Osteocyte Cell Death Research Articles

Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.

Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H₂O₂-induced oxidative stress. H₂O₂ increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K⁺, H⁺, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress.

Non-bone metastatic cancers promote osteocyte-induced bone destruction.

The effects of bone metastatic cancer on the skeleton are well described, whereas less is known regarding the effects of non-metastatic bone cancer on bone. Here we investigated the effects of three non-bone metastatic cancer cachexia models, namely Colon-26 adenocarcinoma (C26), ES-2 ovarian cancer (ES-2), and Lewis lung carcinoma (LLC). Even though C26, ES-2 and LLC tumor growth resulted in comparable weight and muscle loss, the ES-2 and LLC hosts exhibited severe bone loss, whereas only modest bone loss was observed in the C26 bearers, correlating with increased TRAP+ osteoclasts in the femurs of ES-2 and LLC but not C26 hosts. Surprisingly, all three showed increased osteocyte lacunar area indicating osteocytic osteolysis and displayed dramatically increased osteocyte death, as well as empty lacunae. To test whether tumor-secreted factors were responsible for the observed effect, IDG-SW3 osteocyte cells were co-cultured with cancer cells in permeable trans-wells. Apoptosis was observed in the osteocyte cells exposed to all three cancer cell lines suggesting that all tumors were cytotoxic for osteocytes. In addition, the expression of the osteoclastic markers, Acp5, CtsK, Atp6v0d2 and Mmp13, was elevated in IDG-SW3 osteocytes exposed to tumor factors, supporting the in vivo observations of increased lacunar size due to osteocytic osteolysis. For the first time, we describe osteocytic bone destruction and extensive osteocyte cell death in non-bone metastatic cancer. These bone alterations, in conjunction with muscle wasting, may create a musculoskeletal system that is incapable of full recovery upon eradication of tumor. Co-treatment with bone preserving therapies should be considered.

Autophagy Prevents Osteocyte Cell Death under Hypoxic Conditions

Hypoxia occurs under important clinical conditions such as cancers, heart disease, and ischemia. However, the relationship between hypoxia and autophagy in osteocytes is still unclear. The objective of the present study was to uncover the regulatory mechanisms that prevent regulated cell death, such as apoptosis, necrosis, and autophagy, under hypoxia. MLO-Y4 cells, a mouse osteocyte cell line, were exposed to various O₂ partial pressures (PO₂). Subsequently, the cells underwent apoptosis, autophagy, autophagic cell death, and/or necrosis, and thereby we designated PO₂ = 2% as a representative hypoxic condition. Immunofluorescence staining showed an increase of LC3 and a decrease of p62 in MLO-Y4 cells exposed to hypoxia, indicating the induction of autophagy. We then hypothesized that β-estradiol (E2) and vitamin D play an important role in apoptosis and autophagy of osteocytes under hypoxia. 1,25α-dihydroxyvitamin D3 (VitD) protected MLO-Y4 cells from cell death and induced autophagy. However, E2 showed little effect. Finally, Western blotting for phosphorylated mTOR and Akt was carried out in order to investigate the altered autophagy signaling pathways affected by the addition of VitD and E2. However, neither E2 nor VitD were capable of recovering the decreased phosphorylation of those factors. Our results indicated that the effects of VitD on autophagy under hypoxia were dependent on the Akt and mTOR pathways. Thus, the results of the present study showed that VitD suppresses osteocyte cell death in an mTOR pathway-dependent manner in hypoxic conditions. This suggests the potential of VitD as a therapeutic intervention for diseases in which the cell death of osteocytes mainly occurs via hypoxia.

Glucocorticoid-Induced Autophagy Protects Osteocytes Against Oxidative Stress Through Activation of MAPK/ERK Signaling.

Autophagy confers protective or detrimental effects on cells depending on the cellular context. We showed here that oxidative stress‐induced cell death in osteocytic MLO‐Y4 cells coincided with decreased autophagy. Decreased autophagy was also observed in osteocytes of superoxide dismutase 1‐ (SOD1‐) deficient mice. Oxidative stress‐induced osteocyte death was exacerbated by an autophagy inhibitor, chloroquine, suggesting a protective function of basal autophagy levels against oxidative stress‐induced cell death. Pretreatment with dexamethasone reduced the susceptibility of osteocytes to oxidative stress‐induced cell death and conferred protection against TNFα/cycloheximide‐induced cell death. Inhibition of MAPK/ERK attenuated the formation of autophagosome, leading to increased osteocyte cell death. Taken together, our results suggest that autophagy, induced by moderate levels of glucocorticoids, leads to the preconditioning of osteocytes and conveys a novel cell‐protective function against cell death induced by oxidative stress and other insults. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Role of Osteocytes in Myeloma Bone Disease: Anti-sclerostin Antibody as New Therapeutic Strategy.

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of anti-myeloma drugs in vitro. Nevertheless, the combination therapy of anti-sclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.

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.

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.

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.

One-month spaceflight compromises the bone microstructure, tissue-level mechanical properties, osteocyte survival and lacunae volume in mature mice skeletons

The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (−64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape (synchrotron radiation analyses), and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties (i.e., hardness and modulus) were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair.

Hypoxia mediates osteocyte ORP150 expression and cell death invitro.

Skeletal unloading leads to hypoxia in the bone microenvironment, resulting in imbalanced bone remodeling that favors bone resorption. Osteocytes, the mechanosensors of bone, have been demonstrated to orchestrate bone homeostasis. Hypoxic osteocytes either undergo apoptosis or actively stimulate osteoclasts to remove bone matrix during hypoxia. Oxygen‑regulated protein150 (ORP150) is an endoplasmic reticulum‑associated chaperone that has been observed to serve an important role in the cellular adaptation to hypoxia and in preventing cellular apoptosis in various tissue types. The current study hypotheses that expression of ORP150 would be increased in osteocytes under hypoxic conditions (1%O2). The MLO‑Y4 osteocyte cell line was cultured under normoxic or hypoxic conditions for up to 72h. It was demonstrated that 1%O2 significantly induced hypoxia after 16h and up to 72h, significantly reduced cell number at 8 and 48h, induced cell death at 8, 24and 48h and induced apoptosis at 16, 24and 48h. Significant differences in ORP150 mRNA were observed at 72h, however no differences were observed in the protein expression levels. The relative increase in ORP150 mRNA observed in hypoxia, compared with normoxia, may support its cytoprotective role in oxygen‑deprived conditions.

Osteocyte cell death in subchondral bone following joint injury correlates with the severity of aggrecan loss in overlying cartilage

Osteocyte cell death in subchondral bone following joint injury correlates with the severity of aggrecan loss in overlying cartilage

Regulatory Mechanisms of RANKL Presentation to Osteoclast Precursors

It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.

Myeloma-Induced Osteocyte Death Was Blunted By Proteasome Inhibitors Through The Modulation Of Autophagy

Osteocytes are critical in the maintenance of bone integrity regulating bone remodeling through the cell death and autophagy, a cellular process stress-induced to prolong cell survival but when induced excessively can cause cell death. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis. However the mechanisms involved in this process as well as the effect of the proteasome inhibitors able to stimulate bone formation are not known and have been investigated in this study.Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a traswell insert. The treatment for 12-24 hours with (BOR) (2nM) and MG262 (10nM) significantly blunted MLO-Y4 and HOB-01 cell death. In addition, dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at high doses (10-5-10-6 M), was reduced by the treatment with proteasome inhibitors. Interestingly, we found that PTH short-term treatment potentiated the in vitro effects of proteasome inhibitors on DEX-induced osteocyte death. To evaluate the presence of autophagy in osteocytes, we checked the expression of the autophagic marker LC3 both by confocal microscopy and western blot analysis in the co-colture system with MLO-Y4 and RPMI-8226. Prevalence of autophagic cell death and in a lesser extent apoptosis was observed in this system. BOR increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors BOR and MG262 blocked autophagic cell death in osteocytes.To translate our in vitro evidence in a clinical perspective, thereafter we performed a histological evaluation on bone biopsies of a cohort of 37 newly diagnosis MM patients 31 of them with symptomatic MM and 6 with smoldering MM (SMM). The 55% of patients with MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment or observation. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). A reduction of both osteocyte apoptosis and autophagy was demonstrated by TUNEL assays and confocal microscopy. On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035).Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy supporting their use to improve bone integrity in MM patients. Disclosures:Giuliani:Celgene Italy: Research Funding.

Connexin 43 Channels Protect Osteocytes Against Oxidative Stress–Induced Cell Death

The increased osteocyte death by oxidative stress (OS) during aging is a major cause contributing to the impairment of bone quality and bone loss. However, the underlying molecular mechanism is largely unknown. Here, we show that H₂O₂ induced cell death of primary osteocytes and osteocytic MLO-Y4 cells, and also caused dose-dependent decreased expression of gap junction and hemichannel-forming connexin 43 (Cx43). The decrease of Cx43 expression was also demonstrated with the treatment of other oxidants, rotenone and menadione. Antioxidant reversed the effects of oxidants on Cx43 expression and osteocyte cell death. Cx43 protein was also much lower in the osteocytes from 20-month-old as opposed to the 5-week-old or 20-week old mice. Dye transfer assay showed that H₂O₂ reduced the gap junction intercellular communication (GJIC). In contrast to the effect on GJIC, there was a dose-dependent increase of hemichannel function by H₂O₂, which was correlated with the increased cell surface expression of Cx43. Cx43(E2) antibody, an antibody that specifically blocks Cx43 hemichannel activity but not gap junctions, completely blocked dye uptake induced by H₂O₂ and further exacerbated H₂O₂-induced osteocytic cell death. In addition, knockdown of Cx43 expression by small interfering RNA (siRNA) increased the susceptibility of the cells to OS-induced death. Together, our study provides a novel cell protective mechanism mediated by osteocytic Cx43 channels against OS.

Osteocyte regulation of bone mineral: a little give and take

Osteocytes actively participate in almost every phase of mineral handling by bone. They regulate the mineralisation of osteoid during bone formation, and they are also a major RANKL-producing cell. Osteocytes are thus able to liberate bone mineral by regulating osteoclast differentiation and activity in response to a range of stimuli, including bone matrix damage, bone disuse and mechanical unloading, oestrogen deficiency, high-dose glucocorticoid and chemotherapeutic agents. At least some of these activities may be regulated by the osteocyte-secreted product, sclerostin. There is also mounting evidence that in addition to regulating phosphate homeostasis systemically, osteocytes contribute directly to calcium homeostasis in the mature skeleton. Osteocyte cell death and the local loss of control of bone mineralisation may be the cause of focal hypermineralisation of bone and osteopetrosis, as seen in aging and pathology. The sheer number of osteocytes in bone means that "a little give and take" in terms of regulation of bone mineral content translates into a powerful whole organism effect.

Skeletal Muscles Maintain Osteocyte Viability

Osteocyte viability is important to maintain normal bone mass as apoptosis of these cells is a triggering event in the development of osteoporosis. As sarcopenia and osteoporosis often occur concurrently, we hypothesized that skeletal muscles potentially secrete factors that may protect osteocytes from apoptosis. To test this hypothesis, we treated MLO-Y4 osteocyte-like cells with conditioned media (CM) from C2C12 myoblasts or myotubes, and primary muscles. Our results demonstrate that dexamethasone-induced apoptosis of MLO-Y4 osteocyte-like cells is blocked by C2C12 myotube CM, but not by C2C12 myoblast CM. Moreover, survival of MLO-Y4 cells in the presence of dexamethasone is increased if cells were pre-exposed to CM from primary skeletal muscles subjected to ex vivo contractility. As little as 1% of CM blocked apoptosis showing high potency of a soluble factor(s). The responsible factor(s) appears to be less than 10kDa and identification studies are underway. These in vitro data show that skeletal muscle cells produce factors that prevent osteocyte cell death suggesting that skeletal muscles promote the maintenance of bone mass independent of loading. Support: NIH RC2 AR058962 and Missouri Life Sciences Research Board.

Bone in vitro 3D osteoblast-osteocyte co-culture model

Event Abstract Back to Event Bone in vitro 3D osteoblast-osteocyte co-culture model M Vazquez1*, BAJ Evans2, J Ralphs3, D Riccardi3 and DJ Mason3 1 Cardiff University, Arthritis Research UK Biomechanics and Bioengineering Centre, School of Biosciences, United Kingdom 2 Cardiff Universit, Department of Child Health, School of Medicine, United Kingdom 3 Cardiff University, Division of Pathophysiology and Repair, School of Biosciences, United Kingdom Background: Osteoporosis occurs when the activities of bone cells become unbalanced leading to increased bone remodelling and weakening of bones. Anabolic therapies are limited, necessitating further investigation of the mechanisms that regulate bone formation. Normal mechanical loading potently induces bone formation via effects on osteocytes. Current investigations of mechanical loading of bone do not reflect the interactions of the cells within it, with most focusing on mechanical loading of osteoblasts in monolayers. Of the 3D models that do exist, none elucidate the osteoblast-osteocyte interactions that regulate mechanically-induced bone formation. We are developing a novel in vitro 3D co-culture model of bone to investigate osteoblast-osteocyte interactions (1) and the role of glutamate, calcium and adenosine signalling in bone mechanotransduction.{BR}Methods: MLO-Y4 cells (1.5x10^6 cells/ml) were incorporated into acid-soluble rat tail tendon type I collagen (2 mg/ml in alpha MEM, pH7.4) gels and MC3T3-E1(1.0x10^5 cells/well) layered on top and cultured at 37C in SMEM (1 mM CaCl2, 2 mM Glutamax, 50 microgram/ml ascorbic acid, 5 % dialysed FBS) for 1 week. Co-cultures were fixed with 1 % paraformaldehyde, infiltrated with OCT, cryosectioned and labelled with phalloidin and DAPI to assess cell morphology, ethidium homodimer and DAPI to assess cell viability or immunostained using anti-connexin 43 antibody to assess cell connectivity. Osteoblast and osteocyte phenotype were determined by RT-PCR of RNA extracted (Trizol) separately from surface osteoblasts and encased osteocytes.{BR}Results: Preliminary data show co-cultures survive, for at least one week, with osteocyte cell death within gels (18 +/- 2.16 %, n=3) comparable to monolayer cultures (2). MLO-Y4 and MC3T3-E1 cells maintain their morphology, form a network through connexin 43, and express osteocyte and osteoblast phenotypic markers respectively, and components of the glutamate, calcium and adenosine signalling pathways.{BR}Conclusion: We have established a mouse osteoblast-osteocyte 3D co-culture system which can be used to investigate co-ordination of mechanical signalling through glutamate, calcium and adenosine delineating their roles in mechanically-induced bone formation.{BR}

The amazing osteocyte

The last decade has provided a virtual explosion of data on the molecular biology and function of osteocytes. Far from being the “passive placeholder in bone,” this cell has been found to have numerous functions, such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. The osteocyte is a source of soluble factors not only to target cells on the bone surface but also to target distant organs, such as kidney, muscle, and other tissues. This cell plays a role in both phosphate metabolism and calcium availability and can remodel its perilacunar matrix. Osteocytes compose 90% to 95% of all bone cells in adult bone and are the longest lived bone cell, up to decades within their mineralized environment. As we age, these cells die, leaving behind empty lacunae that frequently micropetrose. In aged bone such as osteonecrotic bone, empty lacunae are associated with reduced remodeling. Inflammatory factors such as tumor necrosis factor and glucocorticoids used to treat inflammatory disease induce osteocyte cell death, but by different mechanisms with potentially different outcomes. Therefore, healthy, viable osteocytes are necessary for proper functionality of bone and other organs. © 2011 American Society for Bone and Mineral Research.

In Vitro and In Vivo Evidences of Osteocyte Involvement In Myeloma-Induced Osteolysis

AbstractAbstract 131Multiple myeloma (MM)–induced osteolysis is characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation. Osteocytes are located in the lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling through the cell death and apoptosis triggering osteoclast recruitment and formation. Actually, the potential involvement of osteocytes in MM-induced osteoclast formation and in the bone remodeling alterations occurring in MM patients is still unknown and was investigated in this study. Firstly we evaluated the effect of MM cells on osteocyte survival in a co-culture transwell system of human myeloma cell lines (JJN3, KMS12, XG-1) and the human pre-osteocytic cell line HOB-01. By Transmission Electron Microscopy (TEM) observations and TUNEL assay we showed the occurrence of either apoptotic cells or degenerated non-apoptotic cells in the monolayer of HOB-01 cells co-cultured with MM cells or treated with their conditioned media (CM) as compared to non-treated cells suggesting that MM cells may increase osteocyte cell death. Second, we investigated whether MM cells could affect the pro-osteoclastogenic profile and the osteoclastogenic properties of HOB-01 in co-culture. The microarray analysis, using Affymetrix GeneChip® HG-U133Plus2.0 platform) identified 47 genes significantly modulated by MM cells in HOB-01, most of them up-regulated; among these, we identified the increased expression of the pro-osteoclastogenic genes IL11 and MMP1, a metalloproteinase involved in tumoral ostelysis. The up-regulation of both IL-11 and MMP-1 was then confirmed at both mRNA and protein level either by real time PCR and ELISA assay, respectively. Furthermore, the mRNA expression and the protein levels of the main pro-osteoclastogenic cytokines CCL3/MIP-1α and RANKL were also measured in the co-cultures showing that CCL3/MIP-1α levels were significantly higher than those observed either in HOB-01 or MM cells cultured alone whereas RANKL levels and RANKL/OPG ratio were unchanged. Analyzing separately HOB-01 and MM cells after the co-culture period, we demonstrated that increased levels of CCL3/MIP-1α were due to its up-regulation in MM cells. In line with the increase of the pro-osteoclastogenic cytokines observed in the co-cultures, we found that the CM of HOB-01 co-cultured with MM cells significantly increased CD14+-derived osteoclastogenesis in presence of RANKL as compared to the CM of HOB-01 or MM cells cultured alone. Interestingly this effect was completely blunted in presence of blocking antibody anti-CCL3/MIP-1α and in a lesser extent of anti-IL-11 and anti-MMP-1.To translate into a clinical perspective such in vitro evidences, then we performed histological analysis on bone biopsies obtained from iliac crest of a cohort of 32 patients with MM at the diagnosis or relapsed (ISS I-III, mean age±SD: 72±10), 55% of them with osteolytic bone lesions and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Ten sex-age matched healthy subjects without hematological malignancies or metabolic bone disease were also analyzed. Consistently with our in vitro observations, we found a significant reduction in the percentage of viable osteocytes with an increase of the number of death osteocytes/empty lacunae in MM patients as compared to healthy subjects (p=0.003) but not MGUS (p=0.4). Under Light Microscopy and TUNEL analysis we demonstrated that osteocyte cell death in MM patients was due, at least in part, to an increase of the osteocyte apoptosis. As regard the skeletal involvement in MM patients we showed significant lower percentage of viable osteocytes (p=0.05) and higher amount of death osteocytes and empty lacunae in osteolytic vs. non-osteolytic MM patients (p=0.05). Finally a significant negative correlation was observed in MM patients between the amount of viable osteocytes and that of osteoclasts [Spearman (r): -0.33, p=0.04].Overall our data suggest a critical role of osteocytes in MM-induced osteolysis in MM patients showing that the interaction of MM cells with osteocytes trigger osteoclastogenesis through the increase of osteocyte apoptosis and the production of pro-osteoclastogenic cytokines. Disclosures:No relevant conflicts of interest to declare.

Jaw Osteonecrosis in Patients Treated with Bisphosphonates: An Ultrastructural Study

Osteonecrosis of the jaw is a severe bone disorder traditionally associated with periodontal disease, local malignancy, chemotherapy, glucocorticoid therapy, or trauma. Recently a growing number of publications reported the occurrence of osteonecrosis of the jaw in patients undergoing treatment with bisphosphonates. The mechanism by which bisphosphonates might contribute to the development of osteonecrosis of the jaw is far from being fully elucidated. Suppression of bone turnover, infection, tissue hypoxia and cellular toxicity were proposed as possible mechanisms by which bisphosphonates may exert adverse effects on bone metabolism. Here, we studied 25 consecutive patients treated with bisphosphonates for osteoporosis or tumoral pathologies. We provide good evidence of hyperactive osteoclastic bone resorption and suggest a direct cytotoxic effect of bisphosphonates on bone tissue through induction of osteocyte cell death. We also demonstrate that bisphosphonates only have limited adverse effects on bone vascular network.