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Sample records for bone cell cultures

  1. Phenotype expression of human bone cells cultured on implant substrates.

    PubMed

    Locci, P; Becchetti, E; Pugliese, M; Rossi, L; Belcastro, S; Calvitti, M; Pietrarelli, G; Staffolani, N

    1997-09-01

    Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [3H]-thymidine to evaluate cellular proliferation, and [3H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH4Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM. PMID:9377794

  2. 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

  3. New bone formation by murine osteoprogenitor cells cultured on corticocancellous allograft bone.

    PubMed

    Nelson, Ehren R; Huang, Zhinong; Ma, Ting; Lindsey, Derek; Jacobs, Christopher; Smith, Robert L; Goodman, Stuart B

    2008-12-01

    The gold standard for bone grafting in orthopedics is autograft, however autograft has a limited supply and is associated with significant morbidity at the harvest site. One alternative, allograft bone, provides an osteoconductive scaffold, is in less limited supply, and it does not require a harvest from the patient. However, allograft lacks both osteogenic cells and osteoinductive proteins that make autograft bone so advantageous. This study provides a model to investigate strategies for augmentation of corticocancellous allograft bone discs with bone marrow-derived osteoprogenitor cells (OPCs) plus exogenous growth factors in vitro. In this model, allograft bone discs were created by cutting 1-mm thick slices from the distal femur and proximal tibia of euthanized mice. The allografts were sterilized and scanned by micro-computed tomography (microCT) to provide the pre-culture graft volume and trabecular characteristics. The discs were then seeded with OPCs harvested from murine bone marrow. The seeded grafts were placed in organ culture until harvest, after which they were re-scanned by microCT and the data compared to the corresponding pre-culture data. In addition, bone morphogenetic protein-7 (BMP-7, also know as osteogenic protein-1 or OP-1), basic fibroblast growth factor (bFGF), and OP-1 combined with bFGF were added on a daily basis to the cultures. After final microCT scanning, all grafts were sectioned and evaluated histologically after hematoxylin and eosin (H&E) staining. microCT scans of cultured allografts with cells at 3, 5, and 9 weeks showed a time-dependent, statistically significant increase in bone volume. The trabecular thickness (Tb.Th.) of grafts, from both groups that were augmented with OP-1, showed a statistically significant increase in trabecular thickness of allografts with OPCs. These data suggest that bone marrow-derived OPCs adhere to, and produce, new bone on corticocancellous allograft in vitro. When exogenous OP-1 is added to

  4. In vivo bone formation by human bone marrow cells: effect of osteogenic culture supplements and cell densities.

    PubMed

    Mendes, S C; Van Den Brink, I; De Bruijn, J D; Van Blitterswijk, C A

    1998-12-01

    Bone marrow is known to contain a population of osteoprogenitor cells that can go through complete differentiation when cultured in a medium containing appropriate bioactive factors. In this study, porous particles of a calcium phosphate material were seeded with adult human bone marrow cells in the second passage. After an additional culture period of 1 wk in the particles, these hybrid constructs were subcutaneouslly implanted in nude mice with a survival period of 4 wk. The cell seeding densities range from 0-200 000 cells per particle and the cell culture system was designed to investigate the single and combined effects of dexamethasone and recombinant human bone morphogenetic protein 2 (rhBMP-2). The hybrid "material/tissue" constructs were processed for histology and the amount of de novo bone formation was quantified, for each culture condition, by histomorphometric techniques. The relative percentage of mineralized bone formation reached a maximal value of 19.77+/-5.06, for samples cultured in the presence of rhBMP-2 and with a seeding density of 200 000 cells/particle, compared to 0.52+/-0.45 for samples in which no cells had been cultured and had been incubated in culture medium supplemented with Dex and rhBMP-2. For the tested conditions and for the low cell numbers used in this study, rhBMP-2 proved to be an essential bioactive factor to obtain in vivo bone formation by our culture system. The results from this study prove the potential of cultured adult human bone marrow cells to initiate and accelerate de novo bone formation after transplantation into an ectopic site. PMID:15348953

  5. 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

  6. Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

    PubMed

    Martinovic, Snjezana; Mazic, Sanja; Kisic, Veronika; Basic, Nikolina; Jakic-Razumovic, Jasminka; Borovecki, Fran; Batinic, Drago; Simic, Petra; Grgurevic, Lovorka; Labar, Boris; Vukicevic, Slobodan

    2004-09-01

    Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors. PMID:15314083

  7. 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 )

    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.

  8. The effects of bone pâté on human osteoblasts cell cultures.

    PubMed

    Quaranta, Nicola; Buccoliero, Cinzia; De Luca, Concetta; Mori, Giorgio; Brunetti, Giacomina; Colucci, Silvia; Colaianni, Graziana; Grano, Maria

    2016-06-01

    The aim of the present study was to evaluate the effect of bone pate on human osteoblast differentiation by measuring cell viability, alkaline phosphatase activity and expression of the transcription factors and of the major components of the extracellular matrix. Although bone paté has been used in ear surgery for many years and when placed in contact with mastoid and external auditory canal bone become viable, the cellular mechanisms that lead to its osteointegration have never been described. Bone paté taken from four patients subjected to mastoidectomy and affected by middle ear and mastoid cholesteatoma was placed in contact with osteoblast-like cell cultures. Four experimental conditions were obtained: cell cultures treated with bone patè, with bone paté mixed with fibrin glue, with fibrin glue and untreated. After 24 h, the viability of the cells was evaluated; after 1 week, alkaline phosphatase activity and the expression of transcription factors and bone matrix proteins were assessed by quantitative polymerase chain reaction. After 24 h osteoblasts showed increased viability when treated with bone paté (19 % increase) and bone pate mixed with fibrin glue (34 % increase). After 1 week, the number of alkaline phosphatase positive cells increased by 97 and 94 % in cultures treated with bone paté alone and bone pate mixed with fibrin glue. Treatment with bone patè upregulated transcription factors and components of the extracellular matrix. The present data show that bone paté has a high osteoinductive potential on human osteoblasts, enhancing their activity. PMID:26133919

  9. Sertoli cells promote proliferation of bone marrow-derived mesenchymal stem cells in co-culture.

    PubMed

    Zhang, Fenxi; Lu, Ming; Liu, Hengxing; Ren, Tongming; Miao, Yingying; Wang, Jingjing

    2016-05-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source for cell transplantation. The proliferative ability of BMSCs is an important determinant of the efficiency of transplant therapy. Sertoli cells are "nurse" cells for development of sperm cells. Our recent study showed that Sertoli cells promoted proliferation of human umbilical cord mesenchymal stem cells (hUCMSCs) in co-culture. Studies by other groups also showed that Sertoli cells promoted growth of endothelial cells and neural stem cells. In this study, we investigated the effect of Sertoli cells on proliferation of BMSCs. Our results showed that Sertoli cells in co-culture significantly enhanced proliferation of BMSCs (P < 0.01). Moreover, co-culture with Sertoli cells also markedly increased mRNA and/or protein expressions of Mdm2, p-Akt and Cyclin D1, and decreased p53 expression in BMSCs (P < 0.01 or < 0.05). These findings indicate that Sertoli cells have the potential to enhance proliferation of BMSCs. PMID:27319049

  10. Vascularized Bone Tissue Formation Induced by Fiber-Reinforced Scaffolds Cultured with Osteoblasts and Endothelial Cells

    PubMed Central

    Liu, Xinhui; Zhang, Guoping; Hou, Chuanyong; Wang, Hua; Yang, Yelin; Guan, Guoping; Dong, Wei; Gao, Hongyang

    2013-01-01

    The repair of the damaged bone tissue caused by damage or bone disease was still a problem. Current strategies including the use of autografts and allografts have the disadvantages, namely, diseases transmission, tissue availability and donor morbidity. Bone tissue engineering has been developed and regarded as a new way of regenerating bone tissues to repair or substitute damaged or diseased ones. The main limitation in engineering in vitro tissues is the lack of a sufficient blood vessel system, the vascularization. In this paper, a new-typed hydroxyapatite/collagen composite scaffold which was reinforced by chitosan fibers and cultured with osteoblasts and endothelial cells was fabricated. General observation, histological observation, detection of the degree of vascularization, and X-ray examination had been done to learn the effect of vascularized bone repair materials on the regeneration of bone. The results show that new vessel and bone formed using implant cultured with osteoblasts and endothelial cells. Nanofiber-reinforced scaffold cultured with osteoblasts and endothelial cells can induce vascularized bone tissue formation. PMID:24369019

  11. 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.

  12. Bone Marrow Derived Eosinophil Cultures

    PubMed Central

    Lu, Thomas X.; Rothenberg, Marc E.

    2016-01-01

    Eosinophils are multifunctional effector cells implicated in the pathogenesis of a variety of diseases including asthma, eosinophil gastrointestinal disorders and helminth infection. Mouse bone marrow derived progenitor cells can be differentiated into eosinophils following IL-5 exposure. These bone marrow derived eosinophils are fully differentiated at the end of a 14 day culture based on morphology and expression of molecular markers.

  13. 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.

  14. Calcitonin receptors as markers for osteoclastic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures.

    PubMed

    Hattersley, G; Chambers, T J

    1989-09-01

    The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, but analysis of lineage and regulation of differentiation has been hampered by lack of a specific marker that enables identification of cells of osteoclastic phenotype. Previously used markers, such as multinuclearity, that are specific for osteoclasts in bone become less specific in culture. Uniquely among bone and bone marrow cells, osteoclasts possess abundant calcitonin (CT) receptors. We therefore tested the correlation between the generation of bone-resorptive function and the formation of CT receptor-positive cells from hemopoietic tissue in vitro. Without 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], a hormone that induces osteoclastic differentiation in vitro, bone marrow cultures showed very little bone resorption, and only small numbers of CT receptor-positive cells developed. When 1,25-(OH)2D3 was added to the cultures, CT receptor-positive cells developed within 1 day and reached a peak after 7 days. Bone resorption commenced within 2 days of hormone addition. There was a strong parallelism between the cumulative number of CT receptor-positive cells and the extent of bone resorption. The capacity of cultures to generate bone-resorptive activity and CT receptor-positive cells declined progressively when 1,25-(OH)2D3 was added to hemopoietic tissue after a 7- to 21-day hormone-free incubation period. The number of CT receptor-positive cells in these cultures correlated strongly (r = 0.96) with bone resorption. The behavior of these cultures suggests that 1,25-(OH)2D3 acts to induce terminal differentiation of osteoclast precursors present in the cultures, and that precursor cell numbers decreased with increasing time in vitro. All of the CT receptor-positive cells in control cultures and all of those seen shortly after 1,25-(OH)2D3 addition were mononuclear, despite considerable bone resorption; the majority of CT receptor-positive cells remained mononuclear

  15. Micropatterned Culture and Differentiation of Human Bone Marrow Mesenchymal Stem Cells Using a Polydimethylsiloxane Microstencil.

    PubMed

    Choi, Jin Ho; Bae, Jae-Sung; Lee, Hyun; Jin, Hee Kyung; Kim, Gyu Man

    2016-02-01

    A method for fabrication of polydimethylsiloxane (PDMS) microstencils was developed and its application to localized culture of human bone marrow mesenchymal stem cells (hMSCs) was tested. Unlike conventional culture methods, which culture cells on an entire surface, microscale cell culture provides precise control of the size and shape of stem cell patterns, and minimizes consumption of cells and culture media. A PDMS microstencil was fabricated by PDMS casting using an SU-8 mold prepared by photolithography. A pattern of 500-µm dots was tested. For the test, a PDMS microstencil was placed on a glass disk and cells were seeded on the stencil at a density of 5 x 10⁴ cells/cm². The hMSCs were cultured for 2 days at 37 °C in a humidified 5% CO2 atmosphere. The PDMS microstencil was removed after 2 days and the hMSC patterns were inspected under a microscope. The results confirmed that stem cells can be cultured using a PDMS microstencil. The micropatterned hMSCs retained their ability to differentiate into osteogenic and adipogenic cells. Thus, using a PDMS microstencil, stem cells can be cultured and differentiated in micropatterns in a precisely controlled manner, in any shape and size, for research and bioengineering applications. PMID:27305770

  16. Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures

    NASA Astrophysics Data System (ADS)

    Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.

    1983-07-01

    Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

  17. Mechanism of initial attachment of cells derived from human bone to commonly used prosthetic materials during cell culture.

    PubMed

    Howlett, C R; Evans, M D; Walsh, W R; Johnson, G; Steele, J G

    1994-02-01

    The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in cell culture. In this study the contribution that serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min of culture was determined for metallic and ceramic surfaces. The requirement for Fn or Vn for attachment and spreading of bone-derived cells onto stainless steel 316 (SS), titanium (Ti) and alumina (Al2O3) and to polyethyleneterephthalate (PET) was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of bone-derived cells onto SS, Ti and Al2O3 surfaces were reduced by 73-83% when the cells were seeded in medium containing serum from which the Vn had been removed. Cell attachment and spreading on these surfaces when seeded in medium containing Fn-depleted serum (which contained Vn) were not reduced to the same extent as in the medium containing Vn-depleted serum. The bone-derived cells failed to attach to the surfaces to the same extent when seeded in medium containing serum depleted of both Vn and Fn. Our results show that for human bone-derived cells, the attachment and spreading of cells onto SS, Ti and Al2O3 as well as PET during the first 90 min of a cell culture attachment assay are a function of adsorption of serum Vn onto the surface. PMID:7515290

  18. Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges.

    PubMed

    Zhou, Shuanhu; Yates, Karen E; Eid, Karim; Glowacki, Julie

    2005-01-01

    Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions. We first confirmed that hMSCs have chondrogenic potential when treated with TGF-beta, either in 2-D monolayer cultures or in 3-D porous collagen sponges. Second, we found that DBP markedly enhanced chondrogenesis in hMSCs in 3-D sponges, as assessed by metachromasia and expression of chondrocyte-specific genes AGGRECAN, COL II, and COL X. Human dermal fibroblasts (hDFs) were used to define mechanisms of chondroinduction because unlike hMSCs they have no inherent chondrogenic potential. In situ hybridization revealed that hDFs vicinal to DBPs express chondrocyte-specific genes AGGRECAN or COL II. Macroarray analysis showed that DBP activates TGF-beta/BMP signaling pathway genes in hDFs. Finally, DBP induced hMSCs to express the osteoblast phenotype when cultured with osteogenic supplements. These studies show how culture conditions can influence the differentiation pathway that human marrow stromal cells follow when stimulated by DBP. These results support the potential to engineer cartilage or bone in vitro by using human bone marrow stromal cells and DBP/collagen scaffolds. PMID:15735899

  19. Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture.

    PubMed

    Shu, Chang; Li, Ting Yu; Tsang, Lai Ling; Fok, Kin Lam; Lo, Pui Shan; Zhu, Jin Xia; Ho, Lo Sze; Chung, Yiu Wa; Chan, Hsiao Chang

    2006-10-01

    We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia. PMID:16877014

  20. Evaluation of Posterolateral Lumbar Fusion in Sheep Using Mineral Scaffolds Seeded with Cultured Bone Marrow Cells

    PubMed Central

    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 (β-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

  1. 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

  2. MDR1 gene expression enhances long-term engraftibility of cultured bone marrow cells

    SciTech Connect

    Rentala, Satyanarayana; Sagar Balla, Murali Mohan; Khurana, Satish; Mukhopadhyay, Asok . E-mail: asok@nii.res.in

    2005-09-30

    Primitive hematopoietic stem cells are responsible for long-term engraftment in irradiated host. Here, we report that multi-drug resistance 1 (mdr1) gene expressing primitive hematopoietic cells were multiplied in ex vivo culture, with the support of extracellular matrix components and cytokines. About 20-fold expansion of total nucleated cells was achieved in a 10-day culture. Lin{sup -}Sca-1{sup +} and long-term culture-initiating cells were increased by 54- and 26-fold, respectively. Expanded cells were long-term multi-lineage engraftible in sub-lethally irradiated mice. Donor-derived peripheral blood chimerism was significantly higher (73.2 {+-} 9.1%, p < 0.01) in expanded cells than in normal and 5-flurouracil-treated bone marrow cells. Most interestingly, the expression of mdr1 gene was significantly enhanced in cultured cells than in other two sources of donor cells. The mdr1 gene was functional since expanded cells effluxed Hoechst 33342 and Rh123 dyes. These results suggest that primitive engraftible stem cells can be expanded in the presence of suitable microenvironments.

  3. In vivo ectopic bone formation by devitalized mineralized stem cell carriers produced under mineralizing culture condition.

    PubMed

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

    2014-12-01

    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 (Ca(2+)) and phosphate (PO4 (3-)) 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

  4. 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

  5. Biochemical analysis of the response in rat bone marrow cell cultures to mechanical stimulation.

    PubMed

    Yoshikawa, T; Peel, S A; Gladstone, J R; Davies, J E

    1997-01-01

    Bone marrow cells obtained from rat femora were subjected to primary culture with 15% fetal bovine serum in the presence of 10(-8) M dexamethasone, and following trypsin treatment 5 days later were seeded on Petriperm dishes which have a flexible bottom. After a 2-day subculture, a cyclic stress consisting of a 1 s stretch (0.3% strain. 0.5 Hz) and a 1 s relaxation for 30 min every day was started. Culture tissue was removed on day 2 of the subculture (immediately prior to start of stimulation), and then on days 5 and 8 (3 and 6 days after the start of stimulation, respectively), at which times dry weight, DNA, alkaline phosphatase (ALP) activity, and bone Gla protein (BGP, osteocalcin) were measured. Both the dry weight and DNA showed a significant increase in the stimulated group by day 8, while the ALP activity showed a significant increase by day 5. The BGP began to increase in the stimulated group on day 5 in contrast to the control group in which it only increased on day 8. These results support the contention that mechanical stimulation promotes the differentiation of osteogenic cells and enhances bone formation. Since in this experimental model the acceleration of bone formation by mechanical stimulation can be reproduced in vitro, it is extremely useful for investigating the mechanisms underlying mechanical stimulation. PMID:9622104

  6. Nonspecific suppressor T cells cause decreased mixed lymphocyte culture reactivity in bone marrow transplant patients

    SciTech Connect

    Harada, M.; Ueda, M.; Nakao, S.; Kondo, K.; Odaka, K.; Shiobara, S.; Matsue, K.; Mori, T.; Matsuda, T.

    1986-07-15

    Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period.

  7. PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: in vitro cell culture studies.

    PubMed

    Milovac, Dajana; Gamboa-Martínez, Tatiana C; Ivankovic, Marica; Gallego Ferrer, Gloria; Ivankovic, Hrvoje

    2014-09-01

    In the present study, we examined the potential of using highly porous poly(ε-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. PMID:25063118

  8. Hematopoiesis on nylon mesh templates. I. Long-term culture of rat bone marrow cells.

    PubMed

    Naughton, B A; Preti, R A; Naughton, G K

    1987-01-01

    Rodent hematopoietic cells have been perpetuated in long-term bone marrow culture (LTBMC), provided that they were plated onto a pre-established layer of bone marrow stromal cells (fibroblasts, reticular cells, adipocytes, macrophages, etc.). This monolayer-type system supports the self-renewal of murine pluripotent stem cells and produces substantial numbers of progenitors and mature cells of the myeloid lineage. In an effort to increase the growth potential of cells in LTBMC, an adherent matrix of stromal cells was established on a pretreated nylon screen template. Subsequent seeding of hematopoietic cells onto this matrix occurred both on the surface of the adherent layer and in the interstices formed by the developing stromal cells. A three dimensional growth pattern of hematopoietic colonies and clusters was observed. Mature cells and late stage precursors of the myeloid and erythroid series were observed in the non-adherent layer for the duration of the experiment (39 weeks). Cells similar in appearance to small lymphocytes were also seen in both the non-adherent and adherent layers. Cells associated with the adherent layer of this LTBMC system displayed the ability to reconstitute hematopoiesis in the irradiated host. It is suggested that because of its three-dimensional nature the nylon mesh LTBMC system possesses a greater hematologic potential per unit area than monolayer-type LTBMC. PMID:3480934

  9. Growth of bone marrow and skeletal muscle side population stem cells in suspension culture.

    PubMed

    Pacak, Christina A; Cowan, Douglas B

    2014-01-01

    The ability to efficiently isolate and expand various stem cell populations in vitro is crucial for successful translation of cell-based therapies to the clinical setting. One such heterogeneous population that possesses a remarkable potential for the development of cell-based treatments for a variety of degenerative diseases and disorders is called the Side Population (SP). For many years, investigators have isolated these primitive cells based upon their ability to efflux the fluorophore Hoechst 33342. This attribute enabled separation of SP cells derived from multiple tissue sources from other endogenous cell populations using fluorescence-activated cell sorting (FACS). While all tissue-specific SP fractions appear to contain cells with multi-potent stem cell activity, the therapeutic utility of these cells has yet to be fully realized because of the scarcity of this fraction in vivo. In view of that, we developed a method to expand adult murine bone marrow and skeletal muscle-derived SP cells in vitro. Here, we describe a spinner-flask culture system that supports the growth of SP cells in suspension when they are combined with feeder cells cultured on spherical microcarriers. In this way, their distinguishing biological characteristics can be maintained, attachment-stimulated differentiation is avoided, and therapeutically relevant quantities of SP cells are generated. Modification of the described procedure may permit expansion of the SP from other relevant tissue sources and our method is amenable to establishing compliance with current good manufacturing practices. PMID:25173160

  10. 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.

  11. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

    PubMed

    Maegawa, Naoki; Kawamura, Kenji; Hirose, Motohiro; Yajima, Hiroshi; Takakura, Yoshinori; Ohgushi, Hajime

    2007-01-01

    It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering. PMID:18038421

  12. The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles.

    PubMed

    Bentivegna, Angela; Roversi, Gaia; Riva, Gabriele; Paoletta, Laura; Redaelli, Serena; Miloso, Mariarosaria; Tredici, Giovanni; Dalprà, Leda

    2016-01-01

    Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures. PMID:26880970

  13. The Effect of Culture on Human Bone Marrow Mesenchymal Stem Cells: Focus on DNA Methylation Profiles

    PubMed Central

    Bentivegna, Angela; Roversi, Gaia; Riva, Gabriele; Paoletta, Laura; Redaelli, Serena; Miloso, Mariarosaria; Tredici, Giovanni; Dalprà, Leda

    2016-01-01

    Human bone marrow mesenchymal stem cells (hBM-MSCs) are the best characterized multipotent adult stem cells. Their self-renewal capacity, multilineage differentiation potential, and immunomodulatory properties have indicated that they can be used in many clinical therapies. In a previous work we studied the DNA methylation levels of hBM-MSC genomic DNA in order to delineate a kind of methylation signature specific for early and late passages of culture. In the present work we focused on the modification of the methylation profiles of the X chromosome and imprinted loci, as sites expected to be more stable than whole genome. We propose a model where cultured hBM-MSCs undergo random modifications at the methylation level of most CGIs, nevertheless reflecting the original methylation status. We also pointed out global genome-wide demethylation connected to the long-term culture and senescence. Modification at CGIs promoters of specific genes could be related to the decrease in adipogenic differentiation potential. In conclusion, we showed important changes in CGIs methylation due to long-term in vitro culture that may affect the differentiation potential of hBM-MSCs. Therefore it is necessary to optimize the experimental conditions for in vitro expansion in order to minimize these epigenetic changes and to standardize safer procedures. PMID:26880970

  14. Proliferation, osteogenic differentiation, and distribution of rat bone marrow stromal cells in nonwoven fabrics by different culture methods.

    PubMed

    Ichinohe, Norihisa; Takamoto, Tomoaki; Tabata, Yasuhiko

    2008-01-01

    The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method. PMID:18333809

  15. Studies on canine bone marrow long-term culture: effect of stem cell factor.

    PubMed

    Neuner, E; Schumm, M; Schneider, E M; Guenther, W; Kremmer, E; Vogl, C; Büttner, M; Thierfelder, S; Kolb, H J

    1998-02-16

    Long-term culture of canine marrow cells allows in vitro studies of the hematopoietic system of the dog and characterization of early progenitor cells. Colonies of fresh marrow cells grew equally good in both agar or methylcellulose supplemented with fetal calf serum, while colonies of long-term cultures required agar-based medium containing human serum. Optimum colony growth was obtained when stem cell factor (SCF) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) were used as growth stimuli of colony forming units (CFU). Similar results were achieved with several cell culture media. Addition of hydrocortisone to long-term cultures improved clonogenic growth of cultured cells. Addition of 2-mercaptoethanol had no effect. Strong differences were observed in long-term culture with different horse serum lots and the addition of fetal calf serum to long-term culture suppressed CFU growth of cultured cells. Recharging of cultures with fresh marrow cells on day 7 of culture improved CFU growth only in the following week but had little effect on the outcome. Adding SCF to long-term cultures led to differentiation of more primitive cells and destruction of the stromal layer. Investigation of purified and cultured cell populations was possible when preestablished long-term cultures as stromal layers were used. Loss of long-term culture-initiating ability could be demonstrated in this system with lineage negative marrow cells expanded ex vivo with SCF and GM-CSF. PMID:9613468

  16. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation

    PubMed Central

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122

  17. New 3D-Culture Approaches to Study Interactions of Bone Marrow Adipocytes with Metastatic Prostate Cancer Cells

    PubMed Central

    Herroon, Mackenzie Katheryn; Diedrich, Jonathan Driscoll; Podgorski, Izabela

    2016-01-01

    Adipocytes are a major component of the bone marrow that can critically affect metastatic progression in bone. Understanding how the marrow fat cells influence growth, behavior, and survival of tumor cells requires utilization of in vitro cell systems that can closely mimic the physiological microenvironment. Herein, we present two new three-dimensional (3D) culture approaches to study adipocyte–tumor cell interactions in vitro. The first is a transwell-based system composed of the marrow-derived adipocytes in 3D collagen I gels and reconstituted basement membrane-overlayed prostate tumor cell spheroids. Tumor cells cultured under these 3D conditions are continuously exposed to adipocyte-derived factors, and their response can be evaluated by morphological and immunohistochemical analyses. We show via immunofluorescence analysis of metabolism-associated proteins that under 3D conditions tumor cells have significantly different metabolic response to adipocytes than tumor cells grown in 2D culture. We also demonstrate that this model allows for incorporation of other cell types, such as bone marrow macrophages, and utilization of dye-quenched collagen substrates for examination of proteolysis-driven responses to adipocyte- and macrophage-derived factors. Our second 3D culture system is designed to study tumor cell invasion toward the adipocytes and the consequent interaction between the two cell types. In this model, marrow adipocytes are separated from the fluorescently labeled tumor cells by a layer of collagen I. At designated time points, adipocytes are stained with BODIPY and confocal z-stacks are taken through the depth of the entire culture to determine the distance traveled between the two cell types over time. We demonstrate that this system can be utilized to study effects of candidate factors on tumor invasion toward the adipocytes. We also show that immunohistochemical analyses can be performed to evaluate the impact of direct interaction of prostate

  18. 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.

  19. 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.

  20. 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.

  1. Anti-epileptic drugs and bone loss: Phenytoin reduces pro-collagen I and alters the electrophoretic mobility of osteonectin in cultured bone cells.

    PubMed

    Wilson, Emma L; Garton, Mark; Fuller, Heidi R

    2016-05-01

    Phenytoin is an antiepileptic drug used in the management of partial and tonic-clonic seizures. In previous studies we have shown that valproate, another antiepileptic drug, reduced the amount of two key bone proteins, pro-collagen I and osteonectin (SPARC, BM-40), in both skin fibroblasts and cultured osteoblast-like cells. Here we show that phenytoin also reduces pro-collagen I production in osteoblast-like cells, but does not appear to cause a decrease in osteonectin message or protein production. Instead, a 24h exposure to a clinically relevant concentration of phenytoin resulted in a dose-dependent change in electrophoretic mobility of osteonectin, which was suggestive of a change in post-translational modification status. The perturbation of these important bone proteins could be one of the mechanisms to explain the bone loss that has been reported following long-term treatment with phenytoin. PMID:26999801

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

    SciTech Connect

    Ninomiya, Yuichi; Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi; Nishiyama, Masahiko

    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.

  3. 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

  4. Culture human mesenchymal stem cells with calcium phosphate cement scaffolds for bone repair.

    PubMed

    Weir, Michael D; Xu, Hockin H K

    2010-04-01

    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 + or - SD; n = 5) of (19.5 + or - 1.4) MPa, higher than (8.0 + or - 1.4) MPa of CPC control (p < 0.05). The percentage of live hMSCs on CPC-chitosan was (90.5 + or - 1.3)% at 8 days, matching (90.7 + or - 3.8)% of CPC control (p > 0.1). The CPC-chitosan surface area covered by the attached hMSCs increased from (51 + or - 11)% at 1 day to (90 + or - 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 + or - 0.93) [(mM pNpp/min)/(microg DNA)] versus control media (3.62 + or - 0.40) (p < 0.05). hMSCs in osteogenic media exhibited greater mineralization area of (47.5 + or - 19.7)% compared with (6.1 + or - 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

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

    SciTech Connect

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

    2010-09-17

    Research highlights: {yields} BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. {yields} Co-culture decreases proliferation by cellular self-regulatory mechanisms. {yields} Co-cultured cells present an activated proangiogenic phenotype. {yields} qRT-PCR and cluster analysis identify new target genes playing important roles. -- Abstract: 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. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

  6. 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

  7. Bone marrow culture

    MedlinePlus

    ... 2015 Updated by: Yi-Bin Chen, MD, Leukemia/Bone Marrow Transplant Program, Massachusetts General Hospital, Boston, MA. Also reviewed ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  8. A study of murine bone marrow cells cultured in bioreactors which create an environment which simulated microgravity

    NASA Technical Reports Server (NTRS)

    Lawless, Brother Desales

    1990-01-01

    Previous research indicated that mouse bone marrow cells could be grown in conditions of simulated microgravity. This environment was created in rotating bioreactor vessels. On three attempts mouse cells were grown successfully in the vessels. The cells reached a stage where the concentrations were doubling daily. Phenotypic analysis using a panel of monoclonal antibodies indicated that the cell were hematopoietic pluripotent stem cells. One unsuccessful attempt was made to reestablish the immune system in immunocompromised mice using these cells. Since last summer, several unsuccessful attempts were made to duplicate these results. It was determined by electron microscopy that the cells successfully grown in 1989 contained virus particles. It was suggested that these virally parasitized cells had been immortalized. The work of this summer is a continuation of efforts to grow mouse bone marrow in these vessels. A number of variations of the protocol were introduced. Certified pathogen free mice were used in the repeat experiments. In some attempts the medium of last summer was used; in others Dexture Culture Medium containing Iscove's Medium supplemented with 20 percent horse serum and 10-6 M hydrocortisone. Efforts this summer were directed solely to repeating the work of last summer. Plans were made for investigations if stem cells were isolated. Immortalization of the undifferentiated stem cell would be attempted by transfection with an oncogenic vector. Selective differentiation would be induced in the stem cell line by growing it with known growth factors and immune response modulators. Interest is in identifying any surface antigens unique to stem cells that would help in their characterization. Another goal was to search for markers on stem cells that would distinguish them from stem cells committed to a particular lineage. If the undifferentiated hematopoietic stem cell was obtained, the pathways that would terminally convert it to myeloid, lyphoid

  9. Cell biological effects of mechanical stimulations generated by focused extracorporeal shock wave applications on cultured human bone marrow stromal cells.

    PubMed

    Suhr, Frank; Delhasse, Yvonne; Bungartz, Gerd; Schmidt, Annette; Pfannkuche, Kurt; Bloch, Wilhelm

    2013-09-01

    Human bone marrow stromal cells (hBMSCs) bear tremendous clinical potential due to their immunomodulatory properties in transplantation settings and their contribution to tissue regeneration. In fact, they are among the most promising types of stem-like cells for therapeutic applications and are the subject of intense research. However, the clinical use of hBMSCs has been confounded by limitations in their availability; they are scarce cells cumbersome to isolate and purify. Additionally, they are difficult to target to the site of injury in regeneration experiments. In order to combat these limitations, focused extracorporeal shock waves (fESW, 0.2/0.3mJ∗mm(-2)) were applied to purified, cultured hBMSCs. fESW (0.2mJ∗mm(-2)) stimulations were found to increase hBMSCs' growth rate (p<0.05), proliferation (p<0.05), migration, cell tracking and wound healing (p<0.05, respectively), as well as to reduce the rate of apoptosis activation (p<0.05). The increase in hBMSC migration behavior was found to be mediated by active remodeling of the actin cytoskeleton as indicated by increased directed stress fiber formations (p<0.05). Furthermore, hBMSCs maintain their differentiation potentials after fESW treatment, whereas 0.2mJ∗mm(-2) is the most effective application. In conclusion, our results establish first-timely that hBMSCs' behavior can be modified and optimized in response to defined mechanical stimulation. These findings appear particularly promising as they suggest that mechanical stress preconditions hBMSCs for improved therapeutic performance without genetic manipulations and that mechanically preconditioned hBMSCs will be advantageous for hBMSC-based tissue regeneration. Therefore, this approach opens the door for exploiting the full potential of these cells in regenerative medicine. PMID:23880536

  10. Strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures.

    PubMed

    Querido, William; Campos, Andrea P C; Martins Ferreira, Erlon H; San Gil, Rosane A S; Rossi, Alexandre M; Farina, Marcos

    2014-09-01

    We evaluate the effects of strontium ranelate on the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures, a system that gave us the advantage of obtaining mineral samples produced exclusively during treatment. Cells were treated with strontium ranelate at concentrations of 0.05 and 0.5 mM Sr(2+). Mineral substances were isolated and analyzed by using a combination of methods: Fourier transform infrared spectroscopy, solid-state (1)H nuclear magnetic resonance, X-ray diffraction, micro-Raman spectroscopy and energy dispersive X-ray spectroscopy. The minerals produced in all cell cultures were typical bone-like apatites. No changes occurred in the local structural order or crystal size of the minerals. However, we noticed several relevant changes in the mineral produced under 0.5 mM Sr(2+): (1) increase in type-B CO3 (2-) substitutions, which often lead to the creation of vacancies in Ca(2+) and OH(-) sites; (2) incorporation of Sr(2+) by substituting slightly less than 10 % of Ca(2+) in the apatite crystal lattice, resulting in an increase in both lattice parameters a and c; (3) change in the PO4 (3-) environments, possibly because of the expansion of the lattice; (4) the Ca/P ratio of this mineral was reduced, but its (Ca+Sr)/P ratio was the same as that of the control, indicating that its overall cation/P ratio was preserved. Thus, strontium ranelate changes the composition and crystal structure of the biological bone-like apatite produced in osteoblast cell cultures. PMID:24859219

  11. 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-1α, 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

  12. Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies

    PubMed Central

    2012-01-01

    Background Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows (“BNP dams”). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. Results Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. Conclusion This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types. PMID:23110710

  13. 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.

  14. 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

  15. Bone Marrow Mesenchymal Stromal Cells from Clinical Scale Culture: In Vitro Evaluation of Their Differentiation, Hematopoietic Support, and Immunosuppressive Capacities.

    PubMed

    Fajardo-Orduña, Guadalupe R; Mayani, Héctor; Castro-Manrreza, Marta E; Flores-Figueroa, Eugenia; Flores-Guzmán, Patricia; Arriaga-Pizano, Lourdes; Piña-Sánchez, Patricia; Hernández-Estévez, Erika; Castell-Rodríguez, Andrés E; Chávez-Rueda, Adriana K; Legorreta-Haquet, María V; Santiago-Osorio, Edelmiro; Montesinos, Juan J

    2016-09-01

    The differentiation capacity, hematopoietic support, and immunomodulatory properties of human bone marrow mesenchymal stromal cells (BM-MSCs) make them attractive therapeutic agents for a wide range of diseases. Clinical scale cultures (CSCs) have been used to expand BM-MSCs for their use in cell therapy protocols; however, little is known about the functionality of the expanded cells. The main goal of the present study was to evaluate the functional characteristics of BM-MSCs expanded from CSCs to determine the quality of the cells for cellular therapy protocols. To address this issue, we analyzed the morphology, immunophenotype, differentiation potential (adipogenic, osteogenic and chondrogenic), hematopoietic support, and immunosuppressive capacity of BM-MSCs from short scale cultures (SSCs) and CSCs in a comparative manner. After 12 days of culture in CSCs (HYPERFlask System), BM-MSCs reached cell numbers of 125.52 × 10(6) ± 25.6 × 10(6) MSCs, which corresponded to the number of cells required for transplantation (∼1.7 × 10(6) MSCs/kg for a 70-kg patient). After expansion, BM-MSCs expressed the characteristic markers CD73, CD90, and CD105; however, expansion decreased their differentiation capacity toward the adipogenic, osteogenic, and chondrogenic lineages and their ability to inhibit T-cell proliferation compared with SSCs-MSCs. Importantly, CSCs-MSCs maintained the ability to support the proliferation and expansion of hematopoietic progenitor cells and the capacity to express the molecules, cytokines, and extracellular matrix proteins involved in the regulation of hematopoiesis. Our study highlights the need to evaluate the functional properties of the expanded BM-MSCs for verification of their quality for cell therapy protocols. PMID:27462977

  16. 3D cell culture and osteogenic differentiation of human bone marrow stromal cells plated onto jet-sprayed or electrospun micro-fiber scaffolds.

    PubMed

    Brennan, Meadhbh Á; Renaud, Audrey; Gamblin, Anne-Laure; D'Arros, Cyril; Nedellec, Steven; Trichet, Valerie; Layrolle, Pierre

    2015-08-01

    A major limitation of the 2D culture systems is that they fail to recapitulate the in vivo 3D cellular microenvironment whereby cell-cell and cell-extracellular matrix (ECM) interactions occur. In this paper, a biomaterial scaffold that mimics the structure of collagen fibers was produced by jet-spraying. This micro-fiber polycaprolactone (PCL) scaffold was evaluated for 3D culture of human bone marrow mesenchymal stromal cells (MSCs) in comparison with a commercially available electrospun scaffold. The jet-sprayed scaffolds had larger pore diameters, greater porosity, smaller diameter fibers, and more heterogeneous fiber diameter size distribution compared to the electrospun scaffolds. Cells on jet-sprayed constructs exhibited spread morphology with abundant cytoskeleton staining, whereas MSCs on electrospun scaffolds appeared less extended with fewer actin filaments. MSC proliferation and cell infiltration occurred at a faster rate on jet-sprayed compared to electrospun scaffolds. Osteogenic differentiation of MSCs and ECM production as measured by ALP, collagen and calcium deposition was superior on jet-sprayed compared to electrospun scaffolds. The jet-sprayed scaffold which mimics the native ECM and permits homogeneous cell infiltration is important for 3D in vitro applications such as bone cellular interaction studies or drug testing, as well as bone tissue engineering strategies. PMID:26238732

  17. The development of a mature collagen network in cartilage from human bone marrow stem cells in Transwell culture.

    PubMed

    Murdoch, Alan D; Hardingham, Timothy E; Eyre, David R; Fernandes, Russell J

    2016-03-01

    Damaged hyaline cartilage shows a limited capacity for innate repair. Potential sources of cells to augment the clinical repair of cartilage defects include autologous chondrocytes and mesenchymal stem cells. We have reported that culture of human bone marrow mesenchymal stem cells with specific growth and differentiation factors as shallow multilayers on Transwell permeable membranes provided ideal conditions for chondrogenesis. Rigid translucent cartilaginous disks formed and expressed cartilage-specific structural proteins aggrecan and type II collagen. We report here the analysis of the collagen network assembled in these cartilage constructs and identify key features of the network as it became mature during 28 days of culture. The type II collagen was co-polymerized with types XI and IX collagens in a fibrillar network stabilized by hydroxylysyl pyridinoline cross-links as in epiphyseal and hyaline cartilages. Tandem ion-trap mass-spectrometry identified 3-hydroxylation of Proline 986 and Proline 944 of the α1(II) chains, a post-translational feature of human epiphyseal cartilage type II collagen. The formation of a type II collagen based hydroxy-lysyl pyridinoline cross-linked network typical of cartilage in 28 days shows that the Transwell system not only produces, secretes and assembles cartilage collagens, but also provides all the extracellular mechanisms to modify and generate covalent cross-links that determine a robust collagen network. This organized assembly explains the stiff, flexible nature of the cartilage constructs developed from hMSCs in this culture system. PMID:26523516

  18. Effects of the bifunctional sulfoxide MMS350, a radiation mitigator, on hematopoiesis in long-term bone marrow cultures and on radioresistance of marrow stromal cell lines.

    PubMed

    Shinde, Ashwin; Epperly, Michael W; Cao, Shaonan; Franicola, Darcy; Shields, Donna; Wang, Hong; Wipf, Peter; Sprachman, Melissa M; Greenberger, Joel S

    2014-01-01

    The ionizing irradiation mitigator MMS350 prolongs survival of mice treated with total-body irradiation and prevents radiation-induced pulmonary fibrosis when added to drinking water at day 100 after thoracic irradiation. The effects of MMS350 on hematopoiesis in long-term bone marrow culture and on the radiobiology of derived bone marrow stromal cell lines were tested. Long-term bone marrow cultures were established from C57BL/6NTac mice and maintained in a high-humidity incubator, with 7% CO2 and the addition of 100 μM MMS350 at the weekly media change. Over 10 weeks in culture, MMS350 had no significant effect on maintenance of hematopoietic stem cell production, or on nonadherent cells or colony-forming units of hematopoietic progenitor cells. Stromal cell lines derived from non MMS350-treated long-term cultures or control stromal cells treated with MMS350 were radioresistant in the clonogenic survival curve assay. MMS350 is a non-toxic, highly water-soluble radiation mitigator that exhibits radioprotective effects on bone marrow stromal cells. PMID:24982210

  19. Osteogenic differentiation of CD271+ cells from rabbit bone marrow cultured on three phase PCL/TZ-HA bioactive scaffolds: comparative study with mesenchymal stem cells (MSCs)

    PubMed Central

    Colosimo, Alessia; Rofani, Cristina; Ciraci, Elisa; Salerno, Aurelio; Oliviero, Maria; Maio, Ernesto Di; Iannace, Salvatore; Netti, Paolo A; Velardi, Francesco; Berardi, Anna C

    2015-01-01

    Tissue engineering is one of the major challenges of orthopedics and trauma surgery for bone regeneration. Biomaterials filled with mesenchymal stem cells (MSCs) are considered the most promising approach in bone tissue engineering. Furthermore, our previous study showed that the multi-phase poly [ε-caprolactone]/thermoplastic zein-hydroxyapatite (PCL/TZ-HA) biomaterials improved rabbit (r) MSCs adhesion and osteoblast differentiation, thus demonstrating high potential of this bioengineered scaffold for bone regeneration. In the recent past, CD271 has been applied as a specific selective marker for the enrichment of MSCs from bone marrow (BM-MSCs). In the present study, we aimed at establishing whether CD271-based enrichment could be an efficient method for the selection of rBM-MSCs, displaying higher ability in osteogenic differentiation than non-selected rBM-MSCs in an in vitro system. CD271+ cells were isolated from rabbit bone marrow and were compared with rMSCs in their proliferation rate and osteogenic differentiation capability. Furthermore, rCD271+ cells were tested in their ability to adhere, proliferate and differentiate into osteogenic lineage, while growing on PCL/TZ-HA scaffolds, in comparison to rMSCs. Our result demonstrate that rCD271+ cells were able to adhere, proliferate and differentiate into osteoblasts when cultured on PCL/TZ-HA scaffolds in significantly higher levels as compared to rMSCs. Based on these findings, CD271 marker might serve as an optimal alternative MSCs selection method for the potential preclinical and clinical application of these cells in bone tissue regeneration. PMID:26550238

  20. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    SciTech Connect

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y. )

    1991-03-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.

  1. Vascular cell adhesion molecule-1 and the integrin VLA-4 mediate adhesion of human B cell precursors to cultured bone marrow adherent cells.

    PubMed Central

    Ryan, D H; Nuccie, B L; Abboud, C N; Winslow, J M

    1991-01-01

    Adhesion of B cell precursors to accessory cells in the bone marrow microenvironment may be required for normal early B cell development. Human bone marrow B cell precursors adhere more avidly than mature B cells to bone marrow-derived fibroblasts. To determine the mechanism of this adhesion, expression of adhesion proteins on human B precursor cells and cell lines was measured by flow cytometry. The very late antigen (VLA) integrins VLA-4 and VLA-5 were the only adhesion proteins expressed at higher levels in B cell precursors than mature B cells. Antibodies to the alpha and beta chains of VLA-4, but not VLA-5, significantly blocked binding to bone marrow-derived fibroblasts of immature B cells and cell lines. Although fibronectin is a ligand for VLA-4, anti-fibronectin antibody and a soluble fibronectin fragment containing the VLA-4 binding domain did not block adhesion, suggesting that VLA-4 is involved in adhesion of B cell precursors, but not as a fibronectin receptor. Vascular cell adhesion molecule-1 (VCAM-1), the other known counterreceptor for VLA-4, was identified on bone marrow-derived fibroblasts, and anti-VCAM-1 significantly blocked adhesion of normal B cell precursors to bone marrow-derived fibroblasts, indicating that VLA-4/VCAM-1 interactions are important in adhesion of B cell precursors to the bone marrow microenvironment. Images PMID:1715889

  2. Beneficial reciprocal effects of bone marrow stromal cells and Schwann cells from adult rats in a dynamic co-culture system in vitro without intercellular contact

    PubMed Central

    ZHOU, LI-NA; CUI, XIAO-JUN; SU, KAI-XIN; WANG, XIAO-HONG; GUO, JIN-HUA

    2015-01-01

    In order to examine how implanted bone marrow stromal cells (BMSCs) encourage peripheral nerve regeneration, the present study investigated the interaction of BMSCs and Schwann cells (SCs) using an indirect in vitro co-culture model. SCs and BMSCs were obtained from adult Sprague-Dawley rats. The passaged BMSCs were CD29- and CD44-positive but CD45-negative and were co-cultured with the primary SCs using a Millicell system, which allows BMSCs and SCs to grow in the same culture medium but without direct contact. Expression of the typical SC markers S-100 and glial fibrillary acidic protein (GFAP) of the treated BMSCs as well as the proliferation capacity of the co-cultured SCs was evaluated by immunocytochemical staining on the 3rd and 5th day of co-culture. Immunocytochemical staining showed that >75% of the BMSCs in the indirect co-culture model were GFAP- and S-100-positive on the 3rd and 5th day after co-culture, as opposed to <5% of the BMSCs in the control group. On the 3rd day after co-culture, only a few co-cultured BMSCs showed the typical SC-like morphology, while most BMSCs still kept their native appearance. By contrast, on the 5th day after co-culture, almost all of the co-cultured BMSCs appeared with the typical SC-like morphology. Furthermore, 70.71% of the SCs in the indirect co-culture model were S-100-positive on the 5th day of co-culture, as opposed to >30.43% of the SCs in the control group. These results indicated that BMSCs may interact synergistically with SCs with regard to promoting peripheral nerve regeneration. PMID:26133460

  3. Bone marrow-derived mesenchymal stem cells in three-dimensional culture promote neuronal regeneration by neurotrophic protection and immunomodulation.

    PubMed

    Han, Sufang; Wang, Bin; Li, Xing; Xiao, Zhifeng; Han, Jin; Zhao, Yannan; Fang, Yongxiang; Yin, Yanyun; Chen, Bing; Dai, Jianwu

    2016-07-01

    Accumulating evidence has revealed three-dimensional (3D) culture could better mimic the stem cell niche in vivo in comparison with conventional two-dimensional (2D) culture. In this study, we found that bone marrow derived mesenchymal stem cells (BMSCs) cultured in 3D collagen scaffold (3D BMSCs) exhibited distinctive features including significantly enhancing neurotrophic factor secretions and reducing macrophage activations challenged by lipopolysaccharide (LPS) in vitro. To further evaluate 3D BMSCs' potential benefits to the regeneration of spinal cord injury (SCI), the 3D and 2D BMSCs were respectively implanted in rat hemisected SCI. Compared with 2D cohort, 3D BMSCs transplantation significantly reduced the expressions of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 at 5 days after transplantation, markedly enhanced axonal regeneration, and promoted motor functional recovery during 8 weeks of observation. When Nocodazole was used to depolymerize the cytoskeleton of 3D BMSCs, the changed expressions of neurotrophic factors and inflammatory cytokines were blunted, at least partially. Thus synergistic effects of neuronal protection and immunomodulation of 3D BMSCs may lead to a better functional recovery of SCI and the underlying mechanism may involve the alteration of their cellular morphology because of 3D culture. This study contributes to a better understanding of the cellular characteristics of 3D BMSCs and provides a novel strategy to promote the repair of the injured spinal cord. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1759-1769, 2016. PMID:26990583

  4. Selective growth of freshly isolated human breast epithelial cells cultured at low concentrations in the presence or absence of bone marrow cells.

    PubMed

    Emerman, J T; Stingl, J; Petersen, A; Shpall, E J; Eaves, C J

    1996-01-01

    In this study, we show that conditions previously found to promote the selective growth of human breast epithelial cells (HBEC) in serum-free primary cultures established from normal or malignant tissue can be extended to cultures initiated at low seeding densities (< 5000 cells/cm2). The epithelial nature of the cells produced was documented by their positive staining with antibodies specific for keratins 8, 14, and 18, and 2 antibodies that recognize epithelial-specific antigens (Ber-EP4 and HB8630). HBEC growth was not affected, either positively or negatively, by the use of a medium containing a combination of fetal calf and horse serum, which promotes the growth of many types of stromal cells and associated hematopoietic precursors, or by the inclusion in the initial cell suspension of marrow cells at HBEC to marrow cell ratios typical of bone marrow samples from patients with metastatic breast cancer. The presence of fibroblast feeders from a variety of sources enhanced the growth of HBEC to different degrees. In cultures initiated with low numbers of cells obtained from samples of breast carcinoma, HBEC growth was generally reduced by comparison to cultures of normal HBEC. With the detection methods used, it was not possible to determine the extent to which this decreased growth was due to a reduced frequency of malignant HBEC with in vitro precursor activity, or the presence of reduced numbers of residual normal HBEC precursors, or both. However, preliminary data indicate that this approach also allows the detection of some breast carcinoma cells with proliferative ability that are present in the marrow or pleural effusions of some breast cancer patients. These studies demonstrate the feasibility of detecting normal and malignant HBEC with growth potential when these are cultured at low density and/or as rare contaminants of marrow cell suspensions, and provide a starting point for their further characterization. PMID:8944333

  5. Bone marrow (stem cell) donation

    MedlinePlus

    Stem cell transplant; Allogeneic-donation ... There are two types of bone marrow donation: Autologous bone marrow transplant is when people donate their own bone marrow. "Auto" means self. Allogenic bone marrow transplant is when another person ...

  6. Culture perfusion schedules influence the metabolic activity and granulocyte-macrophage colony-stimulating factor production rates of human bone marrow stromal cells.

    PubMed

    Caldwell, J; Palsson, B O; Locey, B; Emerson, S G

    1991-05-01

    The metabolic function and GM-CSF production rates of adherent human bone marrow stromal cells were investigated as functions of medium and serum feeding rates. A range of medium exchange schedules was studied, ranging from a typical Dexter culture protocol of one weekly medium exchange to a full media exchange daily, which more closely approximates what bone marrow cells experience in situ. Glucose consumption was found to be significantly higher at full daily exchange rate than at any other exchange schedule examined. However, the lactate yield on glucose was a constant, at 1.8 mol/mol, under all conditions considered. Differential serum vs. medium exchange experiment showed that both serum supply and medium nutrients were responsible for the altered behavior at high exchange rates. Glutamine consumption was found to be insignificant under all culture conditions examined. A change in exchange schedule from 50% daily medium exchange to full daily medium exchange after 14 days of culture was found to result in a transient production of GM-CSF and a change in metabolic behavior to resemble that of cultures which had full daily exchange from day one. These results suggest that both stromal cell metabolism and GM-CSF production are sensitive to medium exchange schedules. Taken together, the data presented indicate that attempts to model the function of human bone marrow in vitro may be well served by beginning with medium exchange schedules that more closely mimic the in vivo physiologic state of bone marrow. PMID:2040665

  7. Strontium ranelate increases the formation of bone-like mineralized nodules in osteoblast cell cultures and leads to Sr incorporation into the intact nodules.

    PubMed

    Querido, William; Farina, Marcos

    2013-11-01

    We describe effects of strontium ranelate treatment on intact mineralized nodules produced in osteoblast cell cultures. We analyzed the matrix directly at the cell culture surfaces following treatment with 0.05 and 0.5 mM Sr(2+). This method allowed for data to be obtained from intact nodules, rather than from extracted samples. The bone-like nature of the matrix was evaluated by using attenuated total reflection Fourier transform infrared spectroscopy and the incorporation of Sr into the nodules was investigated by using both energy dispersive X-ray spectroscopy and synchrotron radiation micro X-ray fluorescence. We observed typical mineralized nodules in all of the cell cultures. However, the formation of these nodules was markedly increased in cultures treated with 0.5 mM Sr(2+). In all of the cultures, the nature of the intact matrix was similar to that described in native bone tissue, being comprised of a poorly crystalline CO3 (2-)-containing apatite and a collagenous matrix. This indicated that treatment had no deleterious effects on the matrix. Moreover, the nodules presented Ca and P as the main chemical components, confirming their bone-like mineralized nature. The incorporation of Sr into the nodules was clearly observed in the treated cultures, with their relative Sr content [Sr/(Ca+Sr) ratio] being markedly increased in a dose-dependent manner. Thus, strontium ranelate promoted an increase in the formation of mineralized nodules in osteoblast cell cultures while preserving the bone-like nature of the matrix at the tissue level. We further demonstrated that Sr was incorporated into the intact nodules formed during treatment. PMID:23774883

  8. Cadium-induced bone loss: Effects in ovariectomized mice and osteoclast-like cells in culture

    SciTech Connect

    Bhattacharyya, M.H.; Seed, T.M.; Peterson, D.P.; Moretti, E.S.; Kuhn, C.; Brice, L.F.; Choi, T.T.

    1988-01-01

    The research reported here was conducted to investigate the possibility that cadmium might be a factor that increases bone loss after the menopause. In our first study, we exposed female CF1 mice to a purified diet containing CdCl2 at either 0.25, 5.0, or 50 ppM Cd starting at 70 days of age. After 12 months of exposure, mice were ovariectomized (OV) or sham-operated (SO). After surgery, they remained on their respective diets for an additional six months before sacrifice. Results showed that neither ovariectomy alone nor dietary Cd exposure alone significantly decrease bone calcium content. However, dietary Cd at 50 ppM in combination with ovariectomy caused a striking decrease in the calcium content of mouse bones. The mice in the above study were quite old (435 days old at ovariectomy; 617 days old at sacrifice) and had been exposed to dietary cadmium for one year prior to removal of the ovaries. Consequently, the follow-up study reported here was conducted in mice whose skeletons were pre-labelled with UVCa. This study was designed to determine whether cadmium exposure would cause as increased release of UVCa from the skeletons of OV mice immediately after the start of cadmium exposure and in the absence of the one-year pre-exposure period present in our first study. Such results would indicate that cadmium might act directly on bone rather than indirectly by way of damage to another organ such as the kidney. 14 refs., 1 fig.

  9. Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering.

    PubMed

    Chang, Zhengqi; Hou, Tianyong; Xing, Junchao; Wu, Xuehui; Jin, Huiyong; Li, Zhiqiang; Deng, Moyuan; Xie, Zhao; Xu, Jianzhong

    2014-01-01

    To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF. PMID:25329501

  10. Umbilical Cord Wharton’s Jelly Repeated Culture System: A New Device and Method for Obtaining Abundant Mesenchymal Stem Cells for Bone Tissue Engineering

    PubMed Central

    Xing, Junchao; Wu, Xuehui; Jin, Huiyong; Li, Zhiqiang; Deng, Moyuan; Xie, Zhao; Xu, Jianzhong

    2014-01-01

    To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton’s jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton’s jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15–20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton’s jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF. PMID:25329501

  11. 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

  12. 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

  13. Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials.

    PubMed

    Wang, Yichao; Uemura, Toshimasa; Dong, Jian; Kojima, Hiroko; Tanaka, Junzo; Tateishi, Tetsuya

    2003-12-01

    Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the

  14. Enhanced neuro-therapeutic potential of Wharton's Jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture.

    PubMed

    Drela, Katarzyna; Lech, Wioletta; Figiel-Dabrowska, Anna; Zychowicz, Marzena; Mikula, Michał; Sarnowska, Anna; Domanska-Janik, Krystyna

    2016-04-01

    Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency. PMID:26971678

  15. A Functional Assay to Assess Connexin 43-Mediated Cell-to-Cell Communication of Second Messengers in Cultured Bone Cells.

    PubMed

    Stains, Joseph P; Civitelli, Roberto

    2016-01-01

    Cell-to-cell transfer of small molecules is a fundamental way by which multicellular organisms coordinate function. Recent work has highlighted the complexity of biologic responses downstream of gap junctions. As the connexin-regulated effectors are coming into focus, there is a need to develop functional assays that allow specific testing of biologically relevant second messengers. Here, we describe a modification of the classic gap junction parachute assay to assess biologically relevant molecules passed through gap junctions. PMID:27207296

  16. 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.; 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)

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

    PubMed Central

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

    2015-01-01

    Gja1Jrt/+ mice carry a mutation in one allele of the gap junction protein α1 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 Gja1Jrt/+ 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. Gja1Jrt/+ 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 Gja1Jrt/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1Jrt/+ 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 Gja1Jrt/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1Jrt/+ mouse model. PMID:25568340

  18. Dynamic perfusion bioreactor system for 3D culture of rat bone marrow mesenchymal stem cells on nanohydroxyapatite/polyamide 66 scaffold in vitro.

    PubMed

    Qian, Xu; Yuan, Fang; Zhimin, Zhu; Anchun, Mo

    2013-08-01

    The aim of the study was to investigate the biocompatibility and osteogenic effectiveness of the porous nanohydroxyapatite/polyamide 66 (n-HA/PA66) scaffold material that was cultured with the rat bone marrow mesenchymal stem cells (rBMSCs), under the static culture condition and the dynamic perfusion culture condition in vitro, and to investigate whether the 3D perfusion culture condition was better in provoking proliferation of rBMSCs than the 3D static culture condition. The Methyl thiazolyl tetrazolium (MTT) assay, alkaline phosphatase (ALP) activity assay, Osteocalcin (OCN) assay and scanning electron microscope (SEM) were used to observe the proliferation and differentiation of rBMSCs. The samples were respectively harvested at 1st, 3rd, 7th, 14th, and 21st days and effect comparisons were made between the two of the culture conditions. The results showed that values of MTT, ALP, and OCN were increased continuously and revealed a significant difference between the two culture conditions (p < 0.05). On the 14th day, SEM revealed calcified nodules 2-8 μm in diameter in the lamellar structure. Under the static culture condition, the pores were covered with the cells looking like a piece of blanket, but under the perfusion culture condition the cells were observed to have a 3D lamellar structure. In conclusion, the porous n-HA/PA66 scaffold material can be used as a good candidate material for the bone scaffold construction in the tissue engineering because of its excellent 3D structure, which can greatly improve the proliferation and differentiation of rBMSCs and make them proliferate and osteogenesis even better under the perfusion culture condition. PMID:23362119

  19. Effects of fluid flow and calcium phosphate coating on human bone marrow stromal cells cultured in a defined 2D model system.

    PubMed

    Scaglione, S; Wendt, D; Miggino, S; Papadimitropoulos, A; Fato, M; Quarto, R; Martin, I

    2008-08-01

    In this study, we investigated the effect of the long-term (10 days) application of a defined and uniform level of fluid flow (uniform shear stress of 1.2 x 10(-3) N/m(2)) on human bone marrow stromal cells (BMSC) cultured on different substrates (i.e., uncoated glass or calcium phosphate coated glass, Osteologictrade mark) in a 2D parallel plate model. Both exposure to flow and culture on Osteologic significantly reduced the number of cell doublings. BMSC cultured under flow were more intensely stained for collagen type I and by von Kossa for mineralized matrix. BMSC exposed to flow displayed an increased osteogenic commitment (i.e., higher mRNA expression of cbfa-1 and osterix), although phenotype changes in response to flow (i.e., mRNA expression of osteopontin, osteocalcin and bone sialoprotein) were dependent on the substrate used. These findings highlight the importance of the combination of physical forces and culture substrate to determine the functional state of differentiating osteoblastic cells. The results obtained using a simple and controlled 2D model system may help to interpret the long-term effects of BMSC culture under perfusion within 3D porous scaffolds, where multiple experimental variables cannot be easily studied independently, and shear stresses cannot be precisely computed. PMID:17969030

  20. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

    PubMed

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2016-08-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use. PMID:25777046

  1. Correlation of ionizing irradiation-induced late pulmonary fibrosis with long-term bone marrow culture fibroblast progenitor cell biology in mice homozygous deletion recombinant negative for endothelial cell adhesion molecules.

    PubMed

    Epperly, Michael W; Guo, Hongliang; Shields, Donna; Zhang, Xichen; Greenberger, Joel S

    2004-01-01

    Ionizing irradiation damage to the lung is associated with an acute inflammatory reaction, followed by a latent period and then late effects including predominantly pulmonary fibrosis. The cells mediating fibrosis have recently been shown to derive from the bone marrow hematopoietic microenvironment. Initiation of late pulmonary irradiation lung damage has been correlated with up-regulation of VCAM-1 and ICAM-1 in pulmonary endothelial cells, followed by infiltration of macrophages and bone marrow-derived fibroblasts forming the fibrotic lesions of organizing alveolitis/fibrosis. To determine whether the absence of expression of VCAM-1, ICAM-1, or other adhesion molecules known to be relevant to inflammatory cell attachment to lung endothelial cells was associated with a decrease in irradiation-induced lung fibrosis, homozygous deletion recombinant knockout mice lacking each of several adhesion molecules were tested compared to littermates for survival and development of organizing alveolitis following 20 Gy irradiation to both lungs. Bone marrow culture longevity has been shown to be a parameter, which correlates with both hematopoietic stem cell reserve and the integrity of fibroblast progenitors of the supportive hematopoietic microenvironment; radiation lung survival data were correlated to longevity of hematopoiesis in long-term bone marrow cultures established from tibia and femur bone marrow of the same mice. Homozygous deletion recombinant negative mice including VCAM-1-/-, ICAM-1-/-, E-Selectin-/-, or L-Selectin-/- were irradiated to 20 Gy to both lungs and followed for survival and percent organizing alveolitis at time of death compared to each normal littermate. A significant increase in survival (median 190 days) was detected with L-Selectin-/- compared to littermate control mice (median 140 days) or other groups. Long-term bone marrow cultures from L-Selectin-/- mice showed no detectable difference in marrow fibroblasts or hematopoietic cell biology

  2. Interactions between Mesenchymal Stem Cells, Adipocytes, and Osteoblasts in a 3D Tri-Culture Model of Hyperglycemic Conditions in the Bone Marrow Microenvironment

    PubMed Central

    Rinker, Torri E.; Hammoudi, Taymour M.; Kemp, Melissa L.; Lu, Hang; Temenoff, Johnna S.

    2014-01-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts in high glucose levels. In contrast, MSCs had no reduction of viability or clonogenicity when cultured with adipocytes in high glucose conditions and adipogenic gene expression indicated that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis. PMID:24463781

  3. Effect of cadmium on bone resorption in cultured fetal bone

    SciTech Connect

    Miyahara, T.; Miyakoshi, M.; Kozuka, H.

    1980-08-01

    Itai-itai disease which occurred in Toyama Prefecture, Japan, was thought to be due, at least partly, to chronic cadmium poisoning. Patients suffered severe pain in the waist, back and joints as well as kyphosis spinal column. In addition, x-ray film of these patients revealed abnormalities in the humerus and ribs. These bone lesions have been considered to be caused secondarily by dysfunction of other tissues, especially that of the kidneys, but there are some reports that the bone lesions appear before the occurrence of pathological changes in the kidneys of Cd-administered rat. It is currently unclear whether bone lesions by Cd are due to the direct action on the bone or indirect action which is caused by dysfunction of the kidney or intestine. To clarify the direct action of Cd on the bone, we studied the effect of Cd on the ossification of chick-embryo cultured bones biochemically and histologically. The results showed that Cd inhibited the bone matrix formation and brought about a malfunction in the ossification process. In the present work the effect of Cd on demineralization was studied using /sup 45/Ca-prelabeled bone in tissue culture and low levels of Cd were found to stimulate /sup 45/Ca from the bone.

  4. 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 β-tricalcium phosphate (β-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 β-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

  5. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao; Li, Ang; Xiao, Zhi-Cheng

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  6. Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-Like Cells In Vitro and Co-Cultured with Biological Scaffold as Transplantation Carrier.

    PubMed

    Yue, Wei; Yan, Feng; Zhang, Yue-Lin; Liu, Shu-Ling; Hou, Shu-Ping; Mao, Guo-Chao; Liu, Ning; Ji, Yong

    2016-01-01

    BACKGROUND Autograft and allograft transplantation are used to prompt the regeneration of axons after nerve injury. However, the poor self-regeneration caused by the glial scar and growth inhibitory factors after neuronal necrosis limit the efficacy of these methods. The purpose of this study was to develop a new chitosan porous scaffold for cell seeding. MATERIAL AND METHODS The bone marrow mesenchymal stem cells (BMSCs) and tissue-engineered biomaterial scaffold compound were constructed and co-cultured in vitro with the differentiated BMSCs of Wistar rats and chitosan scaffold in a 3D environment. The purity of the third-generation BMSCs culture was identified using flow cytometry and assessment of induced neuronal differentiation. The scaffolds were prepared by the freeze-drying method. The internal structure of scaffolds and the change of cells' growth and morphology were observed under a scanning electron microscope. The proliferation of cells was detected with the MTT method. RESULTS On day 5 there was a significant difference in the absorbance value of the experimental group (0.549±0.0256) and the control group (0.487±0.0357) (P>0.05); but on day 7 there was no significant difference in the proliferation of the experimental group (0.751±0.011) and the control group and (0.78±0.017) (P>0.05). CONCLUSIONS Tissue engineering technology can provide a carrier for cells seeding and is expected to become an effective method for the regeneration and repair of nerve cells. Our study showed that chitosan porous scaffolds can be used for such purposes. PMID:27225035

  7. Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation.

    PubMed

    Oliveira, J M; Kotobuki, N; Tadokoro, M; Hirose, M; Mano, J F; Reis, R L; Ohgushi, H

    2010-05-01

    Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10(-8) M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quantified, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be beneficial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation. PMID:20152952

  8. In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells, employing a two-chambered co-culture well design.

    PubMed

    Chen, Kelei; Ng, Kian Siang; Ravi, Sujata; Goh, James C H; Toh, Siew Lok

    2016-04-01

    The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd. PMID:23495238

  9. Differentiation of Rat Bone Marrow Mesenchymal Stem Cells Into Neuron-Like Cells In Vitro and Co-Cultured with Biological Scaffold as Transplantation Carrier

    PubMed Central

    Yue, Wei; Yan, Feng; Zhang, Yue-Lin; Liu, Shu-Ling; Hou, Shu-Ping; Mao, Guo-Chao; Liu, Ning; Ji, Yong

    2016-01-01

    Background Autograft and allograft transplantation are used to prompt the regeneration of axons after nerve injury. However, the poor self-regeneration caused by the glial scar and growth inhibitory factors after neuronal necrosis limit the efficacy of these methods. The purpose of this study was to develop a new chitosan porous scaffold for cell seeding. Material/Methods The bone marrow mesenchymal stem cells (BMSCs) and tissue-engineered biomaterial scaffold compound were constructed and co-cultured in vitro with the differentiated BMSCs of Wistar rats and chitosan scaffold in a 3D environment. The purity of the third-generation BMSCs culture was identified using flow cytometry and assessment of induced neuronal differentiation. The scaffolds were prepared by the freeze-drying method. The internal structure of scaffolds and the change of cells’ growth and morphology were observed under a scanning electron microscope. The proliferation of cells was detected with the MTT method. Results On day 5 there was a significant difference in the absorbance value of the experimental group (0.549±0.0256) and the control group (0.487±0.0357) (P>0.05); but on day 7 there was no significant difference in the proliferation of the experimental group (0.751±0.011) and the control group and (0.78±0.017) (P>0.05). Conclusions Tissue engineering technology can provide a carrier for cells seeding and is expected to become an effective method for the regeneration and repair of nerve cells. Our study showed that chitosan porous scaffolds can be used for such purposes. PMID:27225035

  10. Co-culture of bone marrow stem cells and macrophages indicates intermediate mechanism between local inflammation and innate immune system in diabetic periodontitis

    PubMed Central

    Wang, Jia; Li, Hao; Li, Bo; Gong, Qiulin; Chen, Xinmin; Wang, Qi

    2016-01-01

    Diabetic periodontitis (DP), which has been shown to cause alveolar bone loss, is among the most common complications associated with diabetes. The precise mechanisms underlying alveolar bone loss in patients with DP remain unclear. Therefore, the present study established a co-culture system of bone marrow stem cells (BMSCs) and macrophages, in order to investigate the potential mechanisms underlying DP-associated alveolar bone loss in vitro. In addition, Porphyromonas gingivalis (PG) periodontal infection and high glucose levels were used to induce DP in mice. The present study evaluated the protein expression levels of various chemokines and the migration of BMSCs and macrophages. The protein expression levels of extracellular signal-regulated kinase 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the BMSCs exposed to high glucose and PG, which may have been due to the activation of MAPK. In addition, DP induction in mice was associated with the release of chemokine (C-C motif) ligand 2 (CCL2) from BMSCs and the secretion of chemokine (C-C Motif) receptor 2 (CCR2) and tumor necrosis factor-α from macrophages, which was associated in turn with enhanced adhesion and chemotaxis of macrophages. The results of the present study suggested that DP led to the upregulation of CCL2 in the periodontal tissues and enhanced macrophage infiltration via the CCL2/CCR2 axis, which in turn promoted alveolar bone loss.

  11. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  12. Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

    PubMed Central

    Yoshikai, Y; Takeda, Y; Ohga, S; Kishihara, K; Matsuzaki, G; Nomoto, K

    1989-01-01

    We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2541071

  13. Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

    PubMed Central

    Gonçalves, Fabiany da Costa; Paz, Ana Helena da Rosa; Lora, Priscila Schmidt; Passos, Eduardo Pandolfi; Cirne-Lima, Elizabeth Obino

    2012-01-01

    AIM: To investigate the interaction between mesenchymal stem cells (MSCs) and bone grafts using two different cultivation methods: static and dynamic. METHODS: MSCs were isolated from rat bone marrow. MSC culture was analyzed according to the morphology, cell differentiation potential, and surface molecular markers. Before cell culture, freeze-dried bone (FDB) was maintained in culture for 3 d in order to verify culture medium pH. MSCs were co-cultured with FDB using two different cultivation methods: static co-culture (two-dimensional) and dynamic co-culture (three-dimensional). After 24 h of cultivation by dynamic or static methods, histological analysis of Cell adhesion on FDB was performed. Cell viability was assessed by the Trypan Blue exclusion method on days 0, 3 and 6 after dynamic or static culture. Adherent cells were detached from FDB surface, stained with Trypan Blue, and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture. Statistical analyses were performed with SPSS and a P < 0.05 was considered significant. RESULTS: The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures. Rat MSCs were positive for CD44, CD90 and CD29 and negative for CD34, CD45 and CD11bc. FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH (P > 0.05). In histological analysis, there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods (P < 0.05). The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method. On day 0, the cell viability in the dynamic system was significantly higher than in the static system (P < 0.05). There was a statistical difference in cell viability between days 0, 3 and 6 after dynamic culture (P < 0.05). In static culture, cell viability on day 6

  14. CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro-osteogenic and pro-inflammatory cues.

    PubMed

    Pontikoglou, Charalampos; Langonné, Alain; Ba, Mamadou Aliou; Varin, Audrey; Rosset, Philippe; Charbord, Pierre; Sensébé, Luc; Deschaseaux, Frédéric

    2016-04-01

    Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200(pos) cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up-regulated in CD200(pos) cells compared to CD200(neg) fraction. At the functional level, CD200(pos) cells were prone to mineralize the extra-cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200(pos) cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down-regulated. As dexamethasone has anti-inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro-inflammatory cytokines interleukin-1β and tumour necrosis factor-α increased CD200 membrane expression but down-regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro-inflammatory or pro-osteogenic, CD200 expression was down-regulated when nuclear-factor (NF)-κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro-inflammatory cytokines through the same pathway: NF-κB. PMID:26773707

  15. In vitro models of periodontal cells: a comparative study of long-term gingival, periodontal ligament and alveolar bone cell cultures in the presence of beta-glycerophosphate and dexamethasone.

    PubMed

    Cabral, Maria Cristina Trigo; Costa, Maria Adelina; Fernandes, Maria Helena

    2007-06-01

    Human gingival (HG), periodontal ligament (HPL) and alveolar bone (HAB) cells (first subculture) were cultured (10(4) cells/cm2) for 35 days in alpha-Minimal Essential Medium supplemented with 10% fetal bovine serum in the presence of (i) ascorbic acid (AA, 50 microg/mL), (ii) AA + beta-glycerophosphate (betaGP, 10 mM) and (iii) AA + betaGP + dexamethasone (Dex, 10 nM). Cultures were assessed for cell attachment and spreading, cell proliferation, alkaline phosphatase (ALP) and acid phosphatase (ACP) activities and matrix mineralization. HG cell cultures presented a high proliferation rate, a low ability to synthesize ALP and ACP and the formation of a non-mineralized extracellular matrix, regardless the experimental situation. HPL cell cultures were very sensitive to the culture conditions and showed a high proliferation rate, synthesis of moderate levels of ALP and ACP and a modest matrix mineralization in the presence of AA + betaGP + Dex. HAB cell cultures presented a growth rate lower than that of HG and HPL cells, a high ALP activity and comparatively low levels of ACP, and the ready formation of a heavy mineralized matrix in the presence of betaGP. In the three periodontal cell cultures, Dex enhanced cell proliferation and expression of osteoblastic markers. Results showed that betaGP and Dex allowed the modulation of the cell proliferation/differentiation behavior within the proposed physiological and regenerative capabilities of these periodontal cells. PMID:17268872

  16. Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C60/Ti Films

    PubMed Central

    Kopova, Ivana; Lavrentiev, Vasily; Vacik, Jiri; Bacakova, Lucie

    2015-01-01

    Thin films of binary C60/Ti composites, with various concentrations of Ti ranging from ~ 25% to ~ 70%, were deposited on microscopic glass coverslips and were tested for their potential use in bone tissue engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies in the combination of Ti atoms (i.e., widely used biocompatible material for the construction of stomatological and orthopedic implants) with atoms of fullerene C60, which can act as very efficient radical scavengers. However, fullerenes and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic effects. In order to stabilize C60 molecules and to prevent their possible cytotoxic effects, deposition in the compact form of Ti/C60 composites (with various Ti concentrations) was chosen. The reactivity of C60/Ti composites may change in time due to the physicochemical changes of molecules in an air atmosphere. In this study, we therefore tested the dependence between the age of C60/Ti films (from one week to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we did not observe any negative influence of fresh or aged C60/Ti layers on cell behavior, including the DNA damage response. The presence of Ti atoms resulted in improved properties of the C60 layers, which became more suitable for cell cultivation. PMID:25875338

  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 Central

    Purroy, Noelia; Abrisqueta, Pau; Carabia, Júlia; Carpio, Cecilia; Palacio, Carles

    2015-01-01

    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. Effect of nicotine in matrix mineralization by human bone marrow and Saos-2 cells cultured on the surface of plasma-sprayed titanium implants.

    PubMed

    Pereira, M L; Carvalho, J C; Peres, F; Fernandes, M H

    2009-01-01

    Smoking has an established negative impact in the clinical outcome of dental implants. This work analyses the response of human osteoblastic cells to nicotine, at the surface of plasma-sprayed commercial titanium implants. Human bone marrow (HBM) and Saos-2 cells, seeded on the surface of titanium implants and cultured in experimental conditions favoring osteoblastic differentiation, were exposed continuously to nicotine (0.0001 to 0.5 mg mL(-1)) and characterized for cell proliferation and function. Exposure of HBM cells resulted in increased cell proliferation, higher alkaline phosphatase (ALP) activity, and earlier onset of matrix mineralization at levels up to 0.2 mg mL(-1), an initial inhibitory effect in cell growth and functional activity followed by a recovery in the presence of 0.3 mg mL(-1) and a dose-dependent deleterious effect at higher levels. By contrast, exposure to nicotine did not affect cell proliferation of Saos-2 cells at levels up to 0.2 mg mL(-1), and caused only a small positive effect in ALP activity in the presence of 0.05 and 0.1 mg mL(-1); however, matrix mineralization by Saos-2 cells also occurred earlier in the cultures exposed to levels of nicotine up to 0.1 mg mL(-1). Higher concentrations caused dose-dependent inhibitory effects. Considering the high diffusion potential of nicotine, results suggest a local role of nicotine in modulating bone formation events at the implant surface. PMID:18260147

  19. Differentiation Potential of Mesenchymal Stem Cells from Equine Bone Marrow Cultured on Hyaluronic Acid-Chitosan Polyelectrolyte Multilayer Biofilm.

    PubMed

    Listoni, Amanda J; Arruda, Isadora; Maia, Leandro; Barberini, Danielle J; Martins, Ian; Vasconcellos, Fernando C; Landim-Alvarenga, Fernanda C

    2015-01-01

    Nanotechnology techniques have a prominent role in the current technical and scientific scene. The layer-by-layer (LbL) deposition allows obtaining nanostructures with sophisticated multilayer, using a simple, but versatile technique. This procedure, which is used to coat and functionalize surfaces with nanometer- thick films, has applications in bioengineering, medicine, chemistry, materials and chemical engineering among other areas. Chitosan is a biomaterial, coming from the chitin, a very abundant polymer in nature, which has been recently tested as scaffolds. In this experiment we test the hypothesis that the hyaluronic acid-chitosan polyelectrolyte multilayer biofilm would be a good substrate to the adherence of equine mesenchymal stem cells derived from bone marrow. The results showed that these biofilms accelerate the process of cell adhesion on smooth surfaces, allowing a constant cell growth and creating a great option to cover surgical materials. PMID:27125135

  20. Characterization of platelet lysate cultured mesenchymal stromal cells and their potential use in tissue-engineered osteogenic devices for the treatment of bone defects.

    PubMed

    Salvadè, Agnese; Della Mina, Pamela; Gaddi, Diego; Gatto, Francesca; Villa, Antonello; Bigoni, Marco; Perseghin, Paolo; Serafini, Marta; Zatti, Giovanni; Biondi, Andrea; Biagi, Ettore

    2010-04-01

    Mesenchymal stromal cells (MSCs), seeded onto a scaffold and associated with platelet-gel, may represent an innovative treatment to improve bone repair. The preparation of MSCs for clinical use requires the fulfillment of Good Manufacturing Practice indications. The aim of this study was to validate a Good Manufacturing Practice-grade protocol of tissue engineering for bone regeneration, seeding platelet lysate (PL)-cultured MSCs onto an hydroxyapatite clinical-grade scaffold. Six large-scale experiments were performed. MSC expansions were performed comparing fetal bovine serum 10% and PL 5%. We demonstrated that PL lots contain high levels of growth factors possibly responsible of accelerated growth rate, since the number of colony-forming unit-fibroblast and population doublings were always significantly higher in PL cultures. MSCs were characterized for their phenotype and multilineage differentiation capacity, demonstrating appropriate features for both conditions. Gene expression analysis revealed higher expression of typical osteogenic genes of PL-cultured MSCs, when compared to fetal bovine serum MSCs. Cell transformation was excluded by analysis of karyotype, absence of growth without anchorage, and p53/c-myc gene expression. Scaffolds were precoated with retronectin before MSC seeding. MSC adhesion, distribution, and proliferation were demonstrated through the whole surface of the scaffold by scanning electron microscopy analysis or by immunofluorescence and MSC osteogenic differentiation through quantitative reverse transcriptase-polymerase chain reaction of typical osteogenic genes. The present report offers a model of an MSC-based bioengineered device, using an hydroxyapatite clinical-grade scaffold, and supports its potential use in tissue engineering to repair bone defects. PMID:19469694

  1. Enhanced hepatic differentiation of rat bone marrow-derived mesenchymal stem cells in spheroidal aggregate culture on a decellularized liver scaffold

    PubMed Central

    Bao, Ji; Wu, Qiong; Wang, Yujia; Li, Yi; Li, Li; Chen, Fei; Wu, Xiujuan; Xie, Mingjun; Bu, Hong

    2016-01-01

    In the present study, we aimed to determine whether the combination of aggregate culture and decellularized liver scaffolds (DLSs) promoted the hepatic differentiation of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into high yields of mature hepatocytes in vitro. Four culturing methods for differentiation [single cell (2D), spheroids (3D), 2D + DLS and 3D + DLS] were studied. To determine the differentiation stages of the MSCs, RT-qPCR of the hepatocyte genes, immunostaining of hepatocyte markers, and functional analyses were all performed. Compared with the other groups, hepatocyte-like cells which differentiated from BM-MSC spheroids on extracellular matrix (ECM) exhibited more intensive staining of stored glycogen, an elevated level of urea biosynthesis and albumin secretion as well as the higher expression of hepatocyte-specific genes. Our results indicated that DLSs combined with spheroidal aggregate culture may be used as an effective method to facilitate the hepatic maturation of BM-MSCs and may have future applications in stem cell-based liver regenerative medicine. PMID:27314916

  2. Enhanced hepatic differentiation of rat bone marrow-derived mesenchymal stem cells in spheroidal aggregate culture on a decellularized liver scaffold.

    PubMed

    Bao, Ji; Wu, Qiong; Wang, Yujia; Li, Yi; Li, Li; Chen, Fei; Wu, Xiujuan; Xie, Mingjun; Bu, Hong

    2016-08-01

    In the present study, we aimed to determine whether the combination of aggregate culture and decellularized liver scaffolds (DLSs) promoted the hepatic differentiation of murine bone marrow-derived mesenchymal stem cells (BM-MSCs) into high yields of mature hepatocytes in vitro. Four culturing methods for differentiation [single cell (2D), spheroids (3D), 2D + DLS and 3D + DLS] were studied. To determine the differentiation stages of the MSCs, RT-qPCR of the hepatocyte genes, immunostaining of hepatocyte markers, and functional analyses were all performed. Compared with the other groups, hepatocyte-like cells which differentiated from BM‑MSC spheroids on extracellular matrix (ECM) exhibited more intensive staining of stored glycogen, an elevated level of urea biosynthesis and albumin secretion as well as the higher expression of hepatocyte-specific genes. Our results indicated that DLSs combined with spheroidal aggregate culture may be used as an effective method to facilitate the hepatic maturation of BM-MSCs and may have future applications in stem cell-based liver regenerative medicine. PMID:27314916

  3. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms.

    PubMed

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

    2007-10-01

    Significant improvement in the understanding of mesenchymal 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 donors and propagated in vitro until reaching either senescence or passage (P) 25. MSCs in the senescence phase were closely monitored for 8 to 12 weeks before interrupting the cultures. The genetic characterization of MSCs was investigated through array-comparative genomic hybridization (array-CGH), conventional karyotyping, and subtelomeric fluorescent in situ hybridization analysis both before and after prolonged culture. MSCs were tested for the expression of telomerase activity, human telomerase reverse transcriptase (hTERT) transcripts, and alternative lengthening of telomere (ALT) mechanism at different passages. A huge variability in terms of proliferative capacity and MSCs life span was noted between donors. In eight of 10 donors, MSCs displayed a progressive decrease in proliferative capacity until reaching senescence. In the remaining two MSC samples, the cultures were interrupted at P25 to pursue data analysis. Array-CGH and cytogenetic analyses showed that MSCs expanded in vitro did not show chromosomal abnormalities. Telomerase activity and hTERT transcripts were not expressed in any of the examined cultures and telomeres shortened during the culture period. ALT was not evidenced in the MSCs tested. BM-derived MSCs can be safely expanded in vitro and are not susceptible to malignant transformation, thus rendering these cells suitable for cell therapy approaches. PMID:17909019

  4. Osteostimulatory effect of bone grafts on fibroblast cultures

    PubMed Central

    Fathima, Hameed; Harish

    2015-01-01

    Objective: We analyzed the morphological changes and alkaline phosphatase (ALP) level in fibroblast, which is indicative of their functional ability when cultured in three different commercially available graft materials with osseoconductive property. Materials and Methods: Fibroblasts obtained from fifth passage were seeded within three different bone substitutes (bovine hydroxyapatite [HA] [Osseo-graft®], β-tricalciumphosphate [RTR®], bovine HA [Bio-oss®]) and incubated under standard cell culture conditions. 10 samples in each group were evaluated for cell morphology and alkaline phosphates activity using scanning electron microscopy and spectrophotometric analysis on the 7th day of culture. Results: Fibroblast cultured with RTR® showed changes in morphology and increase in ALP activity when compared to fibroblast cultured with Osseo-graft® and Bio-oss®. Conclusion: Alkaline phosphatase activity was observed in fibroblasts when cultured with three types of commercially available bone grafts. ALP activity was highest when cultured with β-tricalcium phosphate graft material indicating its better bone regenerating capacity of this graft material. PMID:26283815

  5. Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

    PubMed

    Wuchter, Patrick; Vetter, Marcel; Saffrich, Rainer; Diehlmann, Anke; Bieback, Karen; Ho, Anthony D; Horn, Patrick

    2016-06-01

    Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion. PMID:26911671

  6. Bone-derived IGF mediates crosstalk between bone and breast cancer cells in bony metastases

    PubMed Central

    Hiraga, Toru; Myoui, Akira; Hashimoto, Nobuyuki; Sasaki, Akira; Hata, Kenji; Morita, Yoshihiro; Yoshikawa, Hideki; Rosen, Clifford J.; Mundy, Gregory R.; Yoneda, Toshiyuki

    2012-01-01

    The continuous release of bone-stored growth factors following bone resorption promotes the colonization of circulating cancer cells. However, the precise role of each of the various growth factors remains unclear. In this study, we investigated the role of bone-derived insulin-like growth factor (IGF) in the development of bone metastases in an animal model of breast cancer. We found that local stimulation of calvarial bone resorption prior to cell inoculation stimulated subsequent bone metastases to that site in vivo, while inhibition of bone resorption inhibited bone metastases. Anchorage-independent growth of cancer cells was stimulated by the culture supernatants from resorbed bones, which contained elevated levels of IGF type I (IGF-1). This stimulation was blocked by IGF-1 receptor (IGF1R) neutralizing antibody, but not antibody targeting other bone-stored growth factors including TGFβ, fibroblast growth factors, and platelet derived growth factors. While recombinant human IGF-I caused IGFIR tyrosine autophosphorylation, followed by activation of Akt and NF-κB in cancer cells, dominant-negative inhibition of IGFIR, Akt, or NF-κB significantly reduced bone metastases with increased apoptosis and decreased mitosis in metastatic cells. Together, our findings suggest that bone-derived IGF-I bridges the crosstalk between bone and metastasized cancer cells via activation of the IGFIR/Akt/NF-κB pathway. Disruption of this pathway therefore may represent a promising therapeutic intervention for bone metastasis. PMID:22738911

  7. Bone scaffold architecture modulates the development of mineralized bone matrix by human embryonic stem cells

    PubMed Central

    Marcos-Campos, Ivan; Marolt, Darja; Petridis, Petros; Bhumiratana, Sarindr; Schmidt, Daniel; Vunjak-Novakovic, Gordana

    2012-01-01

    Decellularized bone has been widely used as a scaffold for bone formation, due to its similarity to the native bone matrix and excellent osteoinductive and biomechanical properties. We have previously shown that human mesenchymal and embryonic stem cells form functional bone matrix on such scaffolds, without the use of growth factors. In this study, we focused on differences in bone matrix that exist even among identical harvesting sites, and the effects of the matrix architecture and mineral content on bone formation by human embryonic stem cells (hESC). Mesenchymal progenitors derived from hESCs were cultured for 5 weeks in decellularized bone scaffolds with three different densities: low (0.281 ± 0.018 mg/mm3), medium (0.434 ± 0.015 mg/mm3) and high (0.618 ± 0.027 mg/mm3). The medium-density group yielded highest densities of cells and newly assembled bone matrix, presumably due to the best balance between the transport of nutrients and metabolites to and from the cells, space for cell infiltration, surface for cell attachment and the mechanical strength of the scaffolds, all of which depend on the scaffold density. Bone mineral was beneficial for the higher expression of bone markers in cultured cells and more robust accumulation of the new bone matrix. PMID:22901965

  8. Uptake of a fluorinated bisphosphonate by cultured bones

    SciTech Connect

    Rowe, D.J.; Etre, L.A.

    1988-01-01

    The uptake of bisphosphonates into bone was studied using 19-day-old fetal rat bones cultured with a new fluorinated bisphosphonate, difluoromethylidene bisphosphonate (F2MBP). F2MBP uptake was assessed by determining the weight percent of fluoride using electron probe microanalysis. By 30 min the weight percent of fluoride was significantly greater in the F2MBP-treated bones than in controls and continually increased throughout the duration of the experiment to reach a fluoride concentration 6-fold greater than controls after 120 h of incubation. When the peripheral cortical bone was analyzed separately from the interior trabecular bone in the F2MBP-treated bones, the fluoride concentration in the periphery increased until 24 h and then remained somewhat constant, while the interior, which is more actively remodeling, showed a continual increase. The uptake of F2MBP during the 1 to 6 h time intervals demonstrated no differences between vital and devitalized bone and, thus, is not cell-mediated. Because analysis of free fluoride in F2MBP media incubated with bones showed that the concentration of fluoride was less than 1% of the total amount of fluoride, the fluoride detected by the probe was most likely that of the intact molecule and not free fluoride. The rapid uptake of the F2MBP molecule was supported by assessing the effects of short-term F2MBP treatment on subsequent bone resorption, as determined by the release of 45Ca from prelabeled bones. Bones treated with F2MBP for only 5 min exhibited reductions in the percentage of 45Ca released during the remainder of the 120 h incubation period similar to that when F2MBP was continuously in the medium.

  9. Bone microenvironment-mediated resistance of cancer cells to bisphosphonates and impact on bone osteocytes/stem cells.

    PubMed

    Alasmari, Abeer; Lin, Shih-Chun; Dibart, Serge; Salih, Erdjan

    2016-08-01

    Anti-resorptive bisphosphonates (BPs) have been clinically used to prevent cancer-bone metastasis and cancer-induced bone pathologies despite the fact that the phenotypic response of the cancer-bone interactions to BP exposure is "uncharted territory". This study offers unique insights into the interplay between cancer stem cells and osteocytes/osteoblasts and mesenchymal stem cells using a three-dimensional (3D) live cancer-bone interactive model. We provide extraordinary cryptic details of the biological events that occur as a result of alendronate (ALN) treatment using 3D live cancer-bone model systems under specific bone remodeling stages. While cancer cells are susceptible to BP treatment in the absence of bone, they are totally unaffected in the presence of bone. Cancer cells colonize live bone irrespective of whether the bone is committed to bone resorption or formation and hence, cancer-bone metastasis/interactions are though to be "independent of bone remodeling stages". In our 3D live bone model systems, ALN inhibited bone resorption at the osteoclast differentiation level through effects of mineral-bound ALN on osteocytes and osteoblasts. The mineral-bound ALN rendered bone incapable of osteoblast differentiation, while cancer cells colonize the bone with striking morphological adaptations which led to a conclusion that a direct anti-cancer effect of BPs in a "live or in vivo" bone microenvironment is implausible. The above studies were complemented with mass spectrometric analysis of the media from cancer-bone organ cultures in the absence and presence of ALN. The mineral-bound ALN impacts the bone organs by limiting transformation of mesenchymal stem cells to osteoblasts and leads to diminished endosteal cell population and degenerated osteocytes within the mineralized bone matrix. PMID:27155840

  10. Expression of mRNAs encoding for growth factors, ECM molecules, and MMP13 in mono-cultures and co-cultures of human periodontal ligament fibroblasts and alveolar bone cells.

    PubMed

    Winter, S; Kohl, A; Huppertz, A; Herold-Mende, C; Wiest, T; Komposch, G; Tomakidi, P

    2005-03-01

    Although the function and effects of many growth factors and extracellular matrix (ECM) molecules have been described for several periodontal tissues in vivo and in vitro, the molecular interactions involved in the communication between cells of the periodontal ligament and the alveolar bone are poorly understood. To contribute to the identification of such interactions, we have generated co-cultures (CCs) of periodontal ligament fibroblasts (PDLs) and alveolar bone cells (ABCs) and compared mRNA expression for various growth factors, ECM molecules, and matrix metalloproteinase13 (MMP13) after 1 and 2 weeks with matched mono-cultures (MCs) by reverse transcription/polymerase chain reaction. Compared with CCs of 1 week, PDLs and ABCs after 2 weeks revealed relatively high levels of all analyzed mRNAs, viz., for EGF, HGF, VEGF, TGFbeta1, collagen-I (COL1), osteonectin (ON), fibronectin (FN1), and MMP13. At week 2, when compared with MCs, CCs showed an elevation of all tested mRNAs in PDLs and ABCs, except for TGFbeta1 and FN1, which only increased in PDLs. After 1 week, when CCs were compared with MCs, mRNAs for HGF and TGFbeta1 were less abundant in PDLs and ABCs, whereas the other genes exhibited lower expression levels in only one of the cell types. Analysis of our data indicated that the expression of mRNAs for growth factors and for COL1, ON, FN1, and MMP13 was modulated in the distinct cell types with respect to culture time and culture type. The differences in the mRNA expression patterns between CCs and MCs suggest that the respective genes are involved in the molecular interactions of PDLs and ABCs. PMID:15668800

  11. A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on β-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro.

    PubMed

    An, Shaofeng; Gao, Yan; Huang, Xiangya; Ling, Junqi; Liu, Zhaohui; Xiao, Yin

    2015-05-01

    The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. β-tricalcium phosphate (β-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous β-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell-Counting kit-8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the β-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the β-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the β-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on β-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects

  12. 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

  13. 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.

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

    PubMed

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

    2015-01-01

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

  15. siDNMT1 Increases γ-globin Expression in Chemical-Inducer-of-Dimerization (CID)-Dependent Mouse βYAC Bone Marrow Cells and in Baboon Erythroid Progenitor Cell Cultures

    PubMed Central

    Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; Ruiz, Maria Armila; Peterson, Kenneth; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult stage erythroid cells. 2) Methods DNMT1 levels were reduced by nucleofection of siRNA targeting DNMT1 in chemical-inducer-of-dimerization (CID)-dependent multipotential mouse bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and in primary cultures of erythroid progenitor cells derived from CD34+ baboon BM cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real time PCR and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabelling of globin chains followed by HPLC analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. 3) Results Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin mRNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. 4) Conclusion DNMT1 is required to maintain DNA methylation of the γ-globin gene promoters and repress γ-globin gene expression in adult-stage erythroid cells. PMID:20974210

  16. In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing.

    PubMed

    Rodriguez, Isaac A; Saxena, Gunjan; Hixon, Katherine R; Sell, Scott A; Bowlin, Gary L

    2016-08-01

    The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2011-2019, 2016. PMID:27038217

  17. Bone regeneration and stem cells

    PubMed Central

    Arvidson, K; Abdallah, B M; Applegate, L A; Baldini, N; Cenni, E; Gomez-Barrena, E; Granchi, D; Kassem, M; Konttinen, Y T; Mustafa, K; Pioletti, D P; Sillat, T; Finne-Wistrand, A

    2011-01-01

    Abstract This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed. PMID:21129153

  18. Resorbing bone stimulates tumor cell growth. A role for the host microenvironment in bone metastasis.

    PubMed

    Manishen, W J; Sivananthan, K; Orr, F W

    1986-04-01

    Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for

  19. The Expression of Bone Morphogenetic Protein 2 and Matrix Metalloproteinase 2 through Retinoic Acid Receptor Beta Induced by All-Trans Retinoic Acid in Cultured ARPE-19 Cells

    PubMed Central

    Gao, Zhenya; Huo, Lijun; Cui, Dongmei; Yang, Xiao; Zeng, Junwen

    2016-01-01

    Purpose All-trans retinoic acid (ATRA) plays an important role in ocular development. Previous studies found that retinoic acid could influence the metabolism of scleral remodeling by promoting retinal pigment epithelium (RPE) cells to secrete secondary signaling factors. The purpose of this study was to investigate whether retinoic acid affected secretion of bone morphogenetic protein 2 (BMP-2) and matrix metalloproteinase 2 (MMP-2) and to explore the signaling pathway of retinoic acid in cultured acute retinal pigment epithelial 19 (ARPE-19) cells. Methods The effects of ATRA (concentrations from 10−9 to 10−5 mol/l) on the expression of retinoic acid receptors (RARs) in ARPE-19 cells were examined at the mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. The effects of treating ARPE-19 cells with ATRA concentrations ranging from 10−9 to 10−5 mol/l for 24 h and 48 h or with 10-6mol/l ATRA at different times ranging from 6h to 72h were assessed using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). The contribution of RARβ-induced activation of ARPE-19 cells was confirmed using LE135, an antagonist of RARβ. Results RARβ mRNA levels significantly increased in the ARPE-19 cells treated with ATRA for 24h and 48h. These increases in RARβ mRNA levels were dose dependent (at concentrations of 10−9 to 10−5 mol/l) with a maximum effect observed at 10−6 mol/l. There were no significant changes in the mRNA levels of RARα and RARγ. Western blot assay revealed that RARβ protein levels were increased significantly in a time-dependent manner in ARPE-19 cells treated with 10−6 mol/l ATRA from 12 h to 72 h, with a marked increase observed at 24 h and 48 h. The upregulation of RARβ and the ATRA-induced secretion in ARPE-19 cells could be inhibited by the RARβ antagonist LE135. Conclusion ATRA induced upregulation of RARβ in ARPE-19 cells and stimulated

  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 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

  1. Stimulation of bone marrow cells and bone formation by nacre: in vivo and in vitro studies.

    PubMed

    Lamghari, M; Almeida, M J; Berland, S; Huet, H; Laurent, A; Milet, C; Lopez, E

    1999-08-01

    There is frequently a loss of vertebral bone due to disease or aging. Nacre (mother of pearl from the oyster Pinctada maxima) stimulates bone cell differentiation and bone formation in vitro and in vivo. Experimental bone defects were prepared in the vertebrae of sheep and used to test the suitability of nacre as an injectable osteogenic biomaterial for treating vertebral bone loss. Twenty-one cavities were prepared in the first four upper lumbar vertebrae of 11 sheep and filled with nacre powder. The lumbar vertebrae were removed after 1 to 12 weeks, embedded undecalcified in methacrylate, and processed for histological studies. The nacre slowly dissolved and the experimental cavities contained a large active cell population. By 12 weeks, the experimental cavity was occupied by newly matured bone trabeculae in contact with or adjacent to the dissolving nacre. The functional new bone trabeculae were covered with osteoid lined with osteoblasts, indicating continuing bone formation. The in vitro study on rat bone marrow explants cultured with a water-soluble extract of the nacre organic matrix also resulted in the stimulation of osteogenic bone marrow cells with enhanced alkaline phosphatase activity. Thus, both the in vivo and in vitro findings suggest that nacre contains one or more signal molecules capable of activating osteogenic bone marrow cells. PMID:10458284

  2. Optimizing stem cell culture.

    PubMed

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  3. Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells.

    PubMed

    Weber, L; Langer, M; Tavella, S; Ruggiu, A; Peyrin, F

    2016-05-01

    In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite(©)), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions. PMID:27054380

  4. Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells

    NASA Astrophysics Data System (ADS)

    Weber, L.; Langer, M.; Tavella, S.; Ruggiu, A.; Peyrin, F.

    2016-05-01

    In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite©), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions.

  5. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  6. Indirect co‑culture of vascular smooth muscle cells with bone marrow mesenchymal stem cells inhibits vascular calcification and downregulates the Wnt signaling pathways.

    PubMed

    Zhu, Meng'en; Fang, Xin; Zhou, Shaoqiong; Li, Wei; Guan, Siming

    2016-06-01

    Vascular calcification (VC) is widely considered to be a crucial clinical indicator of cardiovascular disease. Recently, certain properties of mesenchymal stem cells (MSCs) have been hypothesized to have potential in treating cardiovascular diseases. However, their effect on the initiation and progression of VC remains controversial. The present study aimed to investigate whether MSCs indirectly mediate VC and their impact on the Wnt signaling pathways. A Transwell system was selected to establish the indirect co‑culture environment, and hence, vascular smooth muscle cells (VSMCs) were indirectly co‑cultured in the presence or absence of MSCs at a ratio of 1:1. Osteogenic medium (OS) was added to imitate a calcifying environment. Fourteen days later, VSMCs in the lower layers of the Transwell plates were harvested. Alkaline phosphatase activity and calcium nodules were markedly increased in calcific VSMCs induced by OS. However, these parameters were significantly decreased in VSMCs by indirectly co‑culturing with MSCs in the same medium. Furthermore, the messenger RNA expression levels of osteopontin and osteoprotegerin were notably increased in VSMCs cultured in OS, but reduced by indirect interaction with MSCs. In addition, the activities of canonical and noncanonical Wnt ligands, wingless‑type MMTV integration site family, number 5A (Wnt5a), receptor tyrosine kinase‑like orphan receptor 2 (Ror2) and β‑catenin, which are important in the process of VC, were downregulated by indirect contact with MSCs in OS. Thus, indirect co‑culture with MSCs inhibits VC and downregulates the Wnt signaling pathways. PMID:27121342

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

    PubMed

    Ghoniem, Amir-Alexander; Açil, Yahya; Wiltfang, Jörg; Gierloff, Matthias

    2015-06-01

    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 PPARγ2, C/EBPα, 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 PPARγ2, C/EBPα, 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. PMID:26140219

  8. Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture.

    PubMed

    Miettinen, Johanna A; Pietilä, Mika; Salonen, Riikka J; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V; Lehenkari, Petri

    2011-04-01

    Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity. PMID:21182837

  9. Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

    SciTech Connect

    Miettinen, Johanna A.; Pietilae, Mika; Salonen, Riikka J.; Ohlmeier, Steffen; Ylitalo, Kari; Huikuri, Heikki V.; Lehenkari, Petri

    2011-04-01

    Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-{alpha}) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-{alpha} exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-{alpha} exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-{alpha} exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-{alpha} exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-{alpha} exposure, which might influence MSC differentiation stage and capacity.

  10. Modeling Selective Elimination of Quiescent Cancer Cells from Bone Marrow

    PubMed Central

    Cavnar, Stephen P.; Rickelmann, Andrew D.; Meguiar, Kaille F.; Xiao, Annie; Dosch, Joseph; Leung, Brendan M.; Cai Lesher-Perez, Sasha; Chitta, Shashank; Luker, Kathryn E.; Takayama, Shuichi; Luker, Gary D.

    2015-01-01

    Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer. PMID:26408255

  11. Immune Cell Isolation from Mouse Femur Bone Marrow

    PubMed Central

    Liu, Xiaoyu; Quan, Ning

    2016-01-01

    The bone marrow is the site of hematopoesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 × 107 cells in 5 ml culture media (1.6 × 104 cells/μl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.

  12. 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 μm. 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 μm transplants. In contrast, maximum transplant strength was greatest among 200 μm transplants. Conclusions Lamellar spacing within scaffolds regulates the extent of bone formation; 500 μm yields the most new bone, whereas 200 μm yields the strongest transplants. PMID:21294634

  13. [Immunoregulatory role of mesenchymal stem cells in bone reparation processes].

    PubMed

    Zubov, D O

    2008-01-01

    Bone marrow contains mesenchymal stem cells (MSC) including osteoblast progenitor cells. When culturedunder conditions promoting an osteoblastic phenotype,MSC proliferate to form colonies that produce alkaline phosphatase and, subsequently, a mature osteoblastic phenotype. Transplantation of cultured autologous MSC to patients with non-healing bone fractures gives a good result leading to complete bone fracture consolidation. The aim of the study is to determine a quantitative production of IL-1beta, IL-2, IL-4, IL-6, IL-8 and TNF-alpha by cultured uncommitted and committed osteogenic MSC. The results showed that the cytokine profile consisting of IL-1beta, IL-2, IL-4, IL-6, IL-8 and TNF-alpha is secreted by cultured MSC. The secretion of IL-1beta and IL-2 by cultured MSC together with hyper production of IL-6 (up to 276.5 pg/ml, p<0.05) and IL-8 (up to 106.6 ng/ml, p<0.05) by osteoinducted MSC are firstly shown. The immunoregulatory role of transplanted autologous cells in inflammation and own bone reparation processes during posttraumatic bone fracture healing is highlighted. In conclusion, the data obtained allow examining of cultured autologous MSC as effective activators of bone resorption, inflammation and some immunological reactions in the process of altered osteoreparation. PMID:18756772

  14. Endotoxins affect bioactivity of chitosan derivatives in cultures of bone marrow-derived human mesenchymal stem cells.

    PubMed

    Lieder, Ramona; Gaware, Vivek S; Thormodsson, Finnbogi; Einarsson, Jon M; Ng, Chuen-How; Gislason, Johannes; Masson, Mar; Petersen, Petur H; Sigurjonsson, Olafur E

    2013-01-01

    Biomaterials research has been expanding over the last decade, in part to provide improved medical devices for the treatment of orthopedic tissue injuries. In the quest to provide the best performance combined with low cost for medical implants, an increasing number of non-chemists have entered the field of biomaterials research without the profound knowledge of chemistry needed to understand the complex interaction mechanisms and characteristics of natural substances. Likewise, non-biologists often lack understanding when it comes to the presence of the contaminating biota frequently found in natural substances. This lack of knowledge by researchers in the field, combined with sensitive in vitro cell-based assays, can lead to inaccurate evaluation of biomaterials. Hence, there should be both an active effort to assemble multi-disciplinary teams and a genuine concern for the possible effects of contamination on in vitro assays. Here, we show that the presence of bacterial endotoxins in chitosan derivatives can result in false-positive results, profoundly altering product performance in in vitro assays. False-positive results through uncritical use of natural substances in vitro can be avoided by proper endotoxin testing and careful evaluation of cytokine secretion patterns. PMID:22947323

  15. 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)

  16. Effects of Kagocel® on the Counts of Multipotent Stromal Cells, Expression of Cytokine Genes in Primary Cultures of Bone Marrow Stromal Cells, and Serum Cytokine Concentrations in CBA Mice.

    PubMed

    Gorskaya, Yu F; Grabko, V I; Konopleva, M V; Suslov, A P; Nesterenko, V G

    2015-06-01

    The efficiency of cloning of bone marrow multipotent stromal cells (ECF-MSC) from CBA mice and the MSC counts in the femoral bone increased 24 h after a single in vivo (but not in vitro) injection of kagocel (active substance of antiviral drug Kagocel (®) ) 1.4 times (in response to 50-80 μg) and 4.6 times (in response to 250 μg). The maximum increase of ECF-MSC in response to 50 μg per mouse was detected just 1 h after Kagocel injection to intact mice and to mice previously receiving the drug for 3 days (2 and 1.7 times, respectively). The increase of ECF-MSC was 3-fold less intense in response to oral Kagocel in a dose of 250 μg/mouse vs. intraperitoneal Kagocel, ECF-MSC corresponding to its level in response to oral Poly (I:C). In vivo Kagocel led to emergence of proinflammatory cytokine IFN-γ, IL-1β, and IL-8 mRNA in primary cultures of bone marrow stromal cells. Serum concentrations of IL-2, IL-5, IL-10, GM-CSF, IFN-γ, TNF-α, IL-4, and IL-12 increased 1.5 and 2 times just 1 h after Kagocel injection in doses of 30-50 and 250 μg, respectively, to intact mice and to animals previously treated with the drug for 3 days. The cytokine concentrations normalized after 3 h and increased again after 24 h, though did not reach the levels recorded 1 h after the drug injection. These data indicated that the therapeutic and preventive effects of Kagocel, together with its previously demonstrated stimulation of α- and β-interferon production during several days, could be due to the capacity of this drug to increase the bone marrow ECF-MSC, serum cytokine concentrations, and induce the expression of proinflammatory cytokine genes in the bone marrow stromal cells 1 h after its injection. PMID:26087752

  17. Property of Human Bone Marrow Stromal Cells Derived From Bone Fragments Removed in Sagittal Split Ramus Osteotomy.

    PubMed

    Yoshida, Chihiro; Yamaguchi, Satoshi; Abe, Shigehiro; Harada, Kiyoshi

    2016-06-01

    Bone tissue engineering is in the process of making the shift from bench to bed. Organ as a cell source is important for tissue engineering. The appropriate cells should be harvested without invasiveness and ethical problems. The authors focused on mandibular cortex bone fragments removed in sagittal split ramus osteotomy as a cell source for bone tissue engineering. These bone fragments were discarded after surgery until now. Bone marrow stromal cells (BMSCs) were harvested from inside of bone fragments, which is an endosteal region. Endosteal region is known to be a hematopoietic stem cell niche and harbors osteoblasts, preosteoblasts, and mesenchymal stem cells (MSCs). Bone marrow stromal cells could be cultured easily, and grew rapidly in vitro under ordinary serum-supplemented culture condition. The expression pattern of surface markers of BMSCs was the same as that of MSCs. Bone marrow stromal cells could differentiated into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes, and smooth muscle cells). These results indicated the existence of MSCs in BMSCs. The osteoblastic characters of BMSCs were examined more closely. Bone marrow stromal cells showed a high alkaline phosphatase activity, and expressed osteoblastic markers (PTHr, bone sialoprotein, Type I collagen, Rnut-related transcription factor 2, and osteocalcin). In transplantation experiments, BMSCs generated ectopic bone tissues on the border of hydroxyapatite scaffold without osteogenic differentiation-inducing agents such as dexamethasone (Dex) or bone morphogenetic protein. The results of this study suggest that mandibular cortex bone fragments removed in sagittal split ramus osteotomy are a good cell source for bone tissue engineering. PMID:27171960

  18. Cell interactions in bone tissue engineering

    PubMed Central

    Pirraco, R P; Marques, A P; Reis, R L

    2010-01-01

    Abstract Bone fractures, where the innate regenerative bone response is compromised, represent between 4 and 8 hundred thousands of the total fracture cases, just in the United States. Bone tissue engineering (TE) brought the notion that, in cases such as those, it was preferable to boost the healing process of bone tissue instead of just adding artificial parts that could never properly replace the native tissue. However, despite the hype, bone TE so far could not live up to its promises and new bottom-up approaches are needed. The study of the cellular interactions between the cells relevant for bone biology can be of essential importance to that. In living bone, cells are in a context where communication with adjacent cells is almost permanent. Many fundamental works have been addressing these communications nonetheless, in a bone TE approach, the 3D perspective, being part of the microenvironment of a bone cell, is as crucial. Works combining the study of cell-to-cell interactions in a 3D environment are not as many as expected. Therefore, the bone TE field should not only gain knowledge from the field of fundamental Biology but also contribute for further understanding the biology of bone. In this review, a summary of the main works in the field of bone TE, aiming at studying cellular interactions in a 3D environment, and how they contributed towards the development of a functional engineered bone tissue, is presented. PMID:20050963

  19. Digital Microfluidic Cell Culture.

    PubMed

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  20. 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

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

    PubMed

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

    2014-07-01

    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

  2. Differentiation of rabbit bone mesenchymal stem cells into endothelial cells in vitro and promotion of defective bone regeneration in vivo.

    PubMed

    Liu, Jinzhong; Liu, Chao; Sun, Bin; Shi, Ce; Qiao, Chunyan; Ke, Xiaoliang; Liu, Shutai; Liu, Xia; Sun, Hongchen

    2014-04-01

    Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects. PMID:23943083

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

    SciTech Connect

    Ecarot-Charrier, B.; Bouchard, F.; Delloye, C. )

    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.

  4. Effect of culture conditions and calcium phosphate coating on ectopic bone formation.

    PubMed

    Vaquette, Cédryck; Ivanovski, Saso; Hamlet, Stephen M; Hutmacher, Dietmar W

    2013-07-01

    This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaP-coated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients. We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation. PMID:23623428

  5. Molecular profile of osteoprogenitor cells seeded on allograft bone.

    PubMed

    Smith, Kierann E; Huang, Zhinong; Ma, Ting; Irani, Afraaz; Lane Smith, R; Goodman, Stuart B

    2011-10-01

    In order to optimize and modulate bone formation it is essential to understand the expression patterns of key bone-specific growth factors, as osteoprogenitor cells undergo the processes of proliferation, differentiation and maturation. This study reports the sequential expression of bone-related growth and transcription factors when bone marrow-derived osteoprogenitor cells from C57BL mice were cultured on allograft bone discs. Mineralization and osteocalcin protein levels were used to track osteogenic differentiation and maturation. Bone-related growth factors, such as Bmp-2, Bmp-7, Ctnnb-1, Fgf-2, Igf-1, Vegf-a and Tgf-β1, and transcription factors, such as Runx-2 and osteocalcin, were examined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Total density of mineralized bone was significantly increased 7.6 ± 0.7% in allografts cultured with cells, compared with a 0.5 ± 2.0% increase in the controls without cells (p < 0.01). Osteocalcin protein levels peaked at day 4. Protein expression showed peaks of BMP-2 and TGF-β1 on day 2, with VEGF peaking on day 8, and IGF-1 decreasing on day 2. mRNA for Pdgf-a peaked on day 2; Bmp-2 on days 4 and 16; Ctnnb-1 on days 8 and 20; Vegf-a, Fgf-2, Runx-2 and Igf-1 on day 12; Tgf-β1 on day 16; and Pdgf-b on day 20. Osteogenic growth factors correlated with Runx-2 and Ctnnb-1, whereas a predominant vascular growth factor, Vegf-a, did not follow this pattern. Specific bone-related genes and proteins were expressed in a time-dependent manner when osteoprogenitor cells were cultured on cortico-cancellous bone discs in vitro. PMID:21953868

  6. Liver Cell Culture Devices

    PubMed Central

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver regeneration) and as in vitro screening systems in the early stages of the drug development process, like assessing hepatotoxicity, hepatic drug metabolism, and induction/inhibition studies. Relevant literature is summarized about artificial human liver cell culture systems by scrutinizing PubMed from 2003 to 2009. Existing devices are divided in 2D configurations (e.g., static monolayer, sandwich, perfused cells, and flat plate) and 3D configurations (e.g., liver slices, spheroids, and different types of bioreactors). The essential features of an ideal liver cell culture system are discussed: different types of scaffolds, oxygenation systems, extracellular matrixes (natural and artificial), cocultures with nonparenchymal cells, and the role of shear stress problems. Finally, miniaturization and high-throughput systems are discussed. All these factors contribute in their own way to the viability and functionality of liver cells in culture. Depending on the aim for which they are designed, several good systems are available for predicting hepatotoxicity and hepatic metabolism within the general population. To predict hepatotoxicity in individual cases genomic analysis might be essential as well. PMID:26998397

  7. [Standardized testing of bone implant surfaces with an osteoblast cell culture cyste. III. PVD hard coatings and Ti6Al4V].

    PubMed

    Steinert, A; Hendrich, C; Merklein, F; Rader, C P; Schütze, N; Thull, R; Eulert, J

    2000-12-01

    The effect of titanium-based PVD coatings and a titanium alloy on the proliferation and differentiation of osteoblasts was investigated using a standardised cell culture system. Human fetal osteoblasts (hFOB 1.19) were cultured on titanium-niobium-nitride ([Ti,Nb]N), titanium-niobium-oxy-nitride coatings ([Ti,Nb]ON) and titanium-aluminium-vanadium alloy (Ti6Al4V) for 17 days. Cell culture polystyrene (PS) was used as reference. For the assessment of proliferation, the numbers and viability of the cells were determined, while alkaline phosphatase activity, collagen I and osteocalcin synthesis served as differentiation parameters. On the basis of the cell culture experiments, a cytotoxic effect of the materials can be excluded. In comparison with the other test surfaces, [Ti,Nb]N showed greater cell proliferation. The [Ti,Nb]N coating was associated with the highest level of osteocalcin production, while all other differentiation parameters were identical on all three surfaces. The test system described reveals the influence of PVD coatings on the osteoblast differentiation cycle. The higher oxygen content of the [Ti,Nb]ON surface does not appear to have any positive impact on cell proliferation. The excellent biocompatibility of the PVD coatings is confirmed by in vivo findings. The possible use of these materials in the fields of osteosynthesis and articular surfaces is still under discussion. PMID:11194641

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

    PubMed Central

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

    2013-01-01

    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

  9. Bone marrow capacity for bone cells and trabecular bone turnover in immobilized tibia after sciatic neurectomy in mice.

    PubMed

    Sakai, A; Nakamura, T; Tsurukami, H; Okazaki, R; Nishida, S; Tanaka, Y; Norimura, T; Suzuki, K

    1996-05-01

    Trabecular bone turnover and bone marrow capacity for the development of bone cells in the tibia were assessed after sciatic neurectomy (NX) in mice. The right hindlimbs of 6-week-old DDY mice were neurectomized and left hindlimbs were sham-operated and served as NX controls. Histomorphometrical analyses of the trabecular bone of the proximal tibia demonstrated the initial decrease in bone formation rate for the first 14 days and the subsequent increase in osteoclast surface for the next 14 days. The number of adherent stromal cells per tibia obtained for the NX limbs was reduced on days 7 and 10 postsurgically, and then recovered on day 12. However, the alkaline phosphatase activity of the cells was persistently depressed. The formation of osteoclast-like multinucleated cells in the marrow cultures obtained from NX limbs at days 10, 12, and 14 showed a significant increase in the medium containing parathyroid hormone (PTH). The number of colonies cultured for colony forming units-fibroblastic (CFU-f) that developed from the marrow cells did not differ in the NX and the contralateral limbs at any time during the period. On the other hand, the number of colonies cultured of colony forming units for granulocytes and macrophages (CFU-GM) was markedly increased for both the NX and the contralateral tibiae at days 12 and 14. This study clearly demonstrates that there are two stages in the development of osteopenia after NX. During the first 14 days, trabecular bone formation and number of marrow stromal cells are reduced. In the second 14 day period, the trabecular osteoclast number is increased and osteoclast formation from the bone marrow cells is enhanced in the presence of PTH. However, neither the CFU-f nor the CFU-GM assay could identify the changes in osteogenic or osteoclastogenic potential of the bone marrow. These in vitro assays provide limited information on the shifts in bone marrow cell lineages and the local environment producing osteopenia in the

  10. Culturing Uveal Melanoma Cells.

    PubMed

    Angi, Martina; Versluis, Mieke; Kalirai, Helen

    2015-04-01

    A major challenge in cancer research is the use of appropriate models with which to study a specific biological question. Cell lines have long been used to study cellular processes and the effects of individual molecules because they are easy to use, grow rapidly, produce reproducible results and have a strong track record in research. In uveal melanoma in particular, the absence of animal models that faithfully replicate the behavior of the human disease has propagated the generation and use of numerous cell lines by individual research groups. This in itself, however, can be viewed as a problem due to the lack of standardization when characterizing these entities to determine how closely they reflect the genetic and phenotypic characteristics of this disease. The alternative is to use in vitro primary cultures of cells obtained directly from uveal melanoma patient samples, but this too has its difficulties. Primary cell cultures are difficult to use, hard to obtain and can show considerable heterogeneity. In this article, we review the following: (1) the uveal melanoma cell lines that are currently available, discussing the importance of establishing a bank of those that represent the molecular heterogeneity of uveal melanoma; (2) the methods used to isolate and perform short-term cultures of primary uveal melanoma cells, and (3) the establishment of 3D tissue culture models that bridge the gap between 2D in vitro systems and in vivo models with which to dissect cancer biology and perform therapeutic screens. PMID:27171555

  11. In Vivo Bone Regeneration Using Tubular Perfusion System Bioreactor Cultured Nanofibrous Scaffolds

    PubMed Central

    Yeatts, Andrew B.; Both, Sanne K.; Yang, Wanxun; Alghamdi, Hamdan S.; Yang, Fang; Jansen, John A.

    2014-01-01

    The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23±0.35 mm2 at 21 days compared to 0.99±0.43 mm2 and 0.50±0.29 mm2 in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72±0.40 mm2. Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26±0.43 mm2 and 1.19±0.33 mm2, respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering. PMID:23865551

  12. Inhibiting and stimulating effects of TGF-. beta. 1 on osteoclastic bone resorption in fetal mouse bone organ cultures

    SciTech Connect

    Dieudonne, S.C.; Foo, P.; van Zoelen, E.J.; Burger, E.H. )

    1991-05-01

    The effects of TGF-{beta} 1 on osteoclastic resorption of fetal mouse calvaria and long bones at various stages of development was studied in organ culture. In resorbing calvariae and long bones with an established marrow cavity TGF-beta 1 (4-10 ng/ml) had a stimulating effect on 45Ca release that was partially inhibited by indomethacin. In primitive long bones, however, which were explanted before osteoclast invasion and excavation of a marrow cavity had started, TGF-beta 1 (1-4 ng/ml) inhibited 45Ca release by an indomethacin-insensitive mechanism. Histomorphometry of long bones after staining for tartrate-resistant acid phosphatase (TRAP) revealed that TGF-beta 1 treatment inhibited the migration of TRAP-positive cells from periosteum to developing marrow cavity and inhibited cell fusion. However, the formation of (mononuclear) TRAP-positive cells in the periosteum-perichondrium was strongly enhanced. These data suggest that TGF-beta 1 modulates various steps in the cascade of osteoclast development, recruitment, and activation in different ways, involving both prostaglandin-mediated and prostaglandin-independent pathways. Therefore the net effect of exogenous TGF-beta 1 on osteoclastic resorption in bone organ cultures depends on the relative prevalence of osteoclast progenitors, precursors, and mature osteoclasts in the tissue under study.

  13. A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

    PubMed Central

    Parikh, Mukti R.; Belch, Andrew R.; Pilarski, Linda M; Kirshner, Julia

    2014-01-01

    Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions

  14. Combined effects of low-level laser therapy and human bone marrow mesenchymal stem cell conditioned medium on viability of human dermal fibroblasts cultured in a high-glucose medium.

    PubMed

    Hendudari, Farzane; Piryaei, Abbas; Hassani, Seyedeh-Nafiseh; Darbandi, Hasan; Bayat, Mohammad

    2016-05-01

    Low-level laser therapy (LLLT) exhibited biostimulatory effects on fibroblasts viability. Secretomes can be administered to culture mediums by using bone marrow mesenchymal stem cells conditioned medium (BM-MSCs CM). This study investigated the combined effects of LLLT and human bone marrow mesenchymal stem cell conditioned medium (hBM-MSCs CM) on the cellular viability of human dermal fibroblasts (HDFs), which was cultured in a high-glucose (HG) concentration medium. The HDFs were cultured either in a concentration of physiologic (normal) glucose (NG; 5.5 mM/l) or in HG media (15 mM/l) for 4 days. LLLT was performed with a continuous-wave helium-neon laser (632.8 nm, power density of 0.00185 W/cm(2) and energy densities of 0.5, 1, and 2 J/cm(2)). About 10% of hBM-MSCs CM was added to the HG HDF culture medium. The viability of HDFs was evaluated using dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. A significantly higher cell viability was observed when laser of either 0.5 or 1 J/cm(2) was used to treat HG HDFs, compared to the control groups. The cellular viability of HG-treated HDFs was significantly lower compared to the LLLT + HG HDFs, hBM-MSCs CM-treated HG HDFs, and LLLT + hBM-MSCs CM-treated HG HDFs. In conclusion, hBM-MSCs CM or LLLT alone increased the survival of HG HDFs cells. However, the combination of hBM-MSCs CM and LLLT improved these results in comparison to the conditioned medium. PMID:26984346

  15. Oscillating Cell Culture Bioreactor

    NASA Technical Reports Server (NTRS)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  16. 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

    -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 β-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-β1). TGF-β1 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 α 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

  17. Hematopoietic effects of benzene inhalation assessed by long-term bone marrow culture

    SciTech Connect

    Abraham, N.G.

    1996-12-01

    The strong and long-lasting hematotoxic effect after benzene exposure in vivo (300 ppm, 6 hr/day, 5 days/week for 2 weeks) was assessed in mice with bone marrow cells grown in long-term bone marrow culture (LTBMC). Bone marrow cultures initiated 1 day after the last benzene exposure did not produce adequate numbers of hematopoietic cells over 3 weeks and, in most cases, no erythroid or myeloid clonogenic cells could be recovered. The adherent cell layer of these cultures had a lower capacity for supporting in vitro hematopoiesis after the second seeding with normal bone marrow cells compared with control cultures. Two weeks after the last benzene exposure, body weight, hematocrit, bone marrow cellularity, and committed hematopoietic progenitor content (BFU-E and CFU-GM) were regenerated to normal or subnormal values, whereas hematopoiesis in LTB MC was very poor. Over 8 weeks, little or no significant committed progenitor production was observed. Treatment of mice exposed to benzene with hemin (three doses of 3 {mu}g/g bw iv over 2 weeks for a total dose of 9 {mu}g/g) partially overcame the toxic effect of benzene on the hematopoietic system as measured by the LTBMC method. Cultures from mice treated with hemin had a modest recovery of BFU-E and CFU-GM clonogenic potential after 5 to 6 weeks in LTBMC. In contrast, little or no recovery was obtained for the adherent cell layer clonogenic capacity, even after hemin treatment. These results clearly indicate a strong, long-lasting toxic effect on the bone marrow stroma and a limited recovery of hematopoietic potential by clonogenic cells of the nonadherent population after in vivo hemin treatment. 35 refs., 3 figs., 1 tab.

  18. Aberrant immunoglobulin synthesis in light chain amyloidosis. Free light chain and light chain fragment production by human bone marrow cells in short-term tissue culture.

    PubMed Central

    Buxbaum, J

    1986-01-01

    Bone marrow cells obtained from 14 patients with light chain amyloid (AL) deposition were examined by biosynthetic labeling techniques. These analyses identified free monoclonal light chain (L-chain) synthesis even in those patients whose serum or urine contained no M protein or free L-chains or only an intact M protein. The experiments also identified a subset of patients whose plasma cells synthesized polypeptides bearing constant region antigenic determinants that migrated more rapidly than intact L-chains on polyacrylamide gels. Since most AL fibrils contain L-chain fragments rather than intact L-chains, these studies suggested that the genesis of the fibril components may reflect aberrant synthesis, proteolytic processing, or both. We also noted that in some individuals the pattern of Ig synthesis normalized after several courses of cytotoxic therapy. Thus, we could use bone marrow Ig synthesis as a sensitive biochemical parameter for monitoring therapy. Finally, the presence of aberrant synthetic products in these clones raised questions about their origin with respect to the normal processes of transcription, translation, and posttranslational modification in Ig-producing cells. Images PMID:3091637

  19. In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone.

    PubMed

    Arrigoni, Chiara; Bersini, Simone; Gilardi, Mara; Moretti, Matteo

    2016-01-01

    Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps. PMID:27571063

  20. Bone development: overview of bone cells and signaling.

    PubMed

    Teti, Anna

    2011-12-01

    Vertebrates evolved elaborating a structure made up of more than 200 bones and cartilages articulated with one another to form the skeleton, through which locomotion, organ protection, lodging of hematopoiesis, and mineral homeostasis are allowed. Skeletogenesis starts at the fetal stage, along with marrow hematopoiesis, and evolves postnatally through modeling and remodeling processes that permit skeletal mass buildup. Preservation of skeletal mass is then implemented by balanced remodeling, which ensures continuous renovation of the tissue to allow its mechanical, structural, and metabolic properties to remain unaltered until ageing or diseases disrupt this equilibrium. Skeletal homeostasis is fulfilled by specialized bone cells in association with systemic and local regulators. Herein I review landmark discoveries that shed light on the intricate mesh connecting bone cells among themselves and with other systems, thus representing the cellular basis of normal and abnormal bone development and homeostasis. PMID:21948208

  1. Physiological effects of microgravity on bone cells.

    PubMed

    Arfat, Yasir; Xiao, Wei-Zhong; Iftikhar, Salman; Zhao, Fan; Li, Di-Jie; Sun, Yu-Long; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

    2014-06-01

    Life on Earth developed under the influence of normal gravity (1g). With evidence from previous studies, scientists have suggested that normal physiological processes, such as the functional integrity of muscles and bone mass, can be affected by microgravity during spaceflight. During the life span, bone not only develops as a structure designed specifically for mechanical tasks but also adapts for efficiency. The lack of weight-bearing forces makes microgravity an ideal physical stimulus to evaluate bone cell responses. One of the most serious problems induced by long-term weightlessness is bone mineral loss. Results from in vitro studies that entailed the use of bone cells in spaceflights showed modification in cell attachment structures and cytoskeletal reorganization, which may be involved in bone loss. Humans exposed to microgravity conditions experience various physiological changes, including loss of bone mass, muscle deterioration, and immunodeficiency. In vitro models can be used to extract valuable information about changes in mechanical stress to ultimately identify the different pathways of mechanotransduction in bone cells. Despite many in vivo and in vitro studies under both real microgravity and simulated conditions, the mechanism of bone loss is still not well defined. The objective of this review is to summarize the recent research on bone cells under microgravity conditions based on advances in the field. PMID:24687524

  2. 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

    -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 β-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-β1). TGF-β1 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 α 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

  3. Osteocytes: The master cells in bone remodelling.

    PubMed

    Prideaux, Matthew; Findlay, David M; Atkins, Gerald J

    2016-06-01

    Bone remodelling is an essential process for shaping and maintaining bone mass in the mature skeleton. During our lifetime bone is constantly being removed by osteoclasts and new bone is formed by osteoblasts. The activities of osteoclasts and osteoblasts must be regulated under a strict balance to ensure that bone homeostasis is maintained. Osteocytes, which form an extensive, multi-functional syncytium throughout the bone, are increasingly considered to be the cells that maintain this balance. Current research is elucidating key signalling pathways by which the osteocyte exerts control over the other cell types in bone and over its own activities, and potential ways in which these pathways may be exploited therapeutically. PMID:26927500

  4. Double-layered cell transfer technology for bone regeneration.

    PubMed

    Akazawa, Keiko; Iwasaki, Kengo; Nagata, Mizuki; Yokoyama, Naoki; Ayame, Hirohito; Yamaki, Kazumasa; Tanaka, Yuichi; Honda, Izumi; Morioka, Chikako; Kimura, Tsuyoshi; Komaki, Motohiro; Kishida, Akio; Izumi, Yuichi; Morita, Ikuo

    2016-01-01

    For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called "cell transfer technology", enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration. PMID:27624174

  5. [Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro].

    PubMed

    Ming, Lei-Guo; Wang, Ming-Gang; Chen, Ke-Ming; Zhou, Jian; Han, Gui-Qiu; Zhu, Rui-Qing

    2012-02-01

    This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption. PMID:22512027

  6. Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression.

    PubMed

    Ishizeki, K; Takigawa, M; Harada, Y; Suzuki, F; Nawa, T

    1996-01-01

    We examined whether Meckel's cartilage of embryonic mice, 17 days in utero, undergo the cellular transformation into the osteocyte-like phenotype under organ culture conditions. Explants were grown by our original pithole method modified Trowell-type cultures for up to 4 weeks at 37 degrees C under 5% CO2 in air. Specimens were examined using histological procedures including immunostaining and electron microscopy. In addition, the effects of beta-glycerophosphate on matrix calcification were also examined in cultures with or without beta-glycerophosphate. Addition of beta-glycerophosphate induced calcification at a higher level, but calcium mineral deposition occurred regardless of the addition of beta-glycerophosphate to the culture medium. Light and electron microscopic analyses showed that freshly isolated chondrocytes prior to cell culture had typical hypertrophic morphology, but shortly after commencement of culture, they showed morphological modifications. The cells showing chondrocytic phenotypes became basophilic elliptical cells, and eventually transformed into flattened osteocyte-like cells. Bone-like features for cellular elements were characterized by spindle-shaped cells with elongated processes accompanying bone-specific thick-banded collagen fibrils. Immunostaining showed that at 2 weeks in culture, type I and type II collagens coexisted in the matrix, but subsequently type II collagen synthesis ceased and was replaced by type I collagen synthesis. Immunofluorescent labeling for osteocalcin was noted first in the peripheral cells by 1 week, but at 3 weeks this reaction spread to the central zone in explants. Alkaline phosphatase activity (ALPase) was expressed on the cells in the central zone prior to calcium mineral deposition as shown by von Kossa's reaction at 3 weeks in culture. These results showed that Meckel's cartilage chondrocytes in organ culture synthesize bone-type proteins accompanying osteocytic phenotype expression. PMID:8838497

  7. Bone Metastasis from Renal Cell Carcinoma

    PubMed Central

    Chen, Szu-Chia; Kuo, Po-Lin

    2016-01-01

    About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastasis that are often osteolytic and cause substantial morbidity, such as pain, pathologic fracture, spinal cord compression and hypercalcemia. The presence of bone metastasis in RCC is also associated with poor prognosis. Bone-targeted treatment using bisphosphonate and denosumab can reduce skeletal complications in RCC, but does not cure the disease or improve survival. Elucidating the molecular mechanisms of tumor-induced changes in the bone microenvironment is needed to develop effective treatment. The “vicious cycle” hypothesis has been used to describe how tumor cells interact with the bone microenvironment to drive bone destruction and tumor growth. Tumor cells secrete factors like parathyroid hormone-related peptide, transforming growth factor-β and vascular endothelial growth factor, which stimulate osteoblasts and increase the production of the receptor activator of nuclear factor κB ligand (RANKL). In turn, the overexpression of RANKL leads to increased osteoclast formation, activation and survival, thereby enhancing bone resorption. This review presents a general survey on bone metastasis in RCC by natural history, interaction among the immune system, bone and tumor, molecular mechanisms, bone turnover markers, therapies and healthcare burden. PMID:27338367

  8. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  9. Bone marrow stromal cells cultured on poly (lactide-co-glycolide)/nano-hydroxyapatite composites with chemical immobilization of Arg-Gly-Asp peptide and preliminary bone regeneration of mandibular defect thereof.

    PubMed

    Huang, Yanxia; Ren, Jie; Ren, Tianbin; Gu, Shuying; Tan, Qinggang; Zhang, Lihong; Lv, Kaige; Pan, Kefeng; Jiang, Xinquan

    2010-12-15

    Polyethyleneimine (PEI) was used to create active groups on the poly (lactide-co-glycolide)/nano-hydroxyapatite (PLGA/NHA) surface and Arg-Gly-Asp (RGD) was grafted on the active groups and novel PLGA/NHA 2-D membranes and 3D scaffolds modified with RGD were obtained. X-ray photoelectron spectrum (XPS) results show that sulfur displays only on the modified surface. The RGD-modified PLGA/NHA materials also have much lower static water contact angle and much higher water-absorption ability, which shows that after chemical treatment, the modified materials show better hydrophilic properties. Atomic force microscope (AFM) shows that after surface modification, the surface morphology of PLGA is greatly changed. All these results indicate that RGD peptide has successfully grafted on the surface of PLGA. Rabbit bone marrow stromal cells (MSCs) were seeded in the 2D membranes and 3D scaffolds materials. The influences of the RGD on the cell attachment, growth and differentiation, and proliferation on the different materials were studied. The modified scaffolds were implanted into rabbits to observe preliminary application in regeneration of mandibular defect. The PLGA/NHA-RGD presents better results in bone regeneration in rabbit mandibular defect. PMID:20872750

  10. 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

  11. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics.

    PubMed

    Sun, Dao-Cai; Li, De-Hua; Ji, Hui-Cang; Rao, Guo-Zhou; Liang, Li-Hua; Ma, Ai-Jie; Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang

    2012-06-01

    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones. PMID:22473318

  12. Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells

    PubMed Central

    Yuan, Jing; Yu, Jian-xiong

    2016-01-01

    Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells. PMID:27335573

  13. Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

    PubMed

    Yuan, Jing; Yu, Jian-Xiong

    2016-05-01

    Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells. PMID:27335573

  14. Make no bones about it: cells could soon be reprogrammed to grow replacement bones?

    PubMed

    de Peppo, Giuseppe Maria; Marolt, Darja

    2014-01-01

    Recent developments in nuclear reprogramming allow the generation of patient-matched stem cells with broad potential for applications in cell therapies, disease modeling and drug discovery. An increasing body of work is reporting the derivation of lineage-specific progenitors from human-induced pluripotent stem cells (hiPSCs), which could in the near future be used to engineer personalized tissue substitutes, including those for reconstructive therapies of bone. Although the potential clinical impact of such technology is not arguable, significant challenges remain to be addressed before hiPSC-derived progenitors can be employed to engineer bone substitutes of clinical relevance. The most important challenge is indeed the construction of personalized multicellular bone substitutes for the treatment of complex skeletal defects that integrate fast, are immune tolerated and display biofunctionality and long-term safety. As recent studies suggest, the merging of iPSC technology with advanced biomaterials and bioreactor technologies offers a way to generate bone substitutes in a controllable, automated manner with potential to meet the needs for scale-up and requirements for translation into clinical practice. It is only via the use of state-of-the-art cell culture technologies, process automation under GMP-compliant conditions, application of appropriate engineering strategies and compliance with regulatory policies that personalized lab-made bone grafts can start being used to treat human patients. PMID:24053578

  15. A perfusion bioreactor system efficiently generates cell-loaded bone substitute materials for addressing critical size bone defects.

    PubMed

    Kleinhans, Claudia; Mohan, Ramkumar Ramani; Vacun, Gabriele; Schwarz, Thomas; Haller, Barbara; Sun, Yang; Kahlig, Alexander; Kluger, Petra; Finne-Wistrand, Anna; Walles, Heike; Hansmann, Jan

    2015-09-01

    Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide-co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans. PMID:26011163

  16. Human progenitor cells for bone engineering applications.

    PubMed

    de Peppo, G M; Thomsen, P; Karlsson, C; Strehl, R; Lindahl, A; Hyllner, J

    2013-06-01

    In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies. PMID:23642054

  17. Response of three murine macrophage populations to particulate debris: bone resorption in organ cultures.

    PubMed

    Glant, T T; Jacobs, J J

    1994-09-01

    Particulate wear debris from bone cement or prosthetic components can stimulate macrophages to cause bone resorption. We compared the effect of particle composition (titanium and polymethylmethacrylate as inherent components of prosthetic materials or bone cement and polystyrene as a reference material) on the secretion of interleukin-1 and prostaglandin E2 by peritoneal macrophages and monocyte/macrophage cell lines (P388D1 and IC-21) and on the bone-resorbing activity of conditioned medium harvested from these particle-challenged macrophages. Titanium particles (1-3 microns) in peritoneal macrophage cultures exhibited significantly enhanced bone-resorbing activity measured as 45Ca release, whereas polymethylmethacrylate and polystyrene exhibited this effect to a greater extent in the P388D1 and IC-21 monocyte/macrophage cultures. Although exogenous prostaglandin E2 and recombinant human interleukin-1 could significantly increase the 45Ca release and indomethacin significantly reduced both the spontaneous calcium efflux and active 45Ca release from calvarial bones labeled in vivo, the levels of interleukin-1 and prostaglandin E2, alone or together, did not always correlate with the bone-resorbing activity of conditioned media. Thus, the actual levels of potent bone-resorbing agents (prostaglandin E2 and interleukin-1) measured in conditioned tissue culture media did not necessarily reflect the bone-resorbing capability. An important result of this study is that different macrophage populations may respond differently to the same microenvironmental signal, which in our investigation was particulate wear debris of differing composition and size. PMID:7931789

  18. Bone marrow-derived stem cells and radiation response.

    PubMed

    Greenberger, Joel S; Epperly, Michael

    2009-04-01

    The recovery of tissues and organs from ionizing irradiation is critically dependent on the repopulation of resident stem cells, defined as the subset of cells with capacity for both self-renewal and differentiation. Stem cells of both hematopoietic and epithelial origin reside in defined areas of the cellular microenvironment (recently defined as the stem cell "niche"). Experiments using serial repopulation assays in serial generations of total body irradiated mice receiving transplanted marrow and in continuous bone marrow cultures both identified specific microanatomic sites that comprise the bone marrow stem cell niche. Supportive cells of the hematopoietic microenvironment not only contribute to stem cell repopulation capacity but also to the maintenance of their quiescent or nonproliferative state, which allows the most primitive hematopoietic stem cells to stay in a noncycling state protected from both direct ionizing radiation-induced cell-cycle phase-specific killing and indirect cytokine and free radical mediated killing. Recent evidence has defined both cell contact and humoral mechanisms of protection of hematopoietic stem cells by stromal cells. There is also recent evidence for multilineage differentiation capacity of cells of the hematopoietic microenvironment termed bone marrow stromal cells (mesenchymal stem cells). Both hematopoietic stem cells and mesenchymal stem cell populations have been shown to be involved in the repair of ionizing irradiation damage of distant epithelial as well as other hematopoietic sites through their capacity to migrate through the circulation. The radiobiology of these 2 bone marrow stem cell populations is the subject of intense investigation. This review defines the status of research in the areas of stem cell quiescence, niche contact, and migratory responses to ionizing irradiation. PMID:19249651

  19. [Inhibitory effect of 8-prenylnaringenin on osteoclastogensis of bone marrow cells and bone resorption activity].

    PubMed

    Lü, Xiang; Zhou, Ying; Chen, Ke-Ming; Zhao, Zhi; Zhou, Jian; Ma, Xiao-Ni

    2013-03-01

    This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts. Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in alpha-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol xL(-1), separately. 17beta-Estradiol (E2, 1 x 10(-7) mol x L(-7)) was used as positive control. T RAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days. Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately. The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR. 8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG-supplemented groups. The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly. All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1 x 10(-5) mol x L(-1) 8-PNG. 8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation. PMID:23724646

  20. Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling

    PubMed Central

    Crane, Janet L.; Cao, Xu

    2014-01-01

    During bone resorption, abundant factors previously buried in the bone matrix are released into the bone marrow microenvironment, which results in recruitment and differentiation of bone marrow mesenchymal stem cells (MSCs) for subsequent bone formation, temporally and spatially coupling bone remodeling. Parathyroid hormone (PTH) orchestrates the signaling of many pathways that direct MSC fate. The spatiotemporal release and activation of matrix TGF-β during osteoclast bone resorption recruits MSCs to bone-resorptive sites. Dysregulation of TGF-β alters MSC fate, uncoupling bone remodeling and causing skeletal disorders. Modulation of TGF-β or PTH signaling may reestablish coupled bone remodeling and be a potential therapy. PMID:24487640

  1. Stem Cells in Teeth and Craniofacial Bones.

    PubMed

    Zhao, H; Chai, Y

    2015-11-01

    Stem cells are remarkable, and stem cell-based tissue engineering is an emerging field of biomedical science aiming to restore damaged tissue or organs. In dentistry and reconstructive facial surgery, it is of great interest to restore lost teeth or craniofacial bone defects using stem cell-mediated therapy. In the craniofacial region, various stem cell populations have been identified with regeneration potential. In this review, we provide an overview of the current knowledge concerning the various types of tooth- and craniofacial bone-related stem cells and discuss their in vivo identities and regulating mechanisms. PMID:26350960

  2. Paracrine Factors Produced by Bone Marrow Stromal Cells Induce Apoptosis and Neuroendocrine Differentiation in Prostate Cancer Cells

    PubMed Central

    Zhang, Chu; Soori, Mehrnoosh; Miles, Fayth; Sikes, Robert A.; Carson, Daniel D.; Chung, Leland L.W.; Farach-Carson, Mary C.

    2010-01-01

    Background Preferential bony metastasis of human prostate cancer (PCa) cells contributes to disease mortality and morbidity. Local factors in bone stromal extracellular matrix microenvironment affect tumor growth through paracrine interactions between tumor and stromal cells. Methods Using co-culture and medium transfer, we used several methods to assess interactions between PCa and bone stromal cells using three PCa cell lines: PC3, LNCaP, and the LNCaP derivative, C4-2B. Results Co-culture of LNCaP and C4-2B cells with bone marrow stromal cell lines, HS27a and HS5, decreased cell number, as did culture with conditioned medium (CM) harvested from these two cell lines suggesting a soluble paracrine factor was responsible. PC3 cell growth was unaffected. CM harvested from bone stromal cell lines triggered apoptosis in LNCaP and C4-2B cell lines, but not in PC3 cells. Surviving C4-2B cells grown in bone stromal cell CM over several days were growth arrested, suggesting presence of a growth inhibitor. Apoptosis induced by CM was dose-dependent. Flow cytometry demonstrated that over a five day culture period in stromal cell CM, LNCaP and C4-2B cell lines, but not PC3 cells, underwent greater apoptosis than parallel cultures in SF medium. The LNCaP and C4-2B cells showed morphology and biomarker expression consistent with transdifferentiation towards a neuroendocrine phenotype after exposure to stromal cell CM. Conclusions The reactive bone stromal microenvironment initially is hostile to PCa cells producing widespread apoptosis. Activation of transdifferentiation in a subset of apoptotic resistant cells may support phenotypic adaptation during disease progression in bone, eventually favoring lethal disease. PMID:20665531

  3. Laser Light Induced Photosensitization Of Lymphomas Cells And Normal Bone Marrow Cells

    NASA Astrophysics Data System (ADS)

    Gulliya, Kirpal S.; Pervaiz, Shazib; Nealon, Don G.; VanderMeulen, David L.

    1988-06-01

    Dye mediated, laser light induced photosensitization was tested in an in vitro model for its efficacy in eliminating the contaminating tumor cells for ex vivo autologous bone marrow purging. Daudi and U-937 cells (3 x 106/ml) in RPMI-1640 supplemented with 0.25% human albumin were mixed with 20 µg/ml and 25 µg/ml of MC-540, respectively. These cell-dye mixtures were then exposed to 514 nm argon laser light. Identical treatment was given to the normal bone marrow cells. Viability was determined by the trypan blue exclusion method. Results show that at 31.2 J/cm2 irradiation, 99.9999% Daudi cells were killed while 87% of the normal bone marrow cells survived. No regrowth of Daudi cells was observed for 30 days in culture. However, a light dose of 93.6 J/cm2 was required to obtain 99.999% U-937 cell kill with 80% normal bone marrow cell survival. Mixing of irradiated bone marrow cells with an equal number of lymphoma cells did not interfere with the photodynamic killing of lymphoma cells. Exposure of cells to low doses of recombinant interferon-alpha prior to photodynamic therapy increased the viability of lymphoma cells.

  4. Anticarcinogenic and antimutagenic activity of Alstonia scholaris on the albino mice bone marrow cells and peripheral human lymphocyte culture against methyl methane sulfonate induced genotoxicity

    PubMed Central

    Ahmad, Md. Sultan; Ahmad, Sheeba; Ali, Afsar; Afzal, Mohammad

    2016-01-01

    Background: The use of medicinal plants in modern medicine for the prevention and treatment of cancer is an important aspect. For this reason, it is important to identify antitumor promoting agents present in medicinal plants commonly used by the human population. Materials and Methods: We used in vivo and in vitro methods using chromosomal aberrations (CAs), sister chromatid exchange (SCE) and replication index (RI) as markers, exposed by methyl methanesulfonate (MMS) as well as alcoholic extract of Alstonia scholaris in five increasing concentrations (200, 250, 300, 350 and 400 mg/kg body weight for in vivo and 150, 200, 250 and 300 μg/ml of culture) and of three different durations of 24, 48 and 72 h in the presence as well absence of S9 mix. Results: Extracts of Alstonia reduces the total aberrant cells ranges from 10.0% to 41.84% and frequencies of aberration in the aberrant cells ranges from 220 to 124 against 290 aberrations causes due to MMS in vivo. Similarly in the in vitro, it reduces CAs (39.62%, 32.83%, and 38.48%) and (45.31%, 44.46%, and 38.34%) at 24, 48, and 72 h of exposure respectively; in the absence as well as presence of liver S9 fraction. It also reduces SCE from 7.70 to 4.20 per cell and enhances RI from 1.45 to 1.64. Conclusion: Extracts of Alstonia significantly reduces the number of aberrant cells and frequency of aberration per cell at each concentration and duration of exposure in vivo; and CAs and SCE in vitro and enhances RI. PMID:27308264

  5. Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells

    PubMed Central

    Florencio-Silva, Rinaldo; Sasso, Gisela Rodrigues da Silva; Sasso-Cerri, Estela; Simões, Manuel Jesus; Cerri, Paulo Sérgio

    2015-01-01

    Bone tissue is continuously remodeled through the concerted actions of bone cells, which include bone resorption by osteoclasts and bone formation by osteoblasts, whereas osteocytes act as mechanosensors and orchestrators of the bone remodeling process. This process is under the control of local (e.g., growth factors and cytokines) and systemic (e.g., calcitonin and estrogens) factors that all together contribute for bone homeostasis. An imbalance between bone resorption and formation can result in bone diseases including osteoporosis. Recently, it has been recognized that, during bone remodeling, there are an intricate communication among bone cells. For instance, the coupling from bone resorption to bone formation is achieved by interaction between osteoclasts and osteoblasts. Moreover, osteocytes produce factors that influence osteoblast and osteoclast activities, whereas osteocyte apoptosis is followed by osteoclastic bone resorption. The increasing knowledge about the structure and functions of bone cells contributed to a better understanding of bone biology. It has been suggested that there is a complex communication between bone cells and other organs, indicating the dynamic nature of bone tissue. In this review, we discuss the current data about the structure and functions of bone cells and the factors that influence bone remodeling. PMID:26247020

  6. 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. )

    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.

  7. Engineering bone tissue substitutes from human induced pluripotent stem cells

    PubMed Central

    de Peppo, Giuseppe Maria; Marcos-Campos, Iván; Kahler, David John; Alsalman, Dana; Shang, Linshan; Vunjak-Novakovic, Gordana; Marolt, Darja

    2013-01-01

    Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease. PMID:23653480

  8. 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

  9. HOW DO BONE CELLS SENSE MECHANICAL LOADING?

    PubMed Central

    Gusmão, Carlos Vinícius Buarque de; Belangero, William Dias

    2015-01-01

    Influenced by gravidity, bone tissue experiences stronger or lighter deformation according to the strength of the activities of daily life. Activities resulting in impact are particularly known to stimulate osteogenesis, thus reducing bone mass loss. Knowing how bone cells recognize the mechanical deformation imposed to the bone and trigger a series of biochemical chain reactions is of crucial importance for the development of therapeutic and preventive practices in orthopaedic activity. There is still a long way to run until we can understand the whole process, but current knowledge has shown a strong progression, with researches being conducted focused on therapies. For a mechanical sign to be transformed into a biological one (mechanotransduction), it must be amplified at cell level by the histological structure of bone tissue, producing tensions in cell membrane proteins (integrins) and changing their spatial structure. Such change activates bindings between these and the cytoskeleton, producing focal adhesions, where cytoplasmatic proteins are recruited to enable easier biochemical reactions. Focal adhesion kinase (FAK) is the most important one being self-activated when its structure is changed by integrins. Activated FAK triggers a cascade of reactions, resulting in the activation of ERK-1/2 and Akt, which are proteins that, together with FAK, regulate the production of bone mass. Osteocytes are believed to be the mechanosensor cells of the bone and to transmit the mechanical deformation to osteoblasts and osteoclasts. Ionic channels and gap junctions are considered as intercellular communication means for biochemical transmission of a mechanical stimulus. These events occur continuously on bone tissue and regulate bone remodeling. PMID:27022510

  10. 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

  11. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  12. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures.

    PubMed

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A; Harris, William A

    2013-04-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

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

    SciTech Connect

    Otsuru, Satoru; Tamai, Katsuto . E-mail: tamai@gts.med.osaka-u.ac.jp; Yamazaki, Takehiko; Yoshikawa, Hideki; Kaneda, Yasufumi

    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.

  14. 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

  15. Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

    SciTech Connect

    Long, G.J.; Rosen, J.F.; Pounds, J.G. )

    1990-02-01

    A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state {sup 210}Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with {sup 210}Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of {sup 210}Pb from the cells over a {sup 210}-min period. The intracellular metabolism of {sup 210}Pb was characterized by three kinetic pools of {sup 210}Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of {sup 210}Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone.

  16. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    PubMed Central

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  17. Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

    PubMed

    Coughlin, T R; Schiavi, J; Alyssa Varsanik, M; Voisin, M; Birmingham, E; Haugh, M G; McNamara, L M; Niebur, G L

    2016-01-01

    Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function. PMID:27434268

  18. A 3D printed nano bone matrix for characterization of breast cancer cell and osteoblast interactions

    NASA Astrophysics Data System (ADS)

    Zhu, Wei; Castro, Nathan J.; Cui, Haitao; Zhou, Xuan; Boualam, Benchaa; McGrane, Robert; Glazer, Robert I.; Zhang, Lijie Grace

    2016-08-01

    Bone metastasis is one of the most prevalent complications of late-stage breast cancer, in which the native bone matrix components, including osteoblasts, are intimately involved in tumor progression. The development of a successful in vitro model would greatly facilitate understanding the underlying mechanism of breast cancer bone invasion as well as provide a tool for effective discovery of novel therapeutic strategies. In the current study, we fabricated a series of in vitro bone matrices composed of a polyethylene glycol hydrogel and nanocrystalline hydroxyapatite of varying concentrations to mimic the native bone microenvironment for the investigation of breast cancer bone metastasis. A stereolithography-based three-dimensional (3D) printer was used to fabricate the bone matrices with precisely controlled architecture. The interaction between breast cancer cells and osteoblasts was investigated in the optimized bone matrix. Using a Transwell® system to separate the two cell lines, breast cancer cells inhibited osteoblast proliferation, while osteoblasts stimulated breast cancer cell growth, whereas, both cell lines increased IL-8 secretion. Breast cancer cells co-cultured with osteoblasts within the 3D bone matrix formed multi-cellular spheroids in comparison to two-dimensional monolayers. These findings validate the use of our 3D printed bone matrices as an in vitro metastasis model, and highlights their potential for investigating breast cancer bone metastasis.

  19. Huanglongbing and psyllid cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We successfully established cell cultures of the Asian citrus psyllid, Diaphorina citri (Psyllidae: Hemiptera), DcHH-1. The cell culture also supported growth of Candidatus Liberibacter asiaticus. This bacterial pathogen is associated with Huanglongbing, known as citrus greening disease. Research on...

  20. Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

    PubMed

    Kitami, Megumi; Kaku, Masaru; Rocabado, Juan Marcelo Rosales; Ida, Takako; Akiba, Nami; Uoshima, Katsumi

    2016-09-01

    Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc. PMID:26754153

  1. Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures.

    PubMed

    Hagmann, Sebastien; Rimmele, Claudia; Bucur, Florin; Dreher, Thomas; Zeifang, Felix; Moradi, Babak; Gotterbarm, Tobias

    2016-01-01

    Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches. PMID:27516777

  2. Mesenchymal Stromal Cells from Osteoarthritic Synovium Are a Distinct Population Compared to Their Bone-Marrow Counterparts regarding Surface Marker Distribution and Immunomodulation of Allogeneic CD4+ T-Cell Cultures

    PubMed Central

    Bucur, Florin; Moradi, Babak

    2016-01-01

    Introduction. The participation of an inflammatory joint milieu has been described in osteoarthritis (OA) pathogenesis. Mesenchymal stromal cells (MSCs) play an important role in modulating inflammatory processes. Based on previous studies in an allogeneic T-cell coculture model, we aimed at further determining the role of synovial MSCs in OA pathogenesis. Methods. Bone-marrow (BM) and synovial membrane (SM) MSCs from hip joints of late stage OA patients and CD4+ T-cells from healthy donors were analysed regarding surface marker expression before and after coculture. Proliferation upon CD3/CD28 stimulation and cytokine analyses were compared between MSCs. Results. SM-MSCs differed from BM-MSCs in several surface markers and their osteogenic differentiation potential. Cocultures of both MSCs with CD4+ T-cells resulted in recruitment of CD45RA+ FoxP3+ regulatory T-cells. Upon stimulation, only SM-MSCs suppressed CD4+ T-cell proliferation, while both SM-MSCs and BM-MSCs modified cytokine profiles through suppressing IL-2 and TNF-α as well as increasing IL-6 secretion. Conclusions. Synovial MSCs from OA joints are a unique fraction that can be distinguished from their bone-marrow derived counterparts. Their unique ability to suppress CD3/CD28 induced CD4+ T-cell proliferation makes them a potential target for future therapeutic approaches. PMID:27516777

  3. Investigation of the effects of aging on homologous recombination in long-term bone marrow cultures.

    PubMed

    Epperly, Michael W; Rugo, Rebecca; Cao, Shaonan; Wang, Hong; Franicola, Darcy; Goff, Julie P; Shen, Hongmei; Zhang, Xichen; Wiktor-Brown, Dominika; Engelward, Bevin P; Greenberger, Joel S

    2009-01-01

    Fluorescent yellow direct repeat (FYDR) mice carry a transgenic reporter for homologous recombination (HR) and have been used to reveal an age-dependent increase in HR in the pancreas. An established in vitro model system for accelerated aging of the marrow is the mouse long-term bone marrow culture (LTBMC) system. To determine whether the FYDR system, in which an HR event can lead to a fluorescent cell, can be used to study the effects of aging in LTBMCs, clonally expanded hematopoietic and marrow stromal cells in FYDR, positive control FYDR-Recombined (FYDR-Rec), and negative control wild-type C57BL/6NHsd (WT) LTBMCs were analysed. All groups of cultures demonstrated equivalent parameters of continuous hematopoiesis including generation of multilineage colony forming CFU-GM progenitor cells for over 22 weeks and age associated senescence of hematopoiesis. Results indicate that low expression of the FYDR transgene in bone marrow cells in vivo and in vitro prevents the use of the FYDR mice to study rare combination events in bone marrow. Using an alternative approach for detecting HR, namely the sister chromatid exchange (SCE) assay, a statistically significant increase in the number of SCEs per chromosome was observed in adherent cells subcultured from 20-week-compared to 4-week-old LTBMCs. These data suggest that adherent marrow stromal cells from LTBMCs become increasingly susceptible to HR events during aging. PMID:19779099

  4. Dexamethasone Enhances Osteogenic Differentiation of Bone Marrow- and Muscle-Derived Stromal Cells and Augments Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

    PubMed Central

    Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Masaoka, Tomokazu; Xuetao, Wei; Yoshii, Toshitaka; Horie, Masaki; Yasuda, Hiroaki; Uemura, Toshimasa; Okawa, Atsushi; Sotome, Shinichi

    2015-01-01

    We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2. PMID:25659106

  5. Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration.

    PubMed

    Colnot, Céline

    2009-02-01

    Bone repair requires the mobilization of adult skeletal stem cells/progenitors to allow deposition of cartilage and bone at the injury site. These stem cells/progenitors are believed to come from multiple sources including the bone marrow and the periosteum. The goal of this study was to establish the cellular contributions of bone marrow and periosteum to bone healing in vivo and to assess the effect of the tissue environment on cell differentiation within bone marrow and periosteum. Results show that periosteal injuries heal by endochondral ossification, whereas bone marrow injuries heal by intramembranous ossification, indicating that distinct cellular responses occur within these tissues during repair. [corrected] Next, lineage analyses were used to track the fate of cells derived from periosteum, bone marrow, and endosteum, a subcompartment of the bone marrow. Skeletal progenitor cells were found to be recruited locally and concurrently from periosteum and/or bone marrow/endosteum during bone repair. Periosteum and bone marrow/endosteum both gave rise to osteoblasts, whereas the periosteum was the major source of chondrocytes. Finally, results show that intrinsic and environmental signals modulate cell fate decisions within these tissues. In conclusion, this study sheds light into the origins of skeletal stem cells/progenitors during bone regeneration and indicates that periosteum, endosteum, and bone marrow contain pools of stem cells/progenitors with distinct osteogenic and chondrogenic potentials that vary with the tissue environment. PMID:18847330

  6. Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells

    NASA Astrophysics Data System (ADS)

    Ma, Jin-Ling; van den Beucken, Jeroen J. J. P.; Pan, Ju-Li; Cui, Fu-Zhai; Chen, Su

    2014-03-01

    Cellular strategies remain a crucial component in bone tissue engineering (BTE). So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the tissue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding (i) critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and (ii) the usage of monocultures in clinical application in BTE.

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

    SciTech Connect

    Cowan, Robert W.; Ghert, Michelle; Singh, Gurmit

    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

  8. Adult stem cells in bone and cartilage tissue engineering.

    PubMed

    Salgado, António J; Oliveira, João T; Pedro, Adriano J; Reis, Rui L

    2006-09-01

    The progressive increase in life expectancy within the last century has led to the appearance of novel health related problems, some of those within the musculoskeletal field. Among the latter, one can find diseases such as osteoporosis, rheumatoid arthritis and bone cancer, just to mention some of the most relevant. Other related problems are those that arise from serious injuries, often leading to non-recoverable critical size defects. The therapies currently used to treat this type of diseases/injuries are based on the use of pharmaceutical agents, auto/allotransplant and synthetic materials. However, such solutions present a number of inconveniences and therefore, there is a constant search for novel therapeutic solutions. The appearance of a novel field of science called Tissue engineering brought some hope for the solution of the above mentioned problems. In this field, it is believed that by combining a 3D porous template--scaffold--with an adequate cell population, with osteo or chondrogenic potential, it will be possible to develop bone and cartilage tissue equivalents that when implanted in vivo, could lead to the total regeneration of the affected area. This ideal cell population should have a series of properties, namely a high osteo and chondrogenic potential and at the same time, should be easily expandable and maintained in cultures for long periods of time. Due to its natural and intrinsic properties, stem cells are one of the best available cell types. However, after this sentence, the readers may ask, "Which Stem Cells?". During the last 10/15 years, the scientific community witnessed and reported the appearance of several sources of stem cells with both osteo and chondrogenic potential. Therefore, the present review intends to make an overview of data reported on different sources of adult stem cells (bone marrow, periosteum, adipose tissue, skeletal muscle and umbilical cord) for bone and cartilage regenerative medicine, namely those focusing on

  9. Differential effects of drugs upon hematopoiesis can be assessed in long-term bone marrow cultures established on nylon screens.

    PubMed

    Naughton, B A; Sibanda, B; Azar, L; San Roman, J

    1992-04-01

    Hematotoxicity is associated with exposure to chemotherapeutic drugs and numerous other agents. Most measurements of the hematopoietic effects of prospective therapeutic drugs and environmental agents have been made in animal models. We tested the influence of various drugs on hematopoiesis in long-term cultures of Long-Evans rat bone marrow cells. These cultures were established on nylon screen-bone marrow stromal cell templates that were suspended in liquid medium. Previous phenotypic analyses of adherent zone cells of suspended nylon screen bone marrow cultures (NSBMC) using monoclonal antibodies and flow cytometry indicated that they maintain a multilineage character for extended periods in culture and display continuous proliferation of hematopoietic progenitors (colony-forming unit culture [CFU-C]). NSBMC of various ages were incubated for 21 hr with several concentrations of beta-D-cytosine arabinofuranoside, 5-fluorouracil, cyclophosphamide, or methotrexate. Adherent zone cells were dissociated enzymatically, phenotyped by flow cytometry, and assayed for colony-forming unit culture content. beta-D-cytosine arabinofuranoside, 5-fluorouracil, and methotrexate treatment of bone marrow cultures resulted in a dose-related diminution in colony-forming unit culture numbers in the adherent zones of NSBMC. Phenotypic analyses revealed similar trends but certain of these drugs manifested lineage specificities. Toxicity was also related to cyclophosphamide dose, but the presence of bone marrow stroma was necessary to demonstrate this effect in vitro. A subpopulation of these cells was found to metabolize ethoxyfluorescein ethyl ester to fluorescein after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an effect which was quantified by flow cytometry. NSBMC may be used to ascertain lineage-specific toxicities and evaluate the effects of drugs on the proliferation of hematopoietic progenitor cells. PMID:1549628

  10. 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.

  11. Candidates Cell Sources to Regenerate Alveolar Bone from Oral Tissue

    PubMed Central

    Nishimura, Masahiro; Takase, Kazuma; Suehiro, Fumio; Murata, Hiroshi

    2012-01-01

    Most of the cases of dental implant surgery, especially the bone defect extensively, are essential for alveolar ridge augmentation. As known as cell therapy exerts valuable effects on bone regeneration, numerous reports using various cells from body to regenerate bone have been published, including clinical reports. Mesenchymal cells that have osteogenic activity and have potential to be harvested from intra oral site might be a candidate cells to regenerate alveolar bone, even dentists have not been harvested the cells outside of mouth. This paper presents a summary of somatic cells in edentulous tissues which could subserve alveolar bone regeneration. The candidate tissues that might have differentiation potential as mesenchymal cells for bone regeneration are alveolar bone chip, bone marrow from alveolar bone, periosteal tissue, and gingival tissue. Understanding their phenotype consecutively will provide a rational approach for alveolar ridge augmentation. PMID:22505911

  12. Cellular attachment and osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone

    PubMed Central

    Kim, Beom-Su; Kim, Jin Seong; Sung, Hark-Mo; You, Hyung-Keun; Lee, Jun

    2012-01-01

    The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. PMID:22447716

  13. 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.

  14. The cell biology of bone growth.

    PubMed

    Price, J S; Oyajobi, B O; Russell, R G

    1994-02-01

    The field of bone cell biology is clearly of relevance to the problem of stunting in children, as in the final analysis the cells of the growing long bone are the ultimate 'regulators'. It is the alterations in the functions of these cells that manifests as a reduction in height. Normal longitudinal growth is achieved by the coordinated recruitment, proliferation, differentiation, maturation and eventual death of the cells of growth plate and bone. Cellular activity is closely regulated by endocrine factors acting directly or indirectly, with factors produced locally and stored within the bone and cartilage microenvironment having a critical role in intercellular communication. Disruption of any of these processes can lead to growth disturbances, since it only requires a defect in a single gene to have profound effects. Studies in recent years have shed light on the biochemical and molecular effects of cytokines and growth factors and have shown that these regulatory molecules may mediate the effects of certain hormones important in controlling growth. However, the complex interrelationship of these molecules is still not clear. Notwithstanding, understanding of the mechanisms involved in bone remodelling is increasing, as this area attracts much research because of the high incidence of metabolic bone disease in Western society. Although studies of adult bone remodelling are of relevance, there is a requirement for increased research directed specifically at the mechanisms of endochondral ossification and its regulation. Longitudinal bone growth is a challenge to the cell biologist, since it is an accelerated cycle of cellular division and differentiation, within which it is not easy to separate events temporally and spatially. In addition, different regulatory mechanisms are probably important at different stages of growth. Another difficulty impeding progress in this field is the lack of appropriate animal models for research. Much information has come from

  15. 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.

  16. Chitosan-poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model.

    PubMed

    Costa-Pinto, A R; Correlo, V M; Sol, P C; Bhattacharya, M; Srouji, S; Livne, E; Reis, R L; Neves, N M

    2012-01-01

    Tissue engineering sustains the need of a three-dimensional (3D) scaffold to promote the regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population, allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our group evidenced significant growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded and cultured on melt-based porous chitosan fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of these in vitro 3D cell constructs. In this study, chitosan-based scaffolds were seeded and cultured in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in osteogenic culture conditions was performed into critical cranial size defects in nude mice. The objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks of implantation, scaffolds were harvested and prepared for micro-computed tomography (µCT) analysis. Retrieved implants showed good integration with the surrounding tissue and significant bone formation, more evident for the scaffolds cultured and implanted with human cells. The results of this work demonstrated that chitosan-based scaffolds, besides supporting in vitro proliferation and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects for their use in bone tissue-engineering therapies. PMID:21312336

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

    SciTech Connect

    Geng, Wenxin; Ma, Dongyang; Yan, Xingrong; Liu, Liangqi; Cui, Jihong; Xie, Xin; Li, Hongmin; Chen, Fulin

    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. Human breast cancer bone metastasis in vitro and in vivo: a novel 3D model system for studies of tumour cell-bone cell interactions.

    PubMed

    Holen, I; Nutter, F; Wilkinson, J M; Evans, C A; Avgoustou, P; Ottewell, Penelope D

    2015-10-01

    Bone is established as the preferred site of breast cancer metastasis. However, the precise mechanisms responsible for this preference remain unidentified. In order to improve outcome for patients with advanced breast cancer and skeletal involvement, we need to better understand how this process is initiated and regulated. As bone metastasis cannot be easily studied in patients, researchers have to date mainly relied on in vivo xenograft models. A major limitation of these is that they do not contain a human bone microenvironment, increasingly considered to be an important component of metastases. In order to address this shortcoming, we have developed a novel humanised bone model, where 1 × 10(5) luciferase-expressing MDA-MB-231 or T47D human breast tumour cells are seeded on viable human subchaodral bone discs in vitro. These discs contain functional osteoclasts 2-weeks after in vitro culture and positive staining for calcine 1-week after culture demonstrating active bone resorption/formation. In vitro inoculation of MDA-MB-231 or T47D cells colonised human bone cores and remained viable for <4 weeks, however, use of matrigel to enhance adhesion or a moving platform to increase diffusion of nutrients provided no additional advantage. Following colonisation by the tumour cells, bone discs pre-seeded with MDA-MB-231 cells were implanted subcutaneously into NOD SCID mice, and tumour growth monitored using in vivo imaging for up to 6 weeks. Tumour growth progressed in human bone discs in 80 % of the animals mimicking the later stages of human bone metastasis. Immunohistochemical and PCR analysis revealed that growing MDA-MB-231 cells in human bone resulted in these cells acquiring a molecular phenotype previously associated with breast cancer bone metastases. MDA-MB-231 cells grown in human bone discs showed increased expression of IL-1B, HRAS and MMP9 and decreased expression of S100A4, whereas, DKK2 and FN1 were unaltered compared with the same cells grown in

  19. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  20. Echistatin is a potent inhibitor of bone resorption in culture.

    PubMed

    Sato, M; Sardana, M K; Grasser, W A; Garsky, V M; Murray, J M; Gould, R J

    1990-10-01

    The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone

  1. Intracellular calcium waves in bone cell networks under single cell nanoindentation.

    PubMed

    Guo, X Edward; Takai, Erica; Jiang, Xingyu; Xu, Qiaobing; Whitesides, George M; Yardley, James T; Hung, Clark T; Chow, Eugene M; Hantschel, Thomas; Costa, Kevin D

    2006-09-01

    In this study, bone cells were successfully cultured into a micropatterned network with dimensions close to that of in vivo osteocyte networks using microcontact printing and self-assembled monolyers (SAMs). The optimal geometric parameters for the formation of these networks were determined in terms of circle diameters and line widths. Bone cells patterned in these networks were also able to form gap junctions with each other, shown by immunofluorescent staining for the gap junction protein connexin 43, as well as the transfer of gap-junction permeable calcein-AM dye. We have demonstrated for the first time, that the intracellular calcium response of a single bone cell indented in this bone cell network, can be transmitted to neighboring bone cells through multiple calcium waves. Furthermore, the propagation of these calcium waves was diminished with increased cell separation distance. Thus, this study provides new experimental data that support the idea of osteocyte network memory of mechanical loading similar to memory in neural networks. PMID:17263256

  2. Stromal cells and stem cells in clinical bone regeneration

    PubMed Central

    Grayson, Warren L.; Bunnell, Bruce A.; Martin, Elizabeth; Frazier, Trivia; Hung, Ben P.; Gimble, Jeffrey M.

    2015-01-01

    Stem-cell-mediated bone repair has been used in clinical trials for the regeneration of large craniomaxillofacial defects, to slow the process of bone degeneration in patients with osteonecrosis of the femoral head and for prophylactic treatment of distal tibial fractures. Successful regenerative outcomes in these investigations have provided a solid foundation for wider use of stromal cells in skeletal repair therapy. However, employing stromal cells to facilitate or enhance bone repair is far from being adopted into clinical practice. Scientific, technical, practical and regulatory obstacles prevent the widespread therapeutic use of stromal cells. Ironically, one of the major challenges lies in the limited understanding of the mechanisms via which transplanted cells mediate regeneration. Animal models have been used to provide insight, but these models largely fail to reproduce the nuances of human diseases and bone defects. Consequently, the development of targeted approaches to optimize cell-mediated outcomes is difficult. In this Review, we highlight the successes and challenges reported in several clinical trials that involved the use of bone-marrow-derived mesenchymal or adipose-tissue-derived stromal cells. We identify several obstacles blocking the mainstream use of stromal cells to enhance skeletal repair and highlight technological innovations or areas in which novel techniques might be particularly fruitful in continuing to advance the field of skeletal regenerative medicine. PMID:25560703

  3. The effects of simulated hypogravity on murine bone marrow cells

    NASA Technical Reports Server (NTRS)

    Lawless, Desales

    1989-01-01

    Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

  4. Cell sheet-engineered bones used for the reconstruction of mandibular defects in an animal model

    PubMed Central

    DU, CHUNHUA; YAO, CHAO; LI, NINGYI; WANG, SHUANGYI; FENG, YUANYONG; YANG, XUECAI

    2015-01-01

    The aim of the present study was to investigate the generation of cell sheet-engineered bones used for the reconstruction of mandibular defects. Bone marrow stem cells (BMSCs) were cultured and induced to generate osteoblasts. Poly(lactic-co-glycolic acid) (PLGA) scaffolds were wrapped with or without cell sheets and then implanted into dogs with mandibular defects in the right side (experimental group) or the left side (control group), respectively. Subsequently, X-ray analyses, and hematoxylin and eosin staining were performed at various time points (at 4, 8, 12 or 16 weeks post-implantation; n=4 at each time point). The osteogenesis in the experimental group was significantly improved compared with that in the control group. At 16 weeks after implantation, numerous Haversian systems and a few lamellar bones were observed at the periphery. In the control group, the engineered bone (without BMSC sheets) presented fewer Haversian systems and no lamellar bones. The optical density of the fresh bone in the experimental group was significantly higher compared with that in the control group (P<0.05). In conclusion, tissue-engineered bone with the structure of lamellar bones can be generated using BMSC sheets and implantation of these bones had an improved effects compared with the control group. Cell sheet transplantation was found to enhance bone formation at the reconstruction site of the mandibular defects. PMID:26668619

  5. Bone marrow stromal cell assays – in vitro and in vivo

    PubMed Central

    Robey, Pamela Gehron; Kuznetsov, Sergei A.; Riminucci, Mara; Bianco, Paolo

    2014-01-01

    Summary Populations of bone marrow stromal cells (BMSCs, also known as bone marrow-derived “mesenchymal stem cells”) contain a a subset of cells that are able to recapitulate the formation of a bone/marrow organ (skeletal stem cells, SSCs). The biological properties of BMSC cultures are assessed by a variety of assays, both in vitro and in vivo. Application of these assays in an appropriate fashion provide a great deal of information on the role of BMSCs, and the subset of SSCs, in health and in disease. PMID:24482181

  6. Isolation of Mouse Bone Marrow Mesenchymal Stem Cells.

    PubMed

    Boregowda, Siddaraju V; Krishnappa, Veena; Phinney, Donald G

    2016-01-01

    Mesenchymal stem cells (MSCs) were initially characterized as connective tissue progenitors resident in bone marrow, but have now been isolated from a variety of tissues and organs and shown to also exhibit potent tissue regenerative properties mediated largely via paracrine actions. These findings have spurred the development of MSC-based therapies for treating a diverse array of nonskeletal diseases. Although genetic and experimental rodent models of disease represent important tools for developing efficacious MSC-based therapies, development of reliable methods to isolate MSCs from mouse bone marrow has been hampered by the unique biological properties of these cells. Indeed, few isolation schemes afford high yields and purity while maintaining the genomic integrity of cells. We recently demonstrated that mouse MSCs are highly sensitive to oxidative stress, and long-term expansion of these cells in atmospheric oxygen selects for immortalized clones that lack a functional p53 protein. Herein, we describe a protocol for the isolation of primary MSCs from mouse bone marrow that couples immunodepletion with culture in a low-oxygen environment and affords high purity and yield while preserving p53 function. PMID:27236673

  7. Dissecting the Role of Bone Marrow Stromal Cells on Bone Metastases

    PubMed Central

    Buenrostro, Denise; Park, Serk In; Sterling, Julie A.

    2014-01-01

    Tumor-induced bone disease is a dynamic process that involves interactions with many cell types. Once metastatic cancer cells reach the bone, they are in contact with many different cell types that are present in the cell-rich bone marrow. These cells include the immune cells, myeloid cells, fibroblasts, osteoblasts, osteoclasts, and mesenchymal stem cells. Each of these cell populations can influence the behavior or gene expression of both the tumor cells and the bone microenvironment. Additionally, the tumor itself can alter the behavior of these bone marrow cells which further alters both the microenvironment and the tumor cells. While many groups focus on studying these interactions, much remains unknown. A better understanding of the interactions between the tumor cells and the bone microenvironment will improve our knowledge on how tumors establish in bone and may lead to improvements in diagnosing and treating bone metastases. This review details our current knowledge on the interactions between tumor cells that reside in bone and their microenvironment. PMID:25054153

  8. 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

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

    PubMed

    Oue, Erika; Lee, Ji-Won; Sakamoto, Kei; Iimura, Tadahiro; Aoki, Kazuhiro; Kayamori, Kou; Michi, Yasuyuki; Yamashiro, Masashi; Harada, Kiyoshi; Amagasa, Teruo; Yamaguchi, Akira

    2012-08-01

    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. PMID:22771802

  10. Cell-induced response by tetracyclines on human bone marrow colonized hydroxyapatite and Bonelike.

    PubMed

    Gomes, P S; Santos, J D; Fernandes, M H

    2008-05-01

    Semi-synthetic tetracyclines are commonly used antibiotics that also seem to play an important role in the modulation of the immuno-inflammatory imbalance, verified in several bone diseases. The association of a therapeutic agent (that prevents bacterial infection and induces tissue formation) to a biomaterial aiming to repair/regenerate bone defects could contribute to a more predictable clinical outcome. The present study intends to evaluate the proliferation and functional activity of osteoblast-induced human bone marrow cells, cultured on the surface of hydroxyapatite (HA) and Bonelike, in the presence of therapeutic concentrations of doxycycline and minocycline. First passage bone marrow cells were cultured for 35 days on the surface of HA and Bonelike discs, in the absence or presence of 1 microg ml(-1) doxycycline and minocycline. Cultures performed in standard tissue culture plates were used as control. Doxycycline or minocycline induced cell proliferation and increased the extent of matrix mineralization in osteoblastic cell cultures established in the three substrates. Also, an improved biological behavior was verified in seeded Bonelike compared with HA. The results suggest that the local delivery of tetracyclines might associate the antimicrobial activity in implant-related bone infection with an eventual induction of osteoblastic proliferation and maintenance of the characteristic biological activity of these cells. PMID:18291737

  11. 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

  12. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    SciTech Connect

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

    2007-12-31

    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  13. Disparate osteogenic response of mandible and iliac crest bone marrow stromal cells to pamidronate

    PubMed Central

    Stefanik, D.; Sarin, J.; Lam, T.; Levin, L.; Leboy, PS; Akintoye, SO.

    2010-01-01

    OBJECTIVE Long-term administration of intravenous bisphosphonates like pamidronate is associated with jaw osteonecrosis but axial and appendicular bones are unaffected. Pathogenesis of bisphosphonate-associated jaw osteonecrosis may relate to skeletal-site specific effects of bisphosphonates on osteogenic differentiation of bone marrow stromal cells (BMSCs) of orofacial and axial/appendicular bones. This study evaluated and compared skeletal site-specific osteogenic response of mandible (orofacial bone) and iliac crest (axial bone) human BMSCs to pamidronate. MATERIALS AND METHODS Mandible and iliac crest BMSCs from six normal healthy volunteers were established in culture and tested with pamidronate to evaluate and compare cell survival, osteogenic marker alkaline phosphatase, osteoclast differentiation in co-cultures with CD34+ hematopoietic stem cells, gene expression of receptor activator of NFκB ligand (RANKL) and osteoprotegerin, and in vivo bone regeneration. BRESULTS Mandible BMSCs were more susceptible to pamidronate than iliac crest BMSCs based on decreased cell survival, lower alkaline phosphatase production and structurally less organized in vivo bone regeneration. Pamidronate promoted higher RANKL gene expression and osteoclast recruitment by mandible BMSCs. CONCLUSION Mandible and iliac crest BMSC survival and osteogenic differentiation are disparately affected by pamidronate to favor dysregulated mandible bone homeostasis. PMID:18938273

  14. Giant Cell Tumor of Bone - An Overview

    PubMed Central

    Sobti, Anshul; Agrawal, Pranshu; Agarwala, Sanjay; Agarwal, Manish

    2016-01-01

    Giant Cell tumors (GCT) are benign tumors with potential for aggressive behavior and capacity to metastasize. Although rarely lethal, benign bone tumors may be associated with a substantial disturbance of the local bony architecture that can be particularly troublesome in peri-articular locations. Its histogenesis remains unclear. It is characterized by a proliferation of mononuclear stromal cells and the presence of many multi- nucleated giant cells with homogenous distribution. There is no widely held consensus regarding the ideal treatment method selection. There are advocates of varying surgical techniques ranging from intra-lesional curettage to wide resection. As most giant cell tumors are benign and are located near a joint in young adults, several authors favor an intralesional approach that preserves anatomy of bone in lieu of resection. Although GCT is classified as a benign lesion, few patients develop progressive lung metastases with poor outcomes. Treatment is mainly surgical. Options of chemotherapy and radiotherapy are reserved for selected cases. Recent advances in the understanding of pathogenesis are essential to develop new treatments for this locally destructive primary bone tumor. PMID:26894211

  15. Doxycycline inhibits bone resorption by human interface membrane cells from aseptically loose hip replacements.

    PubMed

    Ong, S M; Taylor, G J S

    2003-04-01

    Matrix metalloproteinases (MMPs) may have a role in the process of aseptic loosening. Doxycycline has been shown to inhibit MMPs. Our aim was to investigate the potential pharmacological effect of doxycycline on aseptic loosening. We used radiolabelled mouse calvariae cultured with human interface membrane cells from aseptically loosened hips. Bone resorption was confirmed in this model. The effect of doxycycline was assessed by culturing dead radiolabelled bone discs with cells from the interface membrane with doxycycline. The control group consisted of the same culture system without doxycycline. Supernatant 45calcium and the total 45calcium remaining in the bone discs at the completion of the culture were used to measure osteolysis. We found that doxycycline can inhibit osteolysis at the interface membrane of aseptically loosened hips. This may have therapeutic implications for the treatment of patients with aseptic loosening of total joint replacements. PMID:12729128

  16. Rare giant cell tumor involvement of the olecranon bone

    PubMed Central

    Yang, Chen; Gong, Yubao; Liu, Jianguo; Qi, Xin

    2014-01-01

    Giant cell tumor (GCT) of bone is a relatively common benign bone lesion and is usually located in long bones, but involvement of the olecranon is extremely rare. Here, we present a case of solitary GCT of bone in the olecranon that was confirmed by preoperative needle biopsy and postoperative histological examination. The treatment included intralesional curettage, allogeneic bone grafting, and plating. At 26 months follow-up, the patient had no local recurrence. PMID:25197303

  17. Adipose derived stem cells for treatment of mandibular bone defects: An autologous study in dogs

    PubMed Central

    Haghighat, Abbas; Akhavan, Ali; Hashemi-Beni, Batool; Deihimi, Parviz; Yadegari, Afshin; Heidari, Fariba

    2011-01-01

    Background: The aim of this research was to evaluate the effect of adipose derived stem cells on bone repair in through and through mandibular bone defects of canine. Materials and Methods: In this prospective comparative study, adipose-derived stem cells were isolated from subcutaneous fat of lateral thoracic area of 4 dogs. The isolated cells were cultured and expanded through 3 passages. The undifferentiated stem cells were seeded in Collatamp and transferred into mandibular bone through-and-through defects. Similar defects on control group were filled with cell-free Collatamp. After 6 weeks, biopsies were taken and histomorphometric analysis was performed. The percentage of new bone formation was measured in each case. The data were subject to statistical analysis using the Wilcoxon test. Differences at P≤0.05 were considered significant. Results: H and E staining of decalcified samples revealed more bone formation in the group, which stem cells were seeded. Cell-free collatamp group revealed an average bone regeneration of %41±13.21, while adipose derived stem cell-seeded collatamp group showed %49±8.24. Conclusion: The use of stem cell seeded collatamp scaffold in mandibular defects caused more bone regeneration. PMID:23372596

  18. Recent advances in bone regeneration using adult stem cells.

    PubMed

    Zigdon-Giladi, Hadar; Rudich, Utai; Michaeli Geller, Gal; Evron, Ayelet

    2015-04-26

    Bone is a highly vascularized tissue reliant on the close spatial and temporal association between blood vessels and bone cells. Therefore, cells that participate in vasculogenesis and osteogenesis play a pivotal role in bone formation during prenatal and postnatal periods. Nevertheless, spontaneous healing of bone fracture is occasionally impaired due to insufficient blood and cellular supply to the site of injury. In these cases, bone regeneration process is interrupted, which might result in delayed union or even nonunion of the fracture. Nonunion fracture is difficult to treat and have a high financial impact. In the last decade, numerous technological advancements in bone tissue engineering and cell-therapy opened new horizon in the field of bone regeneration. This review starts with presentation of the biological processes involved in bone development, bone remodeling, fracture healing process and the microenvironment at bone healing sites. Then, we discuss the rationale for using adult stem cells and listed the characteristics of the available cells for bone regeneration. The mechanism of action and epigenetic regulations for osteogenic differentiation are also described. Finally, we review the literature for translational and clinical trials that investigated the use of adult stem cells (mesenchymal stem cells, endothelial progenitor cells and CD34(+) blood progenitors) for bone regeneration. PMID:25914769

  19. Assessing the repair of critical size bone defects performed in a goat tibia model using tissue-engineered constructs cultured in a bidirectional flow perfusion bioreactor.

    PubMed

    Gardel, Ls; Afonso, M; Frias, C; Gomes, Me; Reis, Rl

    2014-01-01

    This work evaluated in vivo performance of a tissue-engineered bone-like matrix obtained by culturing cell-scaffold constructs in a flow perfusion bioreactor, designed to enable culture of large constructs, envisioning the regeneration of critical-sized defects. A blend of starch with polycaprolactone scaffolds was seeded with goat bone marrow stromal cells (GBMSCs) cultured in the perfusion bioreactor for 14 days using osteogenic medium. Cell seeded scaffolds cultured in static conditions acted as controls. After 14 days, constructs (42 mm length and 16 mm in diameter) were implanted in critical size defects performed in the tibial bone of six adult goats from which the bone marrow had been collected previously. Explants were retrieved after six and 12 weeks of implantation and characterized using scanning electron microscopy, energy-dispersive spectroscopy, micro-computed tomography and radiographic analysis to assess tissue morphology and calcification. Explants were histologically analyzed, using Hematoxylin & Eosin and Masson Trichrome staining. Results provided relevant information about the performance and functionality of starch with polycaprolactone-goat bone marrow stromal cell constructs in a critical size orthotopic defect performed in a large animal model and demonstrated that culture of the starch with polycaprolactone scaffolds with the autologous cells in perfusion culture provide a good therapy for the healing and regenerative process of bone defects. PMID:24413026

  20. [CHARACTERISTICS OF OSTEOCYTE CELL LINES FROM BONES FORMED AS A RESULT OF MEMBRANOUS (SKULL BONES) AND CHONDRAL (LONG BONES) OSSIFICATION].

    PubMed

    Avrunin, A S; Doktorov, A A

    2016-01-01

    The aim of this work was to analyze the literature data and the results of authors' own research, to answer the question--if the osteocytes of bone tissues resulting from membranous and chondral ossification, belong to one or to different cell lines. The differences between the cells of osteocyte lines derived from bones resulting from membranous and chondral ossification were established in: 1) the magnitude of the mechanical signal, initiating the development of the process of mechanotransduction; 2) the nature of the relationship between the magnitude of the mechanical signal that initiates the reorganization of the architecture of bone structures and the resource of their strength; in membranous bones significantly lower mechanical signal caused a substantially greater increment of bone strength resource; 3) the biological activity of bone structures, bone fragments formed from membranous tissue were more optimal for transplantation; 4) the characteristics of expression of functional markers of bone cells at different stages of their differentiation; 5) the nature of the reaction of bone cells to mechanical stress; 6) the sensitivity of bone cells to one of the factors controlling the process of mechanotransduction (PGI2); 7) the functioning of osteocytes during lactation. These differences reflect the functional requirements to the bones of the skeleton--the supporting function in the bones of the limbs and the shaping and protection in the bones of the cranial vault. These data suggest that the results of research conducted on the bones of the skull, should not be transferred to the entire skeleton as a whole. PMID:27487656

  1. Aseptic technique for cell culture.

    PubMed

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here. PMID:18228291

  2. Nanostructured magnesium increases bone cell density

    NASA Astrophysics Data System (ADS)

    Weng, Lucy; Webster, Thomas J.

    2012-12-01

    Magnesium has attracted some attention in orthopedics due to its biodegradability and mechanical properties. Since magnesium is an essential natural mineral for bone growth, it can be expected that as a biomaterial, it would support bone formation. However, upon degradation in the body, magnesium releases OH- which results in an alkaline pH that can be detrimental to cell density (for example, osteoblasts or bone forming cells). For this reason, modification of magnesium may be necessary to compensate for such detrimental effects to cells. This study created biologically inspired nanoscale surface features on magnesium by soaking magnesium in various concentrations of NaOH (from 1 to 10 N) and for various periods of time (from 10 to 30 min). The results provided the first evidence of increased roughness, surface energy, and consequently greater osteoblast adhesion, after 4 h as well as density up to 7 days on magnesium treated with any concentration of NaOH for any length of time compared to untreated controls. For these reasons, this study suggests that soaking magnesium in NaOH could be an inexpensive, simple and effective manner to promote osteoblast functions for numerous orthopedic applications and, thus, should be further studied.

  3. 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

  4. Sonic hedgehog signaling promotes growth of oral squamous cell carcinoma cells associated with bone destruction.

    PubMed

    Honami, Tatsuki; Shimo, Tsuyoshi; Okui, Tatsuo; Kurio, Naito; Hassan, Nur Mohammad Monsur; Iwamoto, Masahiro; Sasaki, Akira

    2012-01-01

    Sonic hedgehog (Shh) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of Shh in bone destruction associated with oral squamous cell carcinomas, which frequently invade the maxilla or the mandible, is still unclear. In this study we show that the use of siRNA for Shh to block SHH secreted by SAS oral squamous cell carcinoma cells suppressed the tumor growth and tumor angiogenesis of subcutaneous SAS xenografts in vivo. Moreover, blockade of Shh in SAS cells decreased tumor growth and osteoclast number in a tibial metaphysis mouse model. Significantly, we clearly show that SHH stimulated osteoclast formation in a co-culture system consisting of murine bone stromal ST2 cells and murine CD11b(+) bone marrow cells. These findings suggest that Shh signaling is a potential target for the treatment of oral squamous cell carcinoma associated with bone destruction. PMID:21945071

  5. Bone Defect Regeneration by a Combination of a β-Tricalcium Phosphate Scaffold and Bone Marrow Stromal Cells in a Non-Human Primate Model

    PubMed Central

    Masaoka, Tomokazu; Yoshii, Toshitaka; Yuasa, Masato; Yamada, Tsuyoshi; Taniyama, Takashi; Torigoe, Ichiro; Shinomiya, Kenichi; Okawa, Atsushi; Morita, Sadao; Sotome, Shinichi

    2016-01-01

    Background: Reconstruction of large bone defects is a great challenge in orthopedic research. In the present study, we prepared composites of bone marrow-derived stromal cells (BMSCs) and β-tricalcium phosphate (β-TCP) with three novel aspects: proliferation of BMSCs with continuous dexamethasone treatment, cell loading under low pressure, and use of autologous plasma as the cell loading medium. The effectiveness of the resulting composite for large bone-defect reconstruction was tested in a non-human primate model, and the bone union capability of the regenerated bones was examined. Materials and Methods: Primary surgery: Bone defects (5 cm long) were created in the left femurs of nine cynomolgus monkeys with resection of the periosteum (five cases) or without resection (four cases), and porous β-TCP blocks were transplanted into the defects. Secondary surgery: Bone marrow aspirates harvested from seven of the nine monkeys were cultured with dexamethasone, and BMSCs were obtained. BMSCs were suspended in autologous plasma and introduced into a porous β-TCP block under low-pressure conditions. The BMSC/β-TCP composites were transplanted into bone defects created at the same sites as the primary surgery. Bone union evaluation: Five regenerated femurs were shortened by osteotomy surgery 8 to 15 months after transplantation of the β-TCP/BMSC composites, and bone union was evaluated radiographically. Results: After the primary surgery and treatment with β-TCP alone, one of the five periosteum-resected monkeys and two of the four periosteum-preserved monkeys exhibited successful bone reconstruction. In contrast, five of the seven cases treated with the β-TCP/MSC composite showed successful bone regeneration. In four of the five osteotomy cases, bone union was confirmed. Conclusion: We validated the effectiveness of a novel β-TCP/BMSC composite for large bone defect regeneration and confirmed the bone union capability of the regenerated bone. PMID:27073583

  6. Cultured Human Renal Cortical Cells

    NASA Technical Reports Server (NTRS)

    1998-01-01

    During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

  7. Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

    PubMed Central

    Bruce, Allison; Evans, Rebecca; Mezan, Ryan; Shi, Lin; Moses, Blake S.; Martin, Karen H.; Gibson, Laura F.; Yang, Yong

    2015-01-01

    Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology. PMID:26488876

  8. Cell culture compositions

    DOEpatents

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  9. Principles of cancer cell culture.

    PubMed

    Cree, Ian A

    2011-01-01

    The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory. PMID:21516394

  10. Langerhans Cell Histiocytosis of the Temporal Bone.

    PubMed

    Ginat, Daniel Thomas; Johnson, Daniel N; Cipriani, Nicole A

    2016-06-01

    Langerhans cell histiocytosis involving the temporal bone region is uncommon and can resemble malignant neoplasms on imaging due to high cellularity. Although recognizing the presence of sharp margins with beveled-edges can be helpful, tissue sampling is often necessary for confirming the diagnosis. Cytology classically demonstrates kidney-bean shaped nuclei within the Langerhans cells and immunohistochemical staining is positive for S-100, peanut agglutinin (PNA), MHC class II, CD1a, and Langerin (CD 207). These features are exemplified in this sine qua non radiology-pathology correlation article. PMID:25903273