Osteocyte Cell Research Articles

In vitro Experiments of Magnetic Field in Human Exposures of Intermediate Frequency

Electromagnetic safety standards are continually updated according to the latest and accumulating scientific pieces of evidence in cell studies, in order to confirm the exact impacts of Electromagnetic Field (EMF) on in-vitro cellular level. Various biological experiments, of both in-vivo and in-vitro, have been conducted for the investigation of the potential effects of EMF, however, these investigations are mostly focused either on the power frequency or microwave EM exposures aiming at the impact on osteocyte or cancer cells. This paper presents an investigation of the bio-electromagnetic impact of the cell viability to HT1080 and UR61 cells and the DNA integrity to the HT1080 cell, under an acute magnetic field of intermediate frequencies (IF) at 100 kHz. IF frequencies are commonly adopted in the wireless power transfer in E-vehicle applications in recent years; this study aims to provide the missing evidence of in-vitro experiments of Magnetic Field in Human Exposures of IF. The impact of the cell viability variation was investigated by MTT assay, while the DNA integrity was investigated by Comet assay. Three identical repeated experiments of prolonging exposure of 6-hours magnetic field of 21A/m, at 100 kHz were carried out for DNA fragmentation; results have indicated that exposure level which is the reference level for the general public of ICNIRP of 21A/m for HT1080, would barely cause any impact on DNA integrity. Three identical repeated experiments of prolonging exposure of 24-hours at the same level of exposure for cell viability investigation, results have indicated that there is a significant statistical difference of cell viability observed in both HT1080 and UR61 cells. The cell viability outcomes were concluded by t-test for a p-value of less than 0.05.

Phenotype and Viability of MLO-Y4 Cells Is Maintained by TGFβ₃ in a Serum-Dependent Manner within a 3D-Co-Culture with MG-63 Cells.

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFβ3. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ3 maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFβ3 on co-cultured cells were serum-dependent. Also, TGFβ3 reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFβ3 maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.

Histology of the endothermic opah (Lampris sp.) suggests a new structure-function relationship in teleost fish bone.

Endothermy, production and retention of heat by the body, appeared convergently in mammals, birds and four spiny-rayed teleost fish lineages. Of these, red-muscle endothermy over most or all of the body has only appeared in two groups: tunas and the opah (Lampris). Hitherto, tunas have been the only spiny-rayed fishes known to have bones containing embedded osteocyte cells; others have acellular bone. We examined bone histology in Lampris for the first time, demonstrating the presence of cellular bone very similar to that of tunas. This contrasts with the acellular condition of its ectothermic close relatives. The distribution of this character suggests that it co-evolved with red-muscle endothermy, hinting at a common physiological mechanism that would link bone histology to endothermy in these distantly related teleost lineages.

Functional and structural characterization of osteocytic MLO-Y4 cell proteins encoded by genes differentially expressed in response to mechanical signals in vitro

The anabolic response of bone to mechanical load is partially the result of osteocyte response to fluid flow-induced shear stress. Understanding signaling pathways activated in osteocytes exposed to fluid flow could identify novel signaling pathways involved in the response of bone to mechanical load. Bioinformatics allows for a unique perspective and provides key first steps in understanding these signaling pathways. We examined proteins encoded by genes differentially expressed in response to fluid flow in murine osteocytic MLO-Y4 cells. We considered structural and functional characteristics including putative intrinsic disorder, evolutionary conservation, interconnectedness in protein-protein interaction networks, and cellular localization. Our analysis suggests that proteins encoded by fluid flow activated genes have lower than expected conservation, are depleted in intrinsic disorder, maintain typical levels of connectivity for the murine proteome, and are found in the cytoplasm and extracellular space. Pathway analyses reveal that these proteins are associated with cellular response to stress, chemokine and cytokine activity, enzyme binding, and osteoclast differentiation. The lower than expected disorder of proteins encoded by flow activated genes suggests they are relatively specialized.

93 - Chemokine MCP-1 feedback loop between adipocytes and mesenchymal stromal cells causes unremitting fat accumulation and contributes to osteocyte and hematopoietic stem cell rarefaction in the bone marrow of diabetic patients

93 - Chemokine MCP-1 feedback loop between adipocytes and mesenchymal stromal cells causes unremitting fat accumulation and contributes to osteocyte and hematopoietic stem cell rarefaction in the bone marrow of diabetic patients

Finite Element Analysis of Osteocytes Mechanosensitivity Under Simulated Microgravity

It was found that the mechanosensitivity of osteocytes could be altered under simulated microgravity. However, how the mechanical stimuli as the biomechanical origins cause the bioresponse in osteocytes under microgravity is unclear yet. Computational studies may help us to explore the mechanical deformation changes of osteocytes under microgravity. Here in this paper, we intend to use the computational simulation to investigate the mechanical behavior of osteocytes under simulated microgravity. In order to obtain the shape information of osteocytes, the biological experiment was conducted under simulated microgravity prior to the numerical simulation The cells were rotated by a clinostat for 6 hours or 5 days and fixed, the cytoskeleton and the nucleus were immunofluorescence stained and scanned, and the cell shape and the fluorescent intensity were measured from fluorescent images to get the dimension information of osteocytes The 3D finite element (FE) cell models were then established based on the scanned image stacks. Several components such as the actin cortex, the cytoplasm, the nucleus, the cytoskeleton of F-actin and microtubules were considered in the model. The cell models in both 6 hours and 5 days groups were then imposed by three magnitudes (0.5, 10 and 15 Pa) of simulating fluid shear stress, with cell total displacement and the internal discrete components deformation calculated. The results showed that under the simulated microgravity: (1) the nuclear area and height statistically significantly increased, which made the ratio of membrane-cortex height to nucleus height statistically significantly decreased; (2) the fluid shear stress-induced maximum displacements and average displacements in the whole cell decreased, with the deformation decreasing amplitude was largest when exposed to 1.5Pa of fluid shear stress; (3) the fluid shear stress-induced deformation of cell membrane-cortex and cytoskeleton decreased, while the fluid shear stress-induced deformation of nucleus increased. The results suggested the mechanical behavior of whole osteocyte cell body was suppressed by simulated microgravity, and this decrement was enlarged with either the increasing amplitude of fluid shear stress or the duration of simulated microgravity. What’s more, the mechanical behavior of membrane-cortex and cytoskeleton was suppressed by the simulated microgravity, which indicated the mechanotransduction process in the cell body may be further inhibited. On the contrary, the cell nucleus deformation increased under simulated microgravity, which may be related to either the decreased amount of cytoskeleton or the increased volume occupied proportion of nucleus in whole cell under the simulated microgravity. The numerical results supported our previous biological experiments, and showed particularly affected cellular components under the simulated microgravity. The computational study here may help us to better understand the mechanism of mechanosensitivity changes in osteocytes under simulated microgravity, and further to explore the mechanism of the bone loss in space flight.

Attraction and Compaction of Migratory Breast Cancer Cells by Bone Matrix Proteins through Tumor-Osteocyte Interactions

Bone is a frequent site of metastasis from breast cancer. To understand the potential role of osteocytes in bone metastasis, we investigated tumor-osteocyte interactions using two cell lines derived from the MDA-MB-231 breast cancer cells, primary breast cancer cells, and MLO-A5/MLO-Y4 osteocyte cells. When three-dimensional (3D) tumor spheroids were grown with osteocyte spheroids, tumor spheroids fused with osteocyte spheroids and shrank. This size reduction was also observed when tumor spheroids were exposed to conditioned medium isolated from osteocyte cells. Mass spectrometry-based analysis predicted that several bone matrix proteins (e.g., collagen, biglycan) in conditioned medium could be responsible for tumor shrinkage. The osteocyte-driven shrinkage was mimicked by type I collagen, the most abundant organic component in bone, but not by hydroxyapatite, a major inorganic component in bone. RNA and protein expression analysis revealed that tumor-osteocyte interactions downregulated Snail, a transcription factor involved in epithelial-to-mesenchymal transition (EMT). An agarose bead assay showed that bone matrix proteins act as a tumor attractant. Collectively, the study herein demonstrates that osteocytes attract and compact migratory breast cancer cells through bone matrix proteins, suppress tumor migration, by Snail downregulation, and promote subsequent metastatic colonization.

Up-regulated MiR-27-3p promotes the G1-S phase transition by targeting inhibitor of growth family member 5 in osteosarcoma

ObjectiveMicroRNAs (miRNAs) play an essential role in regulating malignant progression of tumour cells by inhibiting translation or stability of messenger RNA. However, the expression pattern and regulatory mechanism of miR-27-3p in osteosarcoma remains unclear. MethodsWe examined the expression of miR-27-3p in 5 osteosarcoma cell lines compared with that in 2 normal osteocyte cell lines. Osteosarcoma cells U-2OS and MG-63 were transduced to up-regulate or down-regulate the expression of miR-27-3p. The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide, or MTT, assay, colony formation assays, BrdUrd labelling, immunofluorescence, anchorage-independent growth ability assay and flow cytometry analysis were used to test the effect of miR-27-3p. Luciferase assays were added to verify the direct relationship between miR-27-3p and the predicted target gene inhibitor of growth family member 5 (ING5). ResultsThe expression of miR-27-3p was significantly increased in examined osteosarcoma cell lines compared with that in normal osteocyte cell lines. Up-regulation of miR-27-3p significantly accelerated osteosarcoma cell growth via promoting G1-S transition. In addition, the opposite result was observed in miR-27-3p-down-regulated cells. Up-regulation of ING5 significantly attenuated the miR-27-3p-induced proliferation in osteosarcoma cells. ConclusionsThese data suggested that miR-27-3p could promote the G1-S phase transition that leads to proliferation by down-regulating the expression of ING5 in osteosarcoma.

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor

Exercise has beneficial effects on metabolism and ontissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of abone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). l-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

RETRACTED: Alpinumisoflavone protects against glucocorticoid-induced osteoporosis through suppressing the apoptosis of osteoblastic and osteocytic cells

The long-term use of glucocorticoids is found to cause osteoporosis. This study is designed to evaluate the protective effect of alpinumisoflavone (AIF), a naturally occurring flavonoid compound, on dexamethasone(Dex)-induced osteoporosis. We use a rat model to investigate the apoptosis of osteoblastic and osteocytic cells. The results indicate that AIF effectively protects against dexamethasone-induced osteoporosis. Moreover, AIF effectively reversed dexamethasone-induced apoptosis in osteoblastic and osteocytic cells through inhibiting ROS overproduction and regulating the Nrf2 pathway. In conclusion, the AIF activated Nrf2 signaling pathway was observed to suppress Dex-induced ROS production in osteoblastic and osteocytic cells, which may explain its anti-osteoporotic effects against dexamethasone-induced osteoporosis.

Potential role for a specialized β3 integrin-based structure on osteocyte processes in bone mechanosensation.

Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αV β3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αV β3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to β3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2 × 7R and the low voltage transiently opened T-type calcium channel CaV3.2-1 all reside in close proximity to β3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:642-652, 2018.

TIEG and estrogen modulate SOST expression in the murine skeleton.

TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone.

A Novel Distal Enhancer Mediates Inflammation-, PTH-, and Early Onset Murine Kidney Disease-Induced Expression of the Mouse Fgf23 Gene.

ABSTRACTFibroblast growth factor 23 (FGF23) production is regulated by both calciotropic hormones and inflammation. Consistent with this, elevated FGF23 levels are associated with inflammatory markers as well as parathyroid hormone (PTH) in various disease states, including chronic kidney disease (CKD). However, the molecular mechanisms underpinning Fgf23 transcription in response to these regulators are largely unknown. We therefore utilized chromatin immunoprecipitation followed by DNA sequencing (ChIP‐seq) data from an osteocyte cell line to identify potential regulatory regions of the Fgf23 gene. Based on ChIP‐seq analysis of enhancer‐associated histone modifications, including H3K4 methylation and H3K9 acetylation, we discovered several potential enhancers for Fgf23, one of which was located 16kb upstream of the gene's transcriptional start site. Deletion of this putative enhancer from the mouse genome using CRISPR‐Cas9 technology led to lower bone, thymus, and spleen expression of Fgf23 mRNA without altering circulating levels of the intact hormone, although as previously reported, only bone displayed significant basal expression. Nevertheless, lack of the −16kb enhancer blunted FGF23 upregulation in a tissue‐specific manner by the acute inflammatory inducers lipopolysaccharide (LPS), interleukin‐1‐beta (IL‐1β), and tumor necrosis factor‐alpha (TNFα) in bone, non‐osseous tissues, and in circulation. Lack of the −16kb enhancer also inhibited PTH‐induced bone Fgf23 mRNA. Moreover, the absence of this Fgf23 enhancer in an oxalate diet‐induced murine CKD model prevented the early onset induction of osseous, renal, and thymic Fgf23 mRNA levels and led to a significant blunting of elevated circulating intact FGF23 levels. These results suggest that −16kb enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD. As exemplified herein, these Fgf23 enhancer‐deleted mice will provide a unique model in which to study the role of FGF23 expression in inflammatory diseases. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Osteopontin as a novel substrate for the proprotein convertase 5/6 (PCSK5) in bone

Seven proprotein convertases cleave the basic amino acid consensus sequence K/R-Xn-K/R↓ (where n=0, 2, 4 or 6 variable amino acids) to activate precursor proteins. Despite similarities in substrate specificity, basic amino acid-specific proprotein convertases have a distinct tissue distribution allowing for enzymatic actions on tissue-resident substrates. Proprotein convertase 5/6 (PC5/6) has two splice variants – soluble PC5/6A and membrane-bound PC5/6B – and is expressed during mouse development in many tissues including bone and tooth, but little is known about the substrates for PC5/6 therein. Osteopontin (OPN) is an abundant bone extracellular matrix protein with roles in mineralization, cell adhesion and cell migration, and it has putative consensus sequence sites for cleavage by PC5/6, which may modify its function in bone. Since PC5/6-knockout mouse embryos show developmental abnormalities, and reduced overall mineralization, we designed this study to determine whether OPN is a substrate of PC5/6. In silico analysis of OPN protein sequences identified four potential PC5/6 consensus cleavage sites in human OPN, and three sites – including a noncanonical sequence – in mouse OPN. Ex vivo co-transfections with human OPN revealed complete OPN cleavage reducing full-length OPN (~70kDa) to an N-terminal fragment migrating at ~50kDa and two C-terminal fragments at ~18kDa and ~16kDa. Direct cleavage of OPN by PC5/6A – the predominant isoform expressed in human osteoblast cells – was confirmed by cell-free enzyme-substrate assays and by mass spectrometry. The latter was also used to investigate potential cleavage sites. Co-transfections of PC5/6 and mouse OPN showed partial cleavage of OPN into a C-terminal OPN fragment migrating at ~30kDa and an N-terminal fragment migrating at ~29kDa. Micro-computed tomography of PC5/6-knockout embryos at E18.5 confirmed a reduction in mineralized bone, and in situ hybridization performed on cryo-sections of normal mouse bone using Pcsk5 and Opn anti-sense and control-sense cRNA probes indicated the co-localization of the expression of these genes in bone cells. This mRNA expression profile was supported by semi-quantitative RT-PCR using osteoblast primary cultures, and cultured MC3T3-E1 osteoblast and MLO-Y4 osteocyte cell lines. Immunoblotting for OPN from mouse bone extracts showed altered OPN processing in PC5/6-knockout mice compared to wildtype mice. OPN fragments migrated at ~25kDa and ~16kDa in wildtype bone and were not present in PC5/6-deficient bone. In conclusion, this study demonstrates that Pcsk5 is expressed in bone-forming cells, and that OPN is a novel substrate for PC5/6. Cleavage of OPN by PC5/6 may modify the function of OPN in bone and/or modulate other enzymatic cleavages of OPN, leading to alterations in the bone phenotype.

The Proteasome and Myeloma-Associated Bone Disease.

Bone disease is the hallmark of multiple myeloma (MM), a hematological malignancy characterized by osteolytic lesions due to a severe uncoupled and unbalanced bone remodeling with pronounced osteoblast suppression. Bone metastasis is also a frequent complication of solid tumors including advanced breast or prostate cancer. In the past years, the ubiquitin-proteasome pathway has been proved critical in regulating the balance between bone formation and bone resorption. Proteasome inhibitors (PIs) are a new class of drugs, currently used in the treatment of MM, that affect both tumor cells and bone microenvironment. Particularly, PIs stimulate osteoblast differentiation by human mesenchymal stromal cells and increase bone regeneration in mice. Interestingly, in vitro data indicate that PIs block MM-induced osteoblast and osteocyte cell death by targeting both apoptosis and autophagy. The preclinical data are supported by the following effects observed in MM patients treated with PIs: increase of bone alkaline phosphatase levels, normalization of the markers of bone turnover, and reduction of the skeletal-related events. Moreover, the histomorphometric data indicate that the treatment with bortezomib stimulates osteoblast formation and maintains osteocyte viability in MM patients. This review updates the evidence on the effects of PIs on bone remodeling and on cancer-induced bone disease while focusing on MM bone disease.

Degeneration of the osteocyte network in the C57BL/6 mouse model of aging.

Age-related bone loss and associated fracture risk are major problems in musculoskeletal health. Osteocytes have emerged as key regulators of bone mass and as a therapeutic target for preventing bone loss. As aging is associated with changes in the osteocyte lacunocanalicular system, we focused on the responsible cellular mechanisms in osteocytes. Bone phenotypic analysis was performed in young-(5mo) and aged-(22mo) C57BL/6 mice and changes in bone structure/geometry correlated with alterations in osteocyte parameters determined using novel multiplexed-3D-confocal imaging techniques. Age-related bone changes analogous to those in humans were observed, including increased cortical diameter, decreased cortical thickness, reduced trabecular BV/TV and cortical porosities. This was associated with a dramatic reduction in osteocyte dendrite number and cell density, particularly in females, where osteocyte dendricity decreased linearly from 5, 12, 18 to 22mo and correlated significantly with cortical bone parameters. Reduced dendricity preceded decreased osteocyte number, suggesting dendrite loss may trigger loss of viability. Age-related degeneration of osteocyte networks may impair bone anabolic responses to loading and gender differences in osteocyte cell body and lacunar fluid volumes we observed in aged mice may lead to gender-related differences in mechanosensitivity. Therapies to preserve osteocyte dendricity and viability may be beneficial for bone health in aging.

Increased sphingosine-1-phosphate production in response to osteocyte mechanotransduction

Over the past few years interest has greatly increased in how the lipid mediator sphingosine-1-phosphate (S1P) influences bone homeostasis. Recent work has postulated multiple effects of S1P on osteoblasts and osteoclasts. Based on these findings, S1P has been proposed as a potential osteoporosis treatment. However, to date, there has been only a single study investigating S1P signalling in the cells that co-ordinate bone metabolism: osteocytes. This study aimed to elucidate the role of S1P signalling in osteocyte mechanotransduction.Utilising 3D cell culture we established the expression profile of all genes related to the S1P signalling system in the Ocy454 osteocyte cell line. Exposure to mechanical loading resulted in a downregulation in Sost, Spns2, the S1P transporter, Sgpl1 and Sgppl1 the enzymes responsible for degradation and dephosphorylation of S1P. These findings, in conjunction with fluid-flow induced upregulation of Sphk1, the kinase responsible for phosphorylation of sphingosine, suggest that mechanical stimulation of osteocytes leads to an increase in intracellular S1P. This was confirmed with mechanical loading of Ocy454 cells rapidly increasing S1P production in conditioned media and protein lysates. These findings strongly suggest an important role for S1P in the response to mechanical loading of bone.

Comparative Histomorphometric Evaluation of Healthy and Ankylosed Mandibular Condylar Process.

Temporomandibular joint (TMJ) ankylosis is one of the most disruptive maladies afflicting the masticatory system. The characteristic feature is the formation of bony mass bridging condyle with glenoid fossa. The exact pathogenesis is, however, not completely understood. To investigate and compare histomorphometric features of ankylosed condylar specimen with normal condylar process. Group I included 17 post-traumatic unilateral TMJ ankylosis patients managed by excision of ankylosed mass and interpositional arthroplasty. Group II included 13 condylar head fracture patients managed by surgical debridement. The bony specimens of both the groups were subjected to histomorphometric examination for assessment of percentage of bone in trabeculae area (%BONE), osteocyte cell density (OSTCD), the presence of inflammation and fibrosis. The mean %BONE, OSTCD, %inflammation, %fibrosis was 60.4%, 340.9mm2, 52.9 and 58.8% in group I and 29.6%, 202.6mm2, 31 and 0% in group II. %BONE, OSTCD and fibrosis in cases of TMJ ankylosis were significantly higher than the controls while no significant difference was observed in the presence of inflammation. The persistence of joint inflammation following condylar head fracture causes aggressive reparative process leading to ankylosis.

Glucose uptake inhibition decreases expressions of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteocalcin in osteocytic MLO-Y4-A2 cells.

Bone and glucose metabolism are closely associated with each other. Both osteoblast and osteoclast functions are important for the action of osteocalcin, which plays pivotal roles as an endocrine hormone regulating glucose metabolism. However, it is unknown whether osteocytes are involved in the interaction between bone and glucose metabolism. We used MLO-Y4-A2, a murine long bone-derived osteocytic cell line, to investigate effects of glucose uptake inhibition on expressions of osteocalcin and bone-remodeling modulators in osteocytes. We found that glucose transporter 1 (GLUT1) is expressed in MLO-Y4-A2 cells and that treatment with phloretin, a GLUT inhibitor, significantly inhibited glucose uptake. Real-time PCR and Western blot showed that phloretin significantly and dose-dependently decreased the expressions of RANKL and osteocalcin, whereas osteoprotegerin or sclerostin was not affected. Moreover, phloretin activated AMP-activated protein kinase (AMPK), an intracellular energy sensor. Coincubation of ara-A, an AMPK inhibitor, with phloretin canceled the phloretin-induced decrease in osteocalcin expression, but not RANKL. In contrast, phloretin suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, and treatments with the p38 inhibitor SB203580 and the MEK inhibitor PD98059, but not the JNK inhibitor SP600125, significantly decreased expressions of RANKL and osteocalcin. These results indicate that glucose uptake by GLUT1 is required for RANKL and osteocalcin expressions in osteocytes, and that inhibition of glucose uptake decreases their expressions through AMPK, ERK1/2, and p38 MAPK pathways. These findings suggest that lowering glucose uptake into osteocytes may contribute to maintain blood glucose levels by decreasing osteocalcin expression and RANKL-induced bone resorption.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.