Sample records for bone cell cultures

  1. Rat bone marrow stem cells isolation and culture as a bone formative experimental system

    PubMed Central

    Smajilagi?, Amer; Alji?evi?, Mufida; Redži?, Amira; Filipovi?, Selma; Lagumdžija, Alena C.

    2013-01-01

    Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with multipotential potential and differentiation in to the different cells types such as are osteoblast, chondroblast, adipoblast. In this research we describe pioneering experiment of tissue engineering in Bosnia and Herzegovina, of the isolation and differentiation rat bone marrow stromal cells in to the osteoblast cells lineages. Rat bone marrow stromal cells were isolated by method described by Maniatopulos using their plastic adherence capatibility. The cells obtained by plastic adherence were cultured and serially passaged in the osteoinductive medium to differentiate into the osteocytes. Bone marrow samples from rats long bones used for isolation of stromal cells (BMSCs). Under determinate culture conditions BMSCs were differentiated in osteogenic cell lines detected by Alizarin red staining three weeks after isolation. BMSCs as autologue cells model showed high osteogenetic potential and calcification capatibility in vitro. In future should be used as alternative method for bone transplantation in Regenerative Medicine. PMID:23448607

  2. Rat bone marrow stem cells isolation and culture as a bone formative experimental system.

    PubMed

    Smajilagi?, Amer; Alji?evi?, Mufida; Redži?, Amira; Filipovi?, Selma; Lagumdžija, Alena

    2013-02-01

    Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with multipotential potential and differentiation in to the different cells types such as are osteoblast, chondroblast, adipoblast. In this research we describe pioneering experiment of tissue engineering in Bosnia and Herzegovina, of the isolation and differentiation rat bone marrow stromal cells in to the osteoblast cells lineages. Rat bone marrow stromal cells were isolated by method described by Maniatopulos using their plastic adherence capatibility. The cells obtained by plastic adherence were cultured and serially passaged in the osteoinductive medium to differentiate into the osteocytes. Bone marrow samples from rats long bones used for isolation of stromal cells (BMSCs). Under determinate culture conditions BMSCs were differentiated in osteogenic cell lines detected by Alizarin red staining three weeks after isolation. BMSCs as autologue cells model showed high osteogenetic potential and calcification capatibility in vitro. In future should be used as alternative method for bone transplantation in Regenerative Medicine. PMID:23448607

  3. Human bone cell cultures in biocompatibility testing. Part I: osteoblastic di!erentiation of serially passaged human bone marrow cells cultured in a-MEM and in DMEM

    Microsoft Academic Search

    M. J. Coelho; A. Trigo Cabral; M. H. Fernandes

    2000-01-01

    Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue\\/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic di!erenti- ation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modi\\

  4. Long-Term Culture of Human Bone Marrow Cells

    Microsoft Academic Search

    Suzanne Gartner; Henry S. Kaplan

    1980-01-01

    A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis, as measured by the production of granulocytic-macrophage progenitor cells (CFUc), continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures, the adherent layer consisted

  5. Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

    Microsoft Academic Search

    Haruki Sekiguchi; Masaaki Ii; Kentaro Jujo; Ayumi Yokoyama; Nobuhisa Hagiwara; Takayuki Asahara

    2011-01-01

    Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in

  6. The Effect of Spaceflight on Bone Cell Cultures

    NASA Technical Reports Server (NTRS)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

  7. Comparisons of mouse mesenchymal stem cells in primary adherent culture of compact bone fragments and whole bone marrow.

    PubMed

    Cai, Yiting; Liu, Tianshu; Fang, Fang; Xiong, Chengliang; Shen, Shiliang

    2015-01-01

    The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture. PMID:25821472

  8. Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

    Microsoft Academic Search

    Pierre J. Marie; Abderrahim Lomri; Ayman Sabbagh; Michel Basle

    1989-01-01

    Summary  We report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments.\\u000a After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration\\u000a and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited\\u000a osteoblastic phenotype. Cultured cells show

  9. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    PubMed Central

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  10. Expansion of endothelial progenitor cells in high density dot culture of rat bone marrow cells.

    PubMed

    Lu, Yang; Gong, Yiyi; Lian, Jie; Wang, Ling; Kretlow, James D; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2 × 10(5) cells/cm(2)) by seeding total 9 × 10(5) cells into six high density dots or cultured in regular density (1.6 × 10(4) cells/cm(2)) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  11. Spaceflight effects on cultured embryonic chick bone cells

    NASA Technical Reports Server (NTRS)

    Landis, W. J.; Hodgens, K. J.; Block, D.; Toma, C. D.; Gerstenfeld, L. C.

    2000-01-01

    A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

  12. Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age

    Microsoft Academic Search

    S. C. Mendes; J. M. Tibbe; M. Veenhof; K. Bakker; S. Both; P. P. Platenburg; F. C. Oner; Bruijn de J. D; Blitterswijk van C. A

    2002-01-01

    At present, it is well known that populations of human bone marrow stromal cells (HBMSCs) can differentiate into osteoblasts and produce bone. However, the amount of cells with osteogenic potential that is ultimately obtained will still be dependent on both patient physiological status and culture system. In addition, to use a cell therapy approach in orthopedics, large cell numbers will

  13. Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo (Toyama Medical and Pharmaceutical Univ. (Japan))

    1991-08-01

    Most of cadmium (Cd)-treated animals have been reported to show osteoporosis-like changes in bones. This suggests that Cd may promote bone loss by a direct action on bone. It was found that Cd stimulated prostaglandin E{sub 2}(PGE{sub 2}) production in the osteoblast-like cell, MC3T3-E1. Therefore, Cd stimulates bone resorption by increasing PGE{sub 2} production. Recently, several bone marrow cell culture systems have been developed for examining the formation of osteoclast-like multinucleated cells in vitro. As osteoblasts produce PGE{sub 2} by Cd-induced cyclooxygenase and may play an important role in osteoclast formation, the present study was undertaken to clarify the possibility that Cd might stimulate osteoclast formation in a mouse bone marrow culture system.

  14. Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

    Microsoft Academic Search

    Claudio Muscari; Chiara Gamberini; Ilaria Basile; Francesca Bonafé; Simond Valgimigli; Ombretta Capitani; Carlo Guarnieri; Claudio Marcello Caldarera

    2010-01-01

    The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the

  15. Differentiation of dendritic cells in cultures of rat bone marrow cells

    PubMed Central

    1986-01-01

    Although dendritic cells (DC) originate from bone marrow, they were not observed in fresh preparations of bone marrow cells (BMC). Likewise, accessory activity was barely measurable in a sensitive assay for this potent function of DC. However, both DC and accessory activity developed when BMC were cultured for 5 d. Based on fractionation before culture, nearly all of the accessory activity could be attributed to only 5% of the total BMC recovered in a low-density (LD) fraction. The LD-DC precursors differed from mature DC in a number of important respects. Removal of Ia+ cells from the LD fraction by panning did not decrease the production of DC when the nonadherent cells were cultured. Thus, the cell from which the DC is derived does not express or minimally expresses Ia antigens, in contrast to the strongly Ia+ DC that is produced in bone marrow cultures. Irradiation of LD cells before culture prevented the development of DC. When irradiation was delayed by daily intervals, progressive increases in the number of DC resulted, up to the fifth day. These findings, together with preliminary autoradiographic data, indicate that cell division has occurred, in contrast to the DC, which does not divide. We conclude that bone marrow-derived DC arise in culture from the division of LD, Ia- precursors. PMID:3512761

  16. Long-term culture of leukemic bone marrow primary cells in biomimetic osteoblast niche

    Microsoft Academic Search

    Li Hou; Ting Liu; Jing Tan; Wentong Meng; Li Deng; Hongtao Yu; Xingli Zou; Yuchun Wang

    2009-01-01

    We constructed a “biomimetic osteoblast niche” with bio-derived bone as a scaffold, on which we seeded marrow mesenchymal\\u000a stem cells (MSCs) from CML patients, and induced the MSCs to differentiate into osteoblasts. Bone marrow mononuclear cells\\u000a from CML patients were cultured in the biomimetic niche (3D culture system) or a 2D culture system with the induced MSCs\\/osteoblasts\\u000a as a feeder

  17. Biological evaluation of an ionomeric bone cement by osteoblast cell culture methods.

    PubMed

    Meyer, U; Szulczewski, D H; Barckhaus, R H; Atkinson, M; Jones, D B

    1993-10-01

    Periosteal derived bovine osteoblast-like cells migrated in culture onto an ionomeric cement. Cell cultures were maintained for 4 weeks and used to study the in vitro behaviour of cells on the ionomeric bone cement (IC). The cells produced bone matrix proteins (osteocalcin, bone sialoprotein II) and were osteoblast-like. The osteoblast-like cells colonized the substrate in monolayers and produced an extracellular matrix as seen by light and scanning electron microscopy. Morphological comparison between cells growing on the ionomeric bone cement and cortical bone revealed no significant difference in phenotypic expression. Staining for aluminium in osteoblasts growing on the IC showed an uptake and storage of aluminium in the cells. Energy dispersive X-ray microanalysis revealed high concentrations of aluminium and silicon in the periosteal tissue. Despite the known toxic effect of aluminium in vivo and in vitro on osteoblasts, no signs of toxicity were apparent on light and scanning electron microscopy analysis. PMID:7505630

  18. Bone Grafts Engineered from Human Adipose-Derived Stem Cells in Perfusion Bioreactor Culture

    PubMed Central

    Fröhlich, Mirjam; Grayson, Warren L.; Marolt, Darja; Gimble, Jeffrey M.; Kregar-Velikonja, Nevenka

    2010-01-01

    We report engineering of half-centimeter–sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), decellularized bone scaffolds, and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4?mm diameter?×?4?mm thick), providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400??m/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-?-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs. PMID:19678762

  19. Biocompatibility studies on fibrin glue cultured with bone marrow mesenchymal stem cells in vitro.

    PubMed

    Fang, Huang; Peng, Songlin; Chen, Anmin; Li, Fengfeng; Ren, Kai; Hu, Ning

    2004-01-01

    By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering. PMID:15315346

  20. Improvement of liver fibrosis by infusion of cultured cells derived from human bone marrow.

    PubMed

    Tanimoto, Haruko; Terai, Shuji; Taro, Takami; Murata, Yasuhiko; Fujisawa, Kouichi; Yamamoto, Naoki; Sakaida, Isao

    2013-12-01

    We develop "autologous bone marrow cell infusion (ABMi) therapy" for the treatment of human decompensated liver cirrhosis and confirm the efficacy and safety of this treatment in multicenter clinical studies. With the goal of further expanding the applications of ABMi, we first cultured human bone marrow cells and then determined whether a cell fraction found to be effective in improving liver fibrosis can be amplified. Cells harvested after two passages (P2 cells) consistently contained approximately 94% mesenchymal stem cells (MSCs); conversely, the cells harvested after only medium change (P0 cells) contained many macrophages. MSCs (2.8?×?10(8)) in P2 cells were harvested from 3.8 × 10(8) bone marrow-derived mononuclear cells after 22 days. DNA-chip analysis also showed during the culturing step that bone marrow-derived cells decreased with macrophage phenotype. The infused 5 × 10(5) P2 cells significantly improved liver fibrosis in the nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse carbon tetrachloride (CCl4) liver cirrhosis model and induced the expression of matrix metalloproteinase (MMP)-9 and suppressed expressions of alpha smooth muscle actin (?SMA), tumor necrosis factor alpha (TNF?) and transforming growth factor beta (TGF?) in the liver. Cultured human bone marrow-derived cells (P2 cells) significantly inhibited liver fibrosis. The increase of MMP-9 and suppressed activation of hepatic stellate cells (HSCs) through the regulation of humoral factors (TNF? and TGF?) contribute to the improvement of liver fibrosis by MSCs comprising about 94% of P2 cells. MSCs in cultured human bone marrow-derived mono-nuclear cells (BM-MNCs) proliferate sufficiently in cell therapy, so we believe our cultured bone marrow-derived cell therapy can lead to expanded clinical applications and enable outpatient therapy. PMID:24104560

  1. Co-culture of human bone marrow stromal cells with endothelial cells alters gene expression profiles

    PubMed Central

    Xue, Ying; Xing, Zhe; Bolstad, Anne Isine; Van Dyke, Thomas E.; Mustafa, Kamal

    2014-01-01

    The intricate relationship between angiogenesis and osteogenesis in vivo must be replicated in bone tissue engineering constructs to ensure the formation of a functional vascular network to support successful bone formation. Although communication between bone marrow stromal cells (MSC) and endothelial cells (EC) is recognized as one of the most important cellular interactions in bone regeneration, the underlying mechanisms of this biological process are not well understood. The purpose of this study was to analyze global gene expression associated with intercellular communication between MSC and EC using HumanWG-6 v3.0 expression BeadChips with a one-channel platform system (Illumina, San Diego, CA, USA). Each array contains more than 48,000 probes derived from human genes. A global map of MSC gene expression was generated following co-culture of MSC with EC for 5 and 15 days, in a direct-contact model. The map was used to determine relative alterations in functional processes and pathways. Co-culturing EC with MSC up-regulated genes related to angiogenesis as von Willebrand factor, platelet/endothelial cell adhesion molecule-1, cadherin 5, angiopoietin-related protein 4, and cell surface antigen CD34, and genes playing important roles in osteogenesis as alkaline phosphatase, FK506 binding protein 5, and bone morphogenetic protein. These findings clearly demonstrated that EC had a significant impact on MSC, particularly the bidirectional regulation of angiogenesis and osteogenesis. Moreover, cell-matrix interactions and TGF-? signal pathways were implicated for a crucial role in endothelial, cell-induced gene regulation in MSCs. A detailed study of the functional correlates of the microarray data is warranted to explore cellular and molecular interactions of importance in bone tissue engineering. PMID:23918270

  2. [Biocompatibility of a new ionomer bone cement in endoprosthetics. In vitro testing using a mixed bone cell culture].

    PubMed

    Meyer, U; Szulczewski, D H; Möller, K; Jones, D B; Wuisman, P

    1996-01-01

    Periosteal derived bovine osteoblasts and osteoclasts migrated in culture onto an ionomeric cement, currently evaluated as a cement for orthopaedic implants. Cell cultures were maintained for 4 weeks and used to study the in-vitro behaviour of cells on the material surface. Osteoblasts and osteoclasts colonized the substrate in monolayers and exhibited their phenotypic morphology. Differentiation into both cell lines were demonstrated by immunostaining. Staining for aluminium in cells growing on the bone cement showed an uptake and storage of aluminium in the cells. EDAX-microanalysis revealed high concentrations of Al and Si in the periosteal tissue. Despite uptake of Al in the cells, signs of toxicity were not apparent in the cell culture system. PMID:8779254

  3. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  4. Differential Cell Surface Expression of the STRO-1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells

    Microsoft Academic Search

    STAN GRONTHOS; ANDREW C. W. ZANNETTINO; STEPHEN E. GRAVES; SHUICHI OHTA; SHELLEY J. HAY; PAUL J. SIMMONS

    Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and

  5. Bone culture research

    NASA Technical Reports Server (NTRS)

    Partridge, Nicola C.

    1993-01-01

    The experiments described are aimed at exploring PTH regulation of production of collagenase and protein inhibitors of collagenase (tissue inhibitors of metalloproteases, TIMP-1 and -2) by osteoblast-like osteosarcoma cells under conditions of weightlessness. The results of this work will contribute to information as to whether a microgravity environment alters the functions and responsiveness of the osteoblast. The objectives of the Bone Culture Research (BCR) experiment are: to observe the effects of microgravity on the morphology, rate of proliferation, and behavior of the osteoblastic cells, UMR 106-01; to determine whether microgravy affects the hormonal sensitivity of osteroblastic cells; and to measure the secretion of collagenase and its inhibitors into the medium under conditions of microgravity. The methods employed will consist of the following: the osteoblast-like cells, UMR-106-01, will be cultured in four NASDA cell culture chambers; two chambers will be subjected to microgravity on SL-J; two chambers will remain on the ground at KSC as ground controls but subjected to an identical set of culture conditions as on the shuttle; media will be changed four times; twice the cells will receive the hormone parathyroid hormone-related protein (PTHrP) and media collected; cells will be photographed under conditions of microgravity; and media and photographs will be analyzed upon return to determine whether functions of the cells changed.

  6. Clonal distribution of osteoprogenitor cells in cultured chick periostea: Functional relationship to bone formation

    SciTech Connect

    McCulloch, C.A.; Fair, C.A.; Tenenbaum, H.C.; Limeback, H.; Homareau, R. (Univ. of Toronto, Ontario (Canada))

    1990-08-01

    Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with (3H)thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of (3H)thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of (3H)thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity (3H)thymidine at 1 and 2 days of culture completely blocked bone formation.

  7. CFU-GM Like Colonies Derived from Embryonic Stem Cells Cultured on the Bone Marrow Stromal Cells

    Microsoft Academic Search

    Ali Akbar Movassagh Pour; Mojdeh Salehnia; Ali Akbar Pourfatollah; Masoud Soleimani

    2004-01-01

    The aim of this study was to isolate mouse embryonic stem cells from late blastocyst stage embryos and to use them as a model system for the study of hematopoietic induction outside the embryo by coculturing of embryonic stem cells with bone marrow stromal cells. Blastocyst stage embryos from pregnant NMRI mice were obtained and cultured for 1-2 days in

  8. Evaluation of posterolateral lumbar fusion in sheep using mineral scaffolds seeded with cultured bone marrow cells.

    PubMed

    Cuenca-López, María D; Andrades, José A; Gómez, Santiago; Zamora-Navas, Plácido; Guerado, Enrique; Rubio, Nuria; Blanco, Jerónimo; Becerra, José

    2014-01-01

    The objective of this study is to investigate the efficacy of hybrid constructs in comparison to bone grafts (autograft and allograft) for posterolateral lumbar fusion (PLF) in sheep, instrumented with transpedicular screws and bars. Hybrid constructs using cultured bone marrow (BM) mesenchymal stem cells (MSCs) have shown promising results in several bone healing models. In particular, hybrid constructs made by calcium phosphate-enriched cells have had similar fusion rates to bone autografts in posterolateral lumbar fusion in sheep. In our study, four experimental spinal fusions in two animal groups were compared in sheep: autograft and allograft (reference group), hydroxyapatite scaffold, and hydroxyapatite scaffold seeded with cultured and osteoinduced bone marrow MSCs (hybrid construct). During the last three days of culture, dexamethasone (dex) and beta-glycerophosphate (?-GP) were added to potentiate osteoinduction. The two experimental situations of each group were tested in the same spinal segment (L4-L5). Spinal fusion and bone formation were studied by clinical observation, X-ray, computed tomography (CT), histology, and histomorphometry. Lumbar fusion rates assessed by CT scan and histology were higher for autograft and allograft (70%) than for mineral scaffold alone (22%) and hybrid constructs (35%). The quantity of new bone formation was also higher for the reference group, quite similar in both (autograft and allograft). Although the hybrid scaffold group had a better fusion rate than the non-hybrid scaffold group, the histological analysis revealed no significant differences between them in terms of quantity of bone formation. The histology results suggested that mineral scaffolds were partly resorbed in an early phase, and included in callus tissues. Far from the callus area the hydroxyapatite alone did not generate bone around it, but the hybrid scaffold did. In nude mice, labeled cells were induced to differentiate in vivo and monitored by bioluminescence imaging (BLI). Although the cultured MSCs had osteogenic potential, their contribution to spinal fusion when seeded in mineral scaffolds, in the conditions disclosed here, remains uncertain probably due to callus interference with the scaffolds. At present, bone autografts are better than hybrid constructs for posterolateral lumbar fusion, but we should continue to seek better conditions for efficient tissue engineering. PMID:25522168

  9. Monitoring Dynamic Interactions between Breast Cancer Cells and Human Bone Tissue in a Co-Culture Model

    PubMed Central

    Contag, Christopher H.; Lie, Wen-Rong; Bammer, Marie C.; Hardy, Jonathan W.; Schmidt, Tobi L.; Maloney, William J.; King, Bonnie L.

    2015-01-01

    Purpose Bone is a preferential site of breast cancer metastasis and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. Procedures Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell division and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX® immunoassays. Results BLI demonstrated increased MDA-MB-231-fLuc proliferation (p<0.001) in the presence vs. absence of bones, and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc colonization of bone, and MILLIPLEX® profiles of culture supernatants suggested breast/bone crosstalk. Conclusions Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays. PMID:24008275

  10. Analysis of cells isolated from bone cultured on collagen gels and polystyrene culture dishes

    SciTech Connect

    Fletcher, K.

    1981-01-01

    Bone is a complex tissue which contains three types of differentiated cells viz., osteoblasts, osteoclasts and osteocytes. In mature bone, these cells are identified both by their location within the tissue and their morphological characteristics. In fetal tissue, one also finds many progenitor cells, fibroblasts and some cartilage cells. Each of these cell types has distinct functions which are reflected in their morphology, metabolic properties and response to hormones. Studies were also undertaken to evaluate the class of problems associated with electron microprobe analysis of the extracellular fluid space in bone. It was determined that differences in elemental composition in a small volume between cells and mineral cannot be quantitatively corrected for fluorescence, atomic number or absorption effects of the mineral. A study of the use of free-flow dialysis in the study of metal binding to protein demonstrates the anomalous behavior of mercury in this experimental approach and emphasizes the importance of a thorough examination of the control situation before protein to metal binding is examined.

  11. Nanocrystalline diamond: In vitro biocompatibility assessment by MG63 and human bone marrow cells cultures.

    PubMed

    Amaral, M; Dias, A G; Gomes, P S; Lopes, M A; Silva, R F; Santos, J D; Fernandes, M H

    2008-10-01

    Nanocrystalline diamond (NCD) has a great potential for prosthetic implants coating. Nevertheless, its biocompatibility still has to be better understood. To do so, we employed several materials characterization techniques (SEM, AFM, micro-Raman spectroscopy) and cell culture assays using MG63 osteoblast-like and human bone marrow cells. Biochemical routines (MTT assays, Lowry's method, ALP activity) supported by SEM and confocal microscopy characterization were carried out. We used silicon nitride (Si3N4) substrates for NCD coatings based on a previous demonstration of the superior adhesion and tribological performance of these NCD coated ceramics. Results demonstrate an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared with standard polystyrene tissue culture plates. The nanometric featuring of NCD, associated to its chemical affinity are key points for bone regeneration purposes. PMID:18085649

  12. In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition

    PubMed Central

    Chai, Yoke Chin; Geris, Liesbet; Bolander, Johanna; Pyka, Grzegorz; Van Bael, Simon; Luyten, Frank P.

    2014-01-01

    Abstract Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43?) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions. PMID:25469312

  13. Tissue culture surface characteristics influence the expansion of human bone marrow cells.

    PubMed

    Koller, M R; Palsson, M A; Manchel, I; Maher, R J; Palsson, B O

    1998-11-01

    Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application. PMID:9863530

  14. Proliferation\\/differentiation of osteoblastic human alveolar bone cell cultures in the presence of stainless steel corrosion products

    Microsoft Academic Search

    M. A. Costa; M. H. Fernandes

    2000-01-01

    Human osteoblastic alveolar bone cells were cultured for 28 days in control conditions and in the presence of three non-lethal concentrations of AISI 316L stainless steel (SS) corrosion products. Cells were exposed to SS corrosion products in two experimental situations: (i) in selected stages of the incubation time (during the first, second, third and fourth week of culture); and (ii)

  15. Desensitization of parathyroid hormone receptors on cultured bone cells

    SciTech Connect

    Pun, K.K.; Ho, P.W.; Nissenson, R.A.; Arnaud, C.D. (Univ. of Hong Kong (Hong Kong))

    1990-12-01

    Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. (Nle8,Nle18,Tyr34)bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with (125I)PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself.

  16. Individual clones of hemopoietic cells in murine long-term bone marrow culture

    SciTech Connect

    Chertkov, J.L.; Deryugina, E.I.; Drize, N.J.; Udalov, G.A.

    1987-06-01

    Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession.

  17. Bone Induction by Implants Coated with Cultured Osteogenic Bone Marrow Cells

    Microsoft Academic Search

    Joost D. DeBruijn; Ineke Van Den Brink; Sandra Mendes; Robert Dekker; Yvonne P. Bovell; Clemens A. Van Blitterswijk

    1999-01-01

    The availability of osteoinductive coatings on dental and orthopedic implants will result in an improved fixation of these devices. Those cases where implants are placed in poor-quality bone or where high failure rates are obtained are especially expected to gain from such coatings. This paper presents a novel, biological approach to obtain bioactive and osteoinductive coatings on bone-replacement implant materials.

  18. Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells

    PubMed Central

    LIU, JUN-FENG; DU, ZHONG-DONG; CHEN, ZHI; HE, ZHI-XU

    2014-01-01

    The number and function of endothelial progenitor cells (EPCs) may be a predictive factor for the severity and outcome of cardiovascular disease. However, the manipulation of bone marrow mononuclear cell (BMMC) cultures for EPCs is an elaborate and difficult procedure in small experimental animals. The present study aimed to assess the feasibility of whole bone marrow cell (WBMC) culture for expanding EPCs in small experimental animals. C57BL/6 mice (age, 3–4 weeks; weight, 9.47±0.76 g) were used as the experimental animals, and WBMCs were isolated from the femora and tibiae and cultured in endothelial cell growth medium-2. A BMMC culture for EPCs was used as a control. EPC growth, phenotype and functions were assessed in vitro and in vivo. The results demonstrated that EPCs were easily obtained from a WBMC culture in vitro. The cells exhibited similar growth and biological characteristics when compared with the EPCs derived from the traditional BMMC culture system. Thus, the cells were able to simultaneously bind to lectin and cause phagocytosis of acetylated-low density lipoproteins. In addition, the cells exhibited high expression levels of cluster of differentiation 34 and fetal liver kinase 1, and possessed similar functional properties to BMMC-derived EPCs, including vascular network formation, proliferation, adhesion and migration abilities in vitro. Thus, WBMC-derived EPCs can improve the outcome of pulmonary vascular disease when transplanted into a monocrotaline-induced pulmonary hypertension mouse model. The results of the present study indicated that the WBMC culture system is a more convenient and effective method of obtaining and expanding EPCs compared with BMMC culture, with the advantage of a simplified procedure. PMID:25120604

  19. Three-dimensional (3D) culture of bone-derived human 786-O renal cell carcinoma retains relevant clinical characteristics of bone metastases.

    PubMed

    Pan, Tianhong; Fong, Eliza L S; Martinez, Mariane; Harrington, Daniel A; Lin, Sue-Hwa; Farach-Carson, Mary C; Satcher, Robert L

    2015-08-28

    Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems. PMID:26004343

  20. Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow

    Microsoft Academic Search

    John P. Chute; Abha A. Saini; Dennis J. Chute; Mark R. Wells; William B. Clark; David M. Harlan; Jenny Park; Margaret K. Stull; Curt Civin; Thomas A. Davis

    2002-01-01

    Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disap- pointing. In this study, we describe a novel culture method

  1. Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: growth factor production.

    PubMed

    Chen, Xiaoguang; Katakowski, Mark; Li, Yi; Lu, Dunyue; Wang, Lei; Zhang, Lijie; Chen, Jieli; Xu, Yongxian; Gautam, Subhash; Mahmood, Asim; Chopp, Michael

    2002-09-01

    Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat. However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated. Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best. Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy. Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue. We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI. hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed. TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs. The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis. PMID:12210835

  2. Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture

    Microsoft Academic Search

    Yi Yang; Fabio M. V. Rossi; Edward E. Putnins

    2007-01-01

    Bone marrow mesenchymal stromal cells (BM-MSC) are attractive candidates for connective tissue regeneration. Currently, their use is limited by poor overall cell survival and high apoptosis rates upon transplantation in vivo. We hypothesized that disruption of cell–extracellular matrix contact either during cell expansion or immediately prior to cell transplantation may impair cell viability and facilitate apoptosis. We therefore investigated whether

  3. TGFß1 limits the expansion of the osteoprogenitor fraction in cultures of human bone marrow stromal cells

    Microsoft Academic Search

    Susan Walsh; Carolyn Jefferiss; Karina Stewart; Jon N. Beresford

    2003-01-01

    Currently, there is considerable interest in the possibility of using cultured human bone marrow stromal cells (BMSCs) for skeletal tissue engineering. However, the factors that regulate their ex vivo expansion and promote their osteogenic maturation remain poorly defined. Using BMSCs obtained from a large cohort of adult donors, the effects of transforming growth factor (TGF)Ї on these processes have been

  4. Prolonged hematopoiesis in a primate bone marrow culture system: characteristics of stem cell production and hematopoietic microenvironment

    Microsoft Academic Search

    M. A. S. Moore; A. P. C. Sheridan; T. D. Allen; T. M. Dexter

    1979-01-01

    Maintenance of myelopoiesis and pluripo- tential stem cell production for prolonged periods in vitro hitherto has been limited to mouse bone marrow culture. In an effort to adapt the system for use in higher species, particularly in human and non-human primates, studies were undertaken using the prosimian species, Tupaia gus (tree shrew). In a number of experiments the duration of

  5. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  6. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  7. Culture-Modified Bone Marrow Cells Attenuate Cardiac and Renal Injury in a Chronic Kidney Disease Rat Model via a Novel Antifibrotic Mechanism

    Microsoft Academic Search

    Darren A. Yuen; Kim A. Connelly; Andrew Advani; Christine Liao; Michael A. Kuliszewski; Judy Trogadis; Kerri Thai; Suzanne L. Advani; Yuan Zhang; Darren J. Kelly; Howard Leong-Poi; Armand Keating; Philip A. Marsden; Duncan J. Stewart; Richard E. Gilbert; Arnold Schwartz

    2010-01-01

    BackgroundMost forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs).Methodology\\/Principal FindingsIn

  8. Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

    PubMed

    Mathivanan, Isai; Trepp, Carolyn; Brunold, Claudio; Baerlocher, Gabriela; Enzmann, Volker

    2015-04-10

    The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases. PMID:25724900

  9. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    SciTech Connect

    Perkins, S.; Fleischman, R.A.

    1988-04-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.

  10. Inhibition of osteoclast-like cell formation by bisphosphonates in long-term cultures of human bone marrow.

    PubMed Central

    Hughes, D E; MacDonald, B R; Russell, R G; Gowen, M

    1989-01-01

    Bisphosphonates inhibit bone resorption in vivo and in vitro by unknown mechanisms. The effect of bisphosphonates on the formation of osteoclasts from their mononuclear hematopoietic precursors was investigated using human long-term marrow cultures in which multinucleated cells form that express most of the known features of the osteoclast phenotype (e.g., bone resorption, tartrate-resistant acid phosphatase, calcitonin responsiveness, and reactivity with specific MAbs). The five bisphosphonates that were tested strongly inhibited 1,25-dihydroxyvitamin D3-stimulated formation of these cells with the same relative potencies as they inhibit bone resorption in vivo. Two representative compounds (3-amino-1-hydroxypropylidene-1,1-bisphosphonate and dichloromethylene bisphosphonate) failed to inhibit the proliferation of precursors of the osteoclast-like cells. However, these compounds decreased the proportion of mononuclear and multinucleated cells expressing an osteoclast antigen, thus suggesting a degree of specificity for cells of the osteoclast lineage. We conclude that bisphosphonates are potent inhibitors of osteoclast-like cell formation in long-term human marrow cultures, and that this may be related to their ability to inhibit bone resorption in vivo. Images PMID:2524504

  11. Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage-like Population in Bone Marrow-Derived Dendritic Cell Cultures.

    PubMed

    Poon, Grace F T; Dong, Yifei; Marshall, Kelsey C; Arif, Arif; Deeg, Christoph M; Dosanjh, Manisha; Johnson, Pauline

    2015-07-15

    Although classical dendritic cells (DCs) arise from distinct progenitors in the bone marrow, the origin of inflammatory DCs and the distinction between monocyte-derived DCs and macrophages is less clear. In vitro culture of mouse bone marrow cells with GM-CSF is a well-established method to generate DCs, but GM-CSF has also been used to generate bone marrow-derived macrophages. In this article, we identify a distinct subpopulation of cells within the GM-CSF bone marrow-derived DC culture based on their ability to bind hyaluronan (HA), a major component of the extracellular matrix and ligand for CD44. HA identified a morphologically distinct subpopulation of cells within the immature DC population (CD11c(+) MHC II(mid/low)) that were CCR5(+)/CCR7(-) and proliferated in response to GM-CSF, but, unlike immature DCs, did not develop into mature DCs expressing CCR7 and high levels of MHC II, even after stimulation with LPS. The majority of these cells produced TNF-? in response to LPS but were unable to activate naive T cells, whereas the majority of mature DCs produced IL-12 and activated naive T cells. This HA binding population shared many characteristics with alveolar macrophages and was retained in the alveolar space after lung instillation even after LPS stimulation, whereas the MHC II(high) mature DCs were found in the draining lymph node. Thus, HA binding in combination with MHC II expression can be used to identify alveolar-like macrophages from GM-CSF-treated bone marrow cultures, which provides a useful in vitro model to study alveolar macrophages. PMID:26085682

  12. Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media.

    PubMed

    Guan, Xiu M; Cheng, Min; Li, Hong; Cui, Xiao D; Li, Xin; Wang, Yu L; Sun, Jin L; Zhang, Xiao Y

    2013-12-01

    Ex vivo expansion of endothelial progenitor cells (EPCs) may be a promising strategy to overcome the clinical problem of limited cell numbers. As the culture medium is the key for the cell characteristics, the effects of different culture media on EPCs were investigated in the present study. Rat bone marrow mononuclear cells were cultured in different media, including M-199 media with 20% fetal bovine serum (FBS) and bovine pituitary extract (M1); M-199 media with 10% FBS, 20 ng/ml vascular endothelial growth factor (VEGF) and 10 ng/ml basic fibroblast growth factor (bFGF; M2) or epidermal growth medium (EGM)-2MV media. The cell morphology and biological functions, such as proliferation, adhesion, migration, tube formation and nitric oxide (NO) production were subsequently assayed in vitro. Moreover, endothelial biomarkers and apoptosis were also analyzed. The results showed that endothelial?like cells appeared in all of the culture systems. First?passage cells, namely early EPCs, tended to form colonies in M2 and EGM-2MV media but showed a fusiform shape in M1 media. The 3rd or 4th generation EPCs, namely late EPCs, cultured in EGM-2MV media exhibited increased adhesion, migration, tube formation and NO production as compared with EPCs in M1 or M2 media. Furthermore, late EPCs cultured in EGM-2MV expressed higher levels of endothelial cell markers, such as von Willibrand factor (vWF)and CD31, but relatively greater levels of apoptosis were observed. In conclusion, cell culture conditions, for example the medium used, affects the biological properties of bone marrow-derived early and late EPCs. PMID:24126824

  13. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    SciTech Connect

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)] [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan)] [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: yamacho@saitama-med.ac.jp [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)] [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  14. Prevention of liver fibrosis by intrasplenic injection of high-density cultured bone marrow cells in a rat chronic liver injury model.

    PubMed

    Lian, Jie; Lu, Yang; Xu, Peng; Ai, Ai; Zhou, Guangdong; Liu, Wei; Cao, Yilin; Zhang, Wen Jie

    2014-01-01

    Endothelial progenitor cells (EPCs) from bone marrow have proven to be functional for the prevention of liver fibrosis in chronic liver injury. However, expansion of EPCs in culture is complicated and expansive. Previously, we have established a simple method that could enrich and expand EPCs by simple seeding bone marrow cells in high density dots. The purpose of this study is to evaluate whether cells derived from high-density (HD) culture of rat bone marrow cells could prevent the liver fibrosis in a chronic liver injury rat model, induced by carbon tetrachloride (CCl4). Flow cytometric analysis showed that cells from HD culture were enriched for EPCs, expressing high levels of EPC markers. Intrasplenic injection of HD cultured bone marrow cells in the CCl4-induced liver injury rat showed an enhanced antifibrogenic effect compared with animals treated with cells from regular-density culture. The antifibrogenic effect was demonstrated by biochemical and histological analysis 4 weeks post-transplantation. Furthermore, cells from HD culture likely worked through increasing neovascularization, stimulating liver cell proliferation, and suppressing pro-fibrogenic factor expression. HD culture, which is a simple and cost-effective procedure, could potentially be used to expand bone marrow cells for the treatment of liver fibrosis. PMID:25255097

  15. Microgravity and bone cell mechanosensitivity

    NASA Astrophysics Data System (ADS)

    Klein-Nulend, J.; Bacabac, R. G.; Veldhuijzen, J. P.; Van Loon, J. J. W. A.

    2003-10-01

    The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone. The in vivo operating cell stress derived from bone loading is likely the flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Earlier studies have shown that the disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity, or better near weightlessness, is associated with the loss of bone in astronauts, and has catabolic effects on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found earlier that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGEZ production. Therefore it is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts. In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone.

  16. Microgravity and Bone Cell Mechanosensitivity

    NASA Astrophysics Data System (ADS)

    Klein-Nulend, J.; Bacabac, R.; Veldhuijzen, J.; van Loon, J.

    The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone.The in vivo operating cell stress derived from bone loading is likely flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction.Microgravity, or better near weightlessness, has catabolic effects on the skeleton of astronauts, and on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGE2 production. Therefore it is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts.In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone.

  17. Bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions.

    PubMed

    Khan, Wasim S; Adesida, Adetola B; Tew, Simon R; Lowe, Emma T; Hardingham, Timothy E

    2010-06-01

    Bone marrow-derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow-derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony-forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow-derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow-derived stem cells. PMID:20058274

  18. Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells

    Microsoft Academic Search

    Cong Toai Tran; Ciro Gargiulo; Huynh Duy Thao; Huynh Minh Tuan; Luis Filgueira; D. Michael Strong

    In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis\\u000a for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes.\\u000a Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical\\u000a regenerative purposes. In bone

  19. CpG oligonucleotides and Astragalus polysaccharides are effective adjuvants in cultures of avian bone-marrow-derived dendritic cells.

    PubMed

    Lin, J; Kang, H; Liang, J; Fu, J; Yu, Q; Yang, Q

    2015-01-01

    1. The potential use of CpG oligodeoxynucleotides and/or Astragalus polysaccharide (APS) as adjuvants for the culture of chicken bone-marrow-derived dendritic cells (chBM-DCs) was investigated. 2. Chicken dendritic cells (DCs) were isolated and cultured in the presence of recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. The chBM-DC displayed typical DC morphology and expressed DC surface markers (MHC-II and CD11c). 3. Cultured chBM-DC showed effective T-cell activation in vitro, based on a mixed lymphocyte response (MLR). Flow cytometry analysis showed an increased proportion of cells expressing CD40 and CD80 in the APS-stimulated culture, compared to the control culture. In the MLR, the APS- and CpG-stimulated chBM-DC could activate T-cells more than control chBM-DC. Real-time PCR assays showed that CpG can activate the TLR21 and an inflammatory response, while APS just reduced the expression of IRF-3. 4. The results demonstrated that in vitro the adjuvant CpG can stimulate chBM-DC to mature by activation of the TLR-signalling pathway, whereas the adjuvant APS stimulates maturation of chBM-DC in vitro to a lesser degree and by another mechanism. PMID:25403700

  20. Culture Human Mesenchymal Stem Cells With Calcium Phosphate Cement Scaffolds for Bone Repair

    PubMed Central

    Weir, Michael D.; Xu, Hockin H. K.

    2010-01-01

    Because of its moldability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for craniofacial and orthopedic applications. The objectives of this study were to investigate the response of human mesenchymal stem cells (hMSCs) to a high-strength CPC-chitosan scaffold and to examine cell proliferation and osteogenic differentiation. hMSCs were seeded onto CPC-chitosan composite, CPC control, and tissue culture polystyrene (TCPS). Alkaline phosphatase activity (ALP) and mineralization of hMSCs were measured. CPC-chitosan had a flexural strength (mean ± SD; n = 5) of (19.5 ± 1.4) MPa, higher than (8.0 ± 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 ± 1.3)% at 8 days, matching (90.7 ± 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 ± 11)% at 1 day to (90 ± 4)% at 8 days (p < 0.05), matching those of CPC control (p > 0.1). Hence, the CPC strength was significantly increased via chitosan without compromising the hMSC response. At 8 days, there was a significant increase in ALP of cells in osteogenic media (10.99 ± 0.93) [(mM pNpp/min)/(?g DNA)] versus control media (3.62 ± 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 ± 19.7)% compared with (6.1 ± 2.3)% in control medium on TCPS (p < 0.05). In conclusion, hMSCs showed excellent attachment and viability on the strong and tough CPC-chitosan scaffold, matching the hMSC response on CPC control. hMSCs were successfully differentiated down the osteogenic lineage. Hence, the strong, in situ hardening CPC-chitosan scaffold may be useful as a moderate load-bearing vehicle to deliver hMSCs for maxillofacial and orthopedic bone tissue engineering. PMID:20091907

  1. Differentiation of multipotent mesenchymal stromal cells of human bone marrow into cells of cartilage tissue by culturing in three-dimensional OPLA scaffolds

    Microsoft Academic Search

    A. S. Teplyashin; S. V. Korjikova; S. Z. Sharifullina; M. S. Rostovskaya; N. I. Chupikova; N. Yu. Vasyunina; N. V. Andronova; E. M. Treshalina; I. P. Savchenkova

    2007-01-01

    Bone marrow multipotent mesenchymal stromal cells show considerable promise for the engineering of human three-dimensional\\u000a transplants of cartilage tissue. We demonstrated the directed differentiation of BM MMSC in cells of cartilage tissue by culturing\\u000a them in OPLA polymer three-dimensional scaffolds in a medium with chondrogenesis inducers. Cells were loaded into porous scaffolds\\u000a by saturating polymer blocks with a cellular suspension.

  2. Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage.

    PubMed

    Murdoch, Alan D; Grady, Lisa M; Ablett, Matthew P; Katopodi, Theoni; Meadows, Roger S; Hardingham, Tim E

    2007-11-01

    Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high-density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8-1 mm in thickness from 0.5 x 10(6) cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies. PMID:17656642

  3. Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing

    PubMed Central

    Moisenovich, M. M.; Arkhipova, A. Yu.; Orlova, A. A.; Drutskaya, M. S; Volkova, S. V.; Zacharov, S. E.; Agapov, I. I.; Kirpichnikov, M. P.

    2014-01-01

    Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems. PMID:24772332

  4. Parathyroid Hormone Stimulates TRANCE and Inhibits Osteoprotegerin Messenger Ribonucleic Acid Expression in Murine Bone Marrow Cultures: Correlation with Osteoclast-Like Cell Formation

    Microsoft Academic Search

    SUN-KYEONG LEE; JOSEPH A. LORENZO

    1999-01-01

    We studied the effects of PTH on the expression of tumor necrosis factor-related activation-induced cytokine (TRANCE), osteoprote- gerin (OPG), and receptor activator of NF kB (RANK) messenger RNA (mRNA) in cultured murine bone marrow, calvaria, and osteoblasts. TRANCE, OPG, and RANK are recently identified regulators of os- teoclast formation. Bone marrow cells were cultured with or without PTH(1-34) for 6

  5. Mouse bone marrow-derived mast cells (BMMC) change their phenotype when cultured with fibroblasts

    SciTech Connect

    Levi-Schaffer, F.; Austen, K.F.; Stevens, R.L.

    1986-03-05

    The heparin-containing mast cells (HP-MC) that reside in the connective tissues of the mouse, but not the chondroitin sulfate containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse BMMC (the presumptive in vitro counterpart of the in vivo differentiated mucosal mast cell) were cultured for 2-14 days with confluent skin-derived 3T3 fibroblasts in RPMI-1640 containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin-3 in the conditioned medium. The mast cells remained viable throughout the period of co-culture, since they failed to release LDG and because they increased their histamine content per cell approx.15-fold. After 8-9 days of co-culture, >50% of the BMMC changed histochemically becoming safranin positive. At this time, 30-50% of the (/sup 35/S)glycosaminoglycans on the proteoglycans synthesized by these co-cultured mass cells were heparin, whereas the initial BMMC synthesized proteoglycans containing only chondroitin sulfate E. That interleukin 3-dependent mouse BMMC can be induced to undergo a phenotypic change so as to express characteristics of a HP-MC suggests that the tissue microenvironment determines the differentiated characteristics of these cells.

  6. Research of osteoblastic induced rat bone marrow mesenchymal stem cells cultured on ?-TCP/PLLA porous scaffold

    PubMed Central

    Yang, Yi; Wu, Jiang; Jin, Gele; Li, Liang; Li, Zhongwei; Li, Cao

    2015-01-01

    Background: Ceramic and polymer composite scaffolds are widely used in tissue engineering for bone tissue regeneration. Composite of ?-tricalcium phosphate (?-TCP) and poly L-lactic acid (PLLA), due to its biocompatibility and biodegradability, is widely used in bioengineering. However, optimal ratio, porosity and pore size of this kind of scaffolds were not very clear yet. Materials and methods: We cultured osteoblastic induced rMSCs on ?-TCP/PLLA scaffolds to investigate the optimum construction, which owned better properties for supporting cells growth, proliferation and differentiation. A total of 24 mice were divided into three groups: rMSCs + ?-TCP/PLLA, osteoblastic rMSCs + ?-TCP/PLLA and ?-TCP/PLLA without cells. 8 rude mice were implanted with rMSCs + ?-TCP/PLLA in the left thighs and ?-TCP/PLLA without cells in the right thighs. 8 rude mice were implanted with osteoblastic rMSCs + ?-TCP/PLLA in the left thighs and the same treatments in the right thighs as the above. After 8 and 12 weeks, the mice were sacrificed and implants with the surrounding tissues were harvested together. Paraffin sections were got and HE stain and Masson-Goldner stain were employed to observe the ectopic bone formation. Results: The scaffolds of ?-TCP/PLLA = 2:1 significantly increased osteocalcin production of the cells. In addition, scaffolds with NaCl = 70 wt%, pore size 200~450 ?m showed better compatibility to these seeding cells. A significantly larger area of bone formation in the osteoblastic rMSCs and ?-TCP/PLLA composite than that in rMSCs/scaffold and in the scaffold without cells in vivo. Conclusion: compounds of osteoblastic induced rMSCs and the scaffold with ?-TCP/PLLA = 2:1, NaCl = 70 wt%, pore size = 200-450 ?m had good properties as a kind of bone substitute.

  7. Influence of Culture Medium on Smooth Muscle Cell Differentiation from Human Bone Marrow–Derived Mesenchymal Stem Cells

    PubMed Central

    Gong, Zhaodi; Calkins, Geoffrey; Cheng, Ee-chun; Krause, Diane

    2009-01-01

    Human bone marrow–derived mesenchymal stem cells (hMSCs) represent an appealing source of smooth muscle cells (SMCs) for engineering small-diameter vascular grafts due to the limited availability and replicative capacity of somatic SMCs. However, lack of standardization of hMSC culture conditions has limited some progress in hMSC research. Because, at the moment, a chemically defined, serum-free medium without growth factors is not capable of amplifying hMSCs in vitro, the usage of serum (either human serum or fetal bovine serum [FBS]) continues in hMSC research. The emergence of commercial hMSCs and hMSC media opened a series of questions regarding the compatibility of commercial and homemade hMSCs and hMSC media. In this study, two types of commonly used FBS-containing hMSC media—MSCGM (containing 10% FBS) and MesenPro (containing 2% FBS), along with our homemade medium (low-glucose Dulbecco's modified Eagle's medium plus 10% selected lot FBS)—were compared in their ability to support SMC differentiation from hMSCs. The effects of FBS level, medium supplements (ascorbic acid, copper, etc.), and growth factors (transforming growth factor ?1) were also examined for their impact on SMC differentiation. It was discovered that MesenPro and transforming growth factor ?1 are the strongest SMC inducers from hMSCs. In contrast, hMSCs grown in homemade (10% Dulbecco's modified Eagle's medium) and commercial MSCGM media remained undifferentiated. FBS concentration did not affect SMC differentiation when 10% FBS was compared with 2%. Finally, the mechanism underlying SMC differentiation from hMSCs grown in FBS-containing medium was explored by following the expression changes of serum response factor during the establishment of hMSC culture. PMID:19115826

  8. Serum-free isolation and culture system to enhance the proliferation and bone regeneration of adipose tissue-derived mesenchymal stem cells.

    PubMed

    Sato, Kazutoshi; Itoh, Takehiro; Kato, Toshiki; Kitamura, Yukiko; Kaul, Sunil C; Wadhwa, Renu; Sato, Fujio; Ohneda, Osamu

    2015-05-01

    Cell therapy using human mesenchymal stem cells (MSCs) is an attractive approach for many refractory diseases. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are considered as a favorable tool due to its abundance in the body, easy proliferation, and high cytokine production potency. In order to avoid the risks associated with the use of fetal bovine serum (FBS) in culture that includes batch variations and contamination with pathogens, development of serum-free culture system has been initiated. We have formulated a completely serum-free culture medium (SFM) that could be used not only for the expansion of AT-MSCs but also for initial isolation. We demonstrate that the AT-MSCs isolated and cultured in serum-free medium (AT-MSCs/SFM) possess high proliferation capacity and differentiation potency to osteoblast, adipocyte, and chondrocyte lineages in vitro. In in vivo bone fraction model analysis, AT-MSCs/SFM showed higher bone repair potency and quality of the regenerated bone than the cells cultured in serum-containing medium (AT-MSCs/SCM). This was attributed to the (i) presence of translated cells in the bone, as evidenced by in vivo imaging of the illuminated translated cells and (ii) high level of expression and induction capacity of AT-MSCs/SFM for cytokine BMP2, CCL2, and CCL5. Taken together, we report a new serum-free culture system for AT-MSCs that is suitable for cell therapy. PMID:25588776

  9. Improved revascularization of islet grafts using an angiogenic monocyte subpopulation derived from spheroid culture of bone marrow mononuclear cells.

    PubMed

    Oh, B J; Jin, S-M; Choi, J-M; Oh, S-H; Shim, W; Lee, M-S; Lee, M-K; Kim, J H

    2015-06-01

    The spheroid culture method is an effective strategy for ex vivo expansion of an autologous therapeutic cell population. We investigated if cotransplantation of bone marrow-derived spheroids (BM-spheroid) formed using 3D culture of BM-derived mononuclear cells (BM-MNCs) could improve the posttransplant outcome of islet grafts using a mouse syngeneic marginal mass renal subcapsular islet transplantation model. Using green fluorescent protein transgenic (GFP-Tg) mice, the role of the BM-spheroids and the contribution of vessels derived from donors and recipients in grafted areas were assessed by immunohistochemistry. Compared to fresh BM-MNCs and nonspheroid remnant cells (BM-nonspheroid), the BM-spheroids, mainly composed of CXCR4(+) CD14(+) myeloid cells, showed higher angiogenic capacity, such as in vitro self-formed vessel structures; increased expression of angiogenic and chemoattractive factors; and incorporation into new vessel formation in basement membrane matrix plugs. BM-spheroid cotransplantation with islets improved the posttransplant outcomes in terms of glucose tolerance, serum insulin level, and diabetes reversal rate when compared with cotransplantation of BM-nonspheroids. Immunohistochemistry revealed that cotransplantation of the BM-spheroids increased vessel density, area of grafted endocrine and non-endocrine tissue, and ? cell proliferation. In conclusion, cotransplantation of islets and BM-spheroids improved islet function through facilitation of revascularization and an increase in cell proliferation and islet cell mass. PMID:25865268

  10. Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration.

    PubMed

    Lee, Jung-Seok; Park, Jung-Chul; Kim, Tae-Wan; Jung, Byung-Joo; Lee, Youngseok; Shim, Eun-Kyung; Park, Soyon; Choi, Eun-Young; Cho, Kyoo-Sung; Kim, Chang-Sung

    2015-09-01

    Human bone marrow mesenchymal stem cells (hBMSCs) were isolated from bone marrow of the vertebral body. The hBMSCs were cultured under either hypoxic (1% O2) or normoxic (21% O2; control) conditions and the characteristics as mesenchymal stem cells were compared. Results revealed that hypoxia reduced proliferative potential and colony-forming efficiency of hBMSCs, and significantly enhanced osteogenic and chondrogenic differentiation. The hBMSCs enhanced the regenerative potential of bone in vivo. In vitro synthesis of soluble and insoluble collagen was significantly increased in the hypoxic condition. In vivo collagen tissue regeneration was also enhanced under the hypoxic condition, with concomitant increased expressions of various subtypes of collagen and lysyl-oxidase family mRNA. MicroRNA assays revealed that miR-155-5p, which negatively regulates HIF-1?, was significantly highly expressed. These observations demonstrate that hBMSCs obtained from human vertebrae exhibit altered characteristics under hypoxic conditions, and each factor contributing to hBMSC-mediated tissue healing should be evaluated with the goal of allowing their clinical application. PMID:25952967

  11. Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies

    NASA Astrophysics Data System (ADS)

    Sun, Jianfei; Liu, Xuan; Huang, Jiqing; Song, Lina; Chen, Zihao; Liu, Haoyu; Li, Yan; Zhang, Yu; Gu, Ning

    2014-05-01

    Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon.

  12. Magnetic assembly-mediated enhancement of differentiation of mouse bone marrow cells cultured on magnetic colloidal assemblies

    PubMed Central

    Sun, Jianfei; Liu, Xuan; Huang, Jiqing; Song, Lina; Chen, Zihao; Liu, Haoyu; Li, Yan; Zhang, Yu; Gu, Ning

    2014-01-01

    Here we reported an interesting phenomenon that the field-induced assemblies of magnetic nanoparticles can promote the differentiation of primary mouse bone marrow cells into osteoblasts. The reason was thought to lie in the remnant magnetic interaction inside the assemblies which resulted from the magnetic field-directed assembly. Influence of the assemblies on the cells was realized by means of interface effect rather than the internalization effect. We fabricated a stripe-like assemblies array on the glass plate and cultured cells on this surface. We characterized the morphology of assemblies and measured the mechanic property as well as the magnetic property. The cellular differentiation was measured by staining and quantitative PCR. Finally, Fe uptake was excluded as the reason to cause the phenomenon. PMID:24874764

  13. Bone tissue engineering with human stem cells

    Microsoft Academic Search

    Darja Marolt; Miomir Knezevic; Gordana Vunjak Novakovic

    2010-01-01

    Treatment of extensive bone defects requires autologous bone grafting or implantation of bone substitute materials. An attractive\\u000a alternative has been to engineer fully viable, biological bone grafts in vitro by culturing osteogenic cells within three-dimensional scaffolds, under conditions supporting bone formation. Such grafts\\u000a could be used for implantation, but also as physiologically relevant models in basic and translational studies of

  14. Guanosine-rich oligodeoxynucleotides induce proliferation of macrophage progenitors in cultures of murine bone marrow cells.

    PubMed

    Lang, R; Hültner, L; Lipford, G B; Wagner, H; Heeg, K

    1999-11-01

    Widely used to specifically inhibit gene expression, synthetic oligodeoxynucleotides (ODN) can exert a plethora of non-antisense effects. Immunostimulation by CpG-ODN has attracted particular attention. ODN rich in the nucleotide guanosine (G-rich ODN) constitute another type of sequences displaying non-antisense-mediated effects. We have examined the effects of CpG- and G-rich ODN on primary mouse bone marrow cells (BMC) in vitro. CpG-ODN induced rapid proliferation of B cells and production of IL-6 and IL-12p40. However, when tested in agar colony assays, CpG-ODN failed to promote the formation of colonies. In marked contrast, G-rich non-CpG-ODN led to sustained proliferation of macrophage-like cells without inducing cytokines or hemopoietic growth factors. Unlike CpG-ODN, G-rich ODN effectively induced the formation of macrophage colonies in agar assays, indicating a direct action on progenitor cells. Electrophoretic mobility shift assays revealed specific binding of G-rich ODN to a non-nuclear protein. The ability of a panel of ODN to compete for binding correlated with their potential to induce proliferation of macrophage-like cells from primary mouse BMC. As such, these data reveal a so far unrecognized potential of G-rich ODN to signal directly outgrowth of macrophage progenitors from BMC. PMID:10556804

  15. Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cells.

    PubMed

    Pal, Rakhi; Hanwate, Madhuri; Jan, Majahar; Totey, Satish

    2009-03-01

    Human adult bone marrow-derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi-lineage differentiation potential. We compare the different basal culture media DMEM-F12, DMEM-high glucose (DMEM-HG), DMEM-low glucose (DMEM-LG), knock-out DMEM (DMEM-KO) and Mesencult on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO-SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up-scaling to provide sufficient numbers for transplantation. PMID:19229888

  16. In vitro generation of functional insulin-producing cells from human bone marrow-derived stem cells, but long-term culture running risk of malignant transformation.

    PubMed

    Tang, Dong-Qi; Wang, Qiwei; Burkhardt, Brant R; Litherland, Sally A; Atkinson, Mark A; Yang, Li-Jun

    2012-06-30

    Efforts involving therapeutic islet cell transplantation have been hampered by limited islet availability and immune rejection. In vitro transdifferentiation of human bone marrow-derived stem (hBMDS) cells into functional insulin-producing cells promises to provide a tissue source for autologous cell transplantation. In this study, we isolated hBMDS cells, developed a single-cell-derived stem cell line, and induced the cells to differentiate into islet-like clusters. These islet-like cells expressed multiple genes related to islet development and beta cell function (e.g., Pdx-1, Ngn-3, Islet-1, Neuro-D, Pax4, IAPP, and insulin) and produced insulin and C-peptide within these cells. These islet-like cells demonstrated time-dependent glucose-stimulated insulin release, and the ability to ameliorate hyperglycemia in chemically induced diabetic mice. However, these transplanted differentiated cells became tumorigenic in diabetic immunocompromised mice and their spontaneous transformation was confirmed by a marked increase in growth rate and inactivation of tumor suppressor genes (P21 and P16) by promoter hypermethylation. In conclusion, while hBMDS cells can be transdifferentiated into competent insulin-producing cells, and while such cell might be a potential source for autologous cell therapy for type 1 diabetes, caution is strongly advised in view of the neoplastic propensity of hBMDS cells, especially after a long-term culture in vitro. PMID:22833839

  17. Optimization of culture condition of human bone marrow stromal cells in terms of purification, proliferation, and pluripotency.

    PubMed

    Zhou, Ying; Yu, Dan; Zhu, Huiyong

    2014-10-01

    Human bone marrow stromal cells (hBMSCs) possess multilineage differentiation potential and play an important role in modern tissue engineering. However, the development of culture media to maintain hBMSCs in an undifferentiated, self-renewing state during their robust proliferation remains a challenge. We developed and tested modified growth medium [medium 1: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), low glucose, 2% fetal calf serum (FCS)] on hBMSCs by comparing primary cell isolation, multipassage expansion, culture morphology, proliferation, and cellular phenotype, and performing an expression analysis of intrinsic-regulated genes to other two media. Cell morphology, proliferation, and phenotype varied among the media, while cells cultured in medium 1 displayed small, spindle-shaped morphology with the highest rate of growth capacities and the expected phenotype. RT-PCR analysis showed that medium 1 displayed the lowest expression levels of osteogenic genes, chondrogenic genes (osteonectin, runt-related transcription factor 2, cartilage oligo matrix protein, and SOX9), and adipogenic genes (lipoprotein lipase). The expression of another adipogenic gene, peroxisome proliferator-activator receptor-?2, was higher in medium 1 but did not reach significance. In addition, hBMSCs expanded in medium 1 showed the highest expression ratio of self-renewing-related genes Krüppel-like factor 2 (KLF2) and KLF5. In conclusion, medium 1 allows for better expansion and pluripotency maintenance of hBMSCs and serves as a preferred alternative to traditional serum-containing media for research applications and future clinical use. PMID:24934232

  18. Human Bone Marrow-Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-term In vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms

    Microsoft Academic Search

    Maria Ester Bernardo; Nadia Zaffaroni; Francesca Novara; Angela Maria Cometa; Maria Antonietta Avanzini; Antonia Moretta; Daniela Montagna; Rita Maccario; Raffaella Villa; Maria Grazia Daidone; Orsetta Zuffardi; Franco Locatelli

    2007-01-01

    Significant improvement in the understanding of mesenchy- mal stem cell (MSC) biology has opened the way to their clinical use. However, concerns regarding the possibility that MSCs undergo malignant transformation have been raised. We investigated the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different in vitro culture time points. MSCs were isolated from BM of 10 healthy

  19. Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

    PubMed Central

    2013-01-01

    Background The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results Relative to control, FBS and HS increased cell number (133?±?5 and 116?±?5%, respectively; P??0.8). Runt-related transcription factor2 expression increased 3-fold (P?culture. Osterix expression increased 9-fold (P?culture. Expression of Tbx3 increased 1.8-fold at d 3 (P?

  20. Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells

    Microsoft Academic Search

    Yudi Bai; Yuxiang Bai; Kenichi Matsuzaka; Sadamitsu Hashimoto; Eitoyo Kokubu; Xiaojing Wang; Takashi Inoue

    2010-01-01

    During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts\\/cementoblasts when they are in contact\\u000a with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could\\u000a induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation\\u000a could occur in a co-culture model. To test this hypothesis,

  1. An Exploratory Clinical Trial for Idiopathic Osteonecrosis of Femoral Head by Cultured Autologous Multipotent Mesenchymal Stromal Cells Augmented with Vascularized Bone Grafts

    PubMed Central

    Aoyama, Tomoki; Goto, Koji; Kakinoki, Ryosuke; Ikeguchi, Ryosuke; Ueda, Michiko; Kasai, Yasunari; Maekawa, Taira; Tada, Harue; Teramukai, Satoshi; Nakamura, Takashi

    2014-01-01

    Idiopathic osteonecrosis of femoral head (ION) is a painful disorder that progresses to collapse of the femoral head and destruction of the hip joint. Although its precise pathology remains unknown, the loss of blood supply causing the loss of living bone-forming cells is a hallmark of the pathophysiology of osteonecrosis. Transplantation of multipotent mesenchymal stromal cells (MSCs) is a promising tool for regenerating the musculoskeletal system. The aim of the present study was to assess the safety and efficacy of transplantation of cultured autologous bone marrow-derived MSCs mixed with ?-tricalcium phosphate (?-TCP) in combination with vascularized bone grafts for the treatment of advanced stage ION in a clinical trial. Ten patients with stage 3 ION were enrolled in this study. Autologous bone marrow-derived MSCs were cultured with autologous serum, and cells (0.5–1.0×108) were transplanted after mixing with ?-TCP granules in combination with vascularized iliac bone grafts. Patients were assessed 24 months after treatment. The primary and secondary endpoints were progression of the radiological stage and changes in bone volume at the femoral head, and clinical score, respectively. Nine of ten patients completed the protocol, seven of whom remained at stage 3, and the remaining two cases progressed to stage 4. The average bone volume increased from 56.5±8.5?cm3 to 57.7±10.6?cm3. The average clinical score according to the Japan Orthopaedic Association improved from 65.6±25.5 points to 87.9±19.0 points. One severe adverse event was observed, which was not related to the clinical trial. Although the efficacy of cell transplantation was still to be determined, all procedures were successfully performed and some young patients with extensive necrotic lesions with pain demonstrated good bone regeneration with amelioration of symptoms. Further improvements in our method using MSCs and the proper selection of patients will open a new approach for the treatment of this refractory disease. PMID:24593258

  2. Direct cell contact influences bone marrow mesenchymal stem cell fate

    Microsoft Academic Search

    Stephen G Ball; Adrian C Shuttleworth; Cay M Kielty

    2004-01-01

    Adult bone marrow-derived mesenchymal stem cells (MSC) can differentiate into various cell types of mesenchymal origin, but mechanisms regulating such cellular changes are unclear. We have conducted co-culture experiments to examine whether mesenchymal stem cell differentiation is influenced by indirect or direct contact with differentiated cells. Cultured adult mesenchymal stem cells showed some characteristics of synthetic state vascular smooth muscle

  3. Titanium containing amorphous hydrogenated carbon films (a-C: H/Ti): surface analysis and evaluation of cellular reactions using bone marrow cell cultures in vitro.

    PubMed

    Schroeder, A; Francz, G; Bruinink, A; Hauert, R; Mayer, J; Wintermantel, E

    2000-03-01

    Amorphous hydrogenated carbon (a-C : H) coatings, also called diamond-like carbon (DLC), have many properties required for a protective coating material in biomedical applications. The purpose of this study is to evaluate a new surface coating for bone-related implants by combining the hardness and inertness of a-C : H films with the biological acceptance of titanium. For this purpose, different amounts of titanium were incorporated into a-C : H films by a combined radio frequency (rf) and magnetron sputtering set-up. The X-ray photoelectron spectroscopy (XPS) of air-exposed a-C : H/titanium (a-C : H/Ti) films revealed that the films were composed of TiO2 and TiC embedded in and connected to an a-C : H matrix. Cell culture tests using primary adult rat bone marrow cell cultures (BMC) were performed to determine effects on cell number and on osteoblast and osteoclast differentiation. By adding titanium to the carbon matrix, cellular reactions such as increased proliferation and reduced osteoclast-like cell activity could be obtained, while these reactions were not seen on pure a-C : H films and on glass control samples. In summary, a-C : H/Ti could be a valuable coating for bone implants, by supporting bone cell proliferation while reducing osteoclast-like cell activation. PMID:10674809

  4. Dynamics of bone marrow-derived endothelial progenitor cell\\/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

    Microsoft Academic Search

    A. Aguirre; J. A. Planell; E. Engel

    2010-01-01

    Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications.

  5. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: Basic science to clinical translation

    Microsoft Academic Search

    Hideaki Kagami; Hideki Agata; Arinobu Tojo

    2011-01-01

    Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. This technology has already been used in several clinical studies and its efficacy has been reported. In this review, we focus on bone marrow stromal cells, which are the most commonly used cell source for bone

  6. Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

    PubMed

    Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Juritz, Stephanie; Birk, Richard; Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Schultz, Johannes; Hörmann, Karl; Kinscherf, Ralf; Faber, Anne

    2014-01-01

    The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-?1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium. PMID:24220225

  7. The Bone Morphogenetic Protein Type Ib Receptor Is a Major Mediator of Glial Differentiation and Cell Survival in Adult Hippocampal Progenitor Cell Culture

    PubMed Central

    Brederlau, A.; Faigle, R.; Elmi, M.; Zarebski, A.; Sjöberg, S.; Fujii, M.; Miyazono, K.; Funa, K.

    2004-01-01

    Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture. PMID:15194807

  8. Bone-Immune Cell Crosstalk: Bone Diseases

    PubMed Central

    Mori, Giorgio; D'Amelio, Patrizia; Faccio, Roberta

    2015-01-01

    Bone diseases are associated with great morbidity; thus, the understanding of the mechanisms leading to their development represents a great challenge to improve bone health. Recent reports suggest that a large number of molecules produced by immune cells affect bone cell activity. However, the mechanisms are incompletely understood. This review aims to shed new lights into the mechanisms of bone diseases involving immune cells. In particular, we focused our attention on the major pathogenic mechanism underlying periodontal disease, psoriatic arthritis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, metastatic solid tumors, and multiple myeloma. PMID:26000310

  9. In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice

    SciTech Connect

    Greenberger, J.S. (Havard Medical School, Boston, MA); Eckner, R.J.; Otten, J.A.; Tennant, R.W.

    1982-07-01

    The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10/sup 6/ cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system. (JMT)

  10. Stromal cells in long-term cultures of liver, spleen, and bone marrow at different developmental ages have different capacities to maintain GM-CFC proliferation.

    PubMed

    Van Den Heuvel, R; Schoeters, G; Leppens, H; Vanderborght, O

    1991-02-01

    Measurements were made of the granulocyte-macrophage colony-forming cell (GM-CFC) yield in long-term cultures established from different combinations of stroma and hemopoietic recharge inocula derived from hemopoietic organs at different stages of their embryological development. Results indicated differences in the supporting capacity of the stroma, related to the hemopoietic activity of the organ of origin. Stroma derived from hemopoietically active organs (adult bone marrow, neonatal spleen, fetal liver) supported the proliferation of GM-CFC to a larger extent than stroma derived from organs with a low hemopoietic activity (neonatal bone marrow liver at 2 days; spleen at 3 weeks). Regardless of the origin of the hemopoietic cells, stroma from adult bone marrow displayed the highest ability to support GM-CFC proliferation. The capacity of GM-CFC from hemopoietic recharge cell populations to proliferate on stroma was not related to the hemopoietic activity of their organ of origin. Regardless of their organ of origin the GM-CFC present in each of the different hemopoietic recharge populations were able to proliferate provided that they were seeded on an appropriate stroma. These experiments showed that stromal cells cultured from hemopoietic organs at different developmental ages determine the hemopoietic activity of long-term cultures as measured via GM-CFC recovery. PMID:1991493

  11. Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

    PubMed

    Zappitelli, Tanya; Chen, Frieda; Aubin, Jane E

    2015-03-01

    Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein ?1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model. PMID:25568340

  12. A porous scaffold for bone tissue engineering\\/45S5 Bioglass ® derived porous scaffolds for co-culturing osteoblasts and endothelial cells

    Microsoft Academic Search

    Sanjukta Deb; Ramin Mandegaran; Lucy Di Silvio

    2010-01-01

    One of the major factors in the therapeutic success of bone tissue engineered scaffolds is the ability of the construct to\\u000a vascularise post implantation. One of the approaches for improving vascularisation within scaffolds has been to co-culture\\u000a human umbilical vein endothelial cells (HUVECS) with human osteoblasts (HOBS), which may then promote vascularisation and\\u000a facilitate tissue regeneration. However, in order to

  13. Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

    PubMed Central

    Chung, H.; Kang, Y. S.; Hwang, C. S.; Moon, I. K.; Yim, C. H.; Choi, K. H.; Han, K. O.; Jang, H. C.; Yoon, H. K.; Han, I. K.

    2001-01-01

    Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells. PMID:11748360

  14. Bone and parathyroid inhibitory effects of S-2(3-aminopropylamino)ethylphosphorothioic acid. Studies in experimental animals and cultured bone cells

    SciTech Connect

    Attie, M.F.; Fallon, M.D.; Spar, B.; Wolf, J.S.; Slatopolsky, E.; Goldfarb, S.

    1985-04-01

    S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR 2721) is a radio- and chemoprotective agent which produces hypocalcemia in humans. Intravenous injection of 30 mg/kg WR 2721 in rats and 15 mg/kg in dogs lowers serum calcium by 19 and 25%, respectively. Hypocalcemia in dogs is associated with a fall in serum immunoreactive parathyroid hormone (PTH), which suggests that the mechanism of its hypocalcemic effect is acute hypoparathyroidism. Despite this effect on PTH, in eight chronically parathyroidectomized rats on a low phosphate diet, WR 2721 reduced serum calcium from 9.4 to 7.7 mg/dl at 3 h. Furthermore, in dogs rendered hypercalcemic by continuous infusion of PTH, WR 2721 reduced serum calcium from 11.0 to 10.6 mg/dl. To determine whether WR 2721 causes hypocalcemia by enhancing the exit of calcium from the circulation or inhibiting its entry, the drug was infused 3 h after administration of /sup 45/Ca to rats. WR 2721 did not significantly increase the rate of disappearance of /sup 45/Ca from the circulation even though serum calcium fell by 19%. In incubations with fetal rat long bone labeled in utero with /sup 45/Ca, 10(-3) M WR 2721 inhibited PTH-stimulated, but not base-line release of /sup 45/Ca. Bone resorption by primary culture of chick osteoclasts was inhibited by WR 2721 at concentrations as low as 10(-4) M in the absence of hormonal stimulation. These studies suggest that WR 2721 lowers serum calcium predominantly by blocking calcium release from bone. This acute hypocalcemic effect is at least in part independent of its effect on the parathyroid glands, and is most likely a direct effect of the agent on bone resorption.

  15. Cell-based therapies for regenerating bone

    PubMed Central

    GOODMAN, S. B.

    2013-01-01

    Cellular therapies to replenish bone lost due to acquired conditions such as trauma, infection, tumor, periprosthetic osteolysis and other etiologies have become widespread. Traditional, open, surgical bone grafting techniques have given way to newer cellular therapies that are potentially less invasive and have a lower complication rate and faster recovery time. These new technologies include bone marrow harvesting with concentration of osteoprogenitor cells with/without cell culture, scaffolds which are both osteoconductive and osteoinductive, attempts to facilitate mesenchymal stem cell and osteoprogenitor cell homing both locally and systemically, genetic engineering of specialized stem cells, and the use of potentially immune-privileged fetal and other types of stem cells. Some of these techniques have already been introduced into the orthopaedic clinic, whereas others are still in the pre-clinical testing phase. Given the limited supply of autologous graft, these new techniques will have a dramatic impact on bone regeneration in the future. PMID:24436510

  16. In vitro investigation of titanium and hydroxyapatite dental implant surfaces using a rat bone marrow stromal cell culture system

    Microsoft Academic Search

    C. Knabe; F. Klar; R. Fitzner; R. J. Radlanski; U. Gross

    2002-01-01

    In this study, rat bone marrow cells (RBM) were used to evaluate different titanium and hydroxyapatite dental implant surfaces. The implant surfaces investigated were: a titanium surface having a porous titanium plasma-sprayed coating (sample code Ti-TPS), a titanium surface with a deep profile structure (sample code Ti-DPS), an uncoated titanium substrate with a machined surface (sample code Ti-ma) and a

  17. Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo.

    PubMed

    Purroy, Noelia; Abrisqueta, Pau; Carabia, Júlia; Carpio, Cecilia; Palacio, Carles; Bosch, Francesc; Crespo, Marta

    2015-04-10

    Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells. PMID:25544766

  18. Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures.

    PubMed

    Weichold, F F; Zella, D; Barabitskaja, O; Maciejewski, J P; Dunn, D E; Sloand, E M; Young, N S

    1998-02-01

    Attempts to clarify the pathophysiology of human immunodeficiency virus (HIV)-mediated bone marrow (BM) dysfunction have yielded inconsistent results regarding the susceptibility of BM progenitors to the viral infection. To specifically address this question, we exposed highly purified subpopulations of human BM progenitor cells to various HIV-1 and HIV-2 strains and assessed (pro)viral gene presence and expression in more-committed (CD34+CD38+) as well as most-primitive (CD34+CD38-) cells in long-term BM cultures. Quantitative analysis of long-term culture-initiating cells (LTCIC) failed to demonstrate adverse effects of exposing hematopoietic stem cells to HIV. Our results show that HIV-2, similar to HIV-1, does not infect hematopoietic stem cells in vitro with any significant frequency and infected cells are not present within LTCICs. Cytofluorometric analysis of CD34+ cells for surface molecules that facilitate HIV entry was consistent with the functional assay in that expression of virus receptors was predominantly on the more-committed subsets of BM progenitors. The failure to detect productive or latent HIV in the most-primitive human BM progenitor and stem cells has important implications for future therapeutic strategies, including those dealing with transduction of these cells with protective genes as a treatment modality for AIDS. PMID:9446651

  19. GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.

    PubMed

    Helft, Julie; Böttcher, Jan; Chakravarty, Probir; Zelenay, Santiago; Huotari, Jatta; Schraml, Barbara U; Goubau, Delphine; Reis E Sousa, Caetano

    2015-06-16

    Dendritic cells (DCs) are key players in the immune system. Much of their biology has been elucidated via culture systems in which hematopoietic precursors differentiate into DCs under the aegis of cytokines. A widely used protocol involves the culture of murine bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate BM-derived DCs (BMDCs). BMDCs express CD11c and MHC class II (MHCII) molecules and share with DCs isolated from tissues the ability to present exogenous antigens to T cells and to respond to microbial stimuli by undergoing maturation. We demonstrate that CD11c(+)MHCII(+) BMDCs are in fact a heterogeneous group of cells that comprises conventional DCs and monocyte-derived macrophages. DCs and macrophages in GM-CSF cultures both undergo maturation upon stimulation with lipopolysaccharide but respond differentially to the stimulus and remain separable entities. These results have important implications for the interpretation of a vast array of data obtained with DC culture systems. PMID:26084029

  20. Effects of water-holding capability of the PVF sponge on the adhesion and differentiation of rat bone marrow stem cell culture.

    PubMed

    Togami, Wakana; Sei, Akira; Okada, Tatsuya; Taniwaki, Takuya; Fujimoto, Toru; Nakamura, Takayuki; Tahata, Shogo; Nakanishi, Yoshitaka; Mizuta, Hiroshi

    2014-01-01

    The aim of the study is to estimate the effects of the water-holding capability of the polyvinyl formal (PVF) sponges on osteogenic response in vitro experiments. The rat bone marrow stem cells (BMCs) were seeded and cultured for up to 4 weeks under static conditions in osteogenic media to evaluate the adhesion, proliferation, differentiation, and mineralization on the Dextran-coated PVF sponges with or without water-holding capability. The BMCs seeded onto the PVF sponges with water-holding capability showed more significant increases in DNA content, alkaline phosphatase (ALP) activity, osteocalcin content, and calcium deposition than those without water-holding capability. These results suggest that the Dextran-coated PVF sponges with high water-holding capability would have potential uses as both a new scaffold to bone tissue engineering and as a new biomaterial. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 247-253, 2014. PMID:23657866

  1. Effects of water-holding capability of the PVF sponge on the adhesion and differentiation of rat bone marrow stem cell culture.

    PubMed

    Togami, Wakana; Sei, Akira; Okada, Tatsuya; Taniwaki, Takuya; Fujimoto, Toru; Nakamura, Takayuki; Tahata, Shogo; Nakanishi, Yoshitaka; Mizuta, Hiroshi

    2013-07-22

    The aim of the study is to estimate the effects of the water-holding capability of the polyvinyl formal (PVF) sponges on osteogenic response in vitro experiments. The rat bone marrow stem cells (BMCs) were seeded and cultured for up to four weeks under static conditions in osteogenic media to evaluate the adhesion, proliferation, differentiation and mineralization on the Dextran-coated PVF sponges with or without water-holding capability. The BMCs seeded onto the PVF sponges with water-holding capability showed more significant increases in DNA content, alkaline phosphatase (ALP) activity, osteocalcin content and calcium deposition than those without water-holding capability. These results suggest that the Dextran-coated PVF sponges with high water-holding capability would have potential uses as both a new scaffold to bone tissue engineering and as a new biomaterial. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A. PMID:23878106

  2. Clonal Characterization of Bone Marrow Derived Stem Cells and Their Application for Bone Regeneration

    PubMed Central

    Xiao, Yin; Mareddy, Shobha; Crawford, Ross

    2010-01-01

    Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single-cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homogeneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous population. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo. PMID:21125790

  3. Development of magnetic particle techniques for long-term culture of bone cells with intermittent mechanical activation

    Microsoft Academic Search

    Sarah H. Cartmell; Jon Dobson; Sarah B. Verschueren; Alicia J. El Haj

    2002-01-01

    Magnetic particles were coated with RGD and adhered to primary human osteoblasts. During a 21-day culture, the osteoblasts plus adhered magnetic particles underwent a daily exposure to a time-varying magnetic field via a permanent NdFeB magnet, thus applying a direct mechanical stress to the cells (Bmax?60 mT). After 21 days, preliminary results show that the cells plus magnetic particles were

  4. Rat mucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpart in vivo.

    PubMed

    MacDonald, A J; Pick, J; Bissonnette, E Y; Befus, A D

    1998-04-01

    Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC. PMID:9659226

  5. Donor Age and Mechanosensitivity of Human Bone Cells

    Microsoft Academic Search

    J. Klein-Nulend; J. G. H. Sterck; C. M. Semeins; P. Lips; M. Joldersma; J. A. Baart; E. H. Burger

    2002-01-01

    :   With increasing age the human skeleton decreases in density, thereby compromising its load-bearing capacity. Mechanical loading\\u000a activates bone formation, but an age-dependent decrease in skeletal mechanoresponsiveness has been described in rats. In this\\u000a paper we examine whether age-related bone loss is reflected by a decrease in the mechanosensitivity of isolated bone cells\\u000a from human donors. Bone cell cultures were

  6. Adult Bone Marrow Stem/Progenitor Cells (MSCs) Are Preconditioned by Microenvironmental "Niches" in Culture: A Two-Stage Hypothesis for Regulation of MSC Fate

    NSDL National Science Digital Library

    Carl A. Gregory (Tulane University Health Sciences Center; Center for Gene Therapy REV)

    2005-07-26

    Mesenchymal stem cells (MSCs) are clonal, plastic adherent cells from bone marrow that can differentiate into various tissue lineages, including osteoblasts, adipocytes, chondrocytes, myoblasts, hepatocytes, and possibly even neural cells. Because MSCs are multipotent and their numbers are easily expanded in culture, there has been much interest in their clinical potential for tissue repair and gene therapy. Consequently, numerous studies have been carried out demonstrating the migration and multiorgan engraftment potential of MSCs in animal models and in human clinical trials. Understanding the mechanisms behind MSC cell fate determination is not easy, because the molecular processes that drive engraftment and differentiation are complex. Even in an in vitro system, the molecular cues necessary to induce differentiation are not easily identified or reproduced. In this Perspective, we emphasize the importance of microenvironmental factors in culture and suggest that MSC differentiation in vitro is regulated by a two-stage mechanism involving preconditioning by factors in the culture microenvironment followed by response to soluble differentiating factors.

  7. Cell Stem Cell Endogenous Bone Marrow MSCs

    E-print Network

    Mootha, Vamsi K.

    ). The existence of multipotent bone marrow stromal cells (BMSCs), or skeletal/mesenchymal stem cells (SSCs.02.003 SUMMARY Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical bone- marrow-derived, Mx1+ stromal cells with ``MSC'' features. These cells respond to tissue stress

  8. Adaptation and Infection of Mouse Bone Marrow (JLS-V9) Cells in Suspension Culture for Production of Rauscher Leukemia Virus

    PubMed Central

    Hodge, Howard M.; Klein, Frederick; Bandyopadhyay, Alok K.; Robinson, Orson R.; Shibley, George P.

    1974-01-01

    JLS-V9 mouse bone marrow cells were readily adapted to suspension culture, chronically infected with Rauscher leukemia virus (RLV), and subsequently grown in 7.5- and 14-liter New Brunswick fermentors. The suspension-type cell system can be modified to produce virus with clearly defined properties, such as high ribonucleic acid-dependent deoxyribonucleic acid polymerase (RDDP) activity, high particle count, and high infectious particle count. Biological and biophysical properties of suspension-produced RLV were not affected by concentration and purification employing continuous-flow and rate-zonal centrifugation procedures. The RDDP assay was standardized and showed a linear incorporation of 3H-thymidine 5?-monophosphate (3H-TMP) up to 30 min. Further characterization indicated that a high percentage of 3H-TMP incorporation was due to RDDP. Images PMID:4129475

  9. Human bone marrow-derived stromal cells cultured with a plasma sprayed CaO-ZrO2-SiO2 coating.

    PubMed

    Yang, Fei; Xie, Youtao; Li, Huiwu; Tang, Tingting; Zhang, Xiaoling; Gan, Yaokai; Zheng, Xuebin; Dai, Kerong

    2010-10-01

    The CaO-ZrO2-SiO2 (CZS) coating was prepared by plasma-spraying chemically synthesized CZS powder onto a Ti-6Al-4V substrate. This CZS coating has been demonstrated to have good bioactivity, high bonding strength with the substrate and a low degradation rate. However, the effect of CZS coating on the osseointegration of bone-implant is still unknown. In this study, human bone marrow-derived stromal cells (hBMSCs) were cultured on CZS coating in vitro, and cell behavior was investigated, with the classical hydroxyapatite (HA) coating as a control. Scanning electron microscopy (SEM) and immunofluorescence studies showed that the hBMSCs on the CZS coating spread well with organized cytoskeleton structure at 24 h following cell seeding. The MTT assay and the Alamar Blue assay indicated that CZS coating promoted the attachment and proliferation of hBMSCs. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes, such as ALP, collagen I (COLI), osteopontin (OPN), and osteocalcin (OCN), demonstrated that the osteoblastic differentiation of hBMSCs was enhanced more by CZS coating than by HA coating. These results suggest that CZS coating possess excellent biological properties and may have potential in biomedical applications. PMID:20737586

  10. Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

    PubMed Central

    2012-01-01

    Introduction Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into several mesenchymal lineages, classically derived from bone marrow (BM) but potentially from umbilical cord blood (UCB). Although they are becoming a good tool for regenerative medicine, they usually need to be expanded in fetal bovine serum (FBS)-supplemented media. Human platelet lysate (HPL) has recently been proposed as substitute for safety reasons, but it is not yet clear how this supplement influences the properties of expanded MSCs. Methods In the present study, we compared the effect of various media combining autologous HPL with or without FBS on phenotypic, proliferative and functional (differentiation, cytokine secretion profile) characteristics of human BM-derived MSCs. Results Despite less expression of adipogenic and osteogenic markers, MSCs cultured in HPL-supplemented media fully differentiated along osteoblastic, adipogenic, chondrogenic and vascular smooth muscle lineages. The analyses of particular specific proteins expressed during osteogenic differentiation (calcium-sensing receptor (CaSR) and parathormone receptor (PTHR)) showed their decrease at D0 before any induction for MSC cultured with HPL mostly at high percentage (10%HPL). The cytokine dosage showed a clear increase of proliferation capacity and interleukin (IL)-6 and IL-8 secretion. Conclusions This study shows that MSCs can be expanded in media supplemented with HPL that can totally replace FBS. HPL-supplemented media not only preserves their phenotype as well as their differentiation capacity, but also shortens culture time by increasing their growth rate. PMID:22333342

  11. Insect Cell Culture

    Microsoft Academic Search

    Oers van M. M; D. E. Lynn

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidopteran insects around 1960. Since then, more than 600 insect cell lines have been described from over 100

  12. The divalent strontium salt S12911 enhances bone cell replication and bone formation in vitro

    Microsoft Academic Search

    E. Canalis; M. Hott; P. Deloffre; Y. Tsouderos; P. J. Marie

    1996-01-01

    In this study, we have determined the effect of the divalent strontium salt S12911 on bone cell replication and bone formation in two culture systems. In the first series of experiments, half-calvariae of newborn rats were cultured with S12911 from 24 to 96 h and labeled with 3H-thymidine for the last 6 h of culture or treated with S12911 for

  13. Cultured Adherent Cells from Marrow can Serve as Long-Lasting Precursor Cells for Bone, Cartilage, and Lung in Irradiated Mice

    Microsoft Academic Search

    R. F. Pereira; K. W. Halford; M. D. O'Hara; D. B. Leeper; B. P. Sokolov; M. D. Pollard; O. Bagasra; D. J. Prockop

    1995-01-01

    Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present

  14. Single cell mutational analysis of PIK3CA in circulating tumor cells and metastases in breast cancer reveals heterogeneity, discordance, and mutation persistence in cultured disseminated tumor cells from bone marrow

    PubMed Central

    2014-01-01

    Background Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. Methods We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients’ primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. Results Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. Conclusions Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies. PMID:24947048

  15. Effects of Mössbauer radiation on bone marrow cultures

    NASA Astrophysics Data System (ADS)

    Ortalli, I.; Pedrazzi, G.; Jiang, K.; Zhang, X.; Carlo-Stella, C.; Mangoni, L.; Rizzoli, V.

    1992-04-01

    A low radiation dose approach to cell eradication would be highly desirable in cancer treatments in order to reduce the side ellects of conventional radiotherapy. In the present work we present a preliminary study on coltures of bone marrow mononuclear cells collected from normal subjects and patients with chronic myelogenous leukaemia (CML). Hematin (104, 10-3, 10°M) has been added to mattow culture cells which were then irradiated with a 3.7 GBq (100 mCi)57Co/Rh Mossbauer source for 4 hours. Significant inbibition has been observed on the cell growth due to hematin and irradiatron.

  16. Tissue Engineered Bone Grafts: Biological Requirements, Tissue Culture and Clinical Relevance

    PubMed Central

    Fröhlich, Mirjam; Grayson, Warren L.; Wan, Leo Q.; Marolt, Darja; Drobnic, Matej; Vunjak-Novakovic, Gordana

    2009-01-01

    The tremendous need for bone tissue in numerous clinical situations and the limited availability of suitable bone grafts are driving the development of tissue engineering approaches to bone repair. In order to engineer viable bone grafts, one needs to understand the mechanisms of native bone development and fracture healing, as these processes should ideally guide the selection of optimal conditions for tissue culture and implantation. Engineered bone grafts have been shown to have capacity for osteogenesis, osteoconduction, osteoinduction and osteointegration - functional connection between the host bone and the graft. Cells from various anatomical sources in conjunction with scaffolds and osteogenic factors have been shown to form bone tissue in vitro. The use of bioreactor systems to culture cells on scaffolds before implantation further improved the quality of the resulting bone grafts. Animal studies confirmed the capability of engineered grafts to form bone and integrate with the host tissues. However, the vascularization of bone remains one of the hurdles that need to be overcome if clinically sized, fully viable bone grafts are to be engineered and implanted. We discuss here the biological guidelines for tissue engineering of bone, the bioreactor cultivation of human mesenchymal stem cells on three-dimensional scaffolds, and the need for vascularization and functional integration of bone grafts following implantation. PMID:19075755

  17. Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells

    Microsoft Academic Search

    Sai-Nan Shu; Lai Wei; Jiang-Hua Wang; Yu-Tao Zhan; Hong-Song Chen; Yu Wang

    2004-01-01

    AIM: To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. METHODS: MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the

  18. The presence of an autologous marrow stromal cell layer increases glucocerebrosidase gene transduction of long-term culture initiating cells (LTCICs) from the bone marrow of a patient with Gaucher disease.

    PubMed

    Wells, S; Malik, P; Pensiero, M; Kohn, D B; Nolta, J A

    1995-10-01

    Gaucher disease is a lysosomal storage disorder resulting form deficiency of the acid beta-glucosidase, glucocerebrosidase (GC). Allogeneic bone marrow transplantation has been beneficial in the treatment of Gaucher patients. Therefore, this disorder may be an ideal candidate for gene therapy by GC gene transduction of hematopoietic stem cells. We sought to increase the extent of gene transfer into CD34+ cells from the marrow of a Gaucher patient using G1GC, a simple retroviral vector containing a normal human GC cDNA. The ability of autologous stromal support and recombinant cytokines to increase the extent of transduction of colony-forming-cells (CFCs) and long-term culture initiating cells (LTCICs) was assessed. The presence of a stromal layer significantly increased the extent of GC gene transfer into 14-day CFCs, as determined by polymerase chain reaction (PCR) of individual colonies (18.8% with stroma versus 5% without, P < 0.001). Stromal support also increased the extent of transduction of LTCICs (10% with stroma versus 0.83% without, P < 0.001). Non-adherent cells from long-term bone marrow cultures initiated with CD34+ progenitors transduced on autologous stroma had higher levels of GC enzyme activity than cultures initiated with cells transduced without stroma. The percentage of cells which were GC positive by immunohistochemistry was also increased (21.1% with stroma versus 2.7% without, P = 0.0003). The addition of cytokines (IL-3, IL-6 and Steel factor) to the transduction, in the presence of stroma, significantly increased the extent of gene transfer into CFCs but not LTCICs. These studies indicate that the GC gene can be effectively transduced into LTCICs by retroviral vectors in the presence of stroma at levels significant for clinical gene therapy trials in patients with Gaucher disease. PMID:8593601

  19. Chromatin Changes at the PPAR-?2 Promoter During Bone Marrow-Derived Multipotent Stromal Cell Culture Correlate With Loss of Gene Activation Potential.

    PubMed

    Lynch, Patrick J; Thompson, Elaine E; McGinnis, Kathleen; Rovira Gonzalez, Yazmin I; Lo Surdo, Jessica; Bauer, Steven R; Hursh, Deborah A

    2015-07-01

    Bone marrow-derived multipotent stromal cells (BM-MSCs) display a broad range of therapeutically valuable properties, including the capacity to form skeletal tissues and dampen immune system responses. However, to use BM-MSCs in a clinical setting, amplification is required, which may introduce epigenetic changes that affect biological properties. Here we used chromatin immunoprecipitation to compare post-translationally modified histones at a subset of gene promoters associated with developmental and environmental plasticity in BM-MSCs from multiple donors following culture expansion. At many locations, we observed localization of both transcriptionally permissive (H3K4me3) and repressive (H3K27me3) histone modifications. These chromatin signatures were consistent among BM-MSCs from multiple donors. Since promoter activity depends on the relative levels of H3K4me3 and H3K27me3, we examined the ratio of H3K4me3 to H3K27me3 (K4/K27) at promoters during culture expansion. The H3K4me3 to H3K27me3 ratios were maintained at most assayed promoters over time. The exception was the adipose-tissue specific promoter for the PPAR-?2 isoform of PPAR-?, which is a critical positive regulator of adipogenesis. At PPAR-?2, we observed a change in K4/K27 levels favoring the repressed chromatin state during culture. This change correlated with diminished promoter activity in late passage cells exposed to adipogenic stimuli. In contrast to BM-MSCs and osteoblasts, lineage-restricted preadipocytes exhibited levels of H3K4me3 and H3K27me3 that favored the permissive chromatin state at PPAR-?2. These results demonstrate that locus-specific changes in H3K4me3 and H3K27me3 levels can occur during BM-MSC culture that may affect their properties. Stem Cells 2015;33:2169-2181. PMID:25640287

  20. Exogenous Nkx2.5? or GATA?4?transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co?culture on the treatment of myocardial infarction in rabbits.

    PubMed

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA?4 transfection with myocardial extracellular environment co?culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA?4 were transfected into myocardial extracellular environment co?cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA?4 exogenous gene transfection with myocardial cell extracellular environment co?culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA?4 alone compared with that of transfection in combination with extracellular environment co?culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA?4 into myocardial cell extracellular environment co?cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA?4 gene transfection into myocardial extracellular environment co?cultured BMSCs induced increased differentiation into myocardial cells compared with that of gene transfection alone. Furthermore, significantly enhanced reparative effects were observed in the myocardium of rabbits following treatment with Nkx2.5? or GATA?4?transfected myocardial cell extracellular environment co?cultured BMSCs compared with those treated with untreated BMSCs. PMID:25975979

  1. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits

    PubMed Central

    LI, PU; ZHANG, LEI

    2015-01-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial cells compared with that of gene transfection alone. Furthermore, significantly enhanced reparative effects were observed in the myocardium of rabbits following treatment with Nkx2.5- or GATA-4-transfected myocardial cell extracellular environment co-cultured BMSCs compared with those treated with untreated BMSCs. PMID:25975979

  2. Long-term Cultures of Bone Marrow-Derived Human Mesenchymal Stem Cells Frequently Undergo Spontaneous Malignant Transformation

    Microsoft Academic Search

    Gro Vatne Røsland; Agnete Svendsen; Anja Torsvik; Ewa Sobala; Emmet McCormack; Heike Immervoll; Josef Mysliwietz; Roland Goldbrunner; Per Eystein Lønning; Rolf Bjerkvig; Christian Schichor

    2009-01-01

    Human mesenchymal stem cells (hMSC) aid in tissue main- tenance and repair by differentiating into specialized cell types. Due to this ability, hMSC are currently being evaluated for cell-based therapies of tissue injury and degenerative diseases. However, extensive expansion ex vivo is a prerequi- site to obtain the cell numbers required for human cell-based therapy protocols. Recent studies indicate that

  3. Transplanted Endothelial Cells Enhance Orthotopic Bone Regeneration

    Microsoft Academic Search

    D. Kaigler; P. H. Krebsbach; Z. Wang; E. R. West; K. Horger; D. J. Mooney

    2006-01-01

    The aim of this study was to determine if endothelial cells could enhance bone marrow stromal-cell-mediated bone regeneration in an osseous defect. Using poly-lactide-co-glycolide scaffolds as cell carriers, we transplanted bone marrow stromal cells alone or with endothelial cells into 8.5-mm calvarial defects created in nude rats. Histological analyses of blood vessel and bone formation were performed, and microcomputed tomography

  4. Bone regeneration: the stem/progenitor cells point of view

    PubMed Central

    Deschaseaux, Frédéric; Pontikoglou, Charalampos; Sensébé, Luc

    2010-01-01

    Abstract After bone injuries, several molecular mechanisms establish bone repair from stem/progenitor cells. Inflammation factors attract regenerative cells which expand and differentiate in order to build up a bone highly similar to that before injury. Bone marrow (BM) mesenchymal stem cells (MSCs) as skeletal stem cells and endothelial progenitors (EPCs) are at the origin of such reparation mechanisms. However, discrepancies exist about their identities. Although cultured MSCs are extensively described, their in vivo native forms are poorly known. In addition, recent experiments show that several types of EPC exist. We therefore review up-to-date data on the characterization of such stem/progenitor cells and propose a new point of view of their function in bone regeneration. PMID:19840188

  5. Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

    PubMed

    Toksoz, D; Zsebo, K M; Smith, K A; Hu, S; Brankow, D; Suggs, S V; Martin, F H; Williams, D A

    1992-08-15

    The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo. PMID:1380155

  6. Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering

    PubMed Central

    Açil, Yahya; Wiltfang, Jörg; Gierloff, Matthias

    2015-01-01

    To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPAR?2, C/EBP?, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPAR?2, C/EBP?, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

  7. Leukemia cells induce changes in human bone marrow stromal cells

    PubMed Central

    2013-01-01

    Background Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-? and TNF-? change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. Methods BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1? and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. Results Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-? signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. Conclusions BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells. PMID:24304929

  8. Lamellar Spacing in Cuboid Hydroxyapatite Scaffolds Regulates Bone Formation by Human Bone Marrow Stromal Cells

    PubMed Central

    Afghani, Shahrzad; Franco, Jaime; Launey, Max; Marshall, Sally; Marshall, Grayson W.; Nissenson, Robert; Lee, Janice; Tomsia, Antoni P.; Saiz, Eduardo

    2011-01-01

    Background A major goal in bone engineering is the creation of large volume constructs (scaffolds and stem cells) that bear load. The scaffolds must satisfy two competing requirements—they need be sufficiently porous to allow nutrient flow to maintain cell viability, yet sufficiently dense to bear load. We studied the effect of scaffold macroporosity on bone formation and scaffold strength, for bone formed by human bone marrow stromal cells. Methods Rigid cubical hydroxyapatite/tricalcium phosphate scaffolds were produced by robo-casting. The ceramic line thickness was held constant, but the distance between adjacent lines was either 50, 100, 200, 500, or 1000??m. Cultured human bone marrow stromal cells were combined with the scaffolds in vitro; transplants were placed into the subcutis of immunodeficient mice. Transplants were harvested 9, 18, 23, 38, or 50 weeks later. Bone formation and scaffold strength were analyzed using histology and compression testing. Results Sixty transplants were evaluated. Cortical bone increased with transplant age, and was greatest among 500??m transplants. In contrast, maximum transplant strength was greatest among 200??m transplants. Conclusions Lamellar spacing within scaffolds regulates the extent of bone formation; 500??m yields the most new bone, whereas 200??m yields the strongest transplants. PMID:21294634

  9. Repair of Segmental Bone Defect Using Totally Vitalized Tissue Engineered Bone Graft by a Combined Perfusion Seeding and Culture System

    PubMed Central

    Feng, Ya-Fei; Li, Xiang; Hu, Yun-Yu; Wang, Zhen; Ma, Zhen-Sheng; Lei, Wei

    2014-01-01

    Background The basic strategy to construct tissue engineered bone graft (TEBG) is to combine osteoblastic cells with three dimensional (3D) scaffold. Based on this strategy, we proposed the “Totally Vitalized TEBG” (TV-TEBG) which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. Methods In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and ?-tricalcium phosphate (?-TCP) scaffold fabricated by Rapid Prototyping (RP) technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC) method, static seeding and perfusion culture (SSPC) method, and static seeding and static culture (SSSC) method for their in vitro performance and bone defect healing efficacy with a rabbit model. Results Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. Conclusion This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and maxillofacial fields. PMID:24728277

  10. Bone Marrow Cells Can Give Rise to Ameloblast-like Cells

    Microsoft Academic Search

    B. Hu; F. Unda; S. Bopp-Kuchler; L. Jimenez; X. J. Wang; Y. Haïkel; S. L. Wang; H. Lesot

    2006-01-01

    Post-eruptive loss of ameloblasts requires identification of alternative sources for these cells to realize tooth-tissue-engineering strategies. Recent reports showed that bone-marrow-derived cells can give rise to different types of epithelial cells, suggesting their potential to serve as a source for ameloblasts. To investigate this potential, we mixed c-Kit+-enriched bone marrow cells with embryonic dental epithelial cells and cultured them in

  11. Association of porous hydroxyapatite and bone marrow cells for bone regeneration

    Microsoft Academic Search

    K Anselme; B Noël; B Flautre; M.-C Blary; C Delecourt; M Descamps; P Hardouin

    1999-01-01

    The preparation of hybrid material with osteoinductive capacity may be achieved by association of cultured autologous bone cells with a porous ceramic vehicle. We optimized culture conditions for rabbit marrow stromal stem cells (MSCs), notably by selection from batches of fetal calf serum. Rabbit MSCs formed colony-forming unit-fibroblastic (CFU-Fs) in vitro. Their alkaline phosphatase (ALP) activity was doubled in the

  12. Stem Cells and Calcium Phosphate Cement Scaffolds for Bone Regeneration

    PubMed Central

    Wang, P.; Zhao, L.; Chen, W.; Liu, X.; Weir, M.D.; Xu, H.H.K.

    2014-01-01

    Calcium phosphate cements (CPCs) have excellent biocompatibility and osteoconductivity for dental, craniofacial, and orthopedic applications. This article reviews recent developments in stem cell delivery via CPC for bone regeneration. This includes: (1) biofunctionalization of the CPC scaffold, (2) co-culturing of osteoblasts/endothelial cells and prevascularization of CPC, (3) seeding of CPC with different stem cell species, (4) human umbilical cord mesenchymal stem cell (hUCMSC) and bone marrow MSC (hBMSC) seeding on CPC for bone regeneration, and (5) human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) seeding with CPC for bone regeneration. Cells exhibited good attachment/proliferation in CPC scaffolds. Stem-cell-CPC constructs generated more new bone and blood vessels in vivo than did the CPC control without cells. hUCMSCs, hESC-MSCs, and hiPSC-MSCs in CPC generated new bone and blood vessels similar to those of hBMSCs; hence, they were viable cell sources for bone engineering. CPC with hESC-MSCs and hiPSC-MSCs generated new bone two- to three-fold that of the CPC control. Therefore, this article demonstrates that: (1) CPC scaffolds are suitable for delivering cells; (2) hUCMSCs, hESCs, and hiPSCs are promising alternatives to hBMSCs, which require invasive procedures to harvest with limited cell quantity; and (3) stem-cell-CPC constructs are highly promising for bone regeneration in dental, craniofacial, and orthopedic applications. PMID:24799422

  13. Bone tissue engineering using marrow stromal cells

    Microsoft Academic Search

    Inho Jo; Jung Min Lee; Hwal Suh; Hyongbum Kim

    2007-01-01

    Bone tissue defects cause a significant socioeconomic problem, and bone is the most frequently transplanted tissue beside\\u000a blood. Autografting is considered the gold standard treatment for bone defects, but its utility is limited due to donor site\\u000a morbidity. Hence much research has focused on bone tissue engineering as a promising alternative method for repair of bone\\u000a defects. Marrow stromal cells

  14. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  15. Cell sourcing for bone tissue engineering: Amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

    Microsoft Academic Search

    Alexandra Peister; Maria A. Woodruff; Jarod J. Prince; Derwin P. Gray; Dietmar W. Hutmacher; Robert E. Guldberg

    2011-01-01

    Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem cells (AFS) and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells

  16. Fetal bone cells for tissue engineering.

    PubMed

    Montjovent, Marc-Olivier; Burri, Nathalie; Mark, Silke; Federici, Ermanno; Scaletta, Corinne; Zambelli, Pierre-Yves; Hohlfeld, Patrick; Leyvraz, Pierre-François; Applegate, Lee L; Pioletti, Dominique P

    2004-12-01

    We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1alpha,25-dihydroxyvitamin D3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering. PMID:15589213

  17. IgE-mediated release of leukotriene C4, chondroitin sulfate E proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrow-derived mouse mast cells

    PubMed Central

    1983-01-01

    Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6- disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class- specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose- response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S- chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan- containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses. PMID:6184439

  18. Conditioned Media from Mesenchymal Stem Cells Enhanced Bone Regeneration in Rat Calvarial Bone Defects

    PubMed Central

    Osugi, Masashi; Yoshimi, Ryoko; Inukai, Takeharu; Hibi, Hideharu; Ueda, Minoru

    2012-01-01

    Tissue engineering has recently become available as a treatment procedure for bone augmentation. However, this procedure has several problems, such as high capital investment and expensive cell culture, complicated safety and quality management issues regarding cell handling, and patient problems with the invasive procedure of cell collection. Moreover, it was reported that stem cells secrete many growth factors and chemokines during their cultivation, which could affect cellular characteristics and behavior. This study investigated the effect of stem-cell-cultured conditioned media on bone regeneration. Cultured conditioned media from human bone marrow–derived mesenchymal stem cells (MSC-CM) enhanced the migration, proliferation, and expression of osteogenic marker genes, such as osteocalcin and Runx2, of rat MSCs (rMSCs) in vitro. MSC-CM includes cytokines such as insulin-like growth factor-1 and vascular endothelial growth factor. In vivo, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC-CM/agarose (MSC-CM), Dulbecco's modified Eagle's medium without serum [DMEM(?)]/agarose [DMEM(?)], PBS/agarose (PBS), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using microcomputed tomography (micro-CT) and histological analysis. Micro-CT analysis indicated that the MSC-CM group had a greater area of newly regenerated bone compared with the other groups (p<0.05) and histological analysis at 8 weeks indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the effects of MSC-CM were stronger than those of the MSC group. In vivo imaging and immunohistochemical staining of transgenic rats expressing green fluorescent protein also showed that migration of rMSCs to the bone defect in the MSC-CM group was greater than in the other groups. These results demonstrated that MSC-CM can regenerate bone through mobilization of endogenous stem cells. The use of stem-cell-cultured conditioned media for bone regeneration is a unique concept that utilizes paracrine factors of stem cells without cell transplantation. PMID:22443121

  19. Mechanical Behavior of Bone Cells micrograph view of bone

    E-print Network

    Gefen, Amit

    of the formation of osteocytes and lining cells from osteoblast From: Burger, 2001, In: Bone Mechanics , S. Cowin, Ed., CRC Press. 3 Morphology of osteocytes, osteoblasts and periosteal fibroblasts From: Burger, 2001, Ed., CRC Press. Schematic representation of how the osteocyte network may regulate bone modeling #12

  20. Mesenchymal Stem Cell-Based HLA-Independent Cell Therapy for Tissue Engineering of Bone and Cartilage

    Microsoft Academic Search

    Philipp Niemeyer; Ulf Krause; Philip Kasten; Peter C. Kreuz; Philipp Henle; Norbert P. Sudkamp; Alexander Mehlhorn

    2006-01-01

    Mesenchymal stem cells (MSC) can be obtained from human bone marrow aspirates and, thanks to their differentiation potential and excellent in vitro culture properties, represent an attractive cell line for the regeneration of mesenchymal tissue. Both in vitro and in vivo , they can differentiate into cartilage, bone, tendons and fat cells, and-in contrast to embryonic stem cells-they are not

  1. Regulation of proliferation and differentiation of human fetal bone cells.

    PubMed

    Krattinger, Nathalie; Applegate, Lee A; Biver, Emmanuel; Pioletti, Dominique P; Caverzasio, Joseph

    2011-01-01

    During the last decade, extensive research has been performed in the field of orthopedic medicine to develop cell-based therapies for the restoration of injured bone tissue. We previously demonstrated that human primary fetal bone cells (HFBCs) associated with porous scaffolds induced a bone formation in critical calvaria defect; however, the environmental factors regulating their behavior in culture have not been identified. HFBCs (human fetal femur,12 week development) were compared to marrow-derived human mesenchymal stem cells (HMSCs) for their capacity to proliferate and differentiate into osteoblasts under various culture conditions. When cultured in standard alphaMEM medium, PDGF and FGF-2 increased cell proliferation of both cell types. Investigation of the differentiating capacity of HFBCs and HMSCs in a normal culture medium indicated that HFBCs expressed higher expression levels of RUNX2, OSX, and osteogenic markers compared with HMSCs, while SOX9 was expressed at very low levels in both cells types. However, HMSCs, but not HFBCs enhanced osteoblastic markers in response to osteogenic factors. Surprisingly, BMP-2 with osteogenic factors increased cell numbers and reduced osteoblastic differentiation in HFBCs with the opposite effect seen in HMSCs. Associated with a higher expression of osteoblastic markers, HFBCs produced a higher calcified extra cellular matrix compared with HMSCs. Taken together, data presented in this study suggest that HFBCs have characteristics of osteoprecursor cells that are more advanced in their osteogenesis development compared with mesenchymal stem cells, making fetal cells an interesting biological tool for treatment of skeletal defects and diseases. PMID:21225594

  2. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate

    SciTech Connect

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. (Shriners Hospital, Montreal, Quebec (Canada))

    1989-11-25

    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.

  3. Endogenous bone morphogenetic proteins in human bone marrow-derived multipotent mesenchymal stromal cells

    Microsoft Academic Search

    F. Philipp Seib; Martina Franke; Duohui Jing; Carsten Werner; Martin Bornhäuser

    2009-01-01

    Primary human multipotent mesenchymal stromal cells (MSCs) are capable of self renewal or differentiation into several different lineages, including osteoblasts, chondrocytes and adipocytes. However, upon prolonged in vitro culture, MSCs tend to undergo spontaneous osteogenic differentiation. Here, we address the possible role of endogenous osteogenic bone morphogenetic proteins (BMPs) in in situ osteoblastic differentiation of human MSCs. Human MSCs consistently

  4. Developmental Cell Bone Ridge Patterning during Musculoskeletal

    E-print Network

    Developmental Cell Article Bone Ridge Patterning during Musculoskeletal Assembly Is Mediated.devcel.2009.10.010 SUMMARY During the assembly of the musculoskeletal system, bone ridges provide a stable-skeleton regulatory interactions during musculoskeletal assembly and bone secondary patterning. INTRODUCTION

  5. Molluscan cells in culture: primary cell cultures and cell lines.

    PubMed

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  6. Prostaglandins regulate acid-induced cell-mediated bone resorption.

    PubMed

    Krieger, N S; Parker, W R; Alexander, K M; Bushinsky, D A

    2000-12-01

    Metabolic acidosis induces bone calcium efflux initially by physicochemical dissolution and subsequently by cell-mediated mechanisms involving inhibition of osteoblasts and stimulation of osteoclasts. In rat kidney, acidosis increases endogenous prostaglandin synthesis, and in bone, prostaglandins are important mediators of resorption. To test the hypothesis that acid-induced bone resorption is mediated by prostaglandins, we cultured neonatal mouse calvariae in neutral or physiologically acidic medium with or without 0.56 microM indomethacin to inhibit prostaglandin synthesis. We measured net calcium efflux and medium PGE(2) levels. Compared with neutral pH medium, acid medium led to an increase in net calcium flux and PGE(2) levels after both 48 h and 51 h, a time at which acid-induced net calcium flux is predominantly cell mediated. Indomethacin inhibited the acid-induced increase in both net calcium flux and PGE(2). Net calcium flux was correlated directly with medium PGE(2) (r = 0.879, n = 29, P < 0.001). Exogenous PGE(2), at a level similar to that found after acid incubation, induced net calcium flux in bones cultured in neutral medium. Acid medium also stimulated an increase in PGE(2) levels in isolated bone cells (principally osteoblasts), which was again inhibited by indomethacin. Thus acid-induced stimulation of cell-mediated bone resorption appears to be mediated by endogenous osteoblastic PGE(2) synthesis. PMID:11097626

  7. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, ? particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by ?H2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l ?-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-?1). TGF-?1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to ? particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cel

  8. Lead Increases Free Ca2+ Concentration in Cultured Osteoblastic Bone Cells: Simultaneous Detection of Intracellular Free Pb2+ by 19F NMR

    Microsoft Academic Search

    Francis A. X. Schanne; Terry L. Dowd; Raj K. Gupta; John F. Rosen

    1989-01-01

    Lead (Pb) has been shown to perturb Ca-mediated cellular processes. However, to date, a direct effect of Pb on intracellular free Ca2+ concentration ([Ca2+]i) has not been demonstrated. 19F NMR in combination with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'- tetraacetic acid (5F-BAPTA) was used to simultaneously measure [Ca2+]i and intracellular free Pb2+ concentration ([Pb2+]i) in the rat osteoblastic bone cell line ROS 17\\/2.8. The basal

  9. Bone marrow: all the cells of the immune system are derived from stem cells in the bone marrow. The bone

    E-print Network

    Morante, Silvia

    Bone marrow: all the cells of the immune system are derived from stem cells in the bone marrow as part of the immune system and as a filter #12;Cells of the Immune System Cells destined to become workings of the immune system, while others are cytotoxic and directly contact infected cells and destroy

  10. Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34 + cord blood and bone marrow cells

    Microsoft Academic Search

    J. Clausen; M. Stockschläder; N. Fehse; H. T. Hassan; C. Gabl; A. R. Zander

    2000-01-01

    Monocytes\\/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM)\\u000a in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells\\u000a that give rise to colony forming cells in a 5-week long-term culture (LTC), we established plastic-adherent macrophage layers\\u000a from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence

  11. Inhibitory effects of ? -interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

    Microsoft Academic Search

    U. H. Lerner; Ö. Ljunggren; M. Ransjö; K. Klaushofer; M. Peterlik

    1991-01-01

    The effects of mouse recombinant?-interferon (?-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg9-bradykinin and prostaglandin E2 (PGE2) have been studied using cultures of neonatal calvarial bones and analyzing the release of45Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of?-IFN and indomethacin on formation of PGE2 in bone cultures stimulated by

  12. Support of Human Hematopoisis in Long-Term Bone Marrow Cultures by Murin Stromal Cells Selectively Expressing the Membrane-Bound and Secreted Forms of the Human Homolog of the Steel Gene Product, Stem Cell Factor

    Microsoft Academic Search

    Deniz Toksoz; Krisztina M. Zsebo; Kent A. Smith; Sylvia Hu; David Brankow; Sydney V. Suggs; Francis H. Martin; David A. William

    1992-01-01

    The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report,

  13. Effects of Monoclonal Antibody and Complement Treatment of Human Marrow on Hematopoiesis in Continuous Bone Marrow Culture1

    Microsoft Academic Search

    Joel S. Greenberger; Lisa Rothstein; Paolo DeFabritiis; Marco Bregni; Robert Bast; Jerome Ritz; Lee M. Nadler; Jeffrey M. Lipton; Mary Ann Sakakeeny

    Long-term bone marrow cultures were established from single- cell suspensions of human bone marrow that had been treated with monoclonal antibodies and complement. Each treated cell suspension was evaluated for production of hematopoietic stem cells over 20 weeks. Treatment with antibody to HLA-DR (la), B1, J2, or J5 did not remove adherent cells including those differentiating to adipocytes in 17-hydroxycorticosteroid.

  14. Development of sup 19 F NMR for measurement of (Ca2+)i and (Pb2+)i in cultured osteoblastic bone cells

    SciTech Connect

    Schanne, F.A.; Dowd, T.L.; Gupta, R.K.; Rosen, J.F. (Albert Einstein College of Medicine, Bronx, NY (USA))

    1990-03-01

    Lead (Pb) has been shown to perturb cellular calcium (Ca) homeostasis, altering sizes and flux rates of cellular pools of exchangeable Ca and impairing Ca-mediated cell processes. To date, however, a direct effect of Pb on intracellular-free Ca2+ has not yet been demonstrated. Heavy metals bind to the commonly used fluorescent Ca ion indicators with greater affinity than does Ca and thereby interfere with the expected Ca-dependent fluorescence. In this study, the fluorinated Ca ion indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane N,N,N',N'-tetraacetic acid (5F-BAPTA), and 19F NMR were used to measure the free intracellular Ca ion concentration ((Ca2+)i) in the rat osteoblastic bone cell line, ROS 17/2.8. Both Pb and Ca bind to 5F-BAPTA with high affinity, but the Pb-5F-BAPTA comple produces a 19F NMR signal at a chemical shift distinct from 5F-BAPTA and the Ca-5F-BAPTA complex. The apparent dissociation constants for Pb-5F-BAPTA and Ca-5F-BAPTA are 2 X 10(-10) M and 5 X 10(-7) M, respectively, at 30 degrees C, pH 7.1, and Mg2+ (0.5 mM). Thus, this methodology allows for the simultaneous identification and quantification of free Pb and free Ca ion concentrations. Determinations of (Ca2+)i were based on 19F NMR measurements of 5F-BAPTA-loaded ROS 17/2.8 osteoblastic bone cells that were attached to collagen-coated microcarrier beads. Cells were continuously superfused with freshly oxygenated medium at 30 degrees C. Under these conditions, the (Ca2+)i of ROS 17/2.8 cells was observed to be 128 +/- 14 nM.

  15. Perfusion Based Cell Culture Chips

    Microsoft Academic Search

    A. Heiskanen; J. Emnéus; M. Dufva

    2010-01-01

    \\u000a Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium\\u000a composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates,\\u000a many issues remain to be identified regarding perfusion cell culture

  16. Late Adherent Human Bone Marrow Stromal Cells Form Bone and Restore the Hematopoietic Microenvironment In Vivo

    PubMed Central

    Vianna, Verônica Fernandes; Bonfim, Danielle Cabral; Cavalcanti, Amanda dos Santos; Fernandes, Marco Cury; Kahn, Suzana Assad; Casado, Priscila Ladeira; Lima, Inayá Correa; Murray, Samuel S.; Murray, Elsa J. Brochmann; Duarte, Maria Eugenia Leite

    2013-01-01

    Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term “stem cell,” this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics. PMID:23710460

  17. Bone Tissue Engineering Using Human Mesenchymal Stem Cells: Effects of Scaffold Material and Medium Flow

    Microsoft Academic Search

    Lorenz Meinel; Vassilis Karageorgiou; Robert Fajardo; Brian Snyder; Vivek Shinde-Patil; Ludwig Zichner; David Kaplan; Robert Langer; Gordana Vunjak-Novakovic

    2004-01-01

    We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo

  18. Remyelination of the Rat Spinal Cord by Transplantation of Identified Bone Marrow Stromal Cells

    Microsoft Academic Search

    Yukinori Akiyama; Christine Radtke; Jeffery D. Kocsis

    2002-01-01

    Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluo- rescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal

  19. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats

    Microsoft Academic Search

    C. Maniatopoulos; J. Sodek; A. H. Melcher

    1988-01-01

    Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro. In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow. We have explored development of bone-like tissue in vitro by bone marrow stromal cells. Marrow stromal cells obtained from 40–43-day-old

  20. Fish oil prevents breast cancer cell metastasis to bone

    PubMed Central

    Mandal, Chandi Charan; Ghosh-Choudhury, Triparna; Yoneda, Toshi; Choudhury, Goutam Ghosh; Ghosh-Choudhury, Nandini

    2010-01-01

    The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ?-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion. PMID:20971068

  1. Fish oil prevents breast cancer cell metastasis to bone.

    PubMed

    Mandal, Chandi Charan; Ghosh-Choudhury, Triparna; Yoneda, Toshi; Choudhury, Goutam Ghosh; Ghosh-Choudhury, Nandini

    2010-11-26

    The data derived from epidemiological and animal models confirm a beneficial effect of fish oil (rich in ?-3 polyunsaturated fatty acids) in the amelioration of tumor growth and progression, including breast cancer. The breast cancer patients often develop bone metastasis evidenced by osteolytic lesions, leading to severe pain and bone fracture. Using a mouse model of MDA-MB-231 human breast cancer cell metastasis to bone, here we show that fish oil diet enriched in DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) prevents the formation of osteolytic lesions in bone, indicating suppression of cancer cell metastasis to bone. These results are supported by our data showing both DHA and EPA significantly attenuate the migration/invasion of MDA-MB-231 breast cancer cells in culture. The mechanism that limits breast cancer cells to selective metastasis to bone remains hitherto unexplored. Aberrant increased expression of CD44 is associated with generation of cancer stem cells, which contribute to metastasis of breast cancer cells. We demonstrate that DHA and EPA significantly inhibit the expression of CD44 protein and mRNA by a transcriptional mechanism. Furthermore, we show markedly reduced levels of CD44 mRNA and protein in the tumors of mice, which were fed fish oil diet than those in control diet. Our data provide the first evidence for a salutary effect of fish oil on breast cancer metastasis to bone. Our results identify a novel function of the fish oil active components, DHA and EPA, which target the cell-intrinsic pro-metastatic molecule CD44 to inhibit migration/invasion. PMID:20971068

  2. Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype

    Microsoft Academic Search

    Cristina Zanini; Stefania Bruno; Giorgia Mandili; Denisa Baci; Francesco Cerutti; Giovanna Cenacchi; Leo Izzi; Giovanni Camussi; Marco Forni

    2011-01-01

    BackgroundRegarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself.Methodology\\/Principal FindingsIn the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium,

  3. OSTEOBLAST DIFFERENTIATION AND BONE FORMATION GENE EXPRESSION IN STRONTIUM-INDUCING BONE MARROW MESENCHYMAL STEM CELL

    Microsoft Academic Search

    Monnipha Sila-asna; Ahnond Bunyaratvej; Sakan Maeda; Hiromichi Kitaguchi; Narong Bunyaratavej

    2007-01-01

    Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. We studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. The mRNA synthesis of various osteoblast specific genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture,

  4. An enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples.

    PubMed

    Schlaefli, Philipp; Bertolo, Alessandro; Malonzo, Cherry; Poetzel, Tobias; Baur, Martin; Steffen, Frank; Stoyanov, Jivko

    2015-01-01

    Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug - clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy. PMID:25938767

  5. Enhanced Bone Marrow Stromal Cell Adhesion and Growth on Segmented Poly(ether ester)s Based on Poly(ethylene oxide) and Poly(butylene terephthalate)

    Microsoft Academic Search

    Menno B. Claase; Mark B. Olde Riekerink; Bruijn de Joost D; Dirk W. Grijpma; Gerard H. M. Engbers; Jan Feijen

    2003-01-01

    In previous studies in rats and goats, hydrophilic compositions of the PEOT\\/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro

  6. Isolation, culture, and differentiation potential of mouse marrow stromal cells.

    PubMed

    Anjos-Afonso, Fernando; Bonnet, Dominique

    2008-10-01

    This unit describes how to isolate and expand mesenchymal stromal cells (MSCs) from mouse bone marrow. For reasons that are not clear, it has been difficult to isolate these cells (also known as mesenchymal stem cells). Furthermore, different mouse strains seem to have specific requirements for successful extraction and culture of these cells. A general and easy protocol is presented here for isolating stromal cells from different inbred and transgenic mice commonly used in the stem cell biology field. PMID:18972375

  7. Concise Review: Cell-Based Strategies in Bone Tissue Engineering and Regenerative Medicine

    PubMed Central

    Ma, Jinling; Both, Sanne K.; Yang, Fang; Cui, Fu-Zhai; Pan, Juli; Meijer, Gert J.; Jansen, John A.

    2014-01-01

    Cellular strategies play an important role in bone tissue engineering and regenerative medicine (BTE/RM). Variability in cell culture procedures (e.g., cell types, cell isolation and expansion, cell seeding methods, and preculture conditions before in vivo implantation) may influence experimental outcome. Meanwhile, outcomes from initial clinical trials are far behind those of animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the BTE/RM constructs as some complex clinical implementations require bone regeneration in too large a quantity. Coculture strategies, in which angiogenic cells are introduced into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, preclinical studies have demonstrated that cell-based tissue-engineered constructs generally induce more bone formation compared with acellular constructs. Further, cocultures have been shown to enhance vascularization and bone formation compared with monocultures. However, translational efficacy from animal studies to clinical use requires improvement, and the role implanted cells play in clinical bone regeneration needs to be further elucidated. In view of this, the present review provides an overview of the critical procedures during in vitro and in vivo phases for cell-based strategies (both monoculture and coculture) in BTE/RM to achieve more standardized culture conditions for future studies, and hence enhance bone formation. PMID:24300556

  8. Biomimetic materials for controlling bone cell responses.

    PubMed

    Drevelle, Olivier; Faucheux, Nathalie

    2013-01-01

    Bone defects that cannot "heal spontaneously during life" will become an ever greater health problem as populations age. Harvesting autografts has several drawbacks, such as pain and morbidity at both donor and acceptor sites, the limited quantity of material available, and frequently its inappropriate shape. Researchers have therefore developed alternative strategies that involve biomaterials to fill bone defects. These biomaterials must be biocompatible and interact with the surrounding bone tissue to allow their colonization by bone cells and blood vessels. The latest generation biomaterials are not inert; they control cell responses like adhesion, proliferation and differentiation. These biomaterials are called biomimetic materials. This review focuses on the development of third generation materials. We first briefly describe the bone tissue with its cells and matrix, and then how bone cells interact with the extracellular matrix. The next section covers the materials currently used to repair bone defects. Finally, we describe the strategies employed to modify the surface of materials, such as coating with hydroxyapatite and grafting biomolecules. PMID:23277057

  9. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388

  10. In vitro cultivation of Anaplasma marginale in bovine bone marrow cells

    E-print Network

    Baradji, Issa

    1986-01-01

    December 1986 Major Subject: Veterinary Microbiology IN VITRO CULTIVATION OF ANAPLASMA MARGINALE IN BOVINE BONE MARROW CELLS A Thesis by ISSA BARAOJI Approved as to content and style by: G. . gne (Chairman of Co 't ee) P. D. Tee (Member) T. M... it 0 cape 1 e t t 1 ti ate ~AA 1 tti ale using bovine bone marrow cells from living and slaughtered animals were conducted. Bone marrow cell cultures were compared with erythrocyte cultures for their ability to sop ~ tthpo thofA~it. sam ~ 11...

  11. Effects of static magnetic fields on bone formation in rat osteoblast cultures.

    PubMed

    Yamamoto, Y; Ohsaki, Y; Goto, T; Nakasima, A; Iijima, T

    2003-12-01

    Although the promotional effects on osteoblasts of pulsed electromagnetic fields have been well-demonstrated, the effects of static magnetic fields (SMF) remain unclear; nevertheless, magnets have been clinically used as a 'force source' in various orthodontic treatments. We undertook the present investigation to study the effects of SMF on osteoblastic differentiation, proliferation, and bone nodule formation using a rat calvaria cell culture. During a 20-day culture, the values of the total area and the number and average size of bone nodules showed high levels in the presence of SMF. In the matrix development and mineralization stages, the calcium content in the matrix and two markers of osteoblastic phenotype (alkaline phosphatase and osteocalcin) also showed a significant increase. Accordingly, these findings suggest that SMF stimulates bone formation by promoting osteoblastic differentiation and/or activation. PMID:14630895

  12. Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

    SciTech Connect

    Otsuru, Satoru [Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Tamai, Katsuto [Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)]. E-mail: tamai@gts.med.osaka-u.ac.jp; Yamazaki, Takehiko [Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Yoshikawa, Hideki [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kaneda, Yasufumi [Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2007-03-09

    Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.

  13. Dormancy and growth of metastatic breast cancer cells in a bone-like microenvironment.

    PubMed

    Sosnoski, Donna M; Norgard, Robert J; Grove, Cassidy D; Foster, Shelby J; Mastro, Andrea M

    2015-04-01

    Breast cancer can reoccur, often as bone metastasis, many years if not decades after the primary tumor has been treated. The factors that stimulate dormant metastases to grow are not known, but bone metastases are often associated with skeletal trauma. We used a dormancy model of MDA-MB-231BRMS1, a metastasis-suppressed human breast cancer cell line, co-cultured with MC3T3-E1 osteoblasts in a long term, three dimensional culture system to test the hypothesis that bone remodeling cytokines could stimulate dormant cells to grow. The cancer cells attached to the matrix produced by MC3T3-E1 osteoblasts but grew slowly or not at all until the addition of bone remodeling cytokines, TNF? and IL-?. Stimulation of cell proliferation by these cytokines was suppressed with indomethacin, an inhibitor of cyclooxygenase and of prostaglandin production, or a prostaglandin E2 (PGE2) receptor antagonist. Addition of PGE2 directly to the cultures also stimulated cell proliferation. MCF-7, non-metastatic breast cancer cells, remained dormant when co-cultured with normal human osteoblast and fibroblast growth factor. Similar to the MDA-MB-231BRMS1 cells, MCF-7 proliferation increased in response to TNF? and IL-?. These findings suggest that changes in the bone microenvironment due to inflammatory cytokines associated with bone repair or excess turnover may trigger the occurrence of latent bone metastasis. PMID:25749879

  14. Stem cells and bone: a historical perspective.

    PubMed

    Bianco, Paolo

    2015-01-01

    Bone physiology and stem cells were tightly intertwined with one another, both conceptually and experimentally, long before the current explosion of interest in stem cells and so-called regenerative medicine. Bone is home to the two best known and best characterized systems of postnatal stem cells, and it is the only organ in which two stem cells and their dependent lineages coordinate the overall adaptive responses of two major physiological systems. All along, the nature and the evolutionary significance of the interplay of bone and hematopoiesis have remained a major scientific challenge, but also allowed for some of the most spectacular developments in cell biology-based medicine, such as hematopoietic stem cell transplantation. This question recurs in novel forms at multiple turning points over time: today, it finds in the biology of the "niche" its popular phrasing. Entirely new avenues of investigation emerge as a new view of bone in physiology and medicine is progressively established. Looking at bone and stem cells in a historical perspective provides a unique case study to highlight the general evolution of science in biomedicine since the end of World War II to the present day. A paradigm shift in science and in its relation to society and policies occurred in the second half of the XXth century, with major implications thereof for health, industry, drug development, market and society. Current interest in stem cells in bone as in other fields is intertwined with that shift. New opportunities and also new challenges arise. This article is part of a Special Issue entitled "Stem cells and bone". PMID:25171959

  15. N-acetyl muramyl dipeptide stimulation of bone resorption in tissue culture.

    PubMed Central

    Dewhirst, F E

    1982-01-01

    N-Acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), a structurally defined fragment of bacterial peptidoglycan, stimulated significant release of previously incorporated 45Ca from fetal rat bones in tissue culture over the concentration range of 0.1 to 10.0 micrograms/ml. MDP-Stimulated bone resorption was not inhibited by the addition of the prostaglandin synthetase inhibitor indomethacin to the culture medium. MDP was neither mitogenic for nor stimulated the release of osteoclast-activating factor from cultured human peripheral blood mononuclear cells. Thus, MDP-stimulated bone resorption in vitro is mediated by a mechanism which is not dependent upon prostaglandins or osteoclast-activating factor. 6-O-Stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, a lipophilic analog of MDP, was slightly more potent than MDP. Two diastereomers of MDP, N-acetyl-muramyl-L-alanyl-L-isoglutamine and N-acetyl-muramyl-D-alanyl-D-isoglutamine, which are inactive as adjuvants, were at least 1,000 times less active than MDP in stimulating bone resorption. The stereochemical specificity for bone-resorptive activity paralleled that required for adjuvant activity, macrophage activation, and activation of the reticuloendothelial system. PMID:7054120

  16. Craniosynostosis-Associated Fgfr2C342Y Mutant Bone Marrow Stromal Cells Exhibit Cell Autonomous Abnormalities in Osteoblast Differentiation and Bone Formation

    PubMed Central

    Liu, J.; Kwon, T.-G.; Nam, H. K.; Hatch, N. E.

    2013-01-01

    We recently reported that cranial bones of Fgfr2C342Y/+ craniosynostotic mice are diminished in density when compared to those of wild type mice, and that cranial bone cells isolated from the mutant mice exhibit inhibited late stage osteoblast differentiation. To provide further support for the idea that craniosynostosis-associated Fgfr mutations lead to cell autonomous defects in osteoblast differentiation and mineralized tissue formation, here we tested bone marrow stromal cells isolated from Fgfr2C342Y/+ mice for their ability to differentiate into osteoblasts. Additionally, to determine if the low bone mass phenotype of Crouzon syndrome includes the appendicular skeleton, long bones were assessed by micro CT. Fgfr2C342Y/+ cells showed increased osteoblastic gene expression during early osteoblastic differentiation but decreased expression of alkaline phosphatase mRNA and enzyme activity, and decreased mineralization during later stages of differentiation, when cultured under 2D in vitro conditions. Cells isolated from Fgfr2C342Y/+ mice also formed less bone when allowed to differentiate in a 3D matrix in vivo. Cortical bone parameters were diminished in long bones of Fgfr2C342Y/+ mice. These results demonstrate that marrow stromal cells of Fgfr2C342Y/+ mice have an autonomous defect in osteoblast differentiation and bone mineralization, and that the Fgfr2C342Y mutation influences both the axial and appendicular skeletons. PMID:23762837

  17. Hemangioblastic characteristics of fetal bone marrow–derived Flk1 +CD31 ?CD34 ? cells

    Microsoft Academic Search

    Hong Guo; Baijun Fang; Lianming Liao; Zhigang Zhao; Jiewen Liu; Huishu Chen; Steven H. Hsu; Qi Cui; Robort Chunhua Zhao

    2003-01-01

    Objective. To investigate whether Flk1+CD31?CD34? cells isolated from fetal bone marrow (BM) have characteristics of hemangioblasts, i.e., progenitors of endothelial and hematopoietic cells.Materials and Methods. Mononuclear cells from fetal BM were negatively sorted by CD45, GlyA, and CD34 micromagnetic beads, then cultured to form cell colonies. A single colony was harvested. Culture-expanded cells were seeded on ECM gel or semisolid

  18. Cardiotonic agent milrinone stimulates resorption in rodent bone organ culture.

    PubMed Central

    Krieger, N S; Stappenbeck, T S; Stern, P H

    1987-01-01

    The cardiotonic agent amrinone inhibits bone resorption in vitro. Milrinone, an amrinone analog, is a more potent cardiotonic agent with lower toxicity. In contrast to amrinone, milrinone stimulated resorption in cultures of neonatal mouse calvaria and fetal rat limb bones. Threshold doses were 0.1 microM in calvaria and 0.1 mM in limb bones; maximal stimulation occurred in calvaria at 0.1 mM. Maximal responses to milrinone and parathyroid hormone were comparable. Milrinone concentrations below 0.1 mM did not affect calvarial cyclic AMP. 0.5 microM indomethacin inhibited milrinone effects in calvaria but usually not in limb bones. 3 nM calcitonin inhibited milrinone-stimulated resorption and there was no escape from this inhibition. Structural homology between milrinone and thyroxine has been reported. We find similarities between milrinone and thyroxine actions on bone, because prostaglandin production was crucial for the effects of both agents in calvaria but not in limb bones, and neither agent exhibited escape from calcitonin inhibition. PMID:3027124

  19. Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture

    SciTech Connect

    Bhattacharyya, M.H.; Whelton, B.D.; Stern, P.H.; Peterson, D.P. (Argonne National Lab., IL (USA))

    1988-11-01

    Loss of bone mineral after ovariectomy was studied in mice exposed to dietary cadmium at 0.25, 5, or 50 ppm. Results show that dietary cadmium at 50 ppm increased bone mineral loss to a significantly greater extent in ovariectomized mice than in sham-operated controls. These results were obtained from two studies, one in which skeletal calcium content was determined 6 months after ovariectomy and a second in which {sup 45}Ca release from {sup 45}Ca-prelabeled bones was measured immediately after the start of dietary cadmium exposure. Furthermore, experiments with {sup 45}Ca-prelabeled fetal rat limb bones in culture demonstrated that Cd at 10 nM in the medium, a concentration estimated to be in the plasma of mice exposed to 50 ppm dietary Cd, strikingly increased bone resorption. These in vitro results indicate that cadmium may enhance bone mineral loss by a direct action on bone. Results of the in vivo studies are consistent with a significant role of cadmium in the etiology of Itai-Itai disease among postmenopausal women in Japan and may in part explain the increased risk of postmenopausal osteoporosis among women who smoke.

  20. Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

    PubMed Central

    Nolta, J A; Yu, X J; Bahner, I; Kohn, D B

    1992-01-01

    Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity. Images PMID:1379609

  1. Interaction among Cells of Bone, Immune System, and Solid Tumors Leads to Bone Metastases

    PubMed Central

    Roato, Ilaria

    2013-01-01

    Bone metastases are a dismal consequence for different types of solid tumors, such as breast, prostate, lung, and kidney cancer. The mechanisms regulating the interactions among bone, immune system, and tumor cells have been deeply investigated, and many studies are ongoing to define the specific role of the different cells in the bone metastatic process. The affinity of some tumors to growth in bone results from the special microenvironment provided by bone. Moreover, immune system and bone have a bidirectional relationship: bone cells express surface molecules ruling the expansion of hemopoietic stem cells from which all cells of the mammalian immune system derive, and various immunoregulatory cytokines influence the fate of bone cells. The last findings allow to extend the concept of vicious cycle and add T cells as mediators of the tumor growth in bone. PMID:23710201

  2. Rat bone marrow cell response to titanium and titanium alloy with different surface roughness.

    PubMed

    Rosa, Adalberto L; Beloti, Márcio M

    2003-02-01

    In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response. PMID:12562364

  3. Cannabinoids Stimulate Fibroblastic Colony Formation by Bone Marrow Cells Indirectly via CB 2 Receptors

    Microsoft Academic Search

    A. Scutt; E. M. Williamson

    2007-01-01

    Recently, the cannabinoid receptors CB1 and CB2 were shown to modulate bone formation and resorption in vivo, although little is known of the mechanisms underlying this. The effects of cannabinoids on mesenchymal stem cell (MSC) recruitment\\u000a in whole bone marrow were investigated using either the fibroblastic colony-forming unit (CFU-f) assay or high-density cultures\\u000a of whole bone marrow. Levels of the

  4. Bone marrow-derived stem cells and \\

    Microsoft Academic Search

    A. Hüttmann; C. L. Li; U. Dührsen

    2003-01-01

    Studies describing plasticity of somatic stem cells have become a focus of interest because clinical applications in the treatment of degenerative diseases would be at hand. In particular, bone marrow-derived cells and their potential to contribute to skeletal and cardiac muscle, liver, neurons and epithelium have recently been studied extensively. Nevertheless, results of these studies have not always been consistent

  5. Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells

    Microsoft Academic Search

    Shinji Isomoto; Koji Hattori; Hajime Ohgushi; Hiroshi Nakajima; Yasuhito Tanaka; Yoshinori Takakura

    2007-01-01

    Background  An autograft of cultured bone marrow-derived mesenchymal stem cells has already been used in clinical practice. In those patients\\u000a whose bone marrow cannot be used, a cell allograft with the use of immunosuppressant drugs will be an option in the future.\\u000a However, little is known about the effects of immunosuppressant drugs on mesenchymal stem cells. This study assessed the effects

  6. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    SciTech Connect

    Cowan, Robert W. [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada) [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Ghert, Michelle [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada) [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Singh, Gurmit, E-mail: gurmit.singh@jcc.hhsc.ca [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada) [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These results suggest that T cells may potentiate the catabolic effect of GCT.

  7. Bone regeneration with mesenchymal stem cells

    PubMed Central

    Kon, Elizaveta; Filardo, Giuseppe; Roffi, Alice; Di Martino, Alessandro; Hamdan, Mohammad; De Pasqual, Laura; Merli, Maria Letizia; Marcacci, Maurilio

    2012-01-01

    Summary Bone possesses the intrinsic regeneration capacity as part of the repair process in response to injury, during skeletal development or continuous remodeling throughout adult life. However, some complex clinical conditions require bone regeneration in too large quantity, and tissue engineering approach was developed to favor the regeneration of a new functional tissue. Mesenchymal stem cells (MSCs) have emerged as a promising alternative to the traditional surgical techniques. The purpose of this mini-review is to investigate the role of MSCs in clinical practice for bone regeneration, documenting the state of art and indentifying future research directions. We performed a search of the literature on PUBMED database between 2001 and 2011 using the key words “MSC and bone regeneration”. Inclusion criteria were clinical studies regarding the use of MSC in bone regeneration, for both bone repair and metabolic bone diseases, and in English language. References from selected papers were also screened. Our search resulted in 516 articles. Among these a total of 18 articles were included: 12 case series, 5 case reports and 1 comparative studies. MSCs represent an exciting and promising stem cell population for regeneration of bone in skeletal diseases, especially when tissue engineering or biomaterials are applied. However, literature results are limited, because of the small number and the low quality of trials, the lack of controls and the short follow-up. Researchers have to perform more high quality studies in order to document results and increase the potential of MSCs use in clinical practice, to develop a minimally invasive treatment to favor high quality bone tissue regeneration. PMID:22783331

  8. Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system

    Microsoft Academic Search

    Dina Lewinson; Gila Maor; Nimrod Rozen; Iaron Rabinovich; Shay Stahl; Adi Rachmiel

    2001-01-01

    An in vivo system of membranous bone formation during distraction has been investigated in order to follow cells that express vascular markers with the objective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and

  9. Ureaplasma infection of cell cultures.

    PubMed Central

    Kotani, H; McGarrity, G J

    1986-01-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium. Images PMID:3699891

  10. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells.

    PubMed

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-08-01

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from ?-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and ?-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both ?-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo. PMID:25206516

  11. Substrates for Human Pluripotent Stem Cell Cultures in Conditioned Medium of Mesenchymal Stem Cells

    Microsoft Academic Search

    Yusuke Ueda; Satoshi Fujita; Tatsuya Nishigaki; Yusuke Arima; Hiroo Iwata

    2012-01-01

    We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC

  12. Bone formation by human umbilical cord perivascular cells.

    PubMed

    Kajiyama, Sohtaro; Ujiie, Yuko; Nishikawa, Sumio; Inoue, Kohji; Shirakawa, Satoshi; Hanada, Nobuhiro; Liddell, Robert; Davies, John E; Gomi, Kasuhiro

    2015-08-01

    We investigated the possibility of employing human umbilical perivascular cells (HUCPVCs) within the context of finding an alternative source of mesenchymal stromal cells (MSC) for bone tissue engineering. Since it has previously been reported that conditioned medium (CM) from osteogenic bone marrow (BM) MSCs can potentiate osteogenic differentiation in a secondary cell population, we also employed BM-MSCs to generate CM to stimulate osteogenesis in the HUCPVCs. The BM-MSCs were a commercially available immortalized human cell line. In vitro assays showed negligible levels of osteogenic gene expression in HUCPVCs compared to BM-MSC, but alkaline phosphatase was detected when HUCPVC were cultured in osteogenic medium in the presence of CM from BM-MSC. An in vivo assay employing a rat calvarial osteotomy defect, together with a collagen sponge scaffold, showed that HUCPVCs provided statistically significant bony repair compared to controls. BM-MSC loaded scaffolds were not statistically different from either controls or HUCPVCs. The addition of BM-MSC CM to HUCPVCs also produced no statistically significant difference to the bone formed by HUCPVCs alone. Our results demonstrate that the in vitro assays employed did not predict in vivo outcomes, and that the BM-MSC cell line employed, or CM from such cells, provided no osteogenic advantage over the use of HUCPVCs alone. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2807-2814, 2015. PMID:25676366

  13. Cultured stem cells are sensitive to gravity changes

    Microsoft Academic Search

    L. B. Buravkova; Yu. A. Romanov; N. A. Konstantinova; S. V. Buravkov; Yu. G. Gershovich; I. A. Grivennikov

    2008-01-01

    Stem and precursor cells play an important role in development and regeneration. The state of these cells is regulated by biochemical substances, mechanical stimuli and cellular interactions. To estimate gravity effects we used two types of cultured stem cells: human mesenchymal stromal cells (hMSCs) from bone marrow and mice embryonic stem (mESC) line R1. Gravity changes were simulated by long-term

  14. Boning up on Wolff's Law: Mechanical regulation of the cells that make and maintain bone

    E-print Network

    Simmons, Craig A.

    a r t i c l e i n f o Article history: Accepted 21 August 2009 Keywords: Osteocyte Osteoprogenitor players in bone mechanobiology: osteocytes, the putative primary mechanosensors in intact bone by the cells in bone: osteocytes, the putative mechanosensors; osteoblasts that deposit bone matrix

  15. Fetal bovine bone cells synthesize bone-specific matrix proteins

    Microsoft Academic Search

    S. WILLIAM WHITSON; WILBUR HARRISON; MARY K. DUNLAP; DANIEL E. BOWERS; LARRY W. FISHER; PAMELA GEHRON ROBEY; JOHN D. TERMINE

    1984-01-01

    We isolatedcellsfrom both calvariaand the outercorticesoflong bones from 3- to 5-mo bovine fetuses. The cellswere identifiedas functionalosteoblastsby indirect immunofluorescence using antibodiesagainstthree bone-specific,noncollagenous matrix proteins(osteonectin,the bone proteoglycan,and the bone sialoprotein)and againsttype I Collagen.Inseparateexperiments,confluentculturesofthecellswere radiolabeledand shown to synthesizeand secreteosteonectin,the bone proteoglycanand the bone sialoproteinby imunoprecipitationand fluorographyofSIDSpolyacrylamidegels.Analysisofthe radiolabeled collagenssynthesizedby theculturesshowed thatthey produced predominantly (-94%) type I collagen,with smallamounts of types IIIand V collagens.

  16. Bone Marrow Mesenchymal Stem Cells Induce Angiogenesis and Promote Bladder Cancer Growth in a Rabbit Model

    Microsoft Academic Search

    Keqin Zhang; Benkang Shi; Jun Chen; Dongqing Zhang; Yaofeng Zhu; Changkuo Zhou; Haifeng Zhao; Xianzhou Jiang; Zhishun Xu

    2010-01-01

    Objectives: To investigate the effect of mesenchymal stem cells (MSCs) in the process of tumor development and the possibility of MSCs differentiating into vascular endothelial cells in the tumor microenvironment. Material and Methods: Twenty male New Zealand rabbits were randomly divided into 2 groups: a test group and a control group. MSCs were isolated and cultured by bone marrow cell

  17. Engineering tubular bone using mesenchymal stem cell sheets and coral particles

    SciTech Connect

    Geng, Wenxin [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China)] [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China); Ma, Dongyang [Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of PLA, BinHe 333 South Road, Lanzhou 730052 (China)] [Department of Oral and Maxillofacial Surgery, Lanzhou General Hospital, Lanzhou Command of PLA, BinHe 333 South Road, Lanzhou 730052 (China); Yan, Xingrong; Liu, Liangqi; Cui, Jihong; Xie, Xin; Li, Hongmin [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China)] [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China); Chen, Fulin, E-mail: chenfl@nwu.edu.cn [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China)] [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, No.229 North Taibai Road, Xi’an 710069 (China)

    2013-04-19

    Highlights: • We developed a novel engineering strategy to solve the limitations of bone grafts. • We fabricated tubular constructs using cell sheets and coral particles. • The composite constructs showed high radiological density and compressive strength. • These characteristics were similar to those of native bone. -- Abstract: The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

  18. High density cell culture system

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (inventor)

    1994-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

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

    NASA Technical Reports Server (NTRS)

    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.

  20. TOPICAL REVIEW Stem cells in bone tissue engineering

    Microsoft Academic Search

    Jeong Min Seong; Byung-Chul Kim; Jae-Hong Park; Il Keun Kwon; Anathathios Mantalaris; Yu-Shik Hwang

    2010-01-01

    Bone tissue engineering has been one of the most promising areas of research, providing a potential clinical application to cure bone defects. Recently, various stem cells including embryonic stem cells (ESCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose tissue-derived stem cells (ADSCs), muscle-derived stem cells (MDSCs) and dental pulp stem cells (DPSCs) have

  1. Cell Biology of Thiazide Bone Effects

    NASA Astrophysics Data System (ADS)

    Gamba, Gerardo; Riccardi, Daniela

    2008-09-01

    The thiazide-sensitive Na+:Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian kidney. The activity of NCC is not only related to salt metabolism, but also to calcium and magnesium homeostasis due to the inverse relationship between NCC activity and calcium reabsorption. Hence, the thiazide-type diuretics that specifically block NCC have been used for years, not only for treatment of hypertension and edematous disease, but also for the management of renal stone disease. Epidemiological studies have shown that chronic thiazide treatment is associated with higher bone mineral density and reduced risk of bone fractures, which can only partly be explained in terms of their effects on the kidney. In this regard, we have recently shown that NCC is expressed in bone cells and that inhibition of NCC in bone, either by thiazides or by reduction of NCC protein with specific siRNA, is associated with increased mineralization in vitro. These observations open a field of study to begin to understand the cell biology of the beneficial effects of thiazides in bone.

  2. Regulation of human bone marrow-derived osteoprogenitor cells by osteogenic growth factors.

    PubMed Central

    Long, M W; Robinson, J A; Ashcraft, E A; Mann, K G

    1995-01-01

    Human bone marrow contains a distinct cell population that expresses bone proteins and responds to transforming growth factor beta 1 (TGF-beta), but not to hematopoietic growth factors (Long, M. W., J. L. Williams, and K. G. Mann. 1990. J. Clin. Invest. 86:1387-1395). We now report the isolation, characterization, and growth factor responsiveness of these precursors to human osteoblasts and the identification of a human osteoprogenitor cell. Immunological separation of human bone marrow nonadherent low-density (NALD) cells results in a marked enrichment of cells that express osteocalcin, osteonectin, and bone alkaline phosphatase. Flow cytometric analyses show that distinct cell subpopulations exist among these isolated cells. The majority of the bone antigen-positive cells are approximately the size of a lymphocyte, whereas other, less frequent antibody-separated subpopulations consist of osteoblast-like cells and osteoprogenitor cells. In serum-free cultures, TGF-beta stimulates the small, antigen-positive cells to become osteoblast-like, as these cells both increase in size, and express increased levels of osteocalcin and alkaline phosphatase. Antibody-separated cells also contain a separate population of clonal progenitor cells that form colonies of osteoblast-like cells when cultured in serum-free, semi-solid media. Two types of human osteoprogenitor cells are observed: a colony-forming cell (CFC) that generates several hundred bone antigen-positive cells, and a more mature cluster-forming cell that has a lesser proliferative potential and thus generates clusters of 20-50 antigen-positive cells. Osteopoietic colony-forming cells and cluster-forming cells have an obligate but differential requirement for osteogenic growth factors. The CFCs respond to TGF-beta, basic fibroblast growth factor (bFGF), bone morphogenic protein-2 (BMP-2), and 1, 25-dihydroxy vitamin D3 (1,25-OH D3). In contrast to the colony-forming cells, cluster-forming cells are regulated predominantly by 1,25-OH D3 and TGF-beta, but fail to respond to bFGF. We conclude that human bone marrow contains a nonhematogenous, heterogeneous population of bone precursor cells among which exists a population of proliferating osteoprogenitor cells. Further characterization of these bone precursor cell populations should yield important information on their role in osteogenesis in both health and disease. Images PMID:7860771

  3. Co-cultured tissue-specific scaffolds for tendon/bone interface engineering

    PubMed Central

    Bumgardner, Joel D; Cole, Judith A; Smith, Richard A; Haggard, Warren O

    2014-01-01

    The tendon/ligament-to-bone interface has a complex organization to enable transfer of forces through the tendon/ligament to the bone. The purpose of this study is to create a co-culture environment enabling a tissue-specific tendon region and tissue-specific bone region on a degradable scaffold, using NIH 3T3 fibroblast–deposited extracellular matrix and MC 3T3 osteoblast–deposited extracellular matrix, respectively. Before full characterization of the deposited extracellular matrix coating can be analyzed, co-culture parameters including culture medium and seeding technique should be addressed. An appropriate medium formulation was developed to reduce fibroblast to osteoblast mineralization by adjusting beta-glycerophosphate concentrations. Standard growth medium with fetal bovine serum + 3 mM beta-glycerophosphate + 25 µg/mL ascorbic acid was found to be the most suitable formulation evaluated in these study conditions. Seeding and cell migration studies of co-cultured fibroblast- and osteoblast-specific scaffolds were performed to identify whether tissue regions could be created on the scaffold. Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding. Finally, a preliminary analysis of basic extracellular matrix components was measured in the fibroblast, osteoblast, and transition regions. Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions. In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

  4. Stem cells today: B1. Bone marrow stem cells

    Microsoft Academic Search

    RG Edwards

    2004-01-01

    This review is the second in a series of four devoted to the analysis of recent studies on stem cells. The first considered embryo stem cells (ES). This review covers bone marrow stem cells. They are analysed initially in a historical perspective, and then in relation to foundation studies in the later 20th century before a detailed analysis is presented

  5. Regulation of Haemopoiesis in Long-term Bone Marrow Cultures. IV. Glycosaminoglycan Synthesis and the Stimulation of Haemopoiesis by\\/ -D-Xylosides

    Microsoft Academic Search

    E. SPOONCER; J. T. GALLAGHER; E. KRIZSA; T. M. DEXTER

    Sulfated glycosaminoglycans (GAGs) are distributed in consistent and distinctive patterns between the cell surface and the growth medium of haemopoietically active long- term bone marrow cultures. Heparan sulfate is the main cell surface component and chon- droitin sulfate is the major sulfated species in the medium. When the cultures are supplemented with \\/Y-D-xylosides a significant increase in chondroitin sulfate synthesis

  6. Bone Repair Cells for Craniofacial Regeneration

    PubMed Central

    Pagni, G; Kaigler, D; Rasperini, G; Avila-Ortiz, G; Bartel, R; Giannobile, WV

    2012-01-01

    Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both Somatic and Stem Cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice. PMID:22433781

  7. Targeted delivery of mesenchymal stem cells to the bone.

    PubMed

    Yao, Wei; Lane, Nancy E

    2015-01-01

    Osteoporosis is a disease of excess skeletal fragility that results from estrogen loss and aging. Age related bone loss has been attributed to both elevated bone resorption and insufficient bone formation. We developed a hybrid compound, LLP2A-Ale in which LLP2A has high affinity for the ?4?1 integrin on mesenchymal stem cells (MSCs) and alendronate has high affinity for bone. When LLP2A-Ale was injected into mice, the compound directed MSCs to both trabecular and cortical bone surfaces and increased bone mass and bone strength. Additional studies are underway to further characterize this hybrid compound, LLP2A-Ale, and how it can be utilized for the treatment of bone loss resulting from hormone deficiency, aging, and inflammation and to augment bone fracture healing. This article is part of a Special Issue entitled "Stem Cells and Bone". PMID:25173607

  8. A novel bidirectional continuous perfusion bioreactor for the culture of large-sized bone tissue-engineered constructs.

    PubMed

    Gardel, Leandro S; Correia-Gomes, Carla; Serra, Luís A; Gomes, Manuela E; Reis, Rui L

    2013-11-01

    This works reports the development and preliminary assessment of a new bioreactor for culturing large-sized three-dimensional constructs in bone tissue engineering. The bidirectional continuous perfusion bioreactor (BCPB) promotes mechanical stimulation of cells through the creation of shear forces induced by flow perfusion. The main innovation consists in the possibility of culturing scaffolds of large dimensions that can be suitable for the regeneration of critical sized defects. The functionality of BCPB was preliminarily evaluated by culturing starch-polycaprolactone scaffolds/goat bone marrow stromal cells for 14 and 21 days. Cylindrical blocks were stacked (42 mm thick). Static culture was used as controls. The samples were collected for DNA, alkaline phosphatase (ALP), scanning electron microscopy (SEM), and histological analysis. The results showed higher ALP levels in the bioreactor cultures than those obtained under static conditions. The number of cells in constructs cultured in the bioreactor showed lower values compared to static cultures, suggesting that static conditions tend to privilege the metabolic path way for cellular proliferation while dynamic conditions tend to privilege the metabolic path for osteogenic differentiation. SEM observations show that, the migration and cell distribution was observed in the bioreactor. These results demonstrate the feasibility and the benefit of culturing constructs in BCPB. PMID:23681695

  9. Value of surveillance cultures in a bone marrow transplantation unit.

    PubMed

    Czirók, E; Prinz, G Y; Dénes, R; Reményi, P; Herendi, A

    1997-09-01

    Because of the increased risk of infection with the associated diagnostic and therapeutic problems in bone marrow transplantation (BMT) patients, the usefulness of surveillance cultures (SC) at the BMT department of the National Institute of Haematology, Blood Transfusion, Transplantation and Immunology, Budapest, was reviewed. Between January 1992 and May 1995, 26 BMT operations were performed; 13 patients had 23 febrile espisodes. In 12 of these episodes infection was clinically documented; however, SC of these patients yielded bacteria identical with those in the blood culture in only two episodes (1 and 6 days before their blood cultures became positive, respectively). Out of a total of 1187 samples from these patients, potentially pathogenic bacteria were isolated from 145 SC and 43 blood cultures (drawn on 31 different days). Suppression of the gastrointestinal flora could be achieved by the department's decontamination regimen; however, overgrowth by gram-positive organisms (mainly coagulase-negative staphylococci) occurred in the intestine and at other body sites. On the basis of these results, SC are of limited value in predicting infection or identifying the causative organisms of fever. On the other hand, SC are useful in confirming the efficiency of suppression of the body flora by antimicrobial agents. Specific treatment was based on suitably sampled materials, and close contact between physicians, infectious disease specialists and microbiologists was essential. PMID:9291891

  10. Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

    PubMed Central

    Akahane, Manabu; Shimizu, Takamasa; Ueha, Tomoyuki; Morita, Yusuke; Nakasaki, Shintaro; Kura, Tomohiko; Tohma, Yasuaki; Kido, Akira; Kawate, Kenji; Tanaka, Yasuhito

    2015-01-01

    Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

  11. Imaging of bone infection with labelled white blood cells role of contemporaneous bone marrow imaging

    Microsoft Academic Search

    A. D. King; A. M. Peters; A. W. J. Stuttle; J. P. Lavender

    1990-01-01

    The uptake of white blood cells (WBC) into normal bone marrow may lead to difficulty in detecting bone infection. Twenty-one patients in whom the WBC scan was equivocal or positive underwent a technetium 99m colloid scan to show the distribution of bone marrow. Six patients had a positive WBC scan, and in five of them a discordant colloid scan confirmed

  12. Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations

    Microsoft Academic Search

    C. G. Bellows; J. E. Aubin; J. N. M. Heersche; M. E. Antosz

    1986-01-01

    Summary  Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture\\u000a in the presence of ascorbic acid, Na ?-glycerophosphate, and dexamethasone to determine the capacity of these populations\\u000a to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and ?-glycerophosphate\\u000a or ascorbic acid alone, three-dimensional nodules (?75 ?m thick)

  13. Osteolytic bone resorption in adult T-cell leukemia/lymphoma.

    PubMed

    Shu, Sherry T; Martin, Chelsea K; Thudi, Nanda K; Dirksen, Wessel P; Rosol, Thomas J

    2010-04-01

    Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1alpha (MIP-1alpha), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL. PMID:20214446

  14. Osteolytic bone resorption in adult T-cell leukemia/lymphoma

    PubMed Central

    Shu, Sherry T.; Martin, Chelsea K.; Thudi, Nanda K.; Dirksen, Wessel P.; Rosol, Thomas J.

    2011-01-01

    Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1? (MIP-1?), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL. PMID:20214446

  15. Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

    PubMed

    Kishimoto, Satoko; Ishihara, Masayuki; Mori, Yasutaka; Takikawa, Megumi; Hattori, Hidemi; Nakamura, Shingo; Sato, Toshinori

    2013-12-01

    Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required. PMID:22473706

  16. Connecting mechanics and bone cell activities in the bone remodeling process: an integrated finite element modeling.

    PubMed

    Hambli, Ridha

    2014-01-01

    Bone adaptation occurs as a response to external loadings and involves bone resorption by osteoclasts followed by the formation of new bone by osteoblasts. It is directly triggered by the transduction phase by osteocytes embedded within the bone matrix. The bone remodeling process is governed by the interactions between osteoblasts and osteoclasts through the expression of several autocrine and paracrine factors that control bone cell populations and their relative rate of differentiation and proliferation. A review of the literature shows that despite the progress in bone remodeling simulation using the finite element (FE) method, there is still a lack of predictive models that explicitly consider the interaction between osteoblasts and osteoclasts combined with the mechanical response of bone. The current study attempts to develop an FE model to describe the bone remodeling process, taking into consideration the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain-damage stimulus function is proposed, which controls the level of autocrine and paracrine factors. The cellular behavior is based on Komarova et al.'s (2003) dynamic law, which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cells dynamic rather than adaptive elasticity approaches. The proposed FE model has been implemented in the FE code Abaqus (UMAT routine). An example of human proximal femur is investigated using the model developed. The model was able to predict final human proximal femur adaptation similar to the patterns observed in a human proximal femur. The results obtained reveal complex spatio-temporal bone adaptation. The proposed FEM model gives insight into how bone cells adapt their architecture to the mechanical and biological environment. PMID:25152881

  17. Culture of human osteoblasts on demineralised human bone. Possible means of graft enhancement.

    PubMed

    Nolan, P C; Nicholas, R M; Mulholland, B J; Mollan, R A; Wilson, D J

    1992-03-01

    We cultured human osteoblasts from trabecular bone explants and confirmed their phenotype by alkaline phosphatase assay, increased cyclic adenosine monophosphate production in response to prostaglandin E2 and radiographic micro-analysis of nodules of calcification. The osteoblasts were seeded on to demineralised human bone fragments and examined at ten-day intervals over a 50-day period by scanning electron microscopy. During this time the bank bone became progressively repopulated by the cultured osteoblasts. This system may offer a means of graft enhancement in elective orthopaedic and maxillofacial surgery by delivery of cultured autologous human osteoblasts to bone defects. PMID:1312095

  18. Immune impairments in multiple myeloma bone marrow mesenchymal stromal cells.

    PubMed

    André, Thibaud; Najar, Mehdi; Stamatopoulos, Basile; Pieters, Karlien; Pradier, Olivier; Bron, Dominique; Meuleman, Nathalie; Lagneaux, Laurence

    2015-02-01

    In multiple myeloma (MM), bone marrow mesenchymal stromal cells (BM-MSCs) play an important role in pathogenesis and disease progression by supporting myeloma cell growth and immune escape. Previous studies have suggested that direct and indirect interactions between malignant cells and BM-MSCs result in constitutive abnormal immunomodulatory capacities in MM BM-MSCs. The aim of this study was to investigate the mechanisms that underlie these MM BM-MSCs abnormalities. We demonstrated that MM BM-MSCs exhibit abnormal expression of CD40/40L, VCAM1, ICAM-1, LFA-3, HO-1, HLA-DR and HLA-ABC. Furthermore, an overproduction of IL-6 (1,806 ± 152.5 vs 719.6 ± 18.22 ng/mL; p = 0.035) and a reduced secretion of IL-10 (136 ± 15.02 vs 346.4 ± 35.32 ng/mL; p = 0.015) were quantified in culture medium when MM BM-MSCs were co-cultured with T lymphocytes compared to co-cultures with healthy donor (HD) BM-MSCs. An increased Th17/Treg ratio was observed when T cells were co-cultured with MM BM-MSCs compared to co-cultures with HD BM-MSCs (0.955 vs 0.055). Together, these observations demonstrated that altered immunomodulation capacities of MM BM-MSCs were linked to variations in their immunogenicity and secretion profile. These alterations lead not only to a reduced inhibition of T cell proliferation but also to a shift in the Th17/Treg balance. We identified factors that are potentially responsible for these alterations, such as IL-6, VCAM-1 and CD40, which could also be associated with MM pathogenesis and progression. PMID:25341809

  19. Adipose-Derived Stem Cells in Functional Bone Tissue Engineering: Lessons from Bone Mechanobiology

    PubMed Central

    Bodle, Josephine C.; Hanson, Ariel D.

    2011-01-01

    This review aims to highlight the current and significant work in the use of adipose-derived stem cells (ASC) in functional bone tissue engineering framed through the bone mechanobiology perspective. Over a century of work on the principles of bone mechanosensitivity is now being applied to our understanding of bone development. We are just beginning to harness that potential using stem cells in bone tissue engineering. ASC are the primary focus of this review due to their abundance and relative ease of accessibility for autologous procedures. This article outlines the current knowledge base in bone mechanobiology to investigate how the knowledge from this area has been applied to the various stem cell-based approaches to engineering bone tissue constructs. Specific emphasis is placed on the use of human ASC for this application. PMID:21338267

  20. Mesenchymal stem cells markedly suppress inflammatory bone destruction in rats with adjuvant-induced arthritis.

    PubMed

    Takano, Toshio; Li, Yin-Ji; Kukita, Akiko; Yamaza, Takayoshi; Ayukawa, Yasunori; Moriyama, Kanako; Uehara, Norihisa; Nomiyama, Hisayuki; Koyano, Kiyoshi; Kukita, Toshio

    2014-03-01

    Mesenchymal stem cells (MSCs) have potential to differentiate into multiple cell lineages. Recently, it was shown that MSCs also have anti-inflammatory and immunomodulatory functions. In this report, we investigated the regulatory function of MSCs in the development of inflammatory bone destruction in rats with adjuvant-induced arthritis (AA rats). MSCs were isolated from rat bone marrow tissues, expanded in the presence of basic FGF, and intraperitoneally injected into AA rats. MSC administration significantly suppressed inflammatory parameters: swelling score, swelling width, and thickness of hind paw. Radiographic evaluation indicated that MSC significantly suppressed bone destruction. Histological analysis showed that administration of MSCs markedly suppressed osteoclastogenesis in AA rats. To further delineate their effects on osteoclastogenesis, MSCs were added to in vitro bone marrow cultures undergoing osteoclastogenesis. MSCs significantly suppressed osteoclastogenesis in this system. Chemokine receptor expression in MSCs was assessed by RT-PCR, and a chemotactic assay was performed using a transwell culture system. MSCs showed significant chemotaxis to MIP-1? (CCL3) and SDF-1? (CXCL12), chemokines preferentially expressed in the area of inflammatory bone destruction. Furthermore, MSCs expressed IL-10 and osteoprotegerin, cytokines that suppress osteoclastogenesis. These data suggest that recruitment of MSC to the area of bone destruction in AA rats could suppress inflammatory bone destruction and raise the possibility that MSCs may have potential for the treatment of inflammatory bone destruction in arthritis. PMID:24395111

  1. In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells.

    PubMed

    Redzi?, Amira; Smajilagi?, Amer; Aljicevi?, Mufida; Berberovi?, Ljubomir

    2010-12-01

    Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autologue bone graft. Activity of the Alkaline Phosphatase enzyme were measurement on 0.5, 14, 21, 30 day and increased activity were detected day 14 on animals treated with bone marrow MSCs compared with day 30 on iliac crest treated animals. That results indicates strong osteoinduction activity of the experimental bone marrow MSCs models exposed to the rhBMP-7 factor Comparing ALP activity, that model showed superiorly results than control group. That result initiates us in opinion that MSCs alone should be alternative for the autolologue bone transplantation and in vitro study we isolated singles MSCs from the bone marrow of rat's tibia and femora and cultivated according to the method of Maniatopoulos et all. The small initial colonies of fibroblast like cells were photo-documented after 2 days of primary culture. Such isolated and cultivated MSCs in future studies will be exposed to the growth factors to differentiate in osteoblast and indicate their clinically potential as alternative for conventional medicine and autologue bone transplantation. That new horizons have potential to minimize surgery and patient donor morbidity, with more success treatment in bone regenerative and metabolism diseases. PMID:21874729

  2. Application of different strain regimes in two-dimensional and three-dimensional adipose tissue-derived stem cell cultures induces osteogenesis: implications for bone tissue engineering.

    PubMed

    Diederichs, Solvig; Böhm, Stefanie; Peterbauer, Anja; Kasper, Cornelia; Scheper, Thomas; van Griensven, Martijn

    2010-09-01

    Mechanical strain has become an important tool in tissue engineering for progenitor cell differentiation. Furthermore, it is used to enhance the mechanical properties of engineered tissue constructs. Although strain amplitude and frequency are well investigated and optimal values are known; application of various strain schemes regarding duration and repetition are not described in literature. In this study, we therefore applied singular and repetitive cyclic strain (1 Hz, 5%) of 15 min short-time strain and longer strain durations up to 8 h. Additionally, a gradually increasing strain scheme starting with short-time strain and consecutive elongated strain periods was applied. The cultivation surface was planar silicone on one hand and a three-dimensionally structured collagen I mesh on the other hand. Adipose tissue-derived mesenchymal stem cells and an osteogenic model cell line (MG-63) were exposed to these strain regimes and post-strain cell viability, osteogenic marker gene expression, and matrix mineralization were investigated. Upregulation of alkaline phosphatase, osteocalcin, osteopontin, and BMP-2/4 revealed that even short-time strain can enhance osteogenic differentiation. Elongation and repetition of strain, however, resulted in a decline of the observed short-time strain effects, which we interpret as positively induced cellular adaptation to the mechanically active surroundings. With regard to cellular adaptation, the gradually increasing strain scheme was especially advantageous. PMID:20730929

  3. A new stretching apparatus for applying anisotropic mechanical strain to bone cells in-vitro

    NASA Astrophysics Data System (ADS)

    Grabner, B.; Varga, F.; Fratzl-Zelman, N.; Luegmayr, E.; Glantschnig, H.; Rumpler, M.; Tatschl, A.; Fratzl, P.; Klaushofer, K.

    2000-09-01

    Bone is adapting to in-vivo loading by modeling and remodeling processes. The sensors of the external forces acting on the bone matrix seem to be the bone cells. Osteocytes, osteoblasts, and bone lining cells have been shown to respond to mechanical forces in-vitro. In this work, we describe a new in-vitro system which applies anisotropic stress conditions to MC3T3-E1, osteoblast-like mouse calvaria derived cells. The system allows stretching of cell cultures under well-defined stretching conditions. Cells are grown on an elastic polyurethane culture support (PUCS) that is subjected to uniaxial tensile stress using a direct current (dc) motor-driven linear positioning stage, situated within the incubator. The physical stretching parameters, the maximum elongation of the PUCS (the maximum strain applied to the cells), the strain rate, and the number of cycles, can be varied. First, the actual strains occurring at different locations of the PUCS were determined using optical methods. The surface strain appeared to be uniform over the PUCS and biaxial with a Poisson contraction nearly 80% in magnitude to the axial extension. Second, we tested the behavior of the MC3T3-E1 cells on PUCS compared to the cells grown in petridishes (PD). After 11 days of culture, cell number per dish on PUCS was significantly reduced to PD cultures (20% of control). At that time, cultures on PUCS reached confluency as compared to day 4 for the PD cultures. However, histochemical staining of alkaline phosphatase (ALP) and multilayer formation of the PUCS cultures appeared to be not significantly different from PD cultures. We also looked at the cytoskeleton by phalloidin staining, at vinculin, a protein of the cell-matrix and cell-cell interaction, and at fibronectin, a protein of the extracellular matrix using immuno staining methods. All these features tested so far seemed not to be different in cells cultured on PUCS compared to cultures in PD. Third, the responsiveness to the external force was tested using confluent cells on PUCS. A strain of 6.8 millistrain (6800 microstrain) was applied to the cells, using a strain rate of 4.9 millistrain/s and 350 cycles/h for a period of 48 h. These loading conditions led to significantly decreased cell proliferation, as measured by [3H] deoxythymidine ([3H] dT) incorporation, and significantly increased ALP activity. These data show that the stretching device introduced in this paper offers new possibilities to study the response of osteoblast-like cells to anisotropic forces.

  4. 3D Porous Calcium-Alginate Scaffolds Cell Culture System Improved Human Osteoblast Cell Clusters for Cell Therapy

    PubMed Central

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects. PMID:25825603

  5. Investigation of In Vitro Bone Cell Adhesion and Proliferation on Ti Using Direct Current Stimulation

    PubMed Central

    Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

    2012-01-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 µA, were used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–materials interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 µA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 µA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell-materials interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. PMID:23144532

  6. In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape.

    PubMed

    Mankani, M H; Kuznetsov, S A; Fowler, B; Kingman, A; Robey, P G

    2001-01-01

    Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques. PMID:11084599

  7. Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: correlation with bone marrow microenvironment

    Microsoft Academic Search

    Diana Campioni; Sabrina Moretti; Luisa Ferrari; Marina Punturieri; Gian Luigi Castoldi; Francesco Lanza

    The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alter- ations in the expression of CD105, CD90, CD184, and HLA-DR molecules.

  8. Radiation oncogenesis in cell culture

    SciTech Connect

    Borek, C.

    1982-01-01

    This review article examines the oncogenic effects of radiation with emphasis on ionizing radiations. Cell transformation in vitro is examined with respect to culture systems currently used in these studies, initiation and phenotypic expression of transformation and criteria for transformation. The section of radiation oncogenesis in vitro includes ionizing and nonionizing radiation studies and cocarcinogens and modulators of radiogenic transformations.

  9. Microfluidic Cell-Culture Devices

    Microsoft Academic Search

    Yasuyuki Sakai; Eric Leclerc; Teruo Fujii

    Microfluidics is the emerging technologies that could bring favorable features to tissue engineering applications. Fundamental\\u000a techniques to fabricated microfluidic cell-culture devices and experimental attempts towards in vitro liver tissue reconstitution\\u000a are presented for further discussion on the possible developments in the field of lab-on-a-chip for cellomics.

  10. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression

    Microsoft Academic Search

    Songtao Shi; Stan Gronthos; Shaoqiong Chen; Anand Reddi; Christopher M. Counter; Pamela G. Robey; Cun-Yu Wang

    2002-01-01

    Human postnatal bone marrow stromal stem cells (BMSSCs) have a limited life-span and progressively lose their stem cell properties during ex vivo expansion. Here we report that ectopic expression of human telomerase reverse transcriptase (hTERT) in BMSSCs extended their life-span and maintained their osteogenic potential. In xenogenic transplants, hTERT-expressing BMSSCs (BMSSC-Ts) generated more bone tissue, with a mineralized lamellar bone

  11. Neural stem cells induce bone-marrow-derived mesenchymal stem cells to generate neural stem-like cells via juxtacrine and paracrine interactions

    Microsoft Academic Search

    Arshak R. Alexanian

    2005-01-01

    Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural

  12. Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma.

    PubMed

    Martin, Chelsea K; Dirksen, Wessel P; Shu, Sherry T; Werbeck, Jillian L; Thudi, Nanda K; Yamaguchi, Mamoru; Wolfe, Tobie D; Heller, Kristin N; Rosol, Thomas J

    2012-06-01

    Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor ?B ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior. PMID:22265717

  13. Characterization of Bone Resorption in Novel In Vitro and In Vivo Models of Oral Squamous Cell Carcinoma

    PubMed Central

    Martin, Chelsea K.; Dirksen, Wessel P.; Shu, Sherry T.; Werbeck, Jillian L.; Thudi, Nanda K.; Yamaguchi, Mamoru; Wolfe, Tobie D.; Heller, Kristin N.; Rosol, Thomas J.

    2012-01-01

    Objectives Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Materials and Methods Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Results Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor ?B ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Conclusion Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL : OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior. PMID:22265717

  14. Bone marrow necrosis – initial presentation in sickle cell anemia

    PubMed Central

    Shafiq, Maria; Ali, Natasha

    2013-01-01

    Patient: Male, 20 Final Diagnosis: Sickle cell anemia Symptoms: Bone marrow necrosis • bone pain • fever • hepatomegaly • icterus • splenomegaly • weakness Medication: — Clinical Procedure: — Specialty: Hematology Objective: Unusual clinical course Background: In sickle cell disease, bone involvement is the commonest clinical presentation in the acute as well as chronic setting presenting as painful vaso-occlusive crisis and avascular necrosis, respectively. Other complications include bone marrow necrosis and infarction. Case Report: We report a case of a 20-year-old male who was referred for bone marrow evaluation due to symptoms of fever, weakness, and repeated episodes of bone pains. Bone trephine biopsy revealed multiple areas of central necrosis surrounded by fibroblasts. Conclusions: Recognition of necrosis through bone trephine biopsy is important for early initiation of therapy. PMID:24167641

  15. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro.

    PubMed

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N; Neutzsky-Wulff, Anita V; Geisler, Carsten; Karlsson, Lars; Söderström, Kalle

    2015-07-01

    Osteoclasts reside on bone and are the main bone resorbing cells playing an important role in bone homeostasis, while natural killer (NK) cells are bone-marrow-derived cells known to play a crucial role in immune defence against viral infections. Although mature NK cells traffic through bone marrow as well as to inflammatory sites associated with enhanced bone erosion, including the joints of patients with rheumatoid arthritis, little is known about the impact NK cells may have on mature osteoclasts and bone erosion. We studied the interaction between human NK cells and autologous monocyte-derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically decreased bone erosion. Suppression of bone erosion requires contact between NK cells and osteoclasts, but soluble factors also play a minor role. Antibodies masking leucocyte function-associated antigen-1, DNAX accessory molecule-1 or tumour necrosis factor-related apoptosis-inducing ligand enhance osteoclast survival when co-cultured with activated NK cells and restore the capacity of osteoclasts to erode bone. These results suggest that interleukin-15-activated NK cells may directly affect bone erosion under physiological and pathological conditions. PMID:25684021

  16. Placental-derived stem cells: Culture, differentiation and challenges.

    PubMed

    Oliveira, Maira S; Barreto-Filho, João B

    2015-05-26

    Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

  17. Intraosseous injection of RM1 murine prostate cancer cells promotes rapid osteolysis and periosteal bone deposition.

    PubMed

    McCabe, N Patrick; Madajka, Maria; Vasanji, Amit; Byzova, Tatiana V

    2008-01-01

    The molecular mechanisms associated with prostate cancer (PCa) progression within bone remain a topic of intense investigation. With the availability of transgenic mouse strains, a model of PCa for use in immune competent/transgenic mice would be highly beneficial. This study was designed to explore the utility of RM1 mouse PCa cells in investigations of tumor:bone interactions. The efficacies of several implantation techniques were examined for reliably producing intra-bone RM1 tumor growth and bone lesion formation in immune competent mice. Longitudinal monitoring of bone remodeling and lesion phenotypes was conducted by microcomputed tomography (muCT) and histological analyses. Our results indicate that direct intrabone injections of RM1 cells are necessary for tumor growth within bone and direct implantation promotes the rapid development of osteolytic bone lesions with periosteal bone deposition post-cortical breach. In vitro, RM1 cells promote the proliferation of osteoblast (MC3T3-E1) and osteoclast (Raw264.7) progenitors in a dose dependent manner. Conditioned culture media from RM1 cells appears to promote earlier expression of genes/proteins associated with osteoblastic differentiation. While clearly stimulating osteoclast function in vivo, RM1 cells had little effect on differentiation and tartate resistant acid phosphatase (TRAP) expression by Raw264.7 cells. These data, coupled with in vivo muCT images, indicate the ability of RM1 cells to induce mixed, yet predominentally osteolytic, responses in bone and illustrate the potential of RM1 cells as a model of investigating prostate tumor:stroma interactions in immune competent/transgenic mice on a C57BL/6 background. PMID:18506587

  18. Bone Marrow Stromal Stem Cells: Nature, Biology, and Potential Applications

    Microsoft Academic Search

    Paolo Bianco; Mara Riminucci; Stan Gronthos; Pamela Gehron Robey

    2001-01-01

    Bone marrow stromal cells are progenitors of skeletal tissue components such as bone, cartilage, the hemato- poiesis-supporting stroma, and adipocytes. In addition, they may be experimentally induced to undergo unortho- dox differentiation, possibly forming neural and myogenic cells. As such, they represent an important paradigm of post-natal nonhematopoietic stem cells, and an easy source for potential therapeutic use. Along with

  19. Trabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats

    Microsoft Academic Search

    H Seto; K Aoki; S Kasugai; K Ohya

    1999-01-01

    Low-calcium-fed animals have been accepted as one of the experimental models showing a reduction in bone mass. However, the effects of short-term low-calcium feeding on bone turnover, the development of osteoprogenitor cells, and gene expression of bone matrix proteins have not been reported. In this study, we examined the effect of a low-calcium diet on rat tibia and analyzed the

  20. Rapid growth and osteogenic differentiation of mesenchymal stem cells isolated from human bone marrow

    PubMed Central

    SHIMA, WAN NAZATUL; ALI, ABDUL MANAF; SUBRAMANI, TAMILSELVAN; MOHAMED ALITHEEN, NOORJAHAN BANU; HAMID, MUHAJIR; SAMSUDIN, ABDUL RANI; YEAP, SWEE KEONG

    2015-01-01

    Mesenchymal stem cells (MSCs) are involved in bone formation in the embryo, bone repair and remodeling. The differentiation of these cells is a complex multistep pathway that involves discrete cellular transitions and is similar to that which occurs during hematopoiesis. MSCs have self-renewal capacity without differentiation in long-term culture. In the present study, MSCs were isolated from human bone marrow and characterized by the presence of cluster of differentiation 105 marker using the labeled streptavidin biotin method. The MSCs were cultured in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, ascorbic acid, ?-glycerol phosphate and dexamethasone to differentiate into osteoblasts. Biological in vitro analysis showed the rapid proliferation of the MSCs. Further evaluation of specific osteogenic markers using von Kossa staining and the alkaline phosphate assay demonstrated that the MSCs were stimulated to differentiate into osteoblast-lineage cells. This mesengenic potential indicated that the bone marrow-derived cells were multipotent MSCs. The findings of this study show that bone marrow can be a legitimate source of MSCs for the production of osteoblasts for utilization in bone replacement therapy. PMID:26136960

  1. Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers.

    PubMed

    Chai, Yoke Chin; Roberts, Scott J; Desmet, Eline; Kerckhofs, Greet; van Gastel, Nick; Geris, Liesbet; Carmeliet, Geert; Schrooten, Jan; Luyten, Frank P

    2012-04-01

    Stem cell-based strategies for bone regeneration, which use calcium phosphate (CaP)-based biomaterials in combination with developmentally relevant progenitor populations, have significant potential for clinical repair of skeletal defects. However, the exact mechanism of action and the stem cell-host-material interactions are still poorly understood. We studied if pre-conditioning of human periosteum-derived cells (hPDCs) in vitro could enhance, in combination with a CaP-based biomaterial carrier, ectopic bone formation in vivo. By culturing hPDCs in a biomimetic calcium (Ca(2+)) and phosphate (P(i)) enriched culture conditions, we observed an enhanced cell proliferation, decreased expression of mesenchymal stem cell (MSC) markers and upregulation of osteogenic genes including osterix, Runx2, osteocalcin, osteopontin, and BMP-2. However, the in vitro pre-conditioning protocols were non-predictive for in vivo ectopic bone formation. Surprisingly, culturing in the presence of Ca(2+) and P(i) supplements resulted in partial or complete abrogation of in vivo ectopic bone formation. Through histological, immunohistochemical and microfocus X-ray computed tomography (?CT) analysis of the explants, we found that in situ proliferation, collagen matrix deposition and the mediation of osteoclastic activity by hPDCs are associated to their ectopic bone forming capacity. These data were validated by the multivariate analysis and partial least square regression modelling confirming the non-predictability of in vitro parameters on in vivo ectopic bone formation. Our series of experiments provided further insights on the stem cell-host-material interactions that govern in vivo ectopic bone induction driven by hPDCs on CaP-based biomaterials. PMID:22269651

  2. CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

    SciTech Connect

    Oue, Erika [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan) [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Lee, Ji-Won; Sakamoto, Kei [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan)] [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Iimura, Tadahiro [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan) [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan); Aoki, Kazuhiro [Section of Pharmacology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan)] [Section of Pharmacology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Kayamori, Kou [Section of Diagnostic Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan) [Section of Diagnostic Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Department of Pathology, Ome Municipal General Hospital, Ome, Tokyo (Japan); Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan)] [Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Yamaguchi, Akira, E-mail: akira.mpa@tmd.ac.jp [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan) [Section of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (Japan); Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo (Japan)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cell lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.

  3. Response and adaptation of bone cells to simulated microgravity

    NASA Astrophysics Data System (ADS)

    Hu, Lifang; Li, Runzhi; Su, Peihong; Arfat, Yasir; Zhang, Ge; Shang, Peng; Qian, Airong

    2014-11-01

    Bone loss induced by microgravity during space flight is one of the most deleterious factors on astronaut's health and is mainly attributed to an unbalance in the process of bone remodeling. Studies from the space microgravity have demonstrated that the disruption of bone remodeling is associated with the changes of four main functional bone cells, including osteoblast, osteoclast, osteocyte, and mesenchymal stem cells. For the limited availability, expensive costs and confined experiment conditions for conducting space microgravity studies, the mechanism of bone cells response and adaptation to microgravity is still unclear. Therefore, some ground-based simulated microgravity methods have been developed to investigate the bioeffects of microgravity and the mechanisms. Here, based on our studies and others, we review how bone cells (osteoblasts, osteoclasts, osteocytes and mesenchymal stem cells) respond and adapt to simulated microgravity.

  4. [Detection of high interleukin 6 concentrations in miniaturized human bone marrow long-term cultures].

    PubMed

    Mergenthaler, H G; Hültner, L; Mempel, W; Dörmer, P

    1991-01-01

    The long-term bone marrow culture system (LTBMC) consists of an inductive environment provided by a marrow-derived adherent layer which induces pluripotent stem cell replication. The activity of IL-6 was analyzed in micro LTBMC as a potential stem cell regulator. Immediately after initiation there was a tremendous increase in IL-6 activity. With each refeeding, IL-6 levels rose until confluency of the stromal layer was reached at week two to three post initiation. As a working hypothesis for further investigations, we suggest the immediate shift of stem cells after refeeding from a non-cycling into a cycling state might be mediated by the steep increase of IL-6 alone or in cooperation with other factors. PMID:1725635

  5. Epithelial cells derived from swine bone marrow express stem cell markers and support influenza virus replication in vitro.

    PubMed

    Khatri, Mahesh; Saif, Yehia M

    2011-01-01

    The bone marrow contains heterogeneous population of cells that are involved in the regeneration and repair of diseased organs, including the lungs. In this study, we isolated and characterized progenitor epithelial cells from the bone marrow of 4- to 5-week old germ-free pigs. Microscopically, the cultured cells showed epithelial-like morphology. Phenotypically, these cells expressed the stem cell markers octamer-binding transcription factor (Oct4) and stage-specific embryonic antigen-1 (SSEA-1), the alveolar stem cell marker Clara cell secretory protein (Ccsp), and the epithelial cell markers pan-cytokeratin (Pan-K), cytokeratin-18 (K-18), and occludin. When cultured in epithelial cell growth medium, the progenitor epithelial cells expressed type I and type II pneumocyte markers. Next, we examined the susceptibility of these cells to influenza virus. Progenitor epithelial cells expressed sialic acid receptors utilized by avian and mammalian influenza viruses and were targets for influenza virus replication. Additionally, differentiated type II but not type I pneumocytes supported the replication of influenza virus. Our data indicate that we have identified a unique population of progenitor epithelial cells in the bone marrow that might have airway reconstitution potential and may be a useful model for cell-based therapies for infectious and non-infectious lung diseases. PMID:22216319

  6. Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages

    Microsoft Academic Search

    Haihong Li; Xiaobing Fu; Yunshu Ouyang; Cunliang Cai; Jun Wang; Tongzhu Sun

    2006-01-01

    Adult bone-marrow-derived mesenchymal stem cells (MSCs) are well-established as having the capacity to differentiate into cells with mesodermal, ectodermal, and endodermal characteristics and can leave their niche to home toward and engraft within foreign tissues. To investigate whether adult MSCs contribute to the repair of skin appendages after injury, BrdU-labeled MSCs were co-cultured with heat-shocked confluent sweat gland cells (SGCs)

  7. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells

    SciTech Connect

    Tu Qisheng [Division of Oral Biology, Tufts University School of Dental Medicine, Boston, MA 02111 (United States); Valverde, Paloma [Division of Oral Biology, Tufts University School of Dental Medicine, Boston, MA 02111 (United States)]. E-mail: paloma.valverde@tufts.edu; Chen, Jake [Division of Oral Biology, Tufts University School of Dental Medicine, Boston, MA 02111 (United States)

    2006-03-24

    Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

  8. Progenitor and stem cells for bone and cartilage regeneration.

    PubMed

    El Tamer, M K; Reis, R L

    2009-07-01

    Research in regenerative medicine is developing at a significantly quick pace. Cell-based bone and cartilage replacement is an evolving therapy aiming at the treatment of patients who suffer from limb amputation, damaged tissues and various bone and cartilage-related disorders. Stem cells are undifferentiated cells with the capability to regenerate into one or more committed cell lineages. Stem cells isolated from multiple sources have been finding widespread use to advance the field of tissue repair. The present review gives a comprehensive overview of the developments in stem cells originating from different tissues and suggests future prospects for functional bone and cartilage tissue regeneration. PMID:19418440

  9. Establishment of primary cell cultures: Experiences with 155 cell strains

    Microsoft Academic Search

    M. Dietel; H. Arps; D. Gerding; M. Trapp; A. Niendorf

    1987-01-01

    Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements

  10. Expression of cell surface markers during differentiation of CD34+, CD38-/lo fetal and adult bone marrow cells.

    PubMed

    Olweus, J; Lund-Johansen, F; Terstappen, L W

    1994-12-01

    Even though there has been considerable progress in the phenotypic characterization of CD34+ bone marrow cells, there is still limited knowledge about the cell phenotypes corresponding to functional terms such as colony-forming cells, burst-forming cells, long-term culture-initiating cells, and high-proliferative potential cells. In this study, we performed a detailed analysis of phenotypic characteristics of subsets of CD34+ cells. We compared cells from adult and fetal bone marrow to investigate whether reported functional differences are reflected in the cellular phenotypes. CD34+, CD38-/lo, HLA-DR+ cells, which have been shown to contain the most immature hematopoietic progenitor cells, stained as a homogeneous population with most monoclonal antibodies (mAbs). The antigens sLex, CD32, and CD7 were, however, heterogeneously expressed in the CD38-/lo population. Phenotypic differences in the CD34+, CD38-/lo population was found when comparing adult and fetal bone marrow cells. Adult bone marrow CD34+, CD38-/lo cells stained more brightly with CD4, Thy-1, and CD49b and more dimly with CD32 than the same population in fetal bone marrow. Certain antigens that have previously been regarded as lineage-specific were found on the CD34+, CD38-/lo, HLA-DR+ cells in both fetal and adult bone marrow. These included CD52, CD13, and CD33. The markers that were found to be most useful in discriminating between subsets of lineage-committed cells within the CD34+, CD38+ population included the B cell marker CD19 and the granulomonocytic marker CD64. The phenotypic analysis presented here should provide a basis for establishing a better link between functional and phenotypic characteristics of hematopoietic progenitor cells. PMID:7540098

  11. Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors

    PubMed Central

    Solakoglu, Oender; Maierhofer, Christine; Lahr, Georgia; Breit, Elisabeth; Scheunemann, Peter; Heumos, Isabella; Pichlmeier, Uwe; Schlimok, Günter; Oberneder, Ralph; Köllermann, Manfred W.; Köllermann, Jens; Speicher, Michael R.; Pantel, Klaus

    2002-01-01

    Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21–102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome. PMID:11854519

  12. Cadherin-11 in renal cell carcinoma bone metastasis.

    PubMed

    Satcher, Robert L; Pan, Tianhong; Cheng, Chien-Jui; Lee, Yu-Chen; Lin, Song-Chang; Yu, Guoyu; Li, Xiaoxia; Hoang, Anh G; Tamboli, Pheroze; Jonasch, Eric; Gallick, Gary E; Lin, Sue-Hwa

    2014-01-01

    Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1?, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis. PMID:24587095

  13. Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

    PubMed Central

    Cheng, Chien-Jui; Lee, Yu-Chen; Lin, Song-Chang; Yu, Guoyu; Li, Xiaoxia; Hoang, Anh G.; Tamboli, Pheroze; Jonasch, Eric; Gallick, Gary E.; Lin, Sue-Hwa

    2014-01-01

    Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1?, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis. PMID:24587095

  14. Beyond gap junctions: Connexin43 and bone cell signaling

    PubMed Central

    Plotkin, Lilian I.; Bellido, Teresita

    2012-01-01

    Connexin 43 (Cx43) is the most abundant gap junction protein expressed in bone cells and plays a central role in cell-to-cell communication in the skeleton. Findings of the last decade uncovered functions of Cx43 hemichannels expressed on unopposed plasma cell membranes as mediators of the communication between bone cells and their extracellular milieu. Additionally, through its cytoplastmic C-terminus domain, Cx43 serves as a scaffolding protein that associates with structural and signaling molecules leading to regulation of intracellular signaling, independently of channel activity. This perspective discusses the evidence demonstrating that via these diverse mechanisms Cx43 is a key component of the intracellular machinery responsible for signal transduction in bone in response to pharmacologic, hormonal and mechanical stimuli. This advance in the knowledge of the role of connexins increases our understanding of the pathophysiological mechanisms that regulate bone cell function and provides new opportunities to treat bone diseases. PMID:23041511

  15. Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor cell content through passive purging

    Microsoft Academic Search

    BI Lundell; JJ Vredenburgh; C Tyer; K DeSombre; AK Smith

    1998-01-01

    High-dose chemotherapy (HDC) with hematopoietic support appears promising in the treatment of breast cancer, although reinfusion of contaminating tumor cells may contribute to disease relapse. Ex vivo expansion may reduce tumor cell content through use of a small inoculum volume and by passive purging during culture. We assessed the ex vivo expansion potential of tumor cell positive bone marrow (BM)

  16. A microfluidic system for automatic cell culture

    NASA Astrophysics Data System (ADS)

    Huang, Chun-Wei; Lee, Gwo-Bin

    2007-07-01

    This study presents a new chip capable of automating the cell culture process by using microfluidic technology. This microfluidic cell culture system comprising microheaters, a micro temperature sensor, micropumps, microvalves, microchannels, a cell culture area and several reservoirs was fabricated by using micro-electro-mechanical-systems' fabrication processes. Traditional manual cell culture processes can be performed on this chip. A uni-directional pneumatic micropump was developed to transport the culture reagents and constraint the solutions to flow only in one direction, safeguarding the entire culture process from contamination. A new micro check valve was also used to prevent the culture solutions from flowing back into the microchannels. The microheaters and the micro temperature sensor were used to maintain a constant temperature during the cell culturing process. The pH value suitable for cell growth was also regulated during the cell culture process. A typical cell culturing process for human lung cancer cells (A549) was successfully performed to demonstrate the capability of the developed microfluidic system. This automatic cell culturing system can be eventually integrated with subsequent microfluidic modules for cell purification, collection, counting and lysis to form a cell-based micro-total-analysis system. Preliminary results have been presented in The Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT), 25-28 June 2006

  17. Bone morphogenetic proteins induce the expression of noggin, which limits their activity in cultured rat osteoblasts.

    PubMed Central

    Gazzerro, E; Gangji, V; Canalis, E

    1998-01-01

    Bone morphogenetic proteins (BMPs) induce the differentiation of cells of the osteoblastic lineage and enhance the function of the osteoblast. Growth factors are regulated by binding proteins, but there is no information about binding proteins for BMPs in skeletal cells. Noggin specifically binds BMPs, but its expression by cells of the osteoblastic lineage has not been reported. We tested for the expression of noggin and its induction by BMP-2 in cultures of osteoblast-enriched cells from 22-d-old fetal rat calvariae (Ob cells). BMP-2 caused a time- and dose-dependent increase in noggin mRNA and polypeptide levels, as determined by Northern and Western blot analyses. The effects of BMP-2 on noggin transcripts were dependent on protein, but independent of DNA synthesis. BMP-2 increased the rates of noggin transcription as determined by nuclear run-on assays. BMP-4, BMP-6, and TGF-beta1 increased noggin mRNA in Ob cells, but basic fibroblast growth factor, platelet- derived growth factor BB, and IGF-I did not. Noggin decreased the stimulatory effects of BMPs on DNA and collagen synthesis and alkaline phosphatase activity in Ob cells. In conclusion, BMPs induce noggin transcription in Ob cells, a probable mechanism to limit BMP action in osteoblasts. PMID:9854046

  18. Matrix Metalloproteinase-9 and Cell Division in Neuroblastoma Cells and Bone Marrow Macrophages

    PubMed Central

    Sans-Fons, M. Gloria; Sole, Sonia; Sanfeliu, Coral; Planas, Anna M.

    2010-01-01

    Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages. PMID:20971732

  19. “Humanized” Stem Cell Culture Techniques: The Animal Serum Controversy

    PubMed Central

    Tekkatte, Chandana; Gunasingh, Gency Ponrose; Cherian, K. M.; Sankaranarayanan, Kavitha

    2011-01-01

    Cellular therapy is reaching a pinnacle with an understanding of the potential of human mesenchymal stem cells (hMSCs) to regenerate damaged tissue in the body. The limited numbers of these hMSCs in currently identified sources, like bone marrow, adipose tissue, and so forth, bring forth the need for their in vitro culture/expansion. However, the extensive usage of supplements containing xenogeneic components in the expansion-media might pose a risk to the post-transplantation safety of patients. This warrants the necessity to identify and develop chemically defined or “humanized” supplements which would make in vitro cultured/processed cells relatively safer for transplantation in regenerative medicine. In this paper, we outline the various caveats associated with conventionally used supplements of xenogenic origin and also portray the possible alternatives/additives which could one day herald the dawn of a new era in the translation of in vitro cultured cells to therapeutic interventions. PMID:21603148

  20. Effective bone engineering with periosteum-derived cells.

    PubMed

    Agata, H; Asahina, I; Yamazaki, Y; Uchida, M; Shinohara, Y; Honda, M J; Kagami, H; Ueda, M

    2007-01-01

    Bone augmentation via tissue engineering has generated significant interest. We hypothesized that periosteum-derived cells could be used in place of bone marrow stromal cells (which are widely used) in bone engineering, but the differences in osteogenic potential between these 2 cell types are unclear. Here, we compared the osteogenic potential of these cells, and investigated the optimal osteoinductive conditions for periosteum-derived cells. Both cell types were induced, via bFGF and BMP-2, to differentiate into osteoblasts. Periosteal cells proliferated faster than marrow stromal cells, and osteogenic markers indicated that bone marrow stromal cells were more osteogenic than periosteal cells. However, pre-treatment with bFGF made periosteal cells more sensitive to BMP-2 and more osteogenic. Transplants of periosteal cells treated with BMP-2 after pre-treatment with bFGF formed more new bone than did marrow stromal cells. Analysis of these data suggests that combined treatment with bFGF and BMP-2 can make periosteum a highly useful source of bone regeneration. PMID:17189468

  1. Stromal cell-derived factor-1 potentiates bone morphogenetic protein-2 induced bone formation.

    PubMed

    Higashino, Kosaku; Viggeswarapu, Manjula; Bargouti, Maggie; Liu, Hui; Titus, Louisa; Boden, Scott D

    2011-02-01

    The mechanisms driving bone marrow stem cell mobilization are poorly understood. A recent murine study found that circulating bone marrow-derived osteoprogenitor cells (MOPCs) were recruited to the site of recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone formation. Stromal cell-derived factor-1? (SDF-1?) and its cellular receptor CXCR4 have been shown to mediate the homing of stem cells to injured tissues. We hypothesized that chemokines, such as SDF-1, are also involved with mobilization of bone marrow cells. The CD45(-) fraction is a major source of MOPCs. In this report we determined that the addition of BMP-2 or SDF-1 to collagen implants increased the number of MOPCs in the peripheral blood. BMP-2-induced mobilization was blocked by CXCR4 antibody, confirming the role of SDF-1 in mobilization. We determined for the first time that addition of SDF-1 to implants containing BMP-2 enhances mobilization, homing of MOPCs to the implant, and ectopic bone formation induced by suboptimal BMP-2 doses. These results suggest that SDF-1 increases the number of osteoprogenitor cells that are mobilized from the bone marrow and then home to the implant. Thus, addition of SDF-1 to BMP-2 may improve the efficiency of BMPs in vivo, making their routine use for orthopaedic applications more affordable and available to more patients. PMID:21043834

  2. Role of magnesium ions on osteogenic response in bone marrow stromal cells.

    PubMed

    Yoshizawa, Sayuri; Brown, Andrew; Barchowsky, Aaron; Sfeir, Charles

    2014-08-01

    Biodegradable magnesium (Mg) alloys have been investigated for craniofacial and orthopedic bone fracture fixation due to their initial mechanical strength and high biocompatibility. Although Mg alloys have been reported to enhance bone regeneration in vivo, and enhanced osteogenic marker expression in human bone marrow stromal cells (hBMSCs) cultured in Mg alloy extract was reported, however, the biological mechanism is not fully understood. Thus, it is important to elucidate which signaling pathway in the hBMSCs are activated by Mg(2+) that enhances bone formation. We investigated possible mechanisms underlying effects of Mg(2+) on bone regeneration by culturing differentiated and undifferentiated hBMSCs in the presence of culture medium containing 10?mM MgSO4 both with or without osteogenic factors. mRNA expression of osteogenic genes was analyzed using quantitative PCR arrays. Quantitative PCR array data indicated increased mRNA expression of collagen type X and insulin-like growth factor 2, and decreased expression of integrin alpha 3 in the presence of 10?mM MgSO4. Moreover, Western blotting analysis showed enhanced expression of collagen type X, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-2?, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1?) in the presence of 10?mM MgSO4. In conclusion, 10?mM of MgSO4 enhanced the production of collagen type X and VEGF by hBMSCs. These results also suggest that Mg(2+) released from bone fixation devices may promote bone regeneration by enhancing the production of collagen type X and VEGF of osteogenic cells in bone tissue. PMID:25158202

  3. A novel whole tooth-in-jaw-bone culture of rat molars: morphological, immunohistochemical, and laser capture microdissection analysis.

    PubMed

    Chokechanachaisakul, Uraiwan; Kaneko, Tomoatsu; Yamanaka, Yusuke; Okiji, Takashi; Suda, Hideaki

    2012-10-01

    In conventional whole-tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth-in-jaw-bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2-positive macrophages were also analyzed for their Class II MHC, interleukin-6 (IL-6), and p53 mRNA expression levels by means of immune-laser capture microdissection (immune-LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline-perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium-perfused explants in contrast to the cultured control groups. The Class II MHC, IL-6, and p53 mRNA expression levels of ED2-positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium-perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth-in-jaw-bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp. PMID:22623030

  4. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion

    Microsoft Academic Search

    Naohiro Terada; Takashi Hamazaki; Masahiro Oka; Masanori Hoki; Diana M. Mastalerz; Yuka Nakano; Edwin M. Meyer; Laurence Morel; Bryon E. Petersen; Edward W. Scott

    2002-01-01

    Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such `transdifferentiation' in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific genes such as Y-chromosome markers. Here we demonstrate that mouse bone

  5. Calcification and cellularity in human aortic heart valve tissue determine the differentiation of bone-marrow-derived cells

    Microsoft Academic Search

    Hannu-Ville Leskelä; Jari Satta; Jani Oiva; Heidi Eriksen; Risteli Juha; Paula Korkiamäki; Kaisa K. Ivaska; Ylermi Soini; Petri Lehenkari

    2006-01-01

    Human bone-marrow-derived mesenchymal stem cells (MSC) are responsible the remodeling of human tissue. However, damaged aortic valves are lack the ability to regenerate which is an active cell-mediated process. Diseased aortic valve remodeling has similarities even to bone formation. In this study, the prerequisites for cultured MSCs to undergo osteoblastic differentiation on aortic valves were explored. An ex vivo model

  6. Osteoblastic Wnts differentially regulate bone remodeling and the maintenance of bone marrow mesenchymal stem cells.

    PubMed

    Wan, Yong; Lu, Cheng; Cao, Jingjing; Zhou, Rujiang; Yao, Yiyun; Yu, Jian; Zhang, Lingling; Zhao, Haixia; Li, Hanjun; Zhao, Jianzhi; Zhu, Xuming; He, Lin; Liu, Yongzhong; Yao, Zhengju; Yang, Xiao; Guo, Xizhi

    2013-07-01

    Wnt signaling has important roles in embryonic bone development and postnatal bone remodeling, but inconsistent impact on bone property is observed in different genetic alterations of Lrp5 and ?-catenin. More importantly, it is still controversial whether Lrp5 regulate bone formation locally or globally through gut-derived serotonin. Here we explored the function of Wnt proteins in osteoblastic niche through inactivation of the Wntless (Wls) gene, which abrogates the secretion of Wnts. The depletion of Wls in osteoblast progenitor cells resulted in severe osteopenia with more profound defects in osteoblastogenesis, osteoclastogenesis and maintenance of bone marrow mesenchymal stem cells (BMSCs) compared to that observed in Lrp5 and ?-catenin mutants. These findings support the point of view that Wnt/Lrp5 signaling locally regulates bone mass accrual through multiple effects of osteoblastic Wnts on osteoblastic bone formation and osteoclastic bone resorption. Moreover, osteoblastic Wnts confer a niche role for maintenance of BMSCs, providing novel cues for the definition of BMSCs niche in bone marrow. PMID:23334081

  7. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

    SciTech Connect

    Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)] [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Jhaveri, Hiral M. [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India)] [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India); Mishra, Gyan C. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)] [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

    2010-03-12

    Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

  8. How B Cells Influence Bone Biology in Health and Disease

    PubMed Central

    Horowitz, Mark C.; Fretz, Jackie A.; Lorenzo, Joseph A.

    2010-01-01

    It is now well established that important regulatory interactions occur between the cells in the hematopoietic, immune and skeletal systems (osteoimmunology). B lymphocytes (B cells) are responsible for the generation and production of antibodies or immunoglobulins in the body. Together with T cells these lymphocytes comprise the adaptive immune system, which allows an individual to develop specific responses to an infection and retain memory of that infection, allowing for a faster and more robust response if that same infection occurs again. In addition to this immune function, B cells have a close and multifaceted relationship with bone cells. B cells differentiate from hematopoietic stem cells (HSCs) in supportive niches found on endosteal bone surfaces. Cells in the osteoblast lineage support HSC and B cell differentiation in these niches. B cell differentiation is regulated, at least in part, by a series of transcription factors that function in a temporal manner. While these transcription factors are required for B cell differentiation, their loss causes profound changes in the bone phenotype. This is due, in part, to the close relationship between macrophage/osteoclast and B cell differentiation. Cross talk between B cells and bone cells is reciprocal with defects in the RANKL-RANK, OPG signaling axis resulting in altered bone phenotypes. While the role of B cells during normal bone remodeling appears minimal, activated B cells play an important role in many inflammatory diseases with associated bony changes. This review examines the relationship between B cells and bone cells and how that relationship affects the skeleton and hematopoiesis during health and disease. PMID:20601290

  9. Three-dimensional polycaprolactone-hydroxyapatite scaffolds combined with bone marrow cells for cartilage tissue engineering.

    PubMed

    Wei, Bo; Yao, Qingqiang; Guo, Yang; Mao, Fengyong; Liu, Shuai; Xu, Yan; Wang, Liming

    2015-08-01

    The goal of this study was to investigate the chondrogenic potential of three-dimensional polycaprolactone-hydroxyapatite (PCL-HA) scaffolds loaded with bone marrow cells in vitro and the effect of PCL-HA scaffolds on osteochondral repair in vivo. Here, bone marrow was added to the prepared PCL-HA scaffolds and cultured in chondrogenic medium for 10 weeks. Osteochondral defects were created in the trochlear groove of 29 knees in 17 New Zealand white rabbits, which were then divided into four groups that underwent: implantation of PCL-HA scaffolds (left knee, n?=?17; Group 1), microfracture (right knee, n?=?6; Group 2), autologous osteochondral transplantation (right knee, n?=?6; Group 3), and no treatment (right knee, n?=?5; Control). Extracellular matrix produced by bone marrow cells covered the surface and filled the pores of PCL-HA scaffolds after 10 weeks in culture. Moreover, many cell-laden cartilage lacunae were observed, and cartilage matrix was concentrated in the PCL-HA scaffolds. After a 12-week repair period, Group 1 showed excellent vertical and lateral integration with host bone, but incomplete cartilage regeneration and matrix accumulation. An uneven surface of regenerated cartilage and reduced distribution of cartilage matrix were observed in Group 2. In addition, abnormal bone growth and unstable integration between repaired and host tissues were detected. For Group 3, the integration between transplanted and host cartilage was interrupted. Our findings indicate that the PCL-HA scaffolds loaded with bone marrow cells improved chondrogenesis in vitro and implantation of PCL-HA scaffolds for osteochondral repairenhanced integration with host bone. However, cartilage regeneration remained unsatisfactory. The addition of trophic factors or the use of precultured cell-PCL-HA constructs for accelerated osteochondral repair requires further investigation. PMID:25766036

  10. Dynamized Preparations in Cell Culture

    PubMed Central

    Sunila, Ellanzhiyil Surendran; Preethi, Korengath Chandran; Kuttan, Girija

    2009-01-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  11. Catechin production in cultured Polygonum hydropiper cells

    Microsoft Academic Search

    Kanji Ono; Mayumi Nakao; Masao Toyota; Yoshimi Terashi; Masashi Yamada; Tetsuya Kohno; Yoshinori Asakawa

    1998-01-01

    Callus and suspension-cultured cells were induced from hypocotyls of Polygonum hydropiper seedlings. Both the callus and suspension-cultured cells produced mainly (+)-catechin accompanied by (?)-epicatechin and (?)-epicatechin-3-O-gallate. The (+)-catechin production of suspension-cultured cells increased with cell growth and reached the maximal value (29.0mgg?1 dry wt) after 6days from the start of subculture. This is the highest value of (+)-catechin content among

  12. Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells

    E-print Network

    Simmons, Craig A.

    by transplanted bone marrow stromal cells Craig A. Simmons,a,b,1 Eben Alsberg,a,1 Susan Hsiong,c Woo J. Kim bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels Inc. All rights reserved. Keywords: Alginate; Mesenchymal stem cells; Bone morphogenetic protein-2

  13. Osteogenic and osteoclastic cell interaction: development of a co-culture system.

    PubMed

    Loomer, P M; Ellen, R P; Tenenbaum, H C

    1998-10-01

    The processes involved in the regulation of bone cell metabolism are complex, including those implicated in bone cell coupling. This study was undertaken to develop a model that would permit real-time interaction between osteoclastic cells and osteoblasts in vitro. Osteogenic bone marrow stromal cells were isolated from 18-day-old embryonic chickens, while osteoclastic cells were isolated from laying White Leghorn hens on calcium-deficient diets. Osteoclastic cells (5x10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1x10(4)) already plated on the bottoms of tissue culture plate wells. The data showed that after 4 days of incubation there was up to a fivefold (P<0.05) reduction in all measured parameters of osteogenesis (mineralization, alkaline phosphatase activity and type I collagen production) in osteogenic cultures grown in the presence of osteoclastic cells. Similarly, osteoclastic cell-induced mineral resorption was reduced up to threefold (P<0.05). Co-culture effects on cellular responses could be manipulated by known antiresorptive agents (e.g., pamidronate) altering either the source or the age of osteoclastic cells. The results indicate that the co-culture model may be useful in the study of bone cell interactions. PMID:9724460

  14. Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture.

    PubMed

    Loomer, P M; Ellen, R P; Tenenbaum, H C

    1998-11-01

    This study was undertaken to determine the direct effects of extracts derived from Porphyromonas gingivalis on bone formation and mineral resorption in an osteogenic/osteoclastic cell in vitro co-culture model. Osteogenic bone marrow derived stromal cells were isolated from 18-day old embryonic chickens, while osteoclastic cells were isolated from laying white Leghorn hens on calcium deficient diets. Osteoclastic cells (5 x 10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1 x 10(4)) already plated on the bottoms of tissue culture plate wells. Sonicated P. gingivalis 2561 extracts were prepared from whole bacterial cells and added in varying proportions (0 to 2 microg/ml) to the co-culture growth medium. These co-cultures, and appropriate mono-culture controls, were incubated for a further 4 days. Parameters of bone forming cell activity including alkaline phosphatase activity, calcium and inorganic phosphate accumulation were performed on the osteogenic cells. Mineral substrate resorption by osteoclastic cells was assessed morphometrically. In their respective mono-cultures, the addition of P. gingivalis sonicate to the culture medium had no effect on osteoclastic mineral resorption, but significantly inhibited osteogenesis (up to 45%; P <0.05). In co-cultures, however, the sonicate induced significant increases in mineral resorption (up to 70%; P <0.05), whereas bone forming cell activity was still inhibited, although to a significantly lesser extent than in mono-cultures (up to 25%; P <0.05). These results suggest that P. gingivalis sonicate induced up-regulation of mineral resorption may be mediated via osteogenic cells. PMID:9848536

  15. Bone resorption by cells isolated from rheumatoid synovium.

    PubMed Central

    Chang, J S; Quinn, J M; Demaziere, A; Bulstrode, C J; Francis, M J; Duthie, R B; Athanasou, N A

    1992-01-01

    Cellular mechanisms accounting for the osteolysis of rheumatoid erosions are poorly understood. Cells were isolated and characterised from the synovium of 16 patients with rheumatoid arthritis (RA) and four patients with osteoarthritis and their ability to resorb bone was assessed using a scanning electron microscope bone resorption assay. Macrophages were the major cell type isolated from the synovium of patients with RA. These produced extensive roughening of the bone surface without resorption pit formation. This low grade type of bone resorption was not affected by systemic (calcitonin, parathyroid hormone, 1,25-dihydroxyvitamin D3) or local (interleukin 1, prostaglandin E2) factors influencing bone resorption. Macrophage mediated bone resorption differs qualitatively and quantitatively from that of osteoclasts but is likely to play an important part in the development of marginal erosions in RA. Images PMID:1334644

  16. CD14- mononuclear stromal cells support (CD14+) monocyte-osteoclast differentiation in aneurysmal bone cyst.

    PubMed

    Taylor, Richard Mi; Kashima, Takeshi G; Hemingway, Francesca K E; Dongre, Arundhati; Knowles, Helen J; Athanasou, Nicholas A

    2012-04-01

    Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor ?B ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism. PMID:22330339

  17. A novel single pulsed electromagnetic field stimulates osteogenesis of bone marrow mesenchymal stem cells and bone repair.

    PubMed

    Fu, Yin-Chih; Lin, Chih-Chun; Chang, Je-Ken; Chen, Chung-Hwan; Tai, I-Chun; Wang, Gwo-Jaw; Ho, Mei-Ling

    2014-01-01

    Pulsed electromagnetic field (PEMF) has been successfully applied to accelerate fracture repair since 1979. Recent studies suggest that PEMF might be used as a nonoperative treatment for the early stages of osteonecrosis. However, PEMF treatment requires a minimum of ten hours per day for the duration of the treatment. In this study, we modified the protocol of the single-pulsed electromagnetic field (SPEMF) that only requires a 3-minute daily treatment. In the in vitro study, cell proliferation and osteogenic differentiation was evaluated in the hBMSCs. In the in vivo study, new bone formation and revascularization were evaluated in the necrotic bone graft. Results from the in vitro study showed no significant cytotoxic effects on the hBMSCs after 5 days of SPEMF treatment (1 Tesla, 30 pulses per day). hBMSC proliferation was enhanced in the SPEMF-treated groups after 2 and 4 days of treatment. The osteogenic differentiation of hBMSCs was significantly increased in the SPEMF-treated groups after 3-7 days of treatment. Mineralization also increased after 10, 15, 20, and 25 days of treatment in SPEMF-treated groups compared to the control group. The 7-day short-course treatment achieved similar effects on proliferation and osteogenesis as the 25-day treatment. Results from the in vivo study also demonstrated that both the 7-day and 25-day treatments of SPEMF increased callus formation around the necrotic bone and also increased new vessel formation and osteocyte numbers in the grafted necrotic bone at the 2nd and 4th weeks after surgery. In conclusion, the newly developed SPEMF accelerates osteogenic differentiation of cultured hBMSCs and enhances bone repair, neo-vascularization, and cell growth in necrotic bone in mice. The potential clinical advantage of the SPEMF is the short daily application and the shorter treatment course. We suggest that SPEMF may be used to treat fractures and the early stages of osteonecrosis. PMID:24632682

  18. A Novel Single Pulsed Electromagnetic Field Stimulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells and Bone Repair

    PubMed Central

    Chang, Je-Ken; Chen, Chung-Hwan; Tai, I-Chun; Wang, Gwo-Jaw; Ho, Mei-Ling

    2014-01-01

    Pulsed electromagnetic field (PEMF) has been successfully applied to accelerate fracture repair since 1979. Recent studies suggest that PEMF might be used as a nonoperative treatment for the early stages of osteonecrosis. However, PEMF treatment requires a minimum of ten hours per day for the duration of the treatment. In this study, we modified the protocol of the single-pulsed electromagnetic field (SPEMF) that only requires a 3-minute daily treatment. In the in vitro study, cell proliferation and osteogenic differentiation was evaluated in the hBMSCs. In the in vivo study, new bone formation and revascularization were evaluated in the necrotic bone graft. Results from the in vitro study showed no significant cytotoxic effects on the hBMSCs after 5 days of SPEMF treatment (1 Tesla, 30 pulses per day). hBMSC proliferation was enhanced in the SPEMF-treated groups after 2 and 4 days of treatment. The osteogenic differentiation of hBMSCs was significantly increased in the SPEMF-treated groups after 3–7 days of treatment. Mineralization also increased after 10, 15, 20, and 25 days of treatment in SPEMF-treated groups compared to the control group. The 7-day short-course treatment achieved similar effects on proliferation and osteogenesis as the 25-day treatment. Results from the in vivo study also demonstrated that both the 7-day and 25-day treatments of SPEMF increased callus formation around the necrotic bone and also increased new vessel formation and osteocyte numbers in the grafted necrotic bone at the 2nd and 4th weeks after surgery. In conclusion, the newly developed SPEMF accelerates osteogenic differentiation of cultured hBMSCs and enhances bone repair, neo-vascularization, and cell growth in necrotic bone in mice. The potential clinical advantage of the SPEMF is the short daily application and the shorter treatment course. We suggest that SPEMF may be used to treat fractures and the early stages of osteonecrosis. PMID:24632682

  19. Bone marrow stem cell transplant into intra-bone cavity prevents type 2 diabetes: Role of heme oxygenase-adiponectin

    E-print Network

    Abraham, Nader G.

    Review Bone marrow stem cell transplant into intra-bone cavity prevents type 2 diabetes: Role Abstract Increase in endothelial cell sloughing and diminished function of endothelial stem cell stem cells (BMSCs) including mesenchymal stem cells but not limited to CD34þ stem cells into type 2

  20. Clinical Application of Human Mesenchymal Stromal Cells for Bone Tissue Engineering

    PubMed Central

    Chatterjea, Anindita; Meijer, Gert; van Blitterswijk, Clemens; de Boer, Jan

    2010-01-01

    The gold standard in the repair of bony defects is autologous bone grafting, even though it has drawbacks in terms of availability and morbidity at the harvesting site. Bone-tissue engineering, in which osteogenic cells and scaffolds are combined, is considered as a potential bone graft substitute strategy. Proof-of-principle for bone tissue engineering using mesenchymal stromal cells (MSCs) has been demonstrated in various animal models. In addition, 7 human clinical studies have so far been conducted. Because the experimental design and evaluation parameters of the studies are rather heterogeneous, it is difficult to draw conclusive evidence on the performance of one approach over the other. However, it seems that bone apposition by the grafted MSCs in these studies is observed but not sufficient to bridge large bone defects. In this paper, we discuss the published human clinical studies performed so far for bone-tissue regeneration, using culture-expanded, nongenetically modified MSCs from various sources and extract from it points of consideration for future clinical studies. PMID:21113294

  1. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

    PubMed

    Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

    2015-06-01

    In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced. PMID:25819548

  2. Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells.

    PubMed

    van Gastel, Nick; Torrekens, Sophie; Roberts, Scott J; Moermans, Karen; Schrooten, Jan; Carmeliet, Peter; Luttun, Aernout; Luyten, Frank P; Carmeliet, Geert

    2012-11-01

    One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies. PMID:22911908

  3. Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

    PubMed Central

    Sasaki, Jun-Ichi; Hashimoto, Masanori; Yamaguchi, Satoshi; Itoh, Yoshihiro; Yoshimoto, Itsumi; Matsumoto, Takuya; Imazato, Satoshi

    2015-01-01

    The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs. PMID:26047122

  4. Alterations in bone forming cells due to reduced weight bearing

    NASA Technical Reports Server (NTRS)

    Doty, S. B.; Morey-Holton, E.

    1984-01-01

    A reduction in new bone formation occurred as a result of space flight (Cosmos 1129) and in the suspended animal model of Morey-Holton (1979, 1980). The results indicate that alkaline phosphatase activity of the bone-forming cells is also reduced under these conditions, and the cells in the diaphysis are more affected than those in the metaphyseal region. In addition, these cells show (1) reduced proline incorporation into bone matrix, and (2) increased intracellular lysosomal activity. A change in the cytoskeleton could be the common factor in explaining these results. This suggestion is futher supported by the previous observations that colchicine injections result in decreased osteoblastic function.

  5. Cell culture techniques in honey bee research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  6. Cell culture model for antiulcerogenic agents.

    PubMed

    Terano, A; Hiraishi, H; Shimada, T; Takahashi, M; Yoshiura, K; Horie-Sakata, K

    2001-06-15

    To elucidate the mechanisms of antiulcerogenic agents, we established the cell culture model derived from rat gastric epithelium. The cultured cells were identified as mucus-producing cells by using histological analysis. This culture model is useful for investigating the untiulcer effect of various agents and to reveal the mechanisms of the drug action. In particular, the ulcer-healing model using the cultured monolayer is promising and convenient for the study of several growth factors such as HGF as well as antiulcerogenic agents. The effect of polaporezinc in the cultured model is introduced. PMID:11525256

  7. Periodontal regeneration using engineered bone marrow mesenchymal stromal cells

    Microsoft Academic Search

    Yi Yang; Fabio M. V. Rossi; Edward E. Putnins

    2010-01-01

    Regeneration of lost periodontium is a challenge in that both hard (alveolar bone, cementum) and soft (periodontal ligament) connective tissues need to be restored to their original architecture. Bone marrow mesenchymal stromal cells (BM-MSCs) appear to be an attractive candidate for connective tissue regeneration. We hypothesized that BM-MSCs are able to sense biological cues from the local microenvironment and organize

  8. Bone-marrow haematopoietic-stem-cell niches

    Microsoft Academic Search

    Anne Wilson; Andreas Trumpp

    2006-01-01

    Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche.

  9. Protection of bone marrow progenitor cells by superoxide dismutase

    Microsoft Academic Search

    A. Petkau

    1988-01-01

    Intravenously administered cuprozinc-superoxide dismutase in X-irradiated mice hastens the recovery of peripheral blood cells. This effect is consistent with protection of the pluripotent stem cells by the enzyme. Amongst the bone marrow cells committed to differentiation along the myeloid pathway, there exists in mice a subpopulation of macrophage progenitor cells that is inactivated by superoxide radicals, generated photochemically or by

  10. Cell Mechanisms of Bone Tissue Loss Under Space Flight Conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia

    Investigations on the space biosatellites has shown that the bone skeleton is one of the most im-portant targets of the effect space flight factors on the organism. Bone tissue cells were studied by electron microscopy in biosamples of rats' long bones flown on the board american station "SLS-2" and in experiments with modelling of microgravity ("tail suspension" method) with using autoradiography. The analysis of data permits to suppose that the processes of remod-eling in bone tissue at microgravity include the following succession of cell-to-cell interactions. Osteocytes as mechanosensory cells are first who respond to a changing "mechanical field". The next stage is intensification of osteolytic processes in osteocytes, leading to a volume en-largement of the osteocytic lacunae and removal of the "excess bone". Then mechanical signals have been transmitted through a system of canals and processes of the osteocytic syncitium to certain superficial bone zones and are perceived by osteoblasts and bone-lining cells (superficial osteocytes), as well as by the bone-marrow stromal cells. The sensitivity of stromal cells, pre-osteoblasts and osteoblasts, under microgravity was shown in a number of works. As a response to microgravity, the system of stromal cells -preosteoblasts -osteoblasts displays retardation of proliferation, differentiation and specific functions of osteogenetic cells. This is supported by the 3H-thymidine studies of the dynamics of differentiation of osteogenetic cells in remodeling zones. But unloading is not adequate and in part of the osteocytes are apoptotic changes as shown by our electron microscopic investigations. An osteocytic apoptosis can play the role in attraction the osteoclasts and in regulation of bone remodeling. The apoptotic bodies with a liquid flow through a system of canals are transferred to the bone surface, where they fulfil the role of haemoattractants for monocytes come here and form osteoclasts. The osteoclasts destroy bone tissue. The macrophages are incorporated into resorption lacunaes and utilize the organic matrix and cellular detritus. The products are secreted to remodeling zones and act as haemoattractants for recruiting and subsequent differentiation here of the osteogenic precursor cells. However, as shown by our results with 3H-glycine, in absence of mechanical stimulus the activization of osteoblastogenesis either doesn't occur, or takes place on a smaller scale. According to our electron-microscopic data a load deficit leads to an adaptive differentiation of fibroblasts and adipocytes in this remodeling zones. This sequence of events is considered as a mechanism of bone tissue loss which underlies the development of osteopenia and osteoporosis under space flight condition.

  11. Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

    SciTech Connect

    Waksman, Ron; Baffour, Richard

    2003-09-01

    Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.

  12. Functional studies of maturing myeloid cells during ex vivo expansion for treatment of aplasia: feasibility of ex vivo expansion from cryopreserved bone marrow cell samples.

    PubMed

    Neildez-Nguyen, T M; Vétillard, J; Drouet, M; Hérodin, F; Brouard, N; Mestries, J C; Thierry, D

    1998-02-01

    Ex vivo expanded CD34+ progenitor cells from fresh or cryopreserved primate bone marrow, induced to granulocytic differentiation with growth factors, were investigated to determine whether myeloid cells produced in liquid cultures have the normal biologic functions needed for the treatment of patients with neutropenia following high-dose chemotherapy or therapeutic or accidental radiation exposure. Human and simian (baboons or macaques) CD34+ cells were cultured with granulocyte-colony stimulating factor (G-CSF), stem cell factor (SCF), interleukin-1 (IL-1), IL-3, and IL-6, and assessed at 14 days of culture for their capacity to respond to different functional tests. Immunostaining revealed that human ex vivo expanded cells contained myeloperoxydase (MPO, 82% +/- 8%) and lactoferrin (LF, 30% +/- 6%) in their granules. Maturation of cultured cells was associated with stimulated chemotactic responsiveness and respiratory burst activity (superoxide anion and hydrogen peroxide production) in expansions from human, baboon, and macaque CD34+ progenitor cells. Mature cells obtained from ex vivo expansion of selected cryopreserved human bone marrow CD34+ cells presented reduced but significant functional activities (chemotactic responsiveness and hydrogen peroxide production) when compared with human peripheral blood neutrophils. The validation of nonhuman primate ex vivo expansion systems may permit their use as models of irradiation. The feasibility of ex vivo expansion from cryopreserved bone marrow cell samples may offer considerable opportunity for banking bone marrow for autologous transfusion. PMID:9507383

  13. [Polysaccharides of cell cultures of Silene vulgaris].

    PubMed

    Giunter, E A; Ovodov, Iu S

    2007-01-01

    Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures. PMID:17345866

  14. Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

    PubMed

    Addison, W N; Nelea, V; Chicatun, F; Chien, Y-C; Tran-Khanh, N; Buschmann, M D; Nazhat, S N; Kaartinen, M T; Vali, H; Tecklenburg, M M; Franceschi, R T; McKee, M D

    2015-02-01

    Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. PMID:25460184

  15. Induction of Ym1\\/2 in mouse bone marrow-derived mast cells by IL4 and identification of Ym1\\/2 in connective tissue type-like mast cells derived from bone marrow cells cultured with IL4 and stem cell factor

    Microsoft Academic Search

    Eunkyung Lee; Jumin Yook; Kyungmi Haa; Hyeun Wook Chang

    2005-01-01

    Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR-selected cDNA subtraction to establish

  16. Characterization of Distinct Classes of Differential Gene Expression in Osteoblast Cultures from Non-Syndromic Craniosynostosis Bone

    PubMed Central

    Rojas-Peña, Monica L.; Olivares-Navarrete, Rene; Hyzy, Sharon; Arafat, Dalia; Schwartz, Zvi; Boyan, Barbara D.; Williams, Joseph; Gibson, Greg

    2014-01-01

    Craniosynostosis, the premature fusion of one or more skull sutures, occurs in approximately 1 in 2500 infants, with the majority of cases non-syndromic and of unknown etiology. Two common reasons proposed for premature suture fusion are abnormal compression forces on the skull and rare genetic abnormalities. Our goal was to evaluate whether different sub-classes of disease can be identified based on total gene expression profiles. RNA-Seq data were obtained from 31 human osteoblast cultures derived from bone biopsy samples collected between 2009 and 2011, representing 23 craniosynostosis fusions and 8 normal cranial bones or long bones. No differentiation between regions of the skull was detected, but variance component analysis of gene expression patterns nevertheless supports transcriptome-based classification of craniosynostosis. Cluster analysis showed 4 distinct groups of samples; 1 predominantly normal and 3 craniosynostosis subtypes. Similar constellations of sub-types were also observed upon re-analysis of a similar dataset of 199 calvarial osteoblast cultures. Annotation of gene function of differentially expressed transcripts strongly implicates physiological differences with respect to cell cycle and cell death, stromal cell differentiation, extracellular matrix (ECM) components, and ribosomal activity. Based on these results, we propose non-syndromic craniosynostosis cases can be classified by differences in their gene expression patterns and that these may provide targets for future clinical intervention. PMID:25184005

  17. Clonal analysis of bone marrow and macrophage cultures

    SciTech Connect

    Stewart, C.C.; Walker, E.B.; Johnson, C.; Little, R.

    1984-01-01

    To establish lineages that can be used to study their functional heterogeneity, the proliferation and differentiation of bone marrow derived mononuclear phagocytes and the lineages derived from them were studied. 28 references, 7 figures, 5 tables. (ACR)

  18. Effects of prostaglandin F2 alpha on bone formation and resorption in cultured neonatal mouse calvariae: Role of prostaglandin E2 production

    SciTech Connect

    Raisz, L.G.; Alander, C.B.; Fall, P.M.; Simmons, H.A. (Univ. of Connecticut Health Center, Farmington (USA))

    1990-02-01

    Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2 alpha may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF2 alpha and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 45Ca, bone formation as incorporation of (3H)proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of (3H)thymidine. PGF2 alpha was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2 alpha had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2 alpha increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2 alpha might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2 alpha, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with (3H)arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2 alpha treatment. These results indicate that PGF2 alpha is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2 alpha on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production.

  19. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  20. Ultrastructural and cytobiological studies on possible interactions between PTHrP-secreting tumor cells, stromal cells, and bone cells

    Microsoft Academic Search

    Masahiro Ito; Norio Amizuka; Shohei Tanaka; Yukiko Funatsu-Ozawa; Shin-ichi Kenmotsu; Kimimitsu Oda; Tamio Nakajima; Hidehiro Ozawa

    2003-01-01

    Parathyroid hormone-related peptide (PTHrP) induces pathological bone resorption in an endocrine manner, resulting in hypercalcemia of malignancy. However, the histopathological aspect of the action of PTHrP secreted by tumor cells on bone resorption has not well been documented. Therefore, we studied cell–cell interactions between bone cells, stromal cells, and PTHrP-secreting tumor cells (EC-GI-10) morphologically. Tumor cells injected subcutaneously into the

  1. The bone marrow niche for haematopoietic stem cells

    PubMed Central

    Morrison, Sean J.; Scadden, David T.

    2015-01-01

    Preface Niches are local tissue microenvironments that maintain and regulate stem cells. Haematopoiesis provides a paradigm for understanding mammalian stem cells and their niches, yet the haematopoietic stem cell (HSC) niche remains incompletely defined and beset by competing models. Here we review progress in elucidating the location and cellular components of the HSC niche in the bone marrow. The niche is perivascular, created partly by mesenchymal stromal cells and endothelial cells and often, but not always, located near trabecular bone. Outstanding questions concern the cellular complexity of the niche, the role of the endosteum, and functional heterogeneity among perivascular microenvironments. PMID:24429631

  2. Remyelination of the Rat Spinal Cord by Transplantation of Identified Bone Marrow Stromal Cells

    PubMed Central

    Akiyama, Yukinori; Radtke, Christine; Kocsis, Jeffery D.

    2008-01-01

    Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal cells were GFP expressing and immunopositive for collagen type I, fibronectin, and CD44. Transplantation of these cells by direct microinjection into the demyelinated spinal cord of the immunosuppressed rat resulted in remyelination. The remyelinated axons showed characteristics of both central and peripheral myelination as observed by electron microscopy; conduction velocity of the axons was improved. GFP-positive cells and myelin profiles were observed in the remyelinated spinal cord region, indicating that the donor-isolated stromal cells were responsible for the formation of the new myelin. The GFP-positive cells were colocalized with myelin basic protein-positive and P0-positive cellular elements. These findings indicate that cells contained within the stromal cell fraction of the mononuclear cell layer of bone marrow can form functional myelin during transplantation into demyelinated spinal cord. PMID:12151541

  3. A cell-based model of bone remodeling for identifying activity of icarrin in the treatment of osteoporosis.

    PubMed

    Liu, Yan-Qiu; Han, Xiao-Fei; Liu, Tiegang; Cheng, Meng-Chun; Xiao, Hong-Bin

    2015-01-01

    The activity of icarrin (a flavonoid from Herba epimedii) was investigated in the regulation of bone remodeling, a process coupled by osteoblast-mediated bone forming and osteoclast-mediated bone resorption. By directly co-culturing mouse bone marrow stromal cells and mouse preosteoclastic RAW264.7, and transwell co-culturing rat ovarian follicular granulosa cells (FGC), a 30 % increase in alkaline phosphatase (ALP) activity and 25 % increase in estradiol level occurred. Compared with the antiresorptive drug, alendronate, and an anabolic drug, PTH1-34, icarrin possessed all of the positive effects on the co-culture by increasing ALP activity, estradiol production and decreasing tartrate-resistant acid phosphatase activity. A similar action of icarrin occurred on co-culture of mesenchymal stem cells, mouse peripheral blood mononuclear cells, and FGC. Overall, by using a co-cultured cell-based in vitro screening assay, icarrin is suggested as a new class of dual-action therapeutic agent for osteoporosis. PMID:25257584

  4. Biomaterials and bone mechanotransduction

    NASA Technical Reports Server (NTRS)

    Sikavitsas, V. I.; Temenoff, J. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Bone is an extremely complex tissue that provides many essential functions in the body. Bone tissue engineering holds great promise in providing strategies that will result in complete regeneration of bone and restoration of its function. Currently, such strategies include the transplantation of highly porous scaffolds seeded with cells. Prior to transplantation the seeded cells are cultured in vitro in order for the cells to proliferate, differentiate and generate extracellular matrix. Factors that can affect cellular function include the cell-biomaterial interaction, as well as the biochemical and the mechanical environment. To optimize culture conditions, good understanding of these parameters is necessary. The new developments in bone biology, bone cell mechanotransduction, and cell-surface interactions are reviewed here to demonstrate that bone mechanotransduction is strongly influenced by the biomaterial properties.

  5. Stem cell-based therapies for bone repair.

    PubMed

    Milner, Peter I; Clegg, Peter D; Stewart, Matthew C

    2011-08-01

    This article provides an overview of the cellular and molecular events involved in bone repair and the current approaches to using stem cells as an adjunct to this process. The article emphasizes the key role of osteoprogenitor cells in the formation of bone and where the clinical applications of current research may lend themselves to large animal orthopaedics. The processes involved in osteogenic differentiation are presented and strategies for bone formation, including induction by osteogenic factors, bioscaffolds, and gene therapy, are reviewed. PMID:21872760

  6. High abundance of CD271(+) multipotential stromal cells (MSCs) in intramedullary cavities of long bones.

    PubMed

    Cox, George; Boxall, Sally A; Giannoudis, Peter V; Buckley, Conor T; Roshdy, Tarek; Churchman, Sarah M; McGonagle, Dennis; Jones, Elena

    2012-02-01

    Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed. Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34(+) hematopoietic stem cells and CD31(+) endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45(-/low) CD271(+) MSC population and revealed a trend for higher MSC numbers (average 5 fold, n=6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73(+)CD105(+)CD90(+)) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM. Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a 'low-tech' method of harvesting potentially large numbers of MSCs for regenerative therapies and research. PMID:21807134

  7. Translational Research: Palatal-derived Ecto-mesenchymal Stem Cells from Human Palate: A New Hope for Alveolar Bone and Cranio-Facial Bone Reconstruction

    PubMed Central

    Grimm, Wolf Dieter; Dannan, Aous; Giesenhagen, Bernd; Schau, Ingmar; Varga, Gabor; Vukovic, Mark Alexander; Sirak, Sergey Vladimirovich

    2014-01-01

    The management of facial defects has rapidly changed in the last decade. Functional and esthetic requirements have steadily increased along with the refinements of surgery. In the case of advanced atrophy or jaw defects, extensive horizontal and vertical bone augmentation is often unavoidable to enable patients to be fitted with implants. Loss of vertical alveolar bone height is the most common cause for a non primary stability of dental implants in adults. At present, there is no ideal therapeutic approach to cure loss of vertical alveolar bone height and achieve optimal pre-implantological bone regeneration before dental implant placement. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from different dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. Our research group has cultured palatal-derived stem cells (paldSCs) as dentospheres and further differentiated into various cells of the neuronal and osteogenic lineage, thereby demonstrating their stem cell state. In this publication will be shown whether paldSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate alveolar bone tissue in vivo in an athymic rat model. Furthermore, using these data we have started a proof of principle clinical- and histological controlled study using stem cell-rich palatal tissues for improving the vertical alveolar bone augmentation in critical size defects. The initial results of the study demonstrate the feasibility of using stem cell-mediated tissue engineering to treat alveolar bone defects in humans. PMID:24921024

  8. Translational Research: Palatal-derived Ecto-mesenchymal Stem Cells from Human Palate: A New Hope for Alveolar Bone and Cranio-Facial Bone Reconstruction.

    PubMed

    Grimm, Wolf Dieter; Dannan, Aous; Giesenhagen, Bernd; Schau, Ingmar; Varga, Gabor; Vukovic, Mark Alexander; Sirak, Sergey Vladimirovich

    2014-05-01

    The management of facial defects has rapidly changed in the last decade. Functional and esthetic requirements have steadily increased along with the refinements of surgery. In the case of advanced atrophy or jaw defects, extensive horizontal and vertical bone augmentation is often unavoidable to enable patients to be fitted with implants. Loss of vertical alveolar bone height is the most common cause for a non primary stability of dental implants in adults. At present, there is no ideal therapeutic approach to cure loss of vertical alveolar bone height and achieve optimal pre-implantological bone regeneration before dental implant placement. Recently, it has been found that specific populations of stem cells and/or progenitor cells could be isolated from different dental resources, namely the dental follicle, the dental pulp and the periodontal ligament. Our research group has cultured palatal-derived stem cells (paldSCs) as dentospheres and further differentiated into various cells of the neuronal and osteogenic lineage, thereby demonstrating their stem cell state. In this publication will be shown whether paldSCs could be differentiated into the osteogenic lineage and, if so, whether these cells are able to regenerate alveolar bone tissue in vivo in an athymic rat model. Furthermore, using these data we have started a proof of principle clinical- and histological controlled study using stem cell-rich palatal tissues for improving the vertical alveolar bone augmentation in critical size defects. The initial results of the study demonstrate the feasibility of using stem cell-mediated tissue engineering to treat alveolar bone defects in humans. PMID:24921024

  9. Application of cultured dermal substitute for amelioration of maxillary bone growth suppression after cleft palate operation in rats.

    PubMed

    Kurokawa, Norifumi; Ueda, Koichi; Tsuji, Motomu; Kuroyanagi, Yoshimitsu

    2008-01-01

    The growth and development of maxillary bone and dentition is often impaired in patients who have undergone the cleft palate operation (push back method). Wound contraction exerts adverse effects on maxillary bone growth. The present study investigates the ability of cultured dermal substitute (CDS) to ameliorate maxillary bone growth suppression in experimental animals. We prepared CDS by incorporating rat fibroblasts into a matrix comprising a spongy hyaluronic acid (HA) sheet combined with collagen gel. The number of fibroblasts in the CDS was adjusted to 1.0 x 10(5) cells/cm(2). Wistar rats were randomly assigned to one of the following groups: I, control (no operation); II, surgically exposed bone without matrix or CDS; III, surgery with matrix; IV, surgery with CDS. Under pentobarbital anesthesia, about 2 x 4 mm of the mucosa and periosteum was surgically removed from the left half of the palate. A silicone sheet was placed on the matrix or CDS and affixed with superglue. The palatal width was measured 9 weeks later in skull preparations as the distance between the cheek side cusps of the second molar. The palates in group IV were significantly wider than those in groups II and III, and did not significantly differ from that in the control group. These findings indicated that CDS has the ability to promote wound healing and reduce scar formation through the synergic effects of fibroblasts and the matrix, and thereby to ameliorate indirectly the growth of maxillary bone. PMID:19184287

  10. Regulating apoptosis in mammalian cell cultures

    Microsoft Academic Search

    Nilou Arden; M. J. Betenbaugh

    2006-01-01

    Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian\\u000a cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor\\u000a determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay\\u000a apoptosis and increase

  11. Giant cell tumour of bone: new treatments in development.

    PubMed

    López-Pousa, A; Broto, J Martín; Garrido, T; Vázquez, J

    2015-06-01

    Giant cell tumour of bone (GCTB) is a benign osteolytic tumour with three main cellular components: multinucleated osteoclast-like giant cells, mononuclear spindle-like stromal cells (the main neoplastic components) and mononuclear cells of the monocyte/macrophage lineage. The giant cells overexpress a key mediator in osteoclastogenesis: the RANK receptor, which is stimulated in turn by the cytokine RANKL, which is secreted by the stromal cells. The RANK/RANKL interaction is predominantly responsible for the extensive bone resorption by the tumour. Historically, standard treatment was substantial surgical resection, with or without adjuvant therapy, with recurrence rates of 20-56 %. Studies with denosumab, a monoclonal antibody that specifically binds to RANKL, resulted in dramatic treatment responses, which led to its approval by the United States Food and Drugs Administration (US FDA). Recent advances in the understanding of GCTB pathogenesis are essential to develop new treatments for this locally destructive primary bone tumour. PMID:25617146

  12. Therapeutic potential of adult bone marrow-derived mesenchymal stem cells in prostate cancer bone metastasis

    PubMed Central

    Chanda, Diptiman; Isayeva, Tatyana; Kumar, Sanjay; Hensel, Jonathan A.; Sawant, Anandi; Ramaswamy, Girish; Siegal, Gene P.; Beatty, Matthew S.; Ponnazhagan, Selvarangan

    2010-01-01

    Purpose Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow-derived mesenchymal stem cells (MSC) have the ability to differentiate into osteoblast, and produce osteoprotegerin (OPG), a decoy receptor for the receptor activator for nuclear factor ? B ligand (RANKL), naturally. The present study examined the potential of unmodified MSC to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. Experimental design The human prostate cancer cell line PC3 was implanted in tibiae of SCID mice. After establishment of the tumor, either unmodified or genetically-engineered MSC overexpressing OPG was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro-CT, immunohistochemistry and histomorphometry. Results Data indicated significant (P<0.001) inhibition of tumor growth and restoration of bone in mice treated with both unmodified and modified MSC. Detailed analysis suggested that the donor MSC inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis. Conclusions Overcoming the limitation of the number of MSC available in the bone can provide significant amelioration for osteolytic damage without further modification. PMID:19920103

  13. Inhibition of hepatic stellate cells by bone marrow-derived mesenchymal stem cells in hepatic fibrosis

    PubMed Central

    Jang, Yoon Ok; Jun, Baek Gyu; Kim, Moon Young; Kwon, Sang Ok

    2015-01-01

    Background/Aims Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease. BM-MSCs have been investigated in regenerative medicine due to their ability to secrete various growth factors and cytokines that regress hepatic fibrosis and enhance hepatocyte functionality. The aim of this study was to determine the antifibrosis effect of BM-MSCs on activated hepatic stellate cells (HSCs) and the mechanism underlying how BM-MSCs modulate the function of activated HSCs. Methods We used HSCs in both direct and indirect co-culture systems with BM-MSCs to evaluate the antifibrosis effect of BM-MSCs. The cell viability and apoptosis were evaluated by a direct co-culture system of activated HSCs with BM-MSCs. The activations of both HSCs alone and HSCs with BM-MSCs in the direct co-culture system were observed by immunocytochemistry for alpha-smooth muscle actin (?-SMA). The levels of growth factors and cytokines were evaluated by an indirect co-culture system of activated HSCs with BM-MSCs. Results The BM-MSCs in the direct co-culture system significantly decreased the production of ?-SMA and the viability of activated HSCs, whereas they induced the apoptosis of activated HSCs. The BM-MSCs in the indirect co-culture system decreased the production of transforming growth factor-?1 and interleukin (IL)-6, whereas they increased the production of hepatocyte growth factor and IL-10. These results confirmed that the juxtacrine and paracrine effects of BM-MSCs can inhibit the proliferative, fibrogenic function of activated HSCs and have the potential to reverse the fibrotic process by inhibiting the production of ?-SMA and inducing the apoptosis of HSCs. Conclusions These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.

  14. The use of rat, rabbit or human bone marrow derived cells for cytocompatibility evaluation of metallic elements.

    PubMed

    Tomás, H; Carvalho, G S; Fernandes, M H; Freire, A P; Abrantes, L M

    1997-04-01

    Rat, rabbit and human bone marrow cells were cultured according to the method previously reported for cells of rat origin [1] and were exposed, or not (control), to corrosion products of a Co-Cr orthopaedic alloy as well as to metal salts containing Co2+, Cr3+ and Cr6+. Cells were cultured for 21 days and analysed for the following biochemical parameters: intracellular MTT reduction (i.e. cell viability/proliferation), alkaline phosphatase (ALP) activity and protein production. Morphological observations included both histochemistry (detection of ALP-positive cells, calcium and phosphate deposits) and scanning electron microscopy (SEM). Control cultures of rat and rabbit cells showed higher proliferation rates than human cells at the start of culture, but they all reached similar values on day 21. Protein production was parallel to cell proliferation. In contrast, ALP activity of rat cultures was much stronger than rabbit or human cultures. All cell types were able to develop the osteogenic phenotype in vitro.Co-Cr extract caused inhibitory effects on cell viability, on ALP activity and, to a lower extent, on protein production of all rat, rabbit and human cell cultures. Compared to rat and rabbit cultures, human cultures were the most sensitive to metal ions exposure. PMID:15348764

  15. Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

    Microsoft Academic Search

    Donald P. Lennon; Stephen E. Haynesworth; Scott P. Bruder; Neelam Jaiswal; Arnold I. Caplan

    1996-01-01

    Summary  An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity\\u000a to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as\\u000a mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented

  16. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone

    E-print Network

    Lu, Helen H.

    April 2001 Abstract The use of biodegradable polymers in the field of orthopaedic surgery has gained the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable behavior and interaction at the bone implant site [14,34]. Furthermore, there are no risks of disease

  17. Tricetin, a dietary flavonoid, suppresses benzo(a)pyrene?induced human non?small cell lung cancer bone metastasis.

    PubMed

    Hung, Jen-Yu; Chang, Wei-An; Tsai, Ying-Ming; Hsu, Ya-Ling; Chiang, Hung-Hsing; Chou, Shah-Hwa; Huang, Ming-Shyan; Kuo, Po-Lin

    2015-05-01

    This is the first study to demonstrate that benzo(a)-pyrene (BaP) was able to enhance the production of parathyroid hormone?related protein (PTHrP) by human non?small cell lung cancer H460 cells. Such effect would further contribute to bone metastasis of lung cancer by increasing osteoclastogenesis. This study is also the first to reveal that tricetin (TCN), a flavonoid derivative found in Myrtaceae pollen and Eucalyptus honey, was able to reverse BaP?mediated bone resorption activity of lung cancer cells. Human non?small cell lung cancer H460 cells were treated with BaP to generate conditioned medium. When osteoblasts were cultured with BaP?H460?CM, their expression of osteoclastogenesis activator macrophage colony?stimulating factor (M?CSF) and receptor activator of nuclear factor ?B ligand (RANKL) was increased. BaP?H460?CM reduced the production of osteoprotegerin (OPG), an osteoclastogenesis inhibitor, in osteoblasts. Osteoclastogenesis and bone resorption activity of H460 cells were increased by BaP?H460?CM. With BaP?mediated PTHrP upregulation, IL?8 secretion in H460 cells was increased contributing to human non?small cell lung cancer?mediated osteoclast differentiation and bone resorption. Moreover, TCN suppressed BaP?mediated bone resorption. Therefore, TCN may be a novel agent for treatment of non?small cell lung cancer patients with bone metastasis. PMID:25738754

  18. Silk Protein Sericin Improves Mammalian Cell Culture

    Microsoft Academic Search

    Satoshi Terada; Naoki Takada; Kazuaki Itoh; Takuya Saitoh; Masahiro Sasaki; Hideyuki Yamada

    Mammalian cell cultures generally require supplementation with fetal bovine serum (FBS), or its replacement, into the culture\\u000a media. Sera contain various unidentified and unknown factors and the risk of infections, including bovine spongiform encephalopathy\\u000a (BSE), is of serious concern. Therefore, the supplementation of sera into culture media is a major obstacle for purification\\u000a to recover cell products and this limits

  19. Osteogenic and osteoclastogenic differentiation of co-cultured cells in polylactic acid-nanohydroxyapatite fiber scaffolds.

    PubMed

    Morelli, Sabrina; Salerno, Simona; Holopainen, Jani; Ritala, Mikko; Bartolo, Loredana De

    2015-06-20

    The design of bone substitutes involves the creation of a microenvironment supporting molecular cross-talk between cells and scaffolds during tissue formation and remodelling. Bone remodelling process includes the cooperation of bone-building cells and bone-resorbing cells. In this paper we developed polylactic acid (PLA) and composite PLA-nanohydroxyapatite (nHA) scaffolds with 20 and 50wt.% of nHA by electrospinning technique to be used in bone tissue engineering. The developed scaffolds have different fiber diameter, porosity with interconnected pores and mechanical properties. Taking cues from the bone environment features we investigated the differentiation of human mesenchymal stem cells (hMSCs) from bone marrow in osteoblasts and the osteoclastogenesis in the developed scaffolds in homotypic and in co-culture up to 46 days. PLA and composite PLA-nHA scaffolds induced osteogenic and osteoclastogenic differentiation. Both osteoblasts and osteoclasts displayed high expression of specific markers (osteopontin, osteocalcin, RANK, RANKL) and functions such as secretion of ALP, cathepsin K and TRAP activity on composite scaffolds especially on PLA-nHA containing 20wt.% of nHA. The heterotypic interactions between osteoblasts and osteoclasts co-cultured in the developed scaffolds triggered their functional differentiation and activation. PMID:25858154

  20. Mutagenesis of haploid cultured frog cells

    SciTech Connect

    Mezger-Freed, L.

    1973-01-01

    From 13th international congress of genetics; Berkeley, California (20 Aug 1973). Haploid cells afford an opportunity to test some of the assumptions from bacterial genetics which have been adopted by somatic cell geneticists. Haploid cultured cell lines derived from the grass frog Rana pipiens were compared to diploid cell lines in order to test a model which predicts that recessive mutations will be expressed in diploid cells with a frequency equal to the square of that in haploid cells. Haploid and diploid monolayer cultures were compared for (1) survival after exposure to compounds known to be mutagenic for bacteria (a measure of the frequency with which lethal mutations are expressed), and (2) the induction of drug-resistant variants (putative mutants) by such compounds. The proportion of cells that survived from diploid cultures was no more than ten times that from haploid cultures, a much smaller difference than predicted. Furthermore, the frequency of drug-resistant variants was independent of ploidy. (auth)

  1. Atypical multinucleated cells form in long-term marrow cultures from patients with Paget's disease.

    PubMed Central

    Kukita, A; Chenu, C; McManus, L M; Mundy, G R; Roodman, G D

    1990-01-01

    Although Paget's disease is the most flagrant example of a primary osteoclast disorder, little is known of osteoclast biology in this disease. In this report we have studied the formation of cells with the osteoclast phenotype in long-term cultures of marrow mononuclear cells derived from patients with Paget's disease, and compared these with similar cells formed in long-term marrow cultures from normal individuals, and with osteoclasts present in pagetic bone. Osteoclasts formed in pagetic marrow cultures resembled osteoclasts present in pagetic bone, but were distinctly different from osteoclasts formed in normal marrow cultures. Osteoclast formation was 10-20-fold greater in pagetic marrow cultures than in normal cultures. The multinucleated cells formed in cultures of pagetic marrow were much larger in size, were hyperresponsive to 1,25(OH)2 vitamin D, had more nuclei per cell, had increased levels of tartrate-resistant acid phosphatase activity and had ultrastructural features which were not seen in multinucleated cells formed from normal marrow mononuclear cells. These pagetic marrow-derived multinucleated cells formed large resorption lacunae on calcified matrices and cross-reacted with monoclonal antibodies which preferentially bind to osteoclasts. The multinucleated cells formed from marrow obtained from uninvolved sites in Paget's patients also displayed these abnormal features. Images PMID:2318982

  2. In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells

    PubMed Central

    2011-01-01

    Background Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. Methods MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. Results After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of ?-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. Conclusions The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of ?-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering. PMID:21281488

  3. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  4. Tumor-host cell interactions in the bone disease of myeloma

    PubMed Central

    Fowler, Jessica A.; Edwards, Claire M.; Croucher, Peter I.

    2010-01-01

    Multiple myeloma is a hematological malignancy that is associated with the development of a destructive osteolytic bone disease, which is a major cause of morbidity for patients with myeloma. Interactions between myeloma cells and cells of the bone marrow microenvironment promote both tumor growth and survival and bone destruction, and the osteolytic bone disease is now recognized as a contributing component to tumor progression. Since myeloma bone disease is associated with both an increase in osteoclastic bone resorption and a suppression of osteoblastic bone formation, research to date has largely focused upon the role of the osteoclast and osteoblast. However, it is now clear that other cell types within the bone marrow, including cells of the immune system, mesenchymal stem cells and bone marrow stromal cells, can contribute to the development of myeloma bone disease. This review discusses the cellular mechanisms and potential therapeutic targets that have been implicated in myeloma bone disease. PMID:20615487

  5. Cell-free and cell-based approaches for bone regeneration

    Microsoft Academic Search

    Ericka M. Bueno; Julie Glowacki

    2009-01-01

    The clinical augmentation of bone currently involves the use of autogenous or allogeneic bone grafts and synthetic materials, all of which are associated with limitations. Research on the safe enhancement of bone formation concerns the potential value of scaffolds, stem cells, gene therapy, and chemical and mechanical signals. Optimal scaffolds are engineered to provide mechanical stability while supporting osteogenesis, osteoconduction

  6. Isolation of mesenchymal stem cells from human placenta: Comparison with human bone marrow mesenchymal stem cells

    Microsoft Academic Search

    Zongning Miao; Jun Jin; Lei Chen; Jianzhong Zhu; Wei Huang; Jidong Zhao; Hanguang Qian; Xueguang Zhang

    2006-01-01

    The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid

  7. [RNA synthesis in bone marrow hematopoietic stem cells].

    PubMed

    Domaratskaia, E I; Polikarpova, S I; Khrushchov, N G

    1978-01-01

    The effect of inhibitors of DNA-dependent RNA synthesis (sibiromycin and actinomycin D) and radio toxic doses of 3H-uridine on the bone marrow stem hemopoietic cells in adult mice was studied by means of spleen colonies. The short-term in vitro preincubation of the bone marrow cell suspension with the antibiotics and 3H-uridine resulted in the practically complete inhibition of colony formation. The bone marrow stem hemopoietic cells incorporated 3H-uridine intensively. In the normal adult animals the number of stem cells with the high level of RNA synthesis attained 40% whereas the rest 60% of cells were capable to activate the ribonucleic metabolism upon their in vitro preincubation. PMID:581403

  8. ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

    SciTech Connect

    Rajalin, Ann-Marie; Pollock, Hanna [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland)] [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland); Aarnisalo, Piia, E-mail: piia.aarnisalo@helsinki.fi [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland) [Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki (Finland); Department of Clinical Chemistry, University of Helsinki and Helsinki University Central Hospital (Finland)

    2010-05-28

    The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

  9. Development of an In Vitro Cell System from Zebrafish Suitable to Study Bone Cell Differentiation and Extracellular Matrix Mineralization

    PubMed Central

    Vijayakumar, Parameswaran; Laizé, Vincent; Cardeira, João; Trindade, Marlene

    2013-01-01

    Abstract Mechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28°C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization. PMID:23909483

  10. Emulsions Containing Perfluorocarbon Support Cell Cultures

    NASA Technical Reports Server (NTRS)

    Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

    1990-01-01

    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

  11. Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.

    PubMed

    Gelain, Fabrizio; Bottai, Daniele; Vescovi, Angleo; Zhang, Shuguang

    2006-01-01

    Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with beta-Tubulin(+), GFAP(+) and Nestin(+) markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology. PMID:17205123

  12. Engineered bone culture in a perfusion bioreactor: a 2D computational study of stationary mass and momentum transport.

    PubMed

    Pierre, J; Oddou, C

    2007-12-01

    Successful bone cell culture in large implants still is a challenge to biologists and requires a strict control of the physicochemical and mechanical environments. This study analyses from the transport phenomena viewpoint the limiting factors of a perfusion bioreactor for bone cell culture within fibrous and porous large implants (2.5 cm in length, a few cubic centimetres in volume, 250 microm in fibre diameter with approximately 60% porosity). A two-dimensional mathematical model, based upon stationary mass and momentum transport in these implants is proposed and numerically solved. Cell oxygen consumption, in accordance theoretically with the Michaelis-Menten law, generates non linearity in the boundary conditions of the convection diffusion equation. Numerical solutions are obtained with a commercial code (Femlab 3.1; Comsol AB, Stockholm, Sweden). Moreover, based on the simplification of transport equations, a simple formula is given for estimating the length of the oxygen penetration within the implant. Results show that within a few hours of culture process and for a perfusion velocity of the order of 10(-4) m s(-1), the local oxygen concentration is everywhere sufficiently high to ensure a suitable cell metabolism. But shear stresses induced by the fluid flow with such a perfusion velocity are found to be locally too large (higher than 10(-3) Pa). Suitable shear stresses are obtained by decreasing the velocity at the inlet to around 2 x 10(-5) m s(-1). But consequently hypoxic regions (low oxygen concentrations) appear at the downstream part of the implant. Thus, it is suggested here that in the determination of the perfusion flow rate within a large implant, a compromise between oxygen supply and shear stress effects must be found in order to obtain a successful cell culture. PMID:17852175

  13. Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics.

    PubMed

    Tang, Zhurong; Wang, Zhe; Qing, Fangzhu; Ni, Yilu; Fan, Yujiang; Tan, Yanfei; Zhang, Xingdong

    2015-03-01

    Porous calcium phosphate ceramics (CaP ceramics) could induce ectopic bone formation which was regulated by various signal molecules. In this work, bone marrow mesenchymal stem cells (MSCs) were cultured on the surface of osteoinductive hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramics in comparison with control (culture plate) for up to 14 days to detect the signal molecules which might be affected by the CaP ceramics. Without adding osteogenic factors, MSCs cultured on HA and BCP both expressed higher Runx2, Osterix, collagen type I, osteopontin, bone sialoprotein, and osteocalcin at various stages compared with control, thus confirmed the osteoblastic differentiation of MSCs. Later study demonstrated the messenger RNA level of bone morphogenetic protein 2 (BMP2) and BMP4 were also significantly enhanced by HA and BCP. Furthermore, Smad1, 4, 5, and Dlx5, the main molecules in the BMP/Smads signaling pathway, were upregulated by HA and BCP. Moreover, the higher expression of Smads and BMP2, 4 in BCP over HA, corresponded to the better performance of BCP in stimulating in vitro osteoblastic differentiation of MSCs. This was in accordance with the better osteoinductivity of BCP over HA in vivo. Altogether, these results implied that the CaP ceramics may initiate the osteoblastic differentiation of MSCs by influencing the expression of molecules in BMP/Smads pathway. PMID:24889783

  14. Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins.

    PubMed

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Haithcock, David A; Cundiff, Caitlin A; Schwartz, Zvi; Boyan, Barbara D

    2015-04-01

    Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4 ?m); sandblasted/acid-etched Ti (SLA, Ra=3.2 ?m); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment. PMID:25554602

  15. Multinucleated giant cells from fibroblast cultures

    PubMed Central

    Holt, DJ; Grainger, DW

    2011-01-01

    Many multinucleated giant cells are well-known to form from macrophage origin. Those formed from other cell types are less described, but may be as prevalent in pathological tissue. Giant multinucleated cells derived from secondary and primary fibroblast sources in various cultures with similar characteristics to foreign body giant cells are reported. Secondary-transformed NIH 3T3 fibroblasts rapidly fuse within 24 hours in contact co-cultures with RAW 264.7 immortalized macrophages, while 3T3 mono-cultures, non-contact (transwell) co-cultures, and macrophage-conditioned media-treated 3T3 mono-cultures all do not fuse. Primary-derived murine fibroblasts also form multinucleated cells, both in the presence or absence of co-cultured macrophages that increase during long-term culture (5–30 days). In contrast to 3T3 fusion, this primary cell phenomenon is not due to fibroblast fusion, but rather to nuclear division without cytokinesis. That these multinucleated fibroblasts can originate via different mechanisms may influence and distinguish their behaviors in conditions under which they may arise, including various in vitro culture assays, and in certain fibroblastic pathologies such as the foreign body response, fibrosis, cancer and aged tissue. PMID:21397323

  16. Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

    PubMed Central

    Renzulli, Joseph F.; Del Tatto, Michael; Dooner, Gerri; Aliotta, Jason; Goldstein, Lisa; Dooner, Mark; Colvin, Gerald; Chatterjee, Devasis; Quesenberry, Peter

    2015-01-01

    Purpose Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. Materials and Methods We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 ?M polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. Results Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. Conclusions Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles. PMID:20850816

  17. Characteristics and response of mouse bone marrow derived novel low adherent mesenchymal stem cells acquired by quantification of extracellular matrix

    PubMed Central

    Zheng, Ri-Cheng; Heo, Seong-Joo; Koak, Jai-Young; Lee, Joo-Hee; Park, Ji-Man

    2014-01-01

    PURPOSE The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM). MATERIALS AND METHODS Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation. RESULTS The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time. CONCLUSION Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic. PMID:25352957

  18. HUMAN BONE MARROW MESENCHYMAL STEM CELLS : A SYSTEMATIC REAPPRAISAL VIA THE GENOSTEM EXPERIENCE

    E-print Network

    Paris-Sud XI, Université de

    1 HUMAN BONE MARROW MESENCHYMAL STEM CELLS : A SYSTEMATIC REAPPRAISAL VIA THE GENOSTEM EXPERIENCE 1 Eloi- INM, Montpellier, France Key words : differentiation ; stem cell ; bone ; cartilage ; tendon Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From

  19. Quantitative image analysis of cell colocalization in murine bone marrow.

    PubMed

    Mokhtari, Zeinab; Mech, Franziska; Zehentmeier, Sandra; Hauser, Anja E; Figge, Marc Thilo

    2015-06-01

    Long-term antibody production is a key property of humoral immunity and is accomplished by long-lived plasma cells. They mainly reside in the bone marrow, whose importance as an organ hosting immunological memory is becoming increasingly evident. Signals provided by stromal cells and eosinophils may play an important role for plasma cell maintenance, constituting a survival microenvironment. In this joint study of experiment and theory, we investigated the spatial colocalization of plasma cells, eosinophils and B cells by applying an image-based systems biology approach. To this end, we generated confocal fluorescence microscopy images of histological sections from murine bone marrow that were subsequently analyzed in an automated fashion. This quantitative analysis was combined with computer simulations of the experimental system for hypothesis testing. In particular, we tested the observed spatial colocalization of cells in the bone marrow against the hypothesis that cells are found within available areas at positions that were drawn from a uniform random number distribution. We find that B cells and plasma cells highly colocalize with stromal cells, to an extent larger than in the simulated random situation. While B cells are preferentially in contact with each other, i.e., form clusters among themselves, plasma cells seem to be solitary or organized in aggregates, i.e., loosely defined groups of cells that are not necessarily in direct contact. Our data suggest that the plasma cell bone marrow survival niche facilitates colocalization of plasma cells with stromal cells and eosinophils, respectively, promoting plasma cell longevity. © 2015 International Society for Advancement of Cytometry. PMID:25652548

  20. Constructing a High Density Cell Culture System

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor)

    1996-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  1. PTH Regulates the Hematopoietic Stem Cell Niche in Bone

    Microsoft Academic Search

    Henry M. Kronenberg

    \\u000a In late fetal life, hematopoiesis moves from the liver to the bone marrow in mammals, and continues to occur throughout postnatal\\u000a life, with extramedullary hematopoiesis occurring only when disease blocks hematopoiesis in the marrow. Hematopoietic stem\\u000a cells (HSCs) are uniquely the cells that can generate cells of all hematopoietic lineages. These cells do so by balancing\\u000a an ability to increase

  2. Bone-marrow-derived stem cells — our key to longevity?

    Microsoft Academic Search

    Mariusz Z. Ratajczak; Ewa K. Zuba-Surma; Bogus?aw Machalinski; Magdalena Kucia

    2007-01-01

    Bone marrow (BM) was for many years primarily regarded as the source of hematopoietic stem cells. In this review we discuss\\u000a current views of the BM stem cell compartment and present data showing that BM contains not only hematopoietic but also heterogeneous\\u000a non-hematopoietic stem cells. It is likely that similar or overlapping populations of primitive non-hematopoietic stem cells\\u000a in BM

  3. Biochemical and histological studies on various bone cell preparations

    Microsoft Academic Search

    P. J. Nijweide; A. van der Plas; J. P. Scherft

    1981-01-01

    Summary  Four different cell populations—designated PF, OB, OC, and PC—were isolated from calvaria of 18-day-old chick embryos for\\u000a analysis of the effects of hormones on bone tissue. The cell populations were studied with histological and biochemical methods.\\u000a Apart from the well-known cell types present in calvaria, a new cell type was found in the noncalcified organic matrix between\\u000a the osteoblastic layer

  4. Potentiation of osteoclastogenesis by adipogenic conversion of bone marrow-derived mesenchymal stem cells.

    PubMed

    Mori, Keisuke; Suzuki, Keiji; Hozumi, Akira; Goto, Hisataka; Tomita, Masato; Koseki, Hironobu; Yamashita, Shunichi; Osaki, Makoto

    2014-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are the indispensable component of the bone marrow, being the common precursors for adipocytes and osteoblasts. We show here that adipogenic differentiation resulted in increase in the production of adipocyte markers, such as adiponectin,fatty-acid binding proteins (FABP4), peroxisome proliferator-activated receptor ? (PPAR?), as well as the receptor activator of nuclear-?B ligand (RANKL). Co-culture of osteoclast precursors (OCPs) with BMSCs-derived adipocytes significantly enhanced osteoclast differentiation with low-dose RANKL, whose levels alone could not promote osteoclastogenesis. These results demonstrate for the first time that adipogenic differentiation of BMSCs plays a pivotal role in maintaining bone homeostasis. PMID:24759183

  5. Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

    PubMed Central

    Desplat, V; Dupuis, F; Trimoreau, F; Dulery, C; Praloran, V; Denizot, Y

    1998-01-01

    The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures. PMID:9839696

  6. Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair

    PubMed Central

    Tamburstuen, Margareth V.; Reseland, Janne E.; Spahr, Axel; Brookes, Steven J.; Kvalheim, Gunnar; Slaby, Ivan; Snead, Malcolm L.; Lyngstadaas, S. Petter

    2015-01-01

    Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remain unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a “proof of concept” animal study. The real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role of this protein in early bone formation and repair. PMID:20854943

  7. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

    PubMed Central

    Handschel, Jörg; Berr, Karin; Depprich, Rita A; Kübler, Norbert R; Naujoks, Christian; Wiesmann, Hans-Peter; Ommerborn, Michelle A; Meyer, Ulrich

    2008-01-01

    Background Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation. Methods Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and ?-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR. Results ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17. Conclusion Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering. PMID:18544155

  8. Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

    PubMed

    Song, Ningxia; Gao, Lei; Qiu, Huiying; Huang, Chongmei; Cheng, Hui; Zhou, Hong; Lv, Shuqing; Chen, Li; Wang, Jianmin

    2015-07-01

    The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft?versus?host disease (aGVHD). However, the role of MSCs in graft?versus?leukemia remains to be determined. In the present study, we co?cultured C57BL/6 mouse bone marrow (BM)?derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit?8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)?10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies. PMID:25901937

  9. Signal transduction pathways involved in mechanotransduction in bone cells

    Microsoft Academic Search

    Astrid. Liedert; Daniela Kaspar; Robert Blakytny; Lutz Claes; Anita Ignatius

    2006-01-01

    Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins,

  10. Interactions between MSCs and Immune Cells: Implications for Bone Healing

    PubMed Central

    Kovach, Tracy K.; Dighe, Abhijit S.; Lobo, Peter I.; Cui, Quanjun

    2015-01-01

    It is estimated that, of the 7.9 million fractures sustained in the United States each year, 5% to 20% result in delayed or impaired healing requiring therapeutic intervention. Following fracture injury, there is an initial inflammatory response that plays a crucial role in bone healing; however, prolonged inflammation is inhibitory for fracture repair. The precise spatial and temporal impact of immune cells and their cytokines on fracture healing remains obscure. Some cytokines are reported to be proosteogenic while others inhibit bone healing. Cell-based therapy utilizing mesenchymal stromal cells (MSCs) is an attractive option for augmenting the fracture repair process. Osteoprogenitor MSCs not only differentiate into bone, but they also exert modulatory effects on immune cells via a variety of mechanisms. In this paper, we review the current literature on both in vitro and in vivo studies on the role of the immune system in fracture repair, the use of MSCs in the enhancement of fracture healing, and interactions between MSCs and immune cells. Insight into this paradigm can provide valuable clues in identifying cellular and noncellular targets that can potentially be modulated to enhance both natural bone healing and bone repair augmented by the exogenous addition of MSCs. PMID:26000315

  11. Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2.

    PubMed

    van Gastel, Nick; Stegen, Steve; Stockmans, Ingrid; Moermans, Karen; Schrooten, Jan; Graf, Daniel; Luyten, Frank P; Carmeliet, Geert

    2014-09-01

    The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects. PMID:24989687

  12. Aminoacid transport into cultured tobacco cells

    Microsoft Academic Search

    S. L. Berry; H. M. Harrington; R. L. Bernsteint; R. R. Henkel

    1981-01-01

    Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca2+ for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not

  13. Bioprocessing technology for plant cell suspension cultures

    Microsoft Academic Search

    Wei wen Su

    1995-01-01

    Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production\\u000a of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several\\u000a largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art\\u000a in bioprocessing technologies pertinent

  14. Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells.

    PubMed

    Shetty, P; Bharucha, K; Tanavde, V

    2007-03-01

    The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC. PMID:17208468

  15. Caffeine regulates osteogenic differentiation and mineralization of primary adipose-derived stem cells and a bone marrow stromal cell line.

    PubMed

    Su, Shu-Jem; Chang, Kee-Lung; Su, Shu-Hui; Yeh, Yao-Tsung; Shyu, Huey-Wen; Chen, Kuan-Ming

    2013-06-01

    Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses. PMID:23301724

  16. Neural differentiation potential of peripheral blood- and bone-marrow-derived precursor cells

    PubMed Central

    Kim, Sangnyon; Honmou, Osamu; Kato, Kazunori; Nonaka, Tadashi; Houkin, Kiyohiro; Hamada, Hirufumi; Kocsis, Jeffery D.

    2008-01-01

    Transplantation of mesenchymal stem cells (MSCs) prepared from adult bone marrow (BMSCs) has been reported to ameliorate functional deficits in several CNS diseases in experimental animal models. Bone marrow was enriched in MSCs by selecting for plastic-adherent cells that were grown to confluency in appropriate culture conditions as flattened fibroblast-like cells. Despite the fact that the stem/precursor cells in peripheral blood are widely used for reconstruction in the hematopoietic system, it is not fully understood whether peripheral blood-derived plastic-adherent precursor/stem cells (PMSCs) can differentiate into a neural lineage. To compare the potential of PMSCs and BMSCs for neural differentiation in vitro, BMSCs and PMSCs were prepared from the adult rat and expanded in culture. Although the growth rate of PMSCs was less than BMSCs, immunocytochemical and RT-PCR analyses indicated that both MSC types were successfully induced to nestin-positive neurospheres in the presence of EGF and bFGF. After withdrawal of the mitogens, these cells could differentiate into neurofilament-positive neurons or GFAP-positive glia. Thus, our findings suggest the potential use of PMSCs for a cell therapy in CNS diseases. PMID:17064670

  17. Cryopreservation of Taxus chinensis suspension cell cultures.

    PubMed

    Kim, S I; Choi, H K; Son, J S; Yun, J H; Jang, M S; Kim, H R; Song, J Y; Kim, J H; Choi, H J; Hong, S S

    2001-01-01

    A simple cryopreservation method for suspension cells of Taxus chinensis was established. In this procedure 7 days old suspension cells were used without any pre-culture treatment. At first, cells were incubated in cryoprotectant solution (0.5M DMSO and 0.5M glycerol) on ice for 30 min and then frozen at a cooling rate of 1 degree C/min to -40 degrees C prior to immersion in liquid nitrogen. The average viability of frozen-thawed cells was between 30 to 40%. The recovery of cryopreserved cells in liquid nitrogen for 1 month was accomplished. After rapid thawing, cells were transferred to solid medium and cultivated for 4-6 weeks. The treatment of trehalose as a cryoprotectant enhanced re-growth of frozen-thawed cells. The stable maintenance of paclitaxel biosynthetic ability in cryopreserved cells was confirmed by comparing with that of regularly sub-cultured suspension cells. PMID:11788843

  18. "In-bone" utricle cultures -A simplified, atraumatic technique for in situ cultures of the adult mouse (Mus musculus) utricle.

    E-print Network

    Rubel, Edwin

    . INTRODUCTION Hair cell death and protection are frequently studied with in vitro preparations. In neonatal mice cells and studying hair cell death and protection. Background: The current in vitro technique and atraumatic method for culturing mature mouse utricles and studying hair cell death and protection

  19. Tissue Engineering Bone Using Autologous Progenitor Cells in the Peritoneum

    PubMed Central

    Shen, Jinhui; Nair, Ashwin; Saxena, Ramesh; Zhang, Cheng Cheng; Borrelli, Joseph; Tang, Liping

    2014-01-01

    Despite intensive research efforts, there remains a need for novel methods to improve the ossification of scaffolds for bone tissue engineering. Based on a common phenomenon and known pathological conditions of peritoneal membrane ossification following peritoneal dialysis, we have explored the possibility of regenerating ossified tissue in the peritoneum. Interestingly, in addition to inflammatory cells, we discovered a large number of multipotent mesenchymal stem cells (MSCs) in the peritoneal lavage fluid from mice with peritoneal catheter implants. The osteogenic potential of these peritoneal progenitor cells was demonstrated by their ability to easily infiltrate decalcified bone implants, produce osteocalcin and form mineralized bone in 8 weeks. Additionally, when poly(l-lactic acid) scaffolds loaded with bone morphogenetic protein-2 (a known osteogenic differentiation agent) were implanted into the peritoneum, signs of osteogenesis were seen within 8 weeks of implantation. The results of this investigation support the concept that scaffolds containing BMP-2 can stimulate the formation of bone in the peritoneum via directed autologous stem and progenitor cell responses. PMID:24681529

  20. Oscillatory behavior of cells in tissue culture.

    NASA Astrophysics Data System (ADS)

    Giaever, Ivar; Linton, Michael F. A.; Keese, Charles R.

    1998-03-01

    Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a small electrode deposited at the bottom of a tissue culture well and immersed in ordinary culture medium. By measuring the changes in the impedance of the electrode as a function of time, many important properties of the cells on the electrode can be inferred, such as motion, morphology changes and membrane capacitance. The impedance oscillations of MDCK cells were observed with highly confluent cell layers, where the approximately 100 cells on the electrode acted in unison. The communication between cells can be demonstrated directly by a variation of the ECIS concept, where cells are cultured on two closely spaced electrodes. The impedance fluctuations are measured independently on each electrode and compared by using a cross-correlation function.

  1. Cell Culture on MEMS Platforms: A Review

    E-print Network

    Ni, Ming

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods ...

  2. Noninvasive Real-Time Monitoring by AlamarBlue® During In Vitro Culture of Three-Dimensional Tissue-Engineered Bone Constructs

    PubMed Central

    Zhou, Xiaohua; Holsbeeks, Inge; Impens, Saartje; Sonnaert, Maarten; Bloemen, Veerle; Luyten, Frank

    2013-01-01

    Bone tissue engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and nonfunctional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop noninvasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the nontoxic alamarBlue® (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was evaluated not only in static cultures, but also in dynamic cultures in a perfusion bioreactor setup. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a noninvasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence, the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct. PMID:23327780

  3. Gene-modified bone marrow cell therapy for prostate cancer.

    PubMed

    Wang, H; Thompson, T C

    2008-05-01

    There is a critical need to develop new and effective cancer therapies that target bone, the primary metastatic site for prostate cancer and other malignancies. Among the various therapeutic approaches being considered for this application, gene-modified cell-based therapies may have specific advantages. Gene-modified cell therapy uses gene transfer and cell-based technologies in a complementary fashion to chaperone appropriate gene expression cassettes to active sites of tumor growth. In this paper, we briefly review potential cell vehicles for this approach and discuss relevant gene therapy strategies for prostate cancer. We further discuss selected studies that led to the conceptual development and preclinical testing of IL-12 gene-modified bone marrow cell therapy for prostate cancer. Finally, we discuss future directions in the development of gene-modified cell therapy for metastatic prostate cancer, including the need to identify and test novel therapeutic genes such as GLIPR1. PMID:18385769

  4. Long-term culture of lymphohematopoietic stem cells.

    PubMed Central

    Palacios, R; Bucana, C; Xie, X

    1996-01-01

    Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy. Images Fig. 1 Fig. 2 PMID:8643561

  5. Culture and transfection of axolotl cells.

    PubMed

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration. PMID:25740487

  6. Focal Adhesion Kinase Regulates the Localization and Retention of Pro-B Cells in Bone Marrow Microenvironments

    PubMed Central

    Park, Shin-Young; Wolfram, Peter; Canty, Kimberly; Harley, Brendan; Nombela-Arrieta, César; Pivarnik, Gregory; Manis, John; Beggs, Hilary E; Silberstein, Leslie E

    2012-01-01

    Progenitor B cells reside in complex bone marrow microenvironments where they receive signals for growth and maturation. We reported previously that the CXCL12-FAK-VLA4 pathway plays an important role in progenitor B cell adhesion and migration. Here we have conditionally targeted in B cells Focal Adhesion Kinase (FAK), and find that the numbers of progenitor pro-B, pre-B and immature B cells are reduced by 30–40% in B cell specific FAK knockout mice. When cultured in methylcellulose with IL-7 ± CXCL12, Fak deleted pro-B cells yield significantly fewer cells and colonies. Utilizing in situ quantitative imaging cytometry, we establish that in longitudinal femoral bone marrow sections, pro-B cells are preferentially localized in close proximity to the endosteum of the metaphyses and the diaphysis. Fak deletion disrupts the non-random distribution of pro-B cells and induces the mobilization of pro-B cells to the periphery in vivo. These effects of Fak deletion on pro-B cell mobilization and localization in bone marrow are amplified under inflammatory stress, i.e. following immunization with nitrophenol-conjugated chicken ?-globulin in alum (NP-CGG-alum). Collectively, these studies suggest the importance of FAK in regulating pro-B cell homeostasis and maintenance of their spatial distribution in bone marrow niches. PMID:23264658

  7. Nerve growth factor induces development of connective tissue-type mast cells in vitro from murine bone marrow cells

    PubMed Central

    1991-01-01

    The effect of nerve growth factor (NGF) on proliferation/differentiation of mast cells was investigated in vitro. Although NGF alone neither supported colony formation of bone marrow- derived cultured mast cells (BMCMC) nor induced development of mast cell colonies from nonadherent bone marrow cells (NBMC), addition of NGF to the suboptimal dose of interleukin 3 (IL-3) significantly increased the numbers of mast cell colonies produced by BMCMC or NBMC in methylcellulose. When stimulated by IL-3 alone, cells in mast cell colonies were not stained by berberine sulfate, a fluorescent dye. In contrast, mast cells developing in methylcellulose cultures obtaining both IL-3 and NGF were stained by berberine sulfate. The fluorescence was abolished by the treatment of heparinase but not of chondroitinase ABC, suggesting that mast cells stimulated by IL-3 and NGF produced and stored heparin proteoglycan. The histamine content of BMCMC maintained by IL-3 was also increased by addition of NGF. Since BMCMC showed mucosal mast cell-like phenotype, NGF appeared to induce the phenotypic change to connective tissue-type mast cells (CTMC). In the culture containing BMCMC, 3T3 fibroblasts, and IL-3, the phenotypic change of BMCMC to CTMC was observed as well. Since NGF was detected in this coculture and since addition of anti-NGF monoclonal antibody suppressed the phenotypic change, NGF produced by fibroblasts appeared to induce the phenotypic change. Neither BMCMC alone nor IL-3 alone increased the concentration of NGF. Therefore, there is a possibility that BMCMC stimulated by IL-3 may induce the production and/or release of NGF by fibroblasts. PMID:1711569

  8. Immune Plasticity of Bone Marrow-Derived Mesenchymal Stromal Cells

    Microsoft Academic Search

    J. Stagg; J. Galipeau

    Isolated from simple bone marrow aspirates, mesenchymal stromal cells (MSCs) can be easily expanded ex vivo and differentiated\\u000a into various cell lineages. Because they are present in humans of all ages, are harvested in the absence of prior mobilization\\u000a and preserve their plasticity following gene modification, MSCs are particularly attractive for cell-based medicine. One of\\u000a the most fascinating properties of

  9. Gap Junctions and Biophysical Regulation of Bone Cells

    PubMed Central

    Lloyd, Shane A. J.

    2013-01-01

    Communication between osteoblasts, osteoclasts, and osteocytes is integral to their ability to build and maintain the skeletal system and respond to physical signals. Various physiological mechanisms, including nerve communication, hormones, and cytokines, play an important role in this process. More recently, the important role of direct, cell–cell communication via gap junctions has been established. In this review, we demonstrate the integral role of gap junctional intercellular communication (GJIC) in skeletal physiology and bone cell mechanosensing. PMID:23762015

  10. Shifts in bone marrow cell phenotypes caused by spaceflight

    PubMed Central

    Ortega, M. Teresa; Pecaut, Michael J.; Gridley, Daila S.; Stodieck, Louis S.; Ferguson, Virginia; Chapes, Stephen K.

    2009-01-01

    Bone marrow cells were isolated from the humeri of C57BL/6 mice after a 13-day flight on the space shuttle Space Transportation System (STS)-118 to determine how spaceflight affects differentiation of cells in the granulocytic lineage. We used flow cytometry to assess the expression of molecules that define the maturation/activation state of cells in the granulocytic lineage on three bone marrow cell subpopulations. These molecules included Ly6C, CD11b, CD31 (platelet endothelial cell adhesion molecule-1), Ly6G (Gr-1), F4/80, CD44, and c-Fos. The three subpopulations were small agranular cells [region (R)1], larger granular cells (R2), which were mostly neutrophils, and very large, very granular cells (R3), which had properties of macrophages. Although there were no composite phenotypic differences between total bone marrow cells isolated from spaceflight and ground-control mice, there were subpopulation differences in Ly6C (R1 and R3), CD11b (R2), CD31 (R1, R2, and R3), Ly6G (R3), F4/80 (R3), CD44high (R3), and c-Fos (R1, R2, and R3). In particular, the elevation of CD11b in the R2 subpopulation suggests neutrophil activation in response to landing. In addition, decreases in Ly6C, c-Fos, CD44high, and Ly6G and an increase in F4/80 suggest that the cells in the bone marrow R3 subpopulation of spaceflight mice were more differentiated compared with ground-control mice. The presence of more differentiated cells may not pose an immediate risk to immune resistance. However, the reduction in less differentiated cells may forebode future consequences for macrophage production and host defenses. This is of particular importance to considerations of future long-term spaceflights. PMID:19056998

  11. Bone Engineering of Maxillary Sinus Bone Deficiencies Using Enriched CD90+ Stem Cell Therapy: A Randomized Clinical Trial.

    PubMed

    Kaigler, Darnell; Avila-Ortiz, Gustavo; Travan, Suncica; Taut, Andrei D; Padial-Molina, Miguel; Rudek, Ivan; Wang, Feng; Lanis, Alejandro; Giannobile, William V

    2015-07-01

    Bone engineering of localized craniofacial osseous defects or deficiencies by stem cell therapy offers strong prospects to improve treatment predictability for patient care. The aim of this phase 1/2 randomized, controlled clinical trial was to evaluate reconstruction of bone deficiencies of the maxillary sinus with transplantation of autologous cells enriched with CD90+ stem cells and CD14+ monocytes. Thirty human participants requiring bone augmentation of the maxillary sinus were enrolled. Patients presenting with 50% to 80% bone deficiencies of the maxillary sinus were randomized to receive either stem cells delivered onto a ?-tricalcium phosphate scaffold or scaffold alone. Four months after treatment, clinical, radiographic, and histologic analyses were performed to evaluate de novo engineered bone. At the time of alveolar bone core harvest, oral implants were installed in the engineered bone and later functionally restored with dental tooth prostheses. Radiographic analyses showed no difference in the total bone volume gained between treatment groups; however, density of the engineered bone was higher in patients receiving stem cells. Bone core biopsies showed that stem cell therapy provided the greatest benefit in the most severe deficiencies, yielding better bone quality than control patients, as evidenced by higher bone volume fraction (BVF; 0.5 versus 0.4; p?=?0.04). Assessment of the relation between degree of CD90+ stem cell enrichment and BVF showed that the higher the CD90 composition of transplanted cells, the greater the BVF of regenerated bone (r?=?0.56; p?=?0.05). Oral implants were placed and restored with functionally loaded dental restorations in all patients and no treatment-related adverse events were reported at the 1-year follow-up. These results provide evidence that cell-based therapy using enriched CD90+ stem cell populations is safe for maxillary sinus floor reconstruction and offers potential to accelerate and enhance tissue engineered bone quality in other craniofacial bone defects and deficiencies (Clinicaltrials.gov NCT00980278). © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research. PMID:25652112

  12. Arthroplasty implant biomaterial particle associated macrophages differentiate into lacunar bone resorbing cells.

    PubMed Central

    Pandey, R; Quinn, J; Joyner, C; Murray, D W; Triffitt, J T; Athanasou, N A

    1996-01-01

    OBJECTIVE: To study the pathogenesis of aseptic loosening: in particular, to determine whether macrophages responding to particles of biomaterials commonly used in arthroplasty surgery for arthritis are capable of differentiating into osteoclastic bone resorbing cells, and the cellular and hormonal conditions required for this to occur. METHODS: Biomaterial particles (polymethylmethacrylate, high density polyethylene, titanium, chromium-cobalt, stainless steel) were implanted subcutaneously into mice. Macrophages were isolated from the foreign body granulomas that resulted, cultured on bone slices and coverslips, and assessed for both cytochemical and functional evidence of osteoclast differentiation. RESULTS: Tartrate resistant acid phosphatase (TRAP) negative macrophages isolated from granulomas containing particles of all types of biomaterial composition were capable of differentiating into TRAP positive cells capable of extensive lacunar bone resorption (assessed by scanning electron microscopy). The presence of both UMR106 rat osteoblast-like cells and 1,25-dihydroxy vitamin D3 was necessary for this to occur. CONCLUSION: All implant materials produce wear particles that are the focus of a heavy foreign body macrophage response in the fibrous membrane between a loose implant component and the host bone undergoing resorption. These findings underline the importance of biomaterial wear particle generation and the macrophage response to different types of biomaterial wear particles in the pathogenesis of aseptic loosening. Images PMID:8694579

  13. Adipose mesenchymal stem cells in the field of bone tissue engineering

    PubMed Central

    Romagnoli, Cecilia; Brandi, Maria Luisa

    2014-01-01

    Bone tissue engineering represents one of the most challenging emergent fields for scientists and clinicians. Current failures of autografts and allografts in many pathological conditions have prompted researchers to find new biomaterials able to promote bone repair or regeneration with specific characteristics of biocompatibility, biodegradability and osteoinductivity. Recent advancements for tissue regeneration in bone defects have occurred by following the diamond concept and combining the use of growth factors and mesenchymal stem cells (MSCs). In particular, a more abundant and easily accessible source of MSCs was recently discovered in adipose tissue. These adipose stem cells (ASCs) can be obtained in large quantities with little donor site morbidity or patient discomfort, in contrast to the invasive and painful isolation of bone marrow MSCs. The osteogenic potential of ASCs on scaffolds has been examined in cell cultures and animal models, with only a few cases reporting the use of ASCs for successful reconstruction or accelerated healing of defects of the skull and jaw in patients. Although these reports extend our limited knowledge concerning the use of ASCs for osseous tissue repair and regeneration, the lack of standardization in applied techniques makes the comparison between studies difficult. Additional clinical trials are needed to assess ASC therapy and address potential ethical and safety concerns, which must be resolved to permit application in regenerative medicine. PMID:24772241

  14. Indoxyl sulfate promotes apoptosis in cultured osteoblast cells

    PubMed Central

    2013-01-01

    Background Indoxyl sulfate (IS), an organic anion uremic toxin, promotes the progression of renal dysfunction. Some studies have suggested that IS inhibits osteoclast differentiation and suppresses parathyroid hormone (PTH)-stimulated intracellular cAMP production, decreases PTH receptor expression, and induces oxidative stress in primary mouse calvaria osteoblast cell culture. However, the direct effects of IS on osteoblast apoptosis have not been fully evaluated. Hence, we investigated whether IS acts as a bone toxin by studying whether IS induces apoptosis and inhibits differentiation in the cultured osteoblast cell line MC3T3-E1. Methods We assessed the direct effect of IS on osteoblast differentiation and apoptosis in the MC3T3-E1 cell line. We examined caspase-3/7 activity, apoptosis-related proteins, free radical production, alkaline phosphatase activity, and mRNA expression of type 1 collagen and osteonectin. Furthermore, we investigated the uptake of IS via organic anion transport (OAT). Results We found that IS increased caspase activity and induced apoptosis. Production of free radicals increased depending on the concentration of IS. Furthermore, IS inhibited the expression of mRNA type 1 collagen and osteonectin and alkaline phosphatase activity. The expression of OAT, which is known to mediate the cellular uptake of IS, was detected in in the MC3T3-E1 cell line. The inhibition of OAT improved cell viability and suppressed the production of reactive oxygen species. These results suggest that IS is transported in MC3T3-E1 cells via OAT, which causes oxidative stress to inhibit osteoblast differentiation. Conclusions IS acts as a bone toxin by inhibiting osteoblast differentiation and inducing apoptosis. PMID:24289746

  15. Kinetics of metastatic breast cancer cell trafficking in bone

    PubMed Central

    Phadke, Pushkar A.; Mercer, Robyn R.; Harms, John F.; Jia, Yujiang; Frost, Andra R.; Jewell, Jennifer L.; Bussard, Karen M.; Nelson, Shakira; Moore, Cynthia; Kappes, John C.; Gay, Carol V.; Mastro, Andrea M.; Welch, Danny R.

    2006-01-01

    Purpose In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), while little is known about earlier events, e.g., arrival, localization, initial colonization. Defining these initial steps may potentially identify critical points susceptible to therapeutic intervention. Experimental Design MDA-MB-435 human breast cancer cells engineered with green fluorescent protein (GFP) were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry and histomorphometry at times ranging from 1 hr to 6 wk. Results Single cells were found in distal metaphyses at 1 hr post-injection and remained as single cells up to 72 hr. Diaphyseal arrest occurred rarely and few cells remained there after 24 hr. At 1 wk, numerous foci (2–10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 wk, fewer but larger foci (?50 cells) were seen. Most bones had a single large mass at 4 wk (originating from a colony or coalescing foci) which extended into the diaphysis by 4–6 wk. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 wk; but, at 4–6 wk osteoblasts were dramatically reduced (8% of control), while osteoclasts were reduced modestly (to ~60% of control). Conclusions Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both inter- and intra-tissue differences governing metastasis location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data also explain why bisphosphonates do heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases. PMID:16533765

  16. Development of a complex bone tissue culture system based on cellulose nanowhisker mechanical strain.

    PubMed

    Kim, Dae Seung; Jung, Sang-Myung; Yoon, Gwang Heum; Lee, Hoo Cheol; Shin, Hwa Sung

    2014-11-01

    In bone tissue engineering, scaffolds have been investigated for their ability to support osteoblast growth and differentiation for recovery of damaged bones. Tunicate cellulose nanowhisker (CNW) film and mechanical strain were assessed for their suitability for osteoblasts. In this study, sulfuric acid hydrolysis extraction of tunicates integuments was conducted to obtain CNWs, which were found to be acceptable for adhering, growing, and differentiating osteoblasts without cytotoxicity. Mechanical stress enhanced osteoblast differentiation, and cell survival rate was recovered at around day 3, although there was a slight increase in cell death at day 1 after stimulation. We also found that intracellular flux of calcium ion was related to increased differentiation of CNWs under mechanical stress. Overall, we demonstrated the suitability of tunicate CNWs as a scaffold for bone tissue engineering and developed a complex system based on CNW for osteoblast growth and differentiation that will be useful for bone substitute fabrication. PMID:25454753

  17. Treatment of Streptozotocin-Induced Diabetes Mellitus in Mice by Intra-Bone Marrow Bone Marrow Transplantation plus Portal Vein Injection of ? Cells Induced from Bone Marrow Cells

    Microsoft Academic Search

    M. Li; M. Inaba; K. Q. Guo; H. Hisha; N. G. Abraham; S. Ikehara

    2007-01-01

    Curative therapy for diabetes mellitus mainly involves pancreas or islet transplantation to recruit insulin-producing cells. This approach is limited, however, because of both the shortage of donor organs and allograft rejection. Intra-bone marrow bone marrow transplantation (IBM-BMT) has recently been shown to be effective in inducing donor-specific tolerance in mice and rats without the use of immunosuppressants. After induction of

  18. Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

    SciTech Connect

    Tang, C.-H., E-mail: chtang@mail.cmu.edu.t [Department of Pharmacology, China Medical University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung Taiwan (China); Chiu, Y.-C. [Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan (China); Department of Orthopaedics, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Nursing, Hungkuang University, Taichung County, Taiwan (China); Huang, C.-F. [School of Chinese Medicine, China Medical University, Taichung, Taiwan (China); Chen, Y.-W. [Department of Physiology, China Medical University, Taichung, Taiwan (China); Chen, P.-C. [Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan (China)

    2009-12-01

    Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

  19. Culturing primary mouse pancreatic ductal cells.

    PubMed

    Reichert, Maximilian; Rhim, Andrew D; Rustgi, Anil K

    2015-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles ductal cells morphologically. To study pancreatic ductal cell (PDC) and pancreatic intraepithelial neoplasia (PanIN)/PDAC biology, it is essential to have reliable in vitro culture conditions. Here we describe a methodology to isolate, culture, and passage PDCs and duct-like cells from the mouse pancreas. It can be used to isolate cells from genetically engineered mouse models (GEMMs), providing a valuable tool to study disease models in vitro to complement in vivo findings. The culture conditions allow epithelial cells to outgrow fibroblast and other "contaminating" cell types within a few passages. However, the resulting cultures, although mostly epithelial, are not completely devoid of fibroblasts. Regardless, this protocol provides guidelines for a robust in vitro culture system to isolate, maintain, and expand primary pancreatic ductal epithelial cells. It can be applied to virtually all GEMMs of pancreatic disease and other diseases and cancers that arise from ductal structures. Because most carcinomas resemble ductal structures, this protocol has utility in the study of other cancers in addition to PDAC, such as breast and prostate cancers. PMID:26034301

  20. Transdifferentiation of autologous bone marrow cells on a collagen-poly(?-caprolactone) scaffold for tissue engineering in complete lack of native urothelium.

    PubMed

    Zhao, J; Zeiai, S; Ekblad, A; Nordenskjöld, A; Hilborn, J; Götherström, C; Fossum, M

    2014-07-01

    Urological reconstructive surgery is sometimes hampered by a lack of tissue. In some cases, autologous urothelial cells (UCs) are not available for cell expansion and ordinary tissue engineering. In these cases, we wanted to explore whether autologous mesenchymal stem cells (MSCs) from bone marrow could be used to create urological transplants. MSCs from human bone marrow were cultured in vitro with medium conditioned by normal human UCs or by indirect co-culturing in culture well inserts. Changes in gene expression, protein expression and cell morphology were studied after two weeks using western blot, RT-PCR and immune staining. Cells cultured in standard epithelial growth medium served as controls. Bone marrow MSCs changed their phenotype with respect to growth characteristics and cell morphology, as well as gene and protein expression, to a UC lineage in both culture methods, but not in controls. Urothelial differentiation was also accomplished in human bone marrow MSCs seeded on a three-dimensional poly(?-caprolactone) (PCL)-collagen construct. Human MSCs could easily be harvested by bone marrow aspiration and expanded and differentiated into urothelium. Differentiation could take place on a three-dimensional hybrid PCL-reinforced collagen-based scaffold for creation of a tissue-engineered autologous transplant for urological reconstructive surgery. PMID:24789561

  1. Transdifferentiation of autologous bone marrow cells on a collagen-poly(?-caprolactone) scaffold for tissue engineering in complete lack of native urothelium

    PubMed Central

    Zhao, J.; Zeiai, S.; Ekblad, Å.; Nordenskjöld, A.; Hilborn, J.; Götherström, C.; Fossum, M.

    2014-01-01

    Urological reconstructive surgery is sometimes hampered by a lack of tissue. In some cases, autologous urothelial cells (UCs) are not available for cell expansion and ordinary tissue engineering. In these cases, we wanted to explore whether autologous mesenchymal stem cells (MSCs) from bone marrow could be used to create urological transplants. MSCs from human bone marrow were cultured in vitro with medium conditioned by normal human UCs or by indirect co-culturing in culture well inserts. Changes in gene expression, protein expression and cell morphology were studied after two weeks using western blot, RT-PCR and immune staining. Cells cultured in standard epithelial growth medium served as controls. Bone marrow MSCs changed their phenotype with respect to growth characteristics and cell morphology, as well as gene and protein expression, to a UC lineage in both culture methods, but not in controls. Urothelial differentiation was also accomplished in human bone marrow MSCs seeded on a three-dimensional poly(?-caprolactone) (PCL)–collagen construct. Human MSCs could easily be harvested by bone marrow aspiration and expanded and differentiated into urothelium. Differentiation could take place on a three-dimensional hybrid PCL-reinforced collagen-based scaffold for creation of a tissue-engineered autologous transplant for urological reconstructive surgery. PMID:24789561

  2. Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

    PubMed

    Sellgren, Katelyn L; Ma, Teng

    2015-08-01

    Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2509-2520, 2015. PMID:25504617

  3. Use of Human Perivascular Stem Cells for Bone Regeneration

    PubMed Central

    Corselli, Mirko; Chiang, Michael; Yuan, Wei; Nguyen, Virginia; Askarinam, Asal; Goyal, Raghav; Siu, Ronald K.; Scott, Victoria; Lee, Min; Ting, Kang; Péault, Bruno; Soo, Chia

    2012-01-01

    Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models. PMID:22664543

  4. Lineage?Sca1+c-Kit?CD25+ Cells Are IL-33–Responsive Type 2 Innate Cells in the Mouse Bone Marrow

    PubMed Central

    Brickshawana, Adipong; Shapiro, Virginia Smith; Kita, Hirohito; Pease, Larry R.

    2015-01-01

    IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and KitW-sh/W-sh mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5– and IL-13–positive cells display a marker profile consistent with the Lineage?Sca-1+c-Kit?CD25+ (LSK?CD25+) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK?CD25+ cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK?CD25+ cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK?CD25+ cells to expand, in both size and frequency, and to upregulate ST2 and ?4?7 integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK?CD25+ cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues. PMID:22048767

  5. Bone marrow stem cells: current and emerging concepts.

    PubMed

    Méndez-Ferrer, Simón; Scadden, David T; Sánchez-Aguilera, Abel

    2015-01-01

    The interactions of stromal cells with hematopoietic cells in the bone marrow have long been a subject of research, but only recently have technologies allowed us to dissect them at the stem cell level. On the other hand, limitations of these technical tools might explain numerous discrepancies in this field. It is becoming increasingly clear that mesenchymal stem cells (MSCs) represent an important component of the hematopoietic stem cell (HSC) niche in the bone marrow. However, there is heterogeneity among HSCs, and many putatively different mesenchymal progenitors identified in the bone marrow using Cre recombinase-driven mouse lines seem to exhibit HSC niche properties. Development of better reporter lines has demonstrated that some of these Cre lines do not always specifically mark the expected cells. Also, characterization of different cell populations has often been partial, and issues of redundancy and compensation might explain apparently contradictory results. Recognizing and overcoming these limitations, while also clearly defining the distinctions between subgroups of mesenchymal cells, will be essential to advance the field. PMID:25573321

  6. Microtechnology for Stem Cell Culture

    Microsoft Academic Search

    Elena Serena; Elisa Cimetta; Camilla Luni; Nicola Elvassore

    \\u000a Advances in stem cell research in recent decades have been aided by progress in the development of novel technologies aimed\\u000a at biological systems. At the same time mimicking stem cell niches in vitro has become crucial for both basic stem cell research\\u000a and the development of innovative therapies based on stem cells. Innovative microscale technologies can contribute to our\\u000a quantitative

  7. Potential Spermatogenesis Recovery with Bone Marrow Mesenchymal Stem Cells in an Azoospermic Rat Model

    PubMed Central

    Zhang, Deying; Liu, Xing; Peng, Jinpu; He, Dawei; Lin, Tao; Zhu, Jing; Li, Xuliang; Zhang, Yuanyuan; Wei, Guanghui

    2014-01-01

    Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90?, integrin?1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy. PMID:25062349

  8. Potential spermatogenesis recovery with bone marrow mesenchymal stem cells in an azoospermic rat model.

    PubMed

    Zhang, Deying; Liu, Xing; Peng, Jinpu; He, Dawei; Lin, Tao; Zhu, Jing; Li, Xuliang; Zhang, Yuanyuan; Wei, Guanghui

    2014-01-01

    Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90?, integrin?1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy. PMID:25062349

  9. Cell culture experiments planned for the space bioreactor

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  10. Hydrogels as extracellular matrix mimics for 3D cell culture

    Microsoft Academic Search

    Mark W. Tibbitt; Kristi S. Anseth

    2009-01-01

    Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three- dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have

  11. In vitro induction of E-rosette formation in human bone marrow cells by the thymic proteins LSHr and LSHh.

    PubMed

    Pierschbacher, M D; Kalden, J R; Luckey, T D

    1979-07-01

    Two thymic factors, LSHh and LSHr, were found to be active in the induction of E-rosette formation. In vitro incubation of human bone marrow cells, isolated by Ficoll gradient centrifugation, with subnanogram quantities of either of these LSH proteins increased the number of E-rosette-forming cells in the bone marrow cultures by a factor of two. Kidney extract and bovine serum albumin did not show significant activity when tested as controls. Commercial thymopoietin II peptide was found to be active in the assay at comparable concentrations. The data allow a comparison of LHS proteins with other preparations tested in this manner. PMID:262453

  12. Isolation and expansion of osteogenic cells from bone marrow and in vivo testing at ectopic sites

    Microsoft Academic Search

    K. Kisioglu; B. Kandler; M. Scheuer; G. Watzek; R. Gruber

    2010-01-01

    Summary  BACKGROUND: Cell therapy is a strategy to enhance bone formation in regenerative dentistry. Osteogenic cells can be isolated\\u000a and expanded from total bone marrow by the use of biodegradable microcarriers. It remains, however, unknown whether the cells\\u000a on the surface of microcarriers can provide bone substitutes with an osteogenic potential. METHODS: We therefore isolated\\u000a and expanded rat bone marrow cells

  13. Characteristics of cardiac cell cultures derived from human myocardial explants.

    PubMed

    Pavlova, S V; Perovskii, P P; Chepeleva, E V; Malakhova, A A; Dement'eva, E V; Pokushalov, E A; Sukhikh, G T; Zakiyan, S M

    2013-11-01

    Primary cell cultures derived from human myocardial explants were obtained and characterized. The explant cultures contained cardiac stem cells (c-kit(+); ? 4%), microvascular cells (endothelial cells and pericytes), fibroblasts, and myofibroblasts. It was demonstrated that culturing of cardiac cells in cardiospheres did not promote enrichment of the cell culture with stem cells. MACS-sorted c-kit(+) cells from the explant culture were characterized by limited proliferative capacity and were capable of cardiomyogenic differentiation. The presence of microvascular cells determined general angiogenic potential of the culture. PMID:24319709

  14. Transplanted Human Bone Marrow Mesenchymal Stem Cells Seeded onto Peptide Hydrogel Decrease Alveolar Bone Loss

    PubMed Central

    Karlström, Erik; Cedervall, Jessica; Wendel, Mikael

    2012-01-01

    Abstract Alveolar bone loss can be caused by periodontitis or periodontal trauma. We have evaluated the effects of transplanted undifferentiated human mesenchymal stem cells (hMSCs) on alveolar bone reaction and periodontal ligament healing in an experimental periodontal wound model. The hMSCs seeded onto a self-assembling peptide hydrogel in combination with collagen sponge were implanted into the right mandible of 12 rats and followed for 1 (n=6) or 4 weeks (n=6) postoperatively. The other 12 sham-treated rats were used as controls. Histological and histomorphometrical methods were used to assess the periodontal tissue reaction. The alveolar bone volume density was significantly higher at 1 week after surgery, and the osteoclast number was significantly lower at both 1 week and 4 weeks postoperatively in the mandibles treated with hMSCs. The implanted cells were detected only at 1 week after surgery. In conclusion, transplanted hMSCs can contribute to alveolar bone preservation after a periodontal surgical trauma at least by decreasing local osteoclast number. PMID:23514848

  15. Transplanted Human Bone Marrow Mesenchymal Stem Cells Seeded onto Peptide Hydrogel Decrease Alveolar Bone Loss.

    PubMed

    Tcacencu, Ion; Karlström, Erik; Cedervall, Jessica; Wendel, Mikael

    2012-10-01

    Alveolar bone loss can be caused by periodontitis or periodontal trauma. We have evaluated the effects of transplanted undifferentiated human mesenchymal stem cells (hMSCs) on alveolar bone reaction and periodontal ligament healing in an experimental periodontal wound model. The hMSCs seeded onto a self-assembling peptide hydrogel in combination with collagen sponge were implanted into the right mandible of 12 rats and followed for 1 (n=6) or 4 weeks (n=6) postoperatively. The other 12 sham-treated rats were used as controls. Histological and histomorphometrical methods were used to assess the periodontal tissue reaction. The alveolar bone volume density was significantly higher at 1 week after surgery, and the osteoclast number was significantly lower at both 1 week and 4 weeks postoperatively in the mandibles treated with hMSCs. The implanted cells were detected only at 1 week after surgery. In conclusion, transplanted hMSCs can contribute to alveolar bone preservation after a periodontal surgical trauma at least by decreasing local osteoclast number. PMID:23514848

  16. Silencing of exogenous DNA in cultured cells.

    PubMed

    Ochiai, Hiroshi; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2006-06-01

    The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was transfected into cultured cells including HeLa by electroporation, and the amounts of intranuclear plasmid DNA and luciferase were quantitated at various time points. Decrease in expression efficiency from one copy of the exogenous DNA over time occurred as the case of mouse liver, and its degrees varied between cell lines. These results suggest that exogenous DNA is 'silenced' in the cultured cells as well as in mouse hepatocytes. PMID:16755038

  17. Temporal expression of calcium channel subunits in satellite cells and bone marrow mesenchymal cells.

    PubMed

    Grajales, Liliana; Lach, Lawrence E; Janisch, Patrick; Geenen, David L; García, Jesús

    2015-06-01

    Bone marrow-derived mesenchymal stem cells (MSC) can be differentiated into myocytes, as well as adipocytes, chondrocytes, and osteocytes in culture. Calcium channels mediate excitation-contraction coupling and are essential for the function of muscle. However, little is known about the expression of calcium channel subunits and calcium handling in stem cells. We examined whether the expression of calcium channel subunits in MSC is similar to that of skeletal muscle satellite cells and if their levels of expression are modified after treatment with bone morphogenetic protein-4 (BMP4). We found that during myogenic differentiation, MSC first express the ?2?1 subunit and the cardiac channel subunit Cav1.2. In contrast to the ?2?1 subunit levels, the Cav1.2 subunit decreases rapidly with time. The skeletal channel subunit Cav1.1 is detected at day 3 but its expression increases considerably, resembling more closely the expression of the subunits in satellite cells. Treatment of MSC with BMP4 caused a significant increase in expression of Cav1.2, a delay in expression of Cav1.1, and a reduction in the duration of calcium transients when extracellular calcium was removed. Calcium currents and transients followed a pattern related to the expression of the cardiac (Cav1.2) or skeletal (Cav1.1) ?1subunits. These results indicate that differentiation of untreated MSC resembles differentiation of skeletal muscle and that BMP4 reduces skeletal muscle calcium channel expression and promotes the expression of cardiac calcium channels during myogenic differentiation. PMID:25277766

  18. Bone disease in testicular and extragonadal germ cell tumours.

    PubMed Central

    Hitchins, R. N.; Philip, P. A.; Wignall, B.; Newlands, E. S.; Begent, R. H.; Rustin, G. J.; Bagshawe, K. D.

    1988-01-01

    Of 297 patients with metastatic testicular and extragonadal germ cell tumours (GCT), bone involvement was detected clinically in 3% (7/251) of those at first presentation and in 9% (4/46) of relapsed cases. This difference was not statistically significant (95% confidence limits -2%; +14%). Concurrent systemic metastases, commonly involving lung (7/11 cases) and para-aortic lymph nodes (6/11), were present in all patients with bone disease. All affected patients had localized bone pain and lumbar spine was the most frequent site involved (9/11). Spinal cord compression occurred in two patients while a third developed progressive vertebral collapse after chemotherapy and required extensive surgical reconstruction. At median follow-up of 4 years, survival among patients presenting with bone disease (6/7) was similar to overall survival in the whole group (84%) and appeared better than in those with liver (18/26, 69%) or central nervous system (6/9) metastases at presentation. Back pain in metastatic germ cell tumours is often due to retroperitoneal lymphadenopathy but lumbar spine osseus metastases must be recognized early if severe potential complications, such as spinal cord compression, are to be avoided. In this series, bone metastases were not seen in the absence of widespread systemic disease suggesting all solitary bony lesions in GCT patients should be biopsied. Images Figure 1 Figure 2 PMID:3224081

  19. Osteodifferentiated Mesenchymal Stem Cells from Bone Marrow and Adipose Tissue Express HLA-G and Display Immunomodulatory Properties in HLA-Mismatched Settings: Implications in Bone Repair Therapy

    PubMed Central

    Montespan, Florent; Deschaseaux, Frédéric; Sensébé, Luc; Carosella, Edgardo D.

    2014-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from several sources such as bone marrow and adipose tissue. Depending on the culture conditions, they can differentiate into osteoblasts, chondroblasts, adipocytes, or neurons. In this regard, they constitute promising candidates for cell-based therapy aimed at repairing damaged tissues. In addition, MSCs display immunomodulatory properties through the expression of soluble factors including HLA-G. We here analyse both immunogenicity and immunosuppressive capacity of MSCs derived from bone marrow and adipose tissue before and after osteodifferentiation. Results show that HLA-G expression is maintained after osteodifferentiation and can be boosted in inflammatory conditions mimicked by the addition of IFN-? and TNF-?. Both MSCs and osteodifferentiated MSCs are hypoimmunogenic and exert immunomodulatory properties in HLA-mismatched settings as they suppress T cell alloproliferation in mixed lymphocyte reactions. Finally, addition of biomaterials that stimulate bone tissue formation did not modify MSC immune properties. As MSCs combine both abilities of osteoregeneration and immunomodulation, they may be considered as allogenic sources for the treatment of bone defects. PMID:24877156

  20. The Effects of 1?, 25-dihydroxyvitamin D3 and Transforming Growth Factor-?3 on Bone Development in an Ex Vivo Organotypic Culture System of Embryonic Chick Femora

    PubMed Central

    Smith, Emma L.; Rashidi, Hassan; Kanczler, Janos M.; Shakesheff, Kevin M.; Oreffo, Richard O. C.

    2015-01-01

    Transforming growth factor-beta3 (TGF-?3) and 1?,25-dihydroxyvitamin D3 (1?,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1?,25 (OH)2D3 and TGF-?3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1?,25 (OH) 2D3 (25 nM) or TGF-?3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (?CT), histology and immunohistochemistry. 1?,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-?3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1?,25 (OH) 2D3 and TGF-?3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1?,25(OH)2D and TGF-?3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life. PMID:25835745

  1. BONE MARROW PROGENITOR CELLS REPAIR RAT HEPATIC SINUSOIDAL ENDOTHELIAL CELLS AFTER LIVER INJURY

    PubMed Central

    Harb, Rula; Xie, Guanhua; Lutzko, Carolyn; Guo, Yumei; Wang, Xiangdong; Hill, Colin K.; Kanel, Gary C.; DeLeve, Laurie D.

    2009-01-01

    Background and Aims Damage to hepatic sinusoidal endothelial cells (SEC) initiates sinusoidal obstruction syndrome (SOS), which is most commonly a consequence of myeloablative chemo-irradiation or ingestion of pyrrolizidine alkaloids such as monocrotaline (Mct). This study examines whether SEC are of bone marrow origin, whether bone marrow repair can be a determinant of severity of liver injury and whether treatment with progenitor cells is beneficial. Methods Mct-treated female rats received infusion of male whole bone marrow or CD133+ cells at the peak of sinusoidal injury. The y-chromosome was identified in isolated SEC by fluorescent in situ hybridization. Bone marrow suppression was induced by irradiation of both lower extremities with shielding of the abdomen. Results SEC in uninjured liver have both hematopoietic (CD45, CD33) and endothelial (CD31) markers. After Mct-induced SOS, infusion of bone marrow derived CD133+ progenitor cells replaces more than one-quarter of SEC. All CD133+ cells recovered from the SEC fraction after injury are CD45+. CD133+/45+ progenitors also repaired central vein endothelium. Mct suppresses CD133+/CD45+ progenitors in bone marrow by 50% and in the circulation by 97%. Irradiation-induced bone marrow suppression elicited SOS from a sub-toxic dose of Mct, whereas infusion of bone marrow during the necrotic phase of SOS nearly eradicates histological features of SOS. Conclusions SEC have both hematopoietic and endothelial markers. Bone marrow-derived CD133+/CD45+ progenitors replace SEC and central vein endothelial cells after injury. Toxicity to bone marrow progenitors impairs repair and contributes to the pathogenesis of SOS, whereas timely infusion of bone marrow has therapeutic benefit. PMID:19447108

  2. Large-scale culture of plant cells.

    PubMed

    Scragg, A H; Fowler, M W

    1990-01-01

    The large-scale or mass cultivation of plant cells is the growth of plant cell suspensions at volumes above those normally produced in shake flasks, that is, above IL. Attempts to grow plant cells in fermenters or bioreactors started in the early 1960s with converted carboys. The area has developed steadily, such that today bioreactors in excess of 5000 L have been used successfully for large-scale plant cell culture (1,2). Much of the early work was carried out using bioreactors designed for microbial culture. It was soon found, however, that although plant cell suspensions appear to be similar in many ways to microbial cultures, there are, in fact, key differences that can have a significant influence on large-scale cultivation. Plant cells are large, 20-40 µM in diameter, and up to 100 PM in length; further, they rarely occur as single cells, but as aggregates of up to 2 mm in diameter (Fig. 1). The individual plant cell soon after division is typically rounded, containing considerable amounts of cytoplasm; however, as it ages, the cell expands and becomes dominated by a large vacuole. In consequence, the overall metabolic activity is low compared with microbial cells, which in turn gives a very slow growth rate (measured in days. PMID:21390630

  3. Mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) growth and adhesion in six different bone graft substitutes

    Microsoft Academic Search

    J. SchultheissC; C. Seebach; D. Henrich; K. Wilhelm; J. H. Barker; J. Frank

    Introduction  Several different synthetic and allograft bone graft substitutes are used clinically to treat large bone defects. In contrast\\u000a to the “gold standard” of autologous bone grafts, these do not contain bone-forming (MSC) or vessel-forming (EPC) cells. In\\u000a order to achieve the same level of success enjoyed by autologous bone grafts, they must be compatible with mesenchymal stem\\u000a cells (MSC) and

  4. Osteogenic differentiation and ectopic bone formation of canine bone marrow-derived mesenchymal stem cells in injectable thermo-responsive polymer hydrogel.

    PubMed

    Liao, Han-Tsung; Chen, Chien-Tzung; Chen, Jyh-Ping

    2011-11-01

    This study describes an injectable, thermo-responsive hyaluronic acid-g-chitosan-g-poly(N-isopropylacrylamide) (HA-CPN) copolymer for bone tissue engineering. The wettability, temperature-dependent change of water content, and volume of HA-CPN hydrogel were measured, together with its biocompatibility in vitro and in vivo. The dried hydrogel morphology shows a three-dimensional, porous structure with interconnected pores. Canine bone marrow-derived mesenchymal stem cells (cBMSCs) were encapsulated in HA-CPN hydrogel and osteoinduction was assessed by comparing samples with different osteogenic differentiation induction times but with the same total cell culture time. Cell proliferation and time-dependent osteogenic differentiation, evident from secretion of extracellular matrix and formation of mineral deposits, were observed. The cells showed better proliferation in HA-CPN hydrogel than on tissue culture polystyrene after osteo-induced for 21 days and higher alkaline phosphatase activity regardless of osteo-induction times. Mineralization extent of cBMSCs in HA-CPN followed by Alizarin red stains showed positive stained nodules after osteo-induced longer than 7 days. The cells/hydrogel construct also showed increased mechanical strength and elasticity after osteogenic differentiation, and the increase could be correlated with osteo-induction time. In vivo studies confirmed the biocompatibility and bioresorption of the HA-CPN hydrogel and ectopic bone formation when the hydrogel was used as a cell carrier for osteo-induced cBMSCs and implanted in nude mice subcutaneously. Taken together, the results indicate the feasibility and efficacy of HA-CPN hydrogel as an injectable bone tissue engineering scaffold with cBMSCs. PMID:21870942

  5. Circulating Bone Marrow Cells Can Contribute to Neointimal Formation

    Microsoft Academic Search

    Chih-lu Han; Gordon R. Campbell; Julie H. Campbell

    2001-01-01

    To examine the source of smooth muscle-like cells during vascular healing, C57BL\\/6 (Ly 5.2) female mice underwent whole body irradiation followed by transfusion with 106 nucleated bone marrow cells from congenic (Ly 5.1) male donors. Successful repopulation (88.4 ± 4.9%) by donor marrow was demonstrated in the female mice by flow cytometry with FITC-conjugated A20.1\\/Ly 5.1 monoclonal antibody after 4

  6. The Role of Circulating Bone Cell Precursors in Fracture Healing

    Microsoft Academic Search

    Patrizia D’Amelio; Maria Angela Cristofaro; Anastasia Grimaldi; Marco Ravazzoli; Fernanda Pluviano; Elena Grosso; Gian Piero Pescarmona; Giovanni Carlo Isaia

    2010-01-01

    Fracture healing is a complex process that involves several cell types; as a previous report suggested an increase in osteoblast\\u000a (OB) precursors in peripheral blood during this process, this paper examines the role of circulating bone cell precursors\\u000a in this process in the light of a prior suggestion that OB precursors are increased. Nine healthy men less than 60 years old

  7. The Manipulation of Mesenchymal Stem Cells for Bone Repair

    Microsoft Academic Search

    Shelley R. Winn

    Bone homeostasis is a dynamic process consisting of mutually dependent interactions between cells, substrates, and molecular\\u000a signals that are, in turn, influenced by hormones, mitogens and differentiation factors. In general, when this environment\\u000a is perturbed as a consequence of disease, including osteoporosis or injury, cell and molecular signals initiate a cascade\\u000a of genetically programmed repair processes. Depending on the molecular

  8. Behaviour of mesenchymal stem cells from bone marrow of untreated advanced breast and lung cancer patients without bone osteolytic metastasis.

    PubMed

    Fernández Vallone, Valeria B; Hofer, Erica L; Choi, Hosoon; Bordenave, Raúl H; Batagelj, Emilio; Feldman, Leonardo; La Russa, Vincent; Caramutti, Daniela; Dimase, Federico; Labovsky, Vivian; Martínez, Leandro M; Chasseing, Norma A

    2013-03-01

    Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30-40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction. PMID:23053744

  9. Ion-exchange polymer nanofibers for enhanced osteogenic differentiation of stem cells and ectopic bone formation.

    PubMed

    Shabani, Iman; Haddadi-Asl, Vahid; Soleimani, Masoud; Seyedjafari, Ehsan; Hashemi, Seyed Mahmoud

    2014-01-01

    Nanofibrous scaffolds with specific modifications have shown promising potential for bone tissue engineering applications. In the present study, poly(ether sulfone) (PES) and sulfonated PES (SPES) nanofibers were fabricated via electrospinning. Calcium ions were then incorporated in SPES by immersion in a Ca(OH)2 solution. The calcium-ion-exchanged SPES (Ca-SPES), PES, and SPES nanofibers were characterized and then evaluated for their osteogenic capacity: both in vitro using stem cell culture and in vivo after subcutaneous implantation in mice. After 7 days of immersion in simulated body fluid, the formation of an apatite layer was only observed on Ca-SPES nanofibers. According to the MTT results, an increasing stem cell population was detected on all scaffolds during the period of study. Using real-time reverse transcriptase-polymerase chain reaction, alkaline phosphatase activity, and calcium content assays, it was demonstrated that the osteogenic differentiation of stem cells was higher on Ca-SPES scaffolds in comparison with PES and SPES nanofibers. Interestingly, Ca-SPES scaffolds were shown to induce ectopic bone formation after 12 weeks of subcutaneous implantation in mice. This was confirmed by mineralization and the production of collagen fibers using van Kossa and Masson's trichrome staining, respectively. Taken together, it was demonstrated that the incorporation of calcium ions into the ion-exchange nanofibrous scaffolds not only gives them the ability to enhance osteogenic differentiation of stem cells in vitro but also to induce ectopic bone formation in vivo. PMID:24328219

  10. The Clinical Approach Toward Giant Cell Tumor of Bone

    PubMed Central

    van der Heijden, Lizz; Dijkstra, P.D. Sander; van de Sande, Michiel A.J.; Kroep, Judith R.; Nout, Remi A.; van Rijswijk, Carla S.P.; Bovée, Judith V.M.G.; Hogendoorn, Pancras C.W.

    2014-01-01

    We provide an overview of imaging, histopathology, genetics, and multidisciplinary treatment of giant cell tumor of bone (GCTB), an intermediate, locally aggressive but rarely metastasizing tumor. Overexpression of receptor activator of nuclear factor ?B ligand (RANKL) by mononuclear neoplastic stromal cells promotes recruitment of numerous reactive multinucleated giant cells. Conventional radiographs show a typical eccentric lytic lesion, mostly located in the meta-epiphyseal area of long bones. GCTB may also arise in the axial skeleton and very occasionally in the small bones of hands and feet. Magnetic resonance imaging is necessary to evaluate the extent of GCTB within bone and surrounding soft tissues to plan a surgical approach. Curettage with local adjuvants is the preferred treatment. Recurrence rates after curettage with phenol and polymethylmethacrylate (PMMA; 8%–27%) or cryosurgery and PMMA (0%–20%) are comparable. Resection is indicated when joint salvage is not feasible (e.g., intra-articular fracture with soft tissue component). Denosumab (RANKL inhibitor) blocks and bisphosphonates inhibit GCTB-derived osteoclast resorption. With bisphosphonates, stabilization of local and metastatic disease has been reported, although level of evidence was low. Denosumab has been studied to a larger extent and seems to be effective in facilitating intralesional surgery after therapy. Denosumab was recently registered for unresectable disease. Moderate-dose radiotherapy (40–55 Gy) is restricted to rare cases in which surgery would lead to unacceptable morbidity and RANKL inhibitors are contraindicated or unavailable. PMID:24718514

  11. Standard operating procedure for the good manufacturing practice-compliant production of human bone marrow mesenchymal stem cells.

    PubMed

    Roseti, Livia; Serra, Marta; Bassi, Alessandra

    2015-01-01

    According to the European Regulation (EC 1394/2007), Mesenchymal Stem Cells expanded in culture for clinical use are considered as Advanced Therapy Medicinal Products. As a consequence, they must be produced in compliance with Good Manufacturing Practice in order to ensure safety, reproducibility, and efficacy. Here, we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implantation in humans. This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval. Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss. PMID:25092055

  12. Cultured stem cells are sensitive to gravity changes

    NASA Astrophysics Data System (ADS)

    Buravkova, L. B.; Romanov, Yu. A.; Konstantinova, N. A.; Buravkov, S. V.; Gershovich, Yu. G.; Grivennikov, I. A.

    2008-09-01

    Stem and precursor cells play an important role in development and regeneration. The state of these cells is regulated by biochemical substances, mechanical stimuli and cellular interactions. To estimate gravity effects we used two types of cultured stem cells: human mesenchymal stromal cells (hMSCs) from bone marrow and mice embryonic stem (mESC) line R1. Gravity changes were simulated by long-term (4-7 days) slow clinorotation and leaded to decreased hMSC proliferation, changes of cell morphology and modified F-actin cytoskeleton. We did not find the shifts in cell phenotype except for decreased expression of HLA 1 and CD105 but excretion of IL-6 into medium increased significantly. Remodeling of cytoskeleton started after first 4 h and was similar to preapoptotic changes. This data suggested the modification in cell adhesion and possible commitment of hMSC. It was observed that expression of alkaline phosphatase by MSC in osteogenic medium was more intensive in control. On the contrary, clinorotation did not change formation of mESC colonies and increased proliferation activity in LIF+-medium. However, the number of embryonic bodies after clinorotation was less than in static control. It is suggested that ESCs kept the viability and proliferative potential but decreased the differentiation ability after changes in gravity stimulation.

  13. Cell Culture on MEMS Platforms: A Review

    PubMed Central

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  14. Bilateral Temporal Bone Langerhans Cell Histiocytosis: Radiologic Pearls

    PubMed Central

    Coleman, Mira A.; Matsumoto, Jane; Carr, Carrie M.; Eckel, Laurence J.; Nageswara Rao, Amulya A.

    2013-01-01

    Langerhans cell histiocytosis (LCH) is a rare histiocytic disorder with an unpredictable clinical course and highly varied clinical presentation ranging from single system to multisystem involvement. Although head and neck involvement is common in LCH, isolated bilateral temporal bone involvement is exceedingly rare. Furthermore, LCH is commonly misinterpreted as mastoiditis, otitis media and otitis externa, delaying diagnosis and appropriate therapeutic management. To improve detection and time to treatment, it is imperative to have LCH in the differential diagnosis for unusual presentations of the aforementioned infectious head and neck etiologies. Any lytic lesion of the temporal bone identified by radiology should raise suspicion for LCH. We hereby describe the radiologic findings of a case of bilateral temporal bone LCH, originally misdiagnosed as mastoiditis. PMID:24478812

  15. Isolation and characterization of mouse bone marrow-derived Lin?/VEGF-R2? progenitor cells.

    PubMed

    Barthelmes, Daniel; Irhimeh, Mohammad R; Gillies, Mark C; Zhu, Ling; Shen, Weiyong

    2013-11-01

    Circulating endothelial progenitor cells (EPCs) in the peripheral blood (PB) have physiological roles in the maintenance of the existing vascular beds and rescue of vascular injury. In this study, we have evaluated the properties of Lin?/VEGF-R2? progenitor cells isolated from the mouse bone marrow (BM) and further studied their distribution and integration in an animal model of laser-induced retinal vascular injury. Lin?/VEGF-R2? cells were enriched from C57BL/6 mice BM using magnetic cell sorting with hematopoietic lineage (Lin) depletion followed by VEGF-R2 positive selection. Lin?/VEGF-R2? BM cells were characterized using flow cytometry and immunocytochemistry and further tested for colony formation during culture and tube formation on Matrigel®. Lin?/VEGF-R2? BM cells possessed typical EPC properties such as forming cobble-stone shaped colonies after 3 to 4 weeks of culture, CD34? expression, take up of Dil-acLDL and binding to Ulex europaeus agglutinin. However, they did not form tube-like structures on Matrigel®. The progenitor cells retained their phenotype over extended period of culture. After intravitreal transplantation in eyes subjected to the laser-induced retinal vascular injury, some Lin?/VEGF-R2? cells were able to integrate into the damaged retinal vasculature but the level of cell integration seemed less efficient when compared with previous reports in which EPCs from the human PB were employed. Our results indicate that Lin?/VEGF-R2? cells isolated from the mouse BM share some similarities to EPCs from the human PB but most of them are at a very early stage of maturation and remain quiescent during culture and after intravitreal transplantation. PMID:23771478

  16. Characterization of Monocyte-Macrophage-Lineage Cells Induced from CD34 + Bone Marrow Cells In Vitro

    Microsoft Academic Search

    Kyoko Suzuki; Nobutaka Kiyokawa; Tomoko Taguchi; Hisami Takenouchi; Masahiro Saito; Toshiaki Shimizu; Hajime Okita; Junichiro Fujimoto

    2007-01-01

    We characterized the expression of cell surface antigens and cytokine-secreting ability of monocyte-macrophage-lineage cells\\u000a induced in vitro from CD34+ bone marrow cells. After cultivation for 3 weeks, we observed 2 distinct cell fractions: a floating small, round cell fraction\\u000a and an adherent large, protruding cell fraction. Both cell fractions expressed myelocyte-monocyte-lineage antigens, but mature-macrophage\\u000a markers such as CD206 were expressed

  17. Interleukin1 beta, transforming growth factor beta 1, prostaglandin E 2 , and fibronectin levels in the conditioned mediums of bone marrow fibroblast cultures from lung and breast cancer patients

    Microsoft Academic Search

    A. E. Honegger; E. L. Hofer; R. I. Barañao; T. A. Mackinlay; D. A. Mackinlay; E. O. Bullorsky; R. H. Bordenave; N. A. Chasseing

    2002-01-01

    We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1#),

  18. Stem Cell Rev . Author manuscript Human bone marrow mesenchymal stem cells: a systematic reappraisal

    E-print Network

    Paris-Sud XI, Université de

    Stem Cell Rev . Author manuscript Page /1 11 Human bone marrow mesenchymal stem cells: a systematic (acronym for Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been

  19. Nanofibrous yet injectable polycaprolactone-collagen bone tissue scaffold with osteoprogenitor cells and controlled release of bone morphogenetic protein-2.

    PubMed

    Subramanian, Gayathri; Bialorucki, Callan; Yildirim-Ayan, Eda

    2015-06-01

    In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O2 plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile. PMID:25842103

  20. Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.

    PubMed

    Isern, Joan; Martín-Antonio, Beatriz; Ghazanfari, Roshanak; Martín, Ana M; López, Juan A; del Toro, Raquel; Sánchez-Aguilera, Abel; Arranz, Lorena; Martín-Pérez, Daniel; Suárez-Lledó, María; Marín, Pedro; Van Pel, Melissa; Fibbe, Willem E; Vázquez, Jesús; Scheding, Stefan; Urbano-Ispizúa, Álvaro; Méndez-Ferrer, Simón

    2013-05-30

    Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs. PMID:23623496

  1. Tracking Circadian Rhythms of Bone Mineral Deposition in Murine Calvarial Organ Cultures

    PubMed Central

    McElderry, John-David P.; Zhao, Guisheng; Khmaladze, Alexander; Wilson, Christopher G.; Franceschi, Renny T.; Morris, Michael D.

    2013-01-01

    Osteoblasts, which orchestrate the deposition of small apatite crystals through the expression of nucleating proteins, have been shown to also express clock genes associated with the circadian signaling pathway. We hypothesized that protein-mediated bone mineralization may be linked to circadian oscillator mechanisms functioning in peripheral bone tissue. In this study, Per1 expression in ex vivo neonatal murine calvaria organ cultures was monitored for 6 days using a Per1-luciferase transgene as a bioluminescent indicator of clock function. Fluctuations in Per1 expression had a period of 25±4 hours (n=14) with early expression at CT09:59±03:37 (circadian time). We also established the kinetics of mineral deposition in developing bone by using non-invasive Raman microscopy to track mineral accumulation in calvarial tissue. The content and quality of newly deposited mineral was continually examined at the interparietal bone/fontanel boundary for a period of 6 days with 1 hour temporal resolution. Using this approach, mineralization over time exhibited bursts of mineral deposition followed by little or no deposition, which was recurrent with a periodicity of 26.8±9.6 hours. As many as 6 near-daily mineralization events were observed in the calvaria before deposition ceased. Earliest mineralization events occurred at CT16:51±03:45, which is 6 hours behind Per1 expression. These findings are consistent with the hypothesis that mineralization in developing bone tissue is regulated by a local circadian oscillator mechanism. PMID:23505073

  2. Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T cell differentiation culture system

    PubMed Central

    Wang, Hongfang; Pierce, L. Jeanne; Spangrude, Gerald J.

    2006-01-01

    Objective The OP9-DL1 culture system is an in vitro model for T cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL7 has recently emerged. We examined the roles played by IL7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. Methods Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. Results IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T cell development from the CD4/CD8 double negative (DN) stage to the CD4/CD8 double positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. Conclusion These results validate the OP9-DL1 model for the analysis of T cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T lineage differentiation. PMID:17157170

  3. Co-culture with cardiomyocytes enhanced the myogenic conversion of mesenchymal stromal cells in a dose-dependent manner

    Microsoft Academic Search

    Xiao-qing He; Min-sheng Chen; Shu-Hong Li; Shi-ming Liu; Yun Zhong; Heather Y. McDonald Kinkaid; Wei-Yang Lu; Richard D. Weisel; Ren-Ke Li

    2010-01-01

    To increase the accessibility of myogenic cells for cell therapy in the infarcted heart, we identified conditions to improve\\u000a the reproducible conversion of bone marrow mesenchymal stromal cells (BMSCs) into myogenic cells. Such cells may permit functional\\u000a regeneration following a myocardial infarction. BMSCs derived from green fluorescent protein (GFP) transgenic rats were co-cultured\\u000a with neonatal rat cardiomyocytes (1:1, 1:10, 1:20,

  4. Circulating Osteogenic Precursor Cells in Heterotopic Bone Formation

    PubMed Central

    Suda, Robin K.; Billings, Paul C.; Egan, Kevin P.; Kim, Jung-Hoon; McCarrick-Walmsley, Ruth; Glaser, David L.; Porter, David L.; Shore, Eileen M.; Pignolo, Robert J.

    2012-01-01

    Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow-derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early pre-osseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from FOP patients with active episodes of HO contain significantly higher numbers of clonally-derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just prior to the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation. PMID:19522009

  5. Cell attachment and proliferation of bone marrow-derived osteoblast on zirconia of various surface treatment

    PubMed Central

    Lee, Heesu; Noh, Kwantae; Woo, Yi-Hyung

    2014-01-01

    PURPOSE This study was performed to characterize the effects of zirconia coated with calcium phosphate and hydroxyapatite compared to smooth zirconia after bone marrow-derived osteoblast culture. MATERIALS AND METHODS Bone marrow-derived osteoblasts were cultured on (1) smooth zirconia, (2) zirconia coated with calcium phosphate (CaP), and (3) zirconia coated with hydroxyapatite (HA). The tetrazolium-based colorimetric assay (MTT test) was used for cell proliferation evaluation. Scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cellular morphology and differentiation rate. X-ray photoelectron spectroscopy (XPS) was employed for the analysis of surface chemistry. The genetic expression of the osteoblasts and dissolution behavior of the coatings were observed. Assessment of the significance level of the differences between the groups was done with analysis of variance (ANOVA). RESULTS From the MTT assay, no significant difference between smooth and surface coated zirconia was found (P>.05). From the SEM image, cells on all three groups of discs were sporadically triangular or spread out in shape with formation of filopodia. From the ALP activity assay, the optical density of osteoblasts on smooth zirconia discs was higher than that on surface treated zirconia discs (P>.05). Most of the genes related to cell adhesion showed similar expression level between smooth and surface treated zirconia. The dissolution rate was higher with CaP than HA coating. CONCLUSION The attachment and growth behavior of bone-marrow-derived osteoblasts cultured on smooth surface coated zirconia showed comparable results. However, the HA coating showed more time-dependent stability compared to the CaP coating. PMID:24843393

  6. Bystander effect in glioma suicide gene therapy using bone marrow stromal cells.

    PubMed

    Li, Shaoyi; Gu, Chunyu; Gao, Yun; Amano, Shinji; Koizumi, Shinichiro; Tokuyama, Tsutomu; Namba, Hiroki

    2012-11-01

    An established rat intracranial glioma was successfully treated through the tumoricidal bystander effect generated by intratumoral injection of rat bone marrow stromal cells (BMSCs) transduced with the herpes simplex virus-thymidine kinase gene (BMSCtk cells) followed by systemic ganciclovir administration. In the present study, we tested the bystander effect of this treatment strategy when using human BMSCs as the vector cells. Human BMSCtk cells were mixed with various kinds of brain tumor cell lines (human and rat glioma cells) and examined in vitro and in vivo tumoricidal bystander effects, by co-culture study and co-implantation study in the nude mouse, respectively. A significant in vitro bystander effect was observed between human BMSCtk cells and any of the tumor cells examined in the ganciclovir-containing medium. A potent in vivo bystander effect against human and rat glioma cells was also demonstrated when ganciclovir was administered. Migratory activity of the human BMSCs toward the tumor cells was enhanced by the conditioned media obtained from both human and rat glioma cells compared to the fresh media. The results of this study have demonstrated that the bystander effect generated by BMSCtk cells and ganciclovir is not cell type-specific, suggesting that the strategy would be quite feasible for clinical use. PMID:23022734

  7. Effect of Low Dose Radiation on Differentiation of Bone Marrow Cells into Dendritic Cells

    PubMed Central

    Chun, Sung Hak; Park, Ga-Young; Han, Yu Kyeong; Kim, Sung Dae; Kim, Joong Sun; Lee, Chang Geun; Yang, Kwangmo

    2013-01-01

    Low dose radiation has been shown to be beneficial to living organisms using several biological systems, including immune and hematopoietic systems. Chronic low dose radiation was shown to stimulate immune systems, resulting in controlling the proliferation of cancer cells, maintain immune balance and induce hematopoietic hormesis. Since dendritic cells are differentiated from bone marrow cells and are key players in maintaining the balance between immune activation and tolerance, it may be important to further characterize whether low dose radiation can influence the capacity of bone marrow cells to differentiate into dendritic cells. We have shown that bone marrow cells from low dose-irradiated (?-radiation, 0.2Gy, 15.44mGy/h) mice can differentiate into dendritic cells that have several different characteristics, such as expression of surface molecules, cytokine secretion and antigen uptake capacity, when compared to dentritic cells differentiated from the control bone marrow cells. These differences observed in the low dose radiation group can be beneficial to living organisms either by activation of immune responses to foreign antigens or tumors, or maintenance of self-tolerance. To the best of our knowledge, this is the first report showing that total-body low dose radiation can modulate the capacity of bone marrow cells to differentiate into dendritic cells. PMID:23983665

  8. Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors

    PubMed Central

    Yang, Jinpu; Kaur, Keerat; Ong, Li Lin; Eisenberg, Carol A.; Eisenberg, Leonard M.

    2015-01-01

    The G9a histone methyltransferase inhibitor BIX01294 was examined for its ability to expand the cardiac capacity of bone marrow cells. Inhibition of G9a histone methyltransferase by gene specific knockdown or BIX01294 treatment was sufficient to induce expression of precardiac markers Mesp1 and brachyury in bone marrow cells. BIX01294 treatment also allowed bone marrow mesenchymal stem cells (MSCs) to express the cardiac transcription factors Nkx2.5, GATA4, and myocardin when subsequently exposed to the cardiogenic stimulating factor Wnt11. Incubation of BIX01294-treated MSCs with cardiac conditioned media provoked formation of phase bright cells that exhibited a morphology and molecular profile resembling similar cells that normally form from cultured atrial tissue. Subsequent aggregation and differentiation of BIX01294-induced, MSC-derived phase bright cells provoked their cardiomyogenesis. This latter outcome was indicated by their widespread expression of the primary sarcomeric proteins muscle ?-actinin and titin. MSC-derived cultures that were not initially treated with BIX01294 exhibited neither a commensurate burst of phase bright cells nor stimulation of sarcomeric protein expression. Collectively, these data indicate that BIX01294 has utility as a pharmacological agent that could enhance the ability of an abundant and accessible stem cell population to regenerate new myocytes for cardiac repair.

  9. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    PubMed Central

    Nikfarjam, Laleh; Farzaneh, Parvaneh

    2012-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture. PMID:23508237

  10. PDGFBB promotes PDGFR{alpha}-positive cell migration into artificial bone in vivo

    SciTech Connect

    Yoshida, Shigeyuki [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan) [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Iwasaki, Ryotaro; Kawana, Hiromasa [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan)] [Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Miyauchi, Yoshiteru [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan) [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Integrated Bone Metabolism and Immunology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hoshi, Hiroko; Miyamoto, Hiroya; Mori, Tomoaki [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan) [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Kanagawa, Hiroya [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan)] [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Katsuyama, Eri; Fujie, Atsuhiro [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan) [Center for Human Metabolomic Systems Biology, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); Hao, Wu [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan)] [Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582 (Japan); and others

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We examined effects of PDGFBB in PDGFR{alpha} positive cell migration in artificial bones. Black-Right-Pointing-Pointer PDGFBB was not expressed in osteoblastic cells but was expressed in peripheral blood cells. Black-Right-Pointing-Pointer PDGFBB promoted PDGFR{alpha} positive cell migration into artificial bones but not osteoblast proliferation. Black-Right-Pointing-Pointer PDGFBB did not inhibit osteoblastogenesis. -- Abstract: Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor {alpha} (PDGFR{alpha})-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGF{beta}) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

  11. Therapeutic effect comparison of hepatocyte-like cells and bone marrow mesenchymal stem cells in acute liver failure of rats

    PubMed Central

    Li, Dongliang; Fan, Jingjing; He, Xiuhua; Zhang, Xia; Zhang, Zhiqiang; Zeng, Zhiyu; Ruan, Mei; Cai, Lirong

    2015-01-01

    Goals: To evaluate the therapeutic efficacy of rat bone marrow mesenchymal stem cells (BMSCs) induced into hepatocyte-like cells and of un-induced BMSCs in acute liver failure rats. Methods: BMSCs in highly homogenous passage 3 were cultured using the whole bone marrow adherent culture method. Hepatic-related characters were confirmed with morphology, RT-PCR analysis, glycogen staining and albumin (ALB) immunofluorescence assay. Carbon tetrachloride (CCl4) was injected intraperitoneally to establish an acute rat liver failure model. Hepatocyte-like cells or un-induced BMSCs were respectively injected into the models to examine rats’ appearance, liver function assay and liver tissue pathology. Results: Hepatocyte-like morphology, higher expression of cytokeratin 18 (CK18) mRNA and ALB protein, and glycogen accumulation were confirmed in the induced BMSCs. The transplanted DAPI-labeled BMSCs were localized in the liver tissue 3-14 days after transplantation. The levels of liver function indicators (AST, ALT, ALP, and TBIL) from transplanted rats were significant decreased and pathology was improved, indicating the recovery of liver function. However, the differences were statistically insignificant. Conclusion: Both hepatocyte-like cells and un-induced BMSCs had a similarly positively therapeutic efficacy on liver regeneration in rat liver failure model. PMID:25755689

  12. Bone marrow mononuclear cells and acute myocardial infarction.

    PubMed

    Arnous, Samer; Mozid, Abdul; Martin, John; Mathur, Anthony

    2012-01-01

    Stem cell transplantation is emerging as a potential therapy to treat heart diseases. Promising results from early animal studies led to an explosion of small, non-controlled clinical trials that created even further excitement by showing that stem cell transplantation improved left ventricular systolic function and enhanced remodelling. However, the specific mechanisms by which these cells improve heart function remain largely unknown. A large variety of cell types have been considered to possess the regenerative ability needed to repair the damaged heart. One of the most studied cell types is the bone marrow-derived mononuclear cells and these form the focus of this review. This review article aims to provide an overview of their use in the setting of acute myocardial infarction, the challenges it faces and the future of stem cell therapy in heart disease. PMID:22264393

  13. Proliferation and differentiation of mesenchymal stem cells in a three- dimensional culture system

    NASA Astrophysics Data System (ADS)

    Sun, S.; Hu, J.; Long, M.; Tao, Z.

    Mesenchymal stem cells (MSCs) have multi-differentiation potential and retain the capacity to proliferate and differentiate in vitro, which in turn holds the promise of being able to repair or replace damaged cells or tissues. Since MSCs are rare in amount in vivo, abundant cells usually need be obtained in time in clinic. T herefore, proliferation and differentiation of MSCs in vitro are necessary and important for future applications. Most current studies o MSCs are focused on the cellular andn molecular biology using a two-dimension (2-D) static culture system at unit gravity. The gravity-induced 2D culture of MSCs could potentially not reflect cell-cell- contacts important for proliferation and differentiation of MSCs in vivo. Here we developed a method to proliferate MSCs by using the rotating three-dimensional (3- D) culture system, which can provide low shear, 3-D environment with simulated microgravity. MSCs from human bone marrow were prepared on microcarrier beads and then were seeded in the 3-D culture system. Various rotation conditions were tested to screen out the most suitable one for proliferation of MSCs. 2-D cultures were prepared in routine cell culture dishes as a control. All cultures were uniformly inoculated and medium exchanged at standard intervals. Results were compared with microscopic and immunochemistrical techniques. The differentiation capacity of proliferated MSCs were also tested through induced differentiation experiments. It is found that simulated microgravity and three-dimensional culture condition is an active factor for proliferation of MSCs.

  14. Chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped, nanocomposite scaffold

    PubMed Central

    Patel, Kavi H; Nayyer, Leila

    2013-01-01

    Reconstruction of the human auricle remains a challenge to plastic surgeons, and current approaches are not ideal. Tissue engineering provides a promising alternative. This study aims to evaluate the chondrogenic potential of bone marrow–derived mesenchymal stem cells on a novel, auricular-shaped polymer. The proposed polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea nanocomposite polymer has already been transplanted in patients as the world’s first synthetic trachea, tear duct and vascular bypass graft. The nanocomposite scaffold was fabricated via a coagulation/salt-leaching method and shaped into an auricle. Adult bone marrow–derived mesenchymal stem cells were isolated, cultured and seeded onto the scaffold. On day 21, samples were sent for scanning electron microscopy, histology and immunofluorescence to assess for neocartilage formation. Cell viability assay confirmed cytocompatability and normal patterns of cellular growth at 7, 14 and 21 days after culture. This study demonstrates the potential of a novel polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea scaffold for culturing bone marrow–derived mesenchymal stem cells in chondrogenic medium to produce an auricular-shaped construct. This is supported by scanning electron microscopy, histological and immunofluorescence analysis revealing markers of chondrogenesis including collagen type II, SOX-9, glycosaminoglycan and elastin. To the best of our knowledge, this is the first report of stem cell application on an auricular-shaped scaffold for tissue engineering purposes. Although many obstacles remain in producing a functional auricle, this is a promising step forward. PMID:24555012

  15. Osteocytes and bone lining cells: Which are the best candidates for mechano-sensors in cancellous bone?

    Microsoft Academic Search

    M. G. Mullender; R. Huiskes

    1997-01-01

    Previously, we have investigated the possible role of osteocytes as mechano-sensors, and mediators of bone turnover. It was found that the proposed regulatory mechanism produced morphologies of trabecular bone, under particular loading conditions, which were consistent with morphogenesis and adaptation as seen in reality. The main objective of this study was to descern whether lining cells or osteoblasts could possibly

  16. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  17. Human Endothelial-Like Differentiated Precursor Cells Maintain Their Endothelial Characteristics When Cocultured with Mesenchymal Stem Cell and Seeded onto Human Cancellous Bone

    PubMed Central

    Wilhelm, Kerstin; Warzecha, Joerg; Frank, Johannes; Barker, John; Marzi, Ingo; Seebach, Caroline

    2013-01-01

    Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1? and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1? gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1? gene expression. PMID:23476102

  18. Isolation and Assessment of Mesenchymal Stem Cells Derived From Bone Marrow: Histologic and Histomorphometric Study in a Canine Periodontal Defect.

    PubMed

    Paknejad, Mojgan; Eslaminejad, Mohamadreza Baghaban; Ghaedi, Baharak; Rokn, Amir-Reza; Khorsand, Afshin; Etemad-Moghadam, Shahroo; Alaeddini, Mojgan; Dehghan, Mohammad Mehdi; Moslemi, Neda; Nowzari, Hessam

    2015-06-01

    The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 10(7) mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations. PMID:24383495

  19. Genotoxicity of sennosides on the bone marrow cells of mice.

    PubMed

    Mukhopadhyay, M J; Saha, A; Dutta, A; De, B; Mukherjee, A

    1998-11-01

    Preparations of a number of plants which contain hydroxyanthraquinones as active constituents are used worldwide for their laxative effect. Anthraquinone glycosides of Cassia angustifolia and C. fistula were investigated for their ability to induce a clastogenic effect on the bone marrow cells of Swiss albino mice. The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral exposure to doses of these anthraquinones and their equivalent amount in leaf and pod extracts did not induce significant numbers of chromosomal aberrations or aberrant cells. The results indicate that anthraquinone sennoside B and rhein are weakly genotoxic. PMID:9771555

  20. Signal transduction pathways involved in mechanotransduction in bone cells

    SciTech Connect

    Liedert, Astrid [Institute of Orthopedic Research and Biomechanics, University of Ulm (Germany)]. E-mail: astrid.liedert@uni-ulm.de; Kaspar, Daniela [Institute of Orthopedic Research and Biomechanics, University of Ulm (Germany); Blakytny, Robert [Institute of Orthopedic Research and Biomechanics, University of Ulm (Germany); Claes, Lutz [Institute of Orthopedic Research and Biomechanics, University of Ulm (Germany); Ignatius, Anita [Institute of Orthopedic Research and Biomechanics, University of Ulm (Germany)

    2006-10-13

    Several in vivo and in vitro studies with different loading regimens showed that mechanical stimuli have an influence on proliferation and differentiation of bone cells. Prerequisite for this influence is the transduction of mechanical signals into the cell, a phenomenon that is termed mechanotransduction, which is essential for the maintenance of skeletal homeostasis in adults. Mechanoreceptors, such as the integrins, cadherins, and stretch-activated Ca{sup 2+} channels, together with various signal transduction pathways, are involved in the mechanotransduction process that ultimately regulates gene expression in the nucleus. Mechanotransduction itself is considered to be regulated by hormones, the extracellular matrix of the osteoblastic cells and the mode of the mechanical stimulus.

  1. In situ delivery of bone marrow cells and mesenchymal stem cells improves cardiovascular function in hypertensive rats submitted to myocardial infarction

    Microsoft Academic Search

    Luisa Maria Gomes de Macedo Braga; Silvia Lacchini; Beatriz D’Agord Schaan; Bruno Rodrigues; Kaleizu Rosa; Kátia De Angelis; Luciano Figueiredo Borges; Maria Cláudia Irigoyen; Nance Beyer Nardi

    2008-01-01

    This work aimed to evaluate cardiac morphology\\/function and histological changes induced by bone marrow cells (BMCs) and cultured\\u000a mesenchymal stem cells (MSCs) injected at the myocardium of spontaneously hypertensive rats (SHR) submitted to surgical coronary\\u000a occlusion. Female syngeneic adult SHR, submitted (MI) or not (C) to coronary occlusion, were treated 24 h later with in situ\\u000a injections of normal medium (NM),

  2. A novel view of the adult bone marrow stem cell hierarchy and stem cell trafficking

    PubMed Central

    Ratajczak, M Z

    2015-01-01

    This review presents a novel view and working hypothesis about the hierarchy within the adult bone marrow stem cell compartment and the still-intriguing question of whether adult bone marrow contains primitive stem cells from early embryonic development, such as cells derived from the epiblast, migrating primordial germ cells or yolk sac-derived hemangioblasts. It also presents a novel view of the mechanisms that govern stem cell mobilization and homing, with special emphasis on the role of the complement cascade as a trigger for egress of hematopoietic stem cells from bone marrow into blood as well as the emerging role of novel homing factors and priming mechanisms that support stromal-derived factor 1-mediated homing of hematopoietic stem/progenitor cells after transplantation. PMID:25486871

  3. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  4. Raman spectroscopy: a powerful tool for the non-contact discrimination of bone marrow mesenchymal stem cells and fibroblasts

    NASA Astrophysics Data System (ADS)

    Pudlas, Marieke; Koch, Steffen; Bolwien, Carsten; Hirth, Thomas; Walles, Heike; Schenke-Layland, Katja

    2011-03-01

    Bone-marrow mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medicine. However, it is challenging to determine whether isolated BM-MSC populations are free of fibroblasts. We employed traditional methods and Raman spectroscopy to distinguish BM-MSCs and human dermal fibroblasts (hDFs). Although in vitro differentiation assays revealed the multipotent character of BM-MSCs, long culture periods are a major disadvantage. Using Raman spectroscopy, we could quickly distinguish between BM-MSCs and hDFs. Therefore we conclude that this method is sufficient for the rapid detection of fibroblastic contaminations in BM-MSC cultures.

  5. Shape memory polymers for active cell culture.

    PubMed

    Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

    2011-01-01

    Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

  6. Cell growth on pore-graded biomimetic TiO2 bone scaffolds.

    PubMed

    Müller, Benjamin; Reseland, Janne Elin; Haugen, Håvard Jostein; Tiainen, Hanna

    2015-04-01

    In order to prevent soft tissue down-growth into osseous defect areas, membranes are used when placing bone graft materials. These membranes still show shortcomings in their performance and applications. In the current study, we choose an approach to integrate micro-porous surface structures into a macro-porous scaffold. Low porous surfaces were fabricated by dip-coatings. Four different material compositions (titanium dioxide, polycaprolactone, polycaprolactone/water, polycaprolactone/?-tricalcium phosphate) were characterised in terms of their appearance, architecture, topographical features and cell response. Titanium dioxide surfaces exhibited rougher and more complex textures, resulting in the highest number of osteosarcoma cells and distinct morphologies in terms of cell spreading. Polycaprolactone-based surfaces showed a smoother topography and enhanced microporosity, but the effect on secretion of the bone markers sclerostin and interleukin-6 from human osteoblasts was lower compared to secretion from cells cultured on titanium dioxide. ?-Tricalcium phosphate modification of polycaprolactone did not show any significant improvement regarding cell-material interaction. Nevertheless, surfaces show potential in the mechanical blockage of epithelial and soft tissue cells and may still permit sufficient nutrient transport. PMID:25394623

  7. Uremic toxins impair human bone marrow-derived mesenchymal stem cells functionality in vitro.

    PubMed

    Idziak, Marta; P?dzisz, Piotr; Burdzi?ska, Anna; Gala, Kamila; P?czek, Leszek

    2014-07-01

    Mesenchymal stem cells (MSCs) are becoming therapeutic agents of interest in many areas of medicine, including renal diseases and kidney transplantations. However, the effect of uremia on cell properties is still unclear. Therefore, we examined the in vitro influence of uremic toxins, p-cresol (PC) and indoxyl sulfate (IS), on human bone marrow-derived MSC functionality. Cultured MSCs were treated with PC and IS at concentrations corresponding to subsequent stages of chronic kidney disease. Cell viability was characterized by metabolic activity (MTT assay) and proliferation rate (BrdU assay). Apoptosis (Annexin V test) and cell membrane damage (LDH assay) were also tested. MSC secretory properties were determined by measuring cytokine/growth factor levels in media from toxin-treated cells (ELISA). Uremic concentrations of PC and IS resulted in significant inhibition of MSC metabolic activity and proliferation. Toxins did not induce apoptosis, but damaged cell membranes. MSC paracrine activity was also altered - a decrease of VEGF and TGF-?1 levels and an increase in IGF-1 and IL-8 secretion was detected. Presented data indicate a negative influence of uremic toxins on functional characteristics of human bone marrow-derived MSCs. Therefore, their use as autologous therapeutic agents for kidney disease may be questionable and requires further investigations. The observed phenomenon may be attributable to many other MSC therapies, because of the high prevalence of chronic kidney disease in adult population. PMID:24548687

  8. High Frequency Retrotransposition in Cultured Mammalian Cells

    Microsoft Academic Search

    John V Moran; Susan E Holmes; Thierry P Naas; Ralph J DeBerardinis; Jef D Boeke; Haig H Kazazian

    1996-01-01

    We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were

  9. Effects of Electromagnetic Stimulation on Cell Density and Neural Markers in Murine Enteric Cell Cultures

    NASA Astrophysics Data System (ADS)

    Carreón-Rodríguez, A.; Belkind-Gerson, J.; Serrano-Luna, G.; Cañedo-Dorantes, L.

    2008-08-01

    Availability of adult stem cells from several organs like bone marrow, umbilical cord blood or peripheral blood has become a powerful therapeutic tool for many chronic diseases. Potential of adult stem cells for regeneration extents to other tissues among them the nervous system. However two obstacles should be resolved before such cells could be currently applied in clinical practice: a) slow growth rate and b) ability to form enough dense colonies in order to populate a specific injury or cellular deficiency. Many approaches have been explored as genetic differentiation programs, growth factors, and supplemented culture media, among others. Electromagnetic field stimulation of differentiation, proliferation, migration, and particularly on neurogenesis is little known. Since the biological effects of ELF-EMF are well documented, we hypothesize ELF-EMF could affect growth and maturation of stem cells derived of enteric tissue.

  10. CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

    Microsoft Academic Search

    Junjie Yang; Masaaki II; Naosuke Kamei; Cantas Alev; Sang-Mo Kwon; Atsuhiko Kawamoto; Hiroshi Akimaru; Haruchika Masuda; Yoshiki Sawa; Takayuki Asahara; Maurizio C. Capogrossi

    2011-01-01

    BackgroundEndothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow.Methodology\\/Principal FindingsCD34+ cells, c-Kit+\\/Sca-1+\\/Lin? (KSL) cells, c-Kit+\\/Lin? (KL) cells and Sca-1+\\/Lin? (SL) cells were isolated from

  11. Tributyltin Engages Multiple Nuclear Receptor Pathways and Suppresses Osteogenesis in Bone Marrow Multipotent Stromal Cells.

    PubMed

    Baker, Amelia H; Watt, James; Huang, Cassie K; Gerstenfeld, Louis C; Schlezinger, Jennifer J

    2015-06-15

    Organotins are members of the environmental obesogen class of contaminants because they activate peroxisome proliferator-activated receptor ? (PPAR?), the essential regulator of adipogenesis. Exposure to thiazolidinediones (PPAR? ligands used to treat type 2 diabetes) is associated with increased fractures. Diminished bone quality likely results from PPAR?'s role in promoting adipogenesis while suppressing osteogenesis of bone marrow multipotent mesenchymal stromal cells (BM-MSC). We hypothesized that tributyltin (TBT) would be a potent modifier of BM-MSC differentiation and a negative regulator of bone formation. Organotins interact with both PPAR? and retinoid X receptors (RXR), suggesting that they activate multiple nuclear receptor pathways. To investigate the role of RXR in the actions of TBT, the effects of PPAR? (rosiglitazone) and RXR (bexarotene, LG100268) agonists were compared to the effects of TBT in BMS2 cells and primary mouse BM-MSC cultures. In BMS2 cells, TBT induced the expression of Fabp4, Abca1, and Tgm2 in an RXR-dependent manner. All agonists suppressed osteogenesis in primary mouse BM-MSC cultures, based on decreased alkaline phosphatase activity, mineralization, and expression of osteoblast-related genes. While rosiglitazone and TBT strongly activated adipogenesis, based on lipid accumulation and expression of adipocyte-related genes, the RXR agonists did not. Extending these analyses to other RXR heterodimers showed that TBT and the RXR agonists activated the liver X receptor pathway, whereas rosiglitazone did not. Application of either a PPAR? antagonist (T0070907) or an RXR antagonist (HX531) significantly reduced rosiglitazone-induced suppression of bone nodule formation. Only the RXR antagonist significantly reduced LG100268- and TBT-induced bone suppression. The RXR antagonist also inhibited LG100268- and TBT-induced expression of Abca1, an LXR target gene, in primary BM-MSC cultures. These results provide novel evidence that TBT activates multiple nuclear receptor pathways in BM-MSCs, activation of RXR is sufficient to suppress osteogenesis, and TBT suppresses osteogenesis largely through its direct interaction with RXR. PMID:25932594

  12. Neural stem cells induce bone-marrow-derived mesenchymal stem cells to generate neural stem-like cells via juxtacrine and paracrine interactions

    SciTech Connect

    Alexanian, Arshak R. [Neuroscience Research Labs, Dept. of Neurosurgery, Medical College of Wisconsin, VAMC, 5000 W. National Ave. 151, Milwaukee, WI 53295 (United States)]. E-mail: aalexan@mcw.edu

    2005-11-01

    Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study, we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2, NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into {beta}-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs.

  13. Interactions of human bone cells with diamond-like carbon polymer hybrid coatings.

    PubMed

    Calzado-Martín, Alicia; Saldaña, Laura; Korhonen, Hannu; Soininen, Antti; Kinnari, Teemu J; Gómez-Barrena, Enrique; Tiainen, Veli-Matti; Lappalainen, Reijo; Munuera, Luis; Konttinen, Yrjö T; Vilaboa, Nuria

    2010-08-01

    Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin-TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration. PMID:20197124

  14. ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anthocyanins (ACNs) are potential oxygen radical scavengers that have coronary vasoactive and vasoprotective properties. Cell or tissue