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Sample records for stromal osteoblast function

  1. TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

    Science.gov (United States)

    Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle

  2. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

    Full Text Available Haematopoietic stem cell (HSC transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK haematopoietic stem/progenitor cells (HPC demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

  3. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

    Science.gov (United States)

    Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  4. Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces.

    NARCIS (Netherlands)

    Sikavitsas, V.I.; Bancroft, G.N.; Holtorf, H.L.; Jansen, J.A.; Mikos, A.G.

    2003-01-01

    In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media

  5. The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts.

    Science.gov (United States)

    Özdal-Kurt, F; Tuğlu, I; Vatansever, H S; Tong, S; Şen, B H; Deliloğlu-Gürhan, S I

    2016-08-01

    We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.

  6. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

  7. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  8. Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

    Science.gov (United States)

    Zhang, Jingzhu; Link, Daniel C

    2016-11-01

    The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

  9. Serotonin regulates osteoblast proliferation and function in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Dai, S.Q.; Yu, L.P. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Shi, X. [Department of Obstetrics and Gynecology, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China); Wu, H. [Emergency Department, The First Affiliated Hospital, Soochow University, Suzhou (China); Shao, P.; Yin, G.Y.; Wei, Y.Z. [Department of Orthopedic Surgery, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu (China)

    2014-08-01

    The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT{sub 1A}, 5-HT{sub 1B}, 5-HT{sub 1D}, 5-HT{sub 2A}, 5-HT{sub 2B}, and 5-HT{sub 2C}) were found to exist in rat osteoblasts. Of these, 5-HT{sub 2A} and 5-HT{sub 1B} receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  10. Serotonin regulates osteoblast proliferation and function in vitro

    Directory of Open Access Journals (Sweden)

    S.Q. Dai

    2014-09-01

    Full Text Available The monoamine serotonin (5-hydroxytryptamine, 5-HT, a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, and 5-HT2C were found to exist in rat osteoblasts. Of these, 5-HT2A and 5-HT1B receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.

  11. MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Holmstrøm, Kim; Qiu, Weimin

    2014-01-01

    of miR-34a. siRNA-mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25 homolog......Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified mi...

  12. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ...... with OP showed a pattern of differentiation similar to those of age-matched controls. In conclusion, MSCs maintain their differentiation potential during aging and in patients with OP. Other mechanisms responsible for age-related decrease in bone formation need to be determined....

  13. Altered osteoblast structure and function in parabolic flight

    Science.gov (United States)

    Zhong-Quan, Dai; Ying-Hui, Li; Fen, Yang; Bai, Ding; Ying-Jun, Tan

    Introduction Bone loss has a significant impact on astronauts during spaceflight being one of the main obstacles preventing interplanetary missions However the exact mechanism is not well understood In the present study we investigated the effects of acute gravitational changes generated by parabolic flight on the structure and function of osteoblasts ROS17 2 8 carried by airbus A300 Methods The alteration of microfilament cytoskeleton was observed by the Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I immunofluorescence stain ALP activity and expression COL1A1 expression osteocalcin secrete which presenting the osteoblast function were detected by modified calcium and cobalt method RT-PCR and radioimmunity methods respectively Results The changed gravity induced the reorganization of microfilament cytoskeleton of osteoblast After 3 hours parabolic flight F-actin of osteoblast cytoskeleton became more thickness and directivity whereas G-actin reduced and relatively concentrated at the edge of nucleus observed by confocal fluorescence microscopy This phenomenon is identical with structure alternation observed in hypergravity but the osteoblast function decrease The excretion of osteocalcin the activity and mRNA expression of ALP decrease but the COL1A1 expression has no changes These results were similar to the changes in simulated or real microgravity Conclusion Above results suggest that short time gravity alternative change induce osteoblast structure and function

  14. CD146/MCAM defines functionality of human bone marrow stromal stem cell populations

    DEFF Research Database (Denmark)

    Harkness, Linda; Zaher, Walid; Ditzel, Nicholas

    2016-01-01

    BACKGROUND: Identification of surface markers for prospective isolation of functionally homogenous populations of human skeletal (stromal, mesenchymal) stem cells (hMSCs) is highly relevant for cell therapy protocols. Thus, we examined the possible use of CD146 to subtype a heterogeneous h......MSC population. METHODS: Using flow cytometry and cell sorting, we isolated two distinct hMSC-CD146(+) and hMSC-CD146(-) cell populations from the telomerized human bone marrow-derived stromal cell line (hMSC-TERT). Cells were examined for differences in their size, shape and texture by using high......-content analysis and additionally for their ability to differentiate toward osteogenesis in vitro and form bone in vivo, and their migrational ability in vivo and in vitro was investigated. RESULTS: In vitro, the two cell populations exhibited similar growth rate and differentiation capacity to osteoblasts...

  15. Genetic analysis of Lrp5 function in osteoblast progenitors.

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    Yadav, Vijay K; Arantes, Henrique Pierotti; Barros, Elizabete Ribeiro; Lazaretti-Castro, Marise; Ducy, Patricia

    2010-05-01

    The low-density lipoprotein receptor-related protein (Lrp)-5 regulates osteoblast proliferation and bone formation through its expression in duodenum by modifying the gut serotonin-bone endocrine axis. However, its direct role, if any, in osteoblast progenitor cells has not been studied thus far. Here, we show that mice with a Dermo1-Cre-mediated disruption of Lrp5 in osteoblast progenitor cells have normal embryonic skeletogenesis and normal skeletal growth and development postnatally. Histomorphometric analysis of 3-month-old adult mice revealed normal osteoblast numbers, bone formation rate, and bone mass in Lrp5(Dermo)(-/-) mice. In addition, analysis of two osteoporosis pseudoglioma (OPPG) patients revealed a three- to fivefold increase in their serum serotonin levels compared to age-matched controls. These results rule out a direct function of Lrp5 in osteoblast progenitor cells and add further support to the notion that dysregulation of serotonin synthesis is involved in bone mass abnormalities observed in OPPG patients.

  16. Nanostructured magnesium has fewer detrimental effects on osteoblast function

    Science.gov (United States)

    Weng, Lucy; Webster, Thomas J

    2013-01-01

    Efforts have been made recently to implement nanoscale surface features on magnesium, a biodegradable metal, to increase bone formation. Compared with normal magnesium, nanostructured magnesium has unique characteristics, including increased grain boundary properties, surface to volume ratio, surface roughness, and surface energy, which may influence the initial adsorption of proteins known to promote the function of osteoblasts (bone-forming cells). Previous studies have shown that one way to increase nanosurface roughness on magnesium is to soak the metal in NaOH. However, it has not been determined if degradation of magnesium is altered by creating nanoscale features on its surface to influence osteoblast density. The aim of the present in vitro study was to determine the influence of degradation of nanostructured magnesium, created by soaking in NaOH, on osteoblast density. Our results showed a less detrimental effect of magnesium degradation on osteoblast density when magnesium was treated with NaOH to create nanoscale surface features. The detrimental degradation products of magnesium are of significant concern when considering use of magnesium as an orthopedic implant material, and this study identified a surface treatment, ie, soaking in NaOH to create nanoscale features for magnesium that can improve its use in numerous orthopedic applications. PMID:23674891

  17. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

    Science.gov (United States)

    Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

    2002-01-01

    Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

  18. Increased osteoblast adhesion on nanoparticulate crystalline hydroxyapatite functionalized with KRSR.

    Science.gov (United States)

    Nelson, Michael; Balasundaram, Ganesan; Webster, Thomas J

    2006-01-01

    The present in vitro study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate for novel orthopedic applications. Specifically, nano-crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by hydrothermal treatment for 2 hours at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of control conventional HA particles (or those currently used in orthopedics with micron diameters), the aforementioned calcium phosphate particles were pressed into compacts and sintered at 1100 degrees C for 2 hours. All calcium phosphate-based particles were fully characterized. Results showed that although there was an initial weight gain for all the compacts studied in this experiment, higher eventual degradation rates up to 3 weeks were observed for nano-amorphous calcium phosphate compared with nano-crystalline HA which was higher than conventional HA. Peptide functionalization (with the cell adhesive peptide lysine-arginine-serine-arginine [KRSR] and the non-cell-adhesive peptide lysine-serine-arginine-arginine [KSRR]) was accomplished by means of a three-step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The peptide functionalization was fully characterized. Results demonstrated increased osteoblast (bone-forming cell) adhesion on non-functionalized and functionalized nano-crystalline HA compacts compared with nano amorphous calcium phosphate compacts; both increased osteoblast adhesion compared with conventional HA. To further exemplify the novel properties of nano crystalline HA, results also showed similar osteoblast adhesion between non-functionalized nano crystalline HA and KRSR functionalized conventional HA. Thus, results provided evidence that nanocrystalline HA should be further studied for

  19. Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells

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    Tang, T; Zhang, G; PY Lau, Carol; Zheng, L Z; Xie, X H; Wang, X L; Patrick, Y; Qin, L; Kumta, Shekhar M [Department of Orthopaedics and Traumatology, Chinese University of Hong Kong (Hong Kong); Wang, X H; He, K, E-mail: kumta@cuhk.edu.hk [Department of Mechanical Engineering, Institute of Bio-manufacturing Engineering, Tsinghua University, Beijing (China)

    2011-02-15

    Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 {yields} 4)-2-deoxy-2-sulfoamino-{beta}-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 {mu}g ml{sup -1} at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 {mu}g ml{sup -1}) stimulates cell proliferation while a higher dose (1000 {mu}g ml{sup -1}) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

  20. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells.

    Science.gov (United States)

    Geyh, S; Oz, S; Cadeddu, R-P; Fröbel, J; Brückner, B; Kündgen, A; Fenk, R; Bruns, I; Zilkens, C; Hermsen, D; Gattermann, N; Kobbe, G; Germing, U; Lyko, F; Haas, R; Schroeder, T

    2013-09-01

    Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

  1. Sulfated glycosaminoglycans support osteoblast functions and concurrently suppress osteoclasts.

    Science.gov (United States)

    Salbach-Hirsch, Juliane; Ziegler, Nicole; Thiele, Sylvia; Moeller, Stephanie; Schnabelrauch, Matthias; Hintze, Vera; Scharnweber, Dieter; Rauner, Martina; Hofbauer, Lorenz C

    2014-06-01

    In order to improve bone regeneration, development and evaluation of new adaptive biomaterials is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major extracellular matrix (ECM) components of bone, and display osteogenic properties that are potentially useful for biomaterial applications. Using native and synthetic sulfate-modified GAGs, we manufactured artificial collagen/GAG ECM (aECMs) coatings, and evaluated how the presence of GAGs and their degree of sulfation affects the differentiation of murine mesenchymal stem cells to osteoblasts (OB) cultivated on these aECMs. GAG sulfation regulated osteogenesis at all key steps of OB development. Adhesion, but not migration, was diminished by 50% (P osteogenesis and suppressing their paracrine support of OC functions, thus displaying a favorable profile on bone remodeling. Whether these cellular properties translate into improved bone regeneration needs to be validated in vivo. © 2014 Wiley Periodicals, Inc.

  2. Focal Adhesion of Osteoblastic Cells on Titanium Surface with Amine Functionalities Formed by Plasma Polymerization

    Science.gov (United States)

    Song, Heesang; Jung, Sang Chul; Kim, Byung Hoon

    2012-08-01

    To enhance the focal adhesion of osteoblastic cells on a titanium surface, plasma polymerized allyl amine (AAm) thin films were deposited by plasma polymerization. This plasma polymer functionalization of titanium is advantageous for osteoblastic focal adhesion formation. Such Ti surfaces are useful for the fabrication of titanium-based dental implants for enhancement of osseointegration.

  3. Evaluation of sericin as a fetal bovine serum-replacing cryoprotectant during freezing of human mesenchymal stromal cells and human osteoblast-like cells.

    Science.gov (United States)

    Verdanova, Martina; Pytlik, Robert; Kalbacova, Marie Hubalek

    2014-04-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

  4. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation.

    Science.gov (United States)

    Selvamurugan, Nagarajan; He, Zhiming; Rifkin, Daniel; Dabovic, Branka; Partridge, Nicola C

    2017-01-01

    Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF- β ) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF- β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF- β signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF- β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  5. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    Science.gov (United States)

    Rifkin, Daniel; Dabovic, Branka

    2017-01-01

    Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs. PMID:28512472

  6. The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis

    Directory of Open Access Journals (Sweden)

    Neiva K.

    2005-01-01

    Full Text Available Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1 and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

  7. Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    Directory of Open Access Journals (Sweden)

    Nagarajan Selvamurugan

    2017-01-01

    Full Text Available Pulsed electromagnetic fields (PEMFs have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-β signaling pathway and microRNA 21 (miR21 were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-β signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-β signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-β signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

  8. [Study on proliferation and function of periodontal ligament fibroblasts and osteoblastic cells under hypoxia].

    Science.gov (United States)

    Kubota, M

    1989-12-01

    There have been many reports recognizing vascular changes on pressure side of periodontal tissues during orthodontic tooth movement. The vascular changes cause local hypoxia which seems to affect the phenotypes of periodontal tissue cells. In order to clarify the effect of hypoxia on proliferation and function of periodontal tissue cells, DNA content, alkaline phosphatase (ALP) activity and prostaglandin E2 (PGE2) production under a hypoxic condition in both periodontal ligament fibroblasts (PLF) and osteoblastic cells (MC3T3-E1 cells) were examined in vitro. PLF were cultured from human periodontium and identified by both morphologic characterization and presence of ALP. The results obtained were as follows: 1. Under 10% O2 condition, the activity of proliferation in PLF did not change but that of osteoblasts was inhibited. 2. ALP activity in PLF was stimulated but that of osteoblasts was inhibited under the hypoxic condition. 3. Production of PGE2 in osteoblasts increased after 7 days of hypoxia though that in PLF decreased. In addition, the enhancement of PGE2 production in osteoblasts was due to activation of both phospholipase A2 and PGE2-synthesizing enzymes. 4. From the orthodontic point of view, hypoxia on the pressure side may induce bone resorption by inhibiting mineralization activity of osteoblasts and enhancing production of PGE2 in osteoblasts.

  9. Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Rattan, Suresh; Clark, Brian F.C.

    2001-01-01

    is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change...... in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic......Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long...

  10. Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insuli...

  11. MiR-214 regulates the function of osteoblast under simulated microgravity by targeting ATF4

    Science.gov (United States)

    Li, Yingxian; Wang, Xiaogang; Li, Qi; Lv, Ke; Wan, Yumin; Li, Yinghui; Bai, Yanqiang

    Background: MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3'-untranslated region (UTR) of mRNAs, resulting in translational repression. Growing evidence shows that microRNAs (miRNAs) regu-late various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; How-ever, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast dif-ferentiation remains elusive. Methods: To study the potential involvement of miRNAs in osteoblast differentiation under stimulated microgravity, we analyzed the expression of 20 bone relative miRNAs using real time PCR platform to find particularly miRNAs whose expression is altered during osteoblast differentiation. TargetScan, miRBase and Miranda were used to predict the target gene of candidate miRNA. To investigate whether ATF4 can be directly targeted by miR-214, we engineered luciferase reporters that have either the wild-type 3'UTRs of these genes, or the mutant UTRs with a 6 base pair (bp) deletion in the target sites. Lastly, to address the in vivo role of miR-214 in bone formation, tail suspension mice model was used to simulate the change of osteoblast function and bone loss. Results: Recent studies have sug-gested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify miR-214 in MC3T3-E1 cells, which is a primary mouse osteoblasts cell line, to promote osteoblast differentiation by repressing Activating Transcription Factor4 (ATF4) ex-pression at the posttranscriptional level. What is more, miR-214 was found to be transcribed in C2C12 cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-214 attenuated BMP2-induced osteoblastogenesis

  12. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

    2007-01-01

    The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganizat......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express u......PAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected...

  13. Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

    Science.gov (United States)

    Rath, Subha N; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E; Kneser, Ulrich

    2012-10-01

    In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  14. P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

    DEFF Research Database (Denmark)

    Agrawal, Ankita; Henriksen, Zanne; Syberg, Susanne

    2017-01-01

    The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells...... however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report...... that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form...

  15. Osteoblast functions in functionally graded Ti-6Al-4 V mesh structures.

    Science.gov (United States)

    Nune, K C; Kumar, A; Misra, R D K; Li, S J; Hao, Y L; Yang, R

    2016-03-01

    We describe here the combined efforts of engineering and biological sciences as a systemic approach to fundamentally elucidate osteoblast functions in functionally graded Ti-6Al-4 V mesh structures in relation to uniform/monolithic mesh arrays. First, the interconnecting porous architecture of functionally graded mesh arrays was conducive to cellular functions including attachment, proliferation, and mineralization. The underlying reason is that the graded fabricated structure with cells seeded from the large pore size side provided a channel for efficient transfer of nutrients to other end of the structure (small pore size), leading to the generation of mineralized extracellular matrix by differentiating pre-osteoblasts. Second, a comparative and parametric study indicated that gradient mesh structure had a pronounced effect on cell adhesion and mineralization, and strongly influenced the proliferation phase. High intensity and near-uniform distribution of proteins (actin and vinculin) on struts of the gradient mesh structure (cells seeded from large pore side) implied signal transduction during cell adhesion and was responsible for superior cellular activity, in comparison to the uniform mesh structure and non-porous titanium alloy. Cells adhered to the mesh struts by forming a sheet, bridging the pores through numerous cytoplasmic extensions, in the case of porous mesh structures. Intercellular interaction in porous structures provided a pathway for cells to communicate and mature to a differentiated phenotype. Furthermore, the capability of cells to migrate through the interconnecting porous architecture on mesh structures led to colonization of the entire structure. Cells were embedded layer-by-layer in the extracellular matrix as the matrix mineralized. The outcomes of the study are expected to address challenges associated with the treatment of segmental bone defects and bone-remodeling through favorable modulation of cellular response. Moreover, the study

  16. Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD.

    Science.gov (United States)

    Balasundaram, Ganesan; Sato, Michiko; Webster, Thomas J

    2006-05-01

    Better materials are needed to promote bone growth. For this reason, the present study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate compacts functionalized with the arginine-glycine-aspartic acid (RGD) peptide sequence. Crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by a hydrothermal treatment for 2 h at 200 degrees C and 70 degrees C, respectively. Resulting particles were then pressed into compacts. For the preparation of conventional HA particles (or those with micron diameters), the aforementioned pressed compacts were sintered at 1,100 degrees C for 2 h. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by a novel 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence technique. For all materials, results showed that the immobilization of the cell adhesive RGD sequence increased osteoblast (bone-forming cell) adhesion compared to those non-functionalized and those functionalized with the noncell adhesive control peptide (RGE) after 4 h. However, surprisingly, results also showed that the adhesion of osteoblasts on non-functionalized amorphous nanoparticulate calcium phosphate was similar to conventional HA functionalized with RGD. Osteoblast adhesion on nanocrystalline HA (unfunctionalized and functionalized with RGD) was below that of the respective functionalized amorphous calcium phosphate but above that of the respective functionalized conventional HA. In this manner, results of this study suggest that decreasing the particulate size into the nanometer regime and reducing crystallinity of calcium phosphate based materials may promote osteoblast adhesion to the same degree as the well

  17. Skeletal (stromal) stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Kermani, Abbas Jafari; Zaher, Walid

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy....../preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone....

  18. Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function.

    Science.gov (United States)

    Mohamad, Safa F; Xu, Linlin; Ghosh, Joydeep; Childress, Paul J; Abeysekera, Irushi; Himes, Evan R; Wu, Hao; Alvarez, Marta B; Davis, Korbin M; Aguilar-Perez, Alexandra; Hong, Jung Min; Bruzzaniti, Angela; Kacena, Melissa A; Srour, Edward F

    2017-12-12

    Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45 + F4/80 + cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

  19. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds

    Science.gov (United States)

    Goldstein, A. S.; Juarez, T. M.; Helmke, C. D.; Gustin, M. C.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Culture of seeded osteoblastic cells in three-dimensional osteoconductive scaffolds in vitro is a promising approach to produce an osteoinductive material for repair of bone defects. However, culture of cells in scaffolds sufficiently large to bridge critical-sized defects is a challenge for tissue engineers. Diffusion may not be sufficient to supply nutrients into large scaffolds and consequently cells may grow preferentially at the periphery under static culture conditions. Three alternative culturing schemes that convect media were considered: a spinner flask, a rotary vessel, and a perfusion flow system. Poly(DL-lactic-co-glycolic acid) (PLGA) foam discs (12.7 mm diameter, 6.0 mm thick, 78.8% porous) were seeded with osteoblastic marrow stromal cells and cultured in the presence of dexamethasone and L-ascorbic acid for 7 and 14 days. Cell numbers per foam were found to be similar with all culturing schemes indicating that cell growth could not be enhanced by convection, but histological analysis indicated that the rotary vessel and flow system produced a more uniform distribution of cells throughout the foams. Alkaline phosphatase (ALP) activity per cell was higher with culture in the flow system and spinner flask after 7 days, while no differences in osteocalcin (OC) activity per cell were observed among culturing methods after 14 days in culture. Based on the higher ALP activity and better cell uniformity throughout the cultured foams, the flow system appears to be the superior culturing method, although equally important is the fact that in none of the tests did any of the alternative culturing techniques underperform the static controls. Thus, this study demonstrates that culturing techniques that utilize fluid flow, and in particular the flow perfusion system, improve the properties of the seeded cells over those maintained in static culture.

  20. Osteoblast response to the surface of amino acid-functionalized hydroxyapatite.

    Science.gov (United States)

    Lee, Wing-Hin; Loo, Ching-Yee; Chrzanowski, Wojciech; Rohanizadeh, Ramin

    2015-06-01

    Interactions between proteins and the surface of biomaterials are crucial for the biological function and success of materials implanted in the human body. In this study, hydroxyapatite (HA) with negative and positive surface charges were fabricated by functionalizing the HA surface with acidic or basic amino acids. The influence of HA surface charge on protein adsorption and cell activities was studied. The crystallinity, morphology, and surface charge of amino acid-functionalized HA (AA-HA) particles and the stability of amino acids on the HA surface were determined. Both AA-HA and unmodified HA were studied for their capacity to adsorb proteins present in biological medium. The results showed that the presence of glutamic acid; Glu (acidic amino acids) and arginine; Arg (basic amino acids) on the HA surface resulted in higher protein adsorption owing to stronger electrostatic attraction between the HA particles and the proteins in medium. Functionalizing HA with Glu and Arg significantly promoted osteoblast adhesion on the surface of treated HA. No significant differences in cell proliferation between negatively and positively charged HA was observed. Significantly higher alkaline phosphatase (ALP) activity of osteoblasts on both charged surfaces was seen as compared to the unmodified HA. The study demonstrated that immobilization of amino acids (Glu and Arg) on the surface of HA promoted osteoblast proliferation and ALP activity. © 2014 Wiley Periodicals, Inc.

  1. Changes in the insulin-like growth factor-system may contribute to in vitro age-related impaired osteoblast functions

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Rattan, Suresh

    2000-01-01

    Age-related bone loss is thought to be due to impaired osteoblast functions. Insulin-like growth factors (IGFs) have been shown to be important stimulators of bone formation and osteoblast activities in vitro and in vivo. We tested the hypothesis that in vitro osteoblast senescence is associated ...

  2. Autophagy relieves the function inhibition and apoptosis-promoting effects on osteoblast induced by glucocorticoid

    Science.gov (United States)

    Han, Yudi; Zhang, Lihai; Xing, Yaling; Zhang, Licheng; Chen, Xiaojuan; Tang, Peifu; Chen, Zhongbin

    2018-01-01

    Autophagy may be a major mechanism by which osteoblasts (OBs) protect against the negative effects of chronic glucocorticoid (GC) usage. OBs are closely associated with the remodeling that occurs in GC-induced osteoporosis (GIO). In osteocytes, in response to stress induced by GCs, several pathways are activated, including cell necrosis, apoptosis and autophagy. However, the role of autophagy in OBs following treatment with excess GCs has not been addressed. In the current study, confocal microscopy observation of green fluorescent protein-microtubule-associated protein 1 light chain 3β (LC3) punctuate, and western blotting for LC3II and Beclin 1 were performed for detection of autophagy in the MC3T3-E1 osteoblastic cell line. Flow cytometry and western blotting were used for the examination of apoptosis and expression of BAX apoptosis regulator (Bax)/apoptosis regulator Bcl-2 (Bcl-2). The expression of genes associated with osteoblastic function, runt-related transcription factor 2, α-1 type 1 collagen and osteocalcin, were measured by reverse transcription-quantitative polymerase chain reaction. The results indicated that autophagy was induced in OBs during dexamethasone (Dex) treatment in a dose-dependent manner. The level of autophagy did not continue to increase over time, but peaked at 48 h and then decreased gradually. Subsequently, flow cytometry was used to demonstrate that inhibition of autophagy induced apoptosis in OBs under Dex treatment, and was associated with the upregulation of Bax and the downregulation of Bcl-2 protein expression. Furthermore, the data suggested that the inhibition of autophagy also suppressed the expression of osteoblastic genes. By contrast, the stimulation of autophagy maintained the gene expression level under Dex treatment. The data revealed that autophagy is an important regulator of osteoblastic apoptosis through its interaction with Bax/Bcl-2, and maintains the osteoblastic function of MC3T3-E1 cells following GC

  3. Glycans modify mesenchymal stem cell differentiation to impact on the function of resulting osteoblasts

    OpenAIRE

    Wilson, Katherine M.; Jagger, Alistair M; Walker, Matthew; Seinkmane, Estere; Fox, James M.; Kröger, Roland; Genever, Paul; Ungar, Daniel

    2018-01-01

    Glycans are inherently heterogeneous, yet glycosylation is essential in eukaryotes, and glycans show characteristic cell type-dependent distributions. By using an immortalized human mesenchymal stromal cell (MSC) line model, we show that both N- and O-glycan processing in the Golgi functionally modulates early steps of osteogenic differentiation. We found that inhibiting O-glycan processing in the Golgi prior to the start of osteogenesis inhibited the mineralization capacity of the formed ost...

  4. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    DEFF Research Database (Denmark)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators......-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore...

  5. Osteocalcin, a marker of differentiated function during calcification of cultured chick osteoblasts

    International Nuclear Information System (INIS)

    Lian, J.; Chipman, S.; Glowacki, J.; Gerstenfeld, L.

    1986-01-01

    The expression of differentiated function was examined in cultured osteoblasts isolated from 17-day embryonic chicken calvarie. Cell cultures grown in the absence (control) or presence of 10 mM β-Glycerol Phosphate (βGP) (stimulus for calcification) were analyzed at 6-day intervals over a 30-day period for total mineral, alkaline phosphatase (AP) activity, osteocalcin levels and collagen. AP was first detected in both cultures between days 6 and 9 when cells became crowded. Control cultures maintained high levels of enzyme activity (30-50 fold) while β GPO 4 culture activity declined after day 18 when extensive mineralization occurred. Osteocalcin, the vitamin K-dependent, bone-specific, calcium-binding protein showed a similar pattern of induction as AP with at 50-100-fold increase in both cultures. Collagen accumulated through out the 30-day experimental period for both β GPO 4 and control cultures while collagen synthesis ( 3 H-proline pulse) peaked at day 15 in culture. These results suggest that with time in culture, osteoblast differentiation may be occurring. The increased mineralization of β GPO 4 cultures appeared to down regulate the enzyme activity of AP in comparison to control culture, while osteocalcin synthesis was enhanced. In conclusion, the chick osteoblast system offers a model to study bone cell differentiation, protein synthesis and matrix calcification

  6. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    DEFF Research Database (Denmark)

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R

    2007-01-01

    PAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. MATERIALS AND METHODS: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected......The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal...... of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. RESULTS: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed...

  7. Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2

    Science.gov (United States)

    Dai, Zhongquan; Tan, Yingjun; Yang, Fen; Qu, Lina; Zhang, Hongyu; Wan, Yumin; Li, Yinghui

    2011-01-01

    Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure

  8. Canine Bone Marrow Stromal Cells Promote Functional Recovery in Mice with Spinal Cord Injury

    OpenAIRE

    ODA, Yasutaka; TANI, Kenji; ASARI, Yusuke; QUINTANILHA, Luiz Fernando; HARAGUCHI, Tomoya; MOMOTA, Yutaka; KATAYAMA, Masaaki; ITAMOTO, Kazuhito; NAKAZAWA, Hiroshi; TAURA, Yasuho

    2014-01-01

    ABSTRACT Regenerative therapy has begun to be clinically applied in humans and dogs to treat neurological disorders, such as spinal cord injury (SCI). Here, we show the therapeutic potential of transplantation of cultured canine bone marrow stromal cells (BMSCs) into mice with SCI. Canine BMSC transplantation therapy was performed, immediately after the spinal cord was injured. Canine BMSC therapy enhanced functional recovery of the hind limbs in mice with SCI. Nestin-positive cells were obse...

  9. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Andersen, H; Jensen, Ole N; Moiseeva, Elena P

    2003-01-01

    Prostate cancer cells metastasize to bone causing a predominantly osteosclerotic response. It has been shown that cells from the human prostate cancer cell line PC3 secrete factors that influence the behavior of osteoblast-like cells. Some of these factors with mitogenic activity have been found...... to be proteins with molecular weights between 20 and 30 kDa, but the identity of the osteoblastic mitogenic factor or factors produced by prostate cancer cells is still unknown. Therefore, the aim of this study was to characterize the protein profile of conditioned medium (CM) from PC3 cells in the molecular......BMS) cells. Furthermore, we tested whether adhesion of PC3 cells to plastic, laminin, fibronectin, and collagen type I was influenced by lactose, which inhibits galectin-1. Galectin-1 (1000 ng/ml) inhibited the proliferation of hBMS cells up to 70 +/- 12% (treated/control) of control in contrast to PC3 CM...

  10. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

    Science.gov (United States)

    Jovanovic, Milena; Schmidt, Felix N; Guterman-Ram, Gali; Khayyeri, Hanifeh; Hiram-Bab, Sahar; Orenbuch, Ayelet; Katchkovsky, Svetlana; Aflalo, Anastasia; Isaksson, Hanna; Busse, Björn; Jähn, Katharina; Levaot, Noam

    2018-01-08

    Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39 -/- ) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39 -/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39 -/- mice. Furthermore, Gpr39 -/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39 -/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39 -/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

  11. Surface functionalization of titanium substrates with chitosan-lauric acid conjugate to enhance osteoblasts functions and inhibit bacteria adhesion.

    Science.gov (United States)

    Zhao, Lu; Hu, Yan; Xu, Dawei; Cai, Kaiyong

    2014-07-01

    Orthopedic implants failures are generally related to poor osseointegration and/or bacterial infection in clinical application. Surface functionalization of an implant is one promising alternative for enhancing osseointegration and/or reducing bacterial infection, thus ensuring the long term survival of the implant. In this study, titanium (Ti) substrates were surface functionalized with a polydopamine (PDOP) film as an intermediate layer for post-immobilization of chitosan-lauric acid (Chi-LA) conjugate. Chi-LA conjugate was synthesized and characterized by Fourier transform infrared spectroscopy (FTIR) and hydrogen proton nuclear magnetic resonance (NMR) spectrometer, respectively. Lauric acid (LA), a natural saturated fatty acid, was used mainly due to its good antibacterial property. Scanning electron microscopy (SEM) and water contact angle measurements were employed to detect the morphology changes and surface wettability of Ti substrates. The results suggested that Chi-LA conjugate was successfully immobilized onto the surfaces of Ti substrates. In vitro tests confirmed that the cell adhesion, cell viability, intracellular alkaline phosphatase activity and mineralization capacity of osteoblasts were remarkably improved when cultured onto Chi-LA surface functionalized Ti substrates. Antibacterial assay against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) showed that the Chi-LA modified Ti substrates efficiently inhibited the adhesion and growth of bacteria. Overall, this study developed a promising approach to fabricate functional Ti-based orthopedic implants, which could enhance the biological functions of osteoblasts and concurrently reduce bacteria adhesion. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Luan, Anna; Aitzetmüller, Matthias M; Brett, Elizabeth A; Atashroo, David; Whittam, Alexander J; Hu, Michael S; Walmsley, Graham G; Houschyar, Khosrow S; Schilling, Arndt F; Machens, Hans-Guenther; Gurtner, Geoffrey C; Longaker, Michael T; Wan, Derrick C

    2017-12-01

    Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34 + CD31 - CD45 - ). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. MEK5 suppresses osteoblastic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kaneshiro, Shoichi [Department of Orthopaedic Surgery, Japan Community Health Care Organization Osaka Hospital, 4-2-78 Fukushima, Fukushima Ward, Osaka City, Osaka 553-0003 (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Higuchi, Chikahisa, E-mail: c-higuchi@umin.ac.jp [Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  14. The role of nanocrystalline titania coating on nanostructured austenitic stainless steel in enhancing osteoblasts functions for regeneration of tissue

    Energy Technology Data Exchange (ETDEWEB)

    Shah, J.S.; Venkatsurya, P.K.C.; Thein-Han, W.W. [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Misra, R.D.K., E-mail: dmisra@louisiana.edu [Biomaterials and Biomedical Engineering Research Laboratory, Center for Structural and Functional Materials, University of Louisiana at Lafayette, P.O. Box 44130, Lafayette, LA 70504 (United States); Pesacreta, T.C. [Department of Biology, University of Louisiana at Lafayette, P.O. Box 42451, Lafayette, LA 70504 (United States); Somani, M.C.; Karjalainen, L.P. [Department of Mechanical Engineering, University of Oulu, P.O. Box 4200, 90014 Oulu (Finland)

    2011-03-12

    In the context of osseointegration of metallic implants, while nanostructuring the surface favorably modulates cellular response, the disinfective attributes required during the healing process are not available. Thus, in the present study, we demonstrate that nanocrystalline titania provides cumulative benefit of enhancing osteoblasts functions to promote the efficacy of metal implants together with the disinfective attributes. To this end, the primary objective here is to examine the select functions of bone forming cells (osteoblasts) on electrocrystallized nanonodular titania-coated nanograined/ultrafine grained (NG/UFG) austenitic stainless steel. The accompanying objective is to study the disinfective/antimicrobial activity. To the best of our understanding this is the first study of nanophase titania on a non-titanium substrate. The osteoblasts functions were investigated in terms of cell attachment, proliferation, and quantitative analysis of proteins, actin and vinculin. In comparison to the bare NG/UFG substrate, the nanophase titania-coated substrate exhibited higher degree of cell attachment and proliferation which are regulated via cellular and molecular interactions with proteins, actin and vinculin. The enhanced functions of osteoblasts suggest that nanophase titania adsorbs extracellular matrix proteins, fibronectin and vitronectin from serum enhancing protein, with subsequent binding of integrins and osteoblasts precursor to titania. The antimicrobial attributes assessed in terms of degradation of methyl orange and effectiveness in killing E. coli supports the viewpoint that large surface area of titania would be instrumental in reducing the detrimental effect of biologically reactive oxygen species produced by macrophages in the vicinity of the metal bone/implant interface. In summary, the study provides some new insights concerning nanostructuring of metallic substrates with specific physical and surface properties for medical devices with

  15. Understanding the response of pulsed electric field on osteoblast functions in three-dimensional mesh structures.

    Science.gov (United States)

    Kumar, A; Nune, K C; Misra, Rdk

    2016-10-01

    The endogenous electric field plays a determining role in impacting biological functions including communication with the physiological system, brain, and bone regeneration by influencing cellular functions. From this perspective, the objective of the study described here is to elucidate the effect of external electric field under dynamic conditions, in providing a guiding cue to osteoblasts in terms of cell-cell interactions and synthesis of prominent adhesion and cytoskeleton proteins. This was accomplished using pulsed direct current electric field of strength 0.1-1 V/cm. The electric field provided guided cue to the cells to migrate toward cathode. Membrane blebbing or necrosis was nearly absent in the vicinity of cathode at 0.1 and 0.5 V/cm electric field strength. Moreover, a higher cell proliferation as well as higher expression of vinculin and densely packed actin stress fibers was observed. At anode, the cells though healthy but expression of actin and vinculin was less. We underscore for the first time that the biological functionality can be favorably modulated on 3D printed scaffolds in the presence of electric field and under dynamic conditions with consequent positive effect on cell proliferation, growth, and expression level of prominent proteins. © The Author(s) 2016.

  16. Functional role of acetylcholine and the expression of cholinergic receptors and components in osteoblasts.

    Science.gov (United States)

    Sato, Tsuyoshi; Abe, Takahiro; Chida, Dai; Nakamoto, Norimichi; Hori, Naoko; Kokabu, Shoichiro; Sakata, Yasuaki; Tomaru, Yasuhisa; Iwata, Takanori; Usui, Michihiko; Aiko, Katsuya; Yoda, Tetsuya

    2010-02-19

    Recent studies have indicated that acetylcholine (ACh) plays a vital role in various tissues, while the role of ACh in bone metabolism remains unclear. Here we demonstrated that ACh induced cell proliferation and reduced alkaline phosphatase (ALP) activity via nicotinic (nAChRs) and muscarinic acetylcholine receptors (mAChRs) in osteoblasts. We detected mRNA expression of several nAChRs and mAChRs. Furthermore, we showed that cholinergic components were up-regulated and subunits/subtypes of acetylcholine receptors altered during osteoblast differentiation. To our knowledge, this is the first report demonstrating that osteoblasts express specific acetylcholine receptors and cholinergic components and that ACh plays a possible role in regulating the proliferation and differentiation of osteoblasts. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

  17. Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

    Directory of Open Access Journals (Sweden)

    Bhavani S. Kowtharapu

    2018-02-01

    Full Text Available Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM on corneal epithelial cell function along with substance P (SP. Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK, paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained

  18. Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations

    DEFF Research Database (Denmark)

    Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco

    2016-01-01

    by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation...... of β-catenin in osteoblasts (Ctnnb1(CAosb)), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5(A214V) mice. Likewise, peripheral blood count analysis in HBM...

  19. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    Full Text Available Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs. The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs. In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29 and negative hematopoietic stem cell markers (CD45 and CD34 expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1 MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2 expression; 2 The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation; 3 Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4 Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  20. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    Science.gov (United States)

    Wang, Jinju; Chen, Shuzhen; Zhang, Cheng; Stegeman, Samantha; Pfaff-Amesse, Teresa; Zhang, Ying; Zhang, Wenfeng; Amesse, Lawrence; Chen, Yanfang

    2012-01-01

    Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  1. Canine Bone Marrow Stromal Cells Promote Functional Recovery in Mice with Spinal Cord Injury

    Science.gov (United States)

    ODA, Yasutaka; TANI, Kenji; ASARI, Yusuke; QUINTANILHA, Luiz Fernando; HARAGUCHI, Tomoya; MOMOTA, Yutaka; KATAYAMA, Masaaki; ITAMOTO, Kazuhito; NAKAZAWA, Hiroshi; TAURA, Yasuho

    2014-01-01

    ABSTRACT Regenerative therapy has begun to be clinically applied in humans and dogs to treat neurological disorders, such as spinal cord injury (SCI). Here, we show the therapeutic potential of transplantation of cultured canine bone marrow stromal cells (BMSCs) into mice with SCI. Canine BMSC transplantation therapy was performed, immediately after the spinal cord was injured. Canine BMSC therapy enhanced functional recovery of the hind limbs in mice with SCI. Nestin-positive cells were observed only in the lesion of mice with SCI that received BMSCs. These results suggest that canine BMSCs promote functional recovery in mice with SCI and that migration of nestin-positive cells may contribute to the efficacy of the BMSC treatment. PMID:24561315

  2. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  3. Activation of Osteoblastic Function on Titanium Surface with Titanium-Doped Hydroxyapatite Nanoparticle Coating: An In Vitro Study.

    Science.gov (United States)

    Nakazawa, Masahiro; Yamada, Masahiro; Wakamura, Masato; Egusa, Hiroshi; Sakurai, Kaoru

    Titanium-doped hydroxyapatite (TiHA) nanoparticles contain titanium atoms in the hydroxyapatite lattice, which can physicochemically functionalize the titanium surface without modification of the surface topography. This study aimed to evaluate the physicochemical properties of machined or microroughened titanium surfaces coated with TiHA nanoparticles and the functions of osteoblasts cultured on them. Titanium disks with commercially available surface topography, such as machined or sandblasted, large-grit, and acid-etched (SLA) surfaces, were coated with TiHA. The disks with original or TiHA-coated surfaces were evaluated in topography, wettability, and chemical composition. Osteoblastic cells from rat femurs were cultured on the disks and evaluated in proliferation and differentiation. TiHA coating changed from hydrophobicity to hydrophilicity on both machined and SLA surfaces. Calcium and phosphate atoms were detected all over the surface with TiHA coating regardless of the surface topography. However, the considerable change in the inherent surface topographies was not observed on both types of surfaces after TiHA coating. Osteoblastic proliferative activity at day 4 was increased by TiHA coating on both types of surfaces. TiHA coating did not enhance expressions of bone matrix-related genes such as osteocalcin, osteopontin, bone sialoprotein, alkaline phosphatase, and collagen I. However, depositions of collagen, osteocalcin, and calcium in the culture at days 7 and 20 were increased on both types of surface topographies with TiHA coating. TiHA coating enhanced extracellular matrix formation on smooth and microroughened titanium surfaces by increasing osteoblastic proliferative activity without the deterioration of differentiation through hydrophilic and chemical functionalization.

  4. Influence of sterilization methods on cell behavior and functionality of osteoblasts cultured on TiO2 nanotubes

    International Nuclear Information System (INIS)

    Oh, Seunghan; Brammer, Karla S.; Moon, Kyung-Suk; Bae, Ji-Myung; Jin, Sungho

    2011-01-01

    We investigated the adhesion, proliferation and osteogenic functionality of osteoblasts cultured on titanium dioxide (TiO 2 ) nanotubes in response to different sterilization methods (dry autoclaving vs. wet autoclaving). We prepared various sizes (30-100 nm diameter) of TiO 2 nanotubes on titanium substrates by anodization, sterilized nanotubes by different conditions, and seeded osteoblast cells onto the nanotube surfaces with two different cell seeding densities (10,000 vs. 50,000 cells/well in 12-culture well). The result of this study indicates that the adhesion, proliferation and alkaline phosphatase activity of osteoblasts cultured on only the larger 70 and 100 nm TiO 2 nanotube arrays were dramatically changed by the different sterilization conditions at a low cell seeding density. However, with a higher cell seeding density (50,000 cells/well in 12-cell culture well), the results revealed no significant difference among altered nanotube geometry, 30-100 nm diameters, nor sterilization methods. Next, it was revealed that the nanofeatures of proteins adhered on nanotubular TiO 2 morphology are altered by the sterilization method. It was determined that this protein adhesion effect, in combination with the cell density of osteoblasts seeded onto such TiO 2 nanotube surfaces, has profound effects on cell behavior. This study clearly shows that these are some of the important in vitro culture factors that need to be taken into consideration, as well as TiO 2 nanotube diameters which play an important role in the improvement of cell behavior and functionality.

  5. Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

    Science.gov (United States)

    Globus, Ruth K.; Searby, Nancy D.; Almeida, Eduardo A. C.; Sutijono, Darrell; Yu, Joon-Ho; Malouvier, Alexander; Doty, Steven B.; Morey-Holton, Emily; Weinstein, Steven L.; Dalton, Bonnie P. (Technical Monitor)

    2000-01-01

    Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We

  6. Functional features of gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression

    International Nuclear Information System (INIS)

    Ostrowski, Jerzy; Dobosz, Anna Jerzak Vel; Jarosz, Dorota; Ruka, Wlodzimierz; Wyrwicz, Lucjan S; Polkowski, Marcin; Paziewska, Agnieszka; Skrzypczak, Magdalena; Goryca, Krzysztof; Rubel, Tymon; Kokoszyñska, Katarzyna; Rutkowski, Piotr; Nowecki, Zbigniew I

    2009-01-01

    Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in KIT or PDGFRA of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations. Total RNA was isolated from 29 frozen gastric GISTs and processed for hybridization on GENECHIP ® HG-U133 Plus 2.0 microarrays (Affymetrix). KIT and PDGFRA were analyzed by sequencing, while related mRNA levels were analyzed by quantitative RT-PCR. Fifteen and eleven tumours possessed mutations in KIT and PDGFRA, respectively; no mutation was found in three tumours. Gene expression analysis identified no discriminative profiles associated with clinical or pathological parameters, even though expression of hundreds of genes differentiated tumour receptor mutation and expression status. Functional features of genes differentially expressed between the two groups of GISTs suggested alterations in angiogenesis and G-protein-related and calcium signalling. Our study has identified novel molecular elements likely to be involved in receptor-dependent GIST development and allowed confirmation of previously published results. These elements may be potential therapeutic targets and novel markers of KIT mutation status

  7. Transient warming events occurring after freezing impairs umbilical cord-derived mesenchymal stromal cells functionality.

    Science.gov (United States)

    Chabot, Dominique; Tremblay, Tony; Paré, Isabelle; Bazin, Renée; Loubaki, Lionel

    2017-08-01

    Mesenchymal stromal cells (MSCs) have shown promising results for the treatment of refractory acute graft-versus-host disease. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSCs in clinical trials has raised concerns regarding the retention of their functional activity. This has led to the recommendation by experts in the field to use freshly harvested MSCs, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC functionality and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impact MSC functionality. Following controlled-rate freezing to -130°C, umbilical cord-derived MSCs were left at room temperature (RT) for 2-10 min or on dry ice for 10 min, before being transferred into liquid nitrogen (LqN 2 ). MSCs of each group were subsequently tested (viability, functionality and cellular damage) and compared with their freshly harvested counterparts. We demonstrated that freshly harvested MSCs as well as cryopreserved MSCs that were left on dry ice following step-down freezing have comparable viability, functionality and integrity. In contrast, cryopreserved MSCs that were left at RT before being transferred into LqN 2 were functionally impaired and showed cellular damage upon thawing even though they exhibited high viability. Warming events after freezing and not cryopreservation per se significantly impair MSC functionality, indicating that cryopreserved MSCs can be an advantageous alternative to freshly harvested cells for therapeutic purposes. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Biomechanical Forces Promote Immune Regulatory Function of Bone Marrow Mesenchymal Stromal Cells.

    Science.gov (United States)

    Diaz, Miguel F; Vaidya, Abishek B; Evans, Siobahn M; Lee, Hyun J; Aertker, Benjamin M; Alexander, Alexander J; Price, Katherine M; Ozuna, Joyce A; Liao, George P; Aroom, Kevin R; Xue, Hasen; Gu, Liang; Omichi, Rui; Bedi, Supinder; Olson, Scott D; Cox, Charles S; Wenzel, Pamela L

    2017-05-01

    Mesenchymal stromal cells (MSCs) are believed to mobilize from the bone marrow in response to inflammation and injury, yet the effects of egress into the vasculature on MSC function are largely unknown. Here we show that wall shear stress (WSS) typical of fluid frictional forces present on the vascular lumen stimulates antioxidant and anti-inflammatory mediators, as well as chemokines capable of immune cell recruitment. WSS specifically promotes signaling through NFκB-COX2-prostaglandin E 2 (PGE 2 ) to suppress tumor necrosis factor-α (TNF-α) production by activated immune cells. Ex vivo conditioning of MSCs by WSS improved therapeutic efficacy in a rat model of traumatic brain injury, as evidenced by decreased apoptotic and M1-type activated microglia in the hippocampus. These results demonstrate that force provides critical cues to MSCs residing at the vascular interface which influence immunomodulatory and paracrine activity, and suggest the potential therapeutic use of force for MSC functional enhancement. Stem Cells 2017;35:1259-1272. © 2017 AlphaMed Press.

  9. Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Roland Meisel

    2014-01-01

    Full Text Available Human mesenchymal stromal cells (MSC possess immunosuppressive and antimicrobial effects that are partly mediated by the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO. Therefore MSC represent a promising novel cellular immunosuppressant which has the potential to control steroid-refractory acute graft versus host disease (GvHD. In addition, MSC are capable of reducing the risk of infection in patients after haematopoietic stem cell transplantation (HST. Recent data indicate that signals from the microenvironment including those from microbes may modulate MSC effector functions. As Cytomegalovirus (CMV represents a prominent pathogen in immunocompromised hosts, especially in patients following HST, we investigated the impact of CMV infection on MSC-mediated effects on the immune system. We demonstrate that CMV-infected MSC lose their cytokine-induced immunosuppressive capacity and are no longer able to restrict microbial growth. IDO expression is substantially impaired following CMV infection of MSC and this interaction critically depends on intact virus and the number of MSC as well as the viral load. Since overt CMV infection may undermine the clinical efficacy of MSC in the treatment of GvHD in transplant patients, we recommend that patients scheduled for MSC therapy should undergo thorough evaluation for an active CMV infection and receive CMV-directed antiviral therapy prior to the administration of MSC.

  10. Despite differential gene expression profiles pediatric MDS derived mesenchymal stromal cells display functionality in vitro.

    Science.gov (United States)

    Calkoen, F G J; Vervat, C; van Pel, M; de Haas, V; Vijfhuizen, L S; Eising, E; Kroes, W G M; 't Hoen, P A C; van den Heuvel-Eibrink, M M; Egeler, R M; van Tol, M J D; Ball, L M

    2015-03-01

    Pediatric myelodysplastic syndrome (MDS) is a heterogeneous disease covering a spectrum ranging from aplasia (RCC) to myeloproliferation (RAEB(t)). In adult-type MDS there is increasing evidence for abnormal function of the bone-marrow microenvironment. Here, we extensively studied the mesenchymal stromal cells (MSCs) derived from children with MDS. MSCs were expanded from the bone-marrow of 17 MDS patients (RCC: n=10 and advanced MDS: n=7) and pediatric controls (n=10). No differences were observed with respect to phenotype, differentiation capacity, immunomodulatory capacity or hematopoietic support. mRNA expression analysis by Deep-SAGE revealed increased IL-6 expression in RCC- and RAEB(t)-MDS. RCC-MDS MSC expressed increased levels of DKK3, a protein associated with decreased apoptosis. RAEB(t)-MDS revealed increased CRLF1 and decreased DAPK1 expressions. This pattern has been associated with transformation in hematopoietic malignancies. Genes reported to be differentially expressed in adult MDS-MSC did not differ between MSC of pediatric MDS and controls. An altered mRNA expression profile, associated with cell survival and malignant transformation, of MSC derived from children with MDS strengthens the hypothesis that the micro-environment is of importance in this disease. Our data support the understanding that pediatric and adult MDS are two different diseases. Further evaluation of the pathways involved might reveal additional therapy targets. Copyright © 2015. Published by Elsevier B.V.

  11. Multiple Functions of the New Cytokine-Based Antimicrobial Peptide Thymic Stromal Lymphopoietin (TSLP

    Directory of Open Access Journals (Sweden)

    Louise Bjerkan

    2016-07-01

    Full Text Available Thymic stromal lymphopoietin (TSLP is a pleiotropic cytokine, hitherto mostly known to be involved in inflammatory responses and immunoregulation. The human tslp gene gives rise to two transcription and translation variants: a long form (lfTSLP that is induced by inflammation, and a short, constitutively-expressed form (sfTSLP, that appears to be downregulated by inflammation. The TSLP forms can be produced by a number of cell types, including epithelial and dendritic cells (DCs. lfTSLP can activate mast cells, DCs, and T cells through binding to the lfTSLP receptor (TSLPR and has a pro-inflammatory function. In contrast, sfTSLP inhibits cytokine secretion of DCs, but the receptor mediating this effect is unknown. Our recent studies have demonstrated that both forms of TSLP display potent antimicrobial activity, exceeding that of many other known antimicrobial peptides (AMPs, with sfTSLP having the strongest effect. The AMP activity is primarily mediated by the C-terminal region of the protein and is localized within a 34-mer peptide (MKK34 that spans the C-terminal α-helical region in TSLP. Fluorescent studies of peptide-treated bacteria, electron microscopy, and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of LL-37. Expression of TSLP in skin, oral mucosa, salivary glands, and intestine is part of the defense barrier that aids in the control of both commensal and pathogenic microbes.

  12. Pellet culture model for human primary osteoblasts

    OpenAIRE

    K Jähn; RG Richards; CW Archer; MJ Stoddart

    2010-01-01

    In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation an...

  13. CD54-Mediated Interaction with Pro-inflammatory Macrophages Increases the Immunosuppressive Function of Human Mesenchymal Stromal Cells

    Directory of Open Access Journals (Sweden)

    Nicolas Espagnolle

    2017-04-01

    Full Text Available Summary: Mesenchymal stromal cells (MSCs sense and modulate inflammation and represent potential clinical treatment for immune disorders. However, many details of the bidirectional interaction of MSCs and the innate immune compartment are still unsolved. Here we describe an unconventional but functional interaction between pro-inflammatory classically activated macrophages (M1MΦ and MSCs, with CD54 playing a central role. CD54 was upregulated and enriched specifically at the contact area between M1MФ and MSCs. Moreover, the specific interaction induced calcium signaling and increased the immunosuppressive capacities of MSCs dependent on CD54 mediation. Our data demonstrate that MSCs can detect an inflammatory microenvironment via a direct and physical interaction with innate immune cells. This finding opens different perspectives for MSC-based cell therapy. : Mesenchymal stromal cells (MSCs are promising for cell-based therapy in inflammatory disorders by switching off the immune response. Varin and colleagues demonstrate that MSCs and inflammatory macrophages communicate via an unconventional but functional interaction that strongly increases the immunosuppressive capacities of MSCs. This new communication between the innate immune system and MSCs opens new perspectives for MSC-based cell therapy. Keywords: macrophages, bone marrow mesenchymal stromal cells, functional interaction, CD54, immunosuppression, indoleamine 2,3-dioxygenase, cell therapy

  14. Primary observation on adherent function of bone marrow stromal cells in mice post combined radiation-burn injury

    International Nuclear Information System (INIS)

    Chen Xinghua; Luo Chengji; Guo Chaohua; Wang Ping; Deng Xuecai

    1999-01-01

    Objective: To investigate the adherent function of bone marrow stromal cells in hematopoietic inductive microenvironment post combined radiation-burn injury. Methods: The expression of cell adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), fibro-connection (Fn), laminin (Ln) and collagen type IV (Col IV) on bone marrow stromal cells cultured in vitro was detected by flow cytometry and the binding capacity of bone marrow mononuclear cells to stromal cell adherence layer was tested by cell binding assay and cell binding blocking assay respectively from mice treated with 5.0 Gy γ-ray 15% of total body surface area (TBSA), third-degree burn injury and combined irradiation-burn injury, respectively. Results: 1. The expression levels of molecules mentioned above in burn-injured mice were the highest. The molecules levels in control mice were greater than those in radiation-injured mice, which were lower than those in mice with combined radiation-burn injury. 2. The binding capacity of stromal cell adherence layer in burn-injured mice was greater than that in control mice, and significantly increased from 3 to 7 days post injury as compared with that in controls, radiation-injured mice and combined radiation-burn-injured mice, respectively (P < 0.05-0.01). Contrarily, the capacity of binding in the radiation-injured and combined radiation-burn-injured mice was the lowest from 3 to 7 days post injury. 3. The binding rate of bone marrow mononuclear cells to stromal cell adherence layer descended in different degrees after pre-treatment with monoclonal antibodies directed to VCAM-1, Fn, Ln, or Col IV respectively or VCAM-1 combined with anti-Fn, anti-Ln or anti-Col IV, respectively, in stromal cell adherence layer. Conclusion: The damage of cell adherent function for bone marrow hematopoietic inductive microenvironment post combined radiation-burn injury might be one of the important factors in hematopoietic disorder in combined radiation-burn injury

  15. Bone Marrow-Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality.

    Science.gov (United States)

    Stenger, Elizabeth O; Chinnadurai, Raghavan; Yuan, Shala; Garcia, Marco; Arafat, Dalia; Gibson, Greg; Krishnamurti, Lakshmanan; Galipeau, Jacques

    2017-05-01

    Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)-MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  16. Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in humans.

    Science.gov (United States)

    Poposki, Julie A; Klingler, Aiko I; Stevens, Whitney W; Peters, Anju T; Hulse, Kathryn E; Grammer, Leslie C; Schleimer, Robert P; Welch, Kevin C; Smith, Stephanie S; Sidle, Douglas M; Conley, David B; Tan, Bruce K; Kern, Robert C; Kato, Atsushi

    2017-05-01

    Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of patients with chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We sought to investigate the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant human TSLP with NP extracts, and determined the protein sequence of the truncated forms of TSLP using Edman protein sequencing and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. We investigated the functional activity of truncated TSLP using a PBMC-based bioassay. Edman sequencing and mass spectrometry results indicated that NP extracts generated 2 major truncated products, TSLP (residues 29-124) and TSLP (131-159). Interestingly, these 2 products remained linked with disulfide bonds and presented as a dimerized form, TSLP (29-124 + 131-159). We identified that members of the proprotein convertase were rate-limiting enzymes in the truncation of TSLP between residues 130 and 131 and generated a heterodimeric unstable metabolite TSLP (29-130 + 131-159). Carboxypeptidase N immediately digested 6 amino acids from the C terminus of the longer subunit of TSLP to generate a stable dimerized form, TSLP (29-124 + 131-159), in NPs. These truncations were homeostatic but primate-specific events. A metabolite TSLP (29-130 + 131-159) strongly activated myeloid dendritic cells and group 2 innate lymphoid cells compared with mature TSLP. Posttranslational modifications control the functional activity of TSLP in humans and overproduction of TSLP may be a key trigger for the amplification of type 2 inflammation in diseases. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  17. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Directory of Open Access Journals (Sweden)

    Teruhito Yamashita

    pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

  18. Arctigenin inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

    Science.gov (United States)

    Yamashita, Teruhito; Uehara, Shunsuke; Udagawa, Nobuyuki; Li, Feng; Kadota, Shigetoshi; Esumi, Hiroyasu; Kobayashi, Yasuhiro; Takahashi, Naoyuki

    2014-01-01

    -forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

  19. Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

    International Nuclear Information System (INIS)

    Moon, Ho-Jin; Yun, Young-Pil; Han, Choong-Wan; Kim, Min Sung; Kim, Sung Eun; Bae, Min Soo; Kim, Gyu-Tae; Choi, Yong-Suk; Hwang, Eui-Hwan; Lee, Joon Woo; Lee, Jin-Moo; Lee, Chang-Hoon; Kim, Duck-Su; Kwon, Il Keun

    2011-01-01

    Highlights: → We examine bone metabolism of engineered alendronate attached to Ti surfaces. → Alendronate-immobilized Ti enhances activation of osteoblast differentiation. → Alendronate-immobilized Ti inhibits osteoclast differentiation. → Alendronate-immobilized Ti may be a bioactive implant with dual functions. -- Abstract: The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue. In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast

  20. Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts.

    Science.gov (United States)

    Lisse, Thomas S; Chun, Rene F; Rieger, Sandra; Adams, John S; Hewison, Martin

    2013-06-01

    When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms. Copyright © 2013 American Society for Bone and

  1. Inflammatory conditions dictate the effect of mesenchymal stem or stromal cells on B cell function

    NARCIS (Netherlands)

    F. Luk (Franka); Carreras-Planella, L. (Laura); S.S. Korevaar (Sander); S.F. De Witte (Samantha Fh); F.E. Borràs (Francesc); M.G.H. Betjes (Michiel); C.C. Baan (Carla); M.J. Hoogduijn (Martin); M. Franquesa (Marcella)

    2017-01-01

    textabstractThe immunomodulatory capacity of mesenchymal stem or stromal cells (MSC) makes them a promising tool for treatment of immune disease and organ transplantation. The effects of MSC on B cells are characterized by an abrogation of plasmablast formation and induction of regulatory B cells

  2. Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells.

    Science.gov (United States)

    Tutak, Wojtek; Jyotsnendu, Giri; Bajcsy, Peter; Simon, Carl G

    2017-07-01

    Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl

  3. Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing.

    Science.gov (United States)

    Jung, Y; Wang, J; Schneider, A; Sun, Y-X; Koh-Paige, A J; Osman, N I; McCauley, L K; Taichman, R S

    2006-04-01

    Stromal derived factor-1 (SDF-1 or CXCL12) controls many aspects of stem cell function including trafficking and proliferation. Previously, it was demonstrated that DNA-damaging agents such as irradiation, cyclophosphamide or 5-fluorouracil increase the expression of SDF-1 by osteoblasts in murine marrow. Here, the production of SDF-1 by osteoblasts in vitro in response to cytokines known to be particularly important in bone physiology was examined using primary human osteoblasts (HOBs), mixed marrow stromal cells (BMSCs), and by, mouse, rat and human osteoblast-like cell lines. From these studies, it was determined that the expression of SDF-1 is an early feature of osteoblastic induction that may be modulated by IL-1beta, PDGF-BB, VEGF, TNF-alpha and PTH. Each of these factors increased SDF-1 synthesis, while TGF-beta1 decreased SDF-1 secretion. Of note, the biodistribution of SDF-1 in culture was equally distributed between the medium and detergent-soluble and -insoluble fractions of the cultures. Immunohistochemistry of developing bones demonstrated that SDF-1 was also a feature of early bone development first beginning in the perichondrium and moving into the marrow cavity of the developing bone analogue. As SDF-1 expression increases in response to PTH in vitro, animals were treated with an anabolic regime of PTH for 21 days. Under these conditions, significant increases in SDF-1 mRNA expression were observed near the growth plate and epiphysis regions of the long bones. Yet, in serum, immunodetectable SDF-1 levels were significantly reduced (24%) in the PTH-treated animals (Vehicle: 408 +/- 25 vs. PTH 308 +/- 20 SDF-1 pg/ml). Together, these data suggest a possible mechanism for localizing stem cells into a developing marrow where increased expression of SDF-1 in the local marrow environment along with decreased SDF-1 in the serum may create a homing gradient.

  4. Transplantation of human bone marrow stromal cell-derived neuroregenrative cells promotes functional recovery after spinal cord injury in mice.

    Science.gov (United States)

    Mannoji, Chikato; Koda, Masao; Kamiya, Koshiro; Dezawa, Mari; Hashimoto, Masayuki; Furuya, Takeo; Okawa, Akihiko; Takahashi, Kazuhisa; Yamazaki, Masashi

    2014-01-01

    Transplantation of bone marrow stromal cells (BMSCs) for spinal cord injury (SCI) has been shown to improve functional outcome. BMSCs can be easily obtained from bone marrow aspirate and have fewer problems in the clinical application for human SCI from the ethical and legal points of view. Recently, we produced cells with neural stem and/or progenitor cell property and neural regeneration supporting capacity from human bone marrow stromal cells (human bone marrow stromal cell-derived neuroregenerative cells: hBMSC-NRs). The aim of the present study was to clarify the effectiveness of transplantation of hBMSC-NRs to injured spinal cord of severe combined immunodeficiency (NOD/SCID) mice. Neurite outgrowth assay of PC-12 cells was performed. One week after a T9-level contusion SCI, hBMSCs or hBMSC-NRs were transplanted into the spinal cord. After the transplantation, functional and histological examinations were performed. Conditioned media of hBMSC-NRs significantly promoted the neurite outgrowth of PC-12 cells in vitro. Transplanted hBMSC-NRs survived in the injured spinal cord 8 weeks after SCI. Immunohistochemistry revealed that the density of serotonin-positive fibers of the transplanted group was significantly higher than that of the control group at the epicenter and caudal segment to the injured site. The recovery of hind limb function of the hBMSC-NRs group was significantly better than that of the control group. In conclusion, hBMSC-NRs can be one of the realistic candidates for cell transplantation therapy for human SCI.

  5. Stromal cell contributions to the homeostasis and functionality of the immune system.

    Science.gov (United States)

    Mueller, Scott N; Germain, Ronald N

    2009-09-01

    A defining characteristic of the immune system is the constant movement of many of its constituent cells through the secondary lymphoid tissues, mainly the spleen and lymph nodes, where crucial interactions that underlie homeostatic regulation, peripheral tolerance and the effective development of adaptive immune responses take place. What has only recently been recognized is the role that non-haematopoietic stromal elements have in many aspects of immune cell migration, activation and survival. In this Review, we summarize our current understanding of lymphoid compartment stromal cells, examine their possible heterogeneity, discuss how these cells contribute to immune homeostasis and the efficient initiation of adaptive immune responses, and highlight how targeting of these elements by some pathogens can influence the host immune response.

  6. In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors.

    Directory of Open Access Journals (Sweden)

    Matteo Vecellio

    Full Text Available Adult human cardiac mesenchymal-like stromal cells (CStC represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS in the presence of 5 µM all-trans Retinoic Acid (ATRA, 5 µM Phenyl Butyrate (PB, and 200 µM diethylenetriamine/nitric oxide (DETA/NO, to create a novel epigenetically active cocktail (EpiC. Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

  7. Transcription factor ZNF25 is associated with osteoblast differentiation of human skeletal stem cells

    DEFF Research Database (Denmark)

    Twine, Natalie A.; Harkness, Linda; Kassem, Moustapha

    2016-01-01

    Background The differentiation of human bone marrow derived skeletal stem cells (known as human bone marrow stromal or mesenchymal stem cells, hMSCs) into osteoblasts involves the activation of a small number of well-described transcription factors. To identify additional osteoblastic transcripti...

  8. Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells

    Science.gov (United States)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

  9. Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts

    Science.gov (United States)

    Globus, R. K.; Amblard, D.; Nishimura, Y.; Iwaniec, U. T.; Kim, J-B; Almeida, E. A. C.; Damsky, C. D.; Wronski, T. J.; van der Meulen, M. C. H.

    2005-01-01

    Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they

  10. Notch 1 impairs osteoblastic cell differentiation.

    Science.gov (United States)

    Sciaudone, Maria; Gazzerro, Elisabetta; Priest, Leah; Delany, Anne M; Canalis, Ernesto

    2003-12-01

    Notch receptors are single pass transmembrane receptors activated by membrane-bound ligands with a role in cell proliferation and differentiation. As Notch 1 and 2 mRNAs are expressed by osteoblasts and induced by cortisol, we postulated that Notch could regulate osteoblastogenesis. We investigated the effects of retroviral vectors directing the constitutive expression of the Notch 1 intracellular domain (NotchIC) in murine ST-2 stromal and in MC3T3 cells. NotchIC overexpression was documented by increased Notch 1 transcripts and activity of the Notch-dependent Hairy Enhancer of Split promoter. In the presence of bone morphogenetic protein-2 (BMP-2), ST-2 cells differentiated toward osteoblasts forming mineralized nodules, and Notch 1 opposed this effect and decreased the expression of osteocalcin, type I collagen, and alkaline phosphatase transcripts and Delta2Delta FosB protein. Further, NotchIC decreased Wnt/beta-catenin signaling. As cells differentiated in the presence of BMP-2, they underwent apoptosis, and Notch opposed this event. In the presence of cortisol, NotchIC induced the formation of mature adipocytes and enhanced the effect of cortisol on adipsin, peroxisome proliferator-activated receptor-gamma2 and CCAAT enhancer binding protein alpha and delta mRNA levels. NotchIC also opposed MC3T3 cell differentiation and the expression of a mature osteoblastic phenotype. In conclusion, NotchIC impairs osteoblast differentiation and enhances adipogenesis in stromal cell cultures.

  11. Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

    International Nuclear Information System (INIS)

    Iso, Yoshitaka; Spees, Jeffrey L.; Serrano, Claudia; Bakondi, Benjamin; Pochampally, Radhika; Song, Yao-Hua; Sobel, Burton E.; Delafontaine, Patrick; Prockop, Darwin J.

    2007-01-01

    The aim of this study was to determine whether intravenously administered multipotent stromal cells from human bone marrow (hMSCs) can improve cardiac function after myocardial infarction (MI) without long-term engraftment and therefore whether transitory paracrine effects or secreted factors are responsible for the benefit conferred. hMSCs were injected systemically into immunodeficient mice with acute MI. Cardiac function and fibrosis after MI in the hMSC-treated group were significantly improved compared with controls. However, despite the cardiac improvement, there was no evident hMSC engraftment in the heart 3 weeks after MI. Microarray assays and ELISAs demonstrated that multiple protective factors were expressed and secreted from the hMSCs in culture. Factors secreted by hMSCs prevented cell death of cultured cardiomyocytes and endothelial cells under conditions that mimicked tissue ischemia. The favorable effects of hMSCs appear to reflect the impact of secreted factors rather than engraftment, differentiation, or cell fusion

  12. Human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

    2012-01-01

    Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

  13. CHIP regulates bone mass by targeting multiple TRAF family members in bone marrow stromal cells.

    Science.gov (United States)

    Wang, Tingyu; Li, Shan; Yi, Dan; Zhou, Guang-Qian; Chang, Zhijie; Ma, Peter X; Xiao, Guozhi; Chen, Di

    2018-01-01

    Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip -/- mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip- deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip -/- mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

  14. Antimicrobial polycaprolactone/polyethylene glycol embedded lysozyme coatings of Ti implants for osteoblast functional properties in tissue engineering

    Science.gov (United States)

    Visan, A.; Cristescu, R.; Stefan, N.; Miroiu, M.; Nita, C.; Socol, M.; Florica, C.; Rasoga, O.; Zgura, I.; Sima, L. E.; Chiritoiu, M.; Chifiriuc, M. C.; Holban, A. M.; Mihailescu, I. N.; Socol, G.

    2017-09-01

    In this study, coatings based on lysozyme embedded into a matrix of polyethylene glycol (PEG) and polycaprolactone (PCL) were fabricated by two different methods (Matrix Assisted Pulsed Laser Evaporation - MAPLE and Dip Coating) for obtaining antimicrobial coatings envisaged for long term medical applications. Coatings with different PEG:PCL compositions (3:1; 1:1; 1:3) were synthesized in order to evaluate the antimicrobial activity of lysozyme embedded into the polymeric matrix. The main surface features, such as roughness and wettability, with impact on the microbial adhesion as well as on the eukaryote cell function were measured. The obtained composite coatings exhibited a significant antibacterial activity against Escherichia coli, Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus strains. As well, specific blended coatings showed appropriate viability, good spreading and normal cell morphology of SaOs2 human osteoblasts and mesenchymal stem cells (MSCs). These investigations highlight the suitability of biodegradable composites as implant coatings for decreasing the risk of bacterial contamination associated with prosthetic procedures.

  15. The Bone Marrow-Derived Stromal Cells

    DEFF Research Database (Denmark)

    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

    diseases. BM stromal cells (also known as skeletal or mesenchymal stem cells) [bone marrow stromal stem cell (BMSC)] are multipotent stem cells located within BM stroma and give rise to osteoblasts and adipocytes. However, cellular and molecular mechanisms of BMSC lineage commitment to adipocytic lineage...... and regulation of BM adipocyte formation are not fully understood. In this review, we will discuss recent findings pertaining to identification and characterization of adipocyte progenitor cells in BM and the regulation of differentiation into mature adipocytes. We have also emphasized the clinical relevance...

  16. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

    Science.gov (United States)

    Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G.

    1998-01-01

    Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study

  17. The survival condition and immunoregulatory function of adipose stromal vascular fraction (SVF in the early stage of nonvascularized adipose transplantation.

    Directory of Open Access Journals (Sweden)

    Ziqing Dong

    Full Text Available INTRODUCTION: Adipose tissue transplantation is one of the standard procedures for soft-tissue augmentation, reconstruction, and rejuvenation. However, it is unknown as to how the graft survives after transplantation. We thus seek out to investigate the roles of different cellular components in the survival of graft. MATERIALS & METHODS: The ratios of stromal vascular fraction (SVF cellular components from human adipose tissue were evaluated using flow cytometry. Human liposuction aspirates that were either mixed with marked SVF cells or PBS were transplanted into nude mice. The graft was harvested and stained on days 1,4,7 and 14. The inflammation level of both SVF group and Fat-only group were also evaluated. RESULTS: Flow cytometric analysis showed SVF cells mainly contained blood-derived cells, adipose-derived stromal cells (ASCs, and endothelial cells. Our study revealed that most cells are susceptible to death after transplantation, although CD34+ ASCs can remain viable for 14 days. Notably, we found that ASCs migrated to the peripheral edge of the graft. Moreover, the RT-PCR and the immuno-fluorescence examination revealed that although the SVF did not reduce the number of infiltrating immune cells (macrophages in the transplant, it does have an immunoregulatory function of up-regulating the expression of CD163 and CD206 and down-regulating that of IL-1β, IL-6. CONCLUSIONS: Our study suggests that the survival of adipose tissue after nonvascularized adipose transplantation may be due to the ASCs in SVF cells. Additionally, the immunoregulatory function of SVF cells may be indirectly contributing to the remolding of adipose transplant, which may lead to SVF-enriched adipose transplantation.

  18. Co-delivery of platelet-derived growth factor (PDGF-BB) and bone morphogenic protein (BMP-2) coated onto heparinized titanium for improving osteoblast function and osteointegration.

    Science.gov (United States)

    Kim, Sung Eun; Yun, Young-Pil; Lee, Jae Yong; Shim, June-Sung; Park, Kyeongsoon; Huh, Jung-Bo

    2015-12-01

    The aim of this study was to improve osteoblast function by delivering two growth factors, PDGF-BB and BMP-2, incorporated onto heparinized titanium (Hep-Ti) substrate. To achieve co-delivery of PDGF-BB and BMP-2, the surface of anodized Ti was immobilized with heparin, and then the two growth factors were coated onto the Hep-Ti surface. Incorporation of the two growth factors onto Hep-Ti was evaluated by SEM and XPS. Incorporated PDGF-BB and BMP-2 were released from the Hep-Ti substrate in a sustained manner. In vitro studies revealed that osteoblasts grown on PDGF-BB- and BMP-2-immobilized Hep-Ti increased ALP activity, calcium deposition, osteocalcin and osteopontin levels as compared to those grown on PDGF-BB alone- or BMP-2 alone-immobilized Hep-Ti. These results suggested that co-delivery of PDGF-BB and BMP-2 using Hep-Ti substrate will be a promising material for the enhancement of osteoblast function and osteointegration. Copyright © 2013 John Wiley & Sons, Ltd.

  19. Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

    Science.gov (United States)

    Terriza, Antonia; Vilches-Pérez, José I.; González-Caballero, Juan L.; de la Orden, Emilio; Yubero, Francisco; Barranco, Angel; Gonzalez-Elipe, Agustín R.; Vilches, José; Salido, Mercedes

    2014-01-01

    New biomaterials for Guided Bone Regeneration (GBR), both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB®) HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide) (PLGA) membrane foil functionalized by a very thin film (around 15 nm) of TiO2 (i.e., TiO2/PLGA membranes), designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors. PMID:28788538

  20. Osteoblasts Interaction with PLGA Membranes Functionalized with Titanium Film Nanolayer by PECVD. In vitro Assessment of Surface Influence on Cell Adhesion during Initial Cell to Material Interaction

    Directory of Open Access Journals (Sweden)

    Antonia Terriza

    2014-03-01

    Full Text Available New biomaterials for Guided Bone Regeneration (GBR, both resorbable and non-resorbable, are being developed to stimulate bone tissue formation. Thus, the in vitro study of cell behavior towards material surface properties turns a prerequisite to assess both biocompatibility and bioactivity of any material intended to be used for clinical purposes. For this purpose, we have developed in vitro studies on normal human osteoblasts (HOB® HOB® osteoblasts grown on a resorbable Poly (lactide-co-glycolide (PLGA membrane foil functionalized by a very thin film (around 15 nm of TiO2 (i.e., TiO2/PLGA membranes, designed to be used as barrier membrane. To avoid any alteration of the membranes, the titanium films were deposited at room temperature in one step by plasma enhanced chemical vapour deposition. Characterization of the functionalized membranes proved that the thin titanium layer completely covers the PLGA foils that remains practically unmodified in their interior after the deposition process and stands the standard sterilization protocols. Both morphological changes and cytoskeletal reorganization, together with the focal adhesion development observed in HOB osteoblasts, significantly related to TiO2 treated PLGA in which the Ti deposition method described has revealed to be a valuable tool to increase bioactivity of PLGA membranes, by combining cell nanotopography cues with the incorporation of bioactive factors.

  1. The synthetic progestin, gestodene, affects functional biomarkers in neonatal rat osteoblasts through an estrogen receptor-related mechanism of action.

    Science.gov (United States)

    Enríquez, Juana; García, Gustavo; Herrero, Bertha; Larrea, Fernando

    2017-11-01

    Clinical studies have shown that gestodene (GDN), a potent third-generation synthetic progestin, affects bone resorption. However, its mode of action in bone cells is not fully understood. The aim of this study was to establish whether GDN affects bone directly or through its bioconversion to other metabolites with different biological activities. In this study, we investigated the effects of GDN and its A-ring reduced metabolites on proliferation, differentiation, and mineralization of calvarial osteoblasts isolated from neonatal rat and their capacity to displace [ 3 H]-E 2 at ER binding sites. In contrast to progesterone, gestodene did exert significant effects on osteoblast activities. The most striking finding was the observation that the A-ring reduced derivatives 3β,5α-tetrahydro-GDN and 3α,5α-tetrahydro-GDN, though to a lesser extent, had greater stimulatory effects on the osteoblast activity than those observed with GDN. The effects on osteoblast proliferation and differentiation induced by GDN-reduced derivatives were abolished by the antiestrogen ICI 182780, consistent with their binding affinities for the estrogen receptor. In addition, the presence of a 5α-reductase inhibitor or inhibitors of aldo-keto hydroxysteroid dehydrogenases abolished the GDN-induced enhancement of osteoblast differentiation. These results indicated that GDN is metabolized to the A-ring reduced metabolites with estrogen-like activities and through this mechanism, GDN may affect the osteoblast activity. Together, the data suggest that synthetic progestins derived from 19-nortestosterone such as GDN, have beneficial effects on bone due to their biotransformation into metabolites with intrinsic estrogenic activity.

  2. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Directory of Open Access Journals (Sweden)

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  3. Osteoinductivity of nanostructured hydroxyapatite-functionalized gelatin modulated by human and endogenous mesenchymal stromal cells.

    Science.gov (United States)

    Della Bella, Elena; Parrilli, Annapaola; Bigi, Adriana; Panzavolta, Silvia; Amadori, Sofia; Giavaresi, Gianluca; Martini, Lucia; Borsari, Veronica; Fini, Milena

    2018-04-01

    The demand of new strategies for the induction of bone regeneration is continuously increasing. Biomimetic porous gelatin-nanocrystalline hydroxyapatite scaffolds with tailored properties were previously developed, showing a positive response in terms of cell adhesion, proliferation, and differentiation. In the present paper, we focused on their osteoinductive properties. The effect of scaffolds on osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was investigated in vitro. hMSCs were seeded on GEL (type A gelatin) and GEL containing 10 wt% hydroxyapatite (GEL-HA) and cultured in osteogenic medium. Results showed that GEL and GEL-HA10 sustained hMSC differentiation, with an increased ALP activity and a higher expression of bone specific genes. The osteoinductive ability of these scaffolds was then studied in vivo in a heterotopic bone formation model in nude mice. The influence of hMSCs within the implants was examined as well. Both GEL and GEL-HA10 scaffolds mineralized when implanted without hMSCs. On the contrary, the presence of hMSC abolished or reduced mineralization of GEL and GEL-HA10 scaffolds. However, we could observe a species-specific response to the presence of HA, which stimulated osteogenic differentiation of human cells only. In conclusion, the scaffolds showed promising osteoinductive properties and may be suitable for use in confined critical defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 914-923, 2018. © 2017 Wiley Periodicals, Inc.

  4. Characterization and function of histamine receptors in human bone marrow stromal cells.

    Science.gov (United States)

    Nemeth, Krisztian; Wilson, Todd; Rada, Balazs; Parmelee, Alissa; Mayer, Balazs; Buzas, Edit; Falus, Andras; Key, Sharon; Masszi, Tamas; Karpati, Sarolta; Mezey, Eva

    2012-02-01

    There are several clinical trials worldwide using bone marrow stromal cells (BMSCs) as a cellular therapy to modulate immune responses in patients suffering from various inflammatory conditions. A deeper understanding of the molecular mechanisms involved in this modulatory effect could help us design better, more effective protocols to treat immune mediated diseases. In this study, we demonstrated that human BMSCs express H1, H2, and H4 histamine receptors and they respond to histamine stimulation with an increased interleukin 6 (IL-6) production both in vitro and in vivo. Using different receptor antagonists, we pinpointed the importance of the H1 histamine receptor, while Western blot analysis and application of various mitogen-activated protein kinase inhibitors highlighted the role of p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase kinases in the observed effect. When BMSCs were pretreated with either histamine or degranulated human mast cells, they exhibited an enhanced IL-6-dependent antiapoptotic effect on neutrophil granulocytes. Based on these observations, it is likely that introduction of BMSCs into a histamine-rich environment (such as any allergic setting) or pretreatment of these cells with synthetic histamine could have a significant modulatory effect on the therapeutic potential of BMSCs. Copyright © 2011 AlphaMed Press.

  5. HIV-1 protein induced modulation of primary human osteoblast differentiation and function via a Wnt/β-catenin-dependent mechanism.

    LENUS (Irish Health Repository)

    Butler, Joseph S

    2013-02-01

    HIV infection is associated with metabolic bone disease resulting in bone demineralization and reduced bone mass. The molecular mechanisms driving this disease process have yet to be elucidated. Wnt\\/β-catenin signaling plays a key role in bone development and remodeling. We attempted to determine the effects of the HIV-1 protein, gp120, on Wnt\\/β-catenin signaling at an intracellular and transcriptional level in primary human osteoblasts (HOBs). This work, inclusive of experimental controls, was part of a greater project assessing the effects of a variety of different agents on Wnt\\/β-catenin signaling (BMC Musculoskelet Disord 2010;11:210).We examined the phenotypic effects of silencing and overexpressing the Wnt antagonist, Dickkopf-1 (Dkk1) in HOBs treated with gp120. HOBs exposed to gp120 displayed a significant reduction in alkaline phosphatase activity (ALP) activity and cell proliferation and increased cellular apoptosis over a 48 h time course. Immunocytochemistry demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to HIV-1 protein exposure. These changes were associated with a reduction of TCF\\/LEF-mediated transcription, the transcriptional outcome of canonical Wnt β-catenin signaling. Silencing Dkk1 expression in HOBs exposed to gp120 resulted in increased ALP activity and cell proliferation, and decreased cellular apoptosis relative to scrambled control. Dkk1 overexpression exacerbated the inhibitory effect of gp120 on HOB function, with decreases in ALP activity and cell proliferation and increased cellular apoptosis relative to vector control. Wnt\\/β-catenin signaling plays a key regulatory role in HIV-associated bone loss, with Dkk1, aputative central mediator in this degenerative process. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 218-226, 2013.

  6. Senescence-associated intrinsic mechanisms of osteoblast dysfunctions

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Trinquier, Anne Marie-Pierre Emilie

    2011-01-01

    factors, and intrinsic mechanisms caused by the osteoblast cellular senescence. The aim of this review is to provide a summary of the intrinsic senescence mechanisms affecting osteoblastic functions and how they can be targeted in order to abolish age-related osteoblastic dysfunction and bone loss...

  7. Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions

    Directory of Open Access Journals (Sweden)

    María de Lourdes Mora-García

    2016-10-01

    Full Text Available Abstract Background In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs from bone marrow and other “classic” sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs on effector T lymphocytes through the purinergic pathway. Methods We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs. We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+ lymphocyte function through the generation of adenosine (Ado. Results We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. Conclusions This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

  8. Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

    International Nuclear Information System (INIS)

    Yang Jieping; Murthy, Sreemala; Winata, Therry; Werner, Sean; Abe, Masumi; Prahalad, Agasanur K.; Hock, Janet M.

    2006-01-01

    The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development

  9. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  10. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    International Nuclear Information System (INIS)

    Godoy-Gallardo, Maria; Guillem-Marti, Jordi; Sevilla, Pablo; Manero, José M.; Gil, Francisco J.

    2016-01-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  11. The interplay between nanostructured carbon-grafted chitosan scaffolds and protein adsorption on the cellular response of osteoblasts: structure-function property relationship.

    Science.gov (United States)

    Depan, D; Misra, R D K

    2013-04-01

    The rapid adsorption of proteins occurs during the early stages of biomedical device implantation into physiological systems. In this regard, the adsorption of proteins is a strong function of the nature of a biomedical device, which ultimately governs the biological functions. The objective of this study was to elucidate the interplay between nanostructured carbon-modified (graphene oxide and single-walled carbon nanohorn) chitosan scaffolds and consequent protein adsorption and biological function (osteoblast function). We compare and contrast the footprint of protein adsorption on unmodified chitosan and nanostructured carbon-modified chitosan. A comparative analysis of cell-substrate interactions using an osteoblast cell line (MC3T3-E1) implied that biological functions were significantly enhanced in the presence of nanostructured carbon, compared with unmodified chitosan. The difference in their respective behaviors is related to the degree and topography of protein adsorption on the scaffolds. Furthermore, there was a synergistic effect of nanostructured carbon and protein adsorption in terms of favorably modulating biological functions, including cell attachment, proliferation and viability, with the effect being greater on nanostructured carbon-modified scaffolds. The study also underscores that protein adsorption is favored in nanostructured carbon-modified scaffolds such that bioactivity and biological function are promoted. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Treadmill exercise with bone marrow stromal cells transplantation potentiates recovery of locomotor function after spinal cord injury in rats.

    Science.gov (United States)

    Kim, You-Mi; Seo, Tae-Beom; Kim, Chang-Ju; Ji, Eun-Sang

    2017-06-01

    Transplantation of bone marrow stromal cells (BMSCs) is regarded as a promising candidate for the spinal cord injury (SCI). In the present study, we investigated whether treadmill exercise potentiate the effect of BM-SCs transplantation on the functional recovery in the SCI rats. The spinal cord contusion injury applied at the T9-T10 level using the impactor. Cultured BMSCs were transplanted into the lesion at 1 week after SCI induction. Treadmill exercise was conducted for 6 weeks. Basso-Beattie-Bresnahan (BBB) scale for locomotor function was determined. Sprouting axons in the lesion cavity were detected by immunofluorescence staining for neurofilament-200. Brain-derived neurotrophic factor (BDNF) and synapsin-I expressions were analyzed using western blotting. BMSCs transplantation improved BBB score and increased expressions of neurofilament-200, BDNF, and synapsin-I in the SCI rats. Treadmill exercise potentiated the improving effect of BMSCs transplantation on BBB score in the SCI rats. This potentiating effect of treadmill exercise could be ascribed to the enhancement of BDNF expression in the SCI rats.

  13. Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury

    Directory of Open Access Journals (Sweden)

    Dasa Cizkova

    2018-03-01

    Full Text Available It was recently shown that the conditioned medium (CM of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM applied intrathecally (IT for spinal cord injury (SCI recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI.

  14. Human mesenchymal stromal cells enhance the immunomodulatory function of CD8+CD28− regulatory T cells

    Science.gov (United States)

    Liu, Qiuli; Zheng, Haiqing; Chen, Xiaoyong; Peng, Yanwen; Huang, Weijun; Li, Xiaobo; Li, Gang; Xia, Wenjie; Sun, Qiquan; Xiang, Andy Peng

    2015-01-01

    One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8+CD28− Treg cells and found that the MSCs could not only increase the proportion of CD8+CD28− T cells, but also enhance CD8+CD28−T cells' ability of hampering naive CD4+ T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4+ T cells and inducing the apoptosis of activated CD4+ T cells. Mechanistically, the MSCs affected the functions of the CD8+CD28− T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8+CD28+ T cells to CD8+CD28− T cells, but did increase the proportion of CD8+CD28− T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8+CD28− Treg cells, shedding new light on MSCs-mediated immune regulation. PMID:25482073

  15. Bone marrow stromal cells transplantation combined with ultrashortwave therapy promotes functional recovery on spinal cord injury in rats.

    Science.gov (United States)

    Yin, Yan-Mei; Lu, Yu; Zhang, Li-Xin; Zhang, Guang-Ping; Zhang, Zhi-Qiang

    2015-03-01

    This study investigated the effects of bone marrow stromal cells (BMSCs) transplantation combined with low dose ultrashortwave (USW) radiation on functional recovery from spinal cord injury (SCI) in rats. Female Sprague-Dawley rats (n = 64) were randomly divided into five groups: sham-operated, as well as control, USW, BMSCs, and BMSCs + USW that were subjected to spinal cord contusion. BMSCs were transplanted into rats in the BMSCs and BMSCs+USW groups, while those in the USW and BMSCs + USW were exposed to radiation; Basso-Beattie-Bresnahan (BBB) tests were performed before the operation and at 1-week intervals after SCI for 7 weeks. 7 weeks later, animals were assessed footprint pattern, motor evoked potential (MEP), histological features, and expression of the ED1 and glial fibrillary acidic protein (GFAP), markers of activated macrophages and astrocytes, respectively, in the lesion area. BBB scores were higher in the BMSCs + USW than in the BMSCs and control groups. The USW and BMSCs + USW groups had improved coordination compared to the BMSCs and control groups (P spinal cord area than the other injured rats, and also had expression of GFAP in transplanted BMSCs. All treatment groups had lower ED1 expression and MEP latency than the controls (P spinal cord repair. © 2015 Wiley Periodicals, Inc.

  16. Evaluation of gene delivery strategies to efficiently overexpress functional HLA-G on human bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Joana S Boura

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSC constitutively express low levels of human leukocyte antigen-G (HLA-G, which has been shown to contribute to their immunomodulatory and anti-inflammatory properties. Here, we hypothesized that overexpression of HLA-G on bone marrow-derived MSC would improve their immunomodulatory function, thus increasing their therapeutic potential. Therefore, we investigated which gene transfer system is best suited for delivering this molecule while maintaining its immunomodulatory effects. We performed a side-by-side comparison between three nonviral plasmid-based platforms (pmax-HLA-G1; MC-HLA-G1; pEP-HLA-G1 and a viral system (Lv-HLA-G1 using gene transfer parameters that yielded similar levels of HLA-G1-expressing MSC. Natural killer (NK cell–mediated lysis assays and T cell proliferation assays showed that MSC modified with the HLA-G1 expressing viral vector had significantly lower susceptibility to NK-lysis and significantly reduced T cell proliferation when compared to nonmodified cells or MSC modified with plasmid. We also show that, in plasmid-modified MSC, an increase in Toll-like receptor (TLR9 expression is the mechanism responsible for the abrogation of HLA-G1's immunomodulatory effect. Although MSC can be efficiently modified to overexpress HLA-G1 using viral and nonviral strategies, only viral-based delivery of HLA-G1 is suitable for improvement of MSC's immunomodulatory properties.

  17. What was the ancestral function of decidual stromal cells? A model for the evolution of eutherian pregnancy.

    Science.gov (United States)

    Chavan, Arun Rajendra; Bhullar, Bhart-Anjan S; Wagner, Günter P

    2016-04-01

    In human and mouse, decidual stromal cells (DSC) are necessary for the establishment (implantation) and the maintenance of pregnancy by preventing inflammation and the immune rejection of the semi-allograft conceptus. DSC originated along the stem lineage of eutherian mammals, coincidental with the origin of invasive placentation. Surprisingly, in many eutherian lineages decidual cells are lost after the implantation phase of pregnancy, making it unlikely that DSC are necessary for the maintenance of pregnancy in these animals. In order to understand this variation, we review the literature on the fetal-maternal interface in all major eutherian clades Euarchontoglires, Laurasiatheria, Xenarthra and Afrotheria, as well as the literature about the ancestral eutherian species. We conclude that maintaining pregnancy may not be a shared derived function of DSC among all eutherian mammals. Rather, we propose that DSC originated to manage the inflammatory reaction associated with invasive implantation. We envision that this happened in a stem eutherian that had invasive placenta but still a short gestation. We further propose that extended gestation evolved independently in the major eutherian clades explaining why the major lineages of eutherian mammals differ with respect to the mechanisms maintaining pregnancy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Reply: "Function of Cryopreserved Mesenchymal Stromal Cells With and Without Interferon-γ Prelicensing Is Context Dependent".

    Science.gov (United States)

    Galipeau, Jacques

    2017-05-01

    Freshly thawed mesenchymal stromal cells (MSCs) transiently display altered cell physiology and a recent report by Chinnadurai et al. [Stem Cells 2016;34:2429-2442] demonstrated that thawed allogeneic MSCs are also susceptible to T-cell mediated lysis in vitro and that this effect can be mitigated by activating MSCs with IFN-γ prior to freeze and thaw. In the letter to the editor by Ankrum et al., "Function of Cryopreserved MSCs with and without IFN-γ pre-licensing is Context Dependent," the author provide data that IFN-γ pretreatment of human MSCs before cryobanking fails to enhance their potency post thaw in a mouse model of retinal disease. Considering that the experimental set up involves the use of human MSCs in mice with normal immune systems, we propose that IFN-γ MSC activation enhances the immune incompatibility of xenogenic human cells in a manner which may confound the predictive value for human translational use. Stem Cells 2017;35:1440-1441. © 2016 AlphaMed Press.

  19. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

    2002-01-01

    . The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions.......Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells...

  20. Functional role of the Ca2+-activated Cl− channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

    International Nuclear Information System (INIS)

    Berglund, Erik; Akcakaya, Pinar; Berglund, David; Karlsson, Fredrik; Vukojević, Vladana; Lee, Linkiat; Bogdanović, Darko; Lui, Weng-Onn; Larsson, Catharina; Zedenius, Jan; Fröbom, Robin; Bränström, Robert

    2014-01-01

    DOG1, a Ca 2+ -activated Cl − channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl − currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target. - Highlights: • Subcellular DOG1 localization varies between GIST cells. • DOG1 in GIST is voltage- and Ca 2+ -activated. • Known TMEM16A modulators, like A01 and Eact, modulate DOG1. • DOG1 has small effects on cell viability and proliferation in vitro. • DOG1 impact early apoptotic GIST cells to undergo late apoptosis

  1. Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

    Science.gov (United States)

    Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

    2016-03-01

    Quercetin (3,3',4',5,7-pentahydroxy-flavone) is a flavonoid known for its pharmacological activities, which include antioxidant and anti-inflammatory properties, as well as possible beneficial action on diseases involving bone loss. In this work, we explored the possibility to functionalize hydroxyapatite (HA) with quercetin in order to obtain new materials for bone repair through local administration of the flavonoid. HA was synthesized in presence of different concentrations of quercetin according to two different procedures: direct synthesis and phase transition from monetite. Direct synthesis lead to composite nanocrystals containing up to 3.1 wt% quercetin, which provokes a reduction of the crystals mean dimensions and of the length of the coherently scattering domains. Synthesis conditions provoke a partial oxidation of quercetin and, as a consequence, a significant reduction of its radical scavenging activity (RSA). On the other hand, synthesis through phase transition yields samples containing up to 1.3 wt% of quercetin incorporated into hydroxyapatite, with minor structural modifications, which exhibit relevant anti-oxidant activities, as testified by their high RSA levels, (slightly lower than that of pure quercetin). The biological response to these materials was tested using an innovative triculture model involving osteoblast, osteoclast and endothelial cells, in order to mimic bone microenvironment. The results show that the presence of quercetin in the composite materials enhances human osteoblast-like MG63 proliferation and differentiation, whereas it downregulates osteoclastogenesis of osteoclast precursors 2T-110, and supports proliferation and differentiation of human umbilical vein endothelial cells (HUVEC). The pharmacological activities of the flavonoid quercetin include anti-oxidant and antiinflammatory properties, as well as capability to prevent bone loss. In this paper, we demonstrate that it is possible to synthesize hydroxyapatite

  2. Pellet culture model for human primary osteoblasts.

    Science.gov (United States)

    Jähn, K; Richards, R G; Archer, C W; Stoddart, M J

    2010-09-06

    In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

  3. Pellet culture model for human primary osteoblasts

    Directory of Open Access Journals (Sweden)

    K Jähn

    2010-09-01

    Full Text Available In vitro monolayer culture of human primary osteoblasts (hOBs often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

  4. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced

  5. Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions.

    Science.gov (United States)

    Pollock, Kathryn; Samsonraj, Rebekah M; Dudakovic, Amel; Thaler, Roman; Stumbras, Aron; McKenna, David H; Dosa, Peter I; van Wijnen, Andre J; Hubel, Allison

    2017-06-01

    Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin, the antiapoptotic marker B cell lymphoma 2, as well as the cell surface adhesion molecules CD106 (vascular cell adhesion molecule 1) and CD54 (intracellular adhesion molecule 1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

  6. Changes in Ovarian Stromal Function and Associated Symptoms in Premenopausal Women Undergoing Chemotherapy for Breast Cancer

    National Research Council Canada - National Science Library

    Frost, Marlene H; Loprinzi, Charles L; Kearns, Ann E; Sloan, Jeff A; Barton, Debra L

    2007-01-01

    ...), mid-treatment, immediate post-treatment, and 6 months later. Questionnaires include the Female Sexual Function Index, Greene Climacteric Scale, Profile of Mood States, Schwartz Fatigue Scale, and a menses diary...

  7. The Spatiotemporal Control of Osteoblast Cell Growth, Behavior, and Function Dictated by Nanostructured Stainless Steel Artificial Microenvironments.

    Science.gov (United States)

    Dhawan, Udesh; Pan, Hsu-An; Shie, Meng-Je; Chu, Ying Hao; Huang, Guewha S; Chen, Po-Chun; Chen, Wen Liang

    2017-12-01

    The successful application of a nanostructured biomaterial as an implant is strongly determined by the nanotopography size triggering the ideal cell response. Here, nanoporous topography on 304L stainless steel substrates was engineered to identify the nanotopography size causing a transition in the cellular characteristics, and accordingly, the design of nanostructured stainless steel surface as orthopedic implants is proposed. A variety of nanopore diameters ranging from 100 to 220 nm were fabricated by one-step electrolysis process and collectively referred to as artificial microenvironments. Control over the nanopore diameter was achieved by varying bias voltage. MG63 osteoblasts were cultured on the nanoporous surfaces for different days. Immunofluorescence (IF) and scanning electron microscopy (SEM) were performed to compare the modulation in cell morphologies and characteristics. Osteoblasts displayed differential growth parameters and distinct transition in cell behavior after nanopore reached a certain diameter. Nanopores with 100-nm diameter promoted cell growth, focal adhesions, cell area, viability, vinculin-stained area, calcium mineralization, and alkaline phosphatase activity. The ability of these nanoporous substrates to differentially modulate the cell behavior and assist in identifying the transition step will be beneficial to biomedical engineers to develop superior implant geometries, triggering an ideal cell response at the cell-nanotopography interface.

  8. The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction.

    Science.gov (United States)

    Özdal-Kurt, F; Tuğlu, I; Vatansever, H S; Tong, S; Deliloğlu-Gürhan, S I

    2015-01-01

    Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell

  9. SDHA loss of function mutations in a subset of young adult wild-type gastrointestinal stromal tumors

    International Nuclear Information System (INIS)

    Italiano, Antoine; Chen, Chun-Liang; Sung, Yun-Shao; Singer, Samuel; DeMatteo, Ronald P; LaQuaglia, Michael P; Besmer, Peter; Socci, Nicholas; Antonescu, Cristina R

    2012-01-01

    A subset of KIT/PDGFRA wild-type gastrointestinal stromal tumors (WT GIST) have been associated with alteration of the succinate dehydrogenase (SDH) complex II function. A recent report identified four non-syndromic, KIT/PDGFRA WT GIST harboring compound heterozygous or homozygous mutations in SDHA encoding the main subunit of the SDH complex II. Next generation sequencing was applied on five pediatric and one young adult WT GIST, by whole exome capture and SOLiD 3-plus system sequencing. The putative mutations were first confirmed by Sanger sequencing and then screened on a larger panel of 11 pediatric and young adult WT GIST, including 5 in the context of Carney triad. A germline p.Arg31X nonsense SDHA mutation was identified in one of the six cases tested by SOLiD platform. An additional p.D38V missense mutation in SDHA exon 2 was identified by Sanger sequencing in the extended KIT/PDGFRA WT GIST patients cohort. Western blotting showed loss of SDHA expression in the two cases harboring SDHA mutations, while expression being retained in the other WT GIST tumors. Results were further confirmed by immunohistochemistry for both SDHA and SDHB, which showed a concurrent loss of expression of both proteins in SDHA-mutant lesions, while the remaining WT tumors showed only loss of SDHB expression. Germline and/or somatic aberrations of SDHA occur in a small subset of KIT/PDGFRA WT GISTs, outside the Carney’s triad and are associated with loss of both SDHA and SDHB protein expression. Mutations of the SDH complex II are more particularly associated with KIT/PDGFRA WT GIST occurring in young adults. Although pediatric GIST consistently display alterations of SDHB protein expression, further molecular studies are needed to identify the crucial genes involved in their tumorigenesis

  10. Stromal Gene Expression and Function in Primary Breast Tumors that Metastasize to Bone Cancer

    Science.gov (United States)

    2006-07-01

    significantly altered gene ontology categories were 7 Description Common Function stefin A1 stfa1 cystatin , cathepsin inhibitor breast cancer ... cancer . Breast Cancer Res, 7: 33-36, 2005. 5. Alcaraz, J., Nelson, C . M., and Bissell, M. J. Biomechanical approaches for studying integration of...analysis of fibroblastic stromagenesis in breast cancer progression. J Mammary Gland Biol Neoplasia, 9: 311-324, 2004. 7. Kuperwasser, C ., Chavarria, T

  11. Mesenchymal Stromal Cells Improve Salivary Function and Reduce Lymphocytic Infiltrates in Mice with Sjogren's-Like Disease

    NARCIS (Netherlands)

    Khalili, Saeed; Liu, Younan; Kornete, Mara; Roescher, Nienke; Kodama, Shohta; Peterson, Alan; Piccirillo, Ciriaco A.; Tran, Simon D.

    2012-01-01

    Background: Non-obese diabetic (NOD) mice develop Sjogren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases

  12. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  13. Limonene protects osteoblasts against methylglyoxal-derived adduct formation by regulating glyoxalase, oxidative stress, and mitochondrial function.

    Science.gov (United States)

    Suh, Kwang Sik; Chon, Suk; Choi, Eun Mi

    2017-12-25

    Methylglyoxal (MG) is a potent protein glycating agent and an important precursor of advanced glycation end products, which are involved in the pathogenesis of diabetic osteopathy. In this study, we investigated the effects of limonene on MG-induced damage in osteoblastic MC3T3-E1 cells. Pretreating cells with limonene prevented MG-induced protein adduct formation, tumor necrosis factor alpha and interleukin-6 release, mitochondrial superoxide production, and cardiolipin peroxidation. In addition, limonene increased glyoxalase I activity, and glutathione and heme oxygenase-1 levels in the presence of MG. Pretreatment with limonene prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss, and reduced the levels of adenosine monophosphate-activated protein kinase, peroxisome proliferator activated receptor γ coactivator 1α, and nitric oxide. These results demonstrate that limonene may prevent the development of diabetic osteopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Adipose-derived perivascular mesenchymal stromal/stem cells promote functional vascular tissue engineering for cardiac regenerative purposes.

    Science.gov (United States)

    Morrissette-McAlmon, Justin; Blazeski, Adriana; Somers, Sarah; Kostecki, Geran; Tung, Leslie; Grayson, Warren L

    2018-02-01

    Cardiac tissue engineering approaches have the potential to regenerate functional myocardium with intrinsic vascular networks. This study compared the relative effects of human adipose-derived stem/stromal cells (hASCs) and human dermal fibroblasts (hDFs) in cocultures with neonatal rat ventricular cardiomyocytes (NRVCMs) and human umbilical vein endothelial cells (HUVECs). At the same ratios of NRVCM:hASC and NRVCM:hDF, the hASC cocultures displayed shorter action potentials and maintained capture at faster pacing rates. Similarly, in coculture with HUVECs, hASC:HUVEC exhibited superior ability to support vascular capillary network formation relative to hDF:HUVEC. Based on these studies, a range of suitable cell ratios were determined to develop a triculture system. Six seeding ratios of NRVCM:hASC:HUVEC were tested and it was found that a ratio of 500:50:25 cells (i.e. 250,000:25,000:12,500 cells/cm 2 ) resulted in the formation of robust vascular networks while retaining action potential durations and propagation similar to pure NRVCM cultures. Tricultures in this ratio exhibited an average conduction velocity of 20 ± 2 cm/s, action potential durations at 80% repolarization (APD 80 ) and APD 30 of 122 ± 5 ms and 59 ± 4 ms, respectively, and maximum capture rate of 7.4 ± 0.6 Hz. The NRVCM control groups had APD 80 and APD 30 of 120 ± 9 ms and 51 ± 5 ms, with a maximum capture rate of 7.3 ± 0.2 Hz. In summary, the combination of hASCs in the appropriate ratios with NRVCMs and HUVECs can facilitate the formation of densely vascularized cardiac tissues that appear not to impact the electrophysiological function of cardiomyocytes negatively. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Constitutive β-catenin activation in osteoblasts impairs terminal osteoblast differentiation and bone quality

    International Nuclear Information System (INIS)

    Bao, Quanwei; Chen, Sixu; Qin, Hao; Feng, Jianquan; Liu, Huayu; Liu, Daocheng; Li, Ang; Shen, Yue; Zhong, Xiaozheng; Li, Junfeng; Zong, Zhaowen

    2017-01-01

    Accumulating evidence suggests that Wnt/β-catenin signaling plays a central role in controlling bone mass. We previously reported that constitutive activation of β-catenin (CA-β-catenin) in osteoblasts potentially has side effects on the bone growth and bone remodeling process, although it could increase bone mass. The present study aimed to observe the effects of osteoblastic CA-β-catenin on bone quality and to investigate possible mechanisms of these effects. It was found that CA-β-catenin mice exhibited lower mineralization levels and disorganized collagen in long bones as confirmed by von Kossa staining and sirius red staining, respectively. Also, bone strength decreased significantly in CA-β-catenin mice. Then the effect of CA-β-catenin on biological functions of osteoblasts were investigated and it was found that the expression levels of osteocalcin, a marker for the late differentiation of osteoblasts, decreased in CA-β-catenin mice, while the expression levels of osterix and alkaline phosphatase, two markers for the early differentiation of osteoblasts, increased in CA-β-catenin mice. Furthermore, higher proliferation rate were revealed in osteoblasts that were isolated from CA-β-catenin mice. The Real-time PCR and western blot examination found that the expression level of c-myc and cyclin D1, two G1 progression-related molecules, increased in osteoblasts that were isolated from the CA-β-catenin mice, and the expression levels of CDK14 and cyclin Y, two mitotic-related molecules that can accelerate cells entering into S and G2/M phases, increased in osteoblasts that were isolated from the CA-β-catenin mice. In summary, osteoblastic CA-β-catenin kept osteoblasts in high proliferative state and impaired the terminal osteoblast differentiation, and this led to changed bone structure and decreased bone strength. - Highlights: • Wnt/β-catenin signaling plays a central role in controlling bone mass. • CA-β-catenin has side effects on the bone

  16. 1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

    2001-01-01

    1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated...... osteoblastic phenotype. The IGF system, including IGFs-I, and -II and IGF binding proteins (IGFBPs), plays an important role in osteoblast cell proliferation and differentiation....

  17. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

    Directory of Open Access Journals (Sweden)

    Justyna Czapla

    Full Text Available The aim of the present study was to isolate mesenchymal stromal cells (MSC with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI. The study aimed also to investigate the feasibility of xenogeneic MSCs implantation.MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO. Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb. MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed.In two murine models (hindlimb ischemia and MI we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

  18. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).

    Science.gov (United States)

    Bourin, Philippe; Bunnell, Bruce A; Casteilla, Louis; Dominici, Massimo; Katz, Adam J; March, Keith L; Redl, Heinz; Rubin, J Peter; Yoshimura, Kotaro; Gimble, Jeffrey M

    2013-06-01

    Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters. Copyright © 2013 International Society for Cellular Therapy. All rights reserved.

  19. Measurement of oxygen consumption rate of osteoblasts from ...

    African Journals Online (AJOL)

    Jane

    2011-05-10

    May 10, 2011 ... By measuring the oxygen consumption rates of osteoblasts in different cultures in vitro, it would provide significant instruction and model function for further fundamental investigation and clinical application in bone tissue engineering. Key words: Bone tissue engineering, osteoblast, respiration rate, in vitro ...

  20. Aryl Hydrocarbon Receptor Antagonists Mitigate the Effects of Dioxin on Critical Cellular Functions in Differentiating Human Osteoblast-Like Cells

    Directory of Open Access Journals (Sweden)

    Chawon Yun

    2018-01-01

    Full Text Available The inhibition of bone healing in humans is a well-established effect associated with cigarette smoking, but the underlying mechanisms are still unclear. Recent work using animal cell lines have implicated the aryl hydrocarbon receptor (AhR as a mediator of the anti-osteogenic effects of cigarette smoke, but the complexity of cigarette smoke mixtures makes understanding the mechanisms of action a major challenge. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin is a high-affinity AhR ligand that is frequently used to investigate biological processes impacted by AhR activation. Since there are dozens of AhR ligands present in cigarette smoke, we utilized dioxin as a prototype ligand to activate the receptor and explore its effects on pro-osteogenic biomarkers and other factors critical to osteogenesis using a human osteoblast-like cell line. We also explored the capacity for AhR antagonists to protect against dioxin action in this context. We found dioxin to inhibit osteogenic differentiation, whereas co-treatment with various AhR antagonists protected against dioxin action. Dioxin also negatively impacted cell adhesion with a corresponding reduction in the expression of integrin and cadherin proteins, which are known to be involved in this process. Similarly, the dioxin-mediated inhibition of cell migration correlated with reduced expression of the chemokine receptor CXCR4 and its ligand, CXCL12, and co-treatment with antagonists restored migratory capacity. Our results suggest that AhR activation may play a role in the bone regenerative response in humans exposed to AhR activators, such as those present in cigarette smoke. Given the similarity of our results using a human cell line to previous work done in murine cells, animal models may yield data relevant to the human setting. In addition, the AhR may represent a potential therapeutic target for orthopedic patients who smoke cigarettes, or those who are exposed to secondhand smoke or other

  1. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells

    DEFF Research Database (Denmark)

    Dokkedahl, Karin Stenderup; Justesen, Jeannette; Clausen, Christian

    2003-01-01

    Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching...... donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells......, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation....

  2. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    Directory of Open Access Journals (Sweden)

    Abbas Jafari

    2017-02-01

    Full Text Available Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.

  3. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    DEFF Research Database (Denmark)

    Jafari Kermani, Abbas; Qanie, Diyako; Andersen, Thomas L

    2017-01-01

    and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin....... In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent......Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells...

  4. Substrate Induced Osteoblast-Like Differentiation of Stromal Stem Cells

    Science.gov (United States)

    Belizar, Jacqueline; Glaser, Reena; Hung, Matthew; Simon, Marcia; Jurukovski, Vladimir; Rafailovich, Miriam; Shih, Alice

    2009-03-01

    We have demonstrated that Adipose-derived stem cells (ASCs) can be induced to biomineralize on a polybutadiene (PB) coated Si substrate. The cells began to generate calcium phosphate deposits after a five-day incubation period in the absence of dexamethasone. Control cells plated on tissue culture PS culture dish (TCP) did not biomineralize. In addition, the biomineralizing culture retained proliferative cells In order to determine whether the induction was transient, we transferred the cells exposed to polybutadiene after 14 and 28-day incubation periods to TCP dishes. These cells continued to biominerlize. Genetic testing is underway which will determine whether differentiation is maintained after transfer.

  5. Transplantation of human bone marrow stromal cell-derived Schwann cells reduces cystic cavity and promotes functional recovery after contusion injury of adult rat spinal cord.

    Science.gov (United States)

    Kamada, Takahito; Koda, Masao; Dezawa, Mari; Anahara, Reiko; Toyama, Yoshiro; Yoshinaga, Katsunori; Hashimoto, Masayuki; Koshizuka, Shuhei; Nishio, Yutaka; Mannoji, Chikato; Okawa, Akihiko; Yamazaki, Masashi

    2011-02-01

    The aim of this study was to evaluate whether transplantation of human bone marrow stromal cell-derived Schwann cells (hBMSC-SC) promotes functional recovery after contusive spinal cord injury of adult rats. Human bone marrow stromal cells (hBMSC) were cultured from bone marrow of adult human patients and induced into Schwann cells (hBMSC-SC) in vitro. Schwann cell phenotype was confirmed by immunocytochemistry. Growth factors secreted from hBMSC-SC were detected using cytokine antibody array. Immunosuppressed rats were laminectomized and their spinal cords were contused using NYU impactor (10 g, 25 mm). Nine days after injury, a mixture of Matrigel and hBMSC-SC (hBMSC-SC group) was injected into the lesioned site. Five weeks after transplantation, cresyl-violet staining revealed that the area of cystic cavity was smaller in the hBMSC-SC group than that in the control group. Immunohistochemistry revealed that the number of anti-growth-associated protein-43-positive nerve fibers was significantly larger in the hBMSC-SC group than that in the control group. At the same time, the number of tyrosine hydroxylase- or serotonin-positive fibers was significantly larger at the lesion epicenter and caudal level in the hBMSC-SC group than that in the control group. In electron microscopy, formation of peripheral-type myelin was recognized near the lesion epicenter in the hBMSC-SC group. Hind limb function recovered significantly in the hBMSC-SC group compared with the control group. In conclusion, the functions of hBMSC-SC are comparable to original Schwann cells in rat spinal cord injury models, and are thus potentially useful treatments for patients with spinal cord injury. © 2010 Japanese Society of Neuropathology.

  6. Simvastatin mobilizes bone marrow stromal cells migrating to injured areas and promotes functional recovery after spinal cord injury in the rat.

    Science.gov (United States)

    Han, Xiaoguang; Yang, Ning; Cui, Yueyi; Xu, Yingsheng; Dang, Gengting; Song, Chunli

    2012-07-19

    This study investigated the therapeutic effects of simvastatin administered by subarachnoid injection after spinal cord injury (SCI) in rats; explored the underlying mechanism from the perspective of mobilization, migration and homing of bone marrow stromal cells (BMSCs) to the injured area induced by simvastatin. Green fluorescence protein labeled-bone marrow stromal cells (GFP-BMSCs) were transplanted into rats through the tail vein for stem cell tracing. Twenty-four hours after transplantation, spinal cord injury (SCI) was produced using weight-drop method (10g 4cm) at the T10 level. Simvastatin (5mg/kg) or vehicle was administered by subarachnoid injection at lumbar level 4 after SCI. Locomotor functional recovery was assessed in the 4 weeks following surgery using the open-field test and inclined-plane test. At the end of the study, MRI was used to evaluate the reparation of the injured spinal cord. Animals were then euthanized, histological evaluation was used to measure lesion cavity volumes. Immunofluorescence for GFP and cell lineage markers (NeuN and GFAP) was used to evaluate simvastatin-mediated mobilization and differentiation of transplanted BMSCs. Western blot and immunohistochemistry were used to assess the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Simvastatin-treated animals showed significantly better locomotor recovery, less signal abnormality in MRI and a smaller cavity volume compared to the control group. Immunofluorescence revealed that simvastatin increased the number of GFP-positive cells in the injured spinal cord, and the number of cells double positive for GFP/NeuN or GFP/GFAP was larger in the simvastatin treated group than the control group. Western blot and immunohistochemistry showed higher expression of BDNF and VEGF in the simvastatin treated group than the control group. In conclusion, simvastatin can help to repair spinal cord injury in rat, where the underlying

  7. Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

    Directory of Open Access Journals (Sweden)

    Ozlem Guzeloglu Kayisli

    Full Text Available Use of long-acting progestin only contraceptives (LAPCs offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s in human endometrial stromal cells (HESCs. Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2 or E2+ medroxyprogesterone acetate (MPA or E2+ etonogestrel (ETO or E2+ progesterone (P4 were analyzed by quantitative Real-time (q-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA treated women and ovariectomized guinea pigs (GPs treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR

  8. Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

    Science.gov (United States)

    Guzeloglu Kayisli, Ozlem; Kayisli, Umit A; Basar, Murat; Semerci, Nihan; Schatz, Frederick; Lockwood, Charles J

    2015-01-01

    Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription

  9. Molecular evolution and functional divergence of the Ca(2+ sensor protein in store-operated Ca(2+ entry: stromal interaction molecule.

    Directory of Open Access Journals (Sweden)

    Xinjiang Cai

    2007-07-01

    Full Text Available Receptor-mediated Ca(2+ signaling in many non-excitable cells initially induces Ca(2+ release from intracellular Ca(2+ stores, followed by Ca(2+ influx across the plasma membrane. Recent findings have suggested that stromal interaction molecules (STIMs function as the Ca(2+ sensor to detect changes of Ca(2+ content in the intracellular Ca(2+ stores. Human STIMs and invertebrate STIM share several functionally important protein domains, but diverge significantly in the C-terminus. To better understand the evolutionary significance of STIM activity, phylogenetic analysis of the STIM protein family was conducted after extensive database searching. Results from phylogeny and sequence analysis revealed early adaptation of the C-terminal divergent domains in Urochordata, before the expansion of STIMs in Vertebrata. STIMs were subsequently subjected to one round of gene duplication as early as in the Euteleostomi lineage in vertebrates, with a second round of fish-specific gene duplication. After duplication, STIM-1 and STIM-2 molecules appeared to have undergone purifying selection indicating strong evolutionary constraints within each group. Furthermore, sequence analysis of the EF-hand Ca(2+ binding domain and the SAM domain, together with functional divergence studies, identified critical regions/residues likely underlying functional changes, and provided evidence for the hypothesis that STIM-1 and STIM-2 might have developed distinct functional properties after duplication.

  10. Functional differentiation of uterine stromal cells involves cross-regulation between bone morphogenetic protein 2 and Kruppel-like factor (KLF) family members KLF9 and KLF13.

    Science.gov (United States)

    Pabona, John Mark P; Zeng, Zhaoyang; Simmen, Frank A; Simmen, Rosalia C M

    2010-07-01

    The inability of the uterine epithelium to enter a state of receptivity for the embryo to implant is a significant underlying cause of early pregnancy loss. We previously showed that mice null for the progesterone receptor (PGR)-interacting protein Krüppel-like factor (KLF) 9 are subfertile and exhibit reduced uterine progesterone sensitivity. KLF9 expression is high in predecidual stroma, undetectable in decidua, and enhanced in uteri of mice with conditional ablation of bone morphogenetic protein 2 (BMP2). Given the individual importance of KLF9 and BMP2 for implantation success, we hypothesized that the establishment of uterine receptivity involves KLF9 and BMP2 functional cross-regulation. To address this, we used early pregnant wild-type and Klf9 null mice and KLF9 small interfering RNA-transfected human endometrial stromal cells (HESCs) induced to differentiate under standard conditions. Loss of KLF9 in mice and HESCs enhanced BMP2 expression, whereas recombinant BMP2 treatment of HESCs attenuated KLF9 mRNA levels. IGFBP1 and KLF9-related KLF13 expression were positively associated with BMP2 and inversely associated with KLF9. Prolonged, but not short-term, knockdown of KLF9 in HESCs reduced IGFBP1 expression. Mouse uterine Igfbp1 expression was similarly reduced with Klf9 ablation. PGR-A and PGR-B expression were positively associated with KLF9 in predecidual HESCs but not decidualizing HESCs. KLF13 knockdown attenuated BMP2 and PGR-B and abrogated BMP2-mediated inhibition of KLF9 expression. Results support cross-regulation among BMP2, KLF9, and KLF13 to maintain progesterone sensitivity in stromal cells undergoing differentiation and suggest that loss of this regulatory network compromises establishment of uterine receptivity and implantation success.

  11. Acellular spinal cord scaffold seeded with bone marrow stromal cells protects tissue and promotes functional recovery in spinal cord-injured rats.

    Science.gov (United States)

    Chen, Jian; Zhang, Zhongmin; Liu, Jia; Zhou, Rongping; Zheng, Xiaochen; Chen, Tianyu; Wang, Liang; Huang, Minjun; Yang, Chengliang; Li, Zhen; Yang, Cheng; Bai, Xiaochun; Jin, Dadi

    2014-03-01

    Therapy using scaffolds seeded with stem cells plays an important role in repair of spinal cord injury (SCI), with the transplanted cells differentiating into nerve cells to replace the lost tissue while releasing neurotrophic factors that contribute to repair following SCI and enhance the function of the damaged nervous system. The present study investigated the ability to extend the survival time of bone marrow stromal cells (BMSCs) to restore the damaged spinal cord and improve functional recovery by grafting acellular spinal cord (ASC) scaffold seeded or not with BMSCs in a rat model of acute hemisected SCI. BBB scores revealed that treatment with BMSCs seeded into ASC scaffold led to an obvious improvement in motor function recovery compared with treatment with ASC scaffold alone or untreated controls. This improvement was evident at 2 and 8 weeks after surgery (P < 0.05). When BMSCs labeled with 5-bromodeoxyuridine were implanted together with ASC scaffold into the injured sites, they differentiated into glial cells, and some BMSCs could be observed within the graft by immunofluorescent staining at 8 weeks after implantation. Evaluation of caspase-3 activation suggested that the graft group was able to reduce apoptosis compared with SCI alone at 8 weeks after operation (P < 0.05). This study suggests that ASC scaffolds have the ability to enhance BMSC survival and improve differentiation and could also reduce native damaged nerve tissue apoptosis, thus protecting host tissue as well as improving functional recovery after implantation. Copyright © 2013 Wiley Periodicals, Inc.

  12. Retention and Functional Effect of Adipose-Derived Stromal Cells Administered in Alginate Hydrogel in a Rat Model of Acute Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Bjarke Follin

    2018-01-01

    Full Text Available Background. Cell therapy for heart disease has been proven safe and efficacious, despite poor cell retention in the injected area. Improving cell retention is hypothesized to increase the treatment effect. In the present study, human adipose-derived stromal cells (ASCs were delivered in an in situ forming alginate hydrogel following acute myocardial infarction (AMI in rats. Methods. ASCs were transduced with luciferase and tested for ASC phenotype. AMI was inducted in nude rats, with subsequent injection of saline (controls, 1 × 106 ASCs in saline or 1 × 106 ASCs in 1% (w/v alginate hydrogel. ASCs were tracked by bioluminescence and functional measurements were assessed by magnetic resonance imaging (MRI and 82rubidium positron emission tomography (PET. Results. ASCs in both saline and alginate hydrogel significantly increased the ejection fraction (7.2% and 7.8% at 14 days and 7.2% and 8.0% at 28 days, resp.. After 28 days, there was a tendency for decreased infarct area and increased perfusion, compared to controls. No significant differences were observed between ASCs in saline or alginate hydrogel, in terms of retention and functional salvage. Conclusion. ASCs improved the myocardial function after AMI, but administration in the alginate hydrogel did not further improve retention of the cells or myocardial function.

  13. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...... bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106 and CD166 as revealed by immunohistochemical staining and flow cytometry (FACS) analysis. Ex vivo differentiation of h...

  14. Allogeneic Adipose-Derived Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Regulating Self-Reactive T Cell Responses and Dendritic Cell Function

    Directory of Open Access Journals (Sweden)

    Per Anderson

    2017-01-01

    Full Text Available Multipotent mesenchymal stromal cells (MSCs have emerged as a promising therapy for autoimmune diseases, including multiple sclerosis (MS. Administration of MSCs to MS patients has proven safe with signs of immunomodulation but their therapeutic efficacy remains low. The aim of the current study has been to further characterize the immunomodulatory mechanisms of adipose tissue-derived MSCs (ASCs in vitro and in vivo using the EAE model of chronic brain inflammation in mice. We found that murine ASCs (mASCs suppress T cell proliferation in vitro via inducible nitric oxide synthase (iNOS and cyclooxygenase- (COX- 1/2 activities. mASCs also prevented the lipopolysaccharide- (LPS- induced maturation of dendritic cells (DCs in vitro. The addition of the COX-1/2 inhibitor indomethacin, but not the iNOS inhibitor L-NAME, reversed the block in DC maturation implicating prostaglandin (PG E2 in this process. In vivo, early administration of murine and human ASCs (hASCs ameliorated myelin oligodendrocyte protein- (MOG35-55- induced EAE in C57Bl/6 mice. Mechanistic studies showed that mASCs suppressed the function of autoantigen-specific T cells and also decreased the frequency of activated (CD11c+CD40high and CD11c+TNF-α+ DCs in draining lymph nodes (DLNs. In summary, these data suggest that mASCs reduce EAE severity, in part, through the impairment of DC and T cell function.

  15. Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis : Caveolin-1 Antagonizes Cyclin D1 Function in Mammary Epithelial Cells

    OpenAIRE

    Williams, Terence M.; Sotgia, Federica; Lee, Hyangkyu; Hassan, Ghada; Di Vizio, Dolores; Bonuccelli, Gloria; Capozza, Franco; Mercier, Isabelle; Rui, Hallgeir; Pestell, Richard G.; Lisanti, Michael P.

    2006-01-01

    Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system ...

  16. Effects of Emdogain on osteoblast gene expression.

    Science.gov (United States)

    Carinci, F; Piattelli, A; Guida, L; Perrotti, V; Laino, G; Oliva, A; Annunziata, M; Palmieri, A; Pezzetti, F

    2006-05-01

    Emdogain (EMD) is a protein extract purified from porcine enamel and has been introduced in clinical practice to obtain periodontal regeneration. EMD is composed mainly of amelogenins (90%), while the remaining 10% is composed of non-amelogenin enamel matrix proteins such as enamelins, tuftelin, amelin and ameloblastin. Enamel matrix proteins seem to be involved in root formation. EMD has been reported to promote proliferation, migration, adhesion and differentiation of cells associated with healing periodontal tissues in vivo. How this protein acts on osteoblasts is poorly understood. We therefore attempted to address this question by using a microarray technique to identify genes that are differently regulated in osteoblasts exposed to enamel matrix proteins. By using DNA microarrays containing 20,000 genes, we identified several upregulated and downregulated genes in the osteoblast-like cell line (MG-63) cultured with enamel matrix proteins (Emd). The differentially expressed genes cover a broad range of functional activities: (i) signaling transduction, (ii) transcription, (iii) translation, (iv) cell cycle regulation, proliferation and apoptosis, (v) immune system, (vi) vesicular transport and lysosome activity, and (vii) cytoskeleton, cell adhesion and extracellular matrix production. The data reported are the first genome-wide scan of the effect of enamel matrix proteins on osteoblast-like cells. These results can contribute to our understanding of the molecular mechanisms of bone regeneration and as a model for comparing other materials with similar clinical effects.

  17. Bone marrow mesenchymal stromal cells and olfactory ensheathing cells transplantation after spinal cord injury--a morphological and functional comparison in rats.

    Science.gov (United States)

    Torres-Espín, Abel; Redondo-Castro, Elena; Hernández, Joaquim; Navarro, Xavier

    2014-05-01

    Cell therapy for spinal cord injury (SCI) is a promising strategy for clinical application. Both bone marrow mesenchymal stromal cells (MSCs; also known as bone marrow-derived 'mesenchymal stem cells') and olfactory ensheathing cells (OECs) have demonstrated beneficial effects following transplantation in animal models of SCI. However, due to the large number of affecting parameters that determine the therapy success and the lack of methodological consensus, the comparison of different works is difficult. Therefore, we compared the effects of MSC and OEC transplants at early or delayed time after a spinal cord contusion injury in the rat. Functional outcomes for locomotion, sensory perception and electrophysiological responses were assessed. Moreover, the grafted cells survival and the amount of cavity and spared tissue were studied. The findings indicate that grafted cells survived until 7 days post-injection, but markedly disappeared in the following 2 weeks. Despite the low survival of the cells, MSC and OEC grafts provided tissue protection after early and delayed transplantation. Nevertheless, only acute MSC grafts improved locomotion recovery in treadmill condition and electrophysiological outcomes with respect to the other injured groups. These results, together with previous works, indicate that the MSC seem a better option than OEC for treatment of contusion injuries. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  18. PDLLA honeycomb-like scaffolds with a high loading of superhydrophilic graphene/multi-walled carbon nanotubes promote osteoblast in vitro functions and guided in vivo bone regeneration.

    Science.gov (United States)

    Silva, Edmundo; Vasconcellos, Luana Marotta Reis de; Rodrigues, Bruno V M; Dos Santos, Danilo Martins; Campana-Filho, Sergio P; Marciano, Fernanda Roberta; Webster, Thomas J; Lobo, Anderson Oliveira

    2017-04-01

    Herein, we developed honeycomb-like scaffolds by combining poly (d, l-lactic acid) (PDLLA) with a high amount of graphene/multi-walled carbon nanotube oxides (MWCNTO-GO, 50% w/w). From pristine multi-walled carbon nanotubes (MWCNT) powders, we produced MWCNTO-GO via oxygen plasma etching (OPE), which promoted their exfoliation and oxidation. Initially, we evaluated PDLLA and PDLLA/MWCNTO-GO scaffolds for tensile strength tests, cell adhesion and cell viability (with osteoblast-like MG-63 cells), alkaline phosphatase (ALP, a marker of osteoblast differentiation) activity and mineralized nodule formation. In vivo tests were carried out using PDLLA and PDLLA/MWCNTO-GO scaffolds as fillers for critical defects in the tibia of rats. MWCNTO-GO loading was responsible for decreasing the tensile strength and elongation-at-break of PDLLA scaffolds, although the high mechanical performance observed (~600MPa) assures their application in bone tissue regeneration. In vitro results showed that the scaffolds were not cytotoxic and allowed for osteoblast-like cell interactions and the formation of mineralized matrix nodules. Furthermore, MG-63 cells grown on PDLLA/MWCNTO-GO significantly enhanced osteoblast ALP activity compared to controls (cells alone), while the PDLLA group showed similar ALP activity when compared to controls and PDLLA/MWCNTO-GO. Most impressively, in vivo tests suggested that compared to PDLLA scaffolds, PDLLA/MWCNTO-GO had a superior influence on bone cell activity, promoting greater new bone formation. In summary, the results of this study highlighted that this novel scaffold (MWCNTO-GO, 50% w/w) is a promising alternative for bone tissue regeneration and, thus, should be further studied. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Fabrication of poly(ethylene glycol) hydrogel micropatterns with osteoinductive growth factors and evaluation of the effects on osteoblast activity and function

    Science.gov (United States)

    Subramani, K.; Birch, M. A.

    2006-09-01

    The aims of this study were to fabricate poly(ethylene glycol) (PEG) hydrogel micropatterns on a biomaterial surface to guide osteoblast behaviour and to study how incorporating vascular endothelial growth factor (VEGF) within the adhered hydrogel influenced cell morphology. Standard photolithographic procedures or photopolymerization through a poly(dimethyl siloxane) (PDMS) mould were used to fabricate patterned PEG hydrogels on the surface of silanized silicon wafers. Hydrogel patterns were evaluated by light microscopy and surface profilometry. Rat osteoblasts were cultured on these surfaces and cell morphology investigated by fluorescence microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Release of protein trapped in the polymerized PEG was evaluated and VEGF-PEG surfaces were characterized for their ability to support cell growth. These studies show that photopolymerized PEG can be used to create anti-adhesive structures on the surface of silicon that completely control where cell interaction with the substrate takes place. Using conventional lithography, structures down to 50 µm were routinely fabricated with the boundaries exhibiting sloping sides. Using the PDMS mould approach, structures were fabricated as small as 10 µm and boundaries were very sharp and vertical. Osteoblasts exhibiting typical morphology only grew on the silicon wafer surface that was not coated with PEG. Adding BSA to the monomer solution showed that protein could be released from the hydrogel for up to 7 days in vitro. Incorporating VEGF in the hydrogel produced micropatterns that dramatically altered osteoblast behaviour. At boundaries with the VEGF-PEG hydrogel, there was striking formation of cellular processes and membrane ruffling indicative of a change in cell morphology. This study has explored the morphogenetic properties of VEGF and the applications of nano/microfabrication techniques for guided tissue (bone) regeneration in dental and

  20. The role of versican G3 domain in regulating breast cancer cell motility including effects on osteoblast cell growth and differentiation in vitro – evaluation towards understanding breast cancer cell bone metastasis

    Directory of Open Access Journals (Sweden)

    Du William

    2012-08-01

    Full Text Available Abstract Background Versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, is predictive of relapse, and negatively impacts overall survival rates. The versican G3 domain is important in breast cancer cell growth, migration and bone metastasis. However, mechanistic studies evaluating versican G3 enhanced breast cancer bone metastasis are limited. Methods A versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe tumor migration and invasion to bone stromal cells and MC3T3-E1 cells. Alkaline phosphatase (ALP staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells. Results In the four mouse breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1 conditioned medium (CM inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of alkaline phosphatase (ALP activity. We exogenously expressed a versican G3 construct in a cell line that expresses low versican levels (66c14, and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor AG1478, selective MEK inhibitor PD 98059, and selective AKT inhibitor Triciribine, but not by selective JNK inhibitor SP 600125. Versican G3 enhanced breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling

  1. Bone marrow stromal cell sheets may promote axonal regeneration and functional recovery with suppression of glial scar formation after spinal cord transection injury in rats.

    Science.gov (United States)

    Okuda, Akinori; Horii-Hayashi, Noriko; Sasagawa, Takayo; Shimizu, Takamasa; Shigematsu, Hideki; Iwata, Eiichiro; Morimoto, Yasuhiko; Masuda, Keisuke; Koizumi, Munehisa; Akahane, Manabu; Nishi, Mayumi; Tanaka, Yasuhito

    2017-03-01

    OBJECTIVE Transplantation of bone marrow stromal cells (BMSCs) is a theoretical potential as a therapeutic strategy in the treatment of spinal cord injury (SCI). Although a scaffold is sometimes used for retaining transplanted cells in damaged tissue, it is also known to induce redundant immunoreactions during the degradation processes. In this study, the authors prepared cell sheets made of BMSCs, which are transplantable without a scaffold, and investigated their effects on axonal regeneration, glial scar formation, and functional recovery in a completely transected SCI model in rats. METHODS BMSC sheets were prepared from the bone marrow of female Fischer 344 rats using ascorbic acid and were cryopreserved until the day of transplantation. A gelatin sponge (GS), as a control, or BMSC sheet was transplanted into a 2-mm-sized defect of the spinal cord at the T-8 level. Axonal regeneration and glial scar formation were assessed 2 and 8 weeks after transplantation by immunohistochemical analyses using anti-Tuj1 and glial fibrillary acidic protein (GFAP) antibodies, respectively. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan scale. RESULTS The BMSC sheets promoted axonal regeneration at 2 weeks after transplantation, but there was no significant difference in the number of Tuj1-positive axons between the sheet- and GS-transplanted groups. At 8 weeks after transplantation, Tuj1-positive axons elongated across the sheet, and their numbers were significantly greater in the sheet group than in the GS group. The areas of GFAP-positive glial scars in the sheet group were significantly reduced compared with those of the GS group at both time points. Finally, hindlimb locomotor function was ameliorated in the sheet group at 4 and 8 weeks after transplantation. CONCLUSIONS The results of the present study indicate that an ascorbic acid-induced BMSC sheet is effective in the treatment of SCI and enables autologous transplantation without requiring a

  2. Functional role of the Ca{sup 2+}-activated Cl{sup −} channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Berglund, Erik, E-mail: erik.berglund@ki.se [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Department of Breast and Endocrine Surgery, Karolinska University Hospital, Stockholm (Sweden); Akcakaya, Pinar [Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Stockholm (Sweden); Berglund, David [Section for Transplantation Surgery, Department of Surgical Sciences, Uppsala University Hospital, Uppsala (Sweden); Karlsson, Fredrik [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Department of Breast and Endocrine Surgery, Karolinska University Hospital, Stockholm (Sweden); Vukojević, Vladana [Department of Clinical Neuroscience, Karolinska Institutet, Stockholm (Sweden); Lee, Linkiat [Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Stockholm (Sweden); Bogdanović, Darko [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Lui, Weng-Onn; Larsson, Catharina [Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Stockholm (Sweden); Zedenius, Jan [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Department of Breast and Endocrine Surgery, Karolinska University Hospital, Stockholm (Sweden); Fröbom, Robin [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Bränström, Robert [Endocrine and Sarcoma Surgery Unit, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm (Sweden); Department of Breast and Endocrine Surgery, Karolinska University Hospital, Stockholm (Sweden)

    2014-08-15

    DOG1, a Ca{sup 2+}-activated Cl{sup −} channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl{sup −} currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target. - Highlights: • Subcellular DOG1 localization varies between GIST cells. • DOG1 in GIST is voltage- and Ca{sup 2+}-activated. • Known TMEM16A modulators, like A01 and Eact, modulate DOG1. • DOG1 has small effects on cell viability and proliferation in vitro. • DOG1 impact early apoptotic GIST cells to undergo late apoptosis.

  3. Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

    Science.gov (United States)

    Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

  4. Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lei [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wu, Zhong [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Yin, Gang; Liu, Haifeng; Guan, Xiaojun; Zhao, Xiaoqiang [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China); Wang, Jianguang, E-mail: jianguangwang@163.com [Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Zhu, Jianguo, E-mail: gehujianguo68@163.com [Department of Orthopedics, Changzhou Wujin People’s Hospital-South Division, Affiliated Hospital of Jiangsu University, Changzhou (China)

    2014-12-12

    Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but little is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.

  5. Induction of a program gene expression during osteoblast differentiation with strontium ranelate

    International Nuclear Information System (INIS)

    Zhu Lingling; Zaidi, Samir; Peng Yuanzhen; Zhou Hang; Moonga, Baljit S.; Blesius, Alexia; Dupin-Roger, Isabelle; Zaidi, Mone; Sun Li

    2007-01-01

    Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate

  6. Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

    Directory of Open Access Journals (Sweden)

    Chiara Sassoli

    2018-01-01

    Full Text Available Bone marrow-derived mesenchymal stromal cell- (BM-MSC- based therapy is a promising option for regenerative medicine. An important role in the control of the processes influencing the BM-MSC therapeutic efficacy, namely, extracellular matrix remodelling and proliferation and secretion ability, is played by matrix metalloproteinase- (MMP- 2. Therefore, the identification of paracrine/autocrine regulators of MMP-2 function may be of great relevance for improving BM-MSC therapeutic potential. We recently reported that BM-MSCs release the bioactive lipid sphingosine 1-phosphate (S1P and, here, we demonstrated an impairment of MMP-2 expression/release when the S1P receptor subtype S1PR1 is blocked. Notably, active S1PR1/MMP-2 signalling is required for F-actin structure assembly (lamellipodia, microspikes, and stress fibers and, in turn, cell proliferation. Moreover, in experimental conditions resembling the damaged/regenerating tissue microenvironment (hypoxia, S1P/S1PR1 system is also required for HIF-1α expression and vinculin reduction. Our findings demonstrate for the first time the trophic role of S1P/S1PR1 signalling in maintaining BM-MSCs’ ability to modulate MMP-2 function, necessary for cytoskeleton reorganization and cell proliferation in both normoxia and hypoxia. Altogether, these data provide new perspectives for considering S1P/S1PR1 signalling a pharmacological target to preserve BM-MSC properties and to potentiate their beneficial potential in tissue repair.

  7. hCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.

    Science.gov (United States)

    Tapia-Pizarro, Alejandro; Archiles, Sebastián; Argandoña, Felipe; Valencia, Cecilia; Zavaleta, Keyla; Cecilia Johnson, M; González-Ramos, Reinaldo; Devoto, Luigi

    2017-06-01

    How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such

  8. The effect of metal ions released from different dental implant-abutment couples on osteoblast function and secretion of bone resorbing mediators.

    Science.gov (United States)

    Alrabeah, Ghada O; Brett, Peter; Knowles, Jonathan C; Petridis, Haralampos

    2017-11-01

    The etiology of the reduced marginal bone loss observed around platform-switched implant-abutment connections is not clear but could be related to the release of variable amounts of corrosion products. The present study evaluated the effect of different concentrations of metal ions released from different implant abutment couples on osteoblastic cell viability, apoptosis and expression of genes related to bone resorption. Osteoblastic cells were exposed to five conditions of culture media prepared containing metal ions (titanium, aluminum, vanadium, cobalt, chromium and molybdenum) in different concentrations representing the amounts released from platform-matched and platform-switched implant-abutment couples as a result of an earlier accelerated corrosion experiment. Cell viability was evaluated over 21days using the Alamar Blue assay. Induction of apoptosis was measured after 24h of exposure using flow cytometry. Expression of interleukin-6, interleukin-8, cyclooxygenase-2, caspase-8, osteoprotegerin and receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblastic cells were analysed after exposure for 1, 3 and 21days using real-time quantitative polymerase chain reaction assay RESULTS: Metal ions in concentrations representing the platform-matched groups led to a reduction in cell viability (PMetal ions up-regulated the expression of interleukin-6, interleukin-8, cyclooxygenase-2 and RANKL in a dose dependent manner after 1day of exposure (Pmetal ions. The change in cytokine levels expressed was directly proportional to the metal ion concentration. The observed biological responses to decreased amounts of metal ions released from platform-switched implant-abutment couples compared to platform-matched couples may partly explain the positive radiographic findings in respect to crestal bone level when utilising the "platform-switching" concept, which highlights the possible role of corrosion products in the mediation of crestal bone loss around

  9. Recovery of renal function after administration of adipose-tissue-derived stromal vascular fraction in rat model of acute kidney injury induced by ischemia/reperfusion injury.

    Science.gov (United States)

    Lee, Chunwoo; Jang, Myoung Jin; Kim, Bo Hyun; Park, Jin Young; You, Dalsan; Jeong, In Gab; Hong, Jun Hyuk; Kim, Choung-Soo

    2017-06-01

    Acute kidney injury (AKI) induced by ischemia/reperfusion (I/R) injury is a major challenge in critical care medicine. The purpose of this study is to determine the therapeutic effects of the adipose-tissue-derived stromal vascular fraction (SVF) and the optimal route for SVF delivery in a rat model of AKI induced by I/R injury. Fifty male Sprague-Dawley rats were randomly divided into five groups (10 animals per group): sham, nephrectomy control, I/R injury control, renal arterial SVF infusion and subcapsular SVF injection. To induce AKI by I/R injury, the left renal artery was clamped with a nontraumatic vascular clamp for 40 min, and the right kidney was removed. Rats receiving renal arterial infusion of SVF had a significantly reduced increase in serum creatinine compared with the I/R injury control group at 4 days after I/R injury. The glomerular filtration rate of the renal arterial SVF infusion group was maintained at a level similar to that of the sham and nephrectomy control groups at 14 days after I/R injury. Masson's trichrome staining showed significantly less fibrosis in the renal arterial SVF infusion group compared with that in the I/R injury control group in the outer stripe (P renal arterial SVF infusion and subcapsular SVF injection groups compared with the I/R injury control group in the outer stripe (P renal function is effectively rescued from AKI induced by I/R injury through the renal arterial administration of SVF in a rat model.

  10. Mechanisms regulating osteoblast response to surface microtopography and vitamin D

    Science.gov (United States)

    Bell, Bryan Frederick, Jr.

    A comprehensive understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is essential in the design of advanced biomaterials that better promote bone growth and osseointegration of implants. Dental implants with roughened surfaces and high surface energy are well known to promote osteoblast differentiation in vitro and promote increased bone-to-implant contact in vivo. In addition, increased surface roughness increases osteoblasts response to the vitamin D metabolite 1alpha,25(OH)2D3. However, the exact mechanisms mediating cell response to surface properties and 1alpha,25(OH)2D3 are still being elucidated. The central aim of the thesis is to investigate whether integrin signaling in response to rough surface microtopography enhances osteoblast differentiation and responsiveness to 1alpha,25(OH)2D3. The hypothesis is that the integrin alpha5beta1 plays a role in osteoblast response to surface microtopography and that 1alpha,25(OH) 2D3 acts through VDR-independent pathways involving caveolae to synergistically enhance osteoblast response to surface roughness and 1alpha,25(OH) 2D3. To test this hypothesis the objectives of the studies performed in this thesis were: (1) to determine if alpha5beta 1 signaling is required for osteoblast response to surface microstructure; (2) to determine if increased responsiveness to 1alpha,25(OH)2D 3 requires the vitamin D receptor, (3) to determine if rough titanium surfaces functionalized with the peptides targeting integrins (RGD) and transmembrane proteoglycans (KRSR) will enhance both osteoblast proliferation and differentiation, and (4) to determine whether caveolae, which are associated with integrin and 1alpha,25(OH)2D3 signaling, are required for enhance osteogenic response to surface microstructure and 1alpha,25(OH)2D 3. The results demonstrate that integrins, VDR, and caveolae play important roles in mediating osteoblast response to surface properties and 1alpha,25

  11. Osteoblast Role in Rheumatic Diseases

    Directory of Open Access Journals (Sweden)

    Addolorata Corrado

    2017-06-01

    Full Text Available Alterations in osteoblast growth, differentiation and activity play a role in the pathogenesis of several rheumatic diseases, such as rheumatoid arthritis, spondyloarthritides, osteoarthritis, and osteoporosis. In fact, in these rheumatic diseases, abnormal activity of Wnt signaling, receptor activator of nuclear factor-κB (RANK-RANK ligand (RANKL-osteoprotegerin (OPG signaling, bone morphogenetic proteins (BMPs pathway and other mechanisms have been described in osteoblasts. This review article is focused on current knowledge on the role of osteoblast dysregulation occurring in rheumatic diseases.

  12. Osteoblast Role in Rheumatic Diseases.

    Science.gov (United States)

    Corrado, Addolorata; Maruotti, Nicola; Cantatore, Francesco Paolo

    2017-06-15

    Alterations in osteoblast growth, differentiation and activity play a role in the pathogenesis of several rheumatic diseases, such as rheumatoid arthritis, spondyloarthritides, osteoarthritis, and osteoporosis. In fact, in these rheumatic diseases, abnormal activity of Wnt signaling, receptor activator of nuclear factor-κB (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) signaling, bone morphogenetic proteins (BMPs) pathway and other mechanisms have been described in osteoblasts. This review article is focused on current knowledge on the role of osteoblast dysregulation occurring in rheumatic diseases.

  13. Platelet-Rich Plasma Influences Expansion and Paracrine Function of Adipose-Derived Stromal Cells in a Dose-Dependent Fashion

    NARCIS (Netherlands)

    Willemsen, Joep C. N.; Spiekman, Maroesjka; Stevens, H. P. Jeroen; van der Lei, Berend; Harmsen, Martin C.

    Background: Lipofilling is a treatment modality to restore tissue volume. Both platelet-rich plasma and adipose-derived stromal cells have been reported to augment the efficacy of lipofilling, yet results are not conclusive. The authors hypothesized that the variation reported in literature is

  14. Stromal cell derived factor-1α (SDF-1α) directed chemoattraction of transiently CXCR4 overexpressing mesenchymal stem cells into functionalized three-dimensional biomimetic scaffolds

    DEFF Research Database (Denmark)

    Thieme, S; Ryser, Martin; Gentsch, Marcus

    2009-01-01

    Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface int...

  15. New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: biotinylated galectins as tool to detect specific bindong sites

    Czech Academy of Sciences Publication Activity Database

    Purkrábková, T.; Smetana Jr., K.; Dvořánková, B.; Holíková, Z.; Böck, C.; Lensch, M.; André, S.; Pytlík, R.; Liu, F.; Klíma, Jiří; Smetana, K.; Motlík, Jan; Gabius, H. J.

    2003-01-01

    Roč. 95, - (2003), s. 535-545 ISSN 0248-4900 R&D Projects: GA MŠk LN00A065 Institutional research plan: CEZ:AV0Z5045916 Keywords : biotinylation * bone marrow stromal cells * galectin Subject RIV: ED - Physiology Impact factor: 2.159, year: 2003

  16. Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

    Science.gov (United States)

    Watkins, Marcus; Grimston, Susan K; Norris, Jin Yi; Guillotin, Bertrand; Shaw, Angela; Beniash, Elia; Civitelli, Roberto

    2011-04-15

    Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

  17. Evolution of the osteoblast: skeletogenesis in gar and zebrafish

    Directory of Open Access Journals (Sweden)

    Eames B Frank

    2012-03-01

    Full Text Available Abstract Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD, or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio--a teleost--and the spotted gar (Lepisosteus oculatus--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group. We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our

  18. Celecoxib inhibits osteoblast differentiation independent of cyclooxygenase activity.

    Science.gov (United States)

    Matsuyama, Atsushi; Higashi, Sen; Tanizaki, Saori; Morotomi, Takahiko; Washio, Ayako; Ohsumi, Tomoko; Kitamura, Chiaki; Takeuchi, Hiroshi

    2018-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) exert their effects primarily by inhibiting the activity of cyclooxygenase (COX), thus suppressing prostaglandin synthesis. Some NSAIDs are known to perform functions other than pain control, such as suppressing tumour cell growth, independent of their COX-inhibiting activity. To identify NSAIDs with COX-independent activity, we examined various NSAIDs for their ability to inhibit osteoblastic differentiation using the mouse pre-osteoblast cell line MC3T3-E1. Only celecoxib and valdecoxib strongly inhibited osteoblastic differentiation, and this effect was not correlated with COX-inhibiting activity. Moreover, 2,5-dimethyl (DM)-celecoxib, a celecoxib analogue that does not inhibit COX activity, also inhibited osteoblastic differentiation. Celecoxib and DM-celecoxib inhibited osteoblastic differentiation induced by bone morphogenetic protein (BMP)-2 in C2C12 mouse myoblast cell line. Although celecoxib suppresses the growth of some tumour cells, the viability and proliferation of MC3T3-E1 cells were not affected by celecoxib or DM-celecoxib. Instead, celecoxib and DM-celecoxib suppressed BMP-2-induced phosphorylation of Smad1/5, a major downstream target of BMP receptor. Although it is well known that COX plays important roles in osteoblastic differentiation, these results suggest that some NSAIDs, such as celecoxib, have targets other than COX and regulate phospho-dependent intracellular signalling, thereby modifying bone remodelling. © 2017 John Wiley & Sons Australia, Ltd.

  19. Engineering stromal-epithelial interactions in vitro for toxicology assessment

    Science.gov (United States)

    Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo t...

  20. The role of stromal cells in inflammatory bone loss.

    Science.gov (United States)

    Wehmeyer, C; Pap, T; Buckley, C D; Naylor, A J

    2017-07-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, local and systemic bone loss and a lack of compensatory bone repair. Fibroblast-like synoviocytes (FLS) are the most abundant cells of the stroma and a key population in autoimmune diseases such as RA. An increasing body of evidence suggests that these cells play not only an important role in chronic inflammation and synovial hyperplasia, but also impact bone remodelling. Under inflammatory conditions FLS release inflammatory cytokines, regulate bone destruction and formation and communicate with immune cells to control bone homeostasis. Other stromal cells, such as osteoblasts and terminally differentiated osteoblasts, termed osteocytes, are also involved in the regulation of bone homeostasis and are dysregulated during inflammation. This review highlights our current understanding of how stromal cells influence the balance between bone formation and bone destruction. Increasing our understanding of these processes is critical to enable the development of novel therapeutic strategies with which to treat bone loss in RA. © 2017 British Society for Immunology.

  1. A novel flavonoid isolated from the steam-bark of Ulmus wallichiana planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation.

    Science.gov (United States)

    Swarnkar, Gaurav; Sharan, Kunal; Siddiqui, Jawed A; Chakravarti, Bandana; Rawat, Preeti; Kumar, Manmeet; Arya, Kamal R; Maurya, Rakesh; Chattopadhyay, Naibedya

    2011-05-11

    (2S,3S)-Aromadendrin-6-C-β-d-glucopyranoside (AG) is a novel flavonol isolated from the extract of Ulmus wallichiana (Himalayan Elm). Extract of U. wallichiana is used as a traditional medicine for rapid fracture repair in India. We characterized the mechanism of action of AG in mouse bone cells by investigating its effect on the precursors of osteoblasts, osteoclasts and adipocytes. At nanomolar concentrations, AG increased differentiation of preosteoblasts obtained from neonatal mouse calvaria. The gene expression of osteogenic markers, including runt-related transcription factor 2 (Runx-2), bone morphogenetic protein-2 (BMP-2), type I collagen and osteocalcin were elevated in the preosteoblasts. The extracellular matrix mineralization was higher in preosteoblast and bone marrow cells when AG was present in the medium. Furthermore, AG protected the differentiated osteoblasts from serum deprivation-induced apoptosis, and increased the expression of the anti-osteoclastogenic cytokine, osteoprotegerin. It inhibited osteoclast differentiation of bone marrow precursor cells to osteoclasts in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and monocyte/macrophage-colony stimulating factor (M-CSF). Additionally, in 3T3-L1 preadipocytes, AG decreased the expression of genes involved in adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP) and CCAAT/enhancer-binding protein alpha (CEBP/α), and induced apoptosis of differentiated adipocytes. Induction of adipogenic differentiation was also inhibited in the presence of AG. AG exhibited no estrogenic/antiestrogenic effect. Together, our data show that AG has potent osteogenic, anti-osteoclastogenic and anti-adipogenic effects, which may translate to a better skeletal outcome in postmenopausal osteoporosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Qiu, Weimin; Kassem, Moustapha

    2014-01-01

    Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling...... in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase...

  3. Equine corneal stromal abscesses

    DEFF Research Database (Denmark)

    Henriksen, M. D. L.; Andersen, P. H.; Plummer, C. E.

    2013-01-01

    The last 30 years have seen many changes in the understanding of the pathogenesis and treatment of equine corneal stromal abscesses (SAs). Stromal abscesses were previously considered an eye problem related to corneal bacterial infection, equine recurrent uveitis, corneal microtrauma and corneal...... foreign bodies in horses. They were more commonly diagnosed in horses living in subtropical climatic areas of the world. Therapeutic recommendations to treat equine SAs were historically nearly always a medical approach directed at bacteria and the often associated severe iridocyclitis. Today...... the pathogenesis of most equine SAs appears to be more often related to fungal inoculation of the anterior corneal stroma followed by posterior migration of the fungi deeper into the corneal stroma. There is also now an increased incidence of diagnosis of corneal SAs in horses living in more temperate climates...

  4. The monitoring of gene functions on a cell-defined siRNA microarray in human bone marrow stromal and U2OS cells

    Directory of Open Access Journals (Sweden)

    Hi Chul Kim

    2016-06-01

    Full Text Available Here, we developed a cell defined siRNA microarray (CDSM for human bone marrow stromal cells (hBMSCs designed to control the culture of cells inside the spot area without reducing the efficiency of siRNA silencing, “Development of a cell-defined siRNA microarray for analysis of gene functionin human bone marrow stromal cells” (Kim et al., 2016 [1]. First, we confirmed that p65 protein inhibition efficiency was maintained when hBMSCs were culture for 7 days on the siRNA spot, and siRNA spot activity remained in spite of long term storage (10 days and 2 months. Additionally, we confirmed p65 protein inhibition in U2OS cells after 48 h reverse transfection.

  5. Molecular characterisation of stromal populations derived from human embryonic stem cells

    DEFF Research Database (Denmark)

    Harkness, L.; Twine, N. A.; Abu Dawud, R.

    2015-01-01

    Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide...... an unlimited source of clinical grade cells for therapy. We have generated MSC-like cells from hESC (called here hESC-stromal) that exhibit surface markers and differentiate to osteoblasts and adipocytes, similar to BM-hMSC. In the present study, we used microarray analysis to compare the molecular phenotype...... of hESC-stromal and immortalised BM-hMSC cells (hMSC-TERT). Of the 7379 genes expressed above baseline, only 9.3% of genes were differentially expressed between undifferentiated hESC-stromal and BM-hMSC. Following ex vivo osteoblast induction, 665 and 695 genes exhibited >. 2-fold change (FC) in h...

  6. Intestinal stromal cells in mucosal immunity and homeostasis.

    Science.gov (United States)

    Owens, B M J; Simmons, A

    2013-03-01

    A growing body of evidence suggests that non-hematopoietic stromal cells of the intestine have multiple roles in immune responses and inflammation at this mucosal site. Despite this, many still consider gut stromal cells as passive structural entities, with past research focused heavily on their roles in fibrosis, tumor progression, and wound healing, rather than their contributions to immune function. In this review, we discuss our current knowledge of stromal cells in intestinal immunity, highlighting the many immunological axes in which stromal cells have a functional role. We also consider emerging data that broaden the potential scope of their contribution to immunity in the gut and argue that these so-called "non-immune" cells are reclassified in light of their diverse contributions to intestinal innate immunity and the maintenance of mucosal homeostasis.

  7. Potential of iPSC-Derived Mesenchymal Stromal Cells for Treating Periodontal Disease

    Directory of Open Access Journals (Sweden)

    K. Hynes

    2018-01-01

    Full Text Available Mesenchymal stromal cell-like populations have been derived from mouse-induced pluripotent stem cells (miPSC-MSC with the capability for tissue regeneration. In this study, murine iPSC underwent differentiation towards an MSC-like immunophenotype. Stable miPSC-MSC cultures expressed the MSC-associated markers, CD73, CD105, and Sca-1, but lacked expression of the pluripotency marker, SSEA1, and hematopoietic markers, CD34 and CD45. Functionally, miPSC-MSC exhibited the potential for trilineage differentiation into osteoblasts, adipocytes, and chondrocytes and the capacity to suppress the proliferation of mitogen-activated splenocytes. The efficacy of miPSC-MSC was assessed in an acute inflammation model following systemic or local delivery into mice with subcutaneous implants containing heat-inactivated P. gingivalis. Histological analysis revealed less inflammatory cellular infiltrate within the sponges in mice treated with miPSC-MSC cells delivered locally rather than systemically. Assessment of proinflammatory cytokines in mouse spleens found that CXCL1 transcripts and protein were reduced in mice treated with miPSC-MSC. In a periodontitis model, mice subjected to oral inoculation with P. gingivalis revealed less bone tissue destruction and inflammation within the jaws when treated with miPSC-MSC compared to PBS alone. Our results demonstrated that miPSC-MSC derived from iPSC have the capacity to control acute and chronic inflammatory responses associated with the destruction of periodontal tissue. Therefore, miPSC-MSC present a promising novel source of stromal cells which could be used in the treatment of periodontal disease and other inflammatory systemic diseases such as rheumatoid arthritis.

  8. Immune senescence: significance of the stromal microenvironment

    Science.gov (United States)

    Masters, A. R.; Haynes, L.; Su, D.‐M.

    2016-01-01

    Summary The immune system undergoes age‐associated changes known as immunosenescence, resulting in increased susceptibility to infections, cancers and autoimmunity in the aged. The basis of our understanding of immunosenescence has been derived primarily from studies examining intrinsic defects within many of the cells of the immune system. While these studies have provided insight into the mechanisms of immunosenescence, a picture is now emerging that the stromal microenvironment within lymphoid organs also contributes significantly to the age‐associated decline of immune function. These extrinsic defects appear to impact the functional activity of immune cells and may offer a potential target to recover immune activity. Indeed, rejuvenation studies which have targeted the stromal niche have restored immune function in aged successfully, highlighting the impact of the microenvironment towards the aetiology of immunosenescence. PMID:27529161

  9. Subcloning of three osteoblastic cell lines with distinct differentiation phenotypes from the mouse osteoblastic cell line KS-4.

    Science.gov (United States)

    Yamashita, T; Ishii, H; Shimoda, K; Sampath, T K; Katagiri, T; Wada, M; Osawa, T; Suda, T

    1996-11-01

    Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

  10. Combination of Local Transplantation of In Vitro Bone-marrow Stromal Cells and Pulsed Electromagnetic Fields Accelerate Functional Recovery of Transected Sciatic Nerve Regeneration: A Novel Approach in Transected Nerve Repair.

    Science.gov (United States)

    Mohammadi, Rahim; Mahmoodzadeh, Sirvan

    2015-01-01

    Effect of combination of undifferentiated bone marrow stromal cells (BMSCs) and pulsed electromagnetic fields (PEMF) on transected sciatic nerve regeneration was assessed in rats. A 10 mm nerve segment was excised and a vein graft was used to bridge the gap. Twenty microliter undifferentiated BMSCs (2× 107 cells /mL) were administered into the graft inBMSC group with no exposure to PEMF. In BMSC/PEMF group the whole body was exposed to PEMF (0.3 mT, 2Hz) for 4h/day within 1-5 days. In PEMF group the transected nerve was bridged and phosphate buffered saline was administered into the graft. In authograft group (AUTO), the transected nervesegments were reimplanted reversely and the whole body was exposed to PEMF. The regenerated nerve fibers were studied within 12 weeks after surgery. Behavioral, functional, electrophysiological, biomechanical, gastrocnemius muscle mass findings, morphometric indices and immuonohistochemical reactions confirmed faster recovery of regenerated axons in BMSC/PEMF group compared to those in the other groups (PPEMF improved functional recovery. Combination of local transplantation of in vitro bone-marrow stromal cells and pulsed electromagnetic fields could be considered as an effective, safe and tolerable treatment for peripheral nerve repair in clinical practice.

  11. Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

    OpenAIRE

    Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of t...

  12. Mesenchymal stromal cells: misconceptions and evolving concepts.

    Science.gov (United States)

    Phinney, Donald G; Sensebé, Luc

    2013-02-01

    Nearly half a century has passed since the publication of the first articles describing plastic-adherent cells from bone marrow, referred to initially as colony-forming unit fibroblasts, then marrow stromal cells, mesenchymal stem cells and most recently multipotent mesenchymal stromal cells (MSCs). As expected, our understanding of the nature and biologic functions of MSCs has undergone major paradigm shifts over this time. Despite significant advances made in deciphering their complex biology and therapeutic potential in both experimental animal models and human clinical trials, numerous misconceptions regarding the nature and function of MSCs have persisted in the field. Continued propagation of these misconceptions in some cases may significantly impede the advancement of MSC-based therapies in clinical medicine. We have identified six prevalent misconceptions about MSCs that we believe affect the field, and we attempt to rectify them based on current available data. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. Feeding blueberry diets in early life prevent senescence of osteoblasts and bone loss in ovariectomized adult female rats.

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    Full Text Available Appropriate nutrition during early development is essential for maximal bone mass accretion; however, linkage between early nutrition, childhood bone mass, peak bone mass in adulthood, and prevention of bone loss later in life has not been studied.In this report, we show that feeding a high quality diet supplemented with blueberries (BB to pre-pubertal rats throughout development or only between postnatal day 20 (PND20 and PND34 prevented ovariectomy (OVX-induced bone loss in adult life. This protective effect of BB is due to suppression of osteoblastic cell senescence associated with acute loss of myosin expression after OVX. Early exposure of pre-osteoblasts to serum from BB-fed rats was found to consistently increase myosin expression. This led to maintenance osteoblastic cell development and differentiation and delay of cellular entrance into senescence through regulation of the Runx2 gene. High bone turnover after OVX results in insufficient collagenous matrix support for new osteoblasts and their precursors to express myosin and other cytoskeletal elements required for osteoblast activity and differentiation.These results indicate: 1 a significant prevention of OVX-induced bone loss from adult rats can occur with only 14 days consumption of a BB-containing diet immediately prior to puberty; and 2 the molecular mechanisms underlying these effects involves increased myosin production which stimulates osteoblast differentiation and reduces mesenchymal stromal cell senescence.

  14. Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad

    2016-01-01

    Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional...

  15. Stimulatory effect of undecylenic acid on mouse osteoblast differentiation.

    Science.gov (United States)

    Kim, Myung Hee; Shim, Ki Shuk; Lee, Su-Ui; Kim, Young Sup; Min, Yong Ki; Kim, Seong Hwan

    2010-04-01

    Natural compounds with bone-forming (or anabolic) activity have been recently focused on in bone research. The present study investigated the effect of undecylenic acid (UA) on osteoblast differentiation in mouse osteoblastic MC3T3-E1 subclone 4 cells and primary mouse calvarial cells. Low concentrations of UA (up to 5 microM) exhibited no cytotoxicity and significantly increased the expression and activity of alkaline phosphatase (early differentiation marker of osteoblast) and calcium deposition with the induction of expression of the osteocalcin gene in both cells. Interestingly, at low concentration of UA, the induction of NF-kappaB p65 translocation into nucleus and the up-regulation of AP-1 and NFATc1 transcript levels were also observed, suggesting that the stimulatory effect of UA on osteoblast differentiation could be mediated through the activation of transcription factors. Additionally, although the patterns of UA-induced activation of MAP kinases (JNK and p38) were not completely consistent with the increase of both ALP activity and calcium deposition by UA, MAP kinases might be partially involved in the biological function of UA during the early and late stages of osteoblast differentiation. Copyright (c) 2009 John Wiley & Sons, Ltd.

  16. Stromal infrastructure of the lymph node and coordination of immunity.

    Science.gov (United States)

    Chang, Jonathan E; Turley, Shannon J

    2015-01-01

    The initiation of adaptive immune responses depends upon the careful maneuvering of lymphocytes and antigen into and within strategically placed lymph nodes (LNs). Non-hematopoietic stromal cells form the cellular infrastructure that directs this process. Once regarded as merely structural features of lymphoid tissues, these cells are now appreciated as essential regulators of immune cell trafficking, fluid flow, and LN homeostasis. Recent advances in the identification and in vivo targeting of specific stromal populations have resulted in striking new insights to the function of stromal cells and reveal a level of complexity previously unrealized. We discuss here recent discoveries that highlight the pivotal role that stromal cells play in orchestrating immune cell homeostasis and adaptive immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Identification and proteomic analysis of osteoblast-derived exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

  18. Gastrointestinal stromal tumors

    International Nuclear Information System (INIS)

    Sufliarsky, J.

    2011-01-01

    Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive tract. Better understanding of the molecular characteristics of GISTs led to the clinical development of imatinib for treating patients with this disease. New immuno markers and mechanisms of primary and secondary resistance were discovered. Adjuvant imatinib in intermediate or high risk GIST has improved the recurrence-free survival. Sunitinib in patients with intolerance or progression on imatinib demonstrated significant improvements in progression-free and overall survival versus placebo. Second-generation tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, have shown activity in patients with imatinib- and sunitinib-resistant GIST. (author)

  19. Regulation of apoptosis in osteoclasts and osteoblastic cells

    International Nuclear Information System (INIS)

    Xing Lianping; Boyce, Brendan F.

    2005-01-01

    In postnatal life, the skeleton undergoes continuous remodeling in which osteoclasts resorb aged or damaged bone, leaving space for osteoblasts to make new bone. The balance of proliferation, differentiation, and apoptosis of bone cells determines the size of osteoclast or osteoblast populations at any given time. Bone cells constantly receive signals from adjacent cells, hormones, and bone matrix that regulate their proliferation, activity, and survival. Thus, the amount of bone and its microarchitecture before and after the menopause or following therapeutic intervention with drugs, such as sex hormones, glucocorticoids, parathyroid hormone, and bisphosphonates, is determined in part by effects of these on survival of osteoclasts, osteoblasts, and osteocytes. Understanding the mechanisms and regulation of bone cell apoptosis will enhance our knowledge of bone cell function and help us to develop better therapeutics for the management of osteoporosis and other bone diseases

  20. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

    Science.gov (United States)

    Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

    2017-02-14

    Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Properties and potential of bone marrow mesenchymal stromal cells from children with hematologic diseases.

    Science.gov (United States)

    Dimitriou, H; Linardakis, E; Martimianaki, G; Stiakaki, E; Perdikogianni, C H; Charbord, P; Kalmanti, M

    2008-01-01

    Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases. BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed. MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone. MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

  2. Loss of Asxl1 Alters Self-Renewal and Cell Fate of Bone Marrow Stromal Cell, Leading to Bohring-Opitz-like Syndrome in Mice.

    Science.gov (United States)

    Zhang, Peng; Xing, Caihong; Rhodes, Steven D; He, Yongzheng; Deng, Kai; Li, Zhaomin; He, Fuhong; Zhu, Caiying; Nguyen, Lihn; Zhou, Yuan; Chen, Shi; Mohammad, Khalid S; Guise, Theresa A; Abdel-Wahab, Omar; Xu, Mingjiang; Wang, Qian-Fei; Yang, Feng-Chun

    2016-06-14

    De novo ASXL1 mutations are found in patients with Bohring-Opitz syndrome, a disease with severe developmental defects and early childhood mortality. The underlying pathologic mechanisms remain largely unknown. Using Asxl1-targeted murine models, we found that Asxl1 global loss as well as conditional deletion in osteoblasts and their progenitors led to significant bone loss and a markedly decreased number of bone marrow stromal cells (BMSCs) compared with wild-type littermates. Asxl1(-/-) BMSCs displayed impaired self-renewal and skewed differentiation, away from osteoblasts and favoring adipocytes. RNA-sequencing analysis revealed altered expression of genes involved in cell proliferation, skeletal development, and morphogenesis. Furthermore, gene set enrichment analysis showed decreased expression of stem cell self-renewal gene signature, suggesting a role of Asxl1 in regulating the stemness of BMSCs. Importantly, re-introduction of Asxl1 normalized NANOG and OCT4 expression and restored the self-renewal capacity of Asxl1(-/-) BMSCs. Our study unveils a pivotal role of ASXL1 in the maintenance of BMSC functions and skeletal development. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Enhanced Hematopoietic Stem Cell Self-Renewal-Promoting Ability of Clonal Primary Mesenchymal Stromal/Stem cells Versus Their Osteogenic Progeny.

    Science.gov (United States)

    He, Qiling; Scott Swindle, Claude; Wan, Chao; Flynn, Robert J; Oster, Robert A; Chen, Dongquan; Zhang, Fengjie; Shu, Yinglan; Klug, Christopher A

    2017-02-01

    Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin + Lepr + Sca-1 + CD146 + and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo. Stem Cells 2017;35:473-484. © 2016 AlphaMed Press.

  4. Synchronous Acromegaly and Gastrointestinal Stromal Tumor: A Case Report

    Directory of Open Access Journals (Sweden)

    Hüsniye Başer

    2014-06-01

    Full Text Available Acromegaly is a rare endocrine disorder characterized by the manifestations of sustained hypersecretion of growth hormone and concomitant elevations in circulating concentrations of insulin-like growth factor-1. It has been reported that patients with acromegaly are at the increased risk of developing malignant tumors, particularly colorectal cancer. Gastrointestinal stromal tumors are mesenchymal tumors of the digestive tract. An association between gastrointestinal stromal tumors and insulin-like growth factor system has been reported. Here, we report a patient diagnosed with synchronous acromegaly and gastrointestinal stromal tumor. A 59-year-old man with iron deficiency anemia presented with enlarged hands, coarse facial feature and several skin tags. Thyroid function tests were within normal range. Growth hormone was 5.14 ng/mL, insulin-like growth factor-1 was 820 ng/mL, and no growth hormone suppression was observed on 75g oral glucose tolerance test. Pituitary magnetic resonance imaging revealed microadenoma, and the patient was diagnosed with acromegaly. Upper gastrointestinal tract endoscopy revealed an ulcerovegetan mass in the duodenum and the results of the histopathologcal analysis was consistent with gastrointestinal stromal tumor. The association of synchronous and asynchronous gastrointestinal stromal tumors with other malignancies have been reported. The most common accompanying neoplasms are colorectal and gastric adenocarcinomas, as well as pancreatic tumors. However, in the literature, the number of reported cases of synchronous acromegaly and gastrointestinal stromal tumor are limited, and there are no sufficient data on this association. Turk Jem 2014; 2: 52-55

  5. Mechanical unloading reduces microtubule actin crosslinking factor 1 expression to inhibit β-catenin signaling and osteoblast proliferation.

    Science.gov (United States)

    Yin, Chong; Zhang, Yan; Hu, Lifang; Tian, Ye; Chen, Zhihao; Li, Dijie; Zhao, Fan; Su, Peihong; Ma, Xiaoli; Zhang, Ge; Miao, Zhiping; Wang, Liping; Qian, Airong; Xian, Cory J

    2017-12-08

    Mechanical unloading was considered a major threat to bone homeostasis, and has been shown to decrease osteoblast proliferation although the underlying mechanism is unclear. Microtubule actin crosslinking factor 1 (MACF1) is a cytoskeletal protein that regulates cellular processes and Wnt/β-catenin pathway, an essential signaling pathway for osteoblasts. However, the relationship between MACF1 expression and mechanical unloading, and the function and the associated mechanisms of MACF1 in regulating osteoblast proliferation are unclear. This study investigated effects of mechanical unloading on MACF1 expression levels in cultured MC3T3-E1 osteoblastic cells and in femurs of mice with hind limb unloading; and it also examined the role and potential action mechanisms of MACF1 in osteoblast proliferation in MACF1-knockdown, overexpressed or control MC3T3-E1 cells treated with or without the mechanical unloading condition. Results showed that the mechanical unloading condition inhibited osteoblast proliferation and MACF1 expression in MC3T3-E1 osteoblastic cells and mouse femurs. MACF1 knockdown decreased osteoblast proliferation, while MACF1 overexpression increased it. The inhibitory effect of mechanical unloading on osteoblast proliferation also changed with MACF1 expression levels. Furthermore, MACF1 was found to enhance β-catenin expression and activity, and mechanical unloading decreased β-catenin expression through MACF1. Moreover, β-catenin was found an important regulator of osteoblast proliferation, as its preservation by treatment with its agonist lithium attenuated the inhibitory effects of MACF1-knockdown or mechanical unloading on osteoblast proliferation. Taken together, mechanical unloading decreases MACF1 expression, and MACF1 up-regulates osteoblast proliferation through enhancing β-catenin signaling. This study has thus provided a mechanism for mechanical unloading-induced inhibited osteoblast proliferation. © 2017 Wiley Periodicals, Inc.

  6. GASTROINTESTINAL STROMAL TUMOR (GIST

    Directory of Open Access Journals (Sweden)

    Luigi eTornillo

    2014-11-01

    Full Text Available Gastrointestinal stromal tumors are the most frequent mesenchymal tumors of the gastrointestinal tract. The discovery that these tumors, formerly thought of smooth muscle origin, are indeed better characterized by specific activating mutation in genes coding for the receptor tyrosine kinases CKIT and PDGFRA and that these mutations are strongly predictive for the response to targeted therapy with receptor tyrosine kinase inhibitors has made GISTs the typical example of the integration of basic molecular knowledge in the daily clinical activity. The information on the mutational status of these tumors is essential to predict (and subsequently to plan the therapy. As resistant cases are frequently wild-type, other possible oncogenic events, defining other entities, have been discovered (e.g. succinil dehydrogenase mutation/dysregulation, insuline growth factor expression, mutations in the RAS-RAF-MAPK pathway. The classification of disease must nowadays rely on the integration of the clinico-morphological characteristics with the molecular data.

  7. The transcription factor early B-cell factor 1 regulates bone formation in an osteoblast-nonautonomous manner.

    Science.gov (United States)

    Zee, Tiffany; Boller, Sören; Györy, Ildiko; Makinistoglu, Munevver P; Tuckermann, Jan P; Grosschedl, Rudolf; Karsenty, Gerard

    2013-03-18

    Early B-cell factor 1 (Ebf1) is a transcription factor whose inactivation in all cells results in high bone mass because of an increase in bone formation. This observation suggests Ebf1 may be an inhibitor of osteoblast differentiation. To test this contention, we analyzed Ebf1 pattern of expression and function in osteoblasts ex vivo and in vivo through osteoblast-specific inactivation in the mouse. We show here that in vivo deletion of Ebf1 in osteoblast progenitors does not affect osteoblast differentiation or bone formation accrual post-natally. These observations indicate that the phenotype described in Ebf1(-/)(-) mice is not osteoblast-autonomous. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    International Nuclear Information System (INIS)

    Muthukumaran, Padmalosini; Lim, Chwee Teck; Lee, Taeyong

    2012-01-01

    Highlights: ► Estradiol induced stiffness changes of osteoblasts were quantified using AFM. ► Estradiol causes significant decrease in the stiffness of osteoblasts. ► Decreased stiffness was caused by decreased density of f-actin network. ► Stiffness changes were not associated with mineralized matrix of osteoblasts. ► Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E ∗ ) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with β-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E ∗ . The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E ∗ of osteoblasts significantly decreased by 43–46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness changes of osteoblasts were not associated with changes in the synthesized mineralized matrix of the cells. Thus, a decrease in osteoblast stiffness with estrogen treatment was

  9. Estradiol influences the mechanical properties of human fetal osteoblasts through cytoskeletal changes

    Energy Technology Data Exchange (ETDEWEB)

    Muthukumaran, Padmalosini [Department of Bioengineering, National University of Singapore (Singapore); Lim, Chwee Teck [Department of Bioengineering, National University of Singapore (Singapore); Department of Mechanical Engineering, National University of Singapore (Singapore); Mechanobiology Institute, National University of Singapore (Singapore); Singapore-MIT Alliance for Research and Technology (SMART), National University of Singapore (Singapore); Lee, Taeyong, E-mail: bielt@nus.edu.sg [Department of Bioengineering, National University of Singapore (Singapore)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Estradiol induced stiffness changes of osteoblasts were quantified using AFM. Black-Right-Pointing-Pointer Estradiol causes significant decrease in the stiffness of osteoblasts. Black-Right-Pointing-Pointer Decreased stiffness was caused by decreased density of f-actin network. Black-Right-Pointing-Pointer Stiffness changes were not associated with mineralized matrix of osteoblasts. Black-Right-Pointing-Pointer Estradiol increases inherent alkaline phosphatase activity of osteoblasts. -- Abstract: Estrogen is known to have a direct effect on bone forming osteoblasts and bone resorbing osteoclasts. The cellular and molecular effects of estrogen on osteoblasts and osteoblasts-like cells have been extensively studied. However, the effect of estrogen on the mechanical property of osteoblasts has not been studied yet. It is important since mechanical property of the mechanosensory osteoblasts could be pivotal to its functionality in bone remodeling. This is the first study aimed to assess the direct effect of estradiol on the apparent elastic modulus (E{sup Asterisk-Operator }) and corresponding cytoskeletal changes of human fetal osteoblasts (hFOB 1.19). The cells were cultured in either medium alone or medium supplemented with {beta}-estradiol and then subjected to Atomic Force Microscopy indentation (AFM) to determine E{sup Asterisk-Operator }. The underlying changes in cytoskeleton were studied by staining the cells with TRITC-Phalloidin. Following estradiol treatment, the cells were also tested for proliferation, alkaline phosphatase activity and mineralization. With estradiol treatment, E{sup Asterisk-Operator} of osteoblasts significantly decreased by 43-46%. The confocal images showed that the changes in f-actin network observed in estradiol treated cells can give rise to the changes in the stiffness of the cells. Estradiol also increases the inherent alkaline phosphatase activity of the cells. Estradiol induced stiffness

  10. The transcription factor early B-cell factor 1 regulates bone formation in an osteoblast-nonautonomous manner

    OpenAIRE

    Zee, Tiffany; Boller, Sören; Györy, Ildiko; Makinistoglu, Munevver P.; Tuckermann, Jan P.; Grosschedl, Rudolf; Karsenty, Gerard

    2013-01-01

    Early B-cell factor 1 (Ebf1) is a transcription factor whose inactivation in all cells results in high bone mass because of an increase in bone formation. This observation suggests Ebf1 may be an inhibitor of osteoblast differentiation. To test this contention, we analyzed Ebf1 pattern of expression and function in osteoblasts ex vivo and in vivo through osteoblast-specific inactivation in the mouse. We show here that in vivo deletion of Ebf1 in osteoblast progenitors does not affect osteobla...

  11. Hydrocarbon Deposition Attenuates Osteoblast Activity on Titanium

    Science.gov (United States)

    Hayashi, R.; Ueno, T.; Migita, S.; Tsutsumi, Y.; Doi, H.; Ogawa, T.; Hanawa, T.; Wakabayashi, N.

    2014-01-01

    Although the reported percentage of bone-implant contact is far lower than 100%, the cause of such low levels of bone formation has rarely been investigated. This study tested the negative biological effect of hydrocarbon deposition onto titanium surfaces, which has been reported to be inevitable. Osteogenic MC3T3-E1 cells were cultured on titanium disks on which the carbon concentration was experimentally regulated to achieve carbon/titanium (C/Ti) ratios of 0.3, 0.7, and 1.0. Initial cellular activities such as cell attachment and cell spreading were concentration-dependently suppressed by the amount of carbon on the titanium surface. The osteoblastic functions of alkaline phosphatase activity and calcium mineralization were also reduced by more than 40% on the C/Ti (1.0) surface. These results indicate that osteoblast activity is influenced by the degree of hydrocarbon contamination on titanium implants and suggest that hydrocarbon decomposition before implant placement may increase the biocompatibility of titanium. PMID:24868012

  12. Type V OI primary osteoblasts display increased mineralization despite decreased COL1A1 expression.

    Science.gov (United States)

    Reich, Adi; Bae, Alison S; Barnes, Aileen M; Cabral, Wayne A; Hinek, Aleksander; Stimec, Jennifer; Hill, Suvimol C; Chitayat, David; Marini, Joan C

    2015-02-01

    Patients with type V osteogenesis imperfecta (OI) are heterozygous for a dominant IFITM5 c.-14C>T mutation, which adds five residues to the N terminus of bone-restricted interferon-induced transmembrane-like protein (BRIL), a transmembrane protein expressed in osteoblasts. Type V OI skeletal findings include hyperplastic callus formation, ossification of the forearm interosseous membrane, and dense metaphyseal bands. The objective of this study was to examine the role of osteoblasts in the active mineralization traits of type V OI and the effect of the IFITM5 mutation on type I collagen. We identified eight patients with the IFITM5 c.-14C>T mutation. Cultured osteoblasts from type V OI patients were used to study osteoblast differentiation and mineralization. We verified the expression and stability of mutant IFITM5 transcripts. In differentiated type V OI primary osteoblasts in culture, the IFITM5 expression and BRIL level is comparable with control. Both early and late markers of osteoblast differentiation are increased in type V OI osteoblasts. Mineralization, assayed by alizarin red staining, was increased in type V OI osteoblasts compared with control. However, type V OI osteoblasts have significantly decreased COL1A1 transcripts in mid- to late differentiation. Type I collagen protein is concomitantly decreased, with decreased cross-linked collagen in matrix and altered appearance of fibrils deposited in culture. This study demonstrates that type V OI mineralization has a gain-of-function mechanism at the osteoblast level, which likely underlies the overactive tissue mineralization seen in patients. Decreased type I collagen expression, secretion, and matrix incorporation establish type V OI as a collagen-related defect.

  13. Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts

    Science.gov (United States)

    Shim, Jae-Hyuck; Greenblatt, Matthew B.; Zou, Weiguo; Huang, Zhiwei; Wein, Marc N.; Brady, Nicholas; Hu, Dorothy; Charron, Jean; Brodkin, Heather R.; Petsko, Gregory A.; Zaller, Dennis; Zhai, Bo; Gygi, Steven; Glimcher, Laurie H.; Jones, Dallas C.

    2013-01-01

    Mice deficient in Schnurri-3 (SHN3; also known as HIVEP3) display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts. Here we identified SHN3 as a dampener of ERK activity that functions in part downstream of WNT signaling in osteoblasts. A D-domain motif within SHN3 mediated the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction resulted in aberrant ERK activation and consequent osteoblast hyperactivity in vivo. Additionally, in vivo genetic interaction studies demonstrated that crossing to Lrp5–/– mice partially rescued the osteosclerotic phenotype of Shn3–/– mice; mechanistically, this corresponded to the ability of SHN3 to inhibit ERK-mediated suppression of GSK3β. Inducible knockdown of Shn3 in adult mice resulted in a high–bone mass phenotype, providing evidence that transient blockade of these pathways in adults holds promise as a therapy for osteoporosis. PMID:23945236

  14. Interleukin-1β Suppresses the Transporter Genes Ank and Ent1 Expression in Stromal Progenitor Cells Retaining Mineralization.

    Science.gov (United States)

    Ezura, Yoichi; Lin, Xin; Hatta, Arina; Izu, Yayoi; Noda, Masaki

    2016-08-01

    Heterotopic ossification (HO) in various tissues evokes clinical problems. Inflammatory responses of the stromal progenitor cells may be involved in its etiology. Previous report indicated that pro-inflammatory cytokines including IL-1β enhanced the in vitro calcification of human mesenchymal stem cells (MSCs), by suppressing the expression of ectonucleotide pyrophosphatase/phosphodiesterase-1 gene (ENPP1). However, possible contribution of other related factors had not been investigated. Here, we investigated the expression of regulators of extracellular pyrophosphate and nucleosides including Enpp1, Nt5e, Ank, Enptds, and Ent1, examining various connective tissue stromal progenitor cells, including bone marrow stromal cells and synovium derived cells from mouse, or bone marrow MSCs from human. Consistent with previous studies, we observed characteristic suppression of the osteoblastic marker genes by IL-1β during the osteogenic culture for 20 days. In addition, we observed a reduced expression of the important transporter genes, Ank and Ent1, whereas the alteration in Enpp1 and Nt5e levels was not always consistent among the cell types. Our results suggest that IL-1β suppresses not only the osteoblastic but also the negative regulators of soft-tissue calcification, including Ank and Ent1 in stromal progenitor cells, which may contribute to the mechanisms of HO in various disorders.

  15. Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

    DEFF Research Database (Denmark)

    Bi, Yanming; Nielsen, Karina L; Kilts, Tina M

    2006-01-01

    Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated...... the effects of Bgn on 1alpha, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate...

  16. Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

    Directory of Open Access Journals (Sweden)

    Michael S Stalvey

    Full Text Available Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF. CF-related bone disease (CFBD is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR, the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/- mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+ littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl mRNA was detected, significantly less osteoprotegerin (Opg was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt

  17. IL-6 alters osteocyte signaling toward osteoblasts but not osteoclasts.

    Science.gov (United States)

    Bakker, A D; Kulkarni, R N; Klein-Nulend, J; Lems, W F

    2014-04-01

    Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.

  18. Myokines (muscle-derived cytokines and chemokines) including ciliary neurotrophic factor (CNTF) inhibit osteoblast differentiation.

    Science.gov (United States)

    Johnson, Rachelle W; White, Jason D; Walker, Emma C; Martin, T John; Sims, Natalie A

    2014-07-01

    Muscle and bone are intimately linked by bi-directional signals regulating both muscle and bone cell gene expression and proliferation. It is generally accepted that muscle cells secrete factors (myokines) that influence adjacent bone cells, but these myokines are yet to be identified. We have previously shown that osteocyte-specific deletion of the co-receptor subunit utilized by IL-6 family cytokines, glycoprotein 130 (gp130), resulted in impaired bone formation in the trabecular bone, but enhanced periosteal expansion, suggesting a gp130-dependent periosteum-specific inhibition of osteoblast function, potentially induced by the local muscle fibres. We report here that differentiated primary calvarial osteoblasts cultured in myotube-conditioned media (CM) from myogenic C2C12 cells show reduced mRNA levels of genes associated with osteoblast differentiation. Alkaline phosphatase protein activity and all mRNA markers of osteoblast differentiation in the tested panel (runx2, osterix, alkaline phosphatase, parathyroid hormone (PTH) receptor, osteoprotegerin, osteocalcin, sclerostin) were reduced following culture with myotube CM. The exception was RANKL, which was significantly elevated in differentiated primary osteoblast cultures expressing osteocytic genes. A cytokine array of the C2C12 myotube-conditioned media identified TIMP-1 and MCP-1 as the most abundant myokines, but treatment with recombinant TIMP-1 or MCP-1 did not inhibit osteoblast gene expression. Rather, the IL-6 family cytokine ciliary neurotrophic factor (CNTF), which we found abundantly expressed by mouse muscle at the transcript and protein level, reduced osteoblast gene expression, although not to the same extent as the myotube-conditioned media. These data indicate that muscle cells secrete abundant TIMP-1, MCP-1, and CNTF, and that of these, only CNTF has the ability to suppress osteoblast function and gene expression in a similar manner to myotube-conditioned medium. This suggests that CNTF is

  19. Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Rongrong Zhang

    Full Text Available Intermittent application of parathyroid hormone (PTH has well established anabolic effects on bone mass in rodents and humans. Although transcriptional mechanisms responsible for these effects are not fully understood, it is recognized that transcriptional factor cAMP response element binding protein (CREB mediates PTH signaling in osteoblasts, and that there is a communication between the PTH-CREB pathway and the BMP2 signaling pathway, which is important for osteoblast differentiation and bone formations. These findings, in conjunction with putative cAMP response elements (CREs in the BMP2 promoter, led us to hypothesize that the PTH-CREB pathway could be a positive regulator of BMP2 transcription in osteoblasts. To test this hypothesis, we first demonstrated that PTH signaling activated CREB by phosphorylation in osteoblasts, and that both PTH and CREB were capable of promoting osteoblastic differentiation of primary mouse osteoblast cells and multiple rodent osteoblast cell lines. Importantly, we found that the PTH-CREB signaling pathway functioned as an effective activator of BMP2 expression, as pharmacologic and genetic modulation of PTH-CREB activity significantly affected BMP2 expression levels in these cells. Lastly, through multiple promoter assays, including promoter reporter deletion, mutation, chromatin immunoprecipitation (ChIP, and electrophoretic mobility shift assay (EMSA, we identified a specific CRE in the BMP2 promoter which is responsible for CREB transactivation of the BMP2 gene in osteoblasts. Together, these results demonstrate that the anabolic function of PTH signaling in bone is mediated, at least in part, by CREB transactivation of BMP2 expression in osteoblasts.

  20. Effects of Nonsteroidal Anti-Inflammatory Drugs on Transforming Growth Factor-β Expression and Bioactivity in Rat Osteoblast-Enriched Cultures

    Directory of Open Access Journals (Sweden)

    Je-Ken Chang

    2003-06-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAIDs have been reported to suppress bone remodeling in normal and repaired bones. Our previous results indicated that ketorolac and indomethacin suppressed proliferation, stimulated early differentiation, and induced apoptosis in cultured osteoblasts. Transforming growth factor-b (TGF-b has been reported to enhance proliferation, suppress differentiation, and prevent apoptosis in osteoblasts. We proposed that one pathway of NSAID effects on osteoblast function might be through inhibition of the expression and/or bioactivity of TGF-b in osteoblasts. We tested the effects of ketorolac and indomethacin on the expression of TGF-b1 mRNA and protein and the bioactivity of TGF-b in osteoblast-enriched cultures derived from fetal calvaria. The effects of prostaglandin E1 (PGE1 and PGE2 on TGF-b expression and bioactivity were also examined in order to understand more about the role of prostaglandins in osteoblast function. Simultaneously, we estimated whether these NSAID effects on osteoblasts were prostaglandin-related. The results showed that 24-hour treatments with both PGEs and theoretic therapeutic concentrations of ketorolac and indomethacin had no significant effects on the levels of either transcription or translation of TGF-b or the post-translational function of TGF-b in osteoblasts. These results suggest that NSAIDs do not affect osteoblast function through changes in TGF-b action in osteoblasts.

  1. MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function

    Science.gov (United States)

    Caicedo, Andrés; Fritz, Vanessa; Brondello, Jean-Marc; Ayala, Mickaël; Dennemont, Indira; Abdellaoui, Naoill; de Fraipont, Florence; Moisan, Anaïck; Prouteau, Claire Angebault; Boukhaddaoui, Hassan; Jorgensen, Christian; Vignais, Marie-Luce

    2015-01-01

    Mitochondrial activity is central to tissue homeostasis. Mitochondria dysfunction constitutes a hallmark of many genetic diseases and plays a key role in tumor progression. The essential role of mitochondria, added to their recently documented capacity to transfer from cell to cell, obviously contributes to their current interest. However, determining the proper role of mitochondria in defined biological contexts was hampered by the lack of suitable experimental tools. We designed a protocol (MitoCeption) to directly and quantitatively transfer mitochondria, isolated from cell type A, to recipient cell type B. We validated and quantified the effective mitochondria transfer by imaging, fluorescence-activated cell sorting (FACS) and mitochondrial DNA analysis. We show that the transfer of minute amounts of mesenchymal stem/stromal cell (MSC) mitochondria to cancer cells, a process otherwise occurring naturally in coculture, results in cancer cell enhanced oxidative phosphorylation (OXPHOS) activity and favors cancer cell proliferation and invasion. The MitoCeption technique, which can be applied to different cell systems, will therefore be a method of choice to analyze the metabolic modifications induced by exogenous mitochondria in host cells. PMID:25766410

  2. Improvement of Mouth Functional Disability in Systemic Sclerosis Patients over One Year in a Trial of Fat Transplantation versus Adipose-Derived Stromal Cells

    Directory of Open Access Journals (Sweden)

    Maria Giuseppina Onesti

    2016-01-01

    Full Text Available Background. Systemic sclerosis (SSc is a multisystem disease characterized by cutaneous and visceral fibrosis. Face and mouth changes include telangiectasia, sicca syndrome, and thinning and reduction of mouth width (microcheilia and opening (microstomia. We applied autologous fat transplantation compared with autologous adipose-derived stromal cells (ADSCs injection to evaluate the clinical improvement of mouth opening. Methods. From February to May 2013 ten consecutive SSc patients were enrolled from the outpatient clinic of Plastic Surgery Department of Sapienza University of Rome. Patients were divided into two groups as follows: 5 patients were treated with fat transplantation and 5 patients received infiltration of ADSCs produced by cell factory of our institution. To value mouth opening, we use the Italian version of Mouth Handicap in Systemic Sclerosis Scale (IvMHISS. Mouth opening was assessed in centimetres (Maximal Mouth Opening, MMO. In order to evaluate compliance and physician and patient satisfaction, we employed a Questionnaire of Satisfaction and the Visual Analogic Scale (VAS performed before starting study and 1 year after the last treatment. Results and Conclusion. We noticed that both procedures obtained significant results but neither one emerged as a first-choice technique. The present clinical experimentation should be regarded as a starting point for further experimental research and clinical trials.

  3. Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

    Science.gov (United States)

    Mishra, Sweta; Tai, Qin; Gu, Xiang; Schmitz, James; Poullard, Ashley; Fajardo, Roberto J; Mahalingam, Devalingam; Chen, Xiaodong; Zhu, Xueqiong; Sun, Lu-Zhe

    2015-12-29

    The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

  4. EFFECTS OF PHOSPHATED TITANIUM AND ENAMEL MATRIX DERIVATIVES ON OSTEOBLAST BEHAVIOR IN VITRO

    Science.gov (United States)

    Dacy, J. Anthony; Spears, Robert; Hallmon, William W.; Kerns, David; Rivera-Hidalgo, Francisco; Minevski, Zoran S.; Nelson, Carl J.; Opperman, Lynne A.

    2011-01-01

    Purpose The purpose of this study was to evaluate the effects of phosphated titanium and EMD on osteoblast function. Materials and Methods Primary rat osteoblasts were cultured on discs of either phosphated or non-phosphated titanium and in half of the samples 180μg of EMD was immediately added. Media was changed every 2 days for 28 days, and then analyzed by TGF-β1 and IL-1β ELISAs. Scanning electron microscopy (SEM) and light microscopy (LM) was used to evaluate nodule formation and mineralization. Results Microscopic evaluation revealed no differences in osteoblast attachment on all discs, regardless of treatment. Osteoblast nodule formation was observed in all groups. In the absence of mineralizing media, nodules on the non-phosphated titanium samples showed no evidence of mineralization. All nodules on the phosphated titanium had evidence of mineralization. ELISA analysis revealed no significant differences in IL-1β production between any of the groups. The EMD treated osteoblasts produced significantly more TGF-β1 than non-EMD treated cells for up to 8 days, and osteoblasts on phosphated titanium produced significantly more Tgf-ß1 at 8 days. Discussion and Conclusion Osteoblast attachment appeared unaffected by surface treatment. EMD initiated early TGF-β1 production, but production decreased to control levels within 10 days. Phosphated titanium increased Tgf-ß1 production at 8 days, and induced nodule mineralization even in the absence of mineralizing medium. PMID:17974103

  5. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts

    International Nuclear Information System (INIS)

    Utting, J.C.; Robins, S.P.; Brandao-Burch, A.; Orriss, I.R.; Behar, J.; Arnett, T.R.

    2006-01-01

    We investigated the effect of hypoxia on rat osteoblast function in long-term primary cultures. Reduction of pO 2 from 20% to 5% and 2% decreased formation of mineralized bone nodules 1.7-fold and 11-fold, respectively. When pO 2 was reduced further to 0.2%, bone nodule formation was almost abolished. The inhibitory effect of hypoxia on bone formation was partly due to decreased osteoblast proliferation, as measured by 3 H-thymidine incorporation. Hypoxia also sharply reduced osteoblast alkaline phosphatase (ALP) activity and expression of mRNAs for ALP and osteocalcin, suggesting inhibition of differentiation to the osteogenic phenotype. Hypoxia did not increase the apoptosis of osteoblasts but induced a reversible state of quiescence. Transmission electron microscopy revealed that collagen fibrils deposited by osteoblasts cultured in 2% O 2 were less organized and much less abundant than in 20% O 2 cultures. Furthermore, collagen produced by hypoxic osteoblasts contained a lower percentage of hydroxylysine residues and exhibited an increased sensitivity to pepsin degradation. These data demonstrate the absolute oxygen requirement of osteoblasts for successful bone formation and emphasize the importance of the vasculature in maintaining bone health. We recently showed that hypoxia also acts in a reciprocal manner as a powerful stimulator of osteoclast formation. Considered together, our results help to explain the bone loss that occurs at the sites of fracture, tumors, inflammation and infection, and in individuals with vascular disease or anemia

  6. Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation.

    Science.gov (United States)

    Lai, Chung-Fang; Cheng, Su-Li

    2005-02-01

    Both integrins and BMP-2 exert similar effects on osteoblasts. We examined the relationship between the alphav-containing integrins (alphavbeta) and BMP-2 in osteoblast function. BMP-2 stimulates alphavbeta expression. BMP-2 receptors co-localize/overlap with alphavbeta integrins, and the intact function of alphavbeta is essential in BMP-2 activity. Bone morphogenetic protein (BMP)-2 not only induces osteoblast differentiation and bone matrix mineralization, but also stimulates osteoblast migration on and adhesion to bone matrix proteins. The alphavbeta- and beta1- (alphabeta1) containing integrins mediate osteoblast interaction with many bone matrix proteins and play important roles in osteoblast adhesion, migration, and differentiation. Because alphavbeta integrins and BMP-2 share common effects on osteoblasts, we analyzed their relationship in osteoblast function. The effects of BMP-2 on integrin expression were determined by surface labeling/immunoprecipitation and cell adhesion to matrix proteins. Confocal analysis of the immunostained cells and co-immunoprecipitation of cell extracts were used to study the spatial relationship between integrins and BMP-2 receptors. A function-blocking anti-alphavbeta integrin antibody (L230) was employed to investigate the roles of alphavbeta integrins in BMP-2 function. Human osteoblasts (HOBs) express alphabeta1, alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 integrins at focal adhesion sites. BMP-2 increases the levels of these integrins on osteoblast surface and enhances HOB adhesion to osteopontin and vitronectin. Immunoprecipitation and immunostaining analyses show that BMP-2 receptors co-localize or overlap with alphavbeta and alphabeta1 integrins. Incubation of HOBs with L230 abolishes the antiproliferative effect of BMP-2 and reduces the capacity of BMP-2 to stimulate alkaline phosphatase activity and the expression of osteocalcin, osteopontin, and bone sialoprotein. Furthermore, L230 prevents BMP-2 induction

  7. Identifying A Molecular Phenotype for Bone Marrow Stromal Cells With In Vivo Bone Forming Capacity

    DEFF Research Database (Denmark)

    Larsen, Kenneth H; Frederiksen, Casper M; Burns, Jorge S

    2009-01-01

    Abstract The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone forming capacity is not known. Thus, we employed DNA microarrays...... comparing two human bone marrow stromal cell (hBMSC) populations: one is capable of in vivo heterotopic bone formation (hBMSC-TERT(+Bone)) and the other is not (hBMSC-TERT(-Bone)). Compared to hBMSC-TERT(-Bone), the hBMSC-TERT(+Bone) cells had an increased over-representation of extracellular matrix genes...... (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT(-Bone) cells expressed a larger number of immune-response related genes (26% versus 8%). In order to test for the predictive...

  8. Placental mesenchymal stromal cells derived from blood vessels or avascular tissues: what is the better choice to support endothelial cell function?

    Science.gov (United States)

    König, Julia; Weiss, Gregor; Rossi, Daniele; Wankhammer, Karin; Reinisch, Andreas; Kinzer, Manuela; Huppertz, Berthold; Pfeiffer, Dagmar; Parolini, Ornella; Lang, Ingrid

    2015-01-01

    Mesenchymal stromal cells (MSCs) are promising tools for therapeutic revascularization of ischemic tissues and for support of vessel formation in engineered tissue constructs. Recently, we could show that avascular-derived MSCs from placental amnion release soluble factors that exhibit survival-enhancing effects on endothelial cells (ECs). We hypothesize that MSCs derived from placental blood vessels might have even more potent angiogenic effects. Therefore, we isolated and characterized MSCs from placental chorionic blood vessels (bv-MSCs) and tested their angiogenic potential in comparison to amnion-derived avascular MSCs (av-MSCs). bv-MSCs express a very similar surface marker profile compared with av-MSCs and could be differentiated toward the adipogenic and osteogenic lineages. bv-MSCs exert immunosuppressive properties on peripheral blood mononuclear cells, suggesting that they are suitable for cell transplantation settings. Conditioned medium (Cdm) from av-MSCs and bv-MSCs significantly enhanced EC viability, whereas only Cdm from bv-MSCs significantly increased EC migration and network formation (Matrigel assay). Angiogenesis array analysis of av- and bv-MSC-Cdm revealed a similar secretion pattern of angiogenic factors, including angiogenin, interleukins-6 and -8, and tissue inhibitors of matrix metalloproteinase-1 and 2. Enzyme-linked immunosorbent assay analysis showed that, in contrast to av-MSCs, bv-MSCs secreted vascular endothelial growth factor. In direct coculture with bv-MSCs, ECs showed a significantly increased formation of vessel-like structures compared with av-MSCs. With regard to therapeutic treatment, bv-MSCs and particularly their Cdm might be valuable to stimulate angiogenesis especially in ischemic tissues. av-MSCs and their Cdm could be beneficial in conditions when it is required to promote the survival and stabilization of blood vessels without the risk of unmeant angiogenesis.

  9. The hetero-transplantation of human bone marrow stromal cells carried by hydrogel unexpectedly demonstrates a significant role in the functional recovery in the injured spinal cord of rats.

    Science.gov (United States)

    Raynald; Li, Yanbin; Yu, Hao; Huang, Hua; Guo, Muyao; Hua, Rongrong; Jiang, Fenjun; Zhang, Kaihua; Li, Hailong; Wang, Fei; Li, Lusheng; Cui, FuZhai; An, Yihua

    2016-03-01

    Spinal cord injury (SCI) often causes a disturbance in the microenvironment in the lesion site resulting in sudden loss of sensory and motor function. Transplantation of stem cells provides a promising strategy in the treatment of SCI. But limited growth and immunological incompatibility of the stem cells with the host limits the application of this strategy. In order to get better survival and integration with the host, we employed a hyaluronic acid (HA) based scaffold covalently modified by poly-l-Lysine (PLL) as a vehicle to deliver the human bone marrow stromal cells (BMSCs) to the injured spinal cord of rats. The BMSCs were chosen as an ideal candidate for its advantage of low expression of major histocompatibility complex II. The data unexpectedly showed that the hetero-transplanted cells survived well in the lesion site even at 8 weeks post injury. Both the immunofluorescent and the electrophysiological assay indicated better survival of the transplanted cells and improved axonal growth in SCI rats transplanted with BMSCs in HA-PLL in contrast to the groups without either BMSCs or the HA scaffold transplantation. These promotions may account for the functional recovery assessed by Basso-Beattie-Bresnahan (BBB) locomotor rating scale in the HA-PLL seeded with BMSCs group. These data suggests that hetero-transplantation of human BMSCs delivered by HA scaffold demonstrates a significant role in the functional recovery in the injured spinal cord of rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Stromal cell regulation of homeostatic and inflammatory lymphoid organogenesis

    Science.gov (United States)

    Kain, Matthew J W; Owens, Benjamin M J

    2013-01-01

    Summary Secondary lymphoid organs function to increase the efficiency of interactions between rare, antigen-specific lymphocytes and antigen presenting cells, concentrating antigen and lymphocytes in a supportive environment that facilitates the initiation of an adaptive immune response. Homeostatic lymphoid tissue organogenesis proceeds via exquisitely controlled spatiotemporal interactions between haematopoietic lymphoid tissue inducer populations and multiple subsets of non-haematopoietic stromal cells. However, it is becoming clear that in a range of inflammatory contexts, ectopic or tertiary lymphoid tissues can develop inappropriately under pathological stress. Here we summarize the role of stromal cells in the development of homeostatic lymphoid tissue, and assess emerging evidence that suggests a critical role for stromal involvement in the tertiary lymphoid tissue development associated with chronic infections and inflammation. PMID:23621403

  11. Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation.

    Science.gov (United States)

    Nicolaije, Claudia; Koedam, Marijke; van Leeuwen, Johannes P T M

    2012-04-01

    Accumulating data show that oxygen tension can have an important effect on cell function and fate. We used the human pre-osteoblastic cell line SV-HFO, which forms a mineralizing extracellular matrix, to study the effect of low oxygen tension (2%) on osteoblast differentiation and mineralization. Mineralization was significantly reduced by 60-70% under 2% oxygen, which was paralleled by lower intracellular levels of reactive oxygen species (ROS) and apoptosis. Following this reduction in ROS the cells switched to a lower level of protection by down-regulating their antioxidant enzyme expression. The downside of this is that it left the cells more vulnerable to a subsequent oxidative challenge. Total collagen content was reduced in the 2% oxygen cultures and expression of matrix genes and matrix-metabolizing enzymes was significantly affected. Alkaline phosphatase activity and RNA expression as well as RUNX2 expression were significantly reduced under 2% oxygen. Time phase studies showed that high oxygen in the first phase of osteoblast differentiation and prior to mineralization is crucial for optimal differentiation and mineralization. Switching to 2% or 20% oxygen only during mineralization phase did not change the eventual level of mineralization. In conclusion, this study shows the significance of oxygen tension for proper osteoblast differentiation, extra cellular matrix (ECM) formation, and eventual mineralization. We demonstrated that the major impact of oxygen tension is in the early phase of osteoblast differentiation. Low oxygen in this phase leaves the cells in a premature differentiation state that cannot provide the correct signals for matrix maturation and mineralization. Copyright © 2011 Wiley Periodicals, Inc.

  12. Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts.

    Science.gov (United States)

    Franke, Sybille; Siggelkow, Heide; Wolf, Gunter; Hein, Gert

    2007-06-01

    Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).

  13. Plasticity of human dental pulp stromal cells with bioengineering platforms: a versatile tool for regenerative medicine.

    Science.gov (United States)

    Barachini, Serena; Danti, Serena; Pacini, Simone; D'Alessandro, Delfo; Carnicelli, Vittoria; Trombi, Luisa; Moscato, Stefania; Mannari, Claudio; Cei, Silvia; Petrini, Mario

    2014-12-01

    In recent years, human dental pulp stromal cells (DPSCs) have received growing attention due to their characteristics in common with other mesenchymal stem cells, in addition to the ease with which they can be harvested. In this study, we demonstrated that the isolation of DPSCs from third molar teeth of healthy individuals allowed the recovery of dental mesenchymal stem cells that showed self-renewal and multipotent differentiation capability. DPSCs resulted positive for CD73, CD90, CD105, STRO-1, negative for CD34, CD45, CD14 and were able to differentiate into osteogenic and chondrogenic cells. We also assayed the angiogenic potential of DPSCs, their capillary tube-like formation was assessed using an in vitro angiogenesis assay and the uptake of acetylated low-density lipoprotein was measured as a marker of endothelial function. Based on these results, DPSCs were capable of differentiating into cells with phenotypic and functional features of endothelial cells. Furthermore, this study investigated the growth and differentiation of human DPSCs under a variety of bioengineering platforms, such as low frequency ultrasounds, tissue engineering and nanomaterials. DPSCs showed an enhanced chondrogenic differentiation under ultrasound application. Moreover, DPSCs were tested on different scaffolds, poly(vinyl alcohol)/gelatin (PVA/G) sponges and human plasma clots. We showed that both PVA/G and human plasma clot are suitable scaffolds for adhesion, growth and differentiation of DPSCs toward osteoblastic lineages. Finally, we evaluated the interactions of DPSCs with a novel class of nanomaterials, namely boron nitride nanotubes (BNNTs). From our investigation, DPSCs have appeared as a highly versatile cellular tool to be employed in regenerative medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

    NARCIS (Netherlands)

    Ritfeld, Gaby Jane

    2014-01-01

    Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic

  15. The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

    Science.gov (United States)

    Turner, R. T.

    1999-01-01

    The purpose of the proposed study is to establish whether changes in gravitational loading have a direct effect on osteoblasts to regulate TGF-6 expression. The effects of spaceflight and reloading on TGF-B MRNA and peptide levels will be studied in a newly developed line of immortalized human fetal osteoblasts (HFOB) transfected with an SV-40 temperature dependent mutant to generate proliferating, undifferentiated hFOB cells at 33-34 C and a non-proliferating, differentiated HFOB cells at 37-39'C. Unlike previous cell culture models, HFOB cells have unlimited proliferative capacity yet can be precisely regulated to differentiate into mature cells which express mature osteoblast function. If isolated osteoblasts respond to changes in mechanical loading in a manner similar to their response in animals, the cell system could provide a powerful model to investigate the signal transduction pathway for gravitational loading.

  16. Comparison of functional and histological outcomes after intralesional, intracisternal, and intravenous transplantation of human bone marrow-derived mesenchymal stromal cells in a rat model of spinal cord injury.

    Science.gov (United States)

    Shin, Dong Ah; Kim, Jin-Myung; Kim, Hyoung-Ihl; Yi, Seong; Ha, Yoon; Yoon, Do Heum; Kim, Keung Nyun

    2013-10-01

    Few studies have compared methods of stem cell transplantation. The aim of the present study was to determine the optimal method of delivery of therapeutic stem cells in spinal cord injury (SCI). We compared functional and histologic outcomes after administration of human bone marrow stromal cells (BMSCs) by intralesional (ILT), intracisternal (ICT), and intravenous transplantation (IVT). A rat model of spinal cord injury was produced by dropping a 10-g weight, 2 mm in diameter, onto the exposed spinal cords of animals from a height of 25 mm. In each treatment group, 24 animals were randomly assigned for functional assessment and 24 for histologic examination. BMSCs (3 × 10(5), ILT; 1 × 10(6), ICT; 2 × 10(6), IVT) were transplanted 1 week after SCI in numbers determined in previous studies. Basso-Beattie-Bresnahan scoring was performed in all animals weekly for 6 weeks. Spinal cord specimens were obtained from eight animals in each group 2, 4, and 6 weeks after SCI. Viable BMSCs were counted in six sagittal sections from each spinal cord. All three treatment groups showed improved functional recovery compared to controls beginning 2 weeks after stem cell injection (P < 0.01). The ICT group showed the best functional recovery, followed by the ILT and IVT groups, respectively (P < 0.01). Histological analysis showed the largest number of viable BMSCs in the ILT group, followed by the ICT and IVT groups, respectively (P < 0.01). ICT may be the safest and most effective method for delivering stem cells and improving functional outcome in SCI when no limits are placed on the number of cells transplanted. As research on enhancing engraftment rates advances, further improvement of functional outcome can be expected.

  17. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  18. Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors.

    Science.gov (United States)

    Goodnough, L Henry; Dinuoscio, Gregg J; Ferguson, James W; Williams, Trevor; Lang, Richard A; Atit, Radhika P

    2014-02-01

    The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

  19. Zebrafish scales respond differently to in vitro dynamic and static acceleration: analysis of interaction between osteoblasts and osteoclasts.

    Science.gov (United States)

    Kitamura, Kei-ichiro; Takahira, Koh; Inari, Masato; Satoh, Yusuke; Hayakawa, Kazuichi; Tabuchi, Yoshiaki; Ogai, Kazuhiro; Nishiuchi, Takumi; Kondo, Takashi; Mikuni-Takagaki, Yuko; Chen, Wenxi; Hattori, Atsuhiko; Suzuki, Nobuo

    2013-09-01

    Zebrafish scales consist of bone-forming osteoblasts, bone-resorbing osteoclasts, and calcified bone matrix. To elucidate the underlying molecular mechanism of the effects induced by dynamic and static acceleration, we investigated the scale osteoblast- and osteoclast-specific marker gene expression involving osteoblast-osteoclast communication molecules. Osteoblasts express RANKL, which binds to the osteoclast surface receptor, RANK, and stimulates bone resorption. OPG, on the other hand, is secreted by osteoblast as a decoy receptor for RANKL, prevents RANKL from binding to RANK and thus prevents bone resorption. Therefore, the RANK-RANKL-OPG pathway contributes to the regulation of osteoclastogenesis by osteoblasts. Semaphorin 4D, in contrast, is expressed on osteoclasts, and binding to its receptor Plexin-B1 on osteoblasts results in suppression of bone formation. In the present study, we found that both dynamic and static acceleration at 3.0×g decreased RANKL/OPG ratio and increased osteoblast-specific functional mRNA such as alkaline phosphatase, while static acceleration increased and dynamic acceleration decreased osteoclast-specific mRNA such as cathepsin K. Static acceleration increased semaphorin 4D mRNA expression, while dynamic acceleration had no effect. The results of the present study indicated that osteoclasts have predominant control over bone metabolism via semaphorin 4D expression induced by static acceleration at 3.0×g. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Low-dose PTH increases osteoblast activity via decreased Mef2c/Sost in senescent osteopenic mice.

    Science.gov (United States)

    Saidak, Zuzana; Le Henaff, Carole; Azzi, Sofia; Marty, Caroline; Marie, Pierre J

    2014-10-01

    Intermittent administration of parathyroid hormone (PTH) 1-34 at a standard dose has been shown to induce anabolic effects in bone. However, whether low-dose PTH promotes bone formation during senescence is unknown. To address this issue, we determined the effects of low-dose PTH and analysed the underlying mechanisms in prematurely senescent mice that display osteopenia. Treatment of 9-week-old Samp6 mice for 6 weeks with PTH at a standard dose (100 μg/kg per day) increased vertebral and femoral bone mass and improved bone microarchitecture as a result of increased bone-forming surfaces and mineral apposition rate (MAR). At a tenfold lower dose (10 μg/kg per day), PTH increased axial bone volume and trabecular thickness, as detected by bone histomorphometry but not by micro-computed tomography analysis. This anabolic effect resulted from increased osteoblast activity, as reflected by increased serum N-terminal propeptide of type 1 procollagen (P1NP) levels and MAR, with unchanged bone-forming surface or osteoblast surface. Mechanistically, low-dose PTH increased the expression of osteoblast markers in bone marrow stromal cells and mature osteoblasts, which was associated with increased expression of the Wnt effector Wisp1. Moreover, low-dose PTH decreased the expression of the Mef2c transcription factor, resulting in decreased Sost expression in osteoblasts/osteocytes. These results indicate that PTH at a low dose is effective at promoting bone formation and increased bone volume in senescent osteopenic mice through increased osteoblast activity and modulation of specific Wnt effectors, which raises the potential therapeutic use of intermittent PTH at low dose to increase bone forming activity and bone mass in skeletal senescence. © 2014 Society for Endocrinology.

  1. Osteoblasts in osteoporosis: past, emerging, and future anabolic targets.

    Science.gov (United States)

    Marie, Pierre J; Kassem, Moustapha

    2011-07-01

    Age-related bone loss is associated with significant changes in bone remodeling characterized by decreased trabecular and periosteal bone formation relative to bone resorption, resulting in bone fragility and increased risk of fractures. Prevention or reversal of age-related decrease in bone mass and increase in bone fragility has been based on inhibition of bone resorption using anticatabolic drugs. The current challenge is to promote osteoblastogenesis and bone formation to prevent age-related bone deterioration. A limited number of approved therapeutic molecules are available to activate bone formation. Therefore, there is a need for anabolic drugs that promote bone matrix apposition at the endosteal, endocortical, and periosteal envelopes by increasing the number of osteoblast precursor cells and/or the function of mature osteoblasts. In this study, we review current therapeutics promoting bone formation and anabolic molecules targeting signaling pathways involved in osteoblastogenesis, based on selected full-text articles searched on Medline search from 1990 to 2010. We present current therapeutic approaches focused on intermittent parathyroid hormone and Wnt signaling agonists/antagonists. We also discuss novel approaches for prevention and treatment of defective bone formation and bone loss associated with aging and osteoporosis. These strategies targeting osteoblastic cell functions may prove to be useful in promoting bone formation and improving bone strength in the aging population.

  2. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses.

    Science.gov (United States)

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka; Marycz, Krzysztof

    2017-08-03

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.

  3. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses

    Science.gov (United States)

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka

    2017-01-01

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)—the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs’ and IECs’ morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome. PMID:28771165

  4. Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells.

    Science.gov (United States)

    González-Vázquez, Arlyng; Planell, Josep A; Engel, Elisabeth

    2014-06-01

    Bone is the main store of calcium and progenitor cells in the body. During the resorption process, the local calcium concentration reaches 8-40mM, and the surrounding cells are exposed to these fluctuations in calcium. This stimulus is a signal that is detected through the calcium sensing receptor (CaSR), which modulates chemotactic and proliferative G protein-dependent signaling pathways. The objective of the present work is to evaluate the roles of extracellular calcium ([Ca(2+)]o) and the CaSR in osteoinduction. Rat bone marrow mesenchymal stromal cells (rBMSCs) were stimulated with 10mM of Ca(2+). Several experiments were conducted to demonstrate the effect of [Ca(2+)]o on chemotaxis, proliferation and differentiation on the osteoblastic lineage. It was found that [Ca(2+)]o induces rBMSCs to migrate and proliferate in a concentration-dependent manner. Real-time polymerase chain reaction and immunofluorescence also revealed that 10mM Ca(2+) stimulates overexpression of osteogenic markers in rBMSCs, including alkaline phosphatase (ALP), bone sialoprotein, collagen Ia1 and osteocalcin. Functional assays determining ALP activity and mineralization tests both corroborate the increased expression of these markers in rBMSCs stimulated with Ca(2+). Moreover, CaSR blockage inhibited the cellular response to stimulation with high concentrations of [Ca(2+)]o, revealing that the CaSR is a key modulator of these cellular responses. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  5. Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System

    Science.gov (United States)

    Hanley, Patrick J.; Mei, Zhuyong; Durett, April G.; Cabreira-Harrison, Marie da Graca; Klis, Mariola; Li, Wei; Zhao, Yali; Yang, Bing; Parsha, Kaushik; Mir, Osman; Vahidy, Farhaan; Bloom, Debra; Rice, R. Brent; Hematti, Peiman; Savitz, Sean I; Gee, Adrian P.

    2014-01-01

    Background The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host-disease, diabetes, ischemic cardiomyopathy, and Crohn's disease has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells needs to be cost effective, safe, and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures, and require prolonged culture times. Methods We evaluated the Quantum Cell Expansion system for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results to a flask-based method currently in clinical trials. Results After only two passages, we were able to expand a mean of 6.6×108 MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts, and adipocytes. The Quantum was able to generate the target cell number of 2.0×108 cells in an average of 9-fewer days and in half the number of passages required during flask-based expansion. We estimated the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. Discussion The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared to flasks. In the Quantum, the risk of contamination is substantially reduced due to the substantial decrease in open procedures. PMID:24726657

  6. Ion channel functional protein kinase TRPM7 regulates Mg ions to promote the osteoinduction of human osteoblast via PI3K pathway: In vitro simulation of the bone-repairing effect of Mg-based alloy implant.

    Science.gov (United States)

    Zhang, Xiuzhi; Zu, Haiyue; Zhao, Dewei; Yang, Ke; Tian, Simiao; Yu, Xiaoming; Lu, Faqiang; Liu, Baoyi; Yu, Xiaobing; Wang, Benjie; Wang, Wei; Huang, Shibo; Wang, Yongxuan; Wang, Zihua; Zhang, Zhaodong

    2017-11-01

    Mg-based alloys, as the potential orthopaedic implant, can self-degrade to avoid second operation for its remove, and enable to promote bone repair; however, the underlying molecular mechanisms remain unclear. In the present study, we examined the effect of Mg ions on osteogenesis, chemotaxis and anti-alkaline stress in hFOB1.19 human osteoblast cells to simulate bone-repairing effect of a biodegradable Mg-based alloy implant in vitro, and explored the regulatory role of the transient receptor potential melastatin 7 (TRPM7)/phosphoinositide 3-kinase (PI3K) signalling pathway in the process of Mg ion-induced bone repair by knockdown of TRPM7 and antagonizing PI3K activity. Results indicate that Mg ions up-regulated the expression of Runx2 and alkaline phosphatase (ALP) through TRPM7/PI3K signalling pathway, which could significantly enhance the osteogenic activity of human osteoblasts. Furthermore, the expression levels of MMP2, MMP9 and vascular endothelial growth factor (VEGF) were increased by TRPM7/PI3K signalling pathway, which recruits osteoblasts from low- to high-Mg ion environments by inducing cell migration. Although an alkaline environment has antibacterial effects, alkaline stress can cause cytotoxicity and induce cell death. Finally, we found that Mg ions could activate PI3K phosphorylation to promote cell growth and survival, protecting cells against the alkaline-stress-induced cytotoxicity caused by the degradation of Mg-based alloy implants. Our study not only revealed the molecular mechanism of Mg in promoting bone repair but also explained the protective effects of Mg ions on osteoblasts in an alkaline environment, which provides a theoretical basis and new directions for the application of Mg-based alloy implant material in orthopaedics fixations and osteosarcoma treatment. As a potential biomaterial for orthopaedic implant, biodegradable magnesium has several advantages including self-degradation and bone repair promotion; however, the underlying

  7. Mineralization and Osteoblast Cells Response of Nanograde Pearl Powders

    Directory of Open Access Journals (Sweden)

    Jian-Chih Chen

    2013-01-01

    Full Text Available The main objective of this study is to characterize the thermal, mineralization, and osteoblast cells response of pearl nanocrystallites. The results obtained from X-ray diffraction, FTIR spectra demonstrate that the pearl nano-crystallites can induce the formation of an HA layer on their surface in SBF, even after only short soaking periods. The in vitro cell response to nano-grade pearl powders is assessed by evaluating the cytotoxicity against a mouse embryonic fibroblast cell line and by characterizing the attachment ability and alkaline phosphatase activity of mouse bone cells (MC3T3-E1, abbreviated to E1 and bone marrow stromal precursor (D1 cells. The cytotoxicities of pearls were tested by the filtration and culture of NIH-3T3 mouse embryonic fibroblast cells. The viability of the cultured cells in media containing pearl crystallites for 24 and 72 h is greater than 90%. The bone cells seen in these micrographs are elongated and align predominately along the pearl specimen. The cells observed in these images also appeared well attached and cover the surface almost completely after 1 h. The pearl nanocrystallites had a positive effect on the osteogenic ability of ALP activity, and this promoted the osteogenic differentiation of MSCs significantly at explanations.

  8. Tenascin C affects mineralization of SaOS2 osteoblast-like cells through matrix vesicles.

    Science.gov (United States)

    Li, Chengzhi; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Li, Haiying; Wang, Huaxin; Shi, Liang; Han, Jinxiang

    Tenascin C (TNC) is an extracellular matrix glycoprotein involved in osteogenesis and bone mineralization. In a previous study, we identified TNC protein located in the matrix vesicles (MVs) of osteoblasts. MVs are determinant in the mineralization formation. Therefore, we hypothesize whether TNC can modulate osteoblast mineralization via MVs. In this study, we demonstrated that the expression level of TNC was increased with osteoblast differentiation of osteoblast-like SaOS2 cells, and down-regulation of TNC expression by siRNA could significantly inhibit SaOS2 differentiation toward osteoblasts and mineralization as evidenced by decreases in ALP activity, mineralized nodule formation, calcium deposition, and down-regulation of osteogenic marker genes ALP, and COL1A1. Furthermore, we validated that TNC located in the MVs of mineralized SaOS2 cells, and that down-regulation of TNC could decrease MVs mineralization ability in vitro, and the decrease of MVs mineralization ability was not associated with annexins. In conclusion, in this study, we extended the role of TNC during osteogenesis previous progresses, and that supported TNC as an important functional MVs component in modulating osteoblast mineralization.

  9. Sepsis-Induced Osteoblast Ablation Causes Immunodeficiency.

    Science.gov (United States)

    Terashima, Asuka; Okamoto, Kazuo; Nakashima, Tomoki; Akira, Shizuo; Ikuta, Koichi; Takayanagi, Hiroshi

    2016-06-21

    Sepsis is a host inflammatory response to severe infection associated with high mortality that is caused by lymphopenia-associated immunodeficiency. However, it is unknown how lymphopenia persists after the accelerated lymphocyte apoptosis subsides. Here we show that sepsis rapidly ablated osteoblasts, which reduced the number of common lymphoid progenitors (CLPs). Osteoblast ablation or inducible deletion of interleukin-7 (IL-7) in osteoblasts recapitulated the lymphopenic phenotype together with a lower CLP number without affecting hematopoietic stem cells (HSCs). Pharmacological activation of osteoblasts improved sepsis-induced lymphopenia. This study demonstrates a reciprocal interaction between the immune and bone systems, in which acute inflammation induces a defect in bone cells resulting in lymphopenia-associated immunodeficiency, indicating that bone cells comprise a therapeutic target in certain life-threatening immune reactions. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The transcription factor protein Sox11 enhances early osteoblast differentiation by facilitating proliferation and the survival of mesenchymal and osteoblast progenitors.

    Science.gov (United States)

    Gadi, Jogeswar; Jung, Seung-Hyun; Lee, Min-Jung; Jami, Ajita; Ruthala, Kalyani; Kim, Kyoung-Min; Cho, Nam-Hoon; Jung, Han-Sung; Kim, Cheol-Hee; Lim, Sung-Kil

    2013-08-30

    Sox11 deletion mice are known to exhibit developmental defects of craniofacial skeletal malformations, asplenia, and hypoplasia of the lung, stomach, and pancreas. Despite the importance of Sox11 in the developing skeleton, the role of Sox11 in osteogenesis has not been studied yet. In this study, we identified that Sox11 is an important transcription factor for regulating the proliferation and survival of osteoblast precursor cells as well as the self-renewal potency of mesenchymal progenitor cells via up-regulation of Tead2. Furthermore, Sox11 also plays an important role in the segregation of functional osteoblast lineage progenitors from osteochondrogenic progenitors. Facilitation of osteoblast differentiation from mesenchymal cells was achieved by enhanced expression of the osteoblast lineage specific transcription factors Runx2 and Osterix. Morpholino-targeted disruption of Sox11 in zebrafish impaired organogenesis, including the bones, which were under mineralized. These results indicated that Sox11 plays a crucial role in the proliferation and survival of mesenchymal and osteoblast precursors by Tead2, and osteogenic differentiation by regulating Runx2 and Osterix.

  11. Marginal reticular cells: a stromal subset directly descended from the lymphoid tissue organizer

    Directory of Open Access Journals (Sweden)

    Tomoya eKatakai

    2012-07-01

    Full Text Available The architecture of secondary lymphoid organs (SLOs is supported by several nonhematopoietic stromal cells. Currently it is established that two distinct stromal subsets, follicular dendritic cells and fibroblastic reticular cells, play crucial roles in the formation of tissue compartments within SLOs, i.e., the follicle and T zone, respectively. Although stromal cells in the anlagen are essential for SLO development, the relationship between these primordial cells and the subsets in adulthood remains poorly understood. In addition, the roles of stromal cells in the entry of antigens into the compartments through some tissue structures peculiar to SLOs remain unclear. A recently identified stromal subset, marginal reticular cells (MRCs, covers the margin of SLOs that are primarily located in the outer edge of follicles and construct a unique reticulum. MRCs are closely associated with specialized endothelial or epithelial structures for antigen transport. The similarities in marker expression profiles and successive localization during development suggest that MRCs directly descend from organizer stromal cells in the anlagen. Therefore, MRCs are thought to be a crucial stromal component for the organization and function of SLOs.

  12. Identification of Rorβ targets in cultured osteoblasts and in human bone

    Energy Technology Data Exchange (ETDEWEB)

    Roforth, Matthew M., E-mail: roforth.matthew@mayo.edu; Khosla, Sundeep, E-mail: khosla.sundeep@mayo.edu; Monroe, David G., E-mail: monroe.david@mayo.edu

    2013-11-01

    Highlights: •We examine the gene expression patterns controlled by Rorβ in osteoblasts. •Genes involved in extracellular matrix regulation and proliferation are affected. •Rorβ mRNA levels increase in aged, human bone biopsies. •Rorβ may affect osteoblast activity by modulation of these pathways. -- Abstract: Control of osteoblastic bone formation involves the cumulative action of numerous transcription factors, including both activating and repressive functions that are important during specific stages of differentiation. The nuclear receptor retinoic acid receptor-related orphan receptor β (Rorβ) has been recently shown to suppress the osteogenic phenotype in cultured osteoblasts, and is highly upregulated in bone marrow-derived osteogenic precursors isolated from aged osteoporotic mice, suggesting Rorβ is an important regulator of osteoblast function. However the specific gene expression patterns elicited by Rorβ are unknown. Using microarray analysis, we identified 281 genes regulated by Rorβ in an MC3T3-E1 mouse osteoblast cell model (MC3T3-Rorβ-GFP). Pathway analysis revealed alterations in genes involved in MAPK signaling, genes involved in extracellular matrix (ECM) regulation, and cytokine-receptor interactions. Whereas the identified Rorβ-regulated ECM genes normally decline during osteoblastic differentiation, they were highly upregulated in this non-mineralizing MC3T3-Rorβ-GFP model system, suggesting that Rorβ may exert its anti-osteogenic effects through ECM disruption. Consistent with these in vitro findings, the expression of both RORβ and a subset of RORβ-regulated genes were increased in bone biopsies from postmenopausal women (73 ± 7 years old) compared to premenopausal women (30 ± 5 years old), suggesting a role for RORβ in human age-related bone loss. Collectively, these data demonstrate that Rorβ regulates known osteogenic pathways, and may represent a novel therapeutic target for age-associated bone loss.

  13. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    International Nuclear Information System (INIS)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-01-01

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy

  14. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO{sub 2} coating with magnesium impregnation

    Energy Technology Data Exchange (ETDEWEB)

    Cecchinato, Francesca, E-mail: francesca.cecchinato@mah.se [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Karlsson, Johan [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Ferroni, Letizia; Gardin, Chiara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Galli, Silvia; Wennerberg, Ann [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Zavan, Barbara [Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova (Italy); Andersson, Martin [Department of Chemical and Biological Engineering, Applied Surface Chemistry, Chalmers University of Technology, Gothenburg (Sweden); Jimbo, Ryo [Department of Prosthodontics, Faculty of Odontology, Malmö University, Malmö (Sweden); Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki (Japan)

    2015-07-01

    The aim of this study was to evaluate the osteogenic response of human adipose-derived stromal cells (ADScs) to mesoporous titania (TiO{sub 2}) coatings produced with evaporation-induced self-assembly method (EISA) and loaded with magnesium. Our emphasis with the magnesium release functionality was to modulate progenitor cell osteogenic differentiation under standard culture conditions. Osteogenic properties of the coatings were assessed for stromal cells by means of scanning electron microscopy (SEM) imaging, colorimetric mitochondrial viability assay (MTT), colorimetric alkaline phosphates activity (ALP) assay and real time RT-polymerase chain reaction (PCR). Using atomic force microscopy (AFM) it was shown that the surface expansion area (S{sub dr}) was strongly enhanced by the presence of magnesium. From MTT results it was shown that ADSc viability was significantly increased on mesoporous surfaces compared to the non-porous one at a longer cell culture time. However, no differences were observed between the magnesium impregnated and non-impregnated surfaces. The alkaline phosphatase activity confirmed that ADSc started to differentiate into the osteogenic phenotype after 2 weeks of culturing. The gene expression profile at 2 weeks of cell growth showed that such coatings were capable to incorporate specific osteogenic markers inside their interconnected nano-pores and, at 3 weeks, ADSc differentiated into osteoblasts. Interestingly, magnesium significantly promoted the osteopontin gene expression, which is an essential gene for the early biomaterial–cell osteogenic interaction. - Highlights: • The magnesium loading presents a transitory effect on mesoporous TiO{sub 2} surface topography • The mesoporous structure promotes cellular attachment and spreading • The mesoporous structure activates osteogenesis of mesenchymal stem cells in absence of osteogenic promoters • The physical adsorbed magnesium is suggested to be involved in the expression of

  15. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  16. Osteogenic potential of human adipose-derived stromal cells on 3-dimensional mesoporous TiO2 coating with magnesium impregnation

    International Nuclear Information System (INIS)

    Cecchinato, Francesca; Karlsson, Johan; Ferroni, Letizia; Gardin, Chiara; Galli, Silvia; Wennerberg, Ann; Zavan, Barbara; Andersson, Martin; Jimbo, Ryo

    2015-01-01

    The aim of this study was to evaluate the osteogenic response of human adipose-derived stromal cells (ADScs) to mesoporous titania (TiO 2 ) coatings produced with evaporation-induced self-assembly method (EISA) and loaded with magnesium. Our emphasis with the magnesium release functionality was to modulate progenitor cell osteogenic differentiation under standard culture conditions. Osteogenic properties of the coatings were assessed for stromal cells by means of scanning electron microscopy (SEM) imaging, colorimetric mitochondrial viability assay (MTT), colorimetric alkaline phosphates activity (ALP) assay and real time RT-polymerase chain reaction (PCR). Using atomic force microscopy (AFM) it was shown that the surface expansion area (S dr ) was strongly enhanced by the presence of magnesium. From MTT results it was shown that ADSc viability was significantly increased on mesoporous surfaces compared to the non-porous one at a longer cell culture time. However, no differences were observed between the magnesium impregnated and non-impregnated surfaces. The alkaline phosphatase activity confirmed that ADSc started to differentiate into the osteogenic phenotype after 2 weeks of culturing. The gene expression profile at 2 weeks of cell growth showed that such coatings were capable to incorporate specific osteogenic markers inside their interconnected nano-pores and, at 3 weeks, ADSc differentiated into osteoblasts. Interestingly, magnesium significantly promoted the osteopontin gene expression, which is an essential gene for the early biomaterial–cell osteogenic interaction. - Highlights: • The magnesium loading presents a transitory effect on mesoporous TiO 2 surface topography • The mesoporous structure promotes cellular attachment and spreading • The mesoporous structure activates osteogenesis of mesenchymal stem cells in absence of osteogenic promoters • The physical adsorbed magnesium is suggested to be involved in the expression of osteopontin

  17. Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells

    Czech Academy of Sciences Publication Activity Database

    Kalbáčová, M.; Brož, A.; Kong, J.; Kalbáč, Martin

    2010-01-01

    Roč. 48, č. 15 (2010), s. 4323-4329 ISSN 0008-6223 R&D Projects: GA AV ČR IAA400400911; GA AV ČR KAN200100801; GA AV ČR KAN400100701; GA ČR GAP204/10/1677; GA MŠk ME09060 Institutional research plan: CEZ:AV0Z40400503 Keywords : carbon nanotubes * graphene substrates * SiO2 Subject RIV: CG - Electrochemistry Impact factor: 4.893, year: 2010

  18. A Cell-Based Self-Assembly Approach for the Production of Human Osseous Tissues from Adipose-Derived Stromal/Stem Cells.

    Science.gov (United States)

    Galbraith, Todd; Clafshenkel, William P; Kawecki, Fabien; Blanckaert, Camille; Labbé, Benoit; Fortin, Michel; Auger, François A; Fradette, Julie

    2017-02-01

    Achieving optimal bone defect repair is a clinical challenge driving intensive research in the field of bone tissue engineering. Many strategies focus on seeding graft materials with progenitor cells prior to in vivo implantation. Given the benefits of closely mimicking tissue structure and function with natural materials, the authors hypothesize that under specific culture conditions, human adipose-derived stem/stromal cells (hASCs) can solely be used to engineer human reconstructed osseous tissues (hROTs) by undergoing osteoblastic differentiation with concomitant extracellular matrix production and mineralization. Therefore, the authors are developing a self-assembly methodology allowing the production of such osseous tissues. Three-dimensional (3D) tissues reconstructed from osteogenically-induced cell sheets contain abundant collagen type I and are 2.7-fold less contractile compared to non-osteogenically induced tissues. In particular, hROT differentiation and mineralization is reflected by a greater amount of homogenously distributed alkaline phosphatase, as well as higher calcium-containing hydroxyapatite (P tissues. Taken together, these findings show that hASC-driven tissue engineering leads to hROTs that demonstrate structural and functional characteristics similar to native osseous tissue. These highly biomimetic human osseous tissues will advantageously serve as a platform for molecular studies as well as for future therapeutic in vivo translation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Deletion of Pkd1 in renal stromal cells causes defects in the renal stromal compartment and progressive cystogenesis in the kidney.

    Science.gov (United States)

    Nie, Xuguang; Arend, Lois J

    2017-12-01

    Autosomal dominant polycystic kidney disease (ADPKD), caused by PKD1 and PKD2 gene mutations, is one of the most common genetic diseases, affecting up to 1 in 500 people. Mutations of PKD1 account for over 85% of ADPKD cases. However, mechanisms of disease progression and explanations for the wide range in disease phenotype remain to be elucidated. Moreover, functional roles of PKD1 in the renal stromal compartment are poorly understood. In this work, we tested if Pkd1 is essential for development and maintenance of the renal stromal compartment and if this role contributes to pathogenesis of polycystic kidney disease using a novel tissue-specific knockout mouse model. We demonstrate that deletion of Pkd1 from renal stromal cells using Foxd1-driven Cre causes a spectrum of defects in the stromal compartment, including excessive apoptosis/proliferation and extracellular matrix deficiency. Renal vasculature was also defective. Further, mutant mice showed epithelial changes and progressive cystogenesis in adulthood modeling human ADPKD. Altogether, we provide robust evidence to support indispensable roles for Pkd1 in development and maintenance of stromal cell derivatives by using a novel ADPKD model. Moreover, stromal compartment defects caused by Pkd1 deletion might serve as an important mechanism for pathogenesis of ADPKD.

  20. Update on gastrointestinal stromal tumors for radiologists

    Energy Technology Data Exchange (ETDEWEB)

    Tirumani, Sree Harsha; O' Neill, Alibhe; Jagannathan, Jyothi P. [Dept. of Imaging, Dana-Farber Cancer Institute, Boston (United States); Baheti, Akahay D. [Dept. of Radiology, Tata Memorial Centre, Mumbai (India); Tirumani, Harika [Dept. of Radiology, University of Arkansas for Medical Sciences, Little Rock (United States)

    2017-01-15

    The management of gastrointestinal stromal tumors (GISTs) has evolved significantly in the last two decades due to better understanding of their biologic behavior as well as development of molecular targeted therapies. GISTs with exon 11 mutation respond to imatinib whereas GISTs with exon 9 or succinate dehydrogenase subunit mutations do not. Risk stratification models have enabled stratifying GISTs according to risk of recurrence and choosing patients who may benefit from adjuvant therapy. Assessing response to targeted therapies in GIST using conventional response criteria has several potential pitfalls leading to search for alternate response criteria based on changes in tumor attenuation, volume, metabolic and functional parameters. Surveillance of patients with GIST in the adjuvant setting is important for timely detection of recurrences.

  1. Imaging of gastrointestinal stromal tumour (GIST)

    Energy Technology Data Exchange (ETDEWEB)

    Lau, S. E-mail: laushunhk@yahoo.com.hk; Tam, K.F.; Kam, C.K.; Lui, C.Y.; Siu, C.W.; Lam, H.S.; Mak, K.L

    2004-06-01

    Gastrointestinal stromal tumour (GIST) represents the most common kind of mesenchymal tumour that arises from the alimentary tract. GIST is currently defined as a gastrointestinal tract mesenchymal tumour containing spindle cells (or less commonly epithelioid cells or rarely both) and showing CD117 (c-kit protein) positivity. Targeted molecular therapy of non-resectable GIST using imatinib, a specific tyrosine kinase receptor inhibitor, represents a real milestone in the management of solid malignancy. Imaging studies, both anatomical and functional, are playing an increasingly important role in management of patients with GIST. This review illustrates the radiological appearance of GISTs and the site-specific roles of each imaging tool. Clinical features and radiological differential diagnosis of GIST are also discussed.

  2. Differential Expression Profiling of Long Noncoding RNA and mRNA during Osteoblast Differentiation in Mouse

    Directory of Open Access Journals (Sweden)

    Minjung Kim

    2018-01-01

    Full Text Available Long noncoding RNAs (lncRNAs are emerging as an important controller affecting metabolic tissue development, signaling, and function. However, little is known about the function and profile of lncRNAs in osteoblastic differentiation in mice. Here, we analyzed the RNA-sequencing (RNA-Seq datasets obtained for 18 days in two-day intervals from neonatal mouse calvarial pre-osteoblast-like cells. Over the course of osteoblast differentiation, 4058 mRNAs and 3948 lncRNAs were differentially expressed, and they were grouped into 12 clusters according to the expression pattern by fuzzy c-means clustering. Using weighted gene coexpression network analysis, we identified 9 modules related to the early differentiation stage (days 2–8 and 7 modules related to the late differentiation stage (days 10–18. Gene ontology and KEGG pathway enrichment analysis revealed that the mRNA and lncRNA upregulated in the late differentiation stage are highly associated with osteogenesis. We also identified 72 mRNA and 89 lncRNAs as potential markers including several novel markers for osteoblast differentiation and activation. Our findings provide a valuable resource for mouse lncRNA study and improves our understanding of the biology of osteoblastic differentiation in mice.

  3. Identification of novel regulators of osteoblast matrix mineralization by time series transcriptional profiling.

    Science.gov (United States)

    Staines, Katherine Ann; Zhu, Dongxing; Farquharson, Colin; MacRae, Vicky Elizabeth

    2014-05-01

    Bone mineralization is a carefully orchestrated process, regulated by a number of promoters and inhibitors that function to ensure effective hydroxyapatite formation. Here we sought to identify new regulators of this process through a time series microarray analysis of mineralising primary osteoblast cultures over a 27 day culture period. To our knowledge this is the first microarray study investigating murine calvarial osteoblasts cultured under conditions that permit both physiological extracellular matrix mineralization through the formation of discrete nodules and the terminal differentiation of osteoblasts into osteocytes. RT-qPCR was used to validate and expand the microarray findings. We demonstrate the significant up-regulation of >6,000 genes during the osteoblast mineralization process, the highest-ranked differentially expressed genes of which were those dominated by members of the PPAR-γ signalling pathway, namely Adipoq, Cd36 and Fabp4. Furthermore, we show that the inhibition of this signalling pathway promotes matrix mineralisation in these primary osteoblast cultures. We also identify Cilp, Phex, Trb3, Sox11, and Psat1 as novel regulators of matrix mineralization. Further studies examining the precise function of the identified genes and their interactions will advance our understanding of the mechanisms underpinning biomineralization.

  4. Secondary Release of Exosomes From Astrocytes Contributes to the Increase in Neural Plasticity and Improvement of Functional Recovery After Stroke in Rats Treated With Exosomes Harvested From MicroRNA 133b-Overexpressing Multipotent Mesenchymal Stromal Cells.

    Science.gov (United States)

    Xin, Hongqi; Wang, Fengjie; Li, Yanfeng; Lu, Qing-E; Cheung, Wing Lee; Zhang, Yi; Zhang, Zheng Gang; Chopp, Michael

    2017-02-16

    We previously demonstrated that multipotent mesenchymal stromal cells (MSCs) that overexpress microRNA 133b (miR-133b) significantly improve functional recovery in rats subjected to middle cerebral artery occlusion (MCAO) compared with naive MSCs and that exosomes generated from naive MSCs mediate the therapeutic benefits of MSC therapy for stroke. Here we investigated whether exosomes isolated from miR-133b-overexpressing MSCs (Ex-miR-133b+) exert amplified therapeutic effects. Rats subjected to 2 h of MCAO were intra-arterially injected with Ex-miR-133b+, exosomes from MSCs infected by blank vector (Ex-Con), or phosphate-buffered saline (PBS) and were sacrificed 28 days after MCAO. Compared with the PBS treatment, both exosome treatment groups exhibited significant improvement of functional recovery. Ex-miR-133b+ treatment significantly increased functional improvement and neurite remodeling/brain plasticity in the ischemic boundary area compared with the Ex-Con treatment. Treatment with Ex-miR-133b+ also significantly increased brain exosome content compared with Ex-Con treatment. To elucidate mechanisms underlying the enhanced therapeutic effects of Ex-miR-133b+, astrocytes cultured under oxygen- and glucose-deprived (OGD) conditions were incubated with exosomes harvested from naive MSCs (Ex-Naive), miR-133b downregulated MSCs (Ex-miR-133b-), and Ex-miR-133b+. Compared with the Ex-Naive treatment, Ex-miR-133b+ significantly increased exosomes released by OGD astrocytes, whereas Ex-miR-133b- significantly decreased the release. Also, exosomes harvested from OGD astrocytes treated with Ex-miR-133b+ significantly increased neurite branching and elongation of cultured cortical embryonic rat neurons compared with the exosomes from OGD astrocytes subjected to Ex-Con. Our data suggest that exosomes harvested from miR-133b-overexpressing MSCs improve neural plasticity and functional recovery after stroke with a contribution from a stimulated secondary release of

  5. Bone-derived mesenchymal stromal cells from HIV transgenic mice exhibit altered proliferation, differentiation capacity and paracrine functions along with impaired therapeutic potential in kidney injury

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Kang; Rai, Partab; Lan, Xiqian; Plagov, Andrei; Malhotra, Ashwani [Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhassett, NY (United States); Gupta, Sanjeev [Departments of Medicine and Pathology, Marion Bessin Liver Research Center, Diabetes Center, Cancer Center, Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Institute for Clinical and Translational Research, Albert Einstein College of Medicine, Bronx, NY (United States); Singhal, Pravin C., E-mail: psinghal@nshs.edu [Feinstein Institute for Medical Research, North Shore-Long Island Jewish Health System, Manhassett, NY (United States)

    2013-08-15

    Mesenchymal stem cells (MSCs) secrete paracrine factors that could be cytoprotective and serve roles in immunoregulation during tissue injury. Although MSCs express HIV receptors, and co-receptors, and are susceptible to HIV infection, whether HIV-1 may affect biological properties of MSCs needs more study. We evaluated cellular proliferation, differentiation and paracrine functions of MSCs isolated from compact bones of healthy control mice and Tg26 HIV-1 transgenic mice. The ability of MSCs to protect against cisplatin toxicity was studied in cultured renal tubular cells as well as in intact mice. We successfully isolated MSCs from healthy mice and Tg26 HIV-1 transgenic mice and found the latter expressed viral Nef, Vpu, NL4-3 and Vif genes. The proliferation and differentiation of Tg26 HIV-1 MSCs was inferior to MSCs from healthy mice. Moreover, transplantation of Tg26 HIV-1 MSCs less effectively improved outcomes compared with healthy MSCs in mice with acute kidney injury. Also, Tg26 HIV-1 MSCs secreted multiple cytokines, but at significantly lower levels than healthy MSCs, which resulted in failure of conditioned medium from these MSCs to protect cultured renal tubular cells from cisplatin toxicity. Therefore, HIV-1 had adverse biological effects on MSCs extending to their proliferation, differentiation, function, and therapeutic potential. These findings will help in advancing mechanistical insight in renal injury and repair in the setting of HIV-1 infection. -- Highlights: •MSCs isolated from HIV mice displayed HIV genes. •MSCs isolated from HIV mice exhibited attenuated growth and paracrine functions. •AKI mice with transplanted HIV-MSC displayed poor outcome. •HIV-1 MSC secreted multiple cytokines but at a lower level.

  6. Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement.

    Science.gov (United States)

    Mantoku, Akiko; Chatani, Masahiro; Aono, Kazushi; Inohaya, Keiji; Kudo, Akira

    2016-01-15

    Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Toxicity of iron oxide nanoparticles against osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Shi Sifeng [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China); Jia Jingfu [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Guo Xiaokui [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Zhao Yaping [Shanghai Jiao Tong University, School of Chemistry and Chemical Technology (China); Liu Boyu [Shanghai Jiao Tong University School of Medicine, Department of Medical Microbiology and Parasitology, Institutes of Medical Sciences (China); Chen Desheng; Guo Yongyuan; Zhang Xianlong, E-mail: zhangxianlong20101@163.com [Shanghai Jiao Tong University, Department of Orthopaedic Surgery, Shanghai Sixth People' s Hospital (China)

    2012-09-15

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 {mu}g/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 {mu}g/mL and 25.9 % in 500 {mu}g/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 {mu}g/mL, 23.40 % of apoptosis in a concentration of 300 {mu}g/mL and 28.49 % in a concentration of 500 {mu}g/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  8. Toxicity of iron oxide nanoparticles against osteoblasts

    International Nuclear Information System (INIS)

    Shi Sifeng; Jia Jingfu; Guo Xiaokui; Zhao Yaping; Liu Boyu; Chen Desheng; Guo Yongyuan; Zhang Xianlong

    2012-01-01

    Magnetic nanoparticles have been widely used for tissue repair, magnetic resonance imaging, immunoassays and drug delivery. They are very promising in orthopaedic applications and several magnetic nanoparticles have been exploited for the treatment of orthopaedic disease. Here, we conducted an in vitro study to examine the interaction of magnetic iron oxide nanoparticles with human osteoblasts to evaluate the dose-related toxicity of the nanoparticles on osteoblasts. A transmission electron microscope was used to visualise the internalised magnetic nanoparticles in osteoblasts. The CCK-8 results revealed increased cell viability (107.5 % vitality compared with the control group) when co-cultured at a low concentration (20 μg/mL) and decreased cell viability (59.5 % vitality in a concentration of 300 μg/mL and 25.9 % in 500 μg/mL) when co-cultured in high concentrations. The flow cytometric detection revealed similar results with 5.48 % of apoptosis in a concentration of 20 μg/mL, 23.40 % of apoptosis in a concentration of 300 μg/mL and 28.49 % in a concentration of 500 μg/mL. The disrupted cytoskeleton of osteoblasts was also revealed using a laser scanning confocal microscope. We concluded that use of a low concentration of magnetic iron oxide nanoparticles is important to avoid damage to osteoblasts.

  9. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Science.gov (United States)

    Ren, Chun-E; Zhu, Xueqiong; Li, Jinping; Lyle, Christian; Dowdy, Sean; Podratz, Karl C.; Byck, David; Chen, Hai-Bin; Jiang, Shi-Wen

    2015-01-01

    Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies. PMID:25782154

  10. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  11. Infection Programs Sustained Lymphoid Stromal Cell Responses and Shapes Lymph Node Remodeling upon Secondary Challenge

    Directory of Open Access Journals (Sweden)

    Julia L. Gregory

    2017-01-01

    Full Text Available Lymph nodes (LNs are constructed of intricate networks of endothelial and mesenchymal stromal cells. How these lymphoid stromal cells (LSCs regulate lymphoid tissue remodeling and contribute to immune responses remains poorly understood. We performed a comprehensive functional and transcriptional analysis of LSC responses to skin viral infection and found that LSC subsets responded robustly, with different kinetics for distinct pathogens. Recruitment of cells to inflamed LNs induced LSC expansion, while B cells sustained stromal responses in an antigen-independent manner. Infection induced rapid transcriptional responses in LSCs. This transcriptional program was transient, returning to homeostasis within 1 month of infection, yet expanded fibroblastic reticular cell networks persisted for more than 3 months after infection, and this altered LN composition reduced the magnitude of LSC responses to subsequent heterologous infection. Our results reveal the complexity of LSC responses during infection and suggest that amplified networks of LN stromal cells support successive immune responses.

  12. Estrogen/ERα signaling axis participates in osteoblast maturation via upregulating chromosomal and mitochondrial complex gene expressions

    Science.gov (United States)

    Lin, Pei-I; Tai, Yu-Ting; Chan, Wing P.; Lin, Yi-Ling; Liao, Mei-Hsiu; Chen, Ruei-Ming

    2018-01-01

    Estrogen deficiency usually leads to bone loss and osteoporosis in postmenopausal women. Osteoblasts play crucial roles in bone formation. However, osteoblast functions are influenced by mitochondrial bioenergetic conditions. In this study, we investigated the roles of the estrogen and estrogen receptor alpha (ERα) axis in mitochondrial energy metabolism and subsequent osteoblast mineralization. Exposure of rat calvarial osteoblasts to estradiol caused substantial improvements in alkaline phosphatase activities and cell calcification. In parallel, treatment of human osteoblast-like U2OS cells, derived from a female osteosarcoma patient, with estradiol specifically augmented ERα levels. Sequentially, estradiol stimulated translocation of ERα to nuclei in human osteoblasts and induced expressions of genomic respiratory chain complex NDUFA10, UQCRC1, cytochrome c oxidase (COX)8A, COX6A2, COX8C, COX6C, COX6B2, COX412, and ATP12A genes. Concurrently, estradiol stimulated translocation of ERα to mitochondria from the cytoplasm. A bioinformatic search found the existence of four estrogen response elements in the 5’-promoter region of the mitochondrial cox i gene. Interestingly, estradiol induced COX I mRNA and protein expressions in human osteoblasts or rat calvarial osteoblasts. Knocking-down ERα translation concurrently downregulated estradiol-induced COX I mRNA expression. Consequently, exposure to estradiol led to successive increases in the mitochondrial membrane potential, the mitochondrial enzyme activity, and cellular adenosine triphosphate levels. Taken together, this study showed the roles of the estradiol/ERα signaling axis in improving osteoblast maturation through upregulating the mitochondrial bioenergetic system due to induction of definite chromosomal and mitochondrial complex gene expressions. Our results provide novel insights elucidating the roles of the estrogen/ERα alliance in regulating bone formation. PMID:29416685

  13. Regulation of Clock Genes by Adrenergic Receptor Signaling in Osteoblasts.

    Science.gov (United States)

    Hirai, Takao

    2018-01-01

    The clock system has been identified as one of the major mechanisms controlling cellular functions. Circadian clock gene oscillations also actively participate in the functions of various cell types including bone-related cells. Previous studies demonstrated that clock genes were expressed in bone tissue and also that their expression exhibited circadian rhythmicity. Recent findings have shown that sympathetic tone plays a central role in biological oscillations in bone. Adrenergic receptor (AR) signaling regulates the expression of clock genes in cancellous bone. Furthermore, α 1 -AR signaling in osteoblasts is known to negatively regulate the expression of bone morphogenetic protein-4 (Bmp4) by up-regulating nuclear factor IL-3 (Nfil3)/e4 promoter-binding protein 4 (E4BP4). The ablation of α 1B -AR signaling also increases the expression of the Bmp4 gene in bone. The findings of transient overexpression and siRNA experiments have supported the involvement of the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, Cebpd) in Nfil3 and Bmp4 expression in MC3T3-E1 cells. These findings suggest that the effects of Cebpd are due to the circadian regulation of Bmp4 expression, at least in part, by the up-regulated expression of the clock gene Nfil3 in response to α 1B -AR signaling in osteoblasts. Therefore, AR signaling appears to modulate cellular functionality through the expression of clock genes that are circadian rhythm regulators in osteoblasts. The expression of clock genes regulated by the sympathetic nervous system and clock-controlled genes that affect bone metabolism are described herein.

  14. Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

    DEFF Research Database (Denmark)

    Al-Nbaheen, May; Vishnubalaji, Radhakrishnan; Ali, Dalia

    2013-01-01

    , but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin...... (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34(-), CD45(-), CD14(-), CD31(-), HLA-DR(-), CD13(+), CD29......Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow...

  15. Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2 on osteoblast cells.

    Science.gov (United States)

    Son, Hyo-Eun; Kim, Eun-Jung; Jang, Won-Gu

    2018-01-15

    Curcumin (diferuloylmethane or [1E,6E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6heptadiene-3,5-dione) is a phenolic natural product derived from the rhizomes of the turmeric plant, Curcuma longa. It is reported to have various biological actions such as anti-oxidative, anti-inflammatory, and anti-cancer effects. However, the molecular mechanism of osteoblast differentiation by curcumin has not yet been reported. The cytotoxicity of curcumin was identified using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Expression of osteogenic markers and endoplasmic reticulum (ER) stress markers in C3H1-T1/2 cells were measured using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity in C3H10T1/2 cells. Transcriptional activity was detected using a luciferase reporter assay. Curcumin increased the expression of genes such as distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OC), which subsequently induced osteoblast differentiation in C3H10T1/2 cells. In addition, ALP activity and mineralization was found to be increased by curcumin treatment. Curcumin also induced mild ER stress similar to bone morphogenetic protein 2 (BMP2) function in osteoblast cells. Next, we confirmed that curcumin increased mild ER stress and osteoblast differentiation similar to BMP2 in C3H10T1/2 mesenchymal stem cells. Transient transfection studies also showed that curcumin increased ATF6-Luc activity, while decreasing the activities of CREBH-Luc and SMILE-Luc. In addition, similar to BMP2, curcumin induced the phosphorylation of Smad 1/5/9. Overall, these results demonstrate that curcumin-induced mild ER stress increases osteoblast differentiation via ATF6 expression in C3H10T1/2 cells. Copyright © 2017. Published by Elsevier Inc.

  16. Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells.

    Science.gov (United States)

    Kim, Eun-Cheol; Park, Jaesuh; Kwon, Il Keun; Lee, Suk-Won; Park, Su-Jung; Ahn, Su-Jin

    2017-10-01

    Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry. The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated. SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

  17. Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells

    Science.gov (United States)

    Wang, Limin; Ott, Lindsey; Seshareddy, Kiran; Weiss, Mark L; Detamore, Michael S

    2011-01-01

    Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton’s jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering. PMID:21175290

  18. Ebf1-Dependent Control of the Osteoblast and Adipocyte Lineages

    OpenAIRE

    Hesslein, David G. T.; Fretz, Jackie A.; Xi, Yougen; Nelson, Tracy; Zhou, Shoaming; Lorenzo, Joseph A.; Schatz, David G.; Horowitz, Mark C.

    2008-01-01

    Ebf1 is a transcription factor essential for B cell fate specification and function and important for the development of olfactory sensory neurons. We show here that Ebf1 also plays an important role in regulating osteoblast and adipocyte development in vivo. Ebf1 mRNA and protein is expressed in MSCs, in OBs at most stages of differentiation, and in adipocytes. Tibiae and femora from Ebf1−/− mice had a striking increase in all bone formation parameters examined including the number of OBs, o...

  19. Engineering epithelial-stromal interactions in vitro for toxicology assessment.

    Science.gov (United States)

    Belair, David G; Abbott, Barbara D

    2017-05-01

    Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. Published by Elsevier B.V.

  20. Inhibition of mouse osteoblast proliferation and prostaglandin E2 synthesis by Ulmus davidiana Planch (Ulmaceae).

    Science.gov (United States)

    Jin, Un-Ho; Suh, Seok-Jong; Park, Sang-Dong; Kim, Kap-Sung; Kwon, Dae Young; Kim, Cheorl-Ho

    2008-06-01

    Ulmus davidiana Planch (Ulmaceae) (UD) is a widely used Korean herbal medicine that has been used historically in anti-inflammatory and anticancer therapy. Since UD has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions, this study was undertaken to address whether the water extract of the bark of UD could modulate proliferation of mouse osteoblasts in vitro and to investigate its effect on cyclooxygenase-2 (COX-2), which converts arachidonic acid to prostaglandin E2 (PGE2). Mouse osteoblasts were tested in vitro for growth inhibition, proliferating cell nuclear antigen (PCNA) expression, and COX-2 activity and expression after treatment with UD extract. Its effects were compared with those of indomethacin (a nonselective COX inhibitor) and celecoxib (a selective COX-2 inhibitor). UD demonstrated a strong growth inhibition in tested mouse osteoblasts. The IC50s were 10microg/ml for UD, 6microM for celecoxib and 42microM for indomethacin. UD, as well as celecoxib and indomethacin, suppressed PCNA expression and PGE2 synthesis in osteoblasts. UD inhibited COX-2 expression, whereas celecoxib inhibited COX-2 activity directly. UD selectively and effectively inhibits osteoblasts cell growth in vitro. Inhibition of PGE2 synthesis via suppression of COX-2 expression may be responsible for its anti-inflammatory activity.

  1. Effects of Intermittent Administration of Parathyroid Hormone (1-34 on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Wang

    2016-01-01

    Full Text Available Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1 has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+ and STRO-1(− hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+ hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+ hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R than STRO-1(− hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+ hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+ hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+ hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.

  2. Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Lee-Chuan C.; Ford, Jeffery J. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); Lee, John C. [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States); Adamo, Martin L., E-mail: adamo@biochem.uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center at San Antonio, TX (United States); The Sam and Ann Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, TX (United States)

    2014-07-18

    Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.

  3. Effects of fluoridation of porcine hydroxyapatite on osteoblastic activity of human MG63 cells

    Science.gov (United States)

    Li, Zhipeng; Huang, Baoxin; Mai, Sui; Wu, Xiayi; Zhang, Hanqing; Qiao, Wei; Luo, Xin; Chen, Zhuofan

    2015-06-01

    Biological hydroxyapatite, derived from animal bones, is the most widely used bone substitute in orthopedic and dental treatments. Fluorine is the trace element involved in bone remodeling and has been confirmed to promote osteogenesis when administered at the appropriate dose. To take advantage of this knowledge, fluorinated porcine hydroxyapatite (FPHA) incorporating increasing levels of fluoride was derived from cancellous porcine bone through straightforward chemical and thermal treatments. Physiochemical characteristics, including crystalline phases, functional groups and dissolution behavior, were investigated on this novel FPHA. Human osteoblast-like MG63 cells were cultured on the FPHA to examine cell attachment, cytoskeleton, proliferation and osteoblastic differentiation for in vitro cellular evaluation. Results suggest that fluoride ions released from the FPHA play a significant role in stimulating osteoblastic activity in vitro, and appropriate level of fluoridation (1.5 to 3.1 atomic percents of fluorine) for the FPHA could be selected with high potential for use as a bone substitute.

  4. Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway.

    Science.gov (United States)

    Liao, Xiao-bo; Zhou, Xin-min; Li, Jian-ming; Yang, Jin-fu; Tan, Zhi-ping; Hu, Zhuo-wei; Liu, Wei; Lu, Ying; Yuan, Ling-qing

    2008-05-01

    Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free beta-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the beta-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor alpha1 (Cbfalpha1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfalpha1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.

  5. Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

    Directory of Open Access Journals (Sweden)

    Yi-Jen Chen

    2017-11-01

    Full Text Available The role of osteoblasts in peri-articular bone loss and bone erosion in rheumatoid arthritis (RA has gained much attention, and microRNAs are hypothesized to play critical roles in the regulation of osteoblast function in RA. The aim of this study is to explore novel microRNAs differentially expressed in RA osteoblasts and to identify genes potentially involved in the dysregulated bone homeostasis in RA. RNAs were extracted from cultured normal and RA osteoblasts for sequencing. Using the next generation sequencing and bioinformatics approaches, we identified 35 differentially expressed microRNAs and 13 differentially expressed genes with potential microRNA–mRNA interactions in RA osteoblasts. The 13 candidate genes were involved mainly in cell–matrix adhesion, as classified by the Gene Ontology. Two genes of interest identified from RA osteoblasts, A-kinase anchoring protein 12 (AKAP12 and leucin rich repeat containing 15 (LRRC15, were found to express more consistently in the related RA synovial tissue arrays in the Gene Expression Omnibus database, with the predicted interactions with miR-183-5p and miR-146a-5p, respectively. The Ingenuity Pathway Analysis identified AKAP12 as one of the genes involved in protein kinase A signaling and the function of chemotaxis, interconnecting with molecules related to neovascularization. The findings indicate new candidate genes as the potential indicators in evaluating therapies targeting chemotaxis and neovascularization to control joint destruction in RA.

  6. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics

    Directory of Open Access Journals (Sweden)

    Mansur Halai

    2014-09-01

    Full Text Available In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow–derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow–derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  7. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics.

    Science.gov (United States)

    Halai, Mansur; Ker, Andrew; Meek, Rm Dominic; Nadeem, Danish; Sjostrom, Terje; Su, Bo; McNamara, Laura E; Dalby, Matthew J; Young, Peter S

    2014-01-01

    In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow-derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow-derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  8. Effect of nanocoating with rhamnogalacturonan-I on surface properties and osteoblasts response

    DEFF Research Database (Denmark)

    Gurzawska, Katarzyna Aleksandra; Svava, Rikke; Syberg, Susanne

    2012-01-01

    -I) on surface properties and osteoblasts response. Three different RG-Is from apple and lupin pectins were modified and coated on amino-functionalized tissue culture polystyrene plates (aminated TCPS). Surface properties were evaluated by scanning electron microscopy, contact angle measurement, atomic force...

  9. Gastrointestinal Stromal Tumors: A Case Report

    Directory of Open Access Journals (Sweden)

    Palankezhe Sashidharan

    2014-03-01

    Full Text Available Advances in the identification of gastrointestinal stromal tumors, its molecular and immunohiostochemical basis, and its management have been a watershed in the treatment of gastrointestinal tumors. This paradigm shift occurred over the last two decades and gastrointestinal stromal tumors have now come to be understood as rare gastrointestinal tract tumors with predictable behavior and outcome, replacing the older terminologies like leiomyoma, schwannoma or leiomyosarcoma. This report presents a case of gastric gastrointestinal stromal tumor operated recently in a 47-year-old female patient and the outcome, as well as literature review of the pathological identification, sites of origin, and factors predicting its behavior, prognosis and treatment.

  10. Towards an intraoperative engineering of osteogenic and vasculogenic grafts from the stromal vascular fraction of human adipose tissue

    Directory of Open Access Journals (Sweden)

    AM Müller

    2010-03-01

    Full Text Available Grafts generated by cultivation of progenitor cells from the stromal vascular fraction of human adipose tissue have been proven to have osteogenic and vasculogenic properties in vivo. However, in vitro manufacture of such implants is challenged by complex, impractical and expensive processes, and requires implantation in a separate surgery. This study investigates the feasibility of an intraoperative approach to engineer cell-based bone grafts with tissue harvest, cell isolation, cell seeding onto a scaffold and subsequent implantation within a few hours. Freshly isolated adipose tissue cells from a total of 11 donors, containing variable fractions of mesenchymal and endothelial progenitors, were embedded at different densities in a fibrin hydrogel, which was wrapped around bone substitute materials based on beta-tricalcium phosphate (ChronOS®, hydroxyapatite (Engipore®, or acellular xenograft (Bio-Oss®. The resulting constructs, generated within 3 hours from biopsy harvest, were immediately implanted ectopically in nude mice and analysed after eight weeks. All explants contained blood vessels formed by human endothelial cells, functionally connected to the recipient’s vasculature. Human origin cells were also found within osteoid structures, positively immunostained for bone sialoprotein and osteocalcin. However, even with the highest loaded cell densities, no frank bone tissue was detected, independently of the material used. These results provide a proof-of-principle that an intraoperative engineering of autologous cell-based vasculogenic bone substitutes is feasible, but highlight that – in the absence of in vitro commitment – additional cues (e.g., low dose of osteogenic factors or orthotopic environmental conditions are likely needed to support complete osteoblastic cell differentiation and bone tissue generation.

  11. Effects of microgravity on osteoblast growth activation

    Science.gov (United States)

    Hughes-Fulford, M.; Lewis, M. L.

    1996-01-01

    Space flight is an environmental condition where astronauts can lose up to 19% of weight-bearing bone during long duration missions. We used the MC3T3-E1 osteoblast to investigate bone cell growth in microgravity (10(-6) to 10(-9)g). Osteoblasts were launched on the STS-56 shuttle flight in a quiescent state with 0.5% fetal calf serum (FCS) medium and growth activation was initiated by adding fresh medium with 10% FCS during microgravity exposure. Four days after serum activation, the cells were fixed before return to normal Earth gravity. Ground controls were treated in parallel with the flight samples in identical equipment. On landing, cell number, cell cytoskeleton, glucose utilization, and prostaglandin synthesis in flight (n = 4) and ground controls (n = 4) were examined. The flown osteoblasts grew slowly in microgravity with total cell number significantly reduced (55 +/- 6 vs 141 +/- 8 cells per microscopic field). The cytoskeleton of the flight osteoblasts had a reduced number of stress fibers and a unique abnormal morphology. Nuclei in the ground controls were large and round with punctate Hoechst staining of the DNA nucleosomes. The flight nuclei were 30% smaller than the controls (P prostaglandin E2 (PGE2) synthesis when compared to controls (57.3 +/- 17 vs 138.3 +/- 41 pmol/ml). Cell viability was normal since, on a per-cell basis, glucose use and prostaglandin synthesis were comparable for flight and ground samples. Taken together, these data suggest that growth activation in microgravity results in reduced growth, causing reduced glucose utilization and reduced prostaglandin synthesis, with significantly altered actin cytoskeleton in osteoblasts.

  12. Cord blood-derived macrophage-lineage cells rapidly stimulate osteoblastic maturation in mesenchymal stem cells in a glycoprotein-130 dependent manner.

    Directory of Open Access Journals (Sweden)

    Tania J Fernandes

    Full Text Available In bone, depletion of osteoclasts reduces bone formation in vivo, as does osteal macrophage depletion. How osteoclasts and macrophages promote the action of bone forming osteoblasts is, however, unclear. Since recruitment and differentiation of multi-potential stromal cells/mesenchymal stem cells (MSC generates new active osteoblasts, we investigated whether human osteoclasts and macrophages (generated from cord blood-derived hematopoietic progenitors induce osteoblastic maturation in adipose tissue-derived MSC. When treated with an osteogenic stimulus (ascorbate, dexamethasone and β-glycerophosphate these MSC form matrix-mineralising, alkaline phosphatase-expressing osteoblastic cells. Cord blood-derived progenitors were treated with macrophage colony stimulating factor (M-CSF to form immature proliferating macrophages, or with M-CSF plus receptor activator of NFκB ligand (RANKL to form osteoclasts; culture medium was conditioned for 3 days by these cells to study their production of osteoblastic factors. Both osteoclast- and macrophage-conditioned medium (CM greatly enhanced MSC osteoblastic differentiation in both the presence and absence of osteogenic medium, evident by increased alkaline phosphatase levels within 4 days and increased mineralisation within 14 days. These CM effects were completely ablated by antibodies blocking gp130 or oncostatin M (OSM, and OSM was detectable in both CM. Recombinant OSM very potently stimulated osteoblastic maturation of these MSC and enhanced bone morphogenetic protein-2 (BMP-2 actions on MSC. To determine the influence of macrophage activation on this OSM-dependent activity, CM was collected from macrophage populations treated with M-CSF plus IL-4 (to induce alternative activation or with GM-CSF, IFNγ and LPS to cause classical activation. CM from IL-4 treated macrophages stimulated osteoblastic maturation in MSC, while CM from classically-activated macrophages did not. Thus, macrophage-lineage cells

  13. Novel actions of bisphosphonates in bone: Preservation of osteoblast and osteocyte viability

    Science.gov (United States)

    Bellido, Teresita; Plotkin, Lilian I.

    2010-01-01

    Bisphosphonates stop bone loss by inhibiting the activity of bone resorbing osteoclasts. However, the effect of bisphosphonates on bone mass cannot completely explain the reduction in fracture incidence observed in patients treated with these agents. Recent research efforts provided an explanation to this dichotomy by demonstrating that part of the beneficial effect of bisphosphonates on the skeleton is due to prevention of osteoblast and osteocyte apoptosis. Work of our group, independently confirmed by other investigators, demonstrated that bisphosphonates are able to prevent osteoblast and osteocyte apoptosis in vitro and in vivo. This pro-survival effect is strictly dependent on the expression of connexin (Cx)43, as demonstrated in vitro using cells lacking Cx43 or expressing dominant negative mutants of the protein as well as in vivo using Cx43 osteoblast/osteocyte-specific conditional knock-out mice. Remarkably, this Cx43-dependent survival effect of bisphosphonates is independent of gap junctions and results from opening of Cx43 hemichannels. Hemichannel opening leads to activation of the kinases Src and extracellular signal-regulated kinases (ERKs), followed by phosphorylation of the ERK cytoplasmic target p90RSK kinase and its substrates BAD and C/EBPβ, resulting in inhibition of apoptosis. The anti-apoptotic effect of bisphosphonates is separate from the effect of the drugs on osteoclasts, as analogs that lack anti-resorptive activity are still able to inhibit osteoblast and osteocyte apoptosis in vitro. Furthermore, a bisphosphonate analog that does not inhibit osteoclast activity prevented osteoblast and osteocyte apoptosis and the loss of bone mass and strength induced by glucocorticoids in mice. Preservation of the bone forming function of mature osteoblasts and maintenance of the osteocytic network, in combination with lack anti-catabolic actions, open new therapeutic possibilities for bisphosphonates in the treatment of osteopenic conditions in

  14. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  15. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Science.gov (United States)

    Pellegrini, Gretel G.; Morales, Cynthya C.; Wallace, Taylor C.; Plotkin, Lilian I.; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  16. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  17. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  18. Are mesenchymal stromal cells immune cells?

    NARCIS (Netherlands)

    M.J. Hoogduijn (Martin)

    2015-01-01

    textabstractMesenchymal stromal cells (MSCs) are considered to be promising agents for the treatment of immunological disease. Although originally identified as precursor cells for mesenchymal lineages, in vitro studies have demonstrated that MSCs possess diverse immune regulatory capacities.

  19. Drugs Approved for Gastrointestinal Stromal Tumors

    Science.gov (United States)

    This page lists cancer drugs approved by the Food and Drug Administration (FDA) for gastrointestinal stromal tumors (GIST). The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

  20. Linarin isolated from Buddleja officinalis prevents hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kim, Young Ho; Lee, Young Soon; Choi, Eun Mi

    2011-01-01

    The flowers and leaves buds of Buddleja officinalis MAXIM (Buddlejaceae) are used to treat eye troubles, hernia, gonorrhea and liver troubles in Asia. To elucidate the protective effects of linarin isolated from B. officinalis on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were pre-incubated with linarin for 1h before treatment with 0.3mM H(2)O(2) for 48h, and markers of osteoblast function and oxidative damage were examined. Linarin significantly (P<0.05) increased cell survival, alkaline phosphatase (ALP) activity, collagen content, calcium deposition, and osteocalcin secretion and decreased the production of receptor activator of nuclear factor-kB ligand (RANKL), protein carbonyl (PCO), and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells in the presence of hydrogen peroxide. These results demonstrate that linarin can protect osteoblasts against hydrogen peroxide-induced osteoblastic dysfunction and may exert anti-resorptive actions, at least in part, via the reduction of RANKL and oxidative damage. 2011 Elsevier Inc. All rights reserved.

  1. Gut Mesenchymal Stromal Cells in Immunity

    Directory of Open Access Journals (Sweden)

    Valeria Messina

    2017-01-01

    Full Text Available Mesenchymal stromal cells (MSCs, first found in bone marrow (BM, are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal or interspersed within intestinal submucosa (intercryptal. In Crohn’s disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC. The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn’s disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer.

  2. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    International Nuclear Information System (INIS)

    Balduino, Alex; Mello-Coelho, Valeria; Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H.; Weeraratna, Ashani T.; Becker, Kevin G.; Mello, Wallace de; Taub, Dennis D.; Borojevic, Radovan

    2012-01-01

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the “quiescent” and “proliferative” niches in which hematopoietic stem cells and progenitors reside.

  3. Molecular signature and in vivo behavior of bone marrow endosteal and subendosteal stromal cell populations and their relevance to hematopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Balduino, Alex, E-mail: balduino@uva.edu.br [School of Dentistry, Veiga de Almeida University, Rio de Janeiro, RJ (Brazil); Mello-Coelho, Valeria [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil); National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H. [Department of Periodontics, Prevention and Geriatrics, University of Michigan School of Dentistry, Ann Arbor, MI (United States); Weeraratna, Ashani T.; Becker, Kevin G. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Mello, Wallace de [Instituto Oswaldo Cruz, Rio de Janeiro, RJ (Brazil); Taub, Dennis D. [National Institute on Aging, National Institute of Health, Baltimore, MD (United States); Borojevic, Radovan [Biomedical Science Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2012-11-15

    In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the 'quiescent' and 'proliferative' niches in which hematopoietic stem cells and progenitors reside.

  4. Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Chi Zhang

    Full Text Available Osterix (Osx is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  5. Synergistic effects of 1,25-Dihydroxyvitamin D3 and TGF-beta1 on the production of insulin-like growth factor binding protein 3 in human bone marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Flyvbjerg, Allan; Kassem, M

    2002-01-01

    1,25-Dihydroxyvitamin D3 (calcitriol), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGFs) are all important bone regulatory factors known to affect proliferation and differentiation of human bone-forming cells (osteoblasts). We have previously shown that TGF-beta1...... increased IGF-I and IGF-binding protein (IGFBP)-3 production in human bone marrow stromal (hMS) osteoblast progenitors and calcitriol stimulated IGFBP-3 and IGFBP-4 production. As interaction between signaling pathways of these factors has been reported, the present study aimed at examining the concerted...... actions on components of the IGF-system. We report that co-treatment with TGF-beta1 and calcitriol resulted in a synergistic increase in IGFBP-3 production, thereby suggesting that the effects of these factors on hMS osteoblast differentiation may involve the observed increase in IGFBP-3....

  6. The Role of MKP-1 in the Anti-Proliferative Effects of Glucocorticoids in Primary Rat Pre-Osteoblasts.

    Directory of Open Access Journals (Sweden)

    Micheline Sanderson

    Full Text Available Glucocorticoid (GC-induced osteoporosis has been attributed to a GC-induced suppression of pre-osteoblast proliferation. Our previous work identified a critical role for mitogen-activated protein kinase (MAPK phosphatase-1 (MKP-1 in mediating the anti-proliferative effects of GCs in immortalized pre-osteoblasts, but we subsequently found that MKP-1 null mice were not protected against the pathological effects of GCs on bone. In order to reconcile this discrepancy, we have assessed the effects of GCs on proliferation, activation of the MAPK ERK1/2 and MKP-1 expression in primary adipose-derived stromal cells (ADSCs and ADSC-derived pre-osteoblasts (ADSC-OBs. ADSCs were isolated by means of collagenase digestion from adipose tissue biopsies harvested from adult male Wistar rats. ADSC-OBs were prepared by treating ADSCs with osteoblast differentiation media for 7 days. The effects of increasing concentrations of the GC dexamethasone on basal and mitogen-stimulated cell proliferation were quantified by tritiated thymidine incorporation. ERK1/2 activity was measured by Western blotting, while MKP-1 expression was quantified on both RNA and protein levels, using semi-quantitative real-time PCR and Western blotting, respectively. GCs were strongly anti-proliferative in both naïve ADSCs and ADSC-OBs, but had very little effect on mitogen-induced ERK1/2 activation and did not upregulate MKP-1 protein expression. These findings suggest that the anti-proliferative effects of GCs in primary ADSCs and ADSC-OBs in vitro do not require the inhibition of ERK1/2 activation by MKP-1, which is consistent with our in vivo findings in MKP-1 null mice.

  7. Human osteoblasts within soft peptide hydrogels promote mineralisation in vitro

    Directory of Open Access Journals (Sweden)

    Luis A Castillo Diaz

    2014-07-01

    Full Text Available Biomaterials that provide three-dimensional support networks for the culture of cells are being developed for a wide range of tissue engineering applications including the regeneration of bone. This study explores the potential of the versatile ionic-complementary peptide, FEFEFKFK, for such a purpose as this peptide spontaneously self-assembles into β-sheet-rich fibres that subsequently self-associate to form self-supporting hydrogels. Via simple live/dead cell assays, we demonstrated that 3 wt% hydrogels were optimal for the support of osteoblast cells. We went on to show that these cells are not only viable within the three-dimensional hydrogel but they also proliferate and produce osteogenic key proteins, that is, they behave like in vivo bone cells, over the 14-day period explored here. The gel elasticity increased over time when cells were present – in comparison to a decrease in control samples – indicating the deposition of matrix throughout the peptide scaffold. Moreover, significant quantities of calcium phosphate were deposited. Collectively, these data demonstrate that ionic-complementary octapeptides offer a suitable three-dimensional environment for osteoblastic cell function.

  8. Ebf1-dependent control of the osteoblast and adipocyte lineages.

    Science.gov (United States)

    Hesslein, David G T; Fretz, Jackie A; Xi, Yougen; Nelson, Tracy; Zhou, Shoaming; Lorenzo, Joseph A; Schatz, David G; Horowitz, Mark C

    2009-04-01

    Ebf1 is a transcription factor essential for B cell fate specification and function and important for the development of olfactory sensory neurons. We show here that Ebf1 also plays an important role in regulating osteoblast and adipocyte development in vivo. Ebf1 mRNA and protein is expressed in MSCs, in OBs at most stages of differentiation, and in adipocytes. Tibiae and femora from Ebf1(-/-) mice had a striking increase in all bone formation parameters examined including the number of OBs, osteoid volume, and bone formation rate. Serum osteocalcin, a marker of bone formation, was significantly elevated in mutant mice. The numbers of osteoclasts in bone were normal in younger (4 week-old) Ebf1(-/-) mice but increased in older (12 week-old) Ebf1(-/-) mice. This correlated well with in vitro osteoclast development from bone marrow cells. In addition to the increased osteoblastogenesis, there was a dramatic increase in adipocyte numbers in the bone marrow of Ebf1(-/-) mice. Increased adiposity was also seen histologically in the liver but not in the spleen of these mice, and accompanied by decreased deposition of adipose to subcutaneous sites. Thus Ebf1-deficient mice appear to be a new model of lipodystrophy. Ebf1 is a rare example of a transcription factor that regulates both the osteoblast and adipocyte lineages similarly.

  9. Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering.

    Science.gov (United States)

    Sethu, Sai Nievethitha; Namashivayam, Subhapradha; Devendran, Saravanan; Nagarajan, Selvamurugan; Tsai, Wei-Bor; Narashiman, Srinivasan; Ramachandran, Murugesan; Ambigapathi, Moorthi

    2017-05-01

    Bone, a highly dynamic connective tissue, consist of a bioorganic phase comprising osteogenic cells and proteins which lies over an inorganic phase predominantly made of CaPO 4 (biological apatite). Injury to bone can be due to mechanical, metabolic or inflammatory agents also owing pathological conditions like fractures, osteomyelitis, osteolysis or cysts may arise in enameloid, chondroid, cementum, or chondroid bone which forms the intermediate tissues of the body. Bone tissue engineering (BTE) applies bioactive scaffolds, host cells and osteogenic signals for restoring damaged or diseased tissues. Various bioceramics used in BTE can be bioactive (like glass ceramics and hydroxyapatite bioactive glass), bioresorbable (like tricalcium phosphates) or bioinert (like zirconia and alumina). Limiting the size of these materials to nano-scale has resulted in a higher surface area to volume ratio thereby improving multi-functionality, solubility, surface catalytic activity, high heat and electrical conductivity. Nanoceramics have been found to induce osteoconduction, osteointegration, osteogenesis and osteoinduction. The present review aims at summarizing the interactions of nanoceramics and osteoblast/stem cells for promoting the proliferation and differentiation of the osteoblast cells by nanoceramics as superior bone substitutes in bone tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Blockade of Drp1 rescues oxidative stress-induced osteoblast dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Gan, Xueqi; Huang, Shengbin; Yu, Qing [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States); State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yu, Haiyang [State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041 (China); Yan, Shirley ShiDu, E-mail: shidu@ku.edu [Department of Pharmacology and Toxicology and Higuchi Bioscience Center, University of Kansas, Lawrence, KS, 66047 (United States)

    2015-12-25

    Osteoblast dysfunction, induced by oxidative stress, plays a critical role in the pathophysiology of osteoporosis. However, the underlying mechanisms remain unclarified. Imbalance of mitochondrial dynamics has been closely linked to oxidative stress. Here, we reveal an unexplored role of dynamic related protein 1(Drp1), the major regulator in mitochondrial fission, in the oxidative stress-induced osteoblast injury model. We demonstrate that levels of phosphorylation and expression of Drp1 significantly increased under oxidative stress. Blockade of Drp1, through pharmaceutical inhibitor or gene knockdown, significantly protected against H{sub 2}O{sub 2}-induced osteoblast dysfunction, as shown by increased cell viability, improved cellular alkaline phosphatase (ALP) activity and mineralization and restored mitochondrial function. The protective effects of blocking Drp1 in H{sub 2}O{sub 2}-induced osteoblast dysfunction were evidenced by increased mitochondrial function and suppressed production of reactive oxygen species (ROS). These findings provide new insights into the role of the Drp1-dependent mitochondrial pathway in the pathology of osteoporosis, indicating that the Drp1 pathway may be targetable for the development of new therapeutic approaches in the prevention and the treatment of osteoporosis. - Highlights: • Oxidative stress is an early pathological event in osteoporosis. • Imbalance of mitochondrial dynamics are linked to oxidative stress in osteoporosis. • The role of the Drp1-dependent mitochondrial pathway in osteoporosis.

  11. CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Al-Shammary, Asma; Skagen, Peter

    2015-01-01

    Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and their progenitors have been identified based on retrospective functional criteria. CD markers are employed to define cell populations with distinct functional characteristics. However, defining and pro...

  12. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Science.gov (United States)

    Rosu-Myles, Michael; She, Yi-Min; Fair, Joel; Muradia, Gauri; Mehic, Jelica; Menendez, Pablo; Prasad, Shiv S; Cyr, Terry D

    2012-01-01

    Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  13. Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells.

    Directory of Open Access Journals (Sweden)

    Michael Rosu-Myles

    Full Text Available Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC (CD105+ and non-multipotent (CD105- stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

  14. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    OpenAIRE

    Jingru Meng; Xue Ma; Ning Wang; Min Jia; Long Bi; Yunying Wang; Mingkai Li; Huinan Zhang; Xiaoyan Xue; Zheng Hou; Ying Zhou; Zhibin Yu; Gonghao He; Xiaoxing Luo

    2016-01-01

    Summary Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These function...

  15. Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

    International Nuclear Information System (INIS)

    Yang Chiming; Frei, Hanspeter; Burt, Helen M.; Rossi, Fabio

    2009-01-01

    Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.

  16. Ultrastructural and radiobiological characterization of stromal cells in continuous, long-term marrow culture

    International Nuclear Information System (INIS)

    Tavassoli, M.

    1982-01-01

    Hemopoietic stromal cells were studied in continuous, long-term marrow culture. A correlative study was carried out involving cytochemistry as well as scanning (SEM), and transmission electron microscopy (TEM) with sections cut either perpendicular or parallel to the substratum. Only two stromal cell types were identified: epithelioid cells and macrophages. The appearance of these cells, however, varied according to their topography in the culture and the method of observation; a finding that may explain the multiplicity of the cell types reported in these cultures. The two cell types displayed considerable interconnections and interactions which may be essential in their support function for the proliferation and maintenance of hemopoietic stem cells. They also demonstrated numerous coated pits and vesicles suggestive of extensive receptor-mediated endocytosis. Stromal cells, generally thought to be relatively radioresistant, demonstrated hitherto unrecognized radiosensitivity in culture. Doses of radiation as low as 500 rads interfered with their support function for the maintenance of the hemopoietic stem cell

  17. AKI Recovery Induced by Mesenchymal Stromal Cell-Derived Extracellular Vesicles Carrying MicroRNAs.

    Science.gov (United States)

    Collino, Federica; Bruno, Stefania; Incarnato, Danny; Dettori, Daniela; Neri, Francesco; Provero, Paolo; Pomatto, Margherita; Oliviero, Salvatore; Tetta, Ciro; Quesenberry, Peter J; Camussi, Giovanni

    2015-10-01

    Phenotypic changes induced by extracellular vesicles have been implicated in mesenchymal stromal cell-promoted recovery of AKI. MicroRNAs are potential candidates for cell reprogramming toward a proregenerative phenotype. The aim of this study was to evaluate whether microRNA deregulation inhibits the regenerative potential of mesenchymal stromal cells and derived extracellular vesicles in a model of glycerol-induced AKI in severe combined immunodeficient mice. We generated mesenchymal stromal cells depleted of Drosha to alter microRNA expression. Drosha-knockdown cells produced extracellular vesicles that did not differ from those of wild-type cells in quantity, surface molecule expression, and internalization within renal tubular epithelial cells. However, these vesicles showed global downregulation of microRNAs. Whereas wild-type mesenchymal stromal cells and derived vesicles administered intravenously induced morphologic and functional recovery in AKI, the Drosha-knockdown counterparts were ineffective. RNA sequencing analysis showed that kidney genes deregulated after injury were restored by treatment with mesenchymal stromal cells and derived vesicles but not with Drosha-knockdown cells and vesicles. Gene ontology analysis showed in AKI an association of downregulated genes with fatty acid metabolism and upregulated genes with inflammation, matrix-receptor interaction, and cell adhesion molecules. These alterations reverted after treatment with wild-type mesenchymal stromal cells and extracellular vesicles but not after treatment with the Drosha-knockdown counterparts. In conclusion, microRNA depletion in mesenchymal stromal cells and extracellular vesicles significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of microRNAs in recovery after AKI. Copyright © 2015 by the American Society of Nephrology.

  18. Staphylococcus aureus vs. Osteoblast: Relationship and Consequences in Osteomyelitis

    Science.gov (United States)

    Josse, Jérôme; Velard, Frédéric; Gangloff, Sophie C.

    2015-01-01

    Bone cells, namely osteoblasts and osteoclasts work in concert and are responsible for bone extracellular matrix formation and resorption. This homeostasis is, in part, altered during infections by Staphylococcus aureus through the induction of various responses from the osteoblasts. This includes the over-production of chemokines, cytokines and growth factors, thus suggesting a role for these cells in both innate and adaptive immunity. S. aureus decreases the activity and viability of osteoblasts, by induction of apoptosis-dependent and independent mechanisms. The tight relationship between osteoclasts and osteoblasts is also modulated by S. aureus infection. The present review provides a survey of the relevant literature discussing the important aspects of S. aureus and osteoblast interaction as well as the ability for antimicrobial peptides to kill intra-osteoblastic S. aureus, hence emphasizing the necessity for new anti-infectious therapeutics. PMID:26636047

  19. Ghrelin and gastrointestinal stromal tumors.

    Science.gov (United States)

    Zhu, Chang-Zhen; Liu, Dong; Kang, Wei-Ming; Yu, Jian-Chun; Ma, Zhi-Qiang; Ye, Xin; Li, Kang

    2017-03-14

    Ghrelin, as a kind of multifunctional protein polypeptide, is mainly produced in the fundus of the stomach and can promote occurrence and development of many tumors, including gastrointestinal tumors, which has been proved by the relevant researches. Most gastrointestinal stromal tumors (GISTs, about 80%), as the most common mesenchymal tumor, also develop in the fundus. Scientific research has confirmed that ghrelin, its receptors and mRNA respectively can be found in GISTs, which demonstrated the existence of a ghrelin autocrine/paracrine loop in GIST tissues. However, no reports to date have specified the mechanism whether ghrelin can promote the occurrence and development of GISTs. Studies of pulmonary artery endothelial cells in a low-oxygen environment and cardiac muscle cells in an ischemic environment have shown that ghrelin can activate the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. Moreover, some studies of GISTs have confirmed that activation of the PI3K/AKT/mTOR pathway can indeed promote the growth and progression of GISTs. Whether ghrelin is involved in the development or progression of GISTs through certain pathways remains unknown. Can we find a new target for the treatment of GISTs? This review explores and summaries the relationship among ghrelin, the PI3K/AKT/mTOR pathway and the development of GISTs.

  20. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

    Science.gov (United States)

    Bouderlique, Thibault; Henault, Emilie; Lebouvier, Angelique; Frescaline, Guilhem; Bierling, Phillipe; Rouard, Helene; Courty, José; Albanese, Patricia; Chevallier, Nathalie

    2014-01-01

    Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  1. Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Thibault Bouderlique

    Full Text Available Pleiotrophin (PTN is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

  2. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  3. Fate of bone marrow mesenchymal stromal cells following autologous transplantation in a rabbit model of osteonecrosis.

    Science.gov (United States)

    Sugaya, Hisashi; Mishima, Hajime; Gao, Ran; Kaul, Sunil C; Wadhwa, Renu; Aoto, Katsuya; Li, Meihua; Yoshioka, Tomokazu; Ogawa, Takeshi; Ochiai, Naoyuki; Yamazaki, Masashi

    2016-02-01

    Internalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. The i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. The i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. These results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Autologous transplants of Adipose-Derived Adult Stromal (ADAS) afford dopaminergic neuroprotection in a model of Parkinson’s disease

    OpenAIRE

    McCoy, Melissa K.; Martinez, Terina N.; Ruhn, Kelly A.; Wrage, Philip C.; Keefer, Edward W.; Botterman, Barry R.; Tansey, Keith E.; Tansey, Malú G.

    2007-01-01

    Adult adipose contains stromal progenitor cells with neurogenic potential. However, the stability of neuronal phenotypes adopted by Adipose-Derived Adult Stromal (ADAS) cells and whether terminal neuronal differentiation is required for their consideration as alternatives in cell replacement strategies to treat neurological disorders is largely unknown. We investigated whether in vitro neural induction of ADAS cells determined their ability to neuroprotect or restore function in a lesioned do...

  5. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  6. gp130 in late osteoblasts and osteocytes is required for PTH-induced osteoblast differentiation.

    Science.gov (United States)

    Standal, Therese; Johnson, Rachelle W; McGregor, Narelle E; Poulton, Ingrid J; Ho, Patricia W M; Martin, T John; Sims, Natalie A

    2014-11-01

    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.gp130(f/f)) and littermate controls (Dmp1Cre.gp130(w/w)) were treated with hPTH(1-34) (30 μg/kg 5× per week for 5 weeks). PTH dramatically increased bone formation in Dmp1Cre.gp130(w/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.gp130(f/f) femora compared with Dmp1Cre.gp130(w/w). Furthermore, lentiviral-Cre infection of gp130(f/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. © 2014 Society for Endocrinology.

  7. Osteoblast-like properties of human periodontal ligament cells: an in vitro analysis.

    Science.gov (United States)

    Basdra, E K; Komposch, G

    1997-12-01

    Identifying the biological properties of the cells residing within the periodontal ligament (PDL) will help in understanding the role that these cells play in the various functions of the periodontal ligament, and will improve the success of clinical procedures such as orthodontic tooth movement. For this purpose, fibroblasts isolated from human periodontium were cultured and characterized both histochemically and biochemically with respect to their putative osteoblast-like properties. Histochemically, cultured PDL fibroblasts showed an intense staining for alkaline phosphatase (ALP). Biochemically, the basal ALP activity increased in culture over time. ALP levels after stimulation with 1 alpha, 25-dihydroxyvitamin D3 were significantly higher than those of control cultures. Moreover, immunofluorescence against osteocalcin (a highly reliable osteoblastic marker) was strongly positive. Von Kossa staining of the cell cultures revealed the formation of mineral-like nodules. These results indicate that human PDL fibroblasts exhibit in vitro phenotypic characteristics consistent with osteoblast-like cells, thus suggesting that such cells have the potential to differentiate into osteoblasts and/or cementoblasts.

  8. The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.

    Directory of Open Access Journals (Sweden)

    Marie Smith

    Full Text Available The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content and differentiation (alkaline phosphatase (ALP, collagen and osteocalcin secretion and calcium deposition were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

  9. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    Science.gov (United States)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  10. Mesenchymal Stromal Cells: Updates and Therapeutic Outlook in Rheumatic Diseases

    Directory of Open Access Journals (Sweden)

    Christian Jorgensen

    2013-10-01

    Full Text Available Multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs are adult stem cells exhibiting functional properties that have opened the way for cell-based clinical therapies. MSCs have been reported to exhibit immunosuppressive as well as healing properties, improving angiogenesis and preventing apoptosis or fibrosis through the secretion of paracrine mediators. This review summarizes recent progress on the clinical application of stem cells therapy in some inflammatory and degenerative rheumatic diseases. To date, most of the available data have been obtained in preclinical models and clinical efficacy needs to be evaluated through controlled randomized double-blind trials.

  11. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    Science.gov (United States)

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

  12. The effect of a new direct Factor Xa inhibitor on human osteoblasts: an in-vitro study comparing the effect of rivaroxaban with enoxaparin

    LENUS (Irish Health Repository)

    Solayar, Gandhi N

    2011-10-28

    Abstract Background Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto™ (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast viability, function and gene expression compared to enoxaparin, a commonly used LMWH. Methods Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg\\/ml) or enoxaparin (1, 10 and 100 μg\\/ml). The effect of each drug on osteoblast function was evaluated by measuring alkaline phosphatase activity. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast viability. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusions These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function.

  13. Priming the Surface of Orthopedic Implants for Osteoblast Attachment in Bone Tissue Engineering.

    Science.gov (United States)

    Chan, Kiat Hwa; Zhuo, Shuangmu; Ni, Ming

    2015-01-01

    The development of better orthopedic implants is incessant. While current implants can function reliably in the human body for a long period of time, there are still a significant number of cases for which the implants can fail prematurely due to poor osseointegration of the implant with native bone. Increasingly, it is recognized that it is extremely important to facilitate the attachment of osteoblasts on the implant so that a proper foundation of extracellular matrix (ECM) can be laid down for the growth of new bone tissue. In order to facilitate the osseointegration of the implant, both the physical nanotopography and chemical functionalization of the implant surface have to be optimized. In this short review, however, we explore how simple chemistry procedures can be used to functionalize the surfaces of three major classes of orthopedic implants, i.e. ceramics, metals, and polymers, so that the attachment of osteoblasts on implants can be facilitated in order to promote implant osseointegration.

  14. Bilateral maxillary sinus floor augmentation with tissue-engineered autologous osteoblasts and demineralized freeze-dried bone

    Directory of Open Access Journals (Sweden)

    Aashish Deshmukh

    2015-01-01

    Full Text Available The pneumatization of the maxillary sinus often results in a lack of sufficient alveolar bone for implant placement. In the last decades, maxillary sinus lift has become a very popular procedure with predictable results. Sinus floor augmentation procedures are generally carried out using autologous bone grafts, bone substitutes, or composites of bone and bone substitutes. However, the inherent limitations associated with each of these, have directed the attention of investigators to new technologies like bone tissue engineering. Bone marrow stromal cells have been regarded as multi-potent cells residing in bone marrow. These cells can be harvested from a person, multiplied outside his body using bioengineering principles and technologies and later introduced into a tissue defect. We present a case where tissue-engineered autologous osteoblasts were used along with demineralized freeze-dried bone for sinus floor augmentation.

  15. Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Ziulkoski, Ana L. [Programa de Pos-Graduacao em Ciencias Biologicas: Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Departamento de Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Instituto de Ciencias da Saude, Centro Universitario Feevale, Novo Hamburgo, RS (Brazil); Santos, Aline X.S. dos; Andrade, Claudia M.B.; Trindade, Vera M.T. [Programa de Pos-Graduacao em Ciencias Biologicas: Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Departamento de Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Daniotti, Jose Luis [Departamento de Quimica Biologica, Faculdad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba (Argentina); Borojevic, Radovan [Departamento de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro (Brazil); Guma, Fatima C.R., E-mail: fatima.guma@ufrgs.br [Programa de Pos-Graduacao em Ciencias Biologicas: Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Departamento de Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)

    2009-10-09

    Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cells in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.

  16. Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling

    Directory of Open Access Journals (Sweden)

    Wei Yu

    2016-12-01

    Full Text Available High dose glucocorticoid (GC administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10−7 M dexamethasone (Dex, Y1 receptor shRNA interference, Y1 receptor agonist [Leu31, Pro34]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8 assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.

  17. Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

    Science.gov (United States)

    Yu, Kai-Wen; Yao, Chung-Chen; Jeng, Jiiang-Huei; Shieh, Hao-Ying; Chen, Yi-Jane

    2018-04-01

    Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells. Copyright © 2017. Published by Elsevier B.V.

  18. Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces

    Directory of Open Access Journals (Sweden)

    George E Aninwene II

    2008-06-01

    Full Text Available George E Aninwene II1, Chang Yao2, Thomas J Webster21Department of Biochemical Engineering, University of Maryland, Baltimore, MD; 2Division of Engineering, Brown University, Providence, RI, USAAbstract: Current orthopedic implants have functional lifetimes of only 10–15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone. To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin and inflammation (dexamethasone using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF. Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.Keywords: anodization, titanium, adhesion, simulated body fluid, nanotubes

  19. Enhanced and suppressed mineralization by acetoacetate and β-hydroxybutyrate in osteoblast cultures.

    Science.gov (United States)

    Saito, Akihiro; Yoshimura, Kentaro; Miyamoto, Yoichi; Kaneko, Kotaro; Chikazu, Daichi; Yamamoto, Matsuo; Kamijo, Ryutaro

    2016-04-29

    It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and β-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and β-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, β-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by β-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or β-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

    Science.gov (United States)

    Nakatsu, Yoshihiro; Nakagawa, Fumio; Higashi, Sen; Ohsumi, Tomoko; Shiiba, Shunji; Watanabe, Seiji; Takeuchi, Hiroshi

    2018-02-01

    N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  1. Chemokine stromal cell-derived factor 1alpha activates basophils by means of CXCR4

    DEFF Research Database (Denmark)

    Jinquan, T; Jacobi, H H; Jing, C

    2000-01-01

    The CXC chemokine receptor 4 (CXCR4) is predominantly expressed on inactivated naive T lymphocytes, B lymphocytes, dendritic cells, and endothelial cells. CXC chemokine stromal cell-derived factor 1alpha (SDF-1alpha) is the only known ligand for CXCR4. To date, the CXCR4 expression and function...... of SDF-1alpha in basophils are unknown....

  2. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis

    DEFF Research Database (Denmark)

    Jafari, Abbas; Qanie, Diyako; Levin Andersen, Thomas

    2017-01-01

    Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells...

  3. Zoledronic acid at subtoxic dose extends osteoblastic stage span of primary human osteoblasts.

    Science.gov (United States)

    Zara, Susi; De Colli, Marianna; di Giacomo, Viviana; Zizzari, Vincenzo Luca; Di Nisio, Chiara; Di Tore, Umberto; Salini, Vincenzo; Gallorini, Marialucia; Tetè, Stefano; Cataldi, Amelia

    2015-04-01

    This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. HOs were treated choosing the highest limit concentration (10(-5) M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.

  4. Bone Resorption Is Regulated by Circadian Clock in Osteoblasts.

    Science.gov (United States)

    Takarada, Takeshi; Xu, Cheng; Ochi, Hiroki; Nakazato, Ryota; Yamada, Daisuke; Nakamura, Saki; Kodama, Ayumi; Shimba, Shigeki; Mieda, Michihiro; Fukasawa, Kazuya; Ozaki, Kakeru; Iezaki, Takashi; Fujikawa, Koichi; Yoneda, Yukio; Numano, Rika; Hida, Akiko; Tei, Hajime; Takeda, Shu; Hinoi, Eiichi

    2017-04-01

    We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH) 2 D 3 -induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

  5. Measurement of oxygen consumption rate of osteoblasts from ...

    African Journals Online (AJOL)

    The oxygen consumption rates of Sprague-Dawley (SD) rat's osteoblasts cultured in different in vitro media were on-line measured by using precision dissolved oxygen and respirometry instrumentation. The osteoblasts, isolated from the calvaria of neonatal SD rat by an enzymatic digestive process of 0.25% trypsin solution ...

  6. Cellular response of Murine Osteoblasts to Cryopreservation: the ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-11-02

    Nov 2, 2006 ... engineered bone. The effects of cell-scaffold interactions and cell-cell interactions on osteoblast viability and attachment to hydroxyapatite (HA) scaffolds following cryopreservation ... servation methods for bio-artificial tissue products can be ... tissue-engineered bone constructs involve osteoblasts attached ...

  7. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells

    NARCIS (Netherlands)

    Varma, Maikel J. Oedayrajsingh; Breuls, Roel G. M.; Schouten, Tabitha E.; Jurgens, Wouter J. F. M.; Bontkes, Hetty J.; Schuurhuis, Gerrit J.; van Ham, S. Marieke; van Milligen, Florine J.

    2007-01-01

    Adipose tissue contains a stromal vascular fraction (SVF) that is a rich source of adipose tissue-derived stem cells (ASCs). ASCs are multipotent and in vitro-expanded ASCs have the capacity to differentiate, into amongst others, adipocytes, chondrocytes, osteoblasts, and myocytes. For tissue

  8. Molecular characterisation of group IVA (cytosolic) phospholipase A2 in murine osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Leis, Hans Jörg; Windischhofer, Werner

    2015-02-01

    Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.

  9. Aluminum trichloride inhibits osteoblastic differentiation through inactivation of Wnt/β-catenin signaling pathway in rat osteoblasts.

    Science.gov (United States)

    Cao, Zheng; Fu, Yang; Sun, Xudong; Zhang, Qiuyue; Xu, Feibo; Li, Yanfei

    2016-03-01

    Exposure to aluminum (Al) suppresses bone formation. Osteoblastic differentiation plays a key role in the process of bone formation. However, the effect of Al on osteoblastic differentiation is still controversial, and the mechanism remains unclear. To investigate the effect of Al on osteoblastic differentiation and whether Wnt signaling pathway was involved in it, the primary rat osteoblasts were exposed to 1/40 IC50, 1/20 IC50 and 1/10 IC50 of aluminum trichloride (AlCl3) for 24h, respectively. The activity analysis of alkaline phosphate, qRT-PCR analysis of type I collagen, alkaline phosphate, Wnt3a and Dkk-1, Western blot analysis of p-GSK3β, GSK3β and β-catenin protein and Immunofluorescence staining for β-catenin suggested that AlCl3 inhibited osteoblastic differentiation and Wnt/β-catenin pathway. Moreover, we found exogenous Wnt3a application reversed the inhibitory effect of AlCl3 on osteoblastic differentiation, accompanied by activating the Wnt/β-catenin pathway. Taken together, these findings suggest that AlCl3 inhibites osteoblastic differentiation through inactivation of Wnt/β-catenin pathway in osteoblasts. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Gastrointestinal stromal tumour presenting as gastroduodenal intussusception.

    LENUS (Irish Health Repository)

    Wilson, Mark H

    2012-08-01

    Gastroduodenal intussusception secondary to gastrointestinal stromal tumour is a very rare cause for intestinal obstruction. The diagnosis of this condition can be challenging, as symptoms are often non-specific and intermittent. This article reports a case where the diagnosis was made preoperatively with abdominal imaging and was treated by a combination of endoscopic reduction and laparoscopic resection.

  11. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs...

  12. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro

    OpenAIRE

    Murakami, Kohei; Kobayashi, Yasuhiro; Uehara, Shunsuke; Suzuki, Takako; Koide, Masanori; Yamashita, Teruhito; Nakamura, Midori; Takahashi, Naoyuki; Kato, Hiroyuki; Udagawa, Nobuyuki; Nakamura, Yukio

    2017-01-01

    The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the pres...

  13. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    International Nuclear Information System (INIS)

    Hasegawa, T.; Hirohashi, Setsuo; Shimoda, Tadakazu; Yokoyama, Ryohei; Beppu, Yasuo; Maeda, Shotaro

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.)

  14. Intracortical osteoblastic osteosarcoma with oncogenic rickets

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, T.; Hirohashi, Setsuo [Pathology Division, National Cancer Center Research Institute, Tokyo (Japan); Shimoda, Tadakazu [Clinical Laboratory Division, National Cancer Center Hospital, Tokyo (Japan); Yokoyama, Ryohei; Beppu, Yasuo [Orthopedic Division, National Cancer Center Hospital, Tokyo (Japan); Maeda, Shotaro [Department of Pathology, Nippon Medical School Hospital, Tokyo (Japan)

    1999-01-01

    Intracortical osteosarcoma is the rarest variant of osteosarcoma, occurring within, and usually confined to, the cortical bone. Oncogenic osteomalacia, or rickets, is an unusual clinicopathologic entity in which vitamin D-resistant osteomalacia, or rickets, occurs in association with some tumors of soft tissue or bone. We present a case of oncogenic rickets associated with intracortical osteosarcoma of the tibia in a 9-year-old boy, whose roentgenographic abnormalities of rickets disappeared and pertinent laboratory data except for serum alkaline phosphatase became normal after surgical resection of the tumor. Histologically, the tumor was an osteosarcoma with a prominent osteoblastic pattern. An unusual microscopic feature was the presence of matrix mineralization showing rounded calcified structures (calcified spherules). Benign osteoblastic tumors, such as osteoid osteoma and osteoblastoma, must be considered in the differential diagnosis because of the relatively low cellular atypia and mitotic activity of this tumor. The infiltrating pattern with destruction or engulfment of normal bone is a major clue to the correct diagnosis of intracortical osteosarcoma. The co-existing radiographic changes of rickets were due to the intracortical osteosarcoma. (orig.) With 8 figs., 25 refs.

  15. Osteoblastic Metastases Mimickers on Contrast Enhanced CT

    Directory of Open Access Journals (Sweden)

    Fahad Al-Lhedan

    2017-01-01

    Full Text Available Secondary osseous involvement in lymphoma is more common compared to primary bone lymphoma. The finding of osseous lesion can be incidentally discovered during the course of the disease. However, osseous metastases are infrequently silent. Detection of osseous metastases is crucial for accurate staging and optimal treatment planning of lymphoma. The aim of imaging is to identify the presence and extent of osseous disease and to assess for possible complications such as pathological fracture of the load-bearing bones and cord compression if the lesion is spinal. We are presenting two patients with treated lymphoma who were in complete remission. On routine follow-up contrast enhanced CT, there were new osteoblastic lesions in the spine worrisome for metastases. Additional studies were performed for further evaluation of both of them which did not demonstrate any corresponding suspicious osseous lesion. The patients have a prior history of chronic venous occlusive thrombosis that resulted in collaterals formation. Contrast enhancement of the vertebral body marrow secondary to collaterals formation and venous flow through the vertebral venous plexus can mimic the appearance of spinal osteoblastic metastases.

  16. Nrp2 deficiency leads to trabecular bone loss and is accompanied by enhanced osteoclast and reduced osteoblast numbers.

    Science.gov (United States)

    Verlinden, Lieve; Kriebitzsch, Carsten; Beullens, Ine; Tan, Biauw Keng; Carmeliet, Geert; Verstuyf, Annemieke

    2013-08-01

    Neuropilin 1 (Nrp1) and Nrp2 are transmembrane receptors that can bind class 3 semaphorins (Sema3A-G) in addition to VEGF family members to play important roles in axonal guidance, vascularization and angiogenesis, as well as immune responses. Moreover, recent evidence implicates Sema3A/Nrp-mediated signaling in bone regulation. However, to date the expression of Nrp2 in bone has not been investigated and a possible role for Nrp2 in the maintenance of bone homeostasis in vivo remains unexplored. Here we show that Nrp2, together with its possible coreceptors (Plexin A family members and Plexin D1) and class 3 semaphorin ligands, were expressed during in vitro osteogenic differentiation of bone marrow stromal cells. Moreover, Nrp2 transcript and protein levels were highly induced in hematopoietic bone marrow cell-derived osteoclast cultures. Osteoblastic as well as osteoclastic Nrp2 expression was confirmed by immunohistochemistry of the long bones of mice. Interestingly, Nrp2 knockout mice were characterized by a low bone mass phenotype which was accompanied by an increased number of osteoclasts and a decreased osteoblast count. Collectively, these data point to a physiological role for Nrp2 in bone homeostasis. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

    Science.gov (United States)

    Marijanovic, Inga; Jiang, Xi; Kronenberg, Mark S.; Stover, Mary Louise; Erceg, Ivana; Lichtler, Alexander C.; Rowe, David W.

    2003-01-01

    AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

  18. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation

    Science.gov (United States)

    Kim, Sungwoo; Kang, Yunqing; Krueger, Chad A.; Sen, Milan; Holcomb, John B.; Chen, Di; Wenke, Joseph C.; Yang, Yunzhi

    2012-01-01

    The purpose of this study was to develop and characterize a chitosan gel/gelatin microspheres (MSs) dual delivery system for sequential release of bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-1 (IGF-1) to enhance osteoblast differentiation in vitro. We made and characterized the delivery system based on its degree of cross-linking, degradation, and release kinetics. We also evaluated the cytotoxicity of the delivery system and the effect of growth factors on cell response using pre-osteoblast W-20-17 mouse bone marrow stromal cells. IGF-1 was first loaded into MSs, and then the IGF-1 containing MSs were encapsulated into the chitosan gel which contained BMP-2. Cross-linking of gelatin with glyoxal via Schiff bases significantly increased thermal stability and decreased the solubility of the MSs, leading to a significant decrease in the initial release of IGF-1. Encapsulation of the MSs into the chitosan gel generated polyelectrolyte complexes by intermolecular interactions, which further affected the release kinetics of IGF-1. This combinational delivery system provided an initial release of BMP-2 followed by a slow and sustained release of IGF-1. Significantly greater alkaline phosphatase activity was found in W-20-17 cells treated with the sequential delivery system than other treatments (p<0.05) after a week of culture. PMID:22293583

  19. Bone marrow stromal cell therapy for ischemic stroke: A meta-analysis of randomized control animal trials.

    Science.gov (United States)

    Wu, Qing; Wang, Yuexiang; Demaerschalk, Bart M; Ghimire, Saruna; Wellik, Kay E; Qu, Wenchun

    2017-04-01

    Background Results of animal studies assessing efficacy of bone marrow stromal cell therapy for ischemic stroke remain inconsistent. Aims The aims are to assess efficacy of bone marrow stromal cell therapy for ischemic stroke in animal studies. Methods Randomized controlled animal trials assessing efficacy of bone marrow stromal cell therapy were eligible. Stroke therapy academic industry round table was used to assess methodologic quality of included studies. Primary outcomes were total infarction volume and modified Neurological Severity Score. Multiple prespecified sensitivity analyses and subgroup analyses were conducted. Random effects models were used for meta-analysis. Results Thirty-three randomized animal trials were included with a total of 796 animals. The median quality score was 6 (interquartile range, 5-7). Bone marrow stromal cell therapy decreased total infarction volume (standardized mean difference, 0.897; 95% confidence interval, 0.553-1.241; P animals treated with bone marrow stromal cell and controls was 2.47 (95% confidence interval, 1.84-3.11; P animal studies. Conclusions Bone marrow stromal cell therapy significantly decreased total infarction volume and increased neural functional recovery in randomized controlled animal models of ischemic stroke.

  20. Corneal stromal dystrophies: a clinical pathologic study

    Directory of Open Access Journals (Sweden)

    Elvira Barbosa Abreu

    2012-12-01

    Full Text Available INTRODUCTION: Corneal dystrophy is defined as bilateral and symmetric primary corneal disease, without previous associated ocular inflammation. Corneal dystrophies are classified according to the involved corneal layer in superficial, stromal, and posterior dystrophy. Incidence of each dystrophy varies according to the geographic region studied. PURPOSE: To evaluate the prevalence of stromal corneal dystrophies among corneal buttons specimens obtained by penetrating keratoplasty (PK in an ocular pathology laboratory and to correlate the diagnosis with patient age and gender. METHODS: Corneal button cases of penetrating keratoplasty from January-1996 to May-2009 were retrieved from the archives of The Henry C. Witelson Ophthalmic Pathology Laboratory and Registry, Montreal, Canada. The cases with histopathological diagnosis of stromal corneal dystrophies were stained with special stains (Peroxid acid Schiff, Masson trichrome, Congo red analyzed under polarized light, and alcian blue for classification and correlated with epidemiological information (age at time of PK and gender from patients' file. RESULTS: 1,300 corneal buttons cases with clinical diagnose of corneal dystrophy were retrieved. Stromal corneal dystrophy was found in 40 (3.1% cases. Lattice corneal dystrophy was the most prevalent with 26 cases (65%. Nineteen were female (73.07% and the PK was performed at average age of 59.3 years old. Combined corneal dystrophy was found in 8 (20% cases, 5 (62.5% of them were female and the average age of the penetrating keratoplasty was 54.8 years old. Granular corneal dystrophy was represented by 5 (12.5% cases, and 2 (40% of them were female. Penetrating keratoplasty was performed at average age of 39.5 years old in granular corneal dystrophy cases. Macular corneal dystrophy was present in only 1 (2.5% case, in a 36 years old female. CONCLUSION: Systematic histopathological approach and evaluation, including special stains in all stromal

  1. Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning.

    Science.gov (United States)

    Olson, Timothy S; Caselli, Anna; Otsuru, Satoru; Hofmann, Ted J; Williams, Richard; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin M

    2013-06-27

    Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.

  2. Construction of a human corneal stromal equivalent with non-transfected human corneal stromal cells and acellular porcine corneal stromata.

    Science.gov (United States)

    Diao, Jin-Mei; Pang, Xin; Qiu, Yue; Miao, Ying; Yu, Miao-Miao; Fan, Ting-Jun

    2015-03-01

    A tissue-engineered human corneal stroma (TE-HCS) has been developed as a promising equivalent to the native corneal stroma for replacement therapy. However, there is still a crucial need to improve the current approaches to render the TE-HCS equivalent more favorable for clinical applications. At the present study, we constructed a TE-HCS by incubating non-transfected human corneal stromal (HCS) cells in an acellular porcine corneal stromata (aPCS) scaffold in 20% fetal bovine serum supplemented DMEM/F12 (1:1) medium at 37 °C with 5% CO2in vitro. After 3 days of incubation, the constructed TE-HCS had a suitable tensile strength for transplantation, and a transparency that is comparable to native cornea. The TE-HCS had a normal histological structure which contained regularly aligned collagen fibers and differentiated HCS cells with positive expression of marker and functional proteins, mimicking a native HCS. After transplantation into rabbit models, the TE-HCS reconstructed normal corneal stroma in vivo and function well in maintaining corneal clarity and thickness, indicating that the completely biological TE-HCS could be used as a HCS equivalent. The constructed TE-HCS has promising potentials in regenerative medicine and treatment of diseases caused by corneal stromal disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

    Directory of Open Access Journals (Sweden)

    Vishnubalaji Radhakrishnan

    2012-01-01

    Full Text Available Abstract Background Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs and adult dermal skin (hADSSCs using explants cultures and were compared with bone marrow (hMSC-TERT and adipose tissue-derived mesenchymal stem cells (hADMSCs for their potential differentiation into osteoblasts, adipocytes, and endothelial cells. Results Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC, with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues. Conclusions Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs

  4. Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Aisha, M.D. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); Nor-Ashikin, M.N.K. [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Sharaniza, A.B.R. [DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor (Malaysia); Nawawi, H. [Center for Pathology Diagnostic and Research Laboratories, Clinical Training Center, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia); Froemming, G.R.A., E-mail: gabriele@salam.uitm.edu.my [Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor (Malaysia); I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor (Malaysia)

    2015-09-10

    Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. - Highlights: • OFS stress transmits anabolic signals to osteoblasts. • Actin and tubulin fibers are rearranged under OFS stress. • OFS stress increases mitochondrial metabolism and proliferation. • Reduced RANKL/OPG ratio in response to OFS inhibits osteoclastogenesis. • OFS stress prevents apoptosis and stimulates ALP and OCN.

  5. Ca uptake and its influence by growth hormone in osteoblasts of fetal rat calvaria

    International Nuclear Information System (INIS)

    Wang Hongfu; Jin Weifang; Sekimoto Haku

    1994-01-01

    Uptake and release of Ca 2+ are important functions of osteoblasts. In the paper we studied the uptake of calcium and influence by Growth Hormone in osteoblasts of fetal rat calvaria by liquid scintillation spectrometry of 45 Ca 2+ . In short-term cultures of the bone derived cells, the uptake of 45 Ca 2+ increased steadily. The activity of 45 Ca 2+ in the cells of 15 minute cultures was 2∼3 times of that in the 0 minute cultures. It continued to increase in the cells of 30 minute cultures. Exposure of the bone cells to GH at 55.3 ng/ml increased the uptake of 45 Ca 2+ by 2.3 times in the 30 minute cultures and 1.5 times in the 60 minute cultures than those of the control

  6. The collective influence of 1, 25-dihydroxyvitamin D3with physiological fluid shear stress on osteoblasts.

    Science.gov (United States)

    Li, Yan; Yuan, Jiafeng; Wang, Qianwen; Sun, Lijie; Sha, Yunying; Li, Yanxiang; Wang, Lizhong; Wang, Zhonghua; Ma, Yonggang; Cao, Hui

    2018-01-01

    1, 25-dihydroxyvitamin D 3 (1, 25 (OH) 2 D 3 ) and mechanical stimuli in physiological environment contributes greatly to osteoporosis pathogenesis. Wide investigations have been conducted on how 1, 25-dihydroxyvitamin D 3 and mechanical stimuli separately impact osteoblasts. This study reports the collective influences of 1, 25-dihydroxyvitamin D 3 and flow shear stress (FSS) on biological functions of osteoblasts. 1, 25 (OH) 2 D 3 were prepared in various kinds of concentrations (0, 1, 10, 100 nmmol/L), while physiological fluid shear stress (12 dynes/cm 2 ) was produced by using a parallel-plate fluid flow system. 1, 25 (OH) 2 D 3 affects the responses of ROBs to FSS, including the inhibition of NO release and cell proliferation as well as the promotion of PGE 2 release and cell differentiation. These findings provide a possible mechanism by which 1, 25(OH) 2 D 3 influences osteoblasts' responses to FSS, thus most probably providing guidance for the selection of 1, 25(OH) 2 D 3 concentration and mechanical loading in order to produce functional bone tissues in vitro. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been....../tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment...... reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion...

  8. Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

    Science.gov (United States)

    Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R.

    2015-01-01

    Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10×100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. PMID:26141836

  9. Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation.

    Science.gov (United States)

    Thompson, Bethtrice; Varticovski, Lyuba; Baek, Songjoon; Hager, Gordon L

    2016-01-01

    Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC). However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.

  10. Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast Differentiation.

    Directory of Open Access Journals (Sweden)

    Bethtrice Thompson

    Full Text Available Bone continuously undergoes remodeling by a tightly regulated process that involves osteoblast differentiation from Mesenchymal Stem Cells (MSC. However, commitment of MSC to osteoblastic lineage is a poorly understood process. Chromatin organization functions as a molecular gatekeeper of DNA functions. Detection of sites that are hypersensitive to Dnase I has been used for detailed examination of changes in response to hormones and differentiation cues. To investigate the early steps in commitment of MSC to osteoblasts, we used a model human temperature-sensitive cell line, hFOB. When shifted to non-permissive temperature, these cells undergo "spontaneous" differentiation that takes several weeks, a process that is greatly accelerated by osteogenic induction media. We performed Dnase I hypersensitivity assays combined with deep sequencing to identify genome-wide potential regulatory events in cells undergoing early steps of commitment to osteoblasts. Massive reorganization of chromatin occurred within hours of differentiation. Whereas ~30% of unique DHS sites were located in the promoters, the majority was outside of the promoters, designated as enhancers. Many of them were at novel genomic sites and need to be confirmed experimentally. We developed a novel method for identification of cellular networks based solely on DHS enhancers signature correlated to gene expression. The analysis of enhancers that were unique to differentiating cells led to identification of bone developmental program encompassing 147 genes that directly or indirectly participate in osteogenesis. Identification of these pathways provided an unprecedented view of genomic regulation during early steps of differentiation and changes related to WNT, AP-1 and other pathways may have therapeutic implications.

  11. Osteoblastic cell behaviour on modified titanium surfaces.

    Science.gov (United States)

    Lukaszewska-Kuska, Magdalena; Wirstlein, Przemysław; Majchrowski, Radomir; Dorocka-Bobkowska, Barbara

    2018-02-01

    The surfaces of endoosseous dental implants have been subjected to numerous modifications in order to create a surface which can provide rapid bone healing and fast implant loading. Each modification has involved changes to the chemical composition and topography of the surfaces which have resulted in various biological reactions to the implanted material. The aim of this study was to evaluate the surface topography and chemistry of various modified titanium surfaces: (1) machined surface (MA), (2) alumina-blasted (Al2O3), (3) alumina-blasted and acid-etched (Al2O3 DE), (4) hydroxyapatite/tricalcium phosphate grit-blasted (HA/TCP) and (5) hydroxyapatite/tricalcium phosphate grit-blasted and acid-etched (HA/TCP DE) and to analyse the effects of surface roughness, and chemical composition on human osteoblast vitality, differentiation, morphology and orientation. The modified surfaces were subjected to topographic analysis using Scanning Electron Microscopy (SEM), optical profilometry, roughness analysis and chemical composition evaluation using Energy Dispersion Spectroscopy (EDS) analysis. The biological effects of the titanium modifications was analysed using human osteoblasts cell culture where the cell morphology, vitality (MTS assay) and differentiation (ALP activity) was analysed. The machined surfaces were classified as anisotropic, smooth and composed of titanium and oxygen. The blasted surface samples along with the blasted and etched samples were found to be isotropic and rough. The grit-blasting procedure resulted in the incorporation of components from the blasting material. In the case of the blasted and etched samples, etching decreased the surface development as indicated by the Sdr and also reduced the amount of chemical compounds incorporated into the surfaces during the blasting procedure. The attached NHOst cells, proliferated the surfaces. With regard to the MA samples, the cells spread close to the titanium surface, with expanded cytoplasmic

  12. Osteoblast adhesion to clodronate-hydroxyapatite composite

    Energy Technology Data Exchange (ETDEWEB)

    Luo, E.; Liu, X. [State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610044 (China); Wei, S.C. [School of Stomatology, Peking University, Beijing 100044 (China); Cai, X.X. [State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610044 (China); Hu, J. [State Key Laboratory of Oral Disease, Sichuan University, Chengdu 610044 (China)], E-mail: donow3000@126.com

    2008-11-15

    The present study combined clodronate, one of bisphosphonates, to the surface of HA scaffold by chelation. The cell culture test and gene expression test were performed and evaluated the effect of clodronate modifying the HA on co-culturing osteoblast with HA scaffold in vitro. Characteristic peaks in XPS and FT-IR spectra indicated clodronate being immobilized on the surface of HA. The cell culture test indicated that the cells actively proliferated on the scaffolds. The results of RT-PCR showed there was no significant difference between two groups in expression of RANKL gene, while the expression of OPG gene showed higher expression in Clodronate-HA group. Clodronate had no obvious effect on the cytocompatibility of HA, and Clodronate-HA might be used as bone scaffold with potential ability to improve osteogenesis.

  13. Emdogain stimulates matrix degradation by osteoblasts.

    Science.gov (United States)

    Goda, S; Inoue, H; Kaneshita, Y; Nagano, Y; Ikeo, T; Ikeo, Y T; Iida, J; Domae, N

    2008-08-01

    Emdogain has been used clinically for periodontal regeneration, although the underlying molecular mechanisms are not clear at present. In this study, we hypothesized that Emdogain stimulated degradation of type I collagen via osteoblasts. We showed that Emdogain enhanced cell-mediated degradation of type I collagen in an MMP-dependent manner. Although MG-63 cells spontaneously produced a zymogen form of MMP-1, treatment with Emdogain significantly induced the generation of the active form of this enzyme. We demonstrated that MMP-3 was produced from MG63 cells in response to Emdogain in a MEK1/2-dependent manner. Concomitantly, blocking of MEK1/2 activation by U0126 significantly inhibited the generation of the active form of MMP-1 without affecting the total production of this collagenase. These results suggest that Emdogain facilitates tissue regeneration through the activation of the collagenase, MMP-1, that degrades matrix proteins in bone tissue microenvironments.

  14. Selective cell affinity of biomimetic micro-nano-hybrid structured TiO2 overcomes the biological dilemma of osteoblasts.

    Science.gov (United States)

    Hori, Norio; Iwasa, Fuminori; Ueno, Takeshi; Takeuchi, Kazuo; Tsukimura, Naoki; Yamada, Masahiro; Hattori, Masami; Yamamoto, Akiko; Ogawa, Takahiro

    2010-04-01

    There is a great demand for dental implant surfaces to accelerate the process of peri-implant bone generation to reduce its healing time and enable early loading. To this end, an inverse correlation between the proliferation and functional maturation (differentiation) in osteoblasts presents a challenge for the rapid generation of greater amounts of bone. For instance, osteoblasts exhibit faster differentiation but slower proliferation on micro-roughened titanium surfaces. Using a unique micro-nano-hierarchical topography of TiO(2) that mimics biomineralized matrices, this study demonstrates that this challenge can be overcome without the use of biological agents. Titanium disks of grade 2 commercially pure titanium were prepared by machining (smooth surface). To create a microtexture with peaks and valleys (micropit surface), titanium disks were acid-etched. To create 200-nm TiO(2) nanonodules within the micropits (nanonodule-in-micropit surface), TiO(2) was sputter-deposited onto the acid-etched surface. Rat bone marrow-derived osteoblasts and NIH3T3 fibroblasts were cultured on machined smooth, micropit, and nanonodule-in-micropit surfaces. Despite the substantially increased surface roughness, the addition of 200-nm nanonodules to micropits increased osteoblast proliferation while enhancing their functional differentiation. In contrast, this nanonodule-in-micropit surface decreased proliferation and function in fibroblasts. The data suggest the establishment of cell-selectively functionalized nano-in-micro smart titanium surfaces that involve a regulatory effect on osteoblast proliferation, abrogating the inhibitory mechanism on the micropitted surface, while enhancing their functional differentiation. Biomimetic and controllable nature of this nanonodules-in-micropits surface may offer a novel micro-to-nanoscale hierarchical platform to biologically optimize nanofeatures of biomaterials. Particularly, this micro-nano-hybrid surface may be an effective approach

  15. Transcript analyses of stromal cell derived factors (SDFs): SDF-2, SDF-4 and SDF-5 reveal a different pattern of expression and prognostic association in human breast cancer

    OpenAIRE

    Kang, H.; Escudero-Esparza, Astrid; Douglas-Jones, A.; Mansel, Robert Edward; Jiang, Wen G.

    2009-01-01

    Stromal derived factors, SDFs, are a loosely defined group of molecules that may be generated by stromal cells. Two of the stromal derived factors, SDF-1 and SDF-4 belong to the chemokine family. Other SDFs, such as SDF-2 and SDF-5 are not well defined and their biological functions are less known. Although SDF-1 and its receptor have been strongly indicated in the progression of various cancers including breast cancer, little is known with regard to the role of other SDFs in malignant condit...

  16. Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology.

    Science.gov (United States)

    Zhan, Han-Xiang; Zhou, Bin; Cheng, Yu-Gang; Xu, Jian-Wei; Wang, Lei; Zhang, Guang-Yong; Hu, San-Yuan

    2017-04-28

    Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide. Increasing evidence has confirmed the pivotal role of stromal components in the regulation of carcinogenesis, invasion, metastasis, and therapeutic resistance in PC. Interaction between neoplastic cells and stromal cells builds a specific microenvironment, which further modulates the malignant properties of cancer cells. Instead of being a "passive bystander", stroma may play a role as a "partner in crime" in PC. However, the role of stromal components in PC is complex and requires further investigation. In this article, we review recent advances regarding the regulatory roles and mechanisms of stroma biology, especially the cellular components such as pancreatic stellate cells, macrophages, neutrophils, adipocytes, epithelial cells, pericytes, mast cells, and lymphocytes, in PC. Crosstalk between stromal cells and cancer cells is thoroughly investigated. We also review the prognostic value and molecular therapeutic targets of stroma in PC. This review may help us further understand the molecular mechanisms of stromal biology and its role in PC development and therapeutic resistance. Moreover, targeting stroma components may provide new therapeutic strategies for this stubborn disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Endometrial Stromal Sarcoma: A Rare Entity

    International Nuclear Information System (INIS)

    Jabeen, S.; Anwar, S.; Fatima, N.

    2015-01-01

    Endometrial Stromal Sarcoma (ESS) is a hormone sensitive tumor. It is a rare gynecological tumor and is considered to occur more often in pre-menopausal women. A proper pre-operative diagnosis is difficult and confirmed in most cases after hysterectomy for a presumed benign disease. Endometrial sampling, ultrasound, and magnetic resonance imaging can provide diagnostic clues. For early disease complete surgical cure is possible, however, adjuvant therapy is available for recurrence. This case of Low Grade Endometrial Stromal Sarcoma (LGESS) in a 21 years old woman was presented as irregular vaginal bleeding. Clinical diagnosis of fibroid was made but analysis of endometrium showed ESS confirmed on hysterectomy specimen. One should consider it in any case with rapid fibroid enlargement. (author)

  18. Gastrointestinal Stromal Tumor: Diagnosis and Prognosis

    International Nuclear Information System (INIS)

    Martin, M. T.; Olmedilla, P.; Gonzalez, S.; Oliver, J. M.

    2003-01-01

    Gastrointestinal stromal tumors (GIST) are mesenquimal tumors derived from cell precursors. They have the capacity for myogenic and neurogenic differentiation and are characterized by expression of KIT protein /tyrosine kinase growth factor). Clinically, they exhibit various biological behaviors. We present 8 cases of GIST, describing both their radiological manifestation through computerized tomography (CT) and most accepted criteria for benignity and malignancy. We also describe the response of one meta statically diagnosed tumor to tyrosine kinase inhibitor. (Author) 9 refs

  19. Extragenital endometrial stromal sarcoma arising in endometriosis.

    Science.gov (United States)

    Alcázar, Juan Luis; Guerriero, Stefano; Ajossa, Silvia; Parodo, Giuseppina; Piras, Bruno; Peiretti, Michele; Jurado, Matías; Idoate, Miguel Ángel

    2012-01-01

    The diagnosis rate of deep pelvic endometriosis is increasing. Endometrial stromal sarcoma (ESS) is a rare neoplasm. Extragenital ESS is an extremely uncommon event. Very few cases of extragenital ESS have been reported to date. The diagnosis of this entity is very difficult in some instances. Knowledge about its management is also limited. In this paper, we review the current literature on the clinical management, histology, immunohistochemistry, treatment and outcome of ESS arising in pelvic endometriosis. Copyright © 2012 S. Karger AG, Basel.

  20. Computed tomography in gastrointestinal stromal tumors

    International Nuclear Information System (INIS)

    Ghanem, Nadir; Altehoefer, Carsten; Winterer, Jan; Schaefer, Oliver; Springer, Oliver; Kotter, Elmar; Langer, Mathias; Furtwaengler, Alex

    2003-01-01

    The aim of this study was to define the imaging characteristics of primary and recurrent gastrointestinal stromal tumors (GIST) in computed tomography with respect to the tumor size. Computed tomography was performed in 35 patients with histologically confirmed gastrointestinal stromal tumors and analyzed retrospectively by two experienced and independent radiologist. The following morphologic tumor characteristics of primary (n=20) and (n=16) recurrent tumors were evaluated according to tumor size, shape, homogeneity, density compared with liver, contrast enhancement, presence of calcifications, ulcerations, fistula or distant metastases and the anatomical relationship to the intestinal wall, and the infiltration of adjacent visceral organs. Small GIST ( 5-10 cm) demonstrated an irregular shape, inhomogeneous density on unenhanced and contrast-enhanced images, a combined intra- and extraluminal tumor growth with aggressive findings, and infiltration of adjacent organs in 9 primary diagnosed and 2 recurrent tumors. Large GIST (>10 cm), which were observed in 8 primary tumors and 11 recurrent tumors, showed an irregular margin with inhomogeneous density and aggressive findings, and were characterized by signs of malignancy such as distant and peritoneal metastases. Small recurrent tumors had a similar appearance as compared with large primary tumors. Computed tomography gives additional information with respect to the relationship of gastrointestinal stromal tumor to the gastrointestinal wall and surrounding organs, and it detects distant metastasis. Primary and recurrent GIST demonstrate characteristic CT imaging features which are related to tumor size. Aggressive findings and signs of malignancy are found in larger tumors and in recurrent disease. Computed tomography is useful in detection and characterization of primary and recurrent tumors with regard to tumor growth pattern, tumor size, and varied appearances of gastrointestinal stromal tumors, and indirectly

  1. Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Stik, Gregoire; Crequit, Simon; Petit, Laurence; Durant, Jennifer; Charbord, Pierre; Jaffredo, Thierry; Durand, Charles

    2017-07-03

    Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine. © 2017 Stik et al.

  2. Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

    Science.gov (United States)

    Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

    2014-01-01

    Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45−/14−). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10−7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10−6 M (Group 1, A), 10−5 M (Group 2, B), 10−4 M (Group 3, A/B), and together with 10−7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34−, CD45−, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). Results Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. Conclusions Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin

  3. The impact of doped silicon quantum dots on human osteoblasts

    Czech Academy of Sciences Publication Activity Database

    Ostrovská, L.; Brož, Antonín; Fučíková, A.; Bělinová, T.; Sugimoto, H.; Kanno, T.; Fujii, M.; Valenta, J.; Kalbáčová, M.H.

    2016-01-01

    Roč. 6, č. 68 (2016), s. 63403-63413 ISSN 2046-2069 Institutional support: RVO:67985823 Keywords : silicon quantum dots * osteoblasts * cytotoxicity * photoluminiscence bioimaging Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.108, year: 2016

  4. Osteoblast cell response to surface-modified carbon nanotubes

    International Nuclear Information System (INIS)

    Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

    2012-01-01

    In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

  5. Increased extracellular and intracellular Ca{sup 2+} lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Ryota, E-mail: hryota@juntendo.ac.jp [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Katoh, Youichi, E-mail: katoyo@juntendo-urayasu.jp [Juntendo University Faculty of International Liberal Arts, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Miyamoto, Yuki [Juntendo University Faculty of Health Care and Nursing, Takasu 2-5-1, Urayasu-shi, Chiba 279-0023 (Japan); Itoh, Seigo; Daida, Hiroyuki [Department of Cardiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Nakazato, Yuji [Center for Environmental Research, Department of Cardiology, Juntendo University Faculty of Medicine Urayasu Hospital, Tomioka 2-1-1, Urayasu-shi, Chiba 279-0022 (Japan); Okada, Takao [Department of Physiology, Juntendo University Faculty of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2015-02-20

    Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca{sup 2+} ([Ca{sup 2+}]{sub o} and [Ca{sup 2+}]{sub i}) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} enhanced adipocyte accumulation, which suggested that increases in [Ca{sup 2+}]{sub o} caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca{sup 2+}]{sub o} and high [Ca{sup 2+}]{sub i} may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca{sup 2+}]{sub i} (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca{sup 2+}]{sub o} (addition of CaCl{sub 2}) leads to increases in [Ca{sup 2+}]{sub i}. Flow cytometric methods revealed that high [Ca{sup 2+}]{sub o} suppressed the phosphorylation of ERK independently of intracellular Ca{sup 2+}. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca{sup 2+} provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca{sup 2+}, which results in BMSC proliferation. - Highlights:

  6. Hydroxyapatite-intertwined hybrid nanofibres for the mineralization of osteoblasts.

    Science.gov (United States)

    Sujana, Andra; Venugopal, Jayarama Reddy; Velmurugan, Bhaarathy; Góra, Aleksander; Salla, Manohar; Ramakrishna, Seeram

    2017-06-01

    Advances in tissue engineering have enabled the development of bioactive composite materials to generate biomimetic nanofibrous scaffolds for bone replacement therapies. Polymeric biocomposite nanofibrous scaffolds architecturally mimic the native extracellular matrix (ECM), delivering tremendous regenerative potential for bone tissue engineering. In the present study, biocompatible poly(l-lactic acid)-co-poly(ε-caprolactone)-silk fibroin-hydroxyapatite-hyaluronic acid (PLACL-SF-HaP-HA) nanofibrous scaffolds were fabricated by electrospinning to mimic the native ECM. The developed nanofibrous scaffolds were characterized in terms of fibre morphology, functional group, hydrophilicity and mechanical strength, using SEM, FTIR, contact angle and tabletop tensile-tester, respectively. The nanofibrous scaffolds showed a higher level of pore size and increased porosity of up to 95% for the exchange of nutrients and metabolic wastes. The fibre diameters obtained were in the range of around 255 ± 13.4-789 ± 22.41 nm. Osteoblasts cultured on PLACL-SF-HaP-HA showed a significantly (p functionalized PLACL-SF-HaP-HA nanofibrous scaffolds proved them to be a potential biocomposite for bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Gene-expression analysis of cementoblasts and osteoblasts.

    Science.gov (United States)

    Matthews, B G; Roguljic, H; Franceschetti, T; Roeder, E; Matic, I; Vidovic, I; Joshi, P; Kum, K-Y; Kalajzic, I

    2016-06-01

    Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts. © 2015 John Wiley

  8. Prostate cancer cells induce osteoblastic differentiation via semaphorin 3A.

    Science.gov (United States)

    Liu, Fuzhou; Shen, Weiwei; Qiu, Hao; Hu, Xu; Zhang, Chao; Chu, Tongwei

    2015-03-01

    Prostate cancer metastasis to bone is the second most commonly diagnosed malignant disease among men worldwide. Such metastatic disease is characterized by the presence of osteoblastic bone lesions, and is associated with high rates of mortality. However, the various mechanisms involved in prostate cancer-induced osteoblastic differentiation have not been fully explored. Semaphorin 3A (Sema 3A) is a newly identified regulator of bone metabolism which stimulates differentiation of pre-osteoblastic cells under physiological conditions. We investigated in this study whether prostate cancer cells can mediate osteoblastic activity through Sema 3A. We cultured osteoprogenitor MC3T3-E1 cells in prostate cancer-conditioned medium, and analyzed levels of Sema 3A protein in diverse prostate cancer cell lines to identify cell lines in which Sema 3A production showed a positive correlation with osteo-stimulation. C4-2 cells were stably transfected with Sema 3A short hairpin RNA to further determine whether Sema 3A contributes to the ability of C4-2 cells to induce osteoblastic differentiation. Down-regulation of Sema 3A expression decreased indicators of C4-2 CM-induced osteoblastic differentiation, including alkaline phosphatase production and mineralization. Additionally, silencing or neutralizing Sema 3A in C4-2 cells resulted in diminished β-catenin expression in osteogenitor MC3T3-E1 cells. Our results suggest that prostate cancer-induced osteoblastic differentiation is at least partially mediated by Sema 3A, and may be regulated by the β-catenin signalling pathway. Sema 3A may represent a novel target for treatment of prostate cancer-induced osteoblastic lesions. © 2014 Wiley Periodicals, Inc.

  9. Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Suh, Seok-Jong; Yun, Woo-Sik; Kim, Kap-Sung; Jin, Un-Ho; Kim, June-Ki; Kim, Myung-Sunny; Kwon, Dae Young; Kim, Cheorl-Ho

    2007-02-12

    Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.

  10. Sclerosing Stromal Tumor of Ovary: A Case Report

    Directory of Open Access Journals (Sweden)

    Menka Khanna

    2012-01-01

    Full Text Available Sclerosing stromal tumor (SST is an extremely rare and distinctive sex cord stromal tumor which occurs predominantly in the second and third decades of life. We report a case of a 32-year-old woman who developed a sclerosing stromal tumor of ovary and presented with irregular menstruation and pelvic pain. Her hormonal status was normal but CA-125 was raised. She was suspected to have a malignant tumor on computed tomography and underwent bilateral salpingo-oopherectomy. It is therefore necessary to keep in mind the possibility of sclerosing stromal tumor in a young woman.

  11. The influence of MicroRNA-150 in Osteoblast Matrix Mineralization.

    Science.gov (United States)

    Dong, Chun-Ling; Liu, Hao-Zhi; Zhang, Zhen-Chun; Zhao, Huan-Li; Zhao, Hui; Huang, Yan; Yao, Jian-Hua; Sun, Tian-Sheng

    2015-12-01

    This study investigated the influence of miR-150 expression on osteoblast matrix mineralization and its mechanisms. The mouse osteoblast cell line MC3T3-E1 was used as an in vitro model of bone formation. On the fifth day of mineralization, transfection experiments using agomiR-150, agomiR-NC, antagomiR-150 antagomiR-NC, and mock groups were set up to test the effects of miR-150 in MC3T3-E1 model. The mRNA and protein levels of OC, ALP, type I collagen, and OPN were measured by qRT-PCR and ELISA. Matrix mineralization was detected by alizarin red S (ARS) staining and flow cytometry was employed to quantify apoptosis in each group. RT-PCR and Western blot were applied to detect the expression of target gene MMP14. Our results demonstrated that the endogenous expression levels of miR-150, OC, ALP, type I collagen, and OPN in MC3T3-E1 cells increased steadily. Exogenous expressions of agomiR-150 and antagomiR-150 can significantly up-/down-regulate, respectively, the expression level of miR-150 in MC3T3-E1 cells. Compared with the mock group, higher expression levels of OC, ALP, type I collagen, and OPN mRNA were observed in the agomiR-150 group, while lower mRNA expression levels of OC, ALP, type I collagen, and OPN were found in the antagomiR-150 group. Based on these results, potential miR-150 targeted genes are discussed. Our results showed that miR-150 supports the osteoblastic phenotype related to osteoblast function and bone mineralization. Thus, miR-150 may have potential therapeutic applications in promoting bone formation in certain disease settings, such as in osteoporosis and in elderly patients. © 2015 Wiley Periodicals, Inc.

  12. Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with bioactive ceramics.

    Science.gov (United States)

    Gupta, Gautam; Kirakodu, Sreenatha; El-Ghannam, Ahmed

    2010-12-01

    In this study, we have investigated the effects of dissolved phosphorus and silicon on osteoblast differentiation in vitro. Neonatal rat calvarial osteoblasts were seeded on silica-calcium phosphate composites (SCPCS), hydroxyapatite (HA-200), and tissue culture polystyrene (TCPS) and incubated over 4 days in media containing 0 {minimal essential medium [MEM] (-)} or 3 mM β-glycerophosphate [MEM (+)]. Inductively coupled plasma analysis showed that P-content in original MEM (+) was 225% higher than that in MEM (-). Moreover, P-content in MEM (+) significantly increased to 3.4-4.4 mM and 3.6-4.7 mM after 2 and 4 days incubation with SCPC, respectively, owing to material dissolution and exogenous phosphate supplementation. In contrast, P-content in MEM (+) showed no change upon incubation with HA or TCPS. The P-content in MEM (-) incubated with SCPC was considerably lower than that in MEM (+). SCPC exhibited controlled Si-release in cell culture media [MEM (-) or MEM (+)], with Si-rich SCPC showing a significantly greater dissolution than Si-poor SCPC. Moreover, SCPC, unlike HA, demonstrated a cell- and solution-mediated dissolution over 4 days. Quantitative real-time PCR showed that in MEM (-), osteocalcin and osteopontin mRNA expression on Si-rich SCPC was significantly greater than that on HA, suggesting that Si plays an important role in enhancing bone-cell differentiation. However, osteoblast phenotypic expression on SCPC was significantly decreased after 4 days incubation in MEM (+), indicating that sustained exposure to elevated P-levels in the media can downregulate osteoblast function. Our results demonstrate that the controlled dissolution of SCPC provides a natural stimulus for bone-cell differentiation in vitro and could obviate the need of exogenous phosphate supplementation. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

  13. Transcriptional Regulation of Frizzled-1 in Human Osteoblasts by Sp1.

    Directory of Open Access Journals (Sweden)

    Shibing Yu

    Full Text Available The wingless pathway has a powerful influence on bone metabolism and is a therapeutic target in skeletal disorders. Wingless signaling is mediated in part through the Frizzled (FZD receptor family. FZD transcriptional regulation is poorly understood. Herein we tested the hypothesis that Sp1 plays an important role in the transcriptional regulation of FZD1 expression in osteoblasts and osteoblast mineralization. To test this hypothesis, we conducted FZD1 promoter assays in Saos2 cells with and without Sp1 overexpression. We found that Sp1 significantly up-regulates FZD1 promoter activity in Saos2 cells. Chromatin immunoprecipitation (ChIP and electrophoretic mobility shift (EMSA assays identified a novel and functional Sp1 binding site at -44 to -40 from the translation start site in the FZD1 promoter. The Sp1-dependent activation of the FZD1 promoter was abolished by mithramycin A (MMA, an antibiotic affecting both Sp1 binding and Sp1 protein levels in Saos2 cells. Similarly, down-regulation of Sp1 in hFOB cells resulted in less FZD1 expression and lower alkaline phosphatase activity. Moreover, over-expression of Sp1 increased FZD1 expression and Saos2 cell mineralization while MMA decreased Sp1 and FZD1 expression and Saos2 cell mineralization. Knockdown of FZD1 prior to Sp1 overexpression partially abolished Sp1 stimulation of osteoblast differentiation markers. Taken together, our results suggest that Sp1 plays a role in human osteoblast differentiation and mineralization, which is at least partially mediated by Sp1-dependent transactivation of FZD1.

  14. Alpinia officinarum Stimulates Osteoblast Mineralization and Inhibits Osteoclast Differentiation.

    Science.gov (United States)

    Shim, Ki-Shuk; Lee, Chung-Jo; Yim, Nam-Hui; Gu, Min Jung; Ma, Jin Yeul

    2016-01-01

    Alpinia officinarum rhizome has been used as a traditional herbal remedy to treat inflammatory and internal diseases. Based on the previously observed inhibitory effect of A. officinarum rhizome in an arthritis model, we evaluated whether a water extract of A. officinarum rhizome (WEAO) would enhance in vitro osteoblast mineralization using calvarial osteoblast precursor cells or would inhibit in vitro osteoclast differentiation and bone resorption using bone marrow derived macrophages. In osteoblasts, WEAO enhanced the mRNA levels of transcription factor (runt-related transcription factor 2, smad1, smad5, and junB) and marker (bone morphogenetic protein-2, collagen type 1alpha1, and osteocalcin) genes related to osteoblast mineralization, consistent with increased alizarin red S staining intensity. WEAO markedly inhibited osteoclast differentiation by suppressing the receptor activator for nuclear factor-[Formula: see text]B ligand-induced downregulation of inhibitor of DNA binding 2 and V-maf musculoaponeurotic fibrosarcoma oncogene homolog B and the phosphorylation of c-Jun N-terminal kinase, p38, nuclear factor-[Formula: see text]B, c-Src, and Bruton's tyrosine kinase to induce nuclear factor of activated T cells cytoplasmic 1 expression. WEAO also suppressed the resorbing activity of mature osteoclasts by altering actin ring formation. Therefore, the results of this study demonstrate that WEAO stimulates osteoblast mineralization and inhibits osteoclast differentiation. Thus, WEAO may be a promising herbal candidate to treat or prevent pathological bone diseases by regulating the balance between osteoclast and osteoblast activity.

  15. The role of osteoblasts in peri-prosthetic osteolysis.

    LENUS (Irish Health Repository)

    O'Neill, S C

    2013-08-01

    Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

  16. Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate

    Energy Technology Data Exchange (ETDEWEB)

    Tasat, D.R. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Orona, N.S. [Universidad Nacional de San Martin, Escuela de Ciencia y Tecnologia, Pcia de Bs.As. (Argentina); Mandalunis, P.M. [Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina); Cabrini, R.L. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Ubios, A.M. [Comision Nacional de Energia Atomica, Departamento de Radiobiologia, Buenos Aires (Argentina); Universidad de Buenos Aires, Catedra de Histologia y Embriologia, Facultad de Odontologia, Buenos Aires (Argentina)

    2007-05-15

    Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 {mu}M) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 {mu}M UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 {mu}M UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage. (orig.)

  17. Treatment of femoral head necrosis with transplantation of stromal cell-derived factor-1: an experimental study

    International Nuclear Information System (INIS)

    Meng Wei; Cao Haili; Bai Bin; Zheng Shangfei; Xu Wei

    2009-01-01

    Objective: To discuss the therapeutic mechanism and efficacy of stromal cell-derived factor-1 (SDF-1) in the treatment of femoral head necrosis. Methods: Experimental models of hydrocortisoneinduced femoral head necrosis were established in 30 Japanese rabbits, which were randomly and equally divided into three groups. Group A was regarded as control group, group B received marrow core decompression and saline injection treatment and group C underwent marrow core decompression and SDF-1 transplantation. Eight weeks after the procedure all the survival rabbits (n = 27) were sacrificed, and the specimens were sent for the measuring of bone mineral density and for histopathologic examination. Results Eight weeks after the treatment, the bone mineral density of rabbits in group C was significantly increased. Pathologically, in SDF-1 treated rabbits the amounts of the blood vessels and osteoblast cells were obviously increased while the empty bone lacunae were markedly decreased. Conclusion: Transplantation of stromal cell-derived factor-1 together with marrow core decompression is very effective for the treatment of femoral head necrosis and this technique has showed a vast and bright prospect in clinical practice. (authors)

  18. Autologous rabbit adipose tissue-derived mesenchymal stromal cells for the treatment of bone injuries with distraction osteogenesis.

    Science.gov (United States)

    Sunay, Ozgur; Can, Geylani; Cakir, Zeynep; Denek, Ziya; Kozanoglu, Ilknur; Erbil, Guven; Yilmaz, Mustafa; Baran, Yusuf

    2013-06-01

    Adipose tissue-derived mesenchymal stromal cells (MSCs) have a higher capacity for proliferation and differentiation compared with other cell lineages. Although distraction osteogenesis is the most important therapy for treating bone defects, this treatment is restricted in many situations. The aim of this study was to examine the therapeutic potential of adipose tissue-derived MSCs and osteoblasts differentiated from adipose tissue-derived MSCs in the treatment of bone defects. Bone defects were produced in the tibias of New Zealand rabbits that had previously undergone adipose tissue extraction. Tibial osteotomy was performed, and a distractor was placed on the right leg of the rabbits. The rabbits were placed in control (group I), stem cell (group II) and osteoblast-differentiated stem cell (group III) treatment groups. The rabbits were sacrificed, and the defect area was evaluated by radiologic, biomechanical and histopathologic tests to examine the therapeutic effects of adipose tissue-derived MSCs. Radiologic analyses revealed that callus density and the ossification rate increased in group III compared with group I and group II. In biomechanical tests, the highest ossification rate was observed in group III. Histopathologic studies showed that the quality of newly formed bone and the number of cells active in bone formation were significantly higher in group III rabbits compared with group I and group II rabbits. These data reveal that osteoblasts differentiated from adipose tissue-derived MSCs shorten the consolidation period of distraction osteogenesis. Stem cells could be used as an effective treatment for bone defects. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  19. Stromal PDGFR-β Expression is Associated with Postoperative Survival of Non-Small Cell Lung Cancer Patients Receiving Preoperative Chemo- or Chemoradiotherapy Followed by Surgery.

    Science.gov (United States)

    Kanzaki, Ryu; Ose, Naoko; Kawamura, Tomohiro; Funaki, Soichiro; Shintani, Yasushi; Minami, Masato; Takakura, Nobuyuki; Okumura, Meinoshin

    2018-03-06

    PDGFR-β is used as a stromal biomarker and is functional in mesenchymal cells of the tumor microenvironment. The significance of stromal PDGFR-β expression in non-small cell lung cancer (NSCLC) in patients undergoing preoperative chemo- or chemoradiotherapy had not been determined. Patients with NSCLC undergoing preoperative chemo- or chemoradiotherapy between 1996 and 2014 were assessed for expression of stromal PDGFR-β by immunohistochemistry using resected specimens. Relationships between stromal PDGFR-β expression and survival after operation were analyzed. Forty-three patients who underwent surgery without preoperative treatment in 2005 were also analyzed as a chemo-naïve control group. The mean age of the 92 patients was 60.2 years. Seventy-eight (85%) were male, and 14 (15%) were female. Fifty-four patients (59%) underwent preoperative chemoradiotherapy, and 38 patients (41%) underwent preoperative chemotherapy. Regimens for preoperative chemotherapy were cisplatin (CDDP) based in 48 patients (52%) and carboplatin (CBDCA) based in 43 (42%). While stromal cells expressed PDGFR-β in 21 chemo-naïve patients (49%), stromal cells expressed PDGFR-β in 65 patients who underwent preoperative therapy (p = 0.02). The 5-year disease-free survival rate (DFS) of the PDGFR-β-positive group was significantly worse than that of the negative group (27 vs. 48%, p = 0.04). The 5-year disease-specific survival rate (DSS) in the stromal PDGFR-β-positive group was also significantly worse than in the negative group (43 vs. 70%, p = 0.01). On the other hand, stromal PDGFR-β expression did not influence survival in chemo-naïve patients. Stromal PDGFR-β expression is negatively associated with DFS and DSS in patients with NSCLC undergoing preoperative chemo- or chemoradiotherapy.

  20. Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

    Science.gov (United States)

    Pavlin, D; Zadro, R; Gluhak-Heinrich, J

    2001-10-01

    ) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.

  1. Nacre extract restores the mineralization capacity of subchondral osteoarthritis osteoblasts.

    Science.gov (United States)

    Brion, A; Zhang, G; Dossot, M; Moby, V; Dumas, D; Hupont, S; Piet, M H; Bianchi, A; Mainard, D; Galois, L; Gillet, P; Rousseau, M

    2015-12-01

    Osteoarthritis (OA) is the most common cause of joint chronic pain and involves the entire joints. Subchondral osteoarthritic osteoblasts present a mineralization defect and, to date, only a few molecules (Vitamin D3 and Bone Morphogenetic Protein2) could improve the mineralization potential of this cell type. In this context, we have tested for the first time the effect of nacre extract on the mineralization capacity of osteoblasts from OA patients. Nacre extract is known to contain osteogenic molecules which have demonstrated their activities notably on the MC3T3 pre-osteoblastic cell line. For this goal, molecules were extracted from nacre (ESM, Ethanol Soluble Matrix) and tested on osteoblasts of the subchondral bone from OA patients undergoing total knee replacement and on MC3T3 cells for comparison. We chose to investigate the mineralization with Alizarin Red staining and with the study of extracellular matrix (ECM) structure and composition. In a complementary way the structure of the ECM secreted during the mineralization phase was investigated using second harmonic generation (SHG). Nacre extract was able to induce the early presence (after 7 days) of precipitated calcium in cells. Raman spectroscopy and electron microscopy showed the presence of nanograins of an early crystalline form of calcium phosphate in OA osteoblasts ECM and hydroxyapatite in MC3T3 ECM. SHG collagen fibers signal was present in both cell types but lower for OA osteoblasts. In conclusion, nacre extract was able to rapidly restore the mineralization capacity of osteoarthritis osteoblasts, therefore confirming the potential of nacre as a source of osteogenic compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. An exceptional collision tumor: gastric calcified stromal tumor and ...

    African Journals Online (AJOL)

    The authors report an exceptional case of collision tumor comprised of a gastric calcified stromal tumor and a pancreatic adenocarcinoma. The pancreatic tumor was detected fortuitously on the histological exam of resection specimen. Key words: Collision tumor, stromal tumor, adenocarcinoma ...

  3. a stromal myoid cell line provokes thymic erythropoiesis between

    African Journals Online (AJOL)

    hi-tech

    81 No. 2 February 2004. A STROMAL MYOID CELL LINE PROVOKES THYMIC ERYTHROPOIESIS BETWEEN 16TH TO 20TH WEEKS OF INTRAUTERINE LIFE ... proliferation and differentiation in different stages of development: the stromal myoid cells. Design: ... human myasthenia gravis (MG) has been suggested(3).

  4. [PROFILE OF THE MARROW-DERIVED STROMAL PRECURSORS POPULATION IN C57BL/6N MICE FLOWN ON BIOSATELLITE BION-M1].

    Science.gov (United States)

    Markina, E A; Bobyleva, P I; Andrianova, I V; Andreeva, E R; Buravkova, L B

    2015-01-01

    The CFU-F number, proliferative activity and spontaneous differentiation potential of stromal cells derived from the tibia marrow of C57BL/6N mice readapted to the 1-g gravity following a long-term flight on biosatellite Bion-M1 were evaluated. The CFU-F number, proliferative activity and spontaneous adipogenic and osteogenic differentiation of marrow-derived stromal cells from the space flown group were no different from the group of vivarium control. However, the proliferative activity and adhesion properties of the cells were down-regulated on day 7 of readaptation. These results suggest that space flight factors did not impact the stromal differon of the mouse marrow. The decline of stromal cells activity indicates the decompensation of their functions under 1g gravity.

  5. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    Directory of Open Access Journals (Sweden)

    Bernadette Sosa-García

    2010-11-01

    Full Text Available The retinoblastoma protein (pRb is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

  6. Mammary fibroadenoma with pleomorphic stromal cells.

    Science.gov (United States)

    Abid, Najla; Kallel, Rim; Ellouze, Sameh; Mellouli, Manel; Gouiaa, Naourez; Mnif, Héla; Boudawara, Tahia

    2015-01-01

    The presence of enlarged and pleomorphic nuclei is usually regarded as a feature of malignancy, but it may on occasion be seen in benign lesions such as mammary fibroadenomas. We present such a case of fibroadenoma occurring in a 37-year-old woman presenting with a self-palpable right breast mass. Histological examination of the tumor revealed the presence of multi and mononucleated giant cells with pleomorphic nuclei. The recognition of the benign nature of these cells is necessary for differential diagnosis from malignant lesions of the breast. fibroadenoma - pleomorphic stromal cells - atypia - breast.

  7. Multilineage differentiation of porcine bone marrow stromal cells associated with specific gene expression pattern

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Chen, Li

    2008-01-01

    genes. However, it is not fully clear whether multilineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) of BMSC is associated with a specific gene expression pattern. In the present study, we investigated the gene expression pattern of representative transcription factors and marker......There are increasing reports regarding differentiation of bone marrow stromal cells (BMSC) from human and various species of animals including pigs. The phenotype and function of BMSC along a mesenchymal lineage differentiation are well characterized by specific transcription factors and marker...

  8. Bone marrow-derived mesenchymal stromal cell treatment in patients with severe ischaemic heart failure

    DEFF Research Database (Denmark)

    Mathiasen, Anders Bruun; Qayyum, Abbas Ali; Jørgensen, Erik

    2015-01-01

    AIMS: Regenerative treatment with mesenchymal stromal cells (MSCs) has been promising in patients with ischaemic heart failure but needs confirmation in larger randomized trials. We aimed to study effects of intra-myocardial autologous bone marrow-derived MSC treatment in patients with severe...... identified. CONCLUSION: Intra-myocardial injections of autologous culture expanded MSCs were safe and improved myocardial function in patients with severe ischaemic heart failure. STUDY REGISTRATION NUMBER: NCT00644410 (ClinicalTrials.gov)....

  9. Exendin-4 Induces Bone Marrow Stromal Cells Migration Through Bone Marrow-Derived Macrophages Polarization via PKA-STAT3 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Ning Wang

    2017-12-01

    Full Text Available Background/Aims: The synthesis and degradation processes involved in bone remodeling are critically regulated by osteoblasts and osteoclasts. The GLP-1 receptor agonist Exendin-4 is beneficial for osteoblast differentiation and increases the number of osteoblasts. Methods: We constructed an ovariectomized model to evaluate the impact of Exendin-4 on bone formation in osteoporosis. A macrophage-depleted model was also created to investigate the effect of macrophages on bone formation. Thirty-two female WT C57BL/6 mice (aged 3 months were randomly assigned to a normal control group and four ovariectomized (OVX subgroups: OVX + vehicle group, OVX + Exendin-4 (4.2 µg/kg/day group, OVX + chloride phosphate liposome group and OVX + chloride phosphate liposome + Exendin-4 group. Results: In this study, we found that Exendin-4 not only increased the number of osteoblasts and decreased the number of osteoclasts, but also increased the number of bone marrow stromal cells (BMSCs at the bone surface. Moreover, we found that OVX mice treated with Exendin-4 increased TGF-β1 levels at the bone surface compared with that in OVX mice. Besides, Exendin-4 promoted the polarization of bone marrow-derived macrophages into M2 subtype and increased TGF-β1 secretion by the M2 subtype. Finally, we found that Exendin-4 induced macrophage polarization via the cAMP-PKA-STAT3 signaling pathway. Conclusion: Exendin-4 promotes bone marrow-derived macrophage polarization to the M2 subtype and induces BMSC migration to the bone surface via PKA-STAT3 signaling.

  10. A diarylheptanoid phytoestrogen from Curcuma comosa, 1,7-diphenyl-4,6-heptadien-3-ol, accelerates human osteoblast proliferation and differentiation.

    Science.gov (United States)

    Tantikanlayaporn, Duangrat; Robinson, Lisa J; Suksamrarn, Apichart; Piyachaturawat, Pawinee; Blair, Harry C

    2013-06-15

    Curcuma comosa Roxb. is ginger-family plant used to relieve menopausal symptoms. Previous work showed that C. comosa extracts protect mice from ovariectomy-induced osteopenia with minimal effects on reproductive organs, and identified the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD) as the major active component of C. comosa rhizomes. At 1-10μM, DPHD increased differentiation in transformed mouse osteoblasts, but the effect of DPHD on normal bone cells was unknown. We examined the concentration dependency and mechanism of action of DPHD relative to 17β-estradiol in nontransformed human osteoblasts (h-OB). The h-OB were 10-100 fold more sensitive to DPHD than transformed osteoblasts: DPHD increased h-OB proliferation at 10nM and, at 100nM, activated MAP kinase signaling within 30 min. In long-term differentiation assays, responses of h-OB to DPHD were significant at 10nM, and optimal response in most cases was at 100 nM. At 7-21 days, DPHD accelerated osteoblast differentiation, indicated by alkaline phosphatase activity and osteoblast-specific mRNA production. Effects of DPHD were eliminated by the estrogen receptor antagonist ICI182780. During differentiation, DPHD promoted early expression of osteoblast transcription factors, RUNX2 and osterix. Subsequently, DPHD accelerated production of bone structural genes, including COL1A1 and osteocalcin comparably to 17β-estradiol. In h-OB, DPHD increased the osteoprotegerin to RANKL ratio and supported mineralization more efficiently than 10nM 17β-estradiol. We conclude that DPHD promotes human osteoblast function in vitro effectively at nanomolar concentrations, making it a promising compound to protect bone in menopausal women. Copyright © 2013 Elsevier GmbH. All rights reserved.

  11. Accelerated cell cycle progression in osteoblasts overexpressing the c-fos proto-oncogene: induction of cyclin A and enhanced CDK2 activity.

    Science.gov (United States)

    Sunters, Andrew; Thomas, David P; Yeudall, W Andrew; Grigoriadis, Agamemnon E

    2004-03-12

    Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cell cycle genes. Here we have generated osteoblast cell lines expressing c-fos under the control of a tetracycline-regulatable promoter to investigate the role of c-Fos in osteoblast cell cycle control in vitro. Three stable subclones, AT9.2, AT9.3, and AT9.7, derived from MC3T3-E1 mouse osteoblasts, expressed high levels of exogenous c-fos mRNA and protein in the absence of tetracycline. Functional contribution of ectopic c-Fos to AP-1 complexes was confirmed by electromobility shift assays and transactivation of AP-1 reporter constructs. Induction of exogenous c-Fos in quiescent AT9.2 cells caused accelerated S-phase entry following serum stimulation, resulting in enhanced growth rate. Ectopic c-Fos resulted in increased expression of cyclins A and E protein levels, and premature activation of cyclin A-, cyclin E-, and cyclin-dependent kinase (CDK) 2-associated kinase activities, although cyclin D levels and CDK4 activity were not affected significantly in these cell lines. The enhanced CDK2 kinase activity was associated with a rapid, concomitant dissociation of p27 from CDK2-containing complexes. Deregulated cyclin A expression and CDK2 activity was also observed in primary mouse osteoblasts overexpressing c-Fos, but not in fibroblasts, and c-Fos transgenic tumor-derived osteosarcoma cells constitutively expressed high levels of cyclin A protein. These data suggest that overexpression of c-Fos in osteoblasts results in accelerated S phase entry as a result of deregulated cyclin A/E-CDK2 activity. This represents a novel role for c-Fos in osteoblast growth control and may provide c-Fos-overexpressing osteoblasts with a growth advantage during tumorigenesis.

  12. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis.

    Science.gov (United States)

    Bronckaers, Annelies; Hilkens, Petra; Martens, Wendy; Gervois, Pascal; Ratajczak, Jessica; Struys, Tom; Lambrichts, Ivo

    2014-08-01

    Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  14. A novel flavonoid C-glucoside from Ulmus wallichiana preserves bone mineral density, microarchitecture and biomechanical properties in the presence of glucocorticoid by promoting osteoblast survival: a comparative study with human parathyroid hormone.

    Science.gov (United States)

    Khan, M P; Mishra, J S; Sharan, K; Yadav, M; Singh, A K; Srivastava, A; Kumar, S; Bhaduaria, S; Maurya, R; Sanyal, S; Chattopadhyay, N

    2013-11-15

    6-C-β-D-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF) is a novel compound isolated from Ulmus wallichiana, reported to have bone anabolic action in ovariectomized rats. Here, we studied the effect of GTDF in glucocorticoid (GC)-induced bone loss and its mode of action. Osteoblasts were cultured from rat calvaria or bone marrow to study apoptosis and differentiation by dexamethasone (Dex), methylprednisolone (MP), GTDF, quercetin and rutin. Female Sprague Dawley rats were treated with Dex or MP with or without GTDF or PTH. Efficacy was evaluated by bone microarchitecture using microcomputed tomography, determination of new bone formation by fluorescent labeling of bone and osteoblast apoptosis by co-labeling bone sections with Runx-2 and TUNEL. Serum osteocalcin was determined by ELISA. GTDF preserved trabecular and cortical bones in the presence of Dex and MP and mitigated the MP-mediated suppression of serum osteocalcin. Co-administration of GTDF to MP rats increased mineral apposition, bone formation rates, bone biomechanical strength, reduced osteoblast apoptosis and increased osteogenic differentiation of bone marrow stromal cells compared to MP group, suggesting in vivo osteogenic effect of GTDF. These effects of GTDF were to a great extent comparable to PTH. GTDF prevented GC-induced osteoblast apoptosis by inhibiting p53 expression and acetylation, and activation of AKT but did not influence transactivation of GC receptor (GR). GTDF protects against GC-induced bone loss by promoting osteoblast survival through p53 inhibition and activation of AKT pathways but not as a GR antagonist. GTDF has the potential in the management of GC-induced osteopenia. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. Similar healthy osteoclast and osteoblast activity on nanocrystalline hydroxyapatite and nanoparticles of tri-calcium phosphate compared to natural bone

    Directory of Open Access Journals (Sweden)

    MacMillan AK

    2014-12-01

    Full Text Available Adam K MacMillan,1 Francis V Lamberti,1 Julia N Moulton,2 Benjamin M Geilich,2 Thomas J Webster2,3 1RTI Surgical, Alachua, FL, USA; 2Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 3Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: While there have been numerous studies to determine osteoblast (bone forming cell functions on nanocrystalline compared to micron crystalline ceramics, there have been few studies which have examined osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and receptor activator of nuclear factor κB [RANK]. This is despite the fact that osteoclasts are an important part of maintaining healthy bone since they resorb bone during the bone remodeling process. Moreover, while it is now well documented that bone formation is enhanced on nanoceramics compared to micron ceramics, some have pondered whether osteoblast functions (such as osteoprotegerin and RANK ligand [RANKL] are normal (ie, non-diseased on such materials compared to natural bone. For these reasons, the objective of the present in vitro study was to determine various functions of osteoclasts and osteoblasts on nanocrystalline and micron crystalline hydroxyapatite as well as tri-calcium phosphate materials and compare such results to cortical and cancellous bone. Results showed for the first time similar osteoclast activity (including tartrate-resistant acid phosphatase, formation of resorption pits, size of resorption pits, and RANK and osteoblast activity (osteoprotegerin and RANKL on nanocrystalline hydroxyapatite compared to natural bone, whereas osteoclast and osteoblast functions on micron crystalline versions of these ceramics were much different than natural bone. In this manner, this study provides additional evidence that nanocrystalline calcium phosphates can serve as suitable synthetic

  16. Adipose-Derived Stromal Cells for Treatment of Patients with Chronic Ischemic Heart Disease (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Mathiasen, Anders Bruun; Mygind, Naja Dam

    2017-01-01

    We aimed to evaluate the effect of intramyocardial injections of autologous VEGF-A165-stimulated adipose-derived stromal cells (ASCs) in patients with refractory angina. MyStromalCell trial is a randomized double-blind placebo-controlled study including sixty patients with CCS/NYHA class II...

  17. Osteoblastic cytokine response to gray and white mineral trioxide aggregate.

    Science.gov (United States)

    Bidar, Maryam; Zarrabi, Mohammad Hasan; Tavakol Afshari, Jalil; Aghasizadeh, Navid; Naghavi, Neda; Forghanirad, Maryam; Attaran, Niloufar

    2011-01-01

    The materials used for root-end filling and perforation repair are in direct contact with live tissues e.g. bone and connective tissue; their effects however, are uncertain. The aim of this ex vivostudy was to evaluate the osteoblastic secretory activity adjacent to gray and white mineral trioxide aggregate (MTA) and Intermediate Restorative Material (IRM). The studied materials were prepared and placed in 24-wells plate. Human MG-63 osteoblasts were introduced to materials after their initial set. The supernatant fluid was collected after 1, 3, and 7 days and the level of interleukin-1β was measured by ELISA test. A microscopic exam was also performed to assess proliferation and viability of the cells. Kruskal-Wallis and Tukey tests were used for analysis. T here were significant higher levels of interleukin-1β in the gray and white MTA groups compared to IRM group (P0.05).Morphologic appearance of osteoblasts adjacent to gray and white MTA was similar to normal osteoblasts in all observation periods, however cells adjacent to IRM were round, signifying cytotoxicity of the adjacent material. Human osteoblasts' has a favorable biologic response to white and gray MTA compared to IRM.

  18. Effect of apatite cements on human osteoblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yuasa, T.; Miyamoto, Y.; Takechi, M.; Momota, Y.; Toh, T.; Nagayama, M. [Tokushima Univ. (Japan). First Dept. of Oral and Maxillofacial Surgery; Ishikawa, K.; Suzuki, K. [Okayama Univ. Dental School (Japan). Dept. of Biomaterials

    2001-07-01

    A conventional apatite cement (c-AC) used for reconstruction of bony defect takes 30 min to set and decays upon exposure to liquid before setting. Therefore, we have developed two types of new ACs; fast-setting AC and anti-washout type AC. The aims of this study are to investigate the effect of ACs on cultured osteoblasts and to compare effects of new ACs on osteoblasts with that of c-AC. Disc shape set ACs with almost same surface roughness were prepared and evaluated by powder X-ray diffraction (XRD). The clonal human osteoblasts were cultured on each specimens. Plastic dish and sintered hydroxyapatite (HAP) were used as control. ACs showed the same apatitic XRD patterns with poor crystallinity when compared with sintered HAP. There were no differences in proliferation of osteoblasts among ACs and sintered HAP. The alkaline phosphatase activity and protein levels for type I collagen and osteocalcin of ACs were greater than those of sintered HAP. There was no difference among ACs. In conclusion, it is suggested that ACs could promote the differentiation of osteoblasts as compared with sintered HAP and plastic. Also, it is suggested that two types of new ACs would show the same cell response as c-AC in vivo (orig.)

  19. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  20. Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

    Science.gov (United States)

    Shalhoub, V.; Conlon, D.; Tassinari, M.; Quinn, C.; Partridge, N.; Stein, G. S.; Lian, J. B.

    1992-01-01

    To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the

  1. Ultrastructural characterization of mesenchymal stromal cells labeled with ultrasmall superparamagnetic iron-oxide nanoparticles for clinical tracking studies

    DEFF Research Database (Denmark)

    Hansen, Louise; Hansen, Alastair B; Mathiasen, Anders B

    2014-01-01

    INTRODUCTION: To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell...

  2. PPAR agonists stimulate adipogenesis at the expense of osteoblast differentiation while inhibiting osteoclast formation and activity.

    Science.gov (United States)

    Patel, Jessal J; Butters, Oliver R; Arnett, Timothy R

    2014-06-01

    Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator-activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR-γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR-α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone-treated or fenofibrate-treated cultures; at the same time, lipid droplet formation was increased by 40-70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR-γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR-α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis. Copyright © 2014 John Wiley & Sons, Ltd.

  3. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

    Directory of Open Access Journals (Sweden)

    Kohei Murakami

    Full Text Available The Janus kinases (Jaks are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL. Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3 and prostaglandin E2 (PGE2 in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6, IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

  4. A Jak1/2 inhibitor, baricitinib, inhibits osteoclastogenesis by suppressing RANKL expression in osteoblasts in vitro.

    Science.gov (United States)

    Murakami, Kohei; Kobayashi, Yasuhiro; Uehara, Shunsuke; Suzuki, Takako; Koide, Masanori; Yamashita, Teruhito; Nakamura, Midori; Takahashi, Naoyuki; Kato, Hiroyuki; Udagawa, Nobuyuki; Nakamura, Yukio

    2017-01-01

    The Janus kinases (Jaks) are hubs in the signaling process of more than 50 cytokine or hormone receptors. However, the function of Jak in bone metabolism remains to be elucidated. Here, we showed that the inhibition of Jak1 and/or Jak2 in osteoblast-lineage cells led to impaired osteoclastogenesis due to the reduced expression of receptor activator of nuclear factor-κB ligand (RANKL). Murine calvaria-derived osteoblasts induced differentiation of bone marrow cells into osteoclasts in the presence of 1,25-dihydroxyvitamin D3 (1,25D3) and prostaglandin E2 (PGE2) in vitro. However, treatment with the Jak1/2 inhibitor, baricitinib, markedly inhibited osteoclastogenesis in the co-culture. On the other hand, baricitinib did not inhibit RANKL-induced osteoclast differentiation of bone marrow macrophages. These results indicated that baricitinib acted on osteoblasts, but not on bone marrow macrophages. Baricitinib suppressed 1,25D3 and PGE2-induced up-regulation of RANKL in osteoblasts, but not macrophage colony-stimulating factor expression. Moreover, the addition of recombinant RANKL to co-cultures completely rescued baricitinib-induced impairment of osteoclastogenesis. shRNA-mediated knockdown of Jak1 or Jak2 also suppressed RANKL expression in osteoblasts and inhibited osteoclastogenesis. Finally, cytokine array revealed that 1,25D3 and PGE2 stimulated secretion of interleukin-6 (IL-6), IL-11, and leukemia inhibitory factor in the co-culture. Hence, Jak1 and Jak2 represent novel therapeutic targets for osteoporosis as well as inflammatory bone diseases including rheumatoid arthritis.

  5. Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone.

    Directory of Open Access Journals (Sweden)

    Jinhu Xiong

    Full Text Available The cytokine receptor activator of nuclear factor kappa B ligand (RANKL, encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

  6. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    Science.gov (United States)

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Carbon nanotube-reinforced hydroxyapatite composite and their interaction with human osteoblast in vitro.

    Science.gov (United States)

    Khalid, P; Hussain, M A; Rekha, P D; Arun, A B

    2015-05-01

    As a bone mineral component, hydroxyapatite (HA) has been an attractive bioceramic for the reconstruction of hard tissues. However, its poor mechanical properties, including low fracture toughness and tensile strength, have been a substantial challenge to the application of HA for the replacement of load-bearing and/or large bone defects. In this study, HA is reinforced with high-purity and well-functionalized multiwalled carbon nanotubes (MWCNTs; >99 wt%) having an average diameter of 15 nm and length from 10 to 20 μm. The cellular response of these functionalized CNTs and its composites were examined in human osteoblast sarcoma cell lines. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4) were used to synthesize HA in situ. MWCNTs were functionalized by heating at 100°C in 3:1 ratio of sulfuric acid and nitric acid for 60 min with stirring and dispersed in sodium dodecyl benzene sulfonate by sonication. HA particles were produced in MWCNTs solution by adding Ca(NO3)2·4H2O and (NH4)2HPO4 under vigorously stirring conditions. The composite was dried and washed in distilled water followed by heat treatment at 250°C to obtain CNT-HA powder. Physiochemical characterization of the composite material was carried out using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectrometer, and X-ray diffractometer. Furthermore, this study investigates the cytotoxic effects of functionalized-MWCNTs (f-MWCNTs) and its composites with HA in human osteoblast sarcoma cell lines. Human osteoblast cells were exposed with different concentrations of f-MWCNTs and its composite with HA. The interactions of f-MWCNT and MWCNT-HA composites were analyzed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The results indicate no detrimental effect on survival or mitochondrial activity of the osteoblast cells. Cell viability decreased with an increase in CNT

  8. Loss of menin in osteoblast lineage affects osteocyte?osteoclast crosstalk causing osteoporosis

    OpenAIRE

    Liu, Peng; Lee, Sooyeon; Knoll, Jeanette; Rauch, Alexander; Ostermay, Susanne; Luther, Julia; Malkusch, Nicole; Lerner, Ulf H; Zaiss, Mario M; Neven, Mona; Wittig, Rainer; Rauner, Martina; David, Jean-Pierre; Bertolino, Philippe; Zhang, Chang X

    2017-01-01

    During osteoporosis bone formation by osteoblasts is reduced and/or bone resorption by osteoclasts is enhanced. Currently, only a few factors have been identified in the regulation of bone integrity by osteoblast-derived osteocytes. In this study, we show that specific disruption of menin, encoded by multiple endocrine neoplasia type 1 (Men1), in osteoblasts and osteocytes caused osteoporosis despite the preservation of osteoblast differentiation and the bone formation rate. Instead, an incre...

  9. Aluminum Trichloride Inhibits the Rat Osteoblasts Mineralization In Vitro.

    Science.gov (United States)

    Song, Miao; Huo, Hui; Cao, Zheng; Han, Yanfei; Gao, Li

    2017-01-01

    Aluminum (Al) is an accumulative toxic metal. Excessive Al accumulation inhibits osteoblasts mineralization and induces osteoporosis. However, the inhibition mechanism of Al on the mineralization is not fully understood. Thus, in this study, the rat osteoblasts were cultured and exposed to 0 mmol L -1 (control group, CG) and 0.52 mmol L -1 aluminum trichloride (AlCl 3 , treatment group, TG) for 7, 14, and 21 days, respectively. We found that mineralized matrix nodules, the activity of bone alkaline phosphatase, the concentration of extracellular calcium, the mRNA expression of type-I collagen, the mRNA and protein expressions of osteopontin, osteocalcin, and bone sialoprotein were all decreased, while the concentration of extracellular phosphorus was increased in TG compared with CG with time prolonged. Taken together, these results indicated that AlCl 3 inhibited osteoblasts mineralization in vitro.

  10. Osteoblast recruitment routes in human cancellous bone remodeling

    DEFF Research Database (Denmark)

    Kristensen, Helene B; Levin Andersen, Thomas; Marcussen, Niels

    2014-01-01

    It is commonly proposed that bone forming osteoblasts recruited during bone remodeling originate from bone marrow perivascular cells, bone remodeling compartment canopy cells, or bone lining cells. However, an assessment of osteoblast recruitment during adult human cancellous bone remodeling...... is lacking. We addressed this question by quantifying cell densities, cell proliferation, osteoblast differentiation markers, and capillaries in human iliac crest biopsy specimens. We found that recruitment occurs on both reversal and bone-forming surfaces, as shown by the cell density and osterix levels...... on these respective surfaces, and that bone formation occurs only above a given cell density. Canopies appeared an important source of osteoprogenitors, because (i) canopy cells proved to be more proliferative and less differentiated than bone surface cells, as shown by the inverse levels of Ki-67 and procollagen-3 N...

  11. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    International Nuclear Information System (INIS)

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C.

    1989-01-01

    Isolated mouse osteoblasts that retain their osteogenic activity in culture were incubated with [35S] sulfate. Two radiolabeled proteins, in addition to proteoglycans, were extracted from the calcified matrix of osteoblast cultures. All the sulfate label in both proteins was in the form of tyrosine sulfate as assessed by amino acid analysis and thin layer chromatography following alkaline hydrolysis. The elution behavior on DEAE-Sephacel of the major sulfated protein and the apparent Mr on sodium dodecyl sulfate gels were characteristic of bone sialoprotein II extracted from rat. This protein was shown to cross-react with an antiserum raised against bovine bone sialoprotein II, indicating that bone sialoprotein II synthesized by cultured mouse osteoblasts is a tyrosine-sulfated protein. The minor sulfated protein was tentatively identified as bone sialoprotein I or osteopontin based on its elution properties on DEAE-Sephacel and anomalous behavior on sodium dodecyl sulfate gels similar to those reported for rat bone sialoprotein I

  12. Effects of space-relevant radiation on pre-osteoblasts

    International Nuclear Information System (INIS)

    Hu, Yueyuan

    2014-01-01

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  13. Effects of space-relevant radiation on pre-osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yueyuan

    2014-02-12

    Until now limited research has been conducted to address the mechanisms leading ionizing radiation exposure induced bone loss. This is relevant for cancer radiotherapy and human spaceflight. Exposure to radiation can result in elevated bone fracture risk in patients receiving cancer radiotherapy. In human spaceflight, astronauts are exposed to space radiation which is a very complex mixture consisting primarily of high-energy charged particles. Osteoblasts are of mesenchymal origin and responsible for creating and maintaining skeletal architecture; these cells produce extracellular matrix proteins and regulators of matrix mineralization during initial bone formation and later bone remodeling. The aim of this work was to investigate the effects of ionizing radiation on pre-osteoblasts including cellular survival, cell cycle regulation and differentiation modification. Experiments with the pre-osteoblast cell line OCT-1 and the mesenchymal stem cell line C3H10T1/2 showed that radiation cell killing depends on dose and linear energy transfer (LET) and is most effective at an LET of ∝150 keV/μm. High-LET radiation has a much more pronounced ability to induce cell cycle arrest in the G2/M phase. After both X-rays and heavy ions exposure, expression of the cell cycle regulator CDKN1A was significantly up-regulated in a dose-dependent manner. The findings suggest that cell cycle regulation is more sensitive to high-LET radiation than cell survival, which is not solely regulated through elevated CDKN1A expression. Radiation exposure enhances osteoblastic differentiation and maturation, and mediates Runx2 and TGF-β1 expression during early differentiation of pre-osteoblasts. Osteogenic differentiation did not alter cellular radiosensitivity, DNA repair of radiation-induced damages and the effects of radiation on proliferation. Further experiments are needed to elucidate possible synergistic effects of microgravity and radiation on osteoblast differentiation. This may

  14. Climbing exercise enhances osteoblast differentiation and inhibits adipogenic differentiation with high expression of PTH/PTHrP receptor in bone marrow cells.

    Science.gov (United States)

    Menuki, Kunitaka; Mori, Toshiharu; Sakai, Akinori; Sakuma, Miyuki; Okimoto, Nobukazu; Shimizu, Yuki; Kunugita, Naoki; Nakamura, Toshitaka

    2008-09-01

    We developed previously a mouse voluntary climbing exercise model as a physiological mechanical loading model and reported that climbing exercise increased bone formation, but its effect on adipogenesis is unknown. We assessed the effects of loading and PTH/PTHrP receptor (PTHR1) on bone marrow adipocyte differentiation in relation with osteoblast differentiation. 8-week-old C57BL/6J male mice were divided into ground control (GC) and climbing exercise (EX) group. Mice were housed in 100-cm towers and climbed up toward a bottle placed at the top of the cage to drink water. The values of bone volume and osteoblast number were significantly higher while those of marrow adipocyte volume and number were significantly lower in the 28dayEX group than 28dayGC group. The mRNA expression levels of adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBP) beta and delta were lower in 4dayEX mice, while the adipocyte specific genes fatty acid binding protein (aP2) and phosphoenolpyruvate carboxykinase (PEPCK) expressions were lower in 7dayEX mice. In primary bone marrow cell cultures, the number of alkaline phosphatase-positive colony forming units-fibroblastic (ALP+ CFU-f) and Oil-red-O-positive cells were both increased in the 4dayEX group. Climbing exercise transiently increases both osteogenic and adipogenic potential in bone marrow stromal cells, and inhibits terminal adipocyte differentiation and promotes osteoblast differentiation. Immunoreactivity for the PTHR1 was intense on osteoblastic cell lineage in the endosteal tibial metaphysis. PTHR1 mRNA expression was increased in 4dayEX mice and PTHR1-positive cells were increased after 7 days in the experimental group. Ex vivo addition of PTHR1 antibody decreased and that of PTHrP(1-34) increased the number of ALP+ CFU-f in bone marrow cell cultures obtained at 4 days after the exercise, while the addition of PTHR1 antibody increased and PTHrP(1-34) decreased the number of Oil-red-O-positive cells. Our

  15. Heat shock protein 27 (HSPB1) suppresses the PDGF-BB-induced migration of osteoblasts

    Science.gov (United States)

    Kainuma, Shingo; Tokuda, Haruhiko; Yamamoto, Naohiro; Kuroyanagi, Gen; Fujita, Kazuhiko; Kawabata, Tetsu; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

    2017-01-01

    Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB-induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild-type HSP27-overexpressing cells and the phospho-mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPK, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPK, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27. PMID:28902366

  16. Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.

    Science.gov (United States)

    Hess, A P; Hamilton, A E; Talbi, S; Dosiou, C; Nyegaard, M; Nayak, N; Genbecev-Krtolica, O; Mavrogianis, P; Ferrer, K; Kruessel, J; Fazleabas, A T; Fisher, S J; Giudice, L C

    2007-01-01

    During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and

  17. Adipose-derived mesenchymal stromal cells for chronic myocardial ischemia (MyStromalCell Trial)

    DEFF Research Database (Denmark)

    Qayyum, Abbas Ali; Haack-Sørensen, Mandana; Mathiasen, Anders Bruun

    2012-01-01

    Adipose tissue represents an abundant, accessible source of multipotent adipose-derived stromal cells (ADSCs). Animal studies have suggested that ADSCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes. This makes ADSCs a promising new cell source....... In addition, we give an introduction to the first-in-man clinical trial, MyStromalCell Trial, which is a prospective, randomized, double-blind, placebo-controlled study using culture-expanded ADSCs obtained from adipose-derived cells from abdominal adipose tissue and stimulated with VEGF-A(165) the week...... for regenerative therapy to replace injured tissue by creating new blood vessels and cardiomyocytes in patients with chronic ischemic heart disease. The aim of this special report is to review the present preclinical data leading to clinical stem cell therapy using ADSCs in patients with ischemic heart disease...

  18. CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells in the tumor capsule of skin sweat gland neoplasms.

    Science.gov (United States)

    Nakayama, Hirofumi; Enzan, Hideaki; Miyazaki, Eriko; Moriki, Toshiaki; Toi, Makoto; Zhang, Yanhu

    2002-01-01

    To elucidate the roles of CD34-positive stromal cells and alpha-smooth muscle actin-positive stromal cells at the tumor border of skin sweat gland neoplasms, we examined expression of stromal cell markers in the tumor capsule of 19 skin sweat gland neoplasms (16 mixed tumors of the skin and three nodular hidradenomas) using monoclonal antibodies to CD34, CD31, cytokeratin 14 (CK14), alpha-smooth muscle actin (ASMA) and high molecular weight caldesmon (HCD). We regarded CD34-positive, CD31-, CK14-, ASMA- and HCD-negative stromal cells to be CD34-positive stromal cells, and ASMA-positive, HCD-, CK14-, CD34- and CD31-negative stromal cells to be ASMA-positive stromal cells. CD34-positive stromal cells were detected in the tumor capsule of all 19 of the tumors examined. In nine of the 16 mixed tumors (56%) and all of the three nodular hidradenomas, ASMA-positive stromal cells were detected at the immediate inner side of the CD34-positive stromal cell layers. These results indicate that cellular components in the tumor capsules of mixed tumors of the skin and nodular hidradenomas are CD34-positive stromal cells and ASMA-positive stromal cells, and suggest that stromal cells of these two cell types are associated with tumor capsule formation of skin sweat gland neoplasms.

  19. Engineering stromal-epithelial interactions in vitro for ...

    Science.gov (United States)

    Background: Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue function. Epithelial-mesenchymal interactions (EMIs) have been examined using mammalian models, ex vivo tissue recombination, and in vitro co-cultures. Although these approaches have elucidated signaling mechanisms underlying morphogenetic processes and adult mammalian epithelial tissue function, they are limited by the availability of human tissue, low throughput, and human developmental or physiological relevance. Objectives: Bioengineering strategies to promote EMIs using human epithelial and mesenchymal cells have enabled the development of human in vitro models of adult epidermal and glandular tissues. In this review, we describe recent bioengineered models of human epithelial tissue and organs that can instruct the design of organotypic models of human developmental processes.Methods: We reviewed current bioengineering literature and here describe how bioengineered EMIs have enabled the development of human in vitro epithelial tissue models.Discussion: Engineered models to promote EMIs have recapitulated the architecture, phenotype, and function of adult human epithelial tissue, and similar engineering principles could be used to develop models of developmental morphogenesis. We describe how bioengineering strategies including bioprinting and spheroid culture could be implemented to

  20. A relativity concept in mesenchymal stromal cell manufacturing.

    Science.gov (United States)

    Martin, Ivan; De Boer, Jan; Sensebe, Luc

    2016-05-01

    Mesenchymal stromal cells (MSCs) are being experimentally tested in several biological systems and clinical settings with the aim of verifying possible therapeutic effects for a variety of indications. MSCs are also known to be heterogeneous populations, with phenotypic and functional features that depend heavily on the individual donor, the harvest site, and the culture conditions. In the context of this multidimensional complexity, a recurrent question is whether it is feasible to produce MSC batches as "standard" therapeutics, possibly within scalable manufacturing systems. Here, we provide a short overview of the literature on different culture methods for MSCs, including those employing innovative technologies, and of some typically assessed functional features (e.g., growth, senescence, genomic stability, clonogenicity, etc.). We then offer our perspective of a roadmap on how to identify and refine manufacturing systems for MSCs intended for specific clinical indications. We submit that the vision of producing MSCs according to a unique standard, although commercially attractive, cannot yet be scientifically substantiated. Instead, efforts should be concentrated on standardizing methods for characterization of MSCs generated by different groups, possibly covering a vast gamut of functionalities. Such assessments, combined with hypotheses on the therapeutic mode of action and associated clinical data, should ultimately allow definition of in-process controls and measurable release criteria for MSC manufacturing. These will have to be validated as predictive of potency in suitable pre-clinical models and of therapeutic efficacy in patients. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  1. Sex cord-gonadal stromal tumor of the rete testis.

    Science.gov (United States)

    Sajadi, Kamran P; Dalton, Rory R; Brown, James A

    2009-01-01

    A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

  2. Gastrointestinal Stromal Tumor – An Evolving Concept

    Science.gov (United States)

    Tornillo, Luigi

    2014-01-01

    Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. The discovery that these tumors, formerly thought of smooth muscle origin, are indeed better characterized by specific activating mutation in genes coding for the receptor tyrosine kinases (RTKs) CKIT and PDGFRA and that these mutations are strongly predictive for the response to targeted therapy with RTK inhibitors has made GISTs the typical example of the integration of basic molecular knowledge in the daily clinical activity. The information on the mutational status of these tumors is essential to predict (and subsequently to plan) the therapy. As resistant cases are frequently wild type, other possible oncogenic events, defining other “entities,” have been discovered (e.g., succinil dehydrogenase mutation/dysregulation, insuline growth factor expression, and mutations in the RAS-RAF-MAPK pathway). The classification of disease must nowadays rely on the integration of the clinico-morphological characteristics with the molecular data. PMID:25593916

  3. Prostatic stromal microenvironment and experimental diabetes

    Directory of Open Access Journals (Sweden)

    DL Ribeiro

    2009-06-01

    Full Text Available The diabetes causes alterations in various organ systems, including the male accessory sex glands. The prostate is very important in the reproductive process and it is a frequent target of malignant changes. The aim of this work was to demonstrate the histochemical and ultrastructural alterations in the prostate of diabetic animals. Two groups of animals were utilized: control and non-obese diabetic mice (NOD. Twelve days after the characterization of diabetic status the ventral prostate was collected, fixed in Karnovsky and paraformaldehyde, processed for histochemistry and TEM associated to stereology. The results showed reduction of the epithelial area and increasing of the stromal area with muscular and collagen hypertrophy in the prostatic gland. It was characterized the development of prostatic intraepithelial neoplasia, inflammatory processes and dilation of the organelles involved in the secretory process. It was concluded that diabetes besides damaging the reproductive process, affects the glandular homeostasis favoring the development of prostatic pathologies.

  4. Cryopreservation and revival of mesenchymal stromal cells

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Kastrup, Jens

    2011-01-01

    Over the past few years, the pace of preclinical stem cell research is astonishing and adult stem cells have become the subject of intense research. Due to the presence of promising supporting preclinical data, human clinical trials for stem cell regenerative treatment of various diseases have been...... initiated. As there has been a precedent for the use of bone marrow stem cells in the treatment of hematological malignancies and ischemic heart diseases through randomized clinical safety and efficacy trials, the development of new therapies based on culture-expanded human mesenchymal stromal cells (MSCs......) opens up new possibilities for cell therapy. To facilitate these applications, cryopreservation and long-term storage of MSCs becomes an absolute necessity. As a result, optimization of this cryopreservation protocol is absolutely critical. The major challenge during cellular cryopreservation...

  5. Ovarian Sex Cord-Stromal Tumors.

    Science.gov (United States)

    Schultz, Kris Ann P; Harris, Anne K; Schneider, Dominik T; Young, Robert H; Brown, Jubilee; Gershenson, David M; Dehner, Louis P; Hill, D Ashley; Messinger, Yoav H; Frazier, A Lindsay

    2016-10-01

    Ovarian sex cord-stromal tumors are clinically significant heterogeneous tumors that include several pathologic types. These tumors are often found in adolescents and young adults and can present with hormonal manifestations as well as signs and symptoms of a pelvic mass. Serum tumor markers may assist in preoperative diagnosis and surveillance. Several subtypes are associated with genetic predisposition, including those observed in patients with Peutz-Jegher syndrome. Recent studies have elucidated the relationship between Sertoli-Leydig cell tumors and DICER1 mutations. When classified as International Federation of Gynecology and Obstetrics stage Ia, most subtypes may be treated with surgery alone. Higher stage or recurrent tumors have variable prognoses that range from a usually rapid course in poorly differentiated Sertoli-Leydig cell tumor to an often prolonged course in adult granulosa cell tumors. New understanding of the molecular pathogenesis of these tumors may pave the way for novel therapeutics.

  6. Primary human osteoblasts with reduced alkaline phosphatase and matrix mineralization baseline capacity are responsive to extremely low frequency pulsed electromagnetic field exposure - Clinical implication possible.

    Science.gov (United States)

    Ehnert, Sabrina; Falldorf, Karsten; Fentz, Anne-Kristin; Ziegler, Patrick; Schröter, Steffen; Freude, Thomas; Ochs, Björn G; Stacke, Christina; Ronniger, Michael; Sachtleben, Jens; Nussler, Andreas K

    2015-12-01

    For many years electromagnetic fields (EMFs) have been used clinically with various settings as an exogenous stimulation method to promote fracture healing. However, underlying mechanisms of action and EMF parameters responsible for certain effects remain unclear. Our aim was to investigate the influence of defined EMFs on human osteoblasts' and osteoclasts' viability and function. Primary human osteoblasts and osteoclasts were treated 3 times weekly for 21 days during their maturation process using the Somagen® device (Sachtleben GmbH, Hamburg, Germany), generating defined extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs). Certain ELF-PEMF treatment significantly increased the total protein content (up to 66%), mitochondrial activity (up to 91.1%) and alkaline phosphatase (AP) activity (up to 129.9%) of human osteoblasts during the entire differentiation process. Furthermore, ELF-PEMF treatment enhanced formation of mineralized matrix (up to 276%). Interestingly, ELF-PEMF dependent induction of AP activity and matrix mineralization was strongly donor dependent - only osteoblasts with a poor initial osteoblast function responded to the ELF-PEMF treatment. As a possible regulatory mechanism, activation of the ERK1/2 signaling pathway was identified. Maturation of osteoclasts from human monocytes was not affected by the ELF-PEMF treatment. In summary the results indicate that a specific ELF-PEMF treatment with the Somagen® device improves viability and maturation of osteoblasts, while osteoclast viability and maturation was not affected. Hence, ELF-PEMF might represent an interesting adjunct to conventional therapy supporting bone formation during fracture healing or even for the treatment of osteoporosis.

  7. Primary human osteoblasts with reduced alkaline phosphatase and matrix mineralization baseline capacity are responsive to extremely low frequency pulsed electromagnetic field exposure — Clinical implication possible

    Directory of Open Access Journals (Sweden)

    Sabrina Ehnert

    2015-12-01

    Full Text Available For many years electromagnetic fields (EMFs have been used clinically with various settings as an exogenous stimulation method to promote fracture healing. However, underlying mechanisms of action and EMF parameters responsible for certain effects remain unclear. Our aim was to investigate the influence of defined EMFs on human osteoblasts' and osteoclasts' viability and function. Primary human osteoblasts and osteoclasts were treated 3 times weekly for 21 days during their maturation process using the Somagen® device (Sachtleben GmbH, Hamburg, Germany, generating defined extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs. Certain ELF-PEMF treatment significantly increased the total protein content (up to 66%, mitochondrial activity (up to 91.1% and alkaline phosphatase (AP