Three‐dimensional characterization of osteocyte volumes at multiple scales,and its relationship with bone biology and genome evolution in ray‐finned fishes

Osteocytes, cells embedded within the bone mineral matrix, inform on key aspects of vertebrate biology. In particular, a relationship between volumes of the osteocytes and bone growth and/or genome size has been proposed for several tetrapod lineages. However, the variation in osteocyte volume across different scales is poorly characterized and mostly relies on incomplete, two‐dimensional information. In this study, we characterize the variation of osteocyte volumes in ray‐finned fishes (Actinopterygii), a clade including more than half of modern vertebrate species in which osteocyte biology is poorly known. We use X‐ray synchrotron micro‐computed tomography (SRµCT) to achieve a three‐dimensional visualization of osteocyte lacunae and direct measurement of their size (volumes). Our specimen sample is designed to characterize variation in osteocyte lacuna morphology at three scales: within a bone, among the bones of one individual and among species. At the intra‐bone scale, we find that osteocyte lacunae vary noticeably in size between zones of organized and woven bone (being up to six times larger in woven bone), and across cyclical bone deposition. This is probably explained by differences in bone deposition rate, with larger osteocyte lacunae contained in bone that deposits faster. Osteocyte lacuna volumes vary 3.5‐fold among the bones of an individual, and this cannot readily be explained by variation in bone growth rate or other currently observable factors. Finally, we find that genome size provides the best explanation of variation in osteocyte lacuna volume among species: actinopterygian taxa with larger genomes (polyploid taxa in particular) have larger osteocyte lacunae (with a ninefold variation in median osteocyte volume being measured). Our findings corroborate previous two‐dimensional studies in tetrapods that also observed similar patterns of intra‐individual variation and found a correlation with genome size. This opens new perspectives for further studies on bone evolution, physiology and palaeogenomics in actinopterygians, and vertebrates as a whole.     

树鼩的骨元和骨细胞穴微观结构超微影像的定性比较

近年来,由于树鼩与灵长类动物的亲缘关系,它们引起了人类发展和疾病研究的兴趣。在这项对树鼩,鼠,狗,狒狒和人类的骨骼超微形态的比较研究中,我们定性分析了骨骼的微观结构和形态,以评估树鼩对人类的亲近程度。在3只成年雄性树鼩 (滇西亚种) (Tupaia belangeri chinensis）的股骨中,使用荧光素异硫氰酸盐样品制备和染色共聚焦成像研究了皮质骨的骨元结构,并使用酸蚀刻SEM观察了骨细胞穴的形态。总体而言,树鼩中骨样形成物的密度和结构以及骨细胞穴的形态更像鼠, 与人类,狗和狒狒都明显不同。这些发现表明,尽管树鼩在系统发育上比鼠更接近人类,但它们的骨骼超微形态仍与鼠接近。这是除狗和狒狒之外,第一次对树鼩的骨元和骨细胞穴进行超微影像的研究。这个比较研究的结果丰富了我们对早期灵长类动物骨骼发育,适应性和进化的理解。未来有必要进行进一步的定量比较研究来表征树鼩骨骼的微观形态。     

Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone

Aging decreases the human femur’s fatigue resistance, impact energy absorption, and the ability to withstand load. Changes in the osteocyte distribution and in their elemental composition might be involved in age‐related bone impairment. To address this question, we carried out a histomorphometric assessment of the osteocyte lacunar distribution in the periosteal and endosteal human femoral cortexes of 16 female and 16 male donors with regard to age‐ and sex‐related bone remodeling. Measurements of the bone mineral density distribution by quantitative backscattered electron imaging and energy dispersive X‐ray analysis were taken to evaluate the osteocyte lacunar mineral composition and characteristics. Age‐dependent decreases in the total osteocyte lacunar number were measured in all of the cases. This change signifies a risk for the bone’s safety. Cortical subdivision into periosteal and endosteal regions of interest emphasized that, in both sexes, primarily the endosteal cortex is affected by age‐dependent reduction in number of osteocyte lacunae, whereas the periosteal compartment showed a less pronounced osteocyte lacunar deficiency. In aged bone, osteocyte lacunae showed an increased amount of hypermineralized calcium phosphate occlusions in comparison with younger cases. With respect to Frost’s early delineation of micropetrosis, our microanalyses revealed that the osteocyte lacunae are subject to hypermineralization. Intralacunar hypermineralization accompanied by a decrease in total osteocyte lacunar density may contribute to failure or delayed bone repair in aging bone. A decreased osteocyte lacunar density may cause deteriorations in the canalicular fluid flow and reduce the detection of microdamage, which counteracts the bone’s structural integrity, while hypermineralized osteocyte lacunae may increase bone brittleness and render the bone fragile.     

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.     

Observations of microdamage around osteocyte lacunae in bone

Tensile microdamage was examined using laser scanning confocal microscopy in beam specimens of bovine, equine and human long bones loaded in vitro and whole specimens of rat ulnae loaded in vivo. Microcracks were observed to initiate frequently at osteocyte lacunae. The implication is that osteocyte lacunae act as stress concentrating features in bone. This association provides a potential mechanism for the detection of strain and/ or damage by osteocytes in bone.     

Effects of Nutritional Deficiency of Boron on the Bones of the Appendicular Skeleton of Mice

Scientific evidence has shown the nutritional importance of boron (B) in the remodeling and repair of cancellous bone tissue. However, the effects of the nutritional deficiency of B on the cortical bone tissue of the appendicular skeleton have not yet been described. Thus, a study was performed to histomorphometrically evaluate the density of osteocyte lacunae of cortical bone of mouse femora under conditions of nutritional deficiency of B and to analyze the effects of the deficiency on the biomechanical properties of mouse tibiae. Weaning, 21-day-old male Swiss mice were assigned to the following two groups: controls (B+; n = 10) and experimental (B−; n = 10). Control mice were fed a basal diet containing 3 mg B/kg, whereas experimental mice were fed a B-deficient diet containing 0.07 mg B/kg for 9 weeks. The histological and histomorphometric evaluations of the mice fed a B-deficient diet showed a decrease in the density of osteocyte lacunae in the femoral cortical bone tissue and the evaluation of biomechanical properties showed lower bone rigidity in the tibia.

     

Osteocyte lacuna size and shape in women with and without osteoporotic fracture

Osteocytes have been hypothesized to control the amount and location of bone tissue which is resorbed or formed, based on the strain magnitude they perceive, and therefore may play a role in the bone loss of osteoporosis. The shape of osteocyte lacunae influences the mechanical strain applied to the osteocyte; thus, it is important to quantify their shape to further understand the mechanical environment of this cell. Previous studies of the size and shape of lacunae have been contradictory and limited to two-dimensional measurements on iliac crest biopsies. This investigation measured the size and shape of osteocyte lacunae in trabecular bone near a typical fracture site from three-dimensional image sets obtained by confocal microscopy. Bone tissue specimens were obtained from individuals undergoing hip replacement subsequent to fracture, and matched cadaveric specimens without fracture. After extensive image processing to differentiate the lacunae from the matrix, the volume and anisotropy of the lacuna were determined. No significant difference was found in the size (volume) or shape (anisotropy) of the lacunae between women with and without osteoporotic fracture, although there was a large range of sizes and shapes in both groups. These results suggest that the size or shape of the lacunae, which influences the strain in osteocytes, does not play a role in osteoporotic fracture. In addition, this study provides geometric measures of lacunae that are important in computational modeling of the mechanical environment of osteocytes.     

Synchrotron ultraviolet microspectroscopy on rat cortical bone: involvement of tyrosine and tryptophan in the osteocyte and its environment

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately.     

Metabolic pathways of the fossil dinosaur bones. Part V. Morphological differentiation of osteocyte lacunae and bone canaliculi and their significance in the system of extracellular communication

The study was carried out on dinosaur bones nearly 80 million years old. Samples for examination were prepared with specially elaborated methods. The light and transmission electron microscopic images permitted two kinds of bone lacunae and two types of paralacunar canalicular endings to be distinguished. The lacunae of the first kind were characterized by their elongated shape, their length exceeding their width several times, their dimensions being 31.2/9.4 microns. The lacunae of the other kind were not so long, their mean measurements amounting to 21.32/9.7 microns. Among the paralacunar canalicular endings those of small diameter were more numerous. The canaliculi of wider, funnel-shaped endings amounted to two or three, they were usually localized in the polar part of the lacuna, and were defined as the axial canaliculi. These were canaliculi of a large diameter. The canalicular wall was constructed of collagen fibres. The same fibres were found in the lacunar wall. Also a relationship between the structure of the lacunar wall and the localization of an osteocyte in the lacuna was analysed in the light and electron microscopes. In regard to the structure of the bone lacuna and the localization of an osteocyte in it, zones A and B were distinguished. Zone A had a characteristic loose and disorderly system of collagen fibres building the lacunar wall. The fibres in this area were by nature open to view. Besides, this region of the lacunar wall revealed specific terraced hollows. Zone B was distinguished by a compact system of parallelly arranged collagen fibres, which formed characteristic ridges in the lacunar wall. The localization of the osteocyte in the lacuna was irregular, the pericellular space around it being of variable width. This space was shown to contain mucopolysaccharides. The images obtained from dinosaur bone were compared with those already known for modern bone. These comparisons permitted it to be ascertained that zone A corresponds to a spot in the lacuna in which the osteocyte exhibits a decreased activity. Zone B is the area of the actual direction of the osteocyte's activity aiming at the shaping of the wall of its lacuna. It can be supposed that the widened endings of the paralacunar canaliculi perform more important functions in conveyance, this being evident from comparisons of analogous areas in modern bone.     

Homogenization theory and digital imaging: A basis for studying the mechanics and design principles of bone tissue

Bone tissue is a complex multilevel composite which has the ability to sense ad respond to its mechanical environment. It is believed that bone cells called osteocytes within the bone matrix sense the mechanical environment and determine whether structural alterations are needed. At present it is not known, however, how loads are transferred from the whole bone level to cells. A computational procedure combining representative volume element (RVE) based homogenization theory with digital imaging is proposed to estimate strains at various levels of bone structure. Bone tissue structural organization and RVE based analysis are briefly reviewed. The digital image based computational procedure was applied to estimate strains in individual trabeculae (first-level microstructure). Homogenization analysis of an idealized model was used to estimate strains at one level of bone structure around osteocyte lacunae (second-level trabecular microstructure). The results showed that strain at one level of bone structure is amplified to a broad range at the next microstructural level. In one case, a zeor-level tensile principal strain of 495 muE engendered strains ranging between -1000 and 7000 muE in individual trabeculae (first-level microstructure). Subsequently, a first-level tensile principal strains of 1325 muE within an inidividual trabecula engendered strains ranging between 782 and 2530 muE around osteocyte lacunae. Lacunar orientation was found to influence strains around osteocyte lacunae much more than lacunar ellipticity. In conclusion, the computational procedure combining homogenization theory with digital imaging can proveide estimates of cell level strains within whole bones. Such results may be used to bridge experimental studies of bone adaptation at the whole bone and cell culture level. (c) 1994 John Wiley & Sons, Inc.     

The RIP1–RIP3 Complex Mediates Osteocyte Necroptosis after Ovariectomy in Rats

Osteocyte apoptosis has been reported to play a central role in bone remodeling. In addition to apoptosis, other mechanisms may be involved in osteocyte loss. This study aimed to investigate the effect of necroptosis on osteocytes in ovariectomized (OVX) rats. Ninety-six female Sprague-Dawley rats were randomly divided into an OVX group and a sham group. At 0, 4, 8 and 12 weeks after surgery, specimens from each group (n = 12 each) were harvested. Bone mineral density (BMD) and body weight were measured. Transmission electron microscopy (TEM) and micro-CT were used to observe the changes in cellular morphology and bone microarchitecture induced by estrogen deficiency. Osteocyte apoptosis and necroptosis were evaluated via TUNEL and immunofluorescence staining for active caspase-3. At 8 weeks after ovariectomy, a greater number of osteocytes with typical necrotic morphological features were TUNEL positive but negative for active caspase-3. Western blotting, quantitative real-time PCR and immunofluorescence assessments demonstrated that the levels of receptor-interacting serine/threonine protein kinase 1 (RIP1) and RIP3 in osteocytes were significantly increased at 8 weeks after ovariectomy. These data are the first to suggest that necroptosis accelerates osteocyte loss under conditions of estrogen deficiency-induced osteoporosis in OVX rats. These findings provide evidence of a potential mechanism through which osteocyte necroptosis is associated with postmenopausal osteoporosis.     

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.     

GeneChip Expression Profiling Reveals the Alterations of Energy Metabolism Related Genes in Osteocytes under Large Gradient High Magnetic Fields

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has recently been applied in life science research. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. Osteocyte, as the most important mechanosensor in bone, takes a pivotal position in mediating the mechano-induced bone remodeling. In this study, the effects of LG-HMF on gene expression profiling of osteocyte-like cell line MLO-Y4 were investigated by Affymetrix DNA microarray. LG-HMF affected osteocyte gene expression profiling. Differentially expressed genes (DEGs) and data mining were further analyzed by using bioinfomatic tools, such as DAVID, iReport. 12 energy metabolism related genes (PFKL, AK4, ALDOC, COX7A1, STC1, ADM, CA9, CA12, P4HA1, APLN, GPR35 and GPR84) were further confirmed by real-time PCR. An integrated gene interaction network of 12 DEGs was constructed. Bio-data mining showed that genes involved in glucose metabolic process and apoptosis changed notablly. Our results demostrated that LG-HMF affected the expression of energy metabolism related genes in osteocyte. The identification of sensitive genes to special environments may provide some potential targets for preventing and treating bone loss or osteoporosis.     

Alcohol suppresses the granulopoietic response to pulmonary Streptococcus pneumoniae infection with enhancement of STAT3 signaling

Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. G-CSF produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27(Kip1) pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the cytochrome P450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27(Kip1) signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.     

Expression of the type 1 lysophosphatidic acid receptor in osteoblastic cell lineage controls both bone mineralization and osteocyte specification

Lysphosphatidic acid (LPA) is a major natural bioactive lipid mediator whose biological functions affect multiple organs. These include bone as demonstrated by global Lpar1-knockout mice (Lpar1^−/−) which present a bone growth defect. LPA acts on all bone cells including osteoblasts, that are responsible for bone formation, and osteoclasts, which are specialized cells that resorb bone. LPA appears as a potential new coupling molecule during bone remodeling. LPA₁ is the most ubiquitous LPA receptor among the six LPA receptor family members (LPA_1–6). To better understand the specific role of LPA via its receptor LPA₁ in osteoblastic cell lineage we generated osteoblast-specific Lpar1 knockout mice (Lpar1-∆Ob) by crossing Lpar1^flox/flox and Osx:Cre⁺ mouse lines. Lpar1-∆Ob mice do not recapitulate the bone defects of Lpar1^−/− mice but revealed reduced bone mineralization and decreased cortical thickness, as well as increased bone porosity associated with an augmentation in the lacunae areas of osteocyte and their apoptotic yield. In vitro, primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1^−/− osteoblasts revealed a remarkable premature expression of alkaline phosphatase, reduced cell proliferation associated with decreased YAP-P nuclear accumulation, and reduced mineralization activity. Osteocyte specification is markedly impaired as demonstrated by reduced expression of early (E11) and late (DMP1, DKK1, SOST) osteocyte markers ex vivo in enriched osteocytic fractions of Lpar1-∆Ob mouse bone explants. In addition, E11 expression and dendrite formation induced by FGF2 are markedly impaired in both primary Lpar1-∆Ob and immortalized cl1-Ob-Lpar1^−/− osteoblasts. Taken together these results suggest a new role for LPA in bone mass control via bone mineralization and osteocyte function.     

Variation of osteocyte lacunae size within the tetrapod skeleton: implications for palaeogenomics

Recent studies have emphasized the ability to reconstruct genome sizes (C-values) of extinct organisms such as dinosaurs, using correlations between known genome sizes and bone cell (osteocyte lacunae) volumes. Because of the established positive relationship between cell size and genome size in extant vertebrates, osteocyte lacunae volume is a viable proxy for reconstructing C-values in the absence of any viable genetic material. However, intra-skeletal osteocyte lacunae size variation, which could cause error in genome size estimation, has remained unexplored. Here, 11 skeletal elements of one individual from each of four major clades (Mammalia, Amphibia, Aves, Reptilia) were examined histologically. Skeletal elements in all four clades exhibit significant differences in the average sizes of their lacunae. This variation, however, generally does not cause a significant difference in the estimated genome size when common phylogenetic estimation methods are employed. On the other hand, the spread of the estimations illustrates that this method may not be precise. High variance in genome size estimations remains an outstanding problem. Additionally, a suite of new methods is introduced to further automate the measurement of bone cells and other microstructural features on histological thin sections.     

Mapping of QTLs for Oral Alcohol Self-Administration in B6.C and B6.I Quasi-Congenic RQI Strains

One strategy to identify neurochemical pathways of addiction is to map the relevant genes. In the present study we used 43 B6.C and 35 B6.I inbred RQI mouse strains, carrying <3% donor genome on C57BL/6ByJ background, for gene mapping. The strains were phenotyped for consumption of alcohol (12% v/v) in a two-bottle-choice paradigm, and genotyped for 396 microsatellite markers. The current mapping study extends our earlier experiment scanning five mouse chromosomes (Vadasz et al. (2000) Scanning of five chromosomes for alcohol consumption loci. Alcohol 22:25–34) to a whole-genome study, and discusses the differences and limitations. Data were analyzed with composite interval (CIM) and multiple interval (MIM) QTL mapping methods. CIM of B6.C strains detected significant QTLs on chrs. 6 and 12. A suggestive, but not significant, locus was detected in the B6.I strains on chr. 12. The best MIM model for B6.C strains confirmed one QTL on chr. 6 and one QTL on chr. 12, while the MIM model for the B6.I strains confirmed the suggestive locus on chr. 12. Some of the QTLs for alcohol consumption are new, while others confirm previously reported QTLs for alcohol preference, and alcohol acceptance.     

Estimation of bone matrix apparent stiffness variation caused by osteocyte lacunar size and density

The role of osteocyte lacunar size and density on the apparent stiffness of bone matrix was predicted using a mechanical model from the literature. Lacunar size and lacunar density for different bones from different gender and age groups were used to predict the range of matrix apparent stiffness values for human cortical and cancellous tissue. The results suggest that bone matrix apparent stiffness depends on tissue type (cortical versus cancellous), age, and gender, the magnitudes of the effects being significant but small in all cases. Males had a higher predicted matrix apparent stiffness than females for vertebral cancellous bone (p< I0(-7)) and the difference increased with age (p =0.0007). In contrast, matrix apparent stiffness was not different between males and females forfemoral cortical bone and increased with age in both males (p < 0.0001) and females (p < 0.0364). Osteocyte lacunar density and size may cause significant gender and age-related variations in bone matrix apparent stiffness. The magnitude of variations in matrix apparent stiffness was small within the physiological range of lacunar size and density for healthy bone, whereas the variations can be profound in certain pathological cases. It was proposed that the mechanical effects of osteocyte density be uncoupled from their biological effects by controlling lacunar size in normal bone.     

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.     

Serotonin acts as a novel regulator of interleukin-6 secretion in osteocytes through the activation of the 5-HT2B receptor and the ERK1/2 signalling pathway

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteocyte secretion of IL-6 plays an important role in bone metabolism. Serotonin (5-HT) has recently been reported to regulate bone metabolism. The aim of this study was to evaluate the effect of serotonin on osteocyte expression of IL-6. The requirement for the 5-HT receptor(s) and the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) in serotonin-induced IL-6 synthesis were examined. In this study, real-time PCR and ELISA were used to analyse IL-6 gene and protein expression in serotonin-stimulated MLO-Y4 cells. ERK1/2 pathway activation was determined by Western blot. We found that serotonin significantly activated the ERK1/2 pathway and induced IL-6 mRNA expression and protein synthesis in cultured MLO-Y4 cells. However, these effects were abolished by pre-treatment of MLO-Y4 cells with a 5-HT_2B receptor antagonist, RS127445 or the ERK1/2 inhibitor, PD98059. Our results indicate that serotonin stimulates osteocyte secretion of IL-6 and that this effect is associated with activation of 5-HT_2B receptor and the ERK1/2 pathway. These findings provide support for a role of serotonin in bone metabolism by indicating serotonin regulates bone remodelling by mediating an inflammatory cytokine.