Journal articles on the topic 'Osteocyte differentiation'
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Kramer, Ina, Christine Halleux, Hansjoerg Keller, Marco Pegurri, Jonathan H. Gooi, Patricia Brander Weber, Jian Q. Feng, Lynda F. Bonewald, and Michaela Kneissel. "Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis." Molecular and Cellular Biology 30, no. 12 (April 19, 2010): 3071–85. http://dx.doi.org/10.1128/mcb.01428-09.
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ABSTRACT β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1 loxP/loxP ; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.
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Zhang, Keqin, Cielo Barragan-Adjemian, Ling Ye, Shiva Kotha, Mark Dallas, Yongbo Lu, Shujie Zhao, et al. "E11/gp38 Selective Expression in Osteocytes: Regulation by Mechanical Strain and Role in Dendrite Elongation." Molecular and Cellular Biology 26, no. 12 (June 15, 2006): 4539–52. http://dx.doi.org/10.1128/mcb.02120-05.
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ABSTRACT Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.
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Skottke, Gelinsky, and Bernhardt. "In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels." International Journal of Molecular Sciences 20, no. 8 (April 23, 2019): 1998. http://dx.doi.org/10.3390/ijms20081998.
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Background: Osteocytes are the key regulator cells in bone tissue, affecting activity of both osteoblasts and osteoclasts. Current in vitro studies on osteocyte-osteoblast interaction are invariably performed with rodent cells, mostly murine cell lines, which diminishes the clinical relevance of the data. Objective: The objective of the present study was to establish an in vitro co-culture system of osteoblasts and osteocytes, which is based solely on human primary cells. Methods: Three different approaches for the generation of human primary osteocytes were compared: direct isolation of osteocytes from bone tissue by multistep digestion, long-time differentiation of human pre-osteoblasts embedded in collagen gels, and short time differentiation of mature human osteoblasts in collagen gels. Co-cultivation of mature osteoblasts with osteocytes, derived from the three different approaches was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoblasts on the basal side of a porous membrane, which allowed the separate gene expression analysis for osteocytes and osteoblasts. Fluorescence microscopic imaging and gene expression analysis were performed separately for osteocytes and osteoblasts. Results: All examined approaches provided cells with typical osteocytic morphology, which expressed osteocyte markers E11, osteocalcin, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and receptor activator of NF-κB Ligand (RANKL). Expression of osteocyte markers was not significantly changed in the presence of osteoblasts. In contrast, osteocalcin gene expression of osteoblasts was significantly upregulated in all examined co-cultures with differentiated osteocytes. Alkaline phosphatase (ALPL), bone sialoprotein II (BSPII), and RANKL expression of osteoblasts was not significantly changed in the co-culture. Conclusion: Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells.
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Du, J. H., S. X. Lin, X. L. Wu, S. M. Yang, L. Y. Cao, A. Zheng, J. N. Wu, and X. Q. Jiang. "The Function of Wnt Ligands on Osteocyte and Bone Remodeling." Journal of Dental Research 98, no. 8 (July 2019): 930–38. http://dx.doi.org/10.1177/0022034519854704.
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Bone homeostasis is continually maintained by the process of bone remodeling throughout life. Recent studies have demonstrated that Wnt signaling pathways play a fundamental role in the process of bone homeostasis and remodeling. Intracellular Wnt signaling cascades are initially triggered by a Wnt ligand–receptor complex formation. In previous studies, the blocking of Wnt ligands from different osteoblastic differentiation stages could cause defective bone development at an early stage. Osteocytes, the most abundant and long-lived type of bone cell, are a crucial orchestrator of bone remodeling. However, the role of Wnt ligands on osteocyte and bone remodeling remains unclear. In our present study, we found that, besides osteoblasts, osteocytes also express multiple Wnt ligands in the bone environment. Then, we used a Dmp1-Cre mouse line, in which there is expression in a subset of osteoblasts but mainly osteocytes, to study the function of Wnt ligands on osteocyte and bone remodeling in vivo. Furthermore, we explored the role of Wnt ligands on osteocytic mineralization ability, as well as the regulatory function of osteocytes on the process of osteoblastic differentiation and osteoclastic migration and maturity in vitro. We concluded that Wnt proteins play an important regulatory role in 1) the process of perilacunar/canalicular remodeling, as mediated by osteocytes, and 2) the balance of osteogenesis and bone resorption at the bone surface, as mediated by osteoblasts and osteoclasts, at least partly through the canonical Wnt/β-catenin signaling pathway and the OPG/RANKL signaling pathway.
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Pesce Viglietti, Ayelén Ivana, Paula Constanza Arriola Benitez, María Virginia Gentilini, Lis Noelia Velásquez, Carlos Alberto Fossati, Guillermo Hernán Giambartolomei, and María Victoria Delpino. "Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion." Infection and Immunity 84, no. 1 (October 12, 2015): 11–20. http://dx.doi.org/10.1128/iai.01049-15.
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Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine ifBrucella abortusinfection modifies osteocyte function. Our results indicate thatB. abortusinfection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants fromB. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants fromB. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival.B. abortusinfection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants fromB. abortus-infected macrophages.B. abortusinfection was not capable of inducing osteocyte apoptosis. However, supernatants fromB. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate thatB. abortusinfection could alter osteocyte function, contributing to bone damage.
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Wu, Qing, Xiaokang Zhou, Danqing Huang, Yingchen JI, and Feiwu Kang. "IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro." Cellular Physiology and Biochemistry 41, no. 4 (2017): 1360–69. http://dx.doi.org/10.1159/000465455.
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Background/Aims: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts. Methods: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes. Results: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL. Conclusion: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.
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Gao, Zheng-Rong, Qiong Liu, Jie Zhao, Ya-Qiong Zhao, Li Tan, Shao-Hui Zhang, Ying-Hui Zhou, Yun Chen, Yue Guo, and Yun-Zhi Feng. "A comprehensive analysis of the circRNA–miRNA–mRNA network in osteocyte-like cell associated with Mycobacterium leprae infection." PLOS Neglected Tropical Diseases 16, no. 5 (May 2, 2022): e0010379. http://dx.doi.org/10.1371/journal.pntd.0010379.
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Background Bone formation and loss are the characteristic clinical manifestations of leprosy, but the mechanisms underlying the bone remodeling with Mycobacterium leprae (M. leprae) infection are unclear. Methodology/Principal findings Osteocytes may have a role through regulating the differentiation of osteogenic lineages. To investigate osteocyte-related mechanisms in leprosy, we treated osteocyte-like cell with N-glycosylated muramyl dipeptide (N.g MDP). RNA-seq analysis showed 724 differentially expressed messenger RNAs (mRNAs) and 724 differentially expressed circular RNA (circRNAs). Of these, we filtered through eight osteogenic-related differentially expressed genes, according to the characteristic of competing endogenous RNA, PubMed databases, and bioinformatic analysis, including TargetScan, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes. Based on these results, we built a circRNA–microRNA (miRNA)–mRNA triple network. Quantitative reverse-transcription polymerase chain reaction and western blots analyses confirmed decreased Clock expression in osteocyte-like cell, while increased in bone mesenchymal stem cells (BMSCs), implicating a crucial factor in osteogenic differentiation. Immunohistochemistry showed obviously increased expression of CLOCK protein in BMSCs and osteoblasts in N.g MDP–treated mice, but decreased expression in osteocytes. Conclusions/Significance This analytical method provided a basis for the relationship between N.g MDP and remodeling in osteocytes, and the circRNA–miRNA–mRNA triple network may offer a new target for leprosy therapeutics.
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Ilas, Dragos C., Sarah M. Churchman, Thomas Baboolal, Peter V. Giannoudis, Joseph Aderinto, Dennis McGonagle, and Elena Jones. "The simultaneous analysis of mesenchymal stem cells and early osteocytes accumulation in osteoarthritic femoral head sclerotic bone." Rheumatology 58, no. 10 (April 16, 2019): 1777–83. http://dx.doi.org/10.1093/rheumatology/kez130.
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Abstract Objective OA subchondral bone is a key target for therapy development. Osteocytes, the most abundant bone cell, critically regulate bone formation and resorption. Their progenitors, mesenchymal stem cells (MSCs), display altered behaviour in osteoarthritic subchondral bone. This study investigated the relationships between native osteocytes and native MSCs in osteoarthritic femoral heads. Methods To avoid culture manipulations, a bone treatment procedure was developed to simultaneously obtain pure osteocyte-enriched fragments and matched native CD45-CD271+ MSCs. Gene expression in osteocytes and MSCs was compared between healthy and OA bone and selected molecules were examined by immunohistochemistry in relation to OA tissue pathology. Cell sorting and standard trilineage differentiation assays were employed to test OA MSC functionality. Results Native osteocyte enrichment was confirmed histologically and by higher-level osteocyte maturation transcripts expression, compared with purified MSCs. Compared with healthy bone, native OA osteocytes expressed 9- and 4-fold more early/embedding osteocyte molecules E11 and MMP14, and 6-fold more osteoprotegerin (P<0.01). CD271+ MSCs accumulated in the regions of bone sclerosis (9-fold, P<0.0001) in close juxtaposition to trabeculae densely populated with morphologically immature E11-positive osteocytes (medians of 76% vs 15% in non-sclerotic areas, P<0.0001), and osteoblasts. Gene expression of OA MSCs indicated their bone formation bias, with retained multipotentiality following culture-expansion. Conclusions In human late-stage OA, osteogenically-committed MSCs and adjacent immature osteocytes exhibit a marked accumulation in sclerotic areas. This hitherto unappreciated MSC-early osteocyte axis could be key to understanding bone abnormalities in OA and represents a potential target for novel therapy development in early disease.
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Cherniack, E. Paul, Sahithi Chekuri, and Heather F. Lee. "Potential Non-neoplastic Applications for Polyphenols in Stem Cell Utilization." Current Drug Targets 20, no. 3 (January 25, 2019): 347–53. http://dx.doi.org/10.2174/1389450119666180731092453.
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While polyphenols may have important effects on pluripotential stem cells that make them noteworthy as potential antineoplastic agents, their action on stem cells may portend other health benefits, such as treatments for cardiovascular and neurocognitive disorders. Resveratrol, the beststudied polyphenol, has been found to enable stem cells to differentiate into cardiomyocytes, neurons, osteocytes, and pancreatic beta cells, as well as facilitating augmentation of stem cell populations and protecting them from toxic injury. Curcumin protects mesenchymal stem cells from toxicity, and prevents them from facilitating chondrocytic hypertrophy. Quercetin enabled osteocytic and pancreatic beta cell differentiation, and protected neuronal stem cells from injury. Epigallocatechin gallate prevented damage to osteocyte precursors and averted differentiation into undesirable adipocytes. Genistein facilitated osteogenesis while preventing adipogenesis. Several other polyphenols, daidzein, caffeic and chlorogenic acid, kaempferol, and piceatannol, protect stems cells from reactive oxygen species and foster stem cells differentiation away from adipocytic and toward osteocytic lineages. Further research should better elucidate the pharmacokinetic profiles of each polyphenol, explore novel delivery systems, and expand investigation beyond rodent models to additional species.
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Zhang, Lanlan, and Chunyi Wen. "Osteocyte Dysfunction in Joint Homeostasis and Osteoarthritis." International Journal of Molecular Sciences 22, no. 12 (June 17, 2021): 6522. http://dx.doi.org/10.3390/ijms22126522.
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Structural disturbances of the subchondral bone are a hallmark of osteoarthritis (OA), including sclerotic changes, cystic lesions, and osteophyte formation. Osteocytes act as mechanosensory units for the micro-cracks in response to mechanical loading. Once stimulated, osteocytes initiate the reparative process by recruiting bone-resorbing cells and bone-forming cells to maintain bone homeostasis. Osteocyte-expressed sclerostin is known as a negative regulator of bone formation through Wnt signaling and the RANKL pathway. In this review, we will summarize current understandings of osteocytes at the crossroad of allometry and mechanobiology to exploit the relationship between osteocyte morphology and function in the context of joint aging and osteoarthritis. We also aimed to summarize the osteocyte dysfunction and its link with structural and functional disturbances of the osteoarthritic subchondral bone at the molecular level. Compared with normal bones, the osteoarthritic subchondral bone is characterized by a higher bone volume fraction, a larger trabecular bone number in the load-bearing region, and an increase in thickness of pre-existing trabeculae. This may relate to the aberrant expressions of sclerostin, periostin, dentin matrix protein 1, matrix extracellular phosphoglycoprotein, insulin-like growth factor 1, and transforming growth factor-beta, among others. The number of osteocyte lacunae embedded in OA bone is also significantly higher, yet the volume of individual lacuna is relatively smaller, which could suggest abnormal metabolism in association with allometry. The remarkably lower percentage of sclerostin-positive osteocytes, together with clustering of Runx-2 positive pre-osteoblasts, may suggest altered regulation of osteoblast differentiation and osteoblast-osteocyte transformation affected by both signaling molecules and the extracellular matrix. Aberrant osteocyte morphology and function, along with anomalies in molecular signaling mechanisms, might explain in part, if not all, the pre-osteoblast clustering and the uncoupled bone remodeling in OA subchondral bone.
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Uzbekov, R. E., D. B. Maurel, P. C. Aveline, S. Pallu, C. L. Benhamou, and G. Y. Rochefort. "Centrosome Fine Ultrastructure of the Osteocyte Mechanosensitive Primary Cilium." Microscopy and Microanalysis 18, no. 6 (November 21, 2012): 1430–41. http://dx.doi.org/10.1017/s1431927612013281.
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AbstractThe centrosome is the principal microtubule organization center in cells, giving rise to microtubule-based organelles (e.g., cilia, flagella). The aim was to study the osteocyte centrosome morphology at an ultrastructural level in relation to its mechanosensitive function. Osteocyte centrosomes and cilia in tibial cortical bone were explored by acetylated alpha-tubulin (AαTub) immunostaining under confocal microscopy. For the first time, fine ultrastructure and spatial orientation of the osteocyte centrosome were explored by transmission electron microscopy on serial ultrathin sections. AαTub-positive staining was observed in 94% of the osteocytes examined (222/236). The mother centriole formed a short primary cilium and was longer than the daughter centriole due to an intermediate zone between centriole and cilium. The proximal end of the mother centriole was connected with the surface of daughter centriole by striated rootlets. The mother centriole exhibited distal appendages that interacted with the cell membrane and formed a particular structure called “cilium membrane prolongation.” The primary cilium was mainly oriented perpendicular to the long axis of bone. Mother and daughter centrioles change their original mutual orientation during the osteocyte differentiation process. The short primary cilium is hypothesized as a novel type of fluid-sensing organelle in osteocytes.
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Oralová, Veronika, Sabina Stouracova, Premysl Bartos, and Eva Matalova. "Myb expression during osteocyte differentiation." Bone Reports 16 (May 2022): 101385. http://dx.doi.org/10.1016/j.bonr.2022.101385.
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Xu, Liangcheng, Xin Song, Gwennyth Carroll, and Lidan You. "Novel in vitro microfluidic platform for osteocyte mechanotransduction studies." Integrative Biology 12, no. 12 (December 2020): 303–10. http://dx.doi.org/10.1093/intbio/zyaa025.
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Abstract Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-μm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.
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Wood, Charles L., Paola Divieti Pajevic, and Jonathan H. Gooi. "Osteocyte secreted factors inhibit skeletal muscle differentiation." Bone Reports 6 (June 2017): 74–80. http://dx.doi.org/10.1016/j.bonr.2017.02.007.
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Kim, Hyung Joon, Ha Jin Kim, YunJeong Choi, Moon-Kyoung Bae, Dae Seok Hwang, Sang-Hun Shin, and Jae-Yeol Lee. "Zoledronate Enhances Osteocyte-Mediated Osteoclast Differentiation by IL-6/RANKL Axis." International Journal of Molecular Sciences 20, no. 6 (March 22, 2019): 1467. http://dx.doi.org/10.3390/ijms20061467.
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Bisphosphonates are one of the most widely used synthetic pyrophosphate analogues for the treatment of bone resorbing diseases such as osteoporosis, multiple myeloma, and bone metastases. Although the therapeutic usefulness of bisphosphonates mainly depends on their anti-osteoclastogenic effect, a severe side-effect of bisphosphonates called bisphosphonate-related osteonecrosis of the jaw (BRONJ) could not be explained by the anti-osteoclastogenic effect of bisphosphonates. In the present study, we have evaluated the changes in osteoclastogenesis- or osteoblastogenesis-supporting activities of osteocytes induced by bisphosphonates. Zoledronate, a nitrogen-containing bisphosphonate, markedly increased both the receptor activator of nuclear factor kB ligand (RANKL) as well as sclerostin in osteocyte-like MLO-Y4 cells, which were functionally revalidated by osteoclast/osteoblast generating activities of the conditioned medium obtained from zoledronate-treated MLO-Y4 cells. Of note, the zoledronate treatment-induced upregulation of the RANKL expression was mediated by autocrine interleukin-6 (IL-6) and subsequent activation of the signal transducer and activator of transcription 3 (STAT3) pathway. These results were evidenced by the blunted RANKL expression in the presence of a Janus activated kinase (JAK2)/STAT3 inhibitor, AG490. Also, the osteoclastogenesis-supporting activity was significantly decreased in zoledronate-treated MLO-Y4 cells in the presence of IL-6 neutralizing IgG compared to that of the control IgG. Thus, our results show previously unanticipated effects of anti-bone resorptive bisphosphonate and suggest a potential clinical importance of osteocytes in BRONJ development.
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Heni, Hannah, Julia Ebner, Gudula Schmidt, Klaus Aktories, and Joachim Orth. "Involvement of Osteocytes in the Action of Pasteurella multocida Toxin." Toxins 10, no. 8 (August 13, 2018): 328. http://dx.doi.org/10.3390/toxins10080328.
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Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.
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Sampaio, R. V., M. R. Chiaratti, F. F. Bressan, J. R. Sangalli, M. S. Miranda, T. H. C. De Bem, T. V. G. Silva, et al. "399 ISOLATION AND CHARACTERIZATION OF BOVINE MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE." Reproduction, Fertility and Development 22, no. 1 (2010): 356. http://dx.doi.org/10.1071/rdv22n1ab399.
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Stem cells have been widely used because of their multi-differentiation ability. Compared with embryonic stem cells, mesenchymal stem cells (MSC) are more easily sourced and cultured, besides being easily obtained from adult individuals. In this regard, bovine MSC is of great interest because of its wide application in basic and applied research, e.g. in somatic cell nuclear transfer (SCNT). Success rates of SCNT are expected to be enhanced with the use of MSC as donor cells because they exhibit a more undifferentiated condition when compared with the most widely used cell type, fibroblast. There are few reports on MSC referring to this species. Therefore, our aim was to isolate and culture adipose MSC from cattle. Mesenchymal stem cells were induced to differentiate into adipocytes and osteocytes to prove their multi-differentiation ability. A small piece of adipose tissue was sourced from the base of the tail of a cow and extensively washed in cold saline solution containing 5% antibiotic. Recovered tissue was minced and digested in 0.001% collagenase 1 and incubated for 3 h. Collagenase was inactivated in alpha minimal essential medium (MEM) supplemented with 15% FCS. The treated tissue was centrifuged and the pellet plated on plastic dishes in alpha MEM supplemented with 15% FCS and 1% antibiotic. Culture medium was replaced every 2 days, and cells were plated on new dishes before reaching 75% confluence. To test their resistance to grow after thawing, cells were frozen after each passage, thawed, and cultured. Cell growth was accompanied through several weeks, and cells were tested for differentiation ability after 3 passages, when adipocyte and osteocyte differentiation was accomplished using a standard protocol. Briefly, adipocyte differentiation was inducted in DMEM containing 2% FCS, isobutyl-methylxanthine, dexamethasone, insulin, and indomethacin. Osteocyte differentiation was carried out in alpha MEM without FCS containing ascorbate-2-phosphate, dexamethasone, and beta-glycerophosphate.To confirm differentiation, a tissue-specific staining was carried out using Oil Red for adipocyte staining and Alizarin Red for osteocyte staining. The cells adhered to plastic dishes shortly after plating, presented fibroblast-like morphology, and showed an exponential growth curve during the first 6 passages. The cells were capable of growing after thawing similarly to unthawed cells. After 1 week under differentiation protocols, standard morphological changes were observed in the cells. Mesenchymal stem cells subjected to differentiation into adipocytes showed an increase in their size, developed lipid-like vesicles, and stained positive for Oil Red. An increase in the cell size was also observed in the cells subjected to differentiation into osteocyte. Moreover, these cells stained positive for Alizarin Red. Altogether these results provide evidence that these cells are multi-potent MSC. Therefore, we conclude that the current methodology was efficient in isolating bovine MSC from adipose tissue, and the cells resemble those isolated from other mammals. Bovine MSC constitute a significant source of nuclear donor cells for future experiments in SCNT. Financial support was provided by FAPESPA and Cnpq.
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Dolan, Eimear B., David Tallon, Wing-Yee Cheung, Mitchell B. Schaffler, Oran D. Kennedy, and Laoise M. McNamara. "Thermally induced osteocyte damage initiates pro-osteoclastogenic gene expression in vivo." Journal of The Royal Society Interface 13, no. 119 (June 2016): 20160337. http://dx.doi.org/10.1098/rsif.2016.0337.
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Bone is often subject to harsh temperatures during orthopaedic procedures resulting in thermally induced bone damage, which may affect the healing response. Postsurgical healing of bone is essential to the success of surgery, therefore, an understanding of the thermally induced responses of bone cells to clinically relevant temperatures in vivo is required. Osteocytes have been shown to be integrally involved in the bone remodelling cascade, via apoptosis, in micro-damage systems. However, it is unknown whether this relationship is similar following thermal damage. Sprague–Dawley rat tibia were exposed to clinically relevant temperatures (47°C or 60°C) to investigate the role of osteocytes in modulating remodelling related factors. Immunohistochemistry was used to quantify osteocyte thermal damage (activated caspase-3). Thermally induced pro-osteoclastogenic genes ( Rankl , Opg and M-csf ), in addition to genes known to mediate osteoblast and osteoclast differentiation via prostaglandin production ( Cox2 ), vascularization ( Vegf ) and inflammatory ( Il1a ) responses, were investigated using gene expression analysis. The results demonstrate that heat-treatment induced significant bone tissue and cellular damage. Pro-osteoclastogenic genes were upregulated depending on the amount of temperature elevation compared with the control. Taken together, the results of this study demonstrate the in vivo effect of thermally induced osteocyte damage on the gene expression profile.
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Giuliani, Nicola, Paola Storti, Manuela Abeltino, Marina Bolzoni, Marzia Ferretti, Mirca Lazzaretti, Benedetta Dalla Palma, et al. "In Vitro and In Vivo Evidences of Osteocyte Involvement In Myeloma-Induced Osteolysis." Blood 116, no. 21 (November 19, 2010): 131. http://dx.doi.org/10.1182/blood.v116.21.131.131.
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Abstract Abstract 131 Multiple 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.
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Jiang, Ningning, Fengyang Guo, Wenshu Xu, Zhongyuan Zhang, Hui Jin, Liqun Shi, Xiuyun Zhang, Jie Gao, and Hui Xu. "Effect of fluoride on osteocyte-driven osteoclastic differentiation." Toxicology 436 (April 2020): 152429. http://dx.doi.org/10.1016/j.tox.2020.152429.
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Palumbo, C., S. Palazzini, and G. Marotti. "Morphological study of intercellular junctions during osteocyte differentiation." Bone 11, no. 6 (1990): 401–6. http://dx.doi.org/10.1016/8756-3282(90)90134-k.
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Opiela, Jolanta, Daniel Lipiński, Joanna Romanek, Wojciech Juzwa, Michał Bochenek, and Piotr Wilczek. "5. MMP-2, TIMP-2, TAZ and MEF2a Transcript Expression in Osteogenic and Adipogenic Differentiation of Porcine Mesenchymal Stem Cells." Annals of Animal Science 16, no. 2 (April 1, 2016): 369–85. http://dx.doi.org/10.1515/aoas-2015-0065.
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Abstract Mesenchymal stem cell (MSC) differentiation is regulated intrinsically by transcription factors and extrinsically by the extracellular matrix. We tested whether matrix metalloproteinase-2 (MMP-2) and its inhibitor TIMP-2, MEF2a and TAZ transcription factors are involved in porcine MSC differentiation towards adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to investigate the expression levels of multipotent cell surface markers CD73 and CD105. Real- time PCR was performed to detect the osteogenic- and adipogenic-specific markers, osteocalcin and aP2, respectively, and to estimate the MMP-2, TIMP-2, MEF2a and TAZ transcript expression levels in three groups of cell, i.e., undifferentiated MSCs, adipocytes (A) and osteocytes (O). We showed that at the transcript level, the differentiation of MSCs towards adipocyte fate may involve MMP-2, TIMP-2 and TAZ. We also show that the differentiation of MSCs toward osteocyte fate may involve TIMP-2, MEF2a and TAZ. Our research provides preliminary data on the possible role of the MMP-2, TIMP-2 and TAZ transcripts in adipogenic differentiation and of the TIMP-2, TAZ and MEF2a transcripts in the osteogenic differentiation of porcine MSCs. We report for the first time the possible involvement of MEF2a in the osteogenesis of porcine MSCs. Our work may provide additional evidence for the MMP-independent function of TIMP-2 during osteogenesis.
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Standal, Therese, Rachelle W. Johnson, Narelle E. McGregor, Ingrid J. Poulton, Patricia W. M. Ho, T. John Martin, and Natalie A. Sims. "gp130 in late osteoblasts and osteocytes is required for PTH-induced osteoblast differentiation." Journal of Endocrinology 223, no. 2 (September 16, 2014): 181–90. http://dx.doi.org/10.1530/joe-14-0424.
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Parathyroid hormone (PTH) treatment stimulates osteoblast differentiation and bone formation, and is the only currently approved anabolic therapy for osteoporosis. In cells of the osteoblast lineage, PTH also stimulates the expression of members of the interleukin 6 (IL-6) cytokine superfamily. Although the similarity of gene targets regulated by these cytokines and PTH suggest cooperative action, the dependence of PTH anabolic action on IL-6 cytokine signaling is unknown. To determine whether cytokine signaling in the osteocyte through glycoprotein 130 (gp130), the common IL-6 superfamily receptor subunit, is required for PTH anabolic action, male mice with conditional gp130 deletion in osteocytes (Dmp1Cre.gp130f/f) and littermate controls (Dmp1Cre.gp130w/w) were treated with hPTH(1–34) (30 μg/kg 5× per week for 5 weeks). PTH dramatically increased bone formation in Dmp1Cre.gp130w/w mice, as indicated by elevated osteoblast number, osteoid surface, mineralizing surface, and increased serum N-terminal propeptide of type 1 collagen (P1NP). However, in mice with Dmp1Cre-directed deletion of gp130, PTH treatment changed none of these parameters. Impaired PTH anabolic action was associated with a 50% reduction in Pth1r mRNA levels in Dmp1Cre.gp130f/f femora compared with Dmp1Cre.gp130w/w. Furthermore, lentiviral-Cre infection of gp130f/f primary osteoblasts also lowered Pth1r mRNA levels to 16% of that observed in infected C57/BL6 cells. In conclusion, osteocytic gp130 is required to maintain PTH1R expression in the osteoblast lineage, and for the stimulation of osteoblast differentiation that occurs in response to PTH.
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Galli, C., G. Passeri, and G. M. Macaluso. "Osteocytes and WNT: the Mechanical Control of Bone Formation." Journal of Dental Research 89, no. 4 (March 3, 2010): 331–43. http://dx.doi.org/10.1177/0022034510363963.
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Mechanical loading is of pivotal importance in the maintenance of skeletal homeostasis, but the players involved in the transduction of mechanical stimuli to promote bone maintenance have long remained elusive. Osteocytes, the most abundant cells in bone, possess mechanosensing appendices stretching through a system of bone canaliculi. Mechanical stimulation plays an important role in osteocyte survival and hence in the preservation of bone mechanical properties, through the maintenance of bone hydratation. Osteocytes can also control the osteoblastic differentiation of mesenchymal precursors in response to mechanical loading by modulating WNT signaling pathways, essential regulators of cell fate and commitment, through the protein sclerostin. Mutations of Sost, the sclerostin-encoding gene, have dramatic effects on the skeleton, indicating that osteocytes may act as master regulators of bone formation and localized bone remodeling. Moreover, the development of sclerostin inhibitors is opening new possibilities for bone regeneration in orthopedics and the dental field.
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Krause, Daniela S., Keertik Fulzele, Kevin Barry, Sutada Lotinun, Roland Baron, Lynda Bonewald, Jian Q. Feng, et al. "Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling." Blood 118, no. 21 (November 18, 2011): 219. http://dx.doi.org/10.1182/blood.v118.21.219.219.
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Abstract Abstract 219 Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated. We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected. In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls. In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling. Disclosures: No relevant conflicts of interest to declare.
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Yang, Jiancheng, Dandan Dong, Xinle Luo, Jianhua Zhou, Peng Shang, and Hao Zhang. "Iron Overload-Induced Osteocyte Apoptosis Stimulates Osteoclast Differentiation Through Increasing Osteocytic RANKL Production In Vitro." Calcified Tissue International 107, no. 5 (September 29, 2020): 499–509. http://dx.doi.org/10.1007/s00223-020-00735-x.
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Xia, Yuhan, Aoi Ikedo, Ji-Won Lee, Tadahiro Iimura, Kazuki Inoue, and Yuuki Imai. "Histone H3K27 demethylase, Utx, regulates osteoblast-to-osteocyte differentiation." Biochemical and Biophysical Research Communications 590 (January 2022): 132–38. http://dx.doi.org/10.1016/j.bbrc.2021.12.102.
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Boukhechba, Florian, Thierry Balaguer, Jean-François Michiels, Karin Ackermann, Danielle Quincey, Jean-Michel Bouler, Walter Pyerin, Georges F. Carle, and Nathalie Rochet. "Human Primary Osteocyte Differentiation in a 3D Culture System." Journal of Bone and Mineral Research 24, no. 11 (November 2009): 1927–35. http://dx.doi.org/10.1359/jbmr.090517.
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Liu, Yangxi, Xiaojie Ruan, Jun Li, Bo Wang, Jie Chen, Xiaofang Wang, Pengtao Wang, and Xiaolin Tu. "The Osteocyte Stimulated by Wnt Agonist SKL2001 Is a Safe Osteogenic Niche Improving Bioactivities in a Polycaprolactone and Cell Integrated 3D Module." Cells 11, no. 5 (February 28, 2022): 831. http://dx.doi.org/10.3390/cells11050831.
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Finding and constructing an osteogenic microenvironment similar to natural bone tissue has always been a frontier topic in orthopedics. We found that osteocytes are targeting cells controlling bone anabolism produced by PTH (JBMR 2017, PMID: 27704638), and osteocytes with activated Wnt signaling orchestrate bone formation and resorption (PNAS 2015, PMID: 25605937). However, methods for taking advantage of the leading role of osteocytes in bone regeneration remain unexplored. Herein, we found that the osteocytes with SKL2001-activated Wnt signaling could be an osteogenic microenvironment (SOOME) which upregulates the expression of bone transcription factor Runx2 and Bglap and promotes the differentiation of bone marrow stromal cell ST2 into osteoblasts. Interestingly, 60 μM SKL2001 treatment of osteocytic MLO-Y4 for 24 h maintained Wnt signaling activation for three days after removal, which was sufficient to induce osteoblast differentiation. Triptonide, a Wnt inhibitor, could eliminate this differentiation. Moreover, on day 5, the Wnt signaling naturally decreased to the level of the control group, indicating that this method of Wnt-signaling induction is safe to use. We quickly verified in vivo function of SOOME to a good proximation in 3D bioprinted modules composed of reciprocally printed polycaprolactone bundles (for support) and cell bundles (for bioactivity). In the cell bundles, SOOME stably supported the growth and development of ST2 cells, the 7-day survival rate was as high as 91.6%, and proliferation ability increased linearly. Similarly, SOOME greatly promoted ST2 differentiation and mineralization for 28 days. In addition, SOOME upregulated the expression of angiopoietin 1, promoted endothelial cell migration and angiogenesis, and increased node number and total length of tubes and branches. Finally, we found that the function of SOOME could be realized through the paracrine pathway. This study reveals that osteocytes with Wnt signaling activated by SKL2001 are a safe osteogenic microenvironment. Both SOOME itself and its cell-free culture supernatant can improve bioactivity for osteoblast differentiation, with composite scaffolds especially bearing application value.
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Ben-awadh, Abdullah N., Jesus Delgado-Calle, Xiaolin Tu, Kali Kuhlenschmidt, Matthew R. Allen, Lilian I. Plotkin, and Teresita Bellido. "Parathyroid Hormone Receptor Signaling Induces Bone Resorption in the Adult Skeleton by Directly Regulating the RANKL Gene in Osteocytes." Endocrinology 155, no. 8 (August 1, 2014): 2797–809. http://dx.doi.org/10.1210/en.2014-1046.
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PTH upregulates the expression of the receptor activator of nuclear factor κB ligand (Rankl) in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell responsible for RANKL-mediated stimulation of bone resorption remains undefined. We report that constitutive activation of PTH receptor signaling only in osteocytes in transgenic mice (DMP1-caPTHR1) was sufficient to increase Rankl expression and bone resorption. Resorption in DMP1-caPTHR1 mice crossed with mice lacking the distal control region regulated by PTH in the Rankl gene (DCR−/−) was similar to DMP1-caPTHR1 mice at 1 month of age, but progressively declined to reach values undistinguishable from wild-type (WT) mice at 5 months of age. Moreover, DMP1-caPTHR1 mice exhibited low tissue material density and increased serum alkaline phosphatase activity at 5 month of age, and these indices of high remodeling were partially and totally corrected in compound DMP1-caPTHR1;DCR−/− male mice, and less affected in female mice. Rankl expression in bones from DMP1-caPTHR1 mice was elevated at both 1 and 5 months of age, whereas it was high, similar to DMP1-caPTHR1 mice at 1 month, but low, similar to WT levels at 5 months in compound mice. Moreover, PTH increased Rankl and decreased Sost and Opg expression in ex vivo bone organ cultures established from WT mice, but only regulated Sost and Opg expression in cultures from DCR−/− mice. PTH also increased RANKL expression in osteocyte-containing primary cultures of calvarial cells, in isolated murine osteocytes, and in WT but not in DCR−/− osteocyte-enriched bones. Thus, PTH upregulates Rankl expression in osteocytes in vitro, ex vivo and in vivo, and resorption induced by PTH receptor signaling in the adult skeleton requires direct regulation of the Rankl gene in osteocytes.
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Canalis, Ernesto, David Bridgewater, Lauren Schilling, and Stefano Zanotti. "Canonical Notch activation in osteocytes causes osteopetrosis." American Journal of Physiology-Endocrinology and Metabolism 310, no. 2 (January 15, 2016): E171—E182. http://dx.doi.org/10.1152/ajpendo.00395.2015.
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Activation of Notch1 in cells of the osteoblastic lineage inhibits osteoblast differentiation/function and causes osteopenia, whereas its activation in osteocytes causes a distinct osteopetrotic phenotype. To explore mechanisms responsible, we established the contributions of canonical Notch signaling (Rbpjκ dependent) to osteocyte function. Transgenics expressing Cre recombinase under the control of the dentin matrix protein-1 ( Dmp1) promoter were crossed with Rbpjκ conditional mice to generate Dmp1-Cre +/−; Rbpjκ Δ/Δ mice. These mice did not have a skeletal phenotype, indicating that Rbpjκ is dispensable for osteocyte function. To study the Rbpjκ contribution to Notch activation, Rosa Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and the NICD coding sequence, were crossed with Dmp1-Cre transgenic mice and studied in the context ( Dmp1-Cre +/−; Rosa Notch; Rbpjκ Δ/Δ) or not ( Dmp1-Cre +/−; Rosa Notch) of Rbpjκ inactivation. Dmp1-Cre +/−; Rosa Notch mice exhibited increased femoral trabecular bone volume and decreased osteoclasts and bone resorption. The phenotype was reversed in the context of the Rbpjκ inactivation, demonstrating that Notch canonical signaling was accountable for the phenotype. Notch activation downregulated Sost and Dkk1 and upregulated Axin2, Tnfrsf11b, and Tnfsf11 mRNA expression, and these effects were not observed in the context of the Rbpjκ inactivation. In conclusion, Notch activation in osteocytes suppresses bone resorption and increases bone volume by utilization of canonical signals that also result in the inhibition of Sost and Dkk1 and upregulation of Wnt signaling.
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Deedwania, Preeti, Dipika Deka, Sujata Mohanty, Vatsla Dadhwal, and Aparna Sharma. "Isolation and characterization of mesenchymal stem cells derived from amniotic fluid: A prospective study." International Journal of Molecular & Immuno Oncology 5 (May 13, 2020): 67–72. http://dx.doi.org/10.25259/ijmio_22_2019.
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Objectives: The study aims to isolate, expand, and check the feasibility and differentiation potential of amniotic fluid mesenchymal stem cell (AF-MSC) from the minimum amount of amniotic fluid. Materials and Methods: Amniotic fluid samples were collected from women undergoing 2nd trimester amniocentesis between 16 and 24 weeks of period of gestation. MSCs were isolated and characterized by MSCs surface marker profiling and were expanded in specific growth media to assess their differentiation capability into osteocytes, chondrocytes, and adipocytes. The differentiation was confirmed using specific staining. Results: The isolated AF-MSCs showed successful stem cell population for 18 samples out of 23. All the isolated AF-MSCs showed positivity for MSCs surface markers. For osteocyte differentiation, cells were cultured in osteogenic induction media for 4 weeks, and the differentiation was confirmed by staining with Alizarin Red S stain, which showed extracellular matrix mineralization. For adipocytes differentiation, the induction media exhibited lipid droplets and positive staining with Oil Red O stain. Similarly, cells cultured in chondrocytes differentiation media, showed positive staining with Alcian Blue. Conclusion: AF-MSCs have the capacity to differentiate into common mesodermal cell types. Considering their easy accessibility, amniotic fluid could be a good source for MSCs with a greater potential for cellular therapy in various chronic disabling diseases, for example, spinal cord injuries, massive bone and cartilage damage, and demyelinating diseases.
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Nieuwoudt, Mechiel, Ian Woods, Kian F. Eichholz, Carolina Martins, Kate McSweeney, Nian Shen, and David A. Hoey. "Functionalization of Electrospun Polycaprolactone Scaffolds with Matrix-Binding Osteocyte-Derived Extracellular Vesicles Promotes Osteoblastic Differentiation and Mineralization." Annals of Biomedical Engineering 49, no. 12 (October 18, 2021): 3621–35. http://dx.doi.org/10.1007/s10439-021-02872-2.
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AbstractSynthetic polymeric materials have demonstrated great promise for bone tissue engineering based on their compatibility with a wide array of scaffold-manufacturing techniques, but are limited in terms of the bioactivity when compared to naturally occurring materials. To enhance the regenerative properties of these materials, they are commonly functionalised with bioactive factors to guide growth within the developing tissue. Extracellular matrix vesicles (EVs) play an important role in facilitating endochondral ossification during long bone development and have recently emerged as important mediators of cell-cell communication coordinating bone regeneration, and thus represent an ideal target to enhance the regenerative properties of synthetic scaffolds. Therefore, in this paper we developed tools and protocols to enable the attachment of MLO-Y4 osteocyte-derived EVs onto electrospun polycaprolactone (PCL) scaffolds for bone repair. Initially, we optimize a method for the functionalization of PCL materials with collagen type-1 and fibronectin, inspired by the behaviour of matrix vesicles during endochondral ossification, and demonstrate that this is an effective method for the adhesion of EVs to the material surface. We then used this functionalization process to attach osteogenic EVs, collected from mechanically stimulated MLO-Y4 osteocytes, to collagen-coated electrospun PCL scaffolds. The EV-functionalized scaffold promoted osteogenic differentiation (measured by increased ALP activity) and mineralization of the matrix. In particular, EV-functionalised scaffolds exhibited significant increases in matrix mineralization particularly at earlier time points compared to uncoated and collagen-coated controls. This approach to matrix-based adhesion of EVs provides a mechanism for incorporating vesicle signalling into polyester scaffolds and demonstrates the potential of osteocyte derived EVs to enhance the rate of bone tissue regeneration.
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Xu, Linshan, Yuyang Wang, Jianping Wang, Jianglong Zhai, Li Ren, and Guoying Zhu. "Radiation-Induced Osteocyte Senescence Alters Bone Marrow Mesenchymal Stem Cell Differentiation Potential via Paracrine Signaling." International Journal of Molecular Sciences 22, no. 17 (August 28, 2021): 9323. http://dx.doi.org/10.3390/ijms22179323.
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Cellular senescence and its senescence-associated secretory phenotype (SASP) are widely regarded as promising therapeutic targets for aging-related diseases, such as osteoporosis. However, the expression pattern of cellular senescence and multiple SASP secretion remains unclear, thus leaving a large gap in the knowledge for a desirable intervention targeting cellular senescence. Therefore, there is a critical need to understand the molecular mechanism of SASP secretion in the bone microenvironment that can ameliorate aging-related degenerative pathologies including osteoporosis. In this study, osteocyte-like cells (MLO-Y4) were induced to cellular senescence by 2 Gy γ-rays; then, senescence phenotype changes and adverse effects of SASP on bone marrow mesenchymal stem cell (BMSC) differentiation potential were investigated. The results revealed that 2 Gy irradiation could hinder cell viability, shorten cell dendrites, and induce cellular senescence, as evidenced by the higher expression of senescence markers p16 and p21 and the elevated formation of senescence-associated heterochromatin foci (SAHF), which was accompanied by the enhanced secretion of SASP markers such as IL-1α, IL-6, MMP-3, IGFBP-6, resistin, and adiponectin. When 0.8 μM JAK1 inhibitors were added to block SASP secretion, the higher expression of SASP was blunted, but the inhibition in osteogenic and adipogenic differentiation potential of BMSCs co-cultured with irradiated MLO-Y4 cell conditioned medium (CM- 2 Gy) was alleviated. These results suggest that senescent osteocytes can perturb BMSCs’ differential potential via the paracrine signaling of SASP, which was also demonstrated by in vivo experiments. In conclusion, we identified the SASP factor partially responsible for the degenerative differentiation of BMSCs, which allowed us to hypothesize that senescent osteocytes and their SASPs may contribute to radiation-induced bone loss.
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Marlina, Marlina, Rizki Rahmadian, Armenia Armenia, Wahyu Widowati, Rizal Rizal, Hanna Sari Widya Kusuma, Satrio Haryo Benowo Wibowo, Wahyu Setia Widodo, and Ika Adhani Sholihah. "Isolation, Characterization, Proliferation and Differentiation of Synovial Membrane-derived Mesenchymal Stem Cells (SM-MSCs) from Osteoarthritis Patients." Molecular and Cellular Biomedical Sciences 4, no. 2 (July 1, 2020): 76. http://dx.doi.org/10.21705/mcbs.v4i2.100.
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Background: Mesenchymal stem cells (MSCs) are the cells which has high renewal capacity and and are capable for differentiating into some types of cells. MSCs can be obtained from several tissues including bone marrow, synovial membrane, blood, adipose tissue and periosteum. The proliferation and self-repair ability of MSCs are the advantages to use as stem cells-based therapy of various diseases. The aim of this study was to determine the differentiation, characterization and priliferation of synovial membrane-derived MSCs (SM-MSCs).Materials and Methods: The cells proliferation capacity was determined by cell counting using trypan blue, characterization of MSCs (cluster of differentiation (CD)90, CD11b, CD73, CD34, CD19, CD45, CD105 and human leukocyte antigen-DR isotype (HLA-DR)) using flow cytometry analysis, and differentiation capability into three lineage cells was determined with red alcian blue, oil red O and alizarin staining.Results: The type culture of SM-MSCs was adherent and showed positive CD44, CD105, CD73, CD90 and negative of CD19, HLA-DR, CD11b, CD45, CD34 surface marker. Based on the result, SM-MSCs P3 showed differentiation potency into adipogenic, chondrogenic, and osteogenic lineage cells. The population doubling time of SM-MSCs has increased from P3 to P8. The population doubling time of SM-MSCs P3 was 1.69 days and SM-MSCs P8 was 3.64 days.Conclusion: The results indicated that SM-MCSCs from osteoarthritis patients are able to differentiate into osteocytes, chondrocytes, adipocytes and highly express of CD105, CD73, CD90, CD44 and negative for CD34, CD45, CD14, CD19.Keywords: synovial membrane, mesenchymal stromal cells, adipocyte, chondrocyte, osteocyte
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Boukhechba, F., J. Michiels, K. Ackermann, D. Quincey, J. Bouler, W. Pyerin, G. F. Carle, and N. Rochet. "Human primary osteocyte differentiation in a three-dimensional culture system." Bone 44 (June 2009): S301. http://dx.doi.org/10.1016/j.bone.2009.03.551.
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Ikpegbu, Ekele, Lena Basta, Dylan N. Clements, Robert Fleming, Tonia L. Vincent, David J. Buttle, Andrew A. Pitsillides, Katherine A. Staines, and Colin Farquharson. "FGF-2 promotes osteocyte differentiation through increased E11/podoplanin expression." Journal of Cellular Physiology 233, no. 7 (January 23, 2018): 5334–47. http://dx.doi.org/10.1002/jcp.26345.
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Prideaux, M., A. Pitsillides, N. Loveridge, and C. Farquharson. "The role of matrix mineralisation in osteoblast to osteocyte differentiation." Bone 47 (June 2010): S147. http://dx.doi.org/10.1016/j.bone.2010.04.334.
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Loy, Thomas Leonhard, David Vehlow, Vivien Kauschke, Martin Müller, Christian Heiss, and Katrin Susanne Lips. "Effects of BDNF and PEC Nanoparticles on Osteocytes." Molecules 25, no. 18 (September 10, 2020): 4151. http://dx.doi.org/10.3390/molecules25184151.
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Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.
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Suvannasankha, Attaya, Colin D. Crean, Douglas R. Tompkins, Jesus Delgado-Calle, Teresita M. Bellido, G. David Roodman, and John M. Chirgwin. "Regulation of Osteoblast Function in Myeloma Bone Disease By Semaphorin 4D." Blood 128, no. 22 (December 2, 2016): 4439. http://dx.doi.org/10.1182/blood.v128.22.4439.4439.
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Abstract Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D. We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease. After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression. Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. Disclosures Roodman: Amgen: Consultancy.
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Mullen, C. A., M. G. Haugh, M. B. Schaffler, R. J. Majeska, and L. M. McNamara. "Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation." Journal of the Mechanical Behavior of Biomedical Materials 28 (December 2013): 183–94. http://dx.doi.org/10.1016/j.jmbbm.2013.06.013.
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Mikuni-Takagaki, Yuko, Yoshio Kakai, Masanori Satoyoshi, Eiji Kawano, Yoshihisa Suzuki, Toshio Kawase, and Shigeru Saito. "Matrix mineralization and the differentiation of osteocyte-like cells in culture." Journal of Bone and Mineral Research 10, no. 2 (December 3, 2009): 231–42. http://dx.doi.org/10.1002/jbmr.5650100209.
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Liu, Shiguang, Jianping Zhou, Wen Tang, Xi Jiang, David W. Rowe, and L. Darryl Quarles. "Pathogenic role of Fgf23 in Hyp mice." American Journal of Physiology-Endocrinology and Metabolism 291, no. 1 (July 2006): E38—E49. http://dx.doi.org/10.1152/ajpendo.00008.2006.
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Inactivating mutations of the PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) endopeptidase, the disease-causing gene in X-linked hypophosphatemia (XLH), results in increased circulating levels of fibroblastic growth factor-23 (FGF23), a bone-derived phosphaturic factor. To determine the causal role of FGF23 in XLH, we generated a combined Fgf23-deficient enhanced green fluorescent protein (eGFP) reporter and Phex-deficient Hyp mouse model ( Fgf23+/−/ Hyp). eGFP expression was expressed in osteocytes embedded in bone that exhibited marked upregulation of eGFP in response to Phex deficiency and in CD31-positive cells in bone marrow venules that expressed low eGFP levels independently of Phex. In bone marrow stromal cells (BMSCs) derived from Fgf23−/−/ Hyp mice, eGFP expression was also selectively increased in osteocyte-like cells within mineralization nodules and detected in low levels in CD31-positive cells. Surprisingly, eGFP expression was not increased in cell surface osteoblasts, indicating that Phex deficiency is necessary but not sufficient for increased Fgf23 expression in the osteoblast lineage. Additional factors, associated with either osteocyte differentiation and/or extracellular matrix, are necessary for Phex deficiency to stimulate Fgf23 gene transcription in bone. Regardless, the deletion of Fgf23 from Hyp mice reversed the hypophosphatemia, abnormal 1,25(OH)2D3 levels, rickets, and osteomalacia associated with Phex deficiency. These results suggest that Fgf23 acts downstream of Phex to cause both the renal and bone phenotypes in Hyp mice.
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Gorabi, Armita Mahdavi, Nasim Kiaie, Saeideh Hajighasemi, Tannaz Jamialahmadi, Muhammed Majeed, and Amirhossein Sahebkar. "The Effect of Curcumin on the Differentiation of Mesenchymal Stem Cells into Mesodermal Lineage." Molecules 24, no. 22 (November 7, 2019): 4029. http://dx.doi.org/10.3390/molecules24224029.
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Curcumin has been placed at the forefront of the researcher’s attention due to its pleiotropic pharmacological effects and health benefits. A considerable volume of articles has pointed out curcumin’s effects on the fate of stem cell differentiation. In this review, a descriptive mechanism of how curcumin affects the outcome of the differentiation of mesenchymal stem cells (MSCs) into the mesodermal lineage—i.e., adipocyte, osteocyte, and chondrocyte differentiation—is compiled from the literature. The sections include the mechanism of inhibition or induction of MSCs differentiation to each lineage, their governing molecular mechanisms, and their signal transduction pathways. The effect of different curcumin doses and its structural modifications on the MSCs differentiation is also discussed.
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Ansari, Sana, Keita Ito, and Sandra Hofmann. "Cell Sources for Human In vitro Bone Models." Current Osteoporosis Reports 19, no. 1 (January 15, 2021): 88–100. http://dx.doi.org/10.1007/s11914-020-00648-6.
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Abstract Purpose of Review One aim in bone tissue engineering is to develop human cell-based, 3D in vitro bone models to study bone physiology and pathology. Due to the heterogeneity of cells among patients, patient’s own cells are needed to be obtained, ideally, from one single cell source. This review attempts to identify the appropriate cell sources for development of such models. Recent Findings Bone marrow and peripheral blood are considered as suitable sources for extraction of osteoblast/osteocyte and osteoclast progenitor cells. Recent studies on these cell sources have shown no significant differences between isolated progenitor cells. However, various parameters such as medium composition affect the cell’s proliferation and differentiation potential which could make the peripheral blood-derived stem cells superior to the ones from bone marrow. Summary Peripheral blood can be considered a suitable source for osteoblast/osteocyte and osteoclast progenitor cells, being less invasive for the patient. However, more investigations are needed focusing on extraction and differentiation of both cell types from the same donor sample of peripheral blood.
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Pulcini, Stefano, Lucia Merolle, Chiara Marraccini, Eleonora Quartieri, Daniele Mori, Davide Schiroli, Pamela Berni, et al. "Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential." International Journal of Molecular Sciences 22, no. 16 (August 16, 2021): 8764. http://dx.doi.org/10.3390/ijms22168764.
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Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.
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Giuliani, Nicola, Marzia Ferretti, Manuela Abeltino, Cristina Mancini, Eugenia Martella, Benedetta Dalla Palma, Marina Bolzoni, et al. "Increased Osteocyte Apoptosis in Multiple Myeloma Patients: A Potential Role in Bone Remodeling Alterations Related to Osteolytic Bone Lesions." Blood 114, no. 22 (November 20, 2009): 830. http://dx.doi.org/10.1182/blood.v114.22.830.830.
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Abstract Abstract 830 Osteocytes are non-proliferative differentiated cells of osteogenic lineage located in the bony lacuna/canalicular system of bone and that have been recently hypothesized to regulate local bone remodeling in part through the cell death and apoptosis. A reduction of osteocyte viability has been demonstrated in osteoporotic bone related to estrogen deficiency and glucorticoid administration. As known, multiple myeloma (MM)–induced osteolysis and/or osteoporosis are characterized by severely imbalanced and uncoupled bone remodeling due to increased osteoclast recruitment and suppressed osteoblast differentiation that occur in close contact with MM cells infiltration. Actually, the potential involvement of osteocytes in bone remodeling alterations occurring in MM patients is not known and it has been investigated in the present study. Firstly we performed histological analysis on bone biopsies obtained from iliac crest in a cohort of 34 patients with MM at the diagnosis (ISS I-III, mean age±SD: 72±10), 52% of them with osteolytic bone lesions at the skeletal survey and 10 patients with monoclonal gammopathy of uncertain significance (MGUS) (mean age± SD: 71±14). Sex-aged matched subjects without hematological malignancies or osteoporosis or metabolic bone disease (n°=10) were also analyzed. Histological analyses were performed on Toluidine blu and Gomori's three-chromic stained sections observed under a Light Microscope (LM). On a total of 500 osteocyte lacunae X section we evaluated both the number of viable osteocytes and the number of dead/degenerated/apoptotic osteocytes together with the number of empty lacunae. We found a significant reduction in the percentage of viable osteocytes in MM patients as compared to healthy controls (% of viable osteocytes: mean ± SD: 25±10% vs. 36±9%; p= 0.003) whereas the difference between MM and MGUS did not reach a statistical significance (25±10% vs. 30.5±16%; p= 0.2). Consistently the number of death osteocytes and empty lacunae was significantly increased in MM vs. healthy subjects (mean ± SD/500 lacunae: 371.3± 52 vs. 315±46; p=0.006) but not as compared to MGUS (371.3±52 vs 337±81; p=0.2). As regard the skeletal involvement in MM patients we found that the percent of viable osteocytes was significantly lower in osteolytic vs. non-osteolytic patients (19±9% vs. 30±7%; p= 0.01) as well as the number of death osteocytes and empty lacunae was higher in osteolytic vs. non-osteolytic patients (mean ± SD/500 lacunae: 394±41 vs. 315±55; p=0.04). Following, in order to verify the occurrence of apoptosis both LM and Transmission Electron Microscopy (TEM) observations were also performed. Under LM, TUNEL analysis showed higher presence of apoptotic cells in those specimens obtained from MM patients in comparison with those obtained from healthy controls. Moreover an increase in the number of apoptotic osteocytes was observed in MM with bone lesions as compared to those without osteolysis. These observations were further confirmed in vitro by ultrastructural analysis on mono-layers of human preosteocyte cells (HOB-01-C1) incubated with or without conditioned media (CM) taken from human myeloma cell lines (HMCLs) KMS12 and JJN3 or co-cultured with them. TEM observations, showed cells in apoptosis with apoptotic bodies and degenerated non-apoptotic cells in preosteocytes treated with HMCL-CM or co-cultured with HMCLs as compared to non-treated cells. Interestingly, we also found that CM of preosteocytes co-cultured with HMCLs but not those of non co-cultured cells significantly increased CD14+ -derived osteoclastogenesis evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit-forming assay. In conclusion our data demonstrate that MM bone is characterized by a reduction of viable osteocytes and an increase of osteocyte apoptosis in relation to the presence of bone lesions that may represent a triggering event to the increase of osteoclast recruitment in MM patients. Disclosures: No relevant conflicts of interest to declare.
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Mc Garrigle, MJ, CA Mullen, MG Haugh, MC Voisin, and LM McNamara. "Osteocyte differentiation and the formation of an interconnected cellular network in vitro." European Cells and Materials 31 (May 24, 2016): 323–40. http://dx.doi.org/10.22203/ecm.v031a21.
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Moriishi, Takeshi, Zenjiro Maruyama, Ryo Fukuyama, Masako Ito, Toshihiro Miyazaki, Hideki Kitaura, Hidetake Ohnishi, et al. "Overexpression of Bcl2 in Osteoblasts Inhibits Osteoblast Differentiation and Induces Osteocyte Apoptosis." PLoS ONE 6, no. 11 (November 17, 2011): e27487. http://dx.doi.org/10.1371/journal.pone.0027487.
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Ikpegbu, E., L. Basta, D. Clements, T. L. Vincent, D. J. Buttle, A. A. Pitsillides, K. A. Staines, and C. Farquharson. "FGF-2 Promotes osteocyte differentiation in vitro through increased e11/podoplanin expression." Osteoarthritis and Cartilage 25 (April 2017): S141—S142. http://dx.doi.org/10.1016/j.joca.2017.02.231.
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