Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

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     

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.     

Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

 Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading. Accepted: 9 July 1996     

Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon--a proposal

The concept of bone remodelling by basic multicellular units is well established, but how the resorbing osteoclasts find their way through the pre-existing bone matrix remains unexplained. The alignment of secondary osteons along the dominant loading direction suggests that remodelling is guided by mechanical strain. This means that adaptation (Wolff's Law) takes place throughout life at each remodelling cycle. We propose that alignment during remodelling occurs as a result of different canalicular flow patterns around cutting cone and reversal zone during loading. Low canalicular flow around the tip of the cutting cone is proposed to reduce NO production by local osteocytes thereby causing their apoptosis. In turn, osteocyte apoptosis could be the mechanism that attracts osteoclasts, leading to further excavation of bone in the direction of loading. At the transition between cutting cone and reversal zone, however, enhanced canalicular flow will stimulate osteocytes to increase NO production, which induces osteoclast retraction and detachment from the bone surface. Together, this leads to a treadmill of attaching and detaching osteoclasts in the tip and the periphery of the cutting cone, respectively, and the digging of a tunnel in the direction of loading.     

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.     

Bone remodelling algorithms incorporating both strain and microdamage stimuli

Biomechanical theories to predict bone remodelling have used either mechanical strain or microdamage as the stimulus driving cellular responses. Even though experimental data have implicated both stimuli in bone cell regulation, a mechano-regulatory system incorporating both stimuli has not yet been proposed. In this paper, we test the hypothesis that bone remodelling may be regulated by signals due to both strain and microdamage. Four mechano-regulation algorithms are studied where the stimulus is: strain, damage, combined strain/damage, and either strain or damage with damage-adaptive remodelling prioritised when damage is above a critical level. Each algorithm is implemented with both bone lining cell (surface) sensors and osteocyte cell (internal) sensors. Each algorithm is applied to prediction of a bone multicellular unit (BMU) remodelling on the surface of a bone trabecula. It is predicted that a regulatory system capable of responding to changes in either strain or microdamage but which prioritises removal of damaged bone when damage is above a critical level, is the only one that provides a plausible prediction of BMU behaviour. A mechanism for this may be that, below a certain damage threshold, osteocyte processes can sense changes in strain and fluid flow but above the threshold damage interferes with the signalling mechanism, or causes osteocyte apoptosis so that a remodelling response occurs to remove the dead osteocytes.     

Mechanosensitivity and intercellular communication in HOBIT osteoblastic cells: a possible role for gap junction hemichannels

Mechanically induced intercellular Ca2+ signalling was investigated in differentiated HOBIT osteoblastic cells. HOBIT cells express connexin43 clustered at the cell-to-cell boundary and display functional intercellular coupling assessed by intercellular transfer of Lucifer yellow. Mechanical stimulation of single cells, besides leading to an intracellular Ca2+ rise, induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading was inhibited by 18alpha-glycyrrhetinic acid, demonstrating the involvement of gap junctions in signal propagation. Suramin and apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signalling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18alpha-glycyrrhetinic acid.     

Strain Concentrations Surrounding an Ellipsoid Model of Lacunae and Osteocytes

Direct cell sensing of tissue matrix strains is one possible signaling mechanism for mechanically mediated bone adaptation. We utilized homogenization theory lo estimate bone tissue matrix strains surrounding osteocytes using two sets of models. The first set of models estimated the strain levels surrounding the lacunae and canaliculi, taking into account variations in lamellar properties. The second set estimated strain levels in the osteocyte and the surrounding matrix for different cellular mechanical properties. The results showed that the strain levels found in and surrounding osteocytes, 1700 to 2700 microstrain (denoted as μe; 1 =.0001% strain), were significantly greater than the trabecular tissue level strains of [1325 μe, 287 μe, 87 μe] used for model input. Variation in lamellar properties did not affect strain levels, except at lamellar boundaries. Strain in and surrounding the osteocyte was not significantly affected by cellular stiffness ranging between 28 and 28,000 Pascals (Pa). Strain levels surrounding lacunae and canaliculi were approximately equivalent.     

Investigation of the regulation of bone mass by mechanical loading: from quantitative cytochemistry to gene array

From the 1860s to the early 1980s, the process that fitted bone architecture and mass to function had been investigated and characterized. It was known that increases in exercise were associated with increased bone mass, and that disuse caused osteopaenia, but the mechanisms by which those processes were regulated was not understood. The idea that osteocytes, the cells embedded in bone, were sensitive to the effects of mechanical loading was attractive, yet there was almost no experimental support for it, at least in part because the cells were considered inaccessible for study. In 1984, the techniques devised by Chayen and his co-workers were focused on this area. By analysis of the activity of the enzyme glucose 6-phosphate dehydrogenase in osteocytes in sections of avian bone that had been subjected to brief periods of applied mechanical loading, we showed for the first time that osteocytes could respond within a few minutes to mechanical stimulation. The lack of elevation of activity of other glycolytic enzymes led to the conclusion that this elevation was due to increased activity of the pentose shunt pathway, which was likely to be associated with increased production of reducing equivalents for biosynthesis, and ribose sugars for RNA synthesis. This was the first demonstration of an ability of osteocytes to respond to an external mechanical event and in effect provided a mechanistic link for the fundamental principle of what is known as Wolff's law of bone remodelling. These studies were dependent on several technical advances brought together in the Chayen Cellular Biology Laboratory at the Kennedy Institute. The ability to make cryosections of undecalcified bone, to perform cytochemical analysis of (soluble) enzyme activities by use of colloid stabilizers in the reaction medium, and finally to measure accurately the coloured reaction products by microdensitometry (which avoided optical heterogeneity errors) combined to provide a powerful way to explore bone cell function in situ. In the intervening years since then, similar studies have become routine, and the impact of molecular biological advances in hard tissues have remained dependent on techniques pioneered in the Chayen laboratory. During such studies, other advances have spun off, so that osteocyte gene expression has been analysed in samples taken from sections where the precise tissue characteristics were known, leading to advances in understanding of intercellular signalling mechanisms in bone by differential display, and the role of apoptosis in osteocytes in regulation of osteoclastic resorption. Still more recently, materials extracted from undecalcified sections have been used in gene array studies to discover new candidate genes with a role in the adaptive mechanism. Without Joe Chayen's involvement in this area, which now impacts on almost all bone biological science either directly or indirectly, our understanding of the pathophysiology of osteoporosis would have been very different.     

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

Strain Concentrations Surrounding an Ellipsoid Model of Lacunae and Osteocytes

Direct cell sensing of tissue matrix strains is one possible signaling mechanism for mechanically mediated bone adaptation. We utilized homogenization theory to estimate bone tissue matrix strains surrounding osteocytes using two sets of models. The first set of models estimated the strain levels surrounding the lacunae and canaliculi, taking into account variations in lamellar properties. The second set estimated strain levels in the osteocyte and the surrounding matrix for different cellular mechanical properties. The results showed that the strain levels found in and surrounding osteocytes, 1700 to 2700 microstrain (denoted as μe; 1 μe =.0001% strain), were significantly greater than the trabecular tissue level strains of {1325 μe, 287 μe, 87 μe} used for model input. Variation in lamellar properties did not affect strain levels, except at lamellar boundaries. Strain in and surrounding the osteocyte was not significantly affected by cellular stiffness ranging between 28 and 28, 000 Pascals (Pa). Strain levels surrounding lacunae and canaliculi were approximately equivalent.     

Fluid flow in the osteocyte mechanical environment: a fluid–structure interaction approach

Osteocytes are believed to be the primary sensor of mechanical stimuli in bone, which orchestrate osteoblasts and osteoclasts to adapt bone structure and composition to meet physiological loading demands. Experimental studies to quantify the mechanical environment surrounding bone cells are challenging, and as such, computational and theoretical approaches have modelled either the solid or fluid environment of osteocytes to predict how these cells are stimulated in vivo. Osteocytes are an elastic cellular structure that deforms in response to the external fluid flow imposed by mechanical loading. This represents a most challenging multi-physics problem in which fluid and solid domains interact, and as such, no previous study has accounted for this complex behaviour. The objective of this study is to employ fluid–structure interaction (FSI) modelling to investigate the complex mechanical environment of osteocytes in vivo. Fluorescent staining of osteocytes was performed in order to visualise their native environment and develop geometrically accurate models of the osteocyte in vivo. By simulating loading levels representative of vigorous physiological activity ( $3,000\,\upmu \upvarepsilon $ compression and 300 Pa pressure gradient), we predict average interstitial fluid velocities $(\sim 60.5\,\upmu \text{ m/s })$ and average maximum shear stresses $(\sim 11\, \text{ Pa })$ surrounding osteocytes in vivo. Interestingly, these values occur in the canaliculi around the osteocyte cell processes and are within the range of stimuli known to stimulate osteogenic responses by osteoblastic cells in vitro. Significantly our results suggest that the greatest mechanical stimulation of the osteocyte occurs in the cell processes, which, cell culture studies have indicated, is the most mechanosensitive area of the cell. These are the first computational FSI models to simulate the complex multi-physics mechanical environment of osteocyte in vivo and provide a deeper understanding of bone mechanobiology.     

Functions of the osteocyte network in the regulation of bone mass

Osteocytes establish an extensive intracellular and extracellular communication system via gap-junction-coupled cell processes and canaliculi throughout bone and the communication system is extended to osteoblasts on the bone surface. The osteocyte network is an ideal mechanosensory system and suitable for mechanotransduction. However, the overall function of the osteocyte network remains to be clarified, since bone resorption is enhanced by osteocyte apoptosis, which is followed by a process of secondary necrosis attributable to the lack of scavengers. The enhanced bone resorption is caused by the release of intracellular content, including immunostimulatory molecules that activate osteoclastogenesis through the canaliculi. Therefore, a mouse model is required in which the osteocyte network is disrupted but in which no bone resorption is induced, in order to evaluate the overall functions of the osteocyte network. One such model is the BCL2 transgenic mouse, in which the osteocyte network, including both intracellular and extracellular networks, is disrupted. Another model is the osteocyte-specific Gja1 knockout mouse, in which intercellular communication through gap junctions is impaired but the canalicular system is intact. Combining the findings from these mouse models with previous histological observations showing the inverse linkage between osteocyte density and bone formation, we conclude that the osteocyte network enhances bone resorption and inhibits bone formation under physiological conditions. Further, studies with BCL2 transgenic mice show that these osteocyte functions are augmented in the unloaded condition. In this condition, Rankl upregulation in osteoblasts and Sost upregulation in osteocytes are, at least in part, responsible for enhanced bone resorption and suppressed bone formation, respectively.     

Shear stress inhibits while disuse promotes osteocyte apoptosis

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.     

Multilevel finite element modeling for the prediction of local cellular deformation in bone

The underlying mechanisms by which bone cells respond to mechanical stimuli or how mechanical loads act on osteocytes housed in lacunae in bone are not well understood. In this study, a multilevel finite element (FE) approach is applied to predict local cell deformations in bone tissue. The local structure of the matrix dictates the local mechanical environment of an osteocyte. Cell deformations are predicted from detailed linear FE analysis of the microstructure, consisting of an arrangement of cells embedded in bone matrix material. This work has related the loads applied to a whole femur during the stance phase of the gait cycle to the strain of a single lacuna and of canaliculi. The predicted bone matrix strains around osteocyte lacunae and canaliculi were nonuniform and differed significantly from the macroscopically measured strains. Peak stresses and strains in the walls of the lacuna were up to six times those in the bulk extracellular matrix. Significant strain concentrations were observed at sites where the process meets the cell body.     

Osteocyte lacunae tissue strain in cortical bone

Current theories suggest that bone modeling and remodeling are controlled at the cellular level through signals mediated by osteocytes. However, the specific signals to which bone cells respond are still unknown. Two primary theories are: (1) osteocytes are stimulated via the mechanical deformation of the perilacunar bone matrix and (2) osteocytes are stimulated via fluid flow generated shear stresses acting on osteocyte cell processes within canaliculi. Recently, much focus has been placed on fluid flow theories since in vitro experiments have shown that bone cells are more responsive to analytically estimated levels of fluid shear stress than to direct mechanical stretching using macroscopic strain levels measured on bone in vivo. However, due to the complex microstructural organization of bone, local perilacunar bone tissue strains potentially acting on osteocytes cannot be reliably estimated from macroscopic bone strain measurements. Thus, the objective of this study was to quantify local perilacunar bone matrix strains due to macroscopically applied bone strains similar in magnitude to those that occur in vivo. Using a digital image correlation strain measurement technique, experimentally measured bone matrix strains around osteocyte lacunae resulting from macroscopic strains of approximately 2000 microstrain are significantly greater than macroscopic strain on average and can reach peak levels of over 30,000 microstrain locally. Average strain concentration factors ranged from 1.1 to 3.8, which is consistent with analytical and numerical estimates. This information should lead to a better understanding of how bone cells are affected by whole bone functional loading.