Osteocyte Network Research Articles

An agent based model for real-time signaling induced in osteocytic networks by mechanical stimuli

We recently observed that insertion of unloaded rest between each load cycle substantially enhanced bone formation induced by mild loading regimens. To begin to explore this result, we have developed an agent based model for real-time signaling induced when osteocytic networks are challenged by mechanical stimuli. In the model, activity induced in individual osteocytes were governed by the following cellular functions: (1) threshold levels of tissue strain magnitudes were required to initiate and maximally activate cells, (2) cell activity beyond thresholds were propagated within localized neighborhoods and influenced recipient cell activity, (3) cellular activity was modulated by ‘molecular’ stores and the rates at which stores were replenished when cells were quiescent. Using this model, the real-time response of osteocyte networks was determined as the average of individual cell activity. While not explicitly embedded within the model, interactions between cellular functions served as positive, negative, and end-point feedback mechanisms and resulted in unique real-time network responses to distinct mechanical stimuli. Specifically, the real-time network response to cyclic stimuli consisted of a large magnitude transient followed by low-level steady state fluctuations, while rest-inserted stimuli induced multiple secondary transients. Analysis of interaction patterns suggested that rest-inserted stimuli induced this enhanced and sustained signaling within osteocytic networks by enabling cell recovery of expended molecular stores and by efficiently utilizing properties inherent to cell–cell communication in bone. Importantly, this emergence based approach suggested mechanisms potentially underlying the benefit of rest-inserted stimuli and provides a unique framework for a broader exploration of mechanotransduction function within bone.

Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy

Osteocytes are surrounded by hard bone matrix. Therefore, it has not previously been possible to demonstrate the real architecture of the osteocyte network in bone. We previously reported that it is possible to observe osteocytes in bone by labeling the cells with fluorescence and using confocal laser scanning (CLS) microscopy. In this study, we for the first time conducted an extensive analysis of the morphology and morphometry of the three-dimensional (3D) osteocyte structure using three-dimensionally reconstructed fluorescent images. Sixteen-day-old embryonic chick calvariae were stained with fluorescently labeled phalloidin and observed using a confocal laser scanning microscope. Morphometry of osteocytes in the calvaria was analyzed using extensive three-dimensional reconstructing software IMARIS, process length measuring software NEURON TRACER and cell surface area-/cell volume-analyzing software SURPASS. From the IMARIS-derived images, we found that the average of 10 osteocytes is 52.7 ± 5.7 processes, and the point-to-point distance between centers of the osteocytes was 24.1 ± 2.8 μm. In addition, we could calculate that each osteocyte spans an average of 4180 ± 673 μm 3 of bone volume. NEURON TRACER showed that the length of osteocyte processes was 0.26 ± 0.02 μm per 1 μm 3 bone compartment. In addition, SURPASS indicated that the surface area of osteocytes was 0.36 ± 0.03 μm 2 per 1 μm 3 bone compartment and that the volume ratio of osteocyte cell body to bone compartment was 9.42% ± 1.18%. Together, the average total length of the processes, the average surface area, and the average volume of one osteocyte were 1070 ± 145 μm, 1509 ± 113 μm 2, and 394 ± 49 μm 3, respectively. It is possible to reconstruct the real architecture of the osteocyte network and obtain morphometric data from fluorescently labeled osteocytes in chick calvaria.

Isolation and Purification of Osteocytes

Abstract An isolation method for osteocytes is described. After removal of the periostea, bone cells were isolated from calvariae of 18-day-old chicken embryos by alternating treatments with collagenase and EDTA. Osteocytes were purified from the heterogeneous bone cell population with the help of the osteocyte-specific MAb OB 7.3 bound to protein G-conjugated magnetic beads. The purity of the osteocyte population ultimately obtained was more than 95%. Osteocytes were found to adhere rapidly to glass or plastic substrates. They showed numerous processes of various types. These processes could branch and make contact with those of other osteocytes. After 1–2 days of culture, the isolated osteocytes formed a network of apparently interconnected cell processes very similar to the osteocyte network in bone.

444 骨細胞によるメカノセンシングを考慮したリモデリングシミュレーション(GS-13 : 骨・軟骨(2))

444 骨細胞によるメカノセンシングを考慮したリモデリングシミュレーション(GS-13 : 骨・軟骨(2))

The Effects of Trabecular-Bone Loading Variables on the Surface Signaling Potential for Bone Remodeling and Adaptation

It is widely believed that mechanical forces affect trabecular bone structure and orientation. The cellular mechanisms involved in this relationship, however, are poorly understood. In earlier work we developed a theoretical, computational framework, coupling bone-cell metabolic expressions to the local mechanical effects of external bone loading. This theory is based on the assumption that osteocytes within the bone tissue control the recruitment of bone-resorbing osteoclasts and bone-forming osteoblasts, by sending strain-energy-density (SED) related signals to trabecular surfaces through the osteocytic, canalicular network. The theory explains the known morphological effects of external bone-loading variations in magnitude and frequency. It also explains the development of osteoporosis, as an effect of increased osteoclast resorption due to estrogen deficiency in postmenopausal women, and to reduced physical activity levels in general. However, the theory uses lumped variables to represent the mechanisms of osteocyte mechano-sensing and signaling. The question is whether these mechanisms could not be specified in a more realistic way. On the one hand, anabolic osteocyte signals might be triggered by the local mechanical loading variables they experience directly, as we assumed in our original theory. On the other hand, osteocyte signals might be triggered by fluid flow in the osteocytic network at large, as was suggested by others. For that purpose we compared the effects of SED, maximal principal strain and volumetric strain as representing local loading variables, to their spatial gradients on the morphological predictions of our computational model. We found that, in concept, they all produced reasonable trabecular structures. However, the predicted trabecular morphologies based on SED as the triggering variable were more realistic in dimensions and relevant metabolic parameters.

507 骨細胞ネットワークの形態的特徴を考慮した骨梁モデリング(J01-2 計算生体力学におけるマルチスケール・マルチフィジックス問題(2),J01 計算生体力学におけるマルチスケール・マルチフィジックス問題)

507 骨細胞ネットワークの形態的特徴を考慮した骨梁モデリング(J01-2 計算生体力学におけるマルチスケール・マルチフィジックス問題(2),J01 計算生体力学におけるマルチスケール・マルチフィジックス問題)

Additive effects of estrogen and mechanical stress on nitric oxide and prostaglandin E2 production by bone cells from osteoporotic donors

Mechanical loading is thought to provoke a cellular response via loading-induced flow of interstitial fluid through the lacuno-canalicular network of osteocytes. This response supposedly leads to an adaptation of local bone mass and architecture. It has been suggested that loss of estrogen during menopause alters the sensitivity of bone tissue to mechanical load, thereby contributing to the rapid loss of bone. The present study aimed to determine whether estrogen modulates the mechanoresponsiveness of bone cells from osteoporotic women. Bone cell cultures from nine osteoporotic women (aged 62-90 years) were pre-cultured for 24 h with 10(-11) mol/l 17beta-estradiol (E2) or vehicle, and subjected to 1 h of pulsating fluid flow (PFF) or static culture. E2 alone enhanced prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production by 2.8-fold and 2.0-fold, respectively, and stimulated endothelial nitric oxide synthase protein expression by 2.5-fold. PFF, in the absence of E2, stimulated PGE(2) production by 3.1-fold and NO production by 3.9-fold. Combined treatment with E2 and PFF increased PGE(2) and NO production in an additive manner. When expressed as PFF-treatment-over-control ratio, the response to fluid shear stress was similar in the absence or presence of E2. These results suggest that E2 does not affect the early response to stress in bone cells. Rather, E2 and shear stress both promote the production of paracrine factors such as NO and PGE(2) in an additive manner.

骨細胞間のメカノセンサーネットワークの数理モデル化(OS16c 生体力学における新しい計算解析手法)

骨細胞間のメカノセンサーネットワークの数理モデル化(OS16c 生体力学における新しい計算解析手法)

Age and distance from the surface but not menopause reduce osteocyte density in human cancellous bone

Previous studies of osteocyte density in human cancellous bone have relied mainly on autopsy samples and have demonstrated an age-related decline in men, but there are insufficient data in women. Using previously obtained transiliac bone biopsies from 94 healthy white women, aged 20–73 years, 38 premenopausal and 56 postmenopausal, we measured osteocytes and lacunae in ten randomly selected areas using 5-μm-thick sections stained with Goldner trichrome. For each subject, the number of osteocytes (Ot.N/B.Ar), empty lacunae (EL.N/B.Ar), and total lacunae (Tt.L.N/B.Ar) per bone area, and the proportion of occupied lacunae (Ot.N/Tt.L.N), were calculated. In 92 cases the measurements were made separately in superficial bone (<25 μm from the surface) and in deep bone (>45 μm from the surface). Mean values and differences between extreme values (DEV) for each variable were computed from the ten measured areas. In addition, confocal microscopic examination was performed on 100 μm sections. We found that Ot.N/B.Ar, Tt.L.N/B.Ar, and Ot.N/Tt.L.N decreased, but EL.N/B.Ar increased significantly with age ( p < 0.001). The rates of decline were most rapid initially, falling exponentially with increasing age; the linear regressions for all four variables were significant in premenopausal, but not postmenopausal, women. At all ages, there were significantly more osteocytes in superficial than in deep bone; there was no significant decline with age in superficial bone, but a steeper exponential decline in deep bone than in whole trabeculae. DEV did not change with age for any variable. Confocal images revealed that the morphology of the osteocyte network was heterogeneous in different regions and trabeculae. The trabeculae with lower osteocyte density contained acellular areas, especially in interstitial bone. We conclude: (1) osteocyte density declines with age in women as it does in men; (2) the decline occurs exclusively in deep bone, not in superficial bone, suggesting that it is the age of the bone rather than the age of the subject that is important; (3) the rate of age-related decline falls exponentially with age and is not significant in postmenopausal women alone; (4) except for the differences between superficial and deep bone, the pattern of osteocyte distribution within and between trabeculae was not affected by age or menopause; and (5) the data raise the possibility that one function of remodeling in iliac cancellous bone is to maintain osteocyte viability.

Aging and accumulation of microdamage in canine bone

Fractures associated with minimal trauma are common in aged human beings. However, bone safety margins are better preserved in aged dogs, which are rarely affected with minimal trauma fractures. Although the hierarchical architecture of canine and human compact bone is similar, the precise reasons for this species difference are unclear. Cyclic loading of bone during normal daily activity leads to the formation of microcracks within the tissue matrix of compact bone. Using a standard bulk-staining technique with basic fuchsin, we examined calcified transverse sections of the mid-diaphysis of the canine humerus from dogs of varying ages. We found that the amount of microdamage and porosity increased exponentially with aging, although the increases were relatively small, compared with human bone. Gender (female, ovariohysterectomized female, male, castrated male) did not have a significant effect on the amount of microdamage or porosity in bone. Alterations to the local material properties of bone tissue, or alterations to the local tissue repair responses, may play a role in the accumulation of microdamage in bone with aging. Determination of osteocyte lacunar density (number of osteocyte lacunae per bone area) and activation frequency (number of actively remodeling osteons per bone area per year) indicated that these variables declined exponentially with aging. There also was a trend for bone from dogs with low osteocyte lacunar density to have a higher microcrack density, but not higher porosity. Furthermore, bones with a high activation frequency did not accumulate microcracks or porosity. Taken together, these data suggest that, in canine bone, although a certain minimum number of osteocytes may be essential for an operational network that forms part of the signaling pathways that orchestrate repair of bone microdamage, increases in porosity with aging may not be directly associated with impaired function of the osteocyte network within bone.

Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip.

An increase in osteoblast and osteocyte apoptosis has been demonstrated in mice and humans receiving glucocorticoids and may be involved in the pathogenesis of the associated osteonecrosis. To examine the spatial relationship between osteocyte apoptosis and glucocorticoid-induced osteonecrosis, we determined the prevalence of osteocyte apoptosis in whole femoral heads obtained from patients who underwent prosthetic hip replacement because of osteonecrosis due to chronic glucocorticoid treatment (n = 5), alcoholism (n = 3), and trauma (n = 1) as well as in femoral neck cores from patients with sickle cell disease (n = 5). Abundant apoptotic osteocytes and cells lining cancellous bone were found juxtaposed to the subchondral fracture crescent in femurs from the patients with glucocorticoid excess. In contrast, apoptotic bone cells were absent from the specimens taken from patients with trauma or sickle cell disease and were rare with alcohol abuse. These results indicate that glucocorticoid-induced osteonecrosis is a misnomer. The bone is not necrotic; instead, it shows prominent apoptosis of cancellous lining cells and osteocytes. Glucocorticoid-induced osteocyte apoptosis, a cumulative and irreparable defect, could uniquely disrupt the mechanosensory function of the osteocyte network and thus start the inexorable sequence of events leading to collapse of the femoral head.

Decline in osteocyte lacunar density in human cortical bone is associated with accumulation of microcracks with age

Despite osteocytes’ ideal position to sense the local environment and thereby influence bone remodeling, the function of osteocytes in bone remains controversial. In this study, histomorphometric examination of male and female femoral middiaphyseal cortical bone was conducted to determine if bone’s remodeling response, indicated by tissue porosity and accumulation of damage, is associated with osteocyte lacunar density (number of osteocyte lacunae per bone area). The results support the sensory role of the osteocyte network as the decline in osteocyte lacunar density in human cortical bone is associated with the accumulation of microcracks and increase in porosity with age. Porosity and microcrack density increased exponentially with a decline in osteocyte lacunar density indicating that a certain minimum number of osteocytes is essential for an “operational” network. No gender-related differences were found in the relationship of osteocyte lacunar density to age, porosity, or microcrack density. The coefficient of variation of osteocyte lacunar density increased linearly with age, indicating that aging bone tissue is characterized by increased heterogeneity in the spatial organization of osteocytes. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability density distribution in the donor population, indicating their regulation by similar biological phenomena.

Responses of bone cells to biomechanical forces in vitro.

In this paper, we review recent studies of the mechanism by which mechanical loading of bone is transduced into cellular signals of bone adaptation. Current biomechanical theory and in vivo as well as in vitro experiments agree that the three-dimensional network of osteocytes and bone-lining cells provides the cellular basis for mechanosensing in bone, leading to adaptive bone (re)modeling. They also agree that flow of interstitial fluid through the lacunar-canalicular porosity of bone, as a result of mechanical loading, most likely provides the stimulus for mechanosensing, and informs the bone cellular network about the adequacy of the existing bone structure. Important signaling molecules involved in in vivo adaptive bone formation, as well as in in vitro cellular response to fluid flow, are nitric oxide and prostaglandins. The expression of key enzymes for nitric oxide and prostaglandin production in bone cells is altered by fluid shear stress in vitro. Together, these studies have increased our understanding of the cell biology underlying Wolff's Law. This may lead to new strategies for combating disuse-related osteoporosis, and may also be of use in understanding and predicting the long-term integration of bone-replacing implants.

The role of estrogen in the control of rat osteocyte apoptosis.

We have previously shown that estrogen withdrawal by gonadotrophin-releasing hormone analogs (GnRHa) induces osteocyte death via apoptosis in human bone. Although it is likely that the increase in osteocyte death via apoptosis was related to the loss of estrogen, these experiments could not rule out a direct role for the GnRHa. Therefore, in this study, we have used a rat model of ovariectomy (OVX) to determine whether the effect of estrogen withdrawal extends to other species and to clarify the role of estrogen in the maintenance of osteocyte viability. Twelve 9-week-old rats were divided into three treatment groups: sham operated (SHAM) (n = 4), OVX (n = 4), and OVX + estrogen (E2) (25 microg/day) (n = 4). At 3 weeks following the start of treatment, tibial bones were removed. The percentage of osteocytes displaying DNA breaks, using an in situ nick-translation method, was significantly higher in the OVX group compared with the SHAM control in both cortical bone (10.04% vs. 2.31%, respectively; p < 0.0001) and trabecular bone (6.44% vs. 1.58%, respectively; p = 0.003). Addition of estrogen in the OVX animals completely abrogated the increase in osteocyte apoptosis in cortical bone (0.78%) and trabecular bone (1.17%). The percentage of apoptotic osteocytes decreased with increasing distance from the primary/secondary spongiosa interface below the growth plate in the OVX model and the OVX + E2 model. Nuclear morphology and electrophoresis of DNA confirmed the presence of apoptotic cells in the samples. In conclusion, OVX in the rat results in an increase in osteocyte apoptosis as a direct or indirect result of E2 loss. Addition of estrogen in the OVX animals prevents this increase in osteocyte apoptosis. These data confirm an important role for estrogen in the control of osteocyte apoptosis and the maintenance of osteocyte viability. Estrogen deficiency might, through compromising the viability of osteocyte networks, reduce the ability of bone to respond appropriately to loading.

Trabecular bone volume and microdamage accumulation in the femoral heads of women with and without femoral neck fractures

Repetitive loading causes fragility and microdamage accumulation in the skeleton, but it is not clear whether microcracks accumulate with age in the human skeleton, or whether older women with femoral neck fractures have more microdamage than older women without fracture. This study tested the hypotheses that: (1) microcracks accumulate in the femoral head with age; and (2) older women with femoral neck fractures have significantly more microcracks than similar aged women without fractures. Nonosteoarthritic femoral heads from 9 young (16–66 years) and 12 old (73–88 years) female cadavers and those from 7 females with femoral neck fractures (56–90 years) were dissected. Midfrontal slabs were block stained with 1% basic fuchsin and 150-μm-thick specimens were measured histomorphometrically. Percent trabecular bone area (Tb.Ar) significantly decreased in older and fractured subjects compared with young subjects ( p ≤ 0.05). Microcrack density (Cr.Dn) was significantly higher in women older than 70 years compared with those younger than 70 ( p = 0.005; Spearman's r = 0.41, p = 0.032), but was not different between older women with and without fractures. Osteocyte lacunar density (Ot.Dn) was significantly less in old and fractured subjects compared with young subjects ( p ≤ 0.01), and inversely correlated to crack density ( r = −0.49, p = 0.011). Cr.Dn was higher in women with low Tb.Ar (Spearman's r = −0.56; p = 0.004), and varied inversely with Ot.Dn (Spearman's r = −0.47; p = 0.014). This is consistent with the idea that cracks accumulate more rapidly in women with low bone density. It is also consistent with the idea that the osteocyte network detects microcracks, and signals for their repair.

Surface remodeling of trabecular bone using a tissue level model.

We propose a two-dimensional mathematical model of trabecular bone remodeling that simulates the surface-based addition and removal of material in the actual physiological process. The model is based on a finite element representation of individual trabecular struts in which the material properties of the subtrabecular elements are constant. The remodeling stimulus is strain energy density, sensed and communicated through the osteocytic network as proposed by Mullender et al. We propose a modified osteocyte communication scheme that incorporates bone-lining cells and examines the implications of set point locations in one or the other of these two cell types. This model produces trabecular struts that align with its general loading direction. Placing the set point in the bone-lining cells rather than in the osteocytes makes the model more sensitive to changes in the other biological parameters. Introduction of a dead zone causes the model to reach a less oscillatory equilibrium in fewer iterations and produces better in-filling of trabecular strut intersections. The model gravitates to equilibrium states in which the average strain energy density is inversely proportional to the bone volume fraction to the 3.2 power.

Function of osteocytes in bone.

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction-coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body-containing lacunae with each other and with the outside world. During differentiation from osteoblasts to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are 1) osteocytes are actively involved in bone turnover; 2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and 3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations.

Variation of the Holmes Method for Histologic Staining of Bone Canaliculi

AbstractThe Holmes method for soft tissue staining of nerve fibers was modified to produce excellent detail of the canalicular network in decalcified bone tissue. With simple changes in timing and the meticulous use of fresh reagents, we were able to stain bone lacunae and canaliculi in a variety of animal and human tissues. This technique produced more uniform and accurate results than the thionin/picric acid stains used previously for visualization of osteocytes. The morphologic results can be used for diagnostic purposes as well as stereological studies where the osteocyte network is of importance. (The J Histotechnol 16:355, 1993)

Bone strain sensation via transmembrane potential changes in surface osteoblasts: Loading rate and microstructural implications

A model is developed in which osteoblasts can sense the strains applied to a small region of bone through electrical coupling between adjacent cells. The stress-generated potentials within bone are assumed to occur through streaming potentials, and the coupled network of osteocytes is assumed to act in a manner similar to the classical cable model for nerve cells. In a one-dimensional model, the linear poroelastic equations for motion of the fluid are solved analytically for sinusoidally varying imposed strains, and the streaming potentials are predicted from the fluid flow. The changes in the osteocyte and osteoblast transmembrane potential are given by an analytical solution to the governing equations, and the dependence of the transmembrane potential changes (TPC) on position, loading rate, manner of loading (compression versus bending), and on the degree of cellular coupling is discussed. The model correctly predicts the rate dependence of remodelling established by other investigators. The influence of the electrical parameters within the model indicate that further study of the cellular coupling in bone can yield important new information on bone remodelling.

Osteocytes, strain detection, bone modeling and remodeling.

One of the characteristic features of mammalian and avian bone is a population of live cells of the osteoblast lineage distributed both on the surface and throughout the matrix. These cells communicate with one another via gap junctions. A number of roles have been proposed for both osteocytes and the lacunar/canalicular labyrinth they occupy. These include arrest of fatigue cracks, mineral exchange, osteocytic osteolysis, renewed remodeling activity after release by resorption, stimulation, and guidance of osteoclastic cutting cones involved in mineral exchange and the repair of microdamage, strain detection, and the control of mechanically related bone modeling/remodeling. The question of whether osteocytes control or influence modeling and remodeling is of major importance. Such influence could be crucial in relation to three importance consequences of remodeling activity: calcium regulation, microdamage repair, and mechanically adaptive control of bone architecture. Mechanically adaptive control of bone architecture requires feedback concerning the relationship between current loading and existing architecture. This feedback is most probably derived from the strain in the matrix. The arrangement of the osteocyte network seems ideally suited to both perceive strain throughout the matrix and to influence adaptive modeling and remodeling in a strain-related manner. The hypothesis that osteocytes perform this role has growing experimental support.