gap junctions and hemichannels. Previous studies have shown that these channels play important roles in regulating biological functions in response to mechanical loading. Here, we characterized the distribution of mRNA and protein of Cx43 in mechanical loading model of tooth movement. The locations of bone formation and resorption have been well defined in this model, which provides unique experimental systems for better understanding of potential roles of Cx43 in bone formation and remodeling under mechanical stimulation. We found that mechanical loading increased Cx43 mRNA expression in osteoblasts and bone lining cells, but not in osteocytes, at both formation and resorption sites. Cx43 protein, however, increased in both osteoblasts and osteocytes in response to loading. Interestingly, the upregulation of Cx43 protein by loading was even more pronounced in osteocytes compared to other bone cells, with an appearance of punctate staining on the cell body and dendritic process. Cx45 was reported to be expressed in several bone cell lines, but here we did not detect the Cx45 protein in the alveolar bone cells. These results further suggest the potential involvement of Cx43-forming gap junctions and hemichannels in the process of mechanically induced bone formation and resorption.     

Evidence for osteocyte regulation of bone homeostasis through RANKL expression

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.     

Mechanically stimulated osteocytes regulate osteoblastic activity via gap junctions

The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm² rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types. osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication     

Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-beta: enhancement by estrogen

Osteocytes are the most abundant cells in bone and distributed throughout the bone matrix. They are connected to the each other and to the cells on the bone surface. Thus, they may also secrete some regulatory factors controlling bone remodeling. Using a newly established osteocyte-like cell line MLO-Y4, we have studied the interactions between osteocytes and osteoclasts. We collected the conditioned medium (CM) from MLO-Y4 cells, and added it into the rat osteoclast cultures. The conditioned medium had no effect on osteoclast number in 24-h cultures, but it dramatically inhibited resorption. With 5, 10, and 20% CM, there was 25, 39, and 42% inhibition of resorption, respectively. Interestingly, the inhibitory effect was even more pronounced, when MLO-Y4 cells were pretreated with 10(-8) M 17-beta-estradiol. With 5, 10, and 20% CM, there was 46, 51, and 58% of inhibition. When the conditioned medium was treated with neutralizing antibody against transforming growth factor-beta (TGF-beta), the inhibitory effect was abolished. This suggests that osteocytes secrete significant amounts of TGF-beta, which inhibits bone resorption and is modulated by estrogen. RT-PCR and Western blot analysis show that in MLO-Y4 cells, the prevalent TGF-beta isoform is TGF-beta3. We conclude that osteocytes have an active, inhibitory role in the regulation of bone resorption. Our results further suggest a novel role for TGF-beta in the regulation of communication between different bone cells and suggest that at least part of the antiresorptive effect of estrogen in bone could be mediated via osteocytes.     

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.     

Interstitial fluid flow in canaliculi as a mechanical stimulus for cancellous bone remodeling: in silico validation

Cancellous bone has a dynamic 3-dimensional architecture of trabeculae, the arrangement of which is continually reorganized via bone remodeling to adapt to the mechanical environment. Osteocytes are currently believed to be the major mechanosensory cells and to regulate osteoclastic bone resorption and osteoblastic bone formation in response to mechanical stimuli. We previously developed a mathematical model of trabecular bone remodeling incorporating the possible mechanisms of cellular mechanosensing and intercellular communication in which we assumed that interstitial fluid flow activates the osteocytes to regulate bone remodeling. While the proposed model has been validated by the simulation of remodeling of a single trabecula, it remains unclear whether it can successfully represent in silico the functional adaptation of cancellous bone with its multiple trabeculae. In the present study, we demonstrated the response of cancellous bone morphology to uniaxial or bending loads using a combination of our remodeling model with the voxel finite element method. In this simulation, cancellous bone with randomly arranged trabeculae remodeled to form a well-organized architecture oriented parallel to the direction of loading, in agreement with the previous simulation results and experimental findings. These results suggested that our mathematical model for trabecular bone remodeling enables us to predict the reorganization of cancellous bone architecture from cellular activities. Furthermore, our remodeling model can represent the phenomenological law of bone transformation toward a locally uniform state of stress or strain at the trabecular level.     

Osteocytes,not Osteoblasts or Lining Cells,are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.     

Computational model for the cell-mechanical response of the osteocyte cytoskeleton based on self-stabilizing tensegrity structures

The mechanism by which mechanical stimulation on osteocytes results in biochemical signals that initiate the remodeling process inside living bone tissue is largely unknown. Even the type of stimulation acting on these cells is not yet clearly identified. However, the cytoskeleton of osteocytes is suggested to play a major role in the mechanosensory process due to the direct connection to the nucleus. In this paper, a computational approach to model and simulate the cell structure of osteocytes based on self-stabilizing tensegrity structures is suggested. The computational model of the cell consists of the major components with respect to mechanical aspects: the integrins that connect the cell with the extracellular bone matrix, and different types of protein fibers (microtubules and intermediate filaments) that form the cytoskeleton, the membrane-cytoskeleton (microfilaments), the nucleus and the centrosome. The proposed geometrical cell models represent the cell in its physiological environment which is necessary in order to give a statement on the cell behavior in vivo. Studies on the mechanical response of osteocytes after physiological loading and in particular the mechanical response of the nucleus show that the load acting on the nucleus is rising with increasing deformation applied to the integrins.     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

Describing force-induced bone growth and adaptation by a mathematical model

This work proposes a mathematical model that qualitative describes the process of mechanically force-induced bone growth and adaptation. The mathematical model includes osteocytes as the key interfacing layer connecting tissue, cellular and molecular signaling levels. Specifically, in the presence of an increase in the mechanical stimuli, osteocytes respond by mechano-transduction releasing the local factors nitric oxide (NO) and prostaglandin E(2) (PGE(2)). These local factors act as the signaling recruitment signals for bone cells progenitors and influence the coupling activity among osteoblasts and osteoclasts during the process of bone remodeling. The model is in agreement with qualitative observations found in the literature concerning the process of bone adaptation and the cellular interactions during a local bone remodeling cycle induced by mechanical stimulation.